Quantitative prediction of histamine H1 receptor occupancy by the sedative and non-sedative antagonists in the human central nervous system based on systemic exposure and preclinical data.

Significant histamine H1 receptor occupation in the central nervous system (CNS) is associated with sedation. Here we examined the time profiles of the H1 receptor occupancy (RO) in the CNS using sedative (diphenhydramine and ketotifen) and non-sedative (bepotastine and olopatadine) antagonists at their therapeutic doses by integrating in vitro and animal data. A pharmacokinetic model was constructed to associate plasma concentrations and receptor binding in the brain. Dissociation and association rate constants with the H1 receptor and plasma and brain unbound fractions were determined in vitro. Passive and active clearances across the blood-brain barrier (BBB) were estimated based on physicochemical properties and microdialysis studies in mice and monkeys. The estimated RO values were comparable with the reported values determined at time to maximum concentration (Tmax) of plasma by positron-emission tomography in humans. The simulation suggested that the predicted maximum ROs by bepotastine and olopatadine were greater than the reported values. Sensitivity analysis showed that active transport across BBB had a significant impact on the RO duration of the H1 antagonists examined. The present study demonstrated that modeling and simulation permits a reasonable RO estimation in the human CNS. Our findings will facilitate the development of CNS-acting drugs.

Application of a continuous square-wave potential program for sub nano molar determination of ketotifen.

An electroanalytical method has been developed for the determination of the ketotifen by continuous square wave adsorptive stripping voltammetry on a ultra-gold microelectrode (Au UME) in aqueous solution with phosphate buffer as supporting electrolyte. The best adsorption conditions were found to be pH 2.3, an accumulation potential of 300 mV (Au vs. Ag: AgCl-KCl 3 M) and an accumulation time of 400 ms. Variation of admittance in the detection process is created by inhibition of oxidation reaction of the electrode surface, by adsorption of ketotifen. Furthermore, signal-to-noise ratio was significantly increased by application of discrete fast Fourier transform (FFT) method, background subtraction and two-dimensional integration of the electrode response over a selected potential range and time window. Also in this work some parameters such as square-wave frequency, eluent pH, and accumulation time were optimized. Effects of square-wave frequency, step potential and pulse amplitude were examined for the optimization of instrumental conditions. The calibration curve is linear in the range 2.0x10(-7)---5.0x10(-12) M with a detection limit of 2.0x10(-12) M (ca. 0.7 pg/ml). The method maybe applied direct determination of the drug in pharmaceutical and biological samples. For a concentration of 5.0x10(-8) M a recovery value of 99.89% is obtained.

Ketotifen-loaded microspheres prepared by spray-drying poly(D,L-lactide) and poly(D,L-lactide-co-glycolide) polymers: characterization and in vivo evaluation.

Ketotifen (KT) was encapsulated into poly(D,L-lactide) (PLA) and poly(D,L-lactide-co-glycolide) (PLGA 50/50) by spray-drying to investigate the use of biodegradable drug-loaded microspheres as delivery systems in the intraperitoneal cavity. Ketotifen stability was evaluated by HPLC, and degradation was not observed. Drug entrapment efficiency was 74 +/- 7% (82 +/- 8 microg KT/mg for PLA) and 81 +/- 6% (90 +/- 7 microg KT/mg for PLGA 50/50). PLA microspheres released ketotifen (57% of encapsulated KT) in 350 h at two release rates (221 microg/h, 15 min to 2 h; 1.13 microg/h, 5-350 h). A quicker release of ketotifen took place from PLGA 50/50 microspheres (67.4% of encapsulated KT) in 50 h (322 microg/h, 15 min to 2 h; 16.18 microg/h, 5-50 h). After intraperitoneal administration (10 mg KT/kg b.w.), microsphere aggregations were detected in adipose tissue. Ketotifen concentration was determined in plasma by HPLC. The drug released from PLA and PLGA 50/50 microspheres was detected at 384 and 336 h, respectively. Noncompartmental analysis was performed to determine pharmacokinetic parameters. The inclusion of ketotifen in PLGA and PLA microspheres resulted in significant changes in the plasma disposition of the drug. Overall, these ketotifen-loaded microspheres yielded an intraperitoneal drug release that may be suitable for use as delivery systems in the treatment of inflammatory response in portal hypertension.

Brain histamine H receptor occupancy of orally administered antihistamines measured by positron emission tomography with (11)C-doxepin in a placebo-controlled crossover study design in healthy subjects: a comparison of olopatadine and ketotifen.

AIMS: The strength of sedation due to antihistamines can be evaluated by using positron emission tomography (PET). The purpose of the present study is to measure histamine H(1) receptor (H(1)R) occupancy due to olopatadine, a new second-generation antihistamine and to compare it with that of ketotifen. METHODS: Eight healthy males (mean age 23.5 years-old) were studied following single oral administration of olopatadine 5 mg or ketotifen 1 mg using PET with (11)C-doxepin in a placebo-controlled crossover study design. Binding potential ratio and H(1)R occupancy were calculated and were compared between olopatadine and ketotifen in the medial prefrontal (MPFC), dorsolateral prefrontal (DLPFC), anterior cingulate (ACC), insular (IC), temporal (TC), parietal (PC), occipital cortices (OC). Plasma drug concentration was measured, and correlation of AUC to H(1)R occupancy was examined. RESULTS: H(1)R occupancy after olopatadine treatment was significantly lower than that after ketotifen treatment in the all cortical regions (P < 0.001). Mean H(1)R occupancies for olopatadine and ketotifen were, respectively: MPFC, 16.7 vs. 77.7; DLPFC, 14.1 vs. 85.9; ACC, 14.7 vs. 76.1; IC, 12.8 vs. 69.7; TC, 12.5 vs. 66.5; PC, 13.9 vs. 65.8; and OC, 19.5 vs. 60.6. Overall cortical mean H(1)R occupancy of olopatadine and ketotifen were 15% and 72%, respectively. H(1)R occupancy of both drugs correlated well with their respective drug plasma concentrations (P < 0.001). CONCLUSION: It is suggested that 5 mg oral olopatadine, with its low H(1)R occupancy and thus minimal sedation, could safely be used an antiallergic treatment for various allergic disorders. Abbreviations histamine H(1) receptor (H(1)R), histamine H(1) receptor occupancy (H(1)RO), dopamine D(2) receptor (D(2)R), positron emission tomography (PET), blood-brain barrier (BBB), binding potential ratio (BPR), distribution volume (DV).

Determination of ketotifen in human plasma by LC-MS.

Analytical validation of a new liquid chromatographic-mass spectrometric (LC-MS) method for determination of total amount of ketotifen (unchanged and conjugated) in human plasma is presented. Pizotifen was used as an internal standard. An enzyme hydrolysis of conjugated ketotifen was conducted with a combination of beta-glucuronidase and aryl sulfatase. After enzyme hydrolysis a liquid-liquid extraction was performed as a cleaning step. The quantitative determination was obtained using selected ion monitoring (SIM) LC-MS. Chromatographic condition was a combination of reverse phase gradient system and a switching column technique. A satisfactory hydrolysis, acceptable accuracy, improved precision in the linear range from 0.5 to 20.0 ng/ml plasma, absolute recovery of 98.04% for ketotifen and 95.13% for pizotifen and stability for 7 months at -20 degrees C have been achieved.

Determination of ketotifen and its conjugated metabolite in human plasma by liquid chromatography/tandem mass spectrometry: application to a pharmacokinetic study.

A sensitive and specific liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the investigation of the pharmacokinetics of ketotifen and its major metabolite, ketotifen N-glucuronide, in human plasma. The plasma samples were treated by liquid-liquid extraction and analyzed using LC/MS/MS with an electrospray ionization interface. Diphenhydramine was used as the internal standard. The method had a lower limit of quantitation of 10 pg/mL for ketotifen, which offered increased sensitivity, selectivity and speed of analysis, compared with existing methods. The intra- and inter-day precision were measured to be below 8.2% and accuracy between -2.4% and 3.4% for all QC samples. Incubation of the plasma samples with beta-glucuronidase allowed the quantitation of ketotifen N-glucuronide. This quantitation method was successfully applied to a pharmacokinetic study of ketotifen and its major metabolite after oral administration of 2 mg ketotifen fumarate to 16 healthy volunteers.

Pharmacokinetics of ketotifen fumarate after intravenous, intranasal, oral and rectal administration in rabbits.

The pharmacokinetics of ketotifen fumarate (KF) was examined after administration in rabbits through four different routes (intravenous, intranasal, oral and rectal). The time-course of the plasma concentration of KF after intravenous administration (1 mg/kg dose) fitted a two-compartment open model. KF was rapidly absorbed and showed a high plasma concentration within 0.33 h after intranasal administration. The absolute bioavailability of KF after intranasal administration was 66%. After oral administration at a dose of 1 mg/kg, the plasma concentration of KF was below the detection limit of HPLC analysis. Even at 5 mg/kg, the value of the area under the plasma concentration-time curve (AUC) after oral administration of KF was significantly lower than that after intranasal administration of 1 mg/kg. Oral bioavailability was only 8%. The very low bioavailability of KF after oral administration might be due to the first-pass effect in the liver. We also prepared suppositories containing KF (1 mg/kg) for rectal administration in rabbits. After rectal administration, KF was rapidly absorbed and its bioavailability was 34%. These results indicated that the intranasal route appears the most effective for administering KF, and that rectal administration may be superior to oral administration in terms of bioavailability.

Gas chromatographic-mass spectrometric method for quantitative determination of ketotifen in human plasma after enzyme hydrolysis of conjugated ketotifen.

A validated method for determination of total amount of ketotifen (unchanged and conjugated) in human plasma has been presented. An enzyme hydrolysis of conjugated ketotifen was conducted with combination of beta-glucuronidase and arylsulfatase. After the enzyme hydrolysis a solid-phase extraction was applied as a cleaning step. The quantitative determination by gas chromatography with mass-spectrometry detection (GC-MS) was performed. Pizotifen has been used as an internal standard. A reliable hydrolysis as well as a satisfactory accuracy, improved precision in the linear region from 0.500 to 10.0 ng/ml plasma, limit of detection of 0.010 ng/ml and prolonged capillary column life have been achieved.

Deuterium-labelling and quantitative measurement of ketotifen in human plasma by gas chromatography/negative ion chemical ionization mass spectrometry.

A gas chromatographic/mass spectrometric assay for the anti-anaphylactic agent Ketotifen is described. The method is based on negative ion chemical ionization, utilizing the electrophoric nature of the underivatized drug. A simple procedure for deuteration of the intact target molecule is presented, thus enhancing specificity by internal standardization via stable isotope dilution. The assay was applied to pharmacokinetics of the drug in man.

Refractory cholinergic urticaria successfully treated with ketotifen.

Four patients with cholinergic urticaria associated with additional cardiorespiratory manifestations are described. Two patients reported cold, in addition to heat and exercise, as a factor inducing symptoms. Prospective exercise challenge documented a rise in in plasma histamine sixfold to 20-fold above baseline values that accompanied onset of symptoms. All four subjects had proved refractory to conventional antihistamine therapy. Institution of ketotifen at doses ranging from 3 to 8 mg per day resulted in symptomatic improvement, and in all four subjects a repeat exercise challenge confirmed clinical improvement. In three subjects exercise challenge with ketotifen demonstrated blockade of mast cell-mediator release. Plasma histamine levels remained at baseline. In the fourth patient, histamine rose to about half the peak observed before ketotifen therapy. These findings confirm the observation that ketotifen is both an H1 histamine-receptor antagonist as well as a stabilizer of mast cell-mediator release. We speculate that ketotifen may prove more effective than conventional antihistamines in the management of severe urticaria.

The determination of pharmacokinetic parameters of ketotifen in steady state in young children.

Ketotifen kinetics at steady state in small children compared to adults show very good absorption for a dose of 2 X daily 1 mg. Relative to adult kinetics, a higher dose in relation to body weight is justified, since a more rapid kinetic pattern was proven for the substance in the volunteer collective tested.

Drug level monitoring of antiasthmatic drugs.

In general assays pertaining to drug level monitoring (DLM) of antiasthmatic agents (except theophylline), published during the period 1978-1983, used mostly high-performance liquid chromatographic (HPLC) methodology (approximately 45%) with mass spectrometric (MS) based assays in second place (approximately 30%) followed by immunochemical techniques (approximately 25%). Whenever nanogram or subnanogram antiasthmatic drug concentrations had to be measured such as for the adrenergic stimulants or for the prophylactic agents, then both HPLC-and MS-based methodologies were employed with about equal frequency. The trend in DLM for the phosphodiesterase inhibitor class (theophyllines) seemed to be shifting towards the HPLC methodologies. In part, this was justified by the need for improved selectivity. This criterion appears to have been better satisfied by HPLC, but for all practical purposes the immunochemical methods are and will probably continue to prevail in the clinical laboratory setting until HPLC procedures become truly automated. In the case of DLM of corticosteroids used for the asthmatic, the situation is in our opinion still unclear. This is caused by the presence of endogenous corticosteroids and metabolites, the levels of which in man are known to vary. The current immunochemical procedures offer a facile but less selective option. The future for selective routine corticosteroid assays may well be in HPLC or gas chromatography coupled with MS.

Determination of the central effects of the asthma prophylactic ketotifen, the bronchodilator theophylline, and both in combination: an application of quantitative electroencephalography to the study of drug interactions.

Ketotifen (Zaditen) is a new, orally active benzocycloheptathiophene derivative for use in the prophylaxis of asthma. Besides possessing anti-allergic properties, it also has H1-receptor mediated antihistaminic activity. Drugs which block H1-receptors are known to have some sedative properties. On the other hand the bronchodilator theophylline is a CNS stimulant. We investigated whether these side-effects could be reduced by concomitant administration. In a balanced-sequence crossover study, 12 healthy males received placebo, 1 mg ketotifen + 300 mg theophylline, 1 mg ketotifen + 600 mg theophylline, and each drug separately at 1-week intervals. Quantified electroencephalograms, cardiovascular and behavioral measurements, symptom reports, and blood drug assays were used to assess the drug interaction. Results showed EEG and behavioral effects with both ketotifen and theophylline alone which were less evident with the drugs in combination. Blood drug levels were not altered by combined drug administration. These findings suggest a mutual attenuation of the CNS effects of ketotifen and theophylline at therapeutic doses and encourage their combined use in asthma therapy. The combined effect may be optimized by modifying formulations or timing. The applicability of quantitative EEG to such problems is well demonstrated.

Quantification of ketotifen and its metabolites in human plasma by gas chromatography mass spectrometry.

Gas chromatography mass spectrometry with selected ion monitoring has been used to develop a method for the quantification of ketotifen and its demethylated, 10-hydroxy and 10-hydroxy demethylated metabolites in human plasma. The minimum detectable concentrations for ketotifen and its demethylated metabolites were 50 pg ml-1 and 300 pg ml-1 for the 10-hydroxy metabolite. The methodology has been applied in studies of the kinetics of the drug in man, and plasma levels of the unchanged drugs and its metabolites in free and conjugated form are reported.