Astemizole metabolite; Histamine H1 Receptor

Tecastemizole, a major metabolite of astemizole, is a potent and selective H1 receptor antagonist. Evidence suggests that this and certain other H1 receptor antagonists may possess anti-inflammatory effects that are, in some cases, independent of H1 receptor antagonism. Objective The aim of this study was to investigate the anti-inflammatory effects of tectastemizole in models of allergic inflammation[1].

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Tecastemizole (10–300 μm) significantly inhibited ICAM-1 expression induced through the stimulation of HUVECs with IL-1β (100 U/mL) for 6 h. Astemizole was also seen to inhibit ICAM-1 expression at the higher concentrations tested (100 and 300 μm; Fig. 3a). Similarly, VCAM-1 expression on these cells was inhibited by tecastemizole, although only at higher concentrations (100 and 300 μm), while astemizole was effective only at 300 μm (Fig. 3b). Tecastemizole (10–300 μm) inhibited MNC adhesion to HUVECs, in a concentrationindependent manner, when co-incubated with HUVECs and the inflammatory stimulus for 6 h, then removed by washing before the addition of the MNC suspension. However, astemizole had no effect on MNC adhesion to stimulated HUVECs.At the same concentrations found to inhibit endothelial adhesion molecule expression and adhesion of mononuclear cells to endothelial monolayers, neither tecastemizole nor astemizole affected TNF-α release from isolated moonuclear cells, in response to LPS stimulation.

Tecastemizole inhibited antigen-induced eosinophil recruitment to the lungs of allergic mice in a dose-dependent manner. Furthermore, combination of a sub-effective dose of tecastemizole, combined with a sub-effective dose of dexamethasone inhibited eosinophil accumulation in this model. Plasma extravasation in PCA reactions was inhibited by tecastemizole, although by a mechanism that would appear to be H1 receptor-dependent. Cytokine-induced endothelial intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression, as well as mononuclear cell adhesion to human umbilical vein endothelial cells was inhibited by tecastemazole in a manner independent of H1 receptor antagonism. Conclusion: These data suggest that tecastemizole may have H1 receptor-independent effects in inhibiting late-phase inflammatory responses, while acute responses appear to be inhibited in a H1 receptor-dependent manner. Furthermore, our data suggest an important potential steroid-sparing role for such drugs in the treatment of allergic inflammatory conditions[2].

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Tecastemizole (1 mg/kg), administered daily by intraperitoneal injection, 24 h before the first challenge and an hour before each challenge thereafter, effectively suppressed eosinophil recruitment to the lungs of OVA-sensitized mice (Fig. 2c), while the lower doses tested (0.1 and 0.5 mg/kg) were without effect (Fig. 2a and b, respectively). Lymphocyte and monocyte (MNC) cell numbers in bronchoalveolar lavage (BAL) samples were not affected by tecastemizole at any of the doses administered. Furthermore, no significant effect on peripheral blood leucocyte levels was observed at any of the doses tested.[1]
Tecastemizole (0.1 mg/kg) significantly inhibited the response to the lower dose of histamine; however, at the higher dose (1 mg/kg), responses to both doses of OVA, as well as to both doses of histamine, were inhibited (Fig. 1a). Tecastemizole had no effect on the bradykinin response[1].

Endothelial adhesion molecule expression[1]
Confluent HUVEC monolayers were stimulated with IL-1β, for 6 h, in the absence and presence of tecastemizole or astemizole (10–300 μm). Culture medium was removed and monolayers washed. ICAM-1 and VCAM-1 expression were measured using a direct ELISA technique described previously. In brief, plates were blocked with 5% powdered milk in PBS before application of primary antibody (mouse anti-human ICAM-1, clone BBIG-I1, 1 μg/mL; mouse anti-human VCAM-1, clone BBIG-V1, 1 μg/mL). After 1 h, plates were washed with blocking solution before application of a secondary goat-anti-mouse horseradish peroxidase (HRP) conjugated antibody. Following a further incubation of 1 h, plates were washed repeatedly and 100 μL per well 3,3′,5,5′-tetramethylbenzidine (TMB) substrate added. After approximately 30 min, the reaction was quenched by addition of 100 μL 2 m sulphuric acid. Plates were analysed spectrophotometrically at 450 nm.

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In vitro leucocyte–endothelial adhesion assay[1]
Monolayers of HUVECs were grown to confluency in the central 60 wells of flat-bottomed 96-well plates and were stimulated with IL-1β (100 U/mL), for 6 h, in the absence and presence of tecastemizole, or the parent drug astemizole.
Peripheral venous blood was collected from healthy volunteers (n=6), under Ethical approval (King's College London), into tubes containing 10% v/v ACD, before centrifugation at 1000 g in tubes containing Histopaque-1077.
The mononuclear cell (MNC) layer was collected and contaminating red blood cells removed by hypotonic lysis. For comparison in these experiments, neutrophils were isolated from the remaining blood fraction by sedimentation on 6% dextran solution, centrifugation of the resulting supernatant then hypotonic lysis of contaminating erythrocytes. In both cases, cells were washed three times in modified (Ca2+-/Mg2+-free) Hank's balanced salts solution (HBSS) and radiolabelled with sodium 51chromate (37 kBq per 10~6 viable cells). Following a labelling period of 60 min, cells were washed a further three times in modified HBSS and resuspended in complete HBSS, at a concentration of 106 cells/mL. HUVEC monolayers were washed and 200 μL radiolabelled cell suspension added per well.
Tecastemizole or astemizole (10–300 μm), or vehicle (DMSO) control, were added to some wells, either immediately before IL-1β and left for 6 h, following which, drugs and stimulus were removed before addition of leucocytes, or to pre-stimulated HUVECs, where drugs were added immediately before the leucocyte suspension. Plates were incubated for 30 min at 37°C/5% CO2. At the end of this period, non-adherent cells were gently aspirated and HUVEC monolayers washed with complete HBSS solution. To each well, 200 μL of 1% Igepal were added and left for 15 min. One hundred microliters samples of lysates were removed from wells and put into scintillation vials for γ-counting. The processes involved in the adhesion assay did not affect MNC or neutrophil viability as measured by trypan blue exclusion. HUVEC cultures were examined by phase contrast microscopy immediately before cell lysis to confirm monolayer integrity before plates were used further.

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Tumor necrosis factor-α release from mononuclear cells[1]
Mononuclear cells, isolated as above, were plated (105 cells/well) into the central 60 wells of 96-well plates, in the absence and presence of tecastemizole or astemizole. Lipopolysaccharide (LPS; 10 ng/mL; Escherichia coli 0111:B4) was added to some wells and plates were incubated for 18 h. Following this period, plates were centrifuged and cell-free supernatants transferred to pre-coated wells of an ELISA plate. Tumour necrosis factor-alpha (TNF-α) concentrations were determined using a commercially available kit, with human recombinant TNF-α used to construct a standard curve.

Cutaneous responses in the guinea-pig[1]
Animals were shaved and sensitized by 0.1 mL intradermal (i.d.) injections of 1 : 100 guinea-pig anti-ovalbumin (OVA) serum. After 24 h, animals (n=5 per group) were injected (intraperitoneal) with vehicle (1% dimethylsulphoxide (DMSO)) or tecastemizole (0.1 or 1 mg/kg). One hour later, animals were anaesthetized (urethane, 1.5 g/kg) and Evans blue dye was injected via a foot vein. OVA (Grade V; 0.1 or 1 μg) was administered i.d. into the serum-pretreated sites and saline (control), histamine (0.3 or 0.6 μg) or bradykinin (1 μg) were administered i.d. into the remaining sites, in a balanced block design. Thirty minutes later, the guinea-pigs were killed by an overdose of urethane. Cutaneous responses were assessed as a function of the area (cm2) and intensity (grey pixels) of extravasated dye in skin sites, by analysis of digital photos of the responses, using ImageJ software.
At the end of experiments, tracheae were removed from the animals and isometric contractile responses to histamine were measured ex vivo. Tracheae were cut into sections which were suspended in an organ bath containing Krebs' solution, maintained at 37°C and aerated with 95% O2/5% CO2. Responses to cumulative concentrations of histamine (10−9–10−3 m) were measured and expressed as % of the response to methacholine (10−5 m). By way of comparison, in some experiments, tracheae were removed from naïve animals and cumulative concentration–response curves to histamine constructed, in the absence and presence of tecastemizole (10−8 and 10−7 m) added to the bath. Results are expressed as mean±standard error of the mean (SEM) and data were analysed by one-way anova.

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Mouse model of allergic lung inflammation[1]
Mice were sensitized by i.p. injection of 10 μg OVA, in aluminium hydroxide gel (3 mg), on day 0 and 7. Mice were then challenged for 20 min by exposure to aerosolized 1% OVA in saline, by ultrasonic nebulization, on days 14, 15 and 16 to induce antigen-driven lung inflammation and were studied 24 h after the final challenge. Tecastemizole and/or dexamethasone were dissolved into 0.1% DMSO. The drugs were administered i.p. in a volume of 100 μL 24 h before the first challenge and an hour before every challenge thereafter.
Twenty-four hours after the final challenge, mice were killed by urethane overdose and cells in the lungs were recovered by flushing 3 × 0.5 mL of saline into the lungs, via the cannulated trachea. Total cell counts were determined, and differential cell counts were performed after staining of thin-layer cytospin preparations (Diff-Quik, Gamidor Ltd., Didcot, Oxon, UK). Blood was collected by cardiac puncture, blood smears were prepared and stained and total and differential cell counts carried out. Results are expressed as mean±SEM and data were analysed by one-way anova.

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Effects of tecastemizole were assessed in a murine model of allergic lung inflammation, in passive cutaneous anaphylaxis (PCA) responses in guinea-pig skin and in in vitro assays measuring endothelial adhesion molecule expression and leucocyte-endothelial adhesion.[1]

Metabolism / Metabolites
Norastemizole is a known human metabolite of astemizole.

[1]. Effect of tecastemizole on pulmonary and cutaneous allergic inflammatory responses. Clin Exp Allergy. 2007 Jun;37(6):909-17.

Drug Indication
Investigated for use/treatment in allergic rhinitis.

C19H21FN4

324.4

324.175

C, 70.35; H, 6.53; F, 5.86; N, 17.27

75970-99-9

75970-64-8 (Hydrobromide);75970-64-8 (Hydrobromide)

123618

Off-white to light yellow solid powder

1.28g/cm3

519.8ºC at 760 mmHg

268.2ºC

1.658

3.138

2

4

4

24

393

0

SFOVDSLXFUGAIV-UHFFFAOYSA-N

InChI=1S/C19H21FN4/c20-15-7-5-14(6-8-15)13-24-18-4-2-1-3-17(18)23-19(24)22-16-9-11-21-12-10-16/h1-8,16,21H,9-13H2,(H,22,23)

1-[(4-fluorophenyl)methyl]-N-piperidin-4-ylbenzimidazol-2-amine

R 43512; Norastemizole; Tecastemizole; Norastemizole; 75970-99-9; Soltara; 1-(4-fluorobenzyl)-N-(piperidin-4-yl)-1H-benzo[d]imidazol-2-amine; T 1348; CHEMBL61301; W5DCO14M05; Tecastemizole

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~308.26 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.71 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.71 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.71 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)