Novel Beads Made of Alpha-cyclodextrin and Oil for Topical Delivery of a Lipophilic Drug
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- Trichard, L., Delgado-Charro, M.B., Guy, R.H. et al. Pharm Res (2008) 25: 435. doi:10.1007/s11095-007-9395-0
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To investigate the potential of a novel lipid carrier, comprising beads of alpha-cyclodextrin and soybean oil, for topical drug delivery. Adapalene was chosen as a model drug to explore the ability of the beads to encapsulate and release a highly lipophilic compound.
Materials and Methods
Adapalene-loaded beads were prepared and characterised. Skin tolerance to unloaded beads was tested on human volunteers, while drug release and delivery into stratum corneum, was evaluated in pig skin ex vivo.
The preparation and physical characteristics of the beads were not dependent on whether adapalene had been previously dissolved or dispersed in soybean oil. Drug encapsulation efficiency was high (>96%) and drug loading on the order of a therapeutic level could be achieved in freeze-dried beads prepared from an oily dispersion of adapalene. After application to human skin, unloaded beads induced no adverse reaction and were better tolerated than an alcoholic gel. Tape-stripping the stratum corneum from treated pig skin showed that adapalene release and penetration from the beads was comparable to that from gel and cream formulations available on the market.
These novel beads may offer a well-tolerated and efficient system for the encapsulation and topical delivery of lipophilic drugs.
Key wordsadapalenebeadcyclodextrinlipid carriertopical formulation
Due to its composition and structure, the skin exhibits excellent barrier function and limits the penetration of a wide range of therapeutic agents into the body. Indeed, only small, neutral and moderately lipophilic molecules can easily cross the skin. To improve the delivery of highly lipophilic drugs, several innovative formulations, such as microemulsions (1), liposomes (2) and solid lipid nanoparticles (3), have been developed in recent years.
To further extend the range of formulation strategies available for highly lipophilic therapeutic agents, a novel type of bead has been conceived. These carriers are prepared by a simple and original process involving a mixture of an aqueous solution of α-cyclodextrin with soybean oil, but without the use of any organic solvent or surface-active agent (4). The spherical beads have a diameter of 1.6 ± 0.2 mm and consist in a partial crystalline matrix of cyclodextrin surrounding micro-domains of oil (4). Freeze-dried beads have high lipid content (80%) and a semi-solid consistency which facilitates their application to the skin.
Adapalene [6-(3-(1-adamantyl)-4-methoxyphenyl)-2-naphtoic acid] is a synthetic derivative of vitamin A (retinol), used for the treatment of acne. This drug is currently formulated as an aqueous gel or a cream, and is marketed as Differin®. Adapalene (412.52 g/mol, pKa = 4.23, logP = 8.04, sparingly water-soluble from SciFinder database) was chosen as a model drug with which to explore the ability of the beads to encapsulate and deliver a highly lipophilic compound. The fluorescence properties of the molecule, and its inherent stability, were additional advantages that allowed its facile quantification and visualisation in formulations and in skin.
The present work focused on the preparation and the characterisation of adapalene-loaded beads. Skin tolerance to the unloaded beads was considered a prerequisite for further investigations and was therefore studied first. Subsequently, adapalene penetration into the outer layer of the skin was then quantified after application of the novel beads, and this uptake was compared to that from currently commercialised formulations.
MATERIALS and METHODS
Alpha-cyclodextrin (α-CD) (Pharma grade) and Cropure® soybean oil were purchased from Wacker-Chemie (München, Germany) and Croda (Trappes, France), respectively. Butylated hydroxytoluene (BHT) was from Sigma Chemical Co. (St Louis, USA) and hydroxypropylcellusose pharmaceutical grade (Klucel M®) was a gift from Aqualon-Hercules Inc (Wilmington, USA). Adapalene was supplied by Sequoia Research Products (Oxford, UK). Differin® gel 0.1% (main excipients: carbomer 940, propylene glycol, poloxamer 182 and water) and Differin® cream 0.1% (main excipients: carbomer 934P, perhydrosqualene, cyclomethicone, glycerin and water) were purchased from Galderma (La Défense, France). Ethanol (96° and pure), tetrahydrofuran, acetonitrile, and trifluoroacetic acid (all HPLC grade) were obtained from Fisher Scientific (Loughborough, UK), while chloroform and methanol (analytical grade) were from Carlo Erba Reagenti (Val de Reuil, France).
Preparation of Adapalene-Loaded Beads
Adapalene was either solubilised (86 μg/ml) or dispersed (4.3 mg/ml) in soybean oil. Of this oily phase, 5.8 ml was added to 20 ml of an aqueous solution (8.1% w/v) of α-CD. The mixture was continuously shaken at 200 rpm (Salvis, Bioblock Scientific, Illkirch, France) at 28°C for several days. The resulting beads were then washed and freeze-dried during 48 h to eliminate water (Christ LDC-1 alpha1-4 freeze-dryer, Bioblock Scientific). Unloaded beads were prepared using the same protocol.
Characterisation of Beads
Bead diameter was determined after freeze-drying (n = 50 beads) using an optical microscope (Leitz Diaplan microscope, Leica Microsystèmes, France) equipped with a Coolsnap ES camera (Roper Scientific, Germany). The physical state of adapalene in crushed beads was observed by fluorescence microscopy. Adapalene was excited with a mercury short arc lamp (HBO 50W, Osram, Germany) coupled with a filter (Leica Microsystèmes, France) having the following characteristics: excitation band-pass filter from 340 to 380 nm and emission long-pass filter >425 nm. Differin® gel and Differin® cream were submitted to the same examination.
To extract adapalene from freeze-dried beads, 40 mg were precisely weighed, gently destroyed and then mixed with 5 ml of (2:1, v/v) chloroform/methanol. Insoluble bead material was eliminated by centrifugation for 5 min at 2,000×g (Capsule HF 120, Tomy Tokyo, Japan) and adapalene content in the supernatant was quantified by fluorescence (Luminescence Spectrometer 50B, Perkin Elmer, Bucks, UK).
Encapsulation efficiency and drug loading were expressed as mean ± standard deviation (n = 3 batches).
Skin Tolerance Study
Skin tolerance of unloaded, freeze-dried beads was assessed on seven healthy human volunteers (one male, six females, aged from 22 to 42 years). An adhesive template (Polyethylene foam tape 1772, 3 M™, St Paul, USA) was placed on the forearm to delimit the application site (diameter 2.0 cm). Taking into account the volumic mass of each formulation, 100 μl/cm2 of the beads (0.2 g/ml) or of an alcoholic gel control (3.0% w/v hydroxypropylcellulose, 0.01%w/v butylated hydroxytoluene and 97% w/v ethanol; 0.9 g/ml) was applied under an occlusive cover comprising a double layer of adhesive (Book tape 845, 3 M™, St Paul, USA).
Another control site was untreated at all and simply occluded as a reference. Skin reaction was evaluated by observation and quantified by skin colour, using a chromameter (CM-2600d, Konica Minolta, Osaka, Japan), and by transepidermal water loss (TEWL) measurements (AquaFlux AF 102, BioX, London, UK). Values were obtained pre-application of formulations and at 24, 48 and 72 h post-treatment. The formulations were carefully removed immediately before each of the daily measurement, and then fresh vehicles were re-applied afterwards. Experiment was stopped in the case of intense cutaneous reaction on volunteer skin. The experimental protocol was approved by the Bath Local Research Ethics Committee.
Two-way ANOVA was used to evaluate the influence of the vehicle and of the duration of application on skin colour. p value less than 0.05 was considered statistically different.
To compare the effects of the beads and the alcoholic gel, the Wilcoxon matched paired test (at p < 0.05) was used. A Friedman test evaluated the influence of application time on TEWL variations (again, at p < 0.05).
Adapalene Penetration in Stratum Corneum
Application of Adapalene Formulations
Abdominal skin from a single domestic pig (750 μm dermatomed) was the model used to assess adapalene absorption. After thawing the tissue, any obvious hair was trimmed with scissors, and four application sites (circular, 1.6 cm diameter) were delimited using an adhesive template (Scotch® Book Tape 845, 3 M, St. Paul, USA ), taking care to avoid any skin abrasions or scars. Differin® gel 0.1%, Differin® cream 0.1%, and freeze-dried adapalene-loaded beads (corresponding to 0.01 mg of adapalene/cm2) were each applied at one of the three identified positions. The skin was then placed in a 7 cm diameter Franz cell, the receptor compartment of which was maintained at 32°C and filled with phosphate-buffered saline (pH 7.4). The skin surface was not covered. Twenty-four hours later, the cell was dismantled and any remaining formulations were carefully removed using a wet tissue. Samples were then submitted either to a tape-stripping procedure or to fluorescent microscopic observation.
For each tape, the adapalene concentration in the stratum corneum was plotted as a function of the cumulative thickness removed. The total amount of adapalene taken up into stratum corneum was calculated as the sum of the drug amounts on the tapes collected at each application site (mean±SD ; n = 5 for the three different vehicles).
Adapalene was visualised in the skin by fluorescence microscopy (Leitz Diaplan microscope (Leica Microsystèmes, France), equipped with a Coolsnap ES camera (Roper Scientific, Germany). Adapalene was excited with a mercury short arc lamp as described above. Blue fluorescence from adapalene was clearly distinct from the autofluorescence of skin and hairs.
RESULTS AND DISCUSSION
Unloaded and Adapalene-Loaded Bead Characteristics (n = 3 Different Batches; Mean±SD)
Process Duration (days)
Fabrication Yield (%)
Bead Size (mm)
Encapsulation Efficiency (%)
Drug Loading (mg of drug/g of beads)
84 ± 4
1.6 ± 0.2
Adapalene dissolved in soybean oil (86 μg/ml)
87 ± 2
2.0 ± 0.2
98 ± 1
0.080 ± 0.003
Adapalene dispersed in soybean oil (4.3 mg/ml)
90 ± 4
2.1 ± 0.2
96 ± 4
3.4 ± 0.2
The bead preparation time was longer, and the bead diameter was greater, when the drug was included in the formulation (Table I). It is possible that adapalene interferes in interactions essential to bead formation between cyclodextrin and the triglycerides at the oil/water interface (4).
As a result of these findings, further experiments were undertaken only with the freeze-dried beads prepared from the oily suspension, in which the adapalene content was similar to that in the Differin® formulations (3.4 ± 0.2 mg/g).
Skin Tolerance to Beads
Skin reaction to drug-free beads was evaluated by three different methods and compared to that provoked by an alcoholic gel. The latter was chosen as an irritant reference and to validate the experimental approach. Indeed, ethanol is known to extract lipids from the stratum corneum under certain conditions (9). The alcoholic gel was prepared according to the composition of a currently commercialised product (but excluding the drug, of course).
The unloaded beads were composed of α-CD (20% w/w) and soybean oil (80% w/w) (4). Cyclodextrins have been reported to be non-irritating when applied to skin but no investigation has ever been conducted with concentrations greater than 10% w/v (10, 11,12). The present work demonstrates that a high cyclodextrin content in beads is not irritant in vivo. Moreover, the oil content of the beads was sufficient to provide an occlusive effect, as shown by the TEWL results. It should be emphasised that the application conditions used in this study, involving a large amount of vehicle administered continuously for 72 h under occlusion, are ‘extreme’ in relation to the expected conditions of use for a dermatological product. It may be reasonably concluded, therefore, that no bead-associated adverse effects would be observed under the clinical use of such a formulation.
Adapalene Penetration Study
This work describes a new strategy for lipophilic drug encapsulation and topical delivery. The bead preparation process is simple and advantageously avoids the use of organic solvent, surface-active agents or elevated temperature. The beads are well-tolerated by skin; micro-domains of oil present within the bead inner structure favour high adapalene loading and confer an occlusive property. The beads release adapalene as efficiently as currently commercialised medicines.