University of Groningen Transdermal Delivery of Anticholinergic
Total Page：16
File Type：pdf, Size：1020Kb
University of Groningen Transdermal delivery of anticholinergic bronchodilators Bosman, Ingrid Jolanda IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 1996 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Bosman, I. J. (1996). Transdermal delivery of anticholinergic bronchodilators: methodological and clinical aspects. s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 28-09-2021 Chapter 2 Selection and evaluation of anticholinergics for transdermal drug delivery 2.0. Summary In Part 1, the receptor affinities of 18 anticholinergics, expressed as dissociation constants, were determined in order to select the most active compounds. Six drugs showed high affinities towards the muscarinic receptor (Kd < 30 nM), and these compounds were selected as possible candidates for transdermal drug delivery because they can theoretically provide therapeutic plasma concentrations by dermal application on a limited area. In Part 2, we describe the physicochemical characteristics of the six selected anticholinergics (atropine sulphate monohydrate, benztropine mesylate, dexetimide hydrochloride, oxyphencyclimine hydrochloride, scopolamine hydrobromide trihydrate, tropicamide) together with atropine base. These characteristics may be useful to explain possible differences in in vitro permeation experiments. The chemical structures, molecular weights, and pKa values were available from literature. The partition coefficients, and the solubility, pH and stability in the dosing and receptor solutions were experimentally determined. 24 Chapter 2 Part 1. Selection of anticholinergics for transdermal drug delivery 2.1. Introduction Anticholinergics can be used as bronchodilators to prevent or diminish airways obstruction in patients with obstructive airways diseases. The only regularly used bronchodilator therapy with anticholinergics is the inhalation of ipratropium bromide [1]. However, because of the relatively short duration of action, it must be given four times a day. The transdermal route of administration may offer the advantage of producing a sustained, constant and controlled level of drug in the blood resulting in a prolonged duration of action [2]. To investigate the suitability of a series of drugs with purported anticholinergic properties (anticholinergics) for transdermal drug delivery, the affinities of 18 tertiary amines towards the muscarinic receptor were determined by radioreceptor assay (RRA). We wanted to select the most active compounds for transdermal drug delivery because only very potent molecules can be delivered through a small skin surface. 2.2. Materials and Methods 2.2.1. Chemicals [N-methyl-3H]Scopolamine methyl chloride ([3H]NMS, 81.5 Ci/mmol) was obtained from Du Pont NEN (Du Pont, Wilmington DE, USA). The tested anticholinergics, adiphenine hydrochloride, atropine sulphate monohydrate, benztropine mesylate, biperiden hydrochloride, dexetimide hydrochloride, dicyclomine hydrochloride, homatropine hydrochloride, methixene hydrochloride, orphenadrine hydrochloride, oxyphencyclimine hydrochloride, piperidolate hydrochloride, pirenzepine dihydrochloride monohydrate, procyclidine hydrochloride, profenamine hydrochloride, scopolamine hydrobromide trihydrate, terodiline hydrochloride, trihexyphenidyl hydrochloride, tropicamide, were all of pharmaceutical quality and obtained from local wholesalers. All other chemicals and solvents were of analytical grade and obtained from Merck (Darmstadt, Germany). Polyethylene tubes (12 ml) were obtained from Greiner (Alphen a/d Rijn, The Netherlands). The GF/B glassfibre filters were from Whatman (Maidstone, UK). Rialuma was used as scintillation liquid, obtained from Lumac (Olen, Belgium), in combination with mini-scintillation counting vials from Packard (Groningen, The Netherlands). 2.2.2. Preparation of solutions The 50 mM sodium phosphate buffer pH 7.4 (assay buffer) was prepared by Selection and evaluation of anticholinergics 25 Table 2.1. Affinities of 18 anticholinergics, expressed as dissociation constants (Kd). Very low affinity Low affinity High affinity (Kd > 1 µM) (Kd >30nM) (Kd <30nM) Adiphenine HCl Biperiden HCl Atropine sulphate.H2O Dicyclomine HCl Pirenzepine 2HCl.2H2O Benztropine mesylate Homatropine HCl Procyclidine HCl Dexetimide HCl Methixene HCl Terodiline HCl Oxyphencyclimine HCl Orphenadrine HCl Trihexyphenidyl HCl Scopolamine HBr.3H2O Piperidolate HCl Tropicamide Profenamine HCl dissolving 1.38 g NaH2PO4.H2O and 7.12 g Na2HPO4.2H2O in 1 l distilled water. Anticholinergics stock solutions of 1x10-3M were prepared in ethanol and stored at -20°C. 2.2.3. Determination of receptor affinities The receptor affinities of eighteen tertiary anticholinergics towards the muscarinic receptor [3], expressed as dissociation constants, Kd, were determined by radioreceptor assays, and [3H]NMS was used as radiolabelled ligand. From the anticholinergic stock solutions, appropriate dilutions were made in assay buffer, concentrations ranging from 1x10-9M to 1x10-5M (calibration curve). Fifty µl aliquots of the solutions of anticholinergics were added to polyethylene tubes in duplicate, giving final assay concentrations ranging from 1x10-10M to 1x10-6M. Then 400 µl receptor preparation, containing 2 mg lyophilized receptors in assay buffer [3], were added. The tubes were vortexed and incubated during 60 min at 0°C before 50 µl of [3H]NMS (4x10-9M) were added. The tubes were vortexed again and incubated for another hour at 0°C. After the addition of 4 ml icecold assay buffer, the samples were immediately filtered through Whatman GF/B glassfibre filters under vacuum using a filtration apparatus (48S, University Centre for Pharmacy, Groningen, The Netherlands). The tubes were rinsed twice with 4 ml icecold assay buffer, which was also filtered. The total filtration and rinsing process, taking place in approximately 15 s, was carried out on each tube in turn. The filters were transferred into mini-scintillation vials and dispersed in 3.5 ml scintillation cocktail by shaking for 120 min. The vials were counted for 40,000 counts or 5 min in a liquid scintillation counter (Minaxi, Packard, Groningen, The Netherlands), whatever came first. Fifty µl of the used [3H]NMS (4x10-9M) solution were added to 2 mini-scintillation vials and counted as well. The curves were fitted with the Ligand curve fitting program to calculate the dissociation constants of the anticholinergics [4]. 26 Chapter 2 2.3. Results and Discussion The receptor affinities of 18 anticholinergics, expressed as dissociation constants, were determined in order to select the most active compounds (Table 2.1). Seven drugs had very low affinities (Kd > 1 µM) to the muscarinic receptor, 5 drugs had low affinities (Kd > 30 nM) and 6 drugs showed high affinities with Kd <30nM. These six compounds were selected for in vitro permeation experiments because they may theoretically provide therapeutic plasma concentrations by dermal application on a limited area. Since we also wanted to examine possible differences between salts and bases, we included atropine base in our further experiments. Part 2. Characteristics of the anticholinergics selected for transdermal drug delivery 2.4. Introduction Transport of drugs into or through the skin depends on a number of factors such as characteristics of the permeant, condition and type of skin, other chemicals present in the dosage form (e.g. enhancers) and external conditions (e.g. temperature). The factor with perhaps the greatest influence is the physicochemical character of the permeant [5, 6]. For the majority of drugs, the main barrier in skin permeation is the stratum corneum. According to Fick’s first law, the permeation of a drug through this layer depends on the permeability coefficient and the concentration gradient of the permeant across the stratum corneum [7, 8]: D K ∆C J K ∆C (2.1) p L where: J = steady-state flux of the permeant through the stratum corneum (µg.cm-2.s-1); -1 Kp = permeability coefficient of the permeant in the stratum corneum (cm.s ); ∆C = concentration gradient of the permeant across the stratum corneum (µg.cm-3); D = diffusion coefficient of the permeant in the stratum corneum (cm2.s-1); K = apparent partition coefficient of the permeant between the stratum corneum and the vehicle; L = the length of the pathway through the stratum corneum (cm). The permeability coefficient is the product of the partition coefficient