Research paper
In vitro permeation studies comparing bovine nasal mucosa, porcine cornea and artificial membrane: androstenedione in microemulsions and their components

https://doi.org/10.1016/j.ejpb.2004.03.010Get rights and content

Abstract

The components of a carrier formulation can interact with an added drug as well as with the membrane surface they were applied on. Therefore, they can influence permeability of the membrane and permeation of the drug. The particular membrane structure might lead to different drug permeation out of one and the same carrier formulation. In this study, in vitro permeability of androstenedione (AD) as a highly lipophilic substance was investigated in excised bovine nasal mucosa, porcine cornea and the artificial cellulose membrane Nephrophan®. Two microemulsions (ME) with either the addition of the co-surfactants hydroxypropyl-γ-cyclodextrin (HP-γ-CD; ME-CD) or propylene glycol (PG, ME-PG) were tested in order to be used as carrier systems. Both MEs also consisted of 5% isopropyl myristate (IPM), 20% Cremophor® EL (CrEL), and water. Buffer solution (EBS) with 0.0025% AD served as control solution and was furthermore compared to 0.0025% AD/buffer-solutions containing the ME components HP-γ-CD in different concentrations (0.012, 0.024, 9%) as well as 20% CrEL.

The AD-permeation behaviour through the three tissues was differently influenced by the MEs. The apparent permeability coefficients (Papp) of nasal mucosa for both ME systems did not differ from the Papp of the AD/buffer solution. In case of the other two barriers (cornea, Nephrophan®), ME-PG as well as ME-CD provoked extended time lags for AD to permeate, so the Papp could not be calculated or declined to zero.

Papp of AD/buffer solution without any additives resulted for cornea, nasal mucosa and Nephrophan® in a ratio of 1:3:4. CrEL and 9% HP-γ-CD diminished the Papp, except HP-γ-CD in molar AD/HP-γ-CD-ratios of 1:1 (0.012%) and 1:2 (0.024%).

It seems that the composition of lipophilic and hydrophilic structures of the carrier systems or the additives had a higher impact on the Papp of cornea than on the Papp of the other tissues. Structure and character of the different membranes are considered to be mainly responsible for the differentiated permeation behaviour.

Introduction

Cornea and nasal mucosa both offer the possibility for simple and comfortable drug administration, but differ in structure: human cornea represents a five-layer barrier (∼500 μm thick) consisting of lipophilic epithelium (∼50 μm), hydrophilic stroma (∼450 μm) and lipophilic endothelium (monolayer) as well as Bowman membrane between epithelium and stroma and Descemet's membrane between stroma and endothelium (both monolayers). In contrast, nasal mucosa possesses a total thickness of only about 100 μm and consists of various cell types, which are ciliated and non-ciliated columnar cells, goblet and basal cells. Both administration sites (conjunctival sack and nasal cavity) are joined by the nasolacrimal duct, where normally lacrimal fluid is able to drain off [1]. But, via this route, drugs for a primarily ophthalmic use may also reach nasal mucosa and will thereby be systemically absorbed.

The amount of drug absorption and binding site could be regulated by varying the carrier composition. Furthermore, galenic formulations can optimize drug solubility or improve membrane permeability and therefore increase the bioavailability of the drug [2], [3], [4]. Additionally, adjuvants or formulations themselves can have positive effects like wetting the ocular or nasal surface or protect them against environmental influence.

In this regard a lipophilic component in the formulation seems to be advantageous, but especially for ocular use, unwanted side-effects like blurred vision and eye irritations should be considered. In case of nasal administration incompatibilities can be indicated for example by impairment of ciliar movement.

Use of a ME may minimize these side-effects by administering a lipophilic component in a transparent, water-continuous system [5], [6]. Microemulsions are multi-component systems, which normally contain a hydrophilic and a lipophilic component together with surfactant and co-surfactant, and represent clear to slightly opalescent and thermodynamically stable systems. In our research group a combination of a cyclodextrin with a microemulsion formulation was developed [14], in which the cyclodextrin replaced the co-surfactant.

Cyclodextrins possess promising advantages in modifying nasal and ocular drug absorption [8], [9]. These substances are cyclic oligosaccharides with a hydrophilic outer surface and a hydrophobic inner cavity, so that they are water-soluble and simultaneously able to take up poorly water-soluble, lipophilic molecules or parts of them into their cavities. These inclusion complexes offer various advantages like increasing water solubility or stability of the drug. However, the complex binding is non-covalent and the guest molecule can rapidly dissociate out of the cavity under suitable conditions [10], [11].

In order to detect diffusion of a drug out of the carrier formulation as well as influence of the formulation components on different biological membranes, permeation behaviour of a lipophilic drug through porcine cornea as well as bovine nasal mucosa was studied in the presented work. Porcine nasal mucosa only came to use as permeation barrier very recently [13], [14], whereas permeation studies using bovine nasal mucosa had been established in our group before [7]. However, use of bovine tissues was restricted and later on not possible to get because of the BSE-crisis. Therefore, porcine cornea was used because of its similar thickness and structure to human cornea.

Androstenedione (AD), the precursor of the male sexual hormone testosterone, was selected as lipophilic model drug component for our permeation studies. This substance may be used for nasal administration in order to achieve systemic effects for healthy ageing in men [17] and also for ocular administration with an indication of dry eye syndromes [18].

For considering bare effects of the preparations and their particular excipients on AD-permeation, permeability of an artificial cellulose membrane, Nephrophan®, was examined under same conditions like the biological tissues. In these studies the AD-permeation was unaffected by influences of biological structures.

Section snippets

Materials

AD was purchased from Schering AG (Berlin, Germany). HP-γ-CD (Mrel=1355, medium substitution grade of 0.6) was a gift from Wacker Chemie GmbH (Burghausen, Germany). Cremophor® EL (Macrogol-1500-glyceroltriricinoleat, CrEL), propylene glycole (PG) and isopropyl myristate (IPM) were purchased from Caesar und Lorentz GmbH (Hilden, Germany). Acetonitrile was used in HPLC grade and purchased from Baker J.T. (Deventer, The Netherlands). All other materials were of analytical grade. Earl's balanced

Results

Permeability coefficients (Papp) of AD through bovine nasal mucosa, porcine cornea and Nephrophan® out of different donor solutions are reported in Table 1. Lag-time, if any, is also listed in Table 1, written in italics beside the corresponding Papp.

Statistical comparison of the two differently designed Nephrophan® permeability studies (90 and 300 min) led to no significant differences, except in case of 9% HP-γ-CD. Therefore, they were not combined and listed up due to the test period,

Discussion

Nasal mucosa represents a weak barrier for lipophilic drugs [21], [22] as well as for hydrophilic substances [23], [24]. Nephrophan® consists of regenerated cellulose without biological structures like cell membranes, ion channels or tight junctions, and limits drug permeation only by pore size and number. In contrast, the considerable thicker cornea shows a five-layer structure with marked lipo- and hydrophilic layers. Therefore, the ranging of Papp (cornea<nasal mucosa<Nephrophan®≈1:3:4)

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