Increased Nuclear FOXP2 Is Related to Reduced Neural Stem Cell Number and Increased Neurogenesis in the Dorsal Telencephalon of Embryos of Diabetic Rats through Histamine H1 Receptors.

Diabetic rat embryos have increased cortical neurogenesis and neuron maturation, and their offspring presented altered neuron polarity, lamination, and diminished neuron excitability. The FOXP2 overexpression results in higher cortical neurogenesis by increasing the transition of radial glia to the intermediate progenitor. Similarly, histamine through H1-receptor activation increases cortical neuron differentiation. Indeed, blocking the H1-receptor by the systemic administration of chlorpheniramine to diabetic pregnant rats prevents increased neurogenesis. Here, we explore the relationship between the H1-receptor and FOXP2 on embryo neurogenesis from diabetic dams. Through qRT-PCR, Western blot, immunohistofluorescence, and flow cytometry, we showed an increased FOXP2 expression and nuclear localization, a reduced Nestin expression and -positive cells number, and a higher PKCα expression in the cortical neuroepithelium of fourteen-day-old embryos from diabetic rats. Interestingly, this scenario was prevented by the chlorpheniramine systemic administration to diabetic pregnant rats at embryo day twelve. These data, together with the bioinformatic analysis, suggest that higher H1-receptor activity in embryos under high glucose increases FOXP2 nuclear translocation, presumably through PKCα phosphorylation, impairing the transition of radial glia to intermediate progenitor and increasing neuron differentiation in embryos of diabetic rats.

Precision Printing of Customized Cylindrical Capsules with Multifunctional Layers for Oral Drug Delivery.

Advances in personalized medicine will require custom drug formulations and delivery mechanisms. Herein, we demonstrate a new type of personalized capsule comprising of printed concentric cylindrical layers with each layer having a distinctive functional drug component. Poly ε-caprolactone (PCL) with paracetamol (APAP) and chlorpheniramine maleate (CM), synergistic drugs commonly used to alleviate influenza symptoms, are printed as an inner layer and outer layer, respectively, via microscaled electrohydrodynamic (EHD) printing. Polyvinylpyrrolidone (PVP) nanofibers are embedded as interlayers between the two printed PCL-drug layers using electrospinning (ES) techniques. The complete concentric cylindrical capsule with a 6 mm inner diameter and 15 mm length can be swallowed for oral drug delivery. After dissolution of the PVP interlayer, the capsule separates in two, with inner and outer capsules for continuous drug dosing and targeting. Imaging was achieved using a 3T MRI system which allowed temporal observations of the targeted release through the incorporation of nanoparticles (Fe3O4). The morphology and structure, chemical composition, mechanical properties, and biocompatibility of the capsules were studied in vitro. In summary, this new type of custom printed and electrospun capsule that enabled component separation, targeted drug release may advance personalized medicine via multidrug oral delivery.

Development and evaluation of an oral fast disintegrating anti-allergic film using hot-melt extrusion technology.

The main objective of this novel study was to develop chlorpheniramine maleate orally disintegrating films (ODF) using hot-melt extrusion technology and evaluate the characteristics of the formulation using in vitro and in vivo methods. Modified starch with glycerol was used as a polymer matrix for melt extrusion. Sweetening and saliva-simulating agents were incorporated to improve palatability and lower the disintegration time of film formulations. A standard screw configuration was applied, and the last zone of the barrel was opened to discharge water vapors, which helped to manufacture non-sticky, clear, and uniform films. The film formulations demonstrated rapid disintegration times (6-11s) and more than 95% dissolution in 5min. In addition, the films had characteristic mechanical properties that were helpful in handling and storage. An animal model was employed to determine the taste masking of melt-extruded films. The lead film formulation was subjected to a human panel for evaluation of extent of taste masking and disintegration.

Physicochemical properties and mechanisms of drug release from melt-extruded granules consisting of chlorpheniramine maleate and Eudragit FS.

The objective of this research project was to characterize the drug release profiles, physicochemical properties and drug-polymer interaction of melt-extruded granules consisting of chlorpheniramine maleate (CPM) and Eudragit® FS. Melt extrusion was performed using a single screw extruder at a processing temperature of 65-75 °C. The melt extrudate was milled, blended with lactose monohydrate and then filled into hard gelatin capsules. Each capsule contained 300 mg CPM granules. The release of CPM was determined with the United States Pharmacopeia dissolution apparatus II using a three-stage dissolution medium testing in order to simulate the pH conditions of the gastrointestinal tract. Pore structure, thermal properties and surface morphologies of CPM granules were studied using mercury and helium pycnometer, differential scanning calorimeter and scanning electron microscope. Sustained release of CPM over 10 h was achieved. The release of CPM was a function of drug loading and the size of the milled granules. The complexation between CPM and Eudragit® FS as the result of counterion condensation was observed, and the interaction was characterized using membrane dialysis and H(1) NMR techniques. In both 0.1 N HCl and phosphate buffer pH 6.8, CPM was released via a diffusion mechanism and the release rate was controlled by the pore structure of the melt-extruded granules. In phosphate buffer pH 7.4, CPM release was controlled by the low pH micro-environment created by CPM, the pore structure of the granules and the in situ complexation between CPM and Eudragit® FS.

Potential use of a megamolecular polysaccharide sacran as a hydrogel-based sustained release system.

A megamolecular polysaccharide sacran was newly extracted from cyanobacterium Aphanothece sacrum. Sacran has many preferable properties for transdermal application, e.g. a safe biomaterial, a high moisturizing effect, a formation of film and hydrogel. Additionally, it was recently discovered that sacran has an anti-inflammatory effect for atopic dermatitis model mice. In this study, in order to evaluate the feasibility of sacran-hydrogel as a novel sustained release system, we prepared a sacran-hydrogel containing 4-biphenyl acetic acid (BPAA, an acidic drug), prednisolone (PD, a neutral drug) or chlorpheniramine maleate (CPM, a basic drug), and performed the in vitro release studies. The sacran-hydrogel containing BPAA, PD or CPM provided a sustained release profile in accordance with a quasi-Fickian diffusion model. Furthermore, the release rate of drugs from sacran-hydrogels can be controlled by adjusting the concentration of aluminum chloride as a cross linker. These results suggest the potential use of sacran-hydrogel as a sustained release system for drugs.

Characterization of xenobiotic metabolizing enzymes in bovine small intestinal mucosa.

The intestinal mucosa plays a capital role in dictating the bioavailability of a large array of orally ingested drugs and toxicants. The activity and the expression of several xenobiotic metabolizing enzymes were measured in subcellular fractions from the duodenal mucosa of male veal calves and beef cattle displaying a functional rumen but differing in both age (about 8 months vs. 18 to 24 months) and dietary regimens (i.e., milk replacer plus hay and straw vs. corn and concentrated meal). Intestinal microsomes showed cytochrome P450 (CYP) 2B, 2C- and 3A-mediated activities and the presence of the corresponding immunorelated proteins, but no proof of CYP1A expression and/or functions could be provided. Intestinal microsomes were also active in performing reactions typically mediated by carboxylesterases (indophenylacetate hydrolysis), flavin-containing monooxygenases (methimazole S-oxidation), and uridindiphosphoglucuronyltransferases (1-naphthol glucuronidation), respectively. Cytosolic fractions displayed the glutathione S-transferase (GST)-dependent conjugation of 1-chloro-2,4-dinitrobenzene; besides, the GST-mediated conjugation of ethacrinic acid (GSTpi) or cumene hydroperoxide (GSTalpha) was matched by the presence of the corresponding immunorelated proteins. Conversely, despite the lack of measurable activity with 3,4-dichloronitrobenzene, a protein cross reacting with anti-rat GSTmu antibodies could be clearly detected. Although, as detected by densitometry, CYPs and GST isoenzymes tended to be more expressed in beef cattle than in veal calf preparations, there was a general poor correlation with the rate of the in vitro metabolism of the selected diagnostic probes.

Quantification of chlorpheniramine maleate enantiomers by ultraviolet spectroscopy and chemometric methods.

Chlorpheniramine maleate (CLOR) enantiomers were quantified by ultraviolet spectroscopy and partial least squares regression. The CLOR enantiomers were prepared as inclusion complexes with beta-cyclodextrin and 1-butanol with mole fractions in the range from 50 to 100%. For the multivariate calibration the outliers were detected and excluded and variable selection was performed by interval partial least squares and a genetic algorithm. Figures of merit showed results for accuracy of 3.63 and 2.83% (S)-CLOR for root mean square errors of calibration and prediction, respectively. The ellipse confidence region included the point for the intercept and the slope of 1 and 0, respectively. Precision and analytical sensitivity were 0.57 and 0.50% (S)-CLOR, respectively. The sensitivity, selectivity, adjustment, and signal-to-noise ratio were also determined. The model was validated by a paired t test with the results obtained by high-performance liquid chromatography proposed by the European pharmacopoeia and circular dichroism spectroscopy. The results showed there was no significant difference between the methods at the 95% confidence level, indicating that the proposed method can be used as an alternative to standard procedures for chiral analysis.

Contribution of alpha4beta1 integrin to the antiallergic effect of levocabastine.

Levocabastine is an antiallergic drug acting as a histamine H1-receptor antagonist. In allergic conjunctivitis (AC), it may also antagonize up-regulation of the intercellular adhesion molecule-1 (ICAM-1) expressed on epithelial conjunctival cells. However, little is known about its effects on eosinophils, important effector cells in AC. The adhesion molecule integrin alpha(4)beta(1) is expressed in eosinophils; it interacts with the vascular cell adhesion molecule-1 (VCAM-1) and fibronectin (FN) in vascular endothelial cells and contributes to eosinophil activation and infiltration in AC. This study provides evidence that in a scintillation proximity assay levocabastine (IC(50) 406 microM), but not the first-generation antihistamine chlorpheniramine, displaced (125)I-FN binding to human integrin alpha(4)beta(1) and, in flow cytometry analysis, levocabastine antagonized the binding of a primary antibody to integrin alpha(4) expressed on the Jurkat cell surface. Levocabastine, but not chlorpheniramine, binds the alpha(4)beta(1) integrin and prevents eosinophil adhesion to VCAM-1, FN or human umbilical vascular endothelial cells (HUVEC) in vitro. Similarly, levocabastine affects alpha(L)beta(2)/ICAM-1-mediated adhesion of Jurkat cells. In a model of AC levocabastine eye drops reduced the clinical aspects of the late-phase reaction and the conjunctival expression of alpha(4)beta(1) integrin by reducing infiltrated eosinophils. We propose that blockade of integrin-mediated cell adhesion might be a target of the antiallergic action of levocabastine and may play a role in preventing eosinophil adhesion and infiltration in AC.

Localized expression of histamine H1 receptors in syncytiotrophoblast cells of human placenta.

The previous Northern blot analysis and in situ hybridization studies showed that histamine H1-receptor (H1R) mRNA is expressed in human placenta and suggested that H(1)R plays some roles in the function of placenta in pregnancy. To investigate further, it is essential to show the precise location of H1R in the placenta. In the present study, we investigated H1R expression in human placenta by radioligand binding assay and immunohistochemical study using an antibody against human H1R. Placentas were obtained from normal uncomplicated deliveries. Membranes prepared from the tissue exhibited saturable [3H]mepyramine binding (K(d) = 4.0 +/- 0.6 nM and B(max) = 91.4 +/- 4.9 fmol/mg of protein). Stereoisomers of chlorpheniramine inhibited [(3)H]mepyramine binding; d-chlorpheniramine inhibited more potently than l-chlorpheniramine, K(i) values being 1.1 +/- 0.4 and 270 +/- 170 nM, respectively. The placenta tissues were positively immunostained with anti-H1R antibody only in the region of the syncytiotrophoblast of chorionic villus. The tissues were double stained with anti-H1R antibody and an antibody against human chorionic gonadotoropin (hCG) that is solely expressed in placental syncytiotrophoblast cells. The results showed that H1R and hCG were expressed on the same cells, that is, syncytiotrophoblast cells. These results indicate that H1Rs are specifically expressed in syncytiotrophoblast cells of human placenta organ.

Carrier mediated transport of chlorpheniramine and chlorcyclizine across bovine olfactory mucosa: implications on nose-to-brain transport.

Delivery to the CNS via the nasal cavity has been pursued as a means to circumvent the blood-brain barrier (BBB), yet the mechanism of drug transport across this novel route is not well understood. Hydroxyzine and triprolidine have been reported to readily reach the CNS following nasal administration, whereas no measurable amounts of chlorcyclizine or chlorpheniramine, structurally similar antihistamines, were observed in the CSF. The permeation of chlorpheniramine and chlorcyclizine in vitro across the bovine olfactory mucosa was studied to investigate the biological and physicochemical characteristics that contribute to the limited CNS disposition of these compounds following nasal administration. The submucosal to mucosal fluxes (J(s-m)) of chlorcyclizine and chlorpheniramine across the olfactory mucosa were significantly greater than the mucosal to submucosal fluxes (J(m-s)). Moreover, the submucosal-mucosal permeability of both compounds was temperature dependent and saturable. In the presence of metabolic inhibitors (ouabain and 2,4-dinitrophenol) and P-glycoprotein (P-gp)/multidrug resistance protein 1 (MRP1) inhibitors (quinidine and verapamil), the J(m-s) increased and J(s-m) decreased significantly. These results indicate that chlorpheniramine and chlorcyclizine are effluxed from the olfactory mucosa by efflux transporters such as P-gp and MRP1. Transport studies across inert polymeric membranes demonstrated that the permeability of chlorpheniramine and chlorcyclizine decreased at donor concentrations higher than 3 mM suggesting that physicochemical properties such as self-aggregation also play a role in the reduced olfactory mucosal permeability of these compounds at higher concentrations.

Identification of a dual histamine H1/H3 receptor ligand based on the H1 antagonist chlorpheniramine.

Combining the first generation H(1) antihistamine chlorpheniramine (1) with H(3) ligands of the alkylamine type has led to the identification of compound 9d, a dual ligand of both the H(1) and H(3) receptors.

The enantioselective determination of chlorpheniramine and its major metabolites in human plasma using chiral chromatography on a beta-cyclodextrin chiral stationary phase and mass spectrometric detection.

A sensitive enantioselective high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of plasma concentrations of (-)(R)- and (+)(S)-chlorpheniramine (CP) and their metabolites, desmethyl-chlorpheniramine (DCP), didesmethyl-chorpheniramine (DDCP) and chlorpheniramine N-oxide (CPNO). Enantioselective separations were achieved on a beta-cyclodextrin chiral stationary phase (CYCLOBOND I 2000) with a mobile phase consisting of diethylamine acetate (0.25%, pH 4.4):methanol:acetonitrile [85:7.5:7.5, (v/v/v)]and a flow-rate of 0.5 ml/min. For CP, the enantioselectivity (alpha) of the separation was 1.12 with a resolution factor (R(s)) of 1.17. The method was validated for CP by using mass spectroscopy detection (MSD). Concentrations of each enantiomer could be measured down to 125 pg/ml from a 1-ml plasma sample. Extracted calibration curves were linear from 0.13 to 50.00 ng/ml for each enantiomer. The method was applied to samples from two clinical studies.

Neuroimaging of histamine H1-receptor occupancy in human brain by positron emission tomography (PET): a comparative study of ebastine, a second-generation antihistamine, and (+)-chlorpheniramine, a classical antihistamine.

AIMS: Sedation induced by antihistamines is widely recognized to be caused by their penetration through the blood-brain-barrier and the consequent occupation of brain histamine H1-receptors. We previously studied the mechanism of sedation caused by antihistamines using positron emission tomography (PET). Recently, we revealed the nonsedative characteristic of ebastine, a second-generation antihistamine, with cognitive performance tests. In the present study, H1-receptor occupation by ebastine was examined in the human brain using PET. METHODS: Ebastine 10 mg and (+)-chlorpheniramine 2 or 6 mg were orally given to healthy male volunteers. PET scans with [11C]-doxepin, a potent H1-receptor antagonist, were conducted near tmax of respective drugs. Other volunteers in the control group also received PET scans. The binding potential of doxepin (BP = Bmax/Kd) for available brain H1-receptors was imaged on a voxel-by-voxel basis through graphical analysis. By setting regions of interest, the H1-receptor occupancy of drugs was calculated in several H1-receptor rich regions. RESULTS: Brain distribution of radioactivity after ebastine treatment was similar to that without any drugs. However, after the oral administration of 2 mg (+)-chlorpheniramine, the level was lower than after ebastine and nondrug treatments. Graphical analysis followed by statistical parametric mapping (SPM96) revealed that H1-receptor rich regions such as cortices, cingulate gyrus and thalamus were regions where the BPs after ebastine were significantly higher than after (+)-chlorpheniramine (2 mg). H1-receptor occupancies in cortex were approximately 10% by ebastine and > or = 50% by either dose of (+)-chlorpheniramine (95% confidence interval for difference in the mean receptor occupancies: 27%, 54% for 2 mg and 35%, 62% for 6 mg vs ebastine, respectively). Receptor occupancies increased with increasing plasma concentration of (+)-chlorpheniramine, but not with concentration of carebastine, an active metabolite of ebastine. CONCLUSIONS: Ebastine (10 mg orally) causes brain histamine H1-receptor occupation of approximately 10%, consistent with its lower incidence of sedative effect, whereas (+)-chlorpheniramine occupied about 50% of brain H1-receptors even at a low but sedative dose of 2 mg; occupancy of (+)-chlorpheniramine was correlated with plasma (+)-chlorpheniramine concentration.

Intestinal absorption and metabolism of chlorpheniramine enantiomers in rat.

Chlorpheniramine (CPAM) is a chiral antihistaminic drug commercialized as a racemic mixture. The intestinal absorption and metabolism of CPAM have been investigated in rat using in vivo (oral and IV administration), in situ (intestinal loop model), and in vitro (everted sac model) experiments. Oral and IV administrations of 20 mg/kg of the racemic mixture show that the pharmacokinetics of CPAM are stereoselective, with higher AUCs for the (+)-S-enantiomer compared to its antipode. The monodesmethyl metabolite (DCPM) was quantifiable in blood and its pharmacokinetics are stereoselective after oral but not after IV administration. Experiments using intestinal loops and everted sacs showed that the absorption is not stereoselective and that in vivo stereoselective formation of DCPM is presumably due to stereoselective hepatic metabolism. Moreover, the in vitro and in situ absorption of CPAM are not modified by modulators of P-glycoprotein and cytochromes P450 (cyclosporin A, ketoconazole).

Prevalence of chlorpheniramine in aviation accident pilot fatalities, 1991-1996.

BACKGROUND: Chlorpheniramine is known to cause drowsiness, and this side effect has a potential to impair performance and could be a factor in accidents. METHODS: Therefore, to establish the prevalence of this drug in pilot fatalities of aviation accidents, a postmortem toxicology database--maintained at the Civil Aeromedical Institute--was examined for the presence of chlorpheniramine in the fatalities, occurred during 1991-1996. RESULTS: There were 47 (2.2%) accidents involving chlorpheniramine. Of these, 16 had only chlorpheniramine at 109 ng.ml-1 (n = 4) in blood and 1412 ng.g-1 (n = 12) in liver. Other drugs were also present in the remaining 31 cases, wherein chlorpheniramine concentrations were 93 ng.ml-1 (n = 18) in blood and 747 ng.g-1 (n = 12) in liver. Ninety-five percent of all quantitated blood values were at or above the therapeutic level (10 ng.ml-1), giving a 100 ng.ml-1 (n = 21) mean blood value. The drug's mean liver concentration from all cases was 1080 ng.g-1 (n = 24). The average blood value was approximately 10 times higher than the therapeutic value. CONCLUSIONS: The presence of other drugs did not appear to significantly alter the blood chlorpheniramine level, but no such correlation could be established with the hepatic value. The approximate 10-fold increase in the liver concentration was consistent with the general trend of drug distribution in the hepatic compartment. However, the contribution of postmortem redistribution of the drug to alter its concentration cannot be entirely ruled out. This study suggests that chlorpheniramine was present in some aviation fatalities at levels higher than therapeutic levels.

Affinities of brompheniramine, chlorpheniramine, and terfenadine at the five human muscarinic cholinergic receptor subtypes.

Anticholinergic effects are presumed to be the mechanism for the efficacy of chlorpheniramine in symptomatic relief of the common cold. Terfenadine, a second-generation antihistamine, reportedly lacks anticholinergic side effects. We evaluated affinities of two commonly used over-the-counter antihistamines, brompheniramine and chlorpheniramine, as well as terfenadine in comparison with atropine at the five human muscarinic cholinergic receptor subtypes using CHO cells stably transfected with the individual subtypes. Atropine was more potent than all three drugs at m1-m5 (p<0.01). No significant difference was observed between chlorpheniramine and brompheniramine. Atropine, brompheniramine, and chlorpheniramine could not discriminate between m1-m5. Terfenadine demonstrated subtype selectivity at m3. In vitro comparisons in human muscarinic receptor subtypes could potentially be used to predict clinical anticholinergic effects of antihistamines and to target receptor-specific effects of such agents.

Capillary electrophoresis, nuclear magnetic resonance and mass spectrometry studies of opposite chiral recognition of chlorpheniramine enantiomers with various cyclodextrins.

Markedly different chiral separation abilities were observed for native beta-cyclodextrin (beta-CD), carboxymethyl-beta-CD (CM-beta-CD) and heptakis (2,3,6-tri-O-methyl)-beta-CD (TM-beta-CD) towards the enantiomers of (+/-)-chlorpheniramine ((+/-)-CHL) in capillary electrophoresis (CE). Native beta-CD afforded almost baseline enantioseparation at a concentration of 18 mg/mL, whereas only 1 mg/mL solution of CM-beta-CD was required for adequate enantioseparation. TM-beta-CD allowed the nearly baseline enantioseparation only at a concentration as high as 80 mg/mL. Moreover, the migration order of (+/-)-CHL in the presence of TM-beta-CD was opposite to that with beta-CD and CM-beta-CD. 1H and 13C-NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS) have been used in order to obtain preliminary information about the stoichiometry and the binding constants in the intermolecular diastereomeric complexes of (+/-)-CHL with these CDs.

DNA-chlorpheniramine interaction studied by spectroscopic techniques.

It was previously studied that the antihistaminic chlorpheniramine elicits a biphasic response on cell growth and regulates polyamine metabolism, as described for polyamines. In part, polyamine effects on macromolecular synthesis and cell growth are attributed to nucleic acid:polyamine interactions. In this work, we have tested the hypothesis of a DNA:chlorpheniramine interaction, using fluorometry, FTIR and Raman spectroscopic techniques. The results indicate that DNA:chlorpheniramine interaction occurs inducing conformational changes in the macromolecule by affecting both phosphodiester bonds and bases. Results open new perspectives for characterization of action mechanisms of natural or synthetic diamines with pharmacological or physiological importance.

In vitro characterization of a controlled-release chlorpheniramine maleate delivery system prepared by the air-suspension technique.

Non-pareil cores were spray-coated with a chlorpheniramine maleate (an alkylamine antihistamine) layer and a Eudragit NE30D overcoat in a Wurster air-suspension apparatus. In vitro dissolution studies demonstrated that drug release was a function of polymer membrane thickness. Polyethylene glycol 6000, as a hydrophillic additive, increased the in vitro release of chlorpheniramine maleate from the pellets. Pellets coated with 8.30% Eudragit NE30D, 0.50% talc and 1.00% polyethylene glycol 6000 were found to display desirable controlled release characteristics for chlorpheniramine maleate over the 8-h testing period, which were also comparable with that of Dykatuss capsules. The controlled release pellets exhibited first-order release characteristics for chlorpheniramine maleate. Reproducibility of the manufacturing conditions employed in the study were confirmed thus ensuring reproducibility of drug release characteristics between batches of chlorpheniramine maleate pellets. Drug release from the pellets was shown to be independent of the dissolution method and medium used. Pellets displayed no significant change in drug release characteristics relative to the initial drug release data when stored for 12 weeks at room temperature (20 +/- 2 degrees C) and for 8 weeks at a low temperature (5 +/- 1 degrees C). However, pellets stored at 37 degrees C with 80% relative humidity and at 40 +/- 2 degrees C showed a slower in vitro drug release after 8-week storage and therefore failed to maintain their initial drug release profile.