Population pharmacokinetic modelling of rupatadine solution in 6-11 year olds and optimisation of the experimental design in younger children.

AIMS: To optimise a pharmacokinetic (PK) study design of rupatadine for 2-5 year olds by using a population PK model developed with data from a study in 6-11 year olds. The design optimisation was driven by the need to avoid children's discomfort in the study. METHODS: PK data from 6-11 year olds with allergic rhinitis available from a previous study were used to construct a population PK model which we used in simulations to assess the dose to administer in a study in 2-5 year olds. In addition, an optimal design approach was used to determine the most appropriate number of sampling groups, sampling days, total samples and sampling times. RESULTS: A two-compartmental model with first-order absorption and elimination, with clearance dependent on weight adequately described the PK of rupatadine for 6-11 year olds. The dose selected for a trial in 2-5 year olds was 2.5 mg, as it provided a Cmax below the 3 ng/ml threshold. The optimal study design consisted of four groups of children (10 children each), a maximum sampling window of 2 hours in two clinic visits for drawing three samples on day 14 and one on day 28 coinciding with the final examination of the study. CONCLUSIONS: A PK study design was optimised in order to prioritise avoidance of discomfort for enrolled 2-5 year olds by taking only four blood samples from each child and minimising the length of hospital stays.

D-Optimal mixture design optimization of an HPLC method for simultaneous determination of commonly used antihistaminic parent molecules and their active metabolites in human serum and urine.

This study describes a specific, precise, sensitive and accurate method for simultaneous determination of hydroxyzine, loratadine, terfenadine, rupatadine and their main active metabolites cetirizine, desloratadine and fexofenadine, in serum and urine using meclizine as an internal standard. Solid-phase extraction method for sample clean-up and preconcentration of analytes was carried out using Phenomenex Strata-X-C and Strata X polymeric cartridges. Chromatographic analysis was performed on a Phenomenex cyano (150 × 4.6 mm i.d., 5 µm) analytical column. A D-optimal mixture design methodology was used to evaluate the effect of changes in mobile phase compositions on dependent variables and optimization of the response of interest. The mixture design experiments were performed and results were analyzed. The region of ideal mobile phase composition consisting of acetonitrile-methanol-ammonium acetate buffer (40 mm; pH 3.8 adjusted with acetic acid): 18:36:46% v/v/v was identified by a graphical optimization technique using an overlay plot. While using this optimized condition all analytes were baseline resolved in <10 min. Solvent mixtures were delivered at 1.5 mL/min flow rate and analytes peaks were detected at 222 nm. The proposed bioanalytical method was validated according to US Food and Drug Administration guidelines. The proposed method was sensitive with detection limits of 0.06-0.15 µg/mL in serum and urine samples. Relative standard deviation for inter- and intra-day precision data was found to be <7%. The proposed method may find application in the determination of selected antihistaminic drugs in biological fluids.

Quantification of cyproheptadine in human plasma by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry in a bioequivalence study.

A rapid, sensitive and specific method to quantify cyproheptadine in human plasma using amitriptyline as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid-liquid extraction using a diethyl-ether/dichloromethane (70/30; v/v) solvent. After removing and drying the organic phase, the extracts were reconstituted with a fixed volume of acetonitrile/water (50/50 v/v)+0.1% of acetic acid. The extracts were analyzed by high performance liquid chromatography coupled to electrospray tandem mass spectrometry (LC-MS/MS). Chromatography was performed isocratically using an Alltech Prevail C18 5 µm analytical column, (150 mm x 4.6 mm I.D.). The method had a chromatographic run time of 4 min and a linear calibration curve ranging from 0.05 to 10 ng/mL (r2 > 0.99). The limit of quantification was 0.05 ng/mL. This HPLC/MS/MS procedure was used to assess the bioequivalence of cyproheptadine in two cyproheptadine + cobamamide (4 mg + 1 mg) tablet formulations (Cobactin® [cyproheptadine + cobamamide] test formulation supplied from Zambon Laboratórios Farmacêuticos Ltda. and Cobavital® from Solvay Farma (standard reference formulation)). A single 4 mg + 1 mg [cyproheptadine + cobamamide] dose of each formulation was administered to healthy volunteers. The study was conducted using an open, randomized, two-period crossover design with a 1-week washout interval. Since the 90% CI for Cmax and AUCs ratios were all within the 80-125% bioequivalence limit proposed by the US Food and Drug Administration, it was concluded that the cyproheptadine test formulation (Cobactin®) is bioequivalent to the Cobavital® formulation for both the rate and the extent of absorption of cyproheptadine.

Determination of azatadine in human plasma by liquid chromatography/tandem mass spectrometry.

A sensitive method using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) was developed and validated for the analysis of antihistamine drug azatadine in human plasma. Loratadine was used as internal standard (IS). Analytes were extracted from human plasma by liquid/liquid extraction using ethyl acetate. The organic phase was reduced to dryness under a stream of nitrogen at 30 °C and the residue was reconstituted with the mobile phase. 5 µL of the resulting solution was injected onto the LC-MS/MS system. A 4.6 mm × 150 mm, I.D. 5 µm, Agilent TC-C(18) column was used to perform the chromatographic analysis. The mobile phase consisted of ammonium formate buffer 0.010 M (adjusted to pH 4.3 with 1M formic acid)/acetonitrile (20:80, v/v) The chromatographic run time was 5 min per injection and flow rate was 0.6 mL/min. The retention time was 2.4 and 4.4 min for azatadine and IS, respectively. The tandem mass spectrometric detection mode was achieved with electrospray ionization (ESI) iron source and the multiple reaction monitoring (MRM) (291.3 → 248.2m/z for azatadine, 383.3 → 337.3m/z for IS) was operated in positive ion modes. The low limit of quantitation (LLOQ) was 0.05 ng/mL. The intra-day and inter-day precision of the quality control (QC) samples was 8.93-11.57% relative standard deviation (RSD). The inter-day accuracy of the QC samples was 96.83-105.07% of the nominal values.

Simultaneous determination of rupatadine and its metabolite desloratadine in human plasma by a sensitive LC-MS/MS method: application to the pharmacokinetic study in healthy Chinese volunteers.

A sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous determination of rupatadine and its metabolite desloratadine in human plasma. After the addition of diphenhydramine, the internal standard (IS), plasma samples were extracted with a mixture of methyl tert-butyl ether and n-hexane (1:1, v/v). The analysis was performed on a Ultimate AQ-C18 (4.6mm x 100mm, 5microm) column using a mobile phase consisting of a 80/20 mixture of methanol/water containing 0.0005% formic acid pumped at 0.3mlmin(-1). The analytes and the IS were detected in positive ionization mode and monitoring their precursor-->product ion combinations of m/z 416-->309, 311-->259, and 256-->167, respectively, in multiple reaction monitoring mode. The linear ranges of the assay were 0.1-50 and 0.1-20ngml(-1) for rupatadine and desloratadine, respectively. The lower limits of reliable quantification for both rupatadine and desloratadine were 0.1ngml(-1), which offered high sensitivity and selectivity. The within- and between-run precision was less than 7.2%. The accuracy ranged from -9.2% to +6.4% and -7.2% to +7.2% for rupatadine and desloratadine in quality control samples at three levels, respectively. The method has been successfully applied to a pharmacokinetic study of rupatadine and its major metabolite after oral administration of 10, 20 and 40mg rupatadine tablets to healthy Chinese volunteers.

High performance liquid chromatography-tandem mass spectrometric determination of rupatadine in human plasma and its pharmacokinetics.

A simple, rapid, sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the quantification of rupatadine in human plasma using estazolam as internal standard (IS). Following liquid-liquid extraction, the analytes were separated using a mobile phase of methanol-ammonium acetate (pH 2.2; 5mM) (50:50, v/v) on a reverse phase C18 column and analyzed by a triple-quadrupole mass spectrometer in the positive ion and multiple reaction monitoring (MRM) mode, m/z 416-->309 for rupatadine and m/z 295-->267 for the IS. The assay exhibited a linear dynamic range of 0.1-100 ng/ml for rupatadine in human plasma. The lower limit of quantification (LLOQ) was 0.1 ng/ml with a relative standard deviation of less than 20%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The validated LC-MS/MS method has been successfully applied to study the pharmacokinetics of rupatadine in healthy volunteers.

Evaluation of the cognitive, psychomotor and pharmacokinetic profiles of rupatadine, hydroxyzine and cetirizine, in combination with alcohol, in healthy volunteers.

INTRODUCTION: The Central Nervous System (CNS) impairment induced by moderate alcohol (ALC) ingestion may be enhanced if other drugs are taken simultaneously. Rupatadine (RUP) is a new H(1)-antihistamine which also inhibits platelet activating factor (PAF) release in inflammatory reactions. OBJECTIVE: The main aim of the study was to assess the effects of ALC 0.8 g/Kg on RUP (10 mg and 20 mg) CNS effects. An evaluation of alcohol and RUP pharmacokinetics was also attained. METHODS: Eighteen healthy young volunteers of both sexes participated in a phase I, randomised, crossover, double-blind, placebo-controlled study. At 2-week intervals they received six treatments: (a) placebo (PLA), (b) ALC alone and ALC in combination with: (c) hydroxyzine 25 mg (HYD), (d) cetirizine 10 mg (CET), (e) RUP 10 mg or (f) RUP 20 mg. At baseline and several times thereafter, seven psychomotor performance tests (finger tapping, fine motoric skills, nystagmus, temporal estimation, critical-flicker-fusion frequency, 'd2' cancellation, simple reaction) and eleven subjective self-reports (drunkenness, sleepiness, alertness, clumsiness, anger, inattentiveness, efficiency, happiness, hostility, interest and extraversion) were carried out. Two-way (treatment, time) ANOVAs for repeated measures to each variable together with a multivariate non-parametric approach were applied. Plasma concentrations of alcohol, and of RUP and its metabolites, were quantified by validated immunofluorescence and LC/MS/MS methods, respectively. Plasma-time curves for all compounds were analysed by means of model-independent methods. RESULTS: The combination of alcohol with HYD, CET and RUP 20 mg produced more cognitive and psychomotor impairment as compared to alcohol alone, being the combination of alcohol and HYD the one which induced the greatest deterioration. The combination of alcohol and RUP 10 mg could not be differentiated from ALC alone. Subjective self-reports reflect effects on metacognition after the combination of alcohol with HYD and CET i.e. the increased objective impairment observed was not subjectively perceived by the subjects. No significant differences were obtained when comparing alcohol plasma concentrations assessed after the treatments evaluated. RUP showed a lineal kinetic relationship after 10 and 20 mg with a higher exposition to both metabolites assayed. CONCLUSIONS: Present results showed that single oral doses of rupatadine 10 mg in combination with alcohol do not produce more cognitive and psychomotor impairment than alcohol alone. Higher doses of rupatadine, in combination with alcohol, may induce cognitive and psychomotor deterioration as hydroxyzine and cetirizine at therapeutic doses.

Evaluation of tricyclic antidepressant false positivity in a pediatric case of cyproheptadine (periactin) overdose.

CASE REPORT: The authors report a case of pediatric cyproheptadine toxicity, initially misdiagnosed as tricyclic toxicity based on the results of a preliminary rapid toxicological serum screen. Although such cross-reactivity has been reported, the chemical basis of this observation has not yet been evaluated. By GC/MS methods and HPLC assays adapted for the detection of tricyclic compounds, the authors confirmed that cyproheptadine was indeed responsible for this patient's toxicity. In addition, the authors identified the presence of a cyproheptadine metabolite in the patient's serum. Further testing in an immunoassay-based toxicologic screen demonstrated some cross-reactivity exhibited by the patient's serum, but not the parent compound. These findings showed that the cross-reactivity correlated with the presence of the cyproheptadine metabolite, highlighting the value of confirmatory toxicologic testing of routine rapid toxicologic screens.

Disposition of cyproheptadine in cats after intravenous or oral administration of a single dose.

OBJECTIVE: To determine disposition of cyproheptadine hydrochloride in cats after intravenous or oral administration of a single dose. ANIMALS: 6 healthy cats. PROCEDURE: A randomized crossover design was used, and each cat was studied after intravenous (2 mg) and oral (8 mg) administration of cyproheptadine. Blood samples were collected at fixed time intervals after drug administration, and serum cyproheptadine concentration was determined by means of polarized immunofluorescence. RESULTS: Mean (+/- SD) residence time was significantly longer after oral (823 +/- 191 minutes) than after intravenous (339 +/- 217 minutes) administration, but no significant differences were detected between other pharmacokinetic parameters after oral and intravenous administration. Mean +/- SD oral bioavailability was 1.01 +/- 0.36. Mean elimination half-life after oral administration was 12.8 +/- 9.9 hours. Peak extrapolated cyproheptadine concentration was 669 +/- 206 ng/ml. Only 1 cat developed adverse effects (transient vocalization). CONCLUSIONS: Cyproheptadine appeared to be well tolerated in cats and had high bioavailability after oral administration. The mean elimination half-life of 12 hours indicated that approximately 2.5 days must elapse to achieve steady-state concentrations of cyproheptadine after oral administration of multiple doses. A 12-hour dosing interval is acceptable, but an 8-hour interval may be indicated for some cats. CLINICAL RELEVANCE: On the basis of pharmacokinetic parameters determined in this study, the oral form of cyproheptadine appears to be suitable for use in clinical trials to treat anorexia in cats. Its half-life is compatible with once or twice daily dosing.

Pharmacokinetics of cyproheptadine and its metabolites in rats.

To investigate the pharmacokinetics of cyproheptadine (CPH) and its metabolites, the plasma concentration and urinary excretion of CPH and its detectable metabolites were determined after intravenous (i.v.) administration of parent or synthesized metabolites to rats. The plasma CPH concentration-time course was subjected to biexponential calculation following the i.v. administration of CPH, producing the temporal and low plasma concentrations of desmethylcyproheptadine (DMCPH) and the sustained plasma concentrations of desmethylcyproheptadine-epoxide (DMCPHepo). DMCPH was also eliminated, according to the biexponential equation, after i.v. administration of performed DMCPH, forming DMCPHepo in plasma. On the other hand, no detectable DMCPHepo was found in plasma after the i.v. administration of cyproheptadine epoxide (CPHepo). All compounds administered had large distribution volumes and were almost entirely excreted as DMCPHepo in urine; this excretion continued for a long time. However, the urinary excretion pattern of DMCPHepo after CPHepo was different from those after CPH and DMCPH. The mean residence times of the epoxidized metabolites estimated from the urinary data were much longer than those from the plasma concentration data, suggesting either a gradual reflux of the metabolites from a tissue depot into systemic circulation under those plasma concentrations close of detection limit, or some interaction which delays excretion into the urine. This study suggests that both metabolic pathways of CPH, through DMCPH and CPHepo, to DMCPHepo are possible, but that the demethylation of CPH largely occurs prior to epoxidation; also that the extensive and persistent distribution of DMCPHepo to tissues may relate to the toxicity of CPH reported in rats.

The pharmacokinetics of loratadine in normal geriatric volunteers.

The pharmacokinetics of loratadine, a non-sedating anti-histamine, were studied in 12 normal geriatric volunteers. In an open label fashion, each volunteer received one 40 mg loratadine capsule. Blood was collected prior to and at specified times (up to 120 h) after dosing. Plasma loratadine concentrations were determined by a specific radioimmunoassay and those of an active metabolite, descarboethoxyloratadine, by high performance liquid chromatography. Concentrations of loratadine in the disposition phase were fitted to a biexponential equation and those of descarboethoxyloratadine to either a monoexponential or biexponential equation for pharmacokinetic analysis. Loratadine was rapidly absorbed, reaching a maximum plasma concentration of 50.5 ng/ml at 1.5 h after dosing. The disposition half-lives of loratadine in the distribution and elimination phases were 1.5 and 18.2 h, respectively. The area under the plasma concentration-time curve, was 146.7 h.ng/ml. Descarboethoxyloratadine had a maximum plasma concentration of 28.0 ng/ml at 2.9 h post-dose and an area under the concentration-time curve of 394.9 h.ng/ml. Its disposition half-lives in the distribution and elimination phases were 2.8 and 17.4 h, respectively. Comparison of these data with those from a previous study of loratadine in young adults showed no clear differences in the disposition half-lives between the two groups. The clearance of loratadine tends to be lower in the elderly, but inter-individual variation within each age group appears greater than any age effect.

GLC determination of (-)-1-cyclopropylmethyl-4-(3-trifluoromethylthio-5H-dibenzo [a,d]cyclohepten-5-ylidene)piperidine in human plasma and urine.

(-)-1-Cyclopropylmethyl-4-(3-trifluoromethylthio-5H-dibenzo [a,d]cyclohepten-5-ylidene)piperidine (MK-160) was extracted from human plasma and urine with petroleum ether and quantitated by GLC using a nitrogen-sensitive detector. A homologue of the drug served as the internal standard. The method is specific for the drug in the presence of potential metabolites and is capable of measuring concentrations in plasma as low as 6 ng/ml.

Disposition of cyproheptadine in rats, mice, and humans and identification of a stable epoxide metabolite.

Radioactivity was excreted in the urine and feces of rats, mice, and humans after a dose of 14C-cyproheptadine. The major metabolite in rat urine was unconjugated, but the majority of radioactive materials in mouse and human urine were conjugated with glucuronic acid. Identification of the rat urinary metabolite of cyproheptadine as an epoxide was accomplished with mass spectrometry and other methods. The rat metabolite was 10.11 -epoxydesmethylcyproheptadine and accounted for about 25% of a 45-mg dose of cyproheptadine per kg. Only a small amount of this epoxide was found in mouse urine, and none was apparent in the urine of two humans who received 5 mg of the drug. Dihydrodiols, which could arise by epoxide hydrase hydrolysis of possible 10.11-epoxy metabolites, were not found in the urine of any of the species studied. The spoxide found in rat urine appears to be unusually stable to in vivo hydrolysis. Possible implications of these results in the species-selective pancreotoxicity of cyproheptadine in the rat are presented.