Identification of a novel selective H1-antihistamine with optimized pharmacokinetic properties for clinical evaluation in the treatment of insomnia.

Analogs of the known H(1)-antihistamine R-dimethindene with suitable selectivity for key GPCRs, P450 enzymes and hERG channel were assessed for metabolism profile and in vivo properties. Several analogs were determined to exhibit diverse metabolism. One of these compounds, 10a, showed equivalent efficacy in a rat EEG/EMG model to a previously identified clinical candidate and a potentially superior pharmacokinetic profile as determined from a human microdose study.

Analytical method for simultaneously measuring ex vivo drug receptor occupancy and dissociation rate: application to (R)-dimethindene occupancy of central histamine H1 receptors.

We introduce a novel experimental method to determine both the extent of ex vivo receptor occupancy of administered compound and its dissociation rate constant (k4). [Here, we reference k4 as the rate of offset of unlabeled ligand in convention with Motulsky and Mahan (1)]. We derived a kinetic rate equation based on the dissociation rate constant for an unlabeled compound competing for the same site as a labeled compound and describe a model to simulate fractional occupancy. To validate our model, we performed in vitro kinetics and ex vivo occupancy experiments in rat cortex with varying concentrations of (R)-dimethindene, a sedating antihistamine. Brain tissue was removed at various times post oral administration, and histamine H1 receptor ligand [3H]-doxepin binding to homogenates from drug-treated or vehicle-treated rats was measured at multiple time points at room temperature. Fractional occupancy and k4 for (R)-dimethindene binding to H1 receptors were calculated by using our proposed model. Rats dosed with 30 and 60 mg/kg (R)-dimethindene showed 42% and 67% occupancy of central H1 receptors, respectively. These results were comparable to occupancy data determined by equilibrium radioligand binding. In addition, drug k4 rate determined by using our ex vivo method was equivalent to k4 determined by in vitro competition kinetics (dissociation half-life t(1/2) approximately 30 min). The outlined method can be used to assess, by simulation and experiment, occupancy for compounds based on dissociation rate constants and contributes to current efforts in drug optimization to profile antagonist efficacy in terms of its kinetic drug-target binding parameters. Data described by the method may be analyzed with commercially available software. Suggested fitting procedures are given in the appendix.

Chiral separation of alkylamine antihistamines in pharmaceuticals by capillary isotachophoresis with charged cyclodextrin.

Cyclodextrin-mediated capillary isotachophoresis (ITP) in cationic regime of the separation was developed for the separation and quantitation of alkylamine antihistamine dimethindene (DIM) and pheniramine (PHM) enantiomers in various pharmaceutical preparations (capsules, oral drops, gel, granulated powder). Several electrolyte systems of different compositions and pH were examined. The optimized chiral ITP electrolyte system was consisted of 10 mmol/L potassium acetate adjusted to pH 4.8 with acetic acid, containing 4 mmol/L negatively charged CE-beta-CD (chiral selector) as the leading electrolyte with electroosmotic flow (EOF) suppressing additive, 0.2% (w/v) methylhydroxyethylcellulose (m-HEC), and 5 mmol/L beta-alanine as the terminating electrolyte. The proposed electrophoretic method was successfully validated. It was convenient for the sensitive, simple, rapid, and highly reproducible assay of these antihistamine enantiomers. The calibration graphs relating the ITP zone length to the concentration of DIM and PHM enantiomers were rectilinear (r = 0.999) in the range 40.0-200.0 mg/L of each enantiomer. The relative standard deviations (RSD) were 0.75% for DIM(1), 0.63% for DIM(2), 1.05% for PHM(1), and 0.83% for PHM(2) (n = 6) when determining 100 mg/L DIM and PHM, respectively, standard solutions. According to the validation procedure based on the standard addition technique the recoveries were 97.66-98.34%. Good quantitation was obtained in short analysis time (a single analysis took about 12 min). The minimal sample pretreatment and low running costs make the proposed ITP method a good alternative to commonly used analytical methods (CZE, HPLC). The obtained results suggest that the proposed method is suitable for routine assay of dimethindene and pheniramine enantiomers in various pharmaceuticals.

[Separation of dimetinden enantiomers in drugs by means of capillary isotachophoresis]. / Separacia enantiomérov dimetindenu v liekoch kapilárnou izotachoforézou.

Capillary isotachophoresis was employed to separate and determine dimethinden enantiomers in various dosage forms. Several types of chiral selectors were tested in various electrolyte systems of different composition and different pH. The optimal leading electrolyte was composed of 10 mmol/l potassium acetate and acetic acid to achieve pH 4.8 with an addition of 4 mmol/l carboxyethyl-beta-cyclodextrin as the chiral selector and 0.2% of methylhydroxyethylcellulose (m-HEC) to suppress the electroosmotic flow. The terminating electrolyte was betaalanine of a concentration of 5 mmol/l. The evaluation included the precision, correctness, linearity, robustness, and selectivity of the elaborated ITP method. The pretreatment of the sample prior to analysis consisted in the dissolution and dilution of the appropriate dimethinden-containing dosage form with demineralized water to achieve the required concentration. Such a pretreated sample was directly dosed into the apparatus.

Influence of methanol on the enantioresolution of antihistamines with carboxymethyl-beta-cyclodextrin in capillary electrophoresis.

According to the model of Wren and Rowe, the separation between two enantiomers in capillary electrophoresis (CE) decreases if an organic modifier is added to the run buffer containing a neutral cyclodextrin (CD) in a concentration below its optimal value in a solvent-free system. In previous work, however, it was observed that the addition of methanol to the background electrolyte (BGE) containing not charged carboxymethyl-beta-CD in a concentration below its optimal value, increased the enantioresolution of dimetindene maleate. The enantioresolution decreased when other organic modifiers (ethanol, isopropanol or acetonitrile) were added and/or when other neutral (beta-CD, hydroxypropyl-beta-CD) or chargeable (carboxyethyl-beta- and succinyl-beta-CD) CDs were used. In this CE study further attempts are made to elucidate the observed phenomena through investigating other basic drugs. The effect of organic modifier and CD concentration on the enantioseparation was studied by means of central composite designs. It is shown that obtaining this increase in enantioresolution depends upon the type of CD, the type of organic modifier, and the structure of the analytes. It was also observed that small differences in the structure of the analytes or the CD could have an influence on the enantioresolution. The addition of methanol also resulted in different effects on the resolution of closely related analytes.

Structure-activity relationships of dimethindene derivatives as new M2-selective muscarinic receptor antagonists.

A series of 2,3-disubstituted indenes, which are analogues of the widely used histamine H(1) receptor antagonist dimethindene, have been synthesized and studied as muscarinic and histamine receptor antagonists. The affinities of these compounds for the five human muscarinic receptor subtypes (M(1)-M(5)) and for human histamine H(1) receptors were determined in radioligand binding studies using membranes from transfected Chinese hamster ovary (CHO) cells and [(3)H]N-methylscopolamine ([(3)H]NMS). The results demonstrate that the diisopropyl analogue 19 has a similar high affinity as (S)-dimethindene at M(2) receptors ((S)-dimethindene: pK(i) = 7.52; (-)-19: pK(i) = 7.37) with an improved selectivity pattern ((S)-dimethindene: M(2)/M(1) = 6-fold, M(2)/M(3) = 5-fold, M(2)/M(4) = 10-fold, M(2)/M(5) = 25-fold; (-)-19: M(2)/M(1) = 36-fold, M(2)/M(3) = 96-fold, M(2)/M(4) = 42-fold, M(2)/M(5) = 275-fold). In addition, compound (-)-19 showed 35-fold lower affinity at histamine H(1) receptors (pK(i) = 5.61) than (S)-dimethindene (pK(i) = 7.16). Another interesting compound is the fluoroethyl derivative 20 (pK(i)/M(2) = 7.49), which also exhibits a higher M(2) selectivity (M(2)/M(1) = 19-fold; M(2)/M(3) = 22-fold; M(2)/M(4) = 13-fold; M(2)/M(5) = 62-fold) than (S)-dimethindene. Unfortunately, compound 20 also shows a high affinity for histamine H(1) receptors (pK(i) = 8.14). The compound with the highest affinity for M(2) receptors (pK(i) = 7.91), the dimethylaminomethylene analogue 31, displayed only a small preference for M(2) receptors. In conclusion, compound (-)-19 might be useful to test the hypothesis that blockade of muscarinic M(2) receptors in the brain is a viable mechanism by which to produce improved cognition. This second-generation dimethindene analogue might also be the starting point for the development of M(2)-selective muscarinic antagonists useful for quantifying M(2) receptors in the central nervous system with positron emission tomography imaging.

Mechanistic study of opposite migration order of dimethindene enantiomers in capillary electrophoresis in the presence of native beta-cyclodextrin and heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin.

The possible mechanisms of the opposite affinity pattern of the enantiomers of dimethindene [(R,S)-N,N-dimethyl-3[1(2-pyridyl)ethyl]indene-2-ethylamine] (DIM) towards native beta-cyclodextrin (beta-CD) and heptakis(2,3,6-tri-O-methyl-)-beta-CD (TM-beta-CD) were studied using capillary electrophoresis (CE), NMR spectrometry, electrospray ionization mass spectrometry (ESI-MS) and X-ray crystallography. NMR spectrometry allowed to estimate the stoichiometry of the complex and to determine the binding constants. As found using ESI-MS, together with more abundant 1:1 complex, a complex with 1:2 stoichiometry may also be present in a rather small amount in a solution of DIM and beta-CD. One-dimensional ROESY experiments indicated that the geometry of the complexes of DIM with native beta-CD depends on the ratio of the components in the solution. In the 1:1 solution of DIM and beta-CD the complex may be formed by inclusion of the indene moiety of DIM into the cavity of beta-CD on the primary side and into the cavity of TM-beta-CD into the secondary side. The most likely structural reason for lower affinity of the enantiomers of DIM towards the cavity of TM-beta-CD compared to native beta-CD could be elucidated. The indene moiety does not enter the cavity of TM-beta-CD as deeply as the cavity of beta-CD. This may be the most likely explanation of significantly higher affinity constants of DIM enantiomers towards the latter CD compared to the former one. The marked difference between the structure of the complexes may also be responsible for the opposite affinity pattern of the DIM enantiomers towards beta-CD and TM-beta-CD.

The design and pharmacology of novel selective muscarinic agonists and antagonists.

The muscarinic pharmacology of C1-methyl-substituted chiral compounds related to McN-A-343 and of (R)- and (S)-dimethindene has been studied. Among the McN-A-343 analogues, the (S)-enantiomers were more potent and had higher affinity than the (R)-isomers. The quaternary compound (S)-BN 228 was found to be the most potent M1-selective agonist known today (pEC50: M1/rabbit vas deferens = 7.83; M2/guinea-pig atria = 6.35; M3/guinea-pig ileum = 6.29). In both the atria and ileum the tertiary carbamate, (S)-4-F-MePyMcN, was a competitive antagonist (pA2 value = 7.39 and 6.82, respectively). In contrast, in rabbit vas deferens (S)-4-F-MePyMcN was a potent partial agonist (pEC50 = 7.22; apparent efficacy = 0.83). These results indicate that (S)-4-F-MePyMcN might be a useful tool to study M1 receptor-mediated effects involved in central cholinergic function. (S)-Dimethindene was a potent M2-selective antagonist (pA2 = 7.86/atria; pKi = 7.8/rat heart) with lower affinities for the M1 (pA2 = 6.36/rat duodenum; pKi = 7.1/NB-OK 1 cells), M3 (pA2 = 6.92/guinea-pig ileum; pKi = 6.7/rat pancreas) and M4 receptors (pKi = 7.0/rat striatum). It was more potent (up to 41-fold) than the (R)-isomer. In contrast, the stereoselectivity was inverse at ileal H1 receptors (pA2: (R)-isomer = 9.42; (S)-isomer = 7.48). Thus, (S)-dimethindene could be a valuable agent to test the hypothesis that M2 antagonists show beneficial effects in the treatment of cognitive disorders. It might also become the starting point for the development of diagnostic tools for quantifying M2 receptors in the CNS with PET imaging.

Simultaneous enantioselective determination and quantification of dimethindene and its metabolite N-demethyl-dimethindene in human urine using cyclodextrins as chiral additives in capillary electrophoresis.

Capillary electrophoresis (CE) has been used for the chiral resolution of the basic racemic drug dimethindene (Fenistil) and its metabolite N-demethyl-dimethindene. After oral administration of 4 mg dimethindene maleate the enantioselective excretion in human urine was determined by capillary electrophoresis using a 50 mM phosphate run buffer (pH 3.3) containing 30 mM hydroxypropyl-beta-cyclodextrin. The determination of very low concentrations of dimethindene and N-demethyl-dimethindene in human urine was possible using sample stacking conditions to increase the amount of analyte in the capillary with good peak forms and resolutions. The detection limit of the method was approximately 1 ng ml-1 urine for each compound.