EP1696855A1 - Dermal drug delivery system - Google Patents

Abstract

A device for brushing the skin prior to applying a topical preparation of a drug enhances the permeability of the stratum corneum to the drug.

Description

DERMAL DRUG DELIVERY SYSTEM

The present invention relates to the delivery of drugs by abrading the skin, and devices for use with such methods.

In order for therapeutic quantities of drug to penetrate the skin, the barrier properties of the stratum corneum must be overcome. The stratum corneum exhibits selective permeability and allows only relatively lipophilic compounds with a molecular weight below 400 Daltons to pass.

Methods of overcoming the barrier properties of the stratum corneum may be divided into chemical, such as penetration enhancers and supersaturated concentrations, and physical, such as iontophoresis, skin electroporation, ultrasound and powder needleless injection, methods. In the case of macromolecules (>1000 Da) the use of physical methods has been shown to be advantageous over chemical methods, and has led to a significant increase in the number of topical / transdermal devices.

The mode of operation of these devices includes skin disruption and microneedles/microblades. Gerstel (1976) was one of the first to describe a drug delivery device for percutaneous administration of drugs. The device consists of a drug reservoir and a plurality of microneedles extending from the reservoir, for penetrating the stratum corneum and epidermis to deliver the drug. This device is the forerunner of many devices on the market today. A recent commercialisation of this microneedle technology is the Macro flux® microprojection array developed by Alza Corporation.

Most transdermal devices are currently based on patch technology, and problems encountered include irritancy and poor adhesion. Most patches only deliver about 10% of the total dose, with the 90% of the drug remaining in the patch being discarded.

Known abrasion devices are relatively expensive and can only be operated by trained personnel, thereby limiting their ability to be used by patients. Since it is also a painful surgical procedure, anaesthesia is administered beforehand.

WO 01/89622 describes the use of an applicator with an abrading surface on which the drug is coated. We have now, surprisingly, found that the properties of the stratum corneum can be substantially altered by a short period of brushing, such that therapeutically effective amounts of drug can pass thereacross.

Thus, in a first aspect, the present invention provides a method for the conditioning of skin to enhance transdermal delivery of drug, the method comprising continuous brushing for a period sufficient to reduce, or perturb, the barrier qualities of the stratum corneum.

In this respect, brushing affects the stratum corneum in such a manner as to facilitate, normally, and preferably temporarily, passage of a drug across it, by physically disrupting the barrier thereby to reduce the resistance to the drug's passage.

By "barrier qualities" is meant those properties of the stratum corneum that inhibit the passage of drug. Reducing the barrier qualities is also referred to herein as "enhancing permeability" and other related terms.

By "transdermal" is meant delivery of drug across the stratum corneum. In general, drugs delivered by such a route are intended for local conditions of the skin, and are not usually intended for systemic delivery. Thus, the drug will cross the stratum corneum where, normally, it will then reach the required site of action.

Any drug may be employed in the present invention, the term "drug" here relating to any pharmaceutically active substance capable of delivery across the stratum corneum in association with the method of the present invention. Suitable examples of drugs are given below.

The "continuous brushing" may be interrupted, if desired, but it is intended that the brushing continue for a generally consistent period ranging between about 10 seconds and 5 minutes, although longer or shorter periods may be employed, depending on the results to be achieved and the nature of the drug, as well as other factors, such as pressure applied to the brush.

Thus, by "continuous" is meant that, between the start and finish of the brushing process, the brushing should take place for greater than 50% of the time, preferably greater than 80%, and is, most preferably, substantially uninterrupted. The nature of the brushing may be a simple backward and forwards action, but is preferably oscillatory, and is particularly preferably circular or rotatory. Not only is a circular brushing motion easy to arrange and employ, but it has also been found that the circular motion results in the greatest enhancement of the permeability of the stratum corneum.

Without being limited by theory, it is believed that the continuous brushing motion, especially when circular, not only serves to stretch the skin slightly, but also serves to warm the surface of the skin, both of these actions serving to enhance penetration of drug.

What is particularly surprising is that this circular brushing is able to increase, temporarily, the permeability of the stratum corneum to such an extent that it is even greater than such treatments as tape stripping, a process which is only generally applicable to isolated skin and which substantially destroys the integrity thereof. By contrast, the brushing of the present invention only has a temporary effect on permeability but, whilst the effect is achieved, the permeability is raised substantially.

The drug may be applied in any suitable form. It is generally preferred to apply the drug in the form of a solution, gel, cream, emulsion, colloidal system, foam, mousse, suspension or ointment, and the preparation may generally comprise further ingredients, such as penetration enhancers and emollients.

Suitable drugs for use in accordance with the present invention include, but are not limited to, those in the following Table, either individually or in combination:

Other suitable drugs include the non-steroidal anti-inflammatories (ΝSAIDs), actinic keratosis treatments, and capsaicin.

The approach of the present invention may also be employed in the delivery of protein and peptide substances, and related compounds, such as peptidomimetics, and is of especial use in immunisation programmes, whether for primary or booster shots, and may be useful in BCG, for example. The invention further extends to the delivery of nucleic acids and their related compounds and derivatives. In the present context, a derivative or related compound is one that has been modified from the original, or prepared independently from the original, and which is used to emulate or to provide an affect associated with the original. A mimetic may simply comprise protecting groups, for example, or the backbone may be modified to inhibit or block digestion of the molecule.

In particular, it has been found that the present invention is especially suited for the delivery of hydrophilic drugs. The invention demonstrably increases the delivery rate of hydrophobic drugs, also, but has an especially surprising effect with drugs having a Log P of < 2, and is even more pronounced and beneficial with drugs having a Log P of < 1, and especially with drugs having a Log P of 0 or below. For example, caffeine can be delivered at substantially increased rates using the present invention, and has a Log P of -0.07, as shown in Table 2, below.

Particularly preferred drugs for delivery using the present invention are selected from; methotrexate, aciclovir, dactinomycin, oxytetracychne, 5-fluorouracil, ipatropium bomide, chlortetracycline, ceterizine, carboplatin, aminophylline, ofloxacin, pravastatin sodium, dichloromethotrexate, isoniaziad, theopylline, doxycyline, metronidazole, procaine, 4-aminosalicyclic acid, baclofen, triamcinolone, lidocaine/lignocaine, minoxidil, or combinations thereof.

The preparation of drug may be applied before, during, or after brushing the skin, but the best effects are generally observed by brushing the area of skin to be treated and then, substantially immediately thereafter, applying the drug to the brushed area. While it is not essential to apply the drug immediately after brushing, it will be appreciated that any substantial delay will permit the skin to recover, so that the enhanced permeability will be reduced, the longer the delay.

The nature of the brush is not critical to the present invention. However, it is preferred that the bristles making up the brush are presented in a substantially planar fashion to the skin, although there may be some advantage to having slightly longer bristles in the centre of the brush.

Where the bristles are presented in a substantially planar fashion to the skin, then the area of skin over which the brush moves will be generally equally treated, and there will be less discrepancy of permeation enhancement with increasing pressure.

The bristles forming the brush should be sufficiently resilient as not to splay substantially under the pressures suitable to put the invention into effect. These pressures will generally range from about 20 g/cm² to about 120 g/cm², and it will be appreciated that tougher, or more resilient, bristles, are required with increasing pressure, in order to avoid substantial bristle deformation. With frequency of use, it is likely that a brush will start to deform, whereon it is generally desirable to replace the brushing element. In any event, it is frequently desirable to replace the brushing element between uses where a device is intended for use on different patients. Where the patient applies the treatment himself, then replacement may only be necessary when the brush remains deformed after use.

It is generally preferred that the bristles not be too hard, as this can lead to irritation and reddening of the skin which, whilst it may be acceptable for an occasional treatment, is undesirable where treatment is required on a more frequent basis, such as daily, for example.

It has been found that the use of bristles having similar qualities to those of hard brushes for use in domestic situations for application to the person, such as nailbrushes, leads to very substantially increased permeation qualities of the stratum corneum, while not disrupting the structure of the stratum corneum to any great extent, and that they can be used without causing any substantial discomfort.

The stiffness of the bristle may also be referred to as the Robertson number, and is commonly used in dentistry to define the hardness of a brush. A Robertson #6 is generally considered to be "soft", and is usually at the low end of the range for what is suitable for use with the present invention, given that such bristles deform under moderate pressure. To an extent, this can be accommodated by packing the bristles tightly, but is not generally as useful as using harder bristles.

In the accompanying Example, bristles of Robertson no. 8 provide excellent results, without having to cause distress to the skin. It will be appreciated that exceedingly hard bristles, such as wires, can be used, but these need to be employed with great care in order not to damage the skin, so that a maximum of a Robertson no. of 11 is preferred.

The most preferred Robertson numbers are from about 6 to about 11, more preferably from about 7 to about 10, particularly about 8 to about 9, especially about 8.

The duration of brushing is dependent on a number of factors, including pressure on the brush, the hardness of the bristles and the nature of the drug to be delivered. However, it has generally been found that an appropriate length of time for brushing skin is between about 10 seconds and about two minutes, preferably between about 20 seconds and one minute, and more preferably between about 30 seconds and about 50 seconds, with an average of about 40 seconds generally providing a good guide. With Robertson Grade 8 brushes, good efficacy is often observed at 15 seconds and above, and 25 seconds is often particularly effective.

The pressure on the brush should preferably be not so great as to cause substantial irritation, but requires to be sufficient to have a permeabilising effect. As such, it has been found that a weight of between 20 and 90 grams is generally effective, although, at 90 grams and above, patient discomfort may be incurred.

In general, it has been established that a weight of about between 30 grams and 80 grams is sufficient, with a weight of between 40 grams and 60 grams being preferred. This weight is generally in terms of the accompanying Example.

In terms of pressure, it is preferred to apply between about 20 gm/cm and about 100 gm/cm², and more preferably between about 40 gm/cm² and about 80 gm/cm².

The pressure applied to the brush may be any that is effective to enhance delivery of the selected drug or drugs across the stratum corneum. In terms of N m^"2, particularly suitable pressures are about 200 to about 1500 N m^"2. Pressures of greater than this may also be used, but generally require careful control, short duration, and higher bristle strength, so are not preferred. A more preferred range is about 200 to about 1000 N m^"2, with pressures of about equal to, or greater than, 300 N m^"2 being especially preferred. A most preferred range is about 300 to about 600 N m^" .

The area of the end of the brush for contact with the skin may be as small or as large as desired. For delivery of potent drugs, for example, it may be desired to apply the formulation in a diffuse fashion to a larger area, such as up to 500 cm², in which case it may be desirable to move the brush over the skin to cover the whole of the target area, where the brush cross-section is smaller than the target area. It may also be desired to target a small area, or simply desired only to apply to a small area, to minimise any possible pain, so that an area of about 1 mm² maybe employed, for example. Smaller areas than this may provide problems in finding bristles thin enough and strong enough to with stand the pressure while abrading the skin without causing damage. A small rotating brush with short bristles will often suffice, under such conditions. In general, an area of skin in the approximate region of 1 cm² will often be the skilled physician's area of choice.

Thus, an area of the end of the brush of between about 1 mm² and about 10 cm² is preferred, with a range of about 4 mm to about 5 cm being more preferred, and a range of about 5 mm² to about 2 cm² generally being sufficient for most applications.

The rate of oscillation, or rotation, of the brushes of the invention may be any that serves, in combination with other parameters, such as stiffness and cross-sectional area, to sufficiently perturb the skin to enhance passage of drug. In the accompanying Example, a rate of rotation of 80 revolutions per minute (rpm) was found to be effective. Rates down to 30 m may be employed, but will generally require stiffer bristles and greater pressure to account for diminished interaction, while rates of greater than 300 are increasingly associated with the risk of damage to the skin by treatment that is too harsh. Thus, rates of between 30 and 300 φm are generally preferred, with rates of 50 to 200 φm being more preferred, and rates of 60 to 120 φm generally being most preferred.

The present invention also extends to brushing devices for the skin adapted to enhance the permeability of the stratum corneum.

In one embodiment, the invention provides a mechanised brushing device, wherein the mechanism of the device is adapted to rotate a brush in contact with the skin of a patient, the device having abutment means for contact with the skin, the brush being movably located in relation to the abutment means to allow it to be introduced to the skin, travel of the brush being limited in relation to the abutment means such that pressure on the brush to contact the skin is limited to a predefined range.

Such a device may be adapted to dispense drug during, or preferably after, brushing.

It will be appreciated that the term "mechanised" is used herein to indicate that the brush is not, directly, manually powered, but may be powered by any suitable means, such as clockwork, solar-powered, battery-powered motor, electrically powered, or spring-loaded, for example. The brush will generally comprise a series of bristles which may be individually mounted, or mounted in clusters, and which may be made of any suitable material. It is preferred that the material be resilient or flexible, in order not excessively to abrade the skin. As a whole, the bristles should be sufficiently resilient to be load-bearing, in that effective pressures may be applied through them without substantial deformation at the recommended operating pressure for the brush. As the bristles will generally be resilient, some deformation under operating pressure is to be expected. The brush will begin to lose efficiency when parts of the bristle adjacent the terminus of the bristle, rather than the terminus itself, begin to impact the skin.

The material from which the bristles is made is not critical to the invention, and many suitable bristle materials are well known in the art. Natural fibres, such as animal bristle, may be employed, but it is generally more convenient to employ synthetic polymers, such as polyamides.

For example, a rotary battery powered brush may be spring mounted in an open ended sleeve. Pressure on the housing causes the brush to move through the sleeve and to protrude beyond the opening, a tongue and groove arrangement preventing the brush protruding too far. The opening in the sleeve abuts the skin such that, when the brush is at full extension and the sleeve is in contact with the skin, then a weight of say 40 g is applied through the brush.

A simple timing mechanism may also be employed for example, to limit the duration to 40 seconds, for example.

One embodiment of this invention compromises a supporting body (rigid structure, preferably made of plastic) from which an array/plurality of bristles project vertically. The device is applied to the patient's skin, in a position such that the tip of the bristles make contact with the stratum corneum. A pre-wound spring is incoφorated into the supporting body, which when released by means of a control button (located on the supporting body) will allow the rotation/movement of the bristles in a clockwise or anticlockwise manner for a defined period of time. The number of rotations can then be controlled by the energy stored in the spring, and the manner in which the supporting body is moulded can also control the pressure exerted on the skin. This allows the degree of attrition/perturbation/stretching of the stratum corneum to be performed in a reproducible manner. Another embodiment of this invention includes a system which will not only reduce the barrier nature of the stratum corneum but also contain a formulation which would be released at the same time.

In an alternative embodiment, a rotatably mounted brush is located in an extensible fashion in a body, with electrically powered motor means being provided to rotate the brush. A battery may suitably provide the electrical power. Actuation of the motor serves to engage the brush, and this may be combined, such that extending the brush from the body actuates the motor. Preferably, a timer device cooperates with the motor, such that the motor switches off after a predetermined interval. The brush may then automatically retract, or may be manually or otherwise retracted.

The advantages offered by this system include the fact that it not only abrades the skin but also stretches it temporarily during treatment. This latter technique has been reported to enhance skin permeation (Cormier et al, 2001).

Using the rotating brush method achieves the puφose of reducing the barrier nature of skin with little or no pain, compared to existing abrasion methods.

The ability of the device to enhance in vitro skin permeability was investigated using the finite and infinite dose technique. Butyl paraben, methyl paraben and caffeine were used as model penetrants due to their similarity in molecular weight but differences in lipophilicity. Changes in the structural properties of the membrane were also probed, using scanning and transmission electron microscopy.

Brief Description of the Drawings

Figure la shows the effect of rotating brush A on skin Permeation profile of CF at various pressures and durations;

Figure lb shows the effect of rotating device brush A on skin Permeation profile of BP at various pressures and durations;

Figure lc shows the effect of rotating device brush A on skin Permeation profile of MP at various pressures and durations;

Figure 2a shows the effect of rotating device brush A and B on skin permeation of CF;

Figure 2b shows the effect of rotating device brush A and B on skin permeation of MP; Figure 2c shows the effect of rotating device brush A and B on skin permeation of BP;

Figure 3 shows the results of a pseudo-finite dose study comparing the effect of a device of the invention using brush B with other established modes of skin permeation enhancement;

Figure 4 shows the effect of treatment time on the skin permeation profile of acyclovir using brush B;

Figure 5 compares the effect of device treatment duration and iontophoretic treatment on skin permeation of radiolabelled ³H-labelled acyclovir after 60 min.; and

Figure 6 compares the effect of a device of the invention, using brush B with other enhancement strategies on the skin permeation of Ang II.

The present invention will now be illustrated by the following non-limiting, Example.

EXAMPLE

A systematic investigation involving permeants of differing lipophilicity and molecular weight was performed to evaluate devices of the invention in enhancing skin permeation of permeants with differing physicochemical properties.

MATERIALS

The materials used in this Example were as listed in Table 1 , below. Table 1 Materials used

Brush details

Brush A Internally manufactured Oscillatory brush, Robertson (6) pure bristles Surface area of brush: 1.01 cm²

Brush B Source: L. Cornelissen & Son Ltd (London, UK) Brand name: Robertson (8) pure bristles Surface area of brush: 1.61 cm²

A summary of the properties of the model penetrants is given in Table 2, below, and a comparative description appears below.

Table 2 Physico-chemical properties of the model penetrants

• BP, MP and CF have similar molecular weights but differ in lipophilicity, thereby allowing the effect of the permeation enhancement method of the invention lipophilicity to be studied. • ACN is an antiviral used in treating virus infections of the skin and mucosa. It is poorly absorbed across the skin due to its hydrophilic nature, hence reducing its therapeutic efficacy. ACN is marketed as a cream and licensed to be used topically for the treatment of cold sores, however, due to its retarded diffusion across the skin, it is recommended to be applied five times daily. Its dermato-pharmacological profile therefore makes it a useful model drug for testing the efficiency of the permeation method or device of the invention. The use of ACN as a model marker in investigating the potential of enhancement strategies such as iontophoresis (Volpato at al, 1995, 1998; Stangi at al, 2004) and ethosomes or ethanol based liposomes (Touitou at α/.,2000) in decreasing the skin barrier to improve ACN diffusion has been reported.

• Ang II is a model hydrophilic, high molecular weight peptide which would not ordinarily be absorbed across the skin by conventional formulation approaches. Compared to the other model penetrants described above, it is the least likely to permeate the skin. Its use as a model marker to investigate the effect of iontophoresis and jet injection methods on the in vitro permeability of large molecular weight solutes across animal skin has also been reported (Clemessy at al, 1995; Sugibayashi at al, 2000).

The effectiveness of the device of the invention in increasing the permeability of the above markers was also compared with some of the established and developmental methods of skin permeation enhancement, such as tape stripping, chemical enhancers, iontophoresis and delipidisation (removal of skin lipids).

ANALYTICAL METHODS

HPLC analysis ofMP, BP and CF

Chromatographic measurements were carried out using a Perkin Elmer 200, LC Pump with Autosampler connected to a UN absorbance Detector 759A (Applied Biosystems, Foster city, CA, U.S. A). The various HPLC methods employed for each penetrant were shown to be fit for the purpose by ensuring reproducibility, repeatability, linearity, and intermediate precision. Standard concentrations of the model penetrants (0.1-20 μg/ml) were prepared in PBS and analysed. The chromatographic conditions for MP were as follows; Hypersil 5μ BDS, C18 (150 x 4.6 mm, 5μm) column (Phenomenex^® Ltd, Cheshire, United Kingdom) with the mobile phase comprising 35% acetonitnle: 65% phosphate buffer (0.05 M KH₂PO₄ containing 1% triethylamine, then adjusted to pH 3.5 with orthophosphoric acid). BP chromatographic conditions employed a Licrospher™, LispRP 18-5-1680, C18 (150 x 4.6 mm, 5 μm) column (Hichrom ^® Ltd, Berkshire, England), with a mobile phase of 50% acetonitrile: 50%o phosphate buffer (0.02 M KH₂PO₄ adjusted to pH 3.0 with orthophosphoric acid). CF chromatographic conditions were as follows; Phenomenex^® Prodigy^™ 5μ ODS 2, C18 (150 x 4.6 mm, 5μm), mobile phase comprising 90% acetonitrile: 10%> phosphate buffer (0.02 M KH₂PO₄ adjusted to pH 3.0 with orthophosphoric acid). Flow rate and injection volumes were set at 1 ml/min and 10 μl respectively for all penetrants. Wavelength of detection was set at 254 nm, 256 nm and 270 nm for MP, BP and CF respectively. Analytical determination of CF was modified where necessary to enhance its detection by increasing injection volume to 50 μl and using a wavelength of 215 nm.

Analytical studies for ACT

The radiochemical purity of the ³H-ACN to be used in this study was determined by HPLC. Chromatographic measurements were carried out using a Perkin Elmer, as described previously. ACN chromatographic conditions were as follows; Phenomenex^® Prodigy^™ 5μ ODS 2, C18 (150 x 4.6 mm, 5μm), mobile phase comprising 90%> acetonitrile: 10% phosphate buffer (0.02 M KH₂PO₄ adjusted to pH 3.0 with orthophosphoric acid). Flow rate, injection volumes and wavelength of detection were set at 1 ml/min, 10 μl and 250 nm respectively.

The purity of the ³H-ACN was determined by accurately spiking a 10 μl aliquot of the radiolabelled drug into 1.0 ml of PBS, which was then injected. The purity of the radioactivity was determined by collecting sample fractions at 1 min time intervals from time 0 to 30 min.

Zovirax^® spiked with ³H-ACV was used as the formulation for the entire study. Briefly, 300 μl of ³H ACN was placed into a 1.5 ml centrifuge tube and evaporated off, over a stream of air. Approximately 1 g of Zovirax^® was weighed into the centrifuge tube and carefully mixed with a fine spatula to achieve a homogenous mix. To test for homogeneity, 3 random samples (top, middle and bottom) were taken and determined for radioactivity via scintillation counting. This process was repeated until the %> coefficient of variation (CN) was below 2.5%.

Analytical development studies for Angiotensin II

Analysis of the peptide was carried out using an enzyme immunoassay (EIA) as described in the EIA booklet. Briefly, a competitive enzyme immunoassay which detects Ang II in biological matrices was employed. The principle of the assay is based upon the competition for antibody binding sites between biotinylated and non- biotinylated peptides. The biotin group on the biotinylated peptide is then conjugated to SA-HRP which in turn reacts with another substrate (TMB) leading to the formation of colour. The absorbance recorded from each well is a direct measure of the extent of binding for each peptide with the antibody. Absorbance was measured using a spectrophotometer. The method was validated by calibrating the response at different concentrations of the peptide (data not shown) and also the ability of the method to assess different concentrations of the penetrant in the presence of any matrices was investigated.

IN VITRO SKIN PERMEATION STUDIES

Preparation of human epidermal sheets

Human skin was obtained from cosmetic surgery with informed consent. The epidermis was removed by the standard heat separation method (Kligman and Christophers, 1963). Following removal of subcutaneous fat, individual portions of skin were immersed in water at 60°C for 45 seconds. The skin was then pinned, dermis side down, on a cork board and the epidermis (comprising stratum corneum and viable epidermis) gently removed from the under lying dermis. The latter was discarded and the epidermal membrane floated onto the surface of water and taken up onto a Whatman no.l filter paper. The resultant epidermal sheet were thoroughly dried and stored flat in aluminium foil at -20°C until use. Tape-stripping procedure Tape-stripping was employed in order to partially remove the upper layers of the skin where the barrier properties are known to reside. Tape-stripped skin sections used were prepared by repeated stripping skin with D-squame adhesive disc. The disc was gently placed on the skin after which a known weight was placed into the adhesive disc skin composite for 20 s. The weight was then lifted and the adhesive disc removed. This was then repeated eight times. The permeability profile across the tape-stripped skin was then investigated via Franz cell studies.

Skin delipidisation procedure

The objective here was to remove the intercellular lipids of the SC in order to investigate the potential pathway employed by the model markers in their bid to cross the skin barrier. Delipidised skin was prepared by immersing skin sections in chloroform and methanol (2:1) for 40 min (Rastogi and Singh, 2001a,b), after which the skin was removed, blotted dry with tissue and dried via vacuum drier at 760 mm Hg, 25°C, for 1 hr, to remove any remaining organic solvent. The delipidised skin was then used for Franz cell studies.

Chemical enhancement procedure (50 % ethanol in PBS)

The ability of ethanol to reduce the barrier property of the SC has been well documented (Williams & Barry, 2004). This was conducted by forming a saturated solution of each penetrant containing 50%v/v ethanol (EtOH), which was allowed to stir overnight. The resulting solution was then introduced into the donor well the Franz cells.

Iontophoresis or post-iontophoresis (pre-treatment)

This is a well established physical method of enhancing skin permeation, in vitro, which involves the use of electric current to permeabilise skin and/or promote the migration of drug ions across skin (Cullander, 1992; Guy et al, 2001; Kalia et al, 2004). Anodal treatment was conducted by placing the anode electrode in the donor compartment and the opposite electrode (cathode) in the receptor compartment of the Franz cell. Cathodal iontophoresis was conducted vice versa. The current treatment protocols employed were either

(a) iontophoresis: dose was administered simultaneously with current or

(b) post-iontophoresis: dose applied immediately after skin exposure to current.

A current intensity of 0.40 mA was employed for a 10 min period during both protocols.

Novel device (rotating brush) procedure

The potential device parameters affecting permeation were identified as follows:

(a) speed of bristles/frequency of movement;

(b) pressure exerted on epidermal membrane surface;

(c) duration of treatment; and

(d) nature of bristle; hard or soft.

The mode of treatment was controlled as follows:

• The piece of epidermal sheet with demarcated regions of interest was laid on to a microscope slide which was then placed onto a weighing balance.

• The balance was supported by a jack (lift) sitting directly under the device the cell was then tarred.

• With the aid of the jack, the device was partially lowered (by increasing the height of the jack using the control knob) ensuring that the bristles did not touch the epidermal surface.

• The device was then switched on, and the slide moved until a position was found where the bristles are directly under the demarcated region of the epidermal sheet.

• The device was then switched off. The slide was then attached firmly to the balance by means of scotch tape (tape extends from non demarcated region of epidermal sheet to balance, avoid contact between tape and demarcated region to ensure no movement of the slide during treatment).

• Once the slide was in a stationary position, the device was switched on, ensuring that the speed dial was at the required position. The device was gently lowered (by raising the jack as described above) until the bristles touched the surface, the reading on the balance then gave an indication of the weight (pressure) exerted on the membrane surface.

• The required pressure was then attained by using the control knob of the j ack to lower or increase the height of the balance. The slide then remained stationary during the duration of treatment. The pressure/weight exerted on the membrane during treatment was then read directly from the balance.

• After treatment the scotch tape was carefully removed. The demarcated circular region was then cut off from the remaining epidermal sheet by means of a cork borer, then placed in a Franz cell.

• An integrity check was also conducted after skin treatment to ensure that the bristles did not create holes in the epidermal layer. This was performed by inversion of the Franz cell to observe whether the receptor fluid liquid was leaking through membrane.

Franz cell studies

Optimisation studies using 3 model penetrants; Infinite dose study

The study was conducted using human epidermal sheets (since the barrier properties reside in the stratum corneum) to determine the amount of the model marker penetrating the membrane over a 4hr period. Calibrated Franz cells of known area (~0.65 cm sq ) and volume (~2 ml) were used. The receptor chamber was filled with PBS (pH 7.4) and stirred throughout the duration of the experiment by a PTFE coated magnetic flea. The membrane was clamped in between the donor cap and receptor chamber of the Franz cell (stratum corneum facing upwards), was then treated with the two types of bristles at different pressures and contact time, whilst maintaining a constant device speed (Table 3, below). Table 3 Device parameters used during optimisation studies

250 μl of saturated solution of the model penetrant in PBS was then directly introduced into the donor chamber of the cell. All experiments were conducted in a water bath at 37°C. In order to determine the enhancement effect of the rotating brush, control experiments involving the use of non-treated (intact), tape stripped, donor solutions of marker containing 50% ethanol and delipidised epidermal membranes were also performed.

Pseudofinite dose studies (Acyclovir and CF)

Finite dose permeation experiments were performed using similar conditions as described above. The Franz cell studies were carried out without the use of a donor well, in order to allow for the placement of the iontophoretic device over the epidermal membrane. In addition, the epidermal membrane was mounted onto the receptor well using cyanoacrylate adhesive and ensuring that there was no contact between the adhesive and the effective surface area available for drug permeation. After a drying time of 15 min, the receptor well was then filled with the receptor fluid (PBS). The effectiveness of the seal was confirmed if leakage of receptor fluid from the region of contact between skin and adhesive was not observed.

The skin was treated with the novel device using optimised parameters obtained from the preceding section of this Example. A formulation with a target dose of approximately 9 ± 1 mg/cm² (ACN) and 20 ± 2 mg/cm² (CF) was applied to the epidermal membrane surface using a previously calibrated positive displacement pipette. Selected control experiments were then performed as described above (anodal and cathodal iontophoresis was performed by simultaneous application of dose and current over a 10 min period for CF and ACV respectively). At certain time intervals 200 μl of the receiver fluid was carefully withdrawn from the receiver fluid (maximum duration of 4 h). Approximately 4 ml of scintillation cocktail was then added and the ACN sample analysed by scintillation counting, whereas the other penetrant was assayed via the HPLC method already described.

Angiotensin II-infinite dose

A similar procedure described in the preceding optimisation section was conducted for Ang II, whereby the diffusion of the peptide across skin following brush treatment was compared against all the treatment methods. A 250 μl solution of Ang II in PBS (1 mg/ml) was then introduced into the donor well. The receptor chamber was filled with EIA buffer, previously treated via ultrasound to prevent the formation of air bubbles. Receptor fluid was sampled after a 4 h and 24 h period and analysed via the EIA method described.

HISTOLOGICAL STUDIES

The effect of bristle perturbation on the epidermis was assessed by scanning and transmission electron microscopy, where the integrity of brush treated and untreated (control) samples were compared. Brush treated and untreated (control) samples were fixed in 2% formaldehyde / 2.5% glutaraldehyde in 0.1M phosphate buffer pH 7.4 overnight. Scanning electron microscopy (SEM) was performed as follows: A 5 x 5 mm square of skin was pinned to a thin piece of cork in order to keep it flat during processing. Samples were immersed 20 min in each of 30, 50, 70, 95, 100, 100, 100% v/v acetone in water, then dried using liquid carbon dioxide in a Polaron E3000 critical point drier. The samples were then removed from the cork and mounted on 12.5 mm aluminium pin stubs using double sided adhesive carbon pads. Samples were sputter coated with approximately 20 nm of gold in a Polaron E5100 sputter coater and examined and photographed using a Philip SEM501B scanning electron microscope. For transmission electron microscopy (TEM), a 1 x 2 mm strip was cut from the fixed skin, then dehydrated by sequential immersion in acetone at increasing concentrations and embedded in spurr resin and polymerised for 48 hr at 60 °C. Ultra-thin sections were cut on a Reichart Jung OMU4 ultra microtome using a diamond knife and then taken up on 200 mesh hexagonal copper grids. Sections were stained for 15 min in 1%> w/w uranyl acetate in 50%) v/v ethanol in water followed by 5 minutes in Reynold's lead citrate. These sections were examined and photographed using a JEOL JEM100CX II transmission electron microscope.

DATA INTERPRETATION & STATISTICAL ANALYSIS

Since the SC is made up of dead cells no active transport processes exist. Transport of the penetrant is therefore solely by passive diffusion, which can be described by Fick's first law (equation 1). The diffusion of a drug across the stratum corneum (J_s) is therefore the rate-determining step in skin permeation.

J_s = D x K x Cv (1)

Where Js represents the flux of the permeant across the membrane; D is the diffusion coefficient of the penetrant in the membrane; K, the stratum corneum- vehicle partition coefficient; C_v, the concentration of the penetrant in the vehicle; and L the diffusional path length across the membrane. Js was determined from the linear portions of the permeation profile obtained from the infinite dose study as in accordance with Fick's law. Permeability coefficient (Kp) which is a product of (D , K and L) was calculated as Js / C_v. The lag time (T_L) was determined by the intercept of the linear portions of the skin permeation profile on the x-axis where applicable. Enhancement factors (EF) were calculated as a ratio of flux of permeant through treated skin to that of untreated skin. All data reported represent a mean of n >3-6 and its standard deviation (s.d.) or error (s.e.) except otherwise stated. Statistical analysis was conducted using the analysis of variance method (ANON A) and student's t-test, the level of significance was taken at p< 0.05. RESULTS AND DISCUSSION

Optimisation studies

The infinite dose method was used to investigate the relationship between the nature of bristles, pressure (weight) exerted on membrane and treatment time on permeation of the 3 markers. This was then compared to already established methods of skin penetration enhancement. The rotational speed of the brush was maintained at 80 φm for each experiment. The limitation of using the soft bristles (brush A) included the fact that the length of the bristles at the periphery of the brush were slightly longer than those in the middle. Therefore, the surface of the brush was not uniform. Hence at low pressures (300 Nm^"2) only the peripheral bristles were in contact with the skin, so that pressures > 450 Nm^" were used, in order to ensure maximum contact with skin on using brush A. As a result of the flat nature of the surface of brush B (hard bristles) such problems were not experienced, thereby more readily allowing the assessment of pressures at 300 Nm^"2.

Permeation ofMP, BP & CF (using soft bristles)

The human skin used in experiments involving brush A was from the same donor. Figures la-c show the effect of using the device with brush A (soft bristles) at different pressures and treatment times for the different permeants. A minimum threshold pressure of 450 Nm ^" and a maximum treatment time of 45 s was employed.

In Figure la, which shows the effect of rotating brush A on skin permeation profile of CF: (O) untreated skin; (■) 450 N m^"2, 20s; (X) 450 N m^"2, 45s; (Δ) 750 N m^"2, 45s; (♦) 1200 N m^"2, 45 s. Data represents, mean ± s.d. (n=3-6). Device speed maintained at 80 φm

In Figure lb, which shows the effect of rotating device brush A on skin permeation profile of BP: (O) untreated skin; (■) 450 N m^"2, 20s; (X) 450 N m^"2, 45s; (Δ) 750 N m^"2, 45s; (♦) 1200 N m^"2, 45 s. Data represents, mean +± s.d. (n=3-6). Device speed maintained at 80 φm In Figure lc, which shows the effect of rotating device brush A on skin permeation profile of MP: (O) untreated skin; (X) 450 N m^"2,45s; (♦) 1200 N m^"2, 45 s. Data represents, mean ± s.d. (n=3-6). Device speed maintained at 80 φm.

Where an enhancement factor (EF) of 2 or more was observed (i.e. significantly different from control) the use of a shorter treatment time of 20 s was investigated (c.f. CF -Table 4a and BP -Table 4b, below). The CF fluxes observed under conditions of 450 Nm ^"2, 20 s, were however not significantly different from that of untreated skin. An increase in treatment time from 20 s to 45 s at constant pressure was found to generally increase the flux of all the markers. The use of brush A was found to enhance the permeation of all the markers by at least an average factor of 2 (Tables 4a-c). Enhancement factors recorded were in the order of CF > BP > MP. Increasing the pressure exerted on the membrane was found to enhance permeation. However, no significant differences (p>0.05) were observed in flux between 450 Nm ^"2 and 1200 Nm ^"2 at a treatment time of 45 s. This may be due to the fact that, at weights greater than 450 Nm ^"2, the nature of the bristles of brush A becomes a limiting factor, such that further increase in applied pressure did not yield an increase in flux.

Table 4a Effect of treatment on in vitro skin permeation parameters of CF using brush A

Skin treatment Js K_P T_L EF (10^"4 μg/cm/s) (10^"8 cm/s) (min)

Untreated 6.96 ± 1.53 3.39 ± 0.75 77.66 ± 12.96 - 450 Nm^"2/ 20 s 10.3 ± 2.67^p 5.04 ± 1.30 68.33 ± 17.76 1.47 450 Nm^"2/45 s 16.3 ± 0.86^* 7.95 ± 0.42 41.21 ± 8.09 2.34 750 Nm^"2/45 s - - - - 1200 Nm^"2/45 s 18.4 ± 8.93^* 8.97 ± 4.06 16.74 ± 6.50 2.64 Delipidised 52.25 ± 25.17^* 25.66 ± 12.28 4.19 ± 1.39 7.47

Data represents mean ± s.d. (n>3) except where otherwise stated. ^p represents n =2 ^* Flux significantly different from that of untreated skin (p<0.05).

Device speed maintained at 80 φm. Table 4b Effect of treatment on in vitro skin permeation parameters of BP using brush A

Skin treatment Js K_P T_L EF (10^"3 μg/cm/s) (10^"5 cm/s) (min)

Untreated 3.80 ±0.46 1.90 ±0.23 33.47 ±8.79 -

450 Nm^"2, 20 s 6.60 ± 0.49^* 3.30 ±0.25 37.10 ±2.26 1.74

450 Nm^"2,45 s 7.50 ±0.75^* 3.75 ± 0.37 25.22 ±2.17 1.97

750 Nm^"2,45 s 8.26 ± 0.89^* 4.13 ±0.45 26.34 ±1.48 2.17

1200Nm^"2,45s 8.62 ±1.05^* 4.31 ±0.52 26.39 ±1.30 2.26

Delipidised 15.95 ±1.55^* 7.98 ± 0.78 18.58±3 4.20

Data represents mean ± s.d. (n>3) except where otherwise stated. Flux significantly different from that of untreated skin (p<0.05). Device speed maintained at 80 φm.

Table 4c Effect of treatment on in vitro skin permeation parameters of MP using brush A.

Skin treatment Js Kp T_L EF (10^"3 μg/cm/s) (10^"6cm/s) (min)

Untreated 7.46 ±0.67 3.83 ±0.35 16.61 ±5.15

450Nm^"2,45s 9.78±0.54^* 5.02±0.27 16.12 ±4.45 1.31

750Nm^"2,45s - - -

1200Nm^"2,45s 11.16 ±1.60^* 5.74 ±0.83 12.43 ±5.46 1.50

Delipidised 28.40 ±0.29^* 14.59 ±1.48 3.87 ±2.09 3.81

Data represents mean ± s.d. (n>3) except where otherwise stated. Flux significantly different from that of untreated skin (p<0.05). Device speed maintained at 80 φm.

The hydrophilic nature of CF results in very long lag times, as observed in this experiment (~70 min) with untreated skin. The effect of the device on the lag times was profound in the case of CF, with altering either the pressure or duration of treatment significantly reducing the lag times by ~ 75% of their original value. The use of delipidised skin was also found to be more effective than use of the device with brush A in enhancing permeation of the 3 markers. The lower EF's recorded for the parabens, compared to CF, signifies the relative ease at which such lipophilic markers permeate the SC, either in the absence or presence of skin lipids.

The electron micrographs obtained (not shown) demonstrated some disruption (attrition) of the Stratum corneum and the associated loosening of these layers. Such perturbation possibly creates channels/disruptions in the membrane, which may account for the increase in flux of the permeants. Disruption is restricted within the upper layers of the skin, whilst the remainder of the epidermis is unaffected. Enhancement factors less of 2 recorded in this part of the study prompted the use of harder bristles (brush B) in order to further enhance skin permeation.

Permeation ofCF, MP andBP using harder bristles (Brush B)

Surface analysis of the epidermis by electron microscopy showed that the extent of barrier disruption or perturbation induced by the device, on using brush B, was slightly greater than that of brush A (data not shown)

Release profiles shown in Figures 2a-c, (Data in Tables 5a-c, below) depict the effect of bristle type on skin absoφtion of the 3 markers. A minimum threshold pressure of 300 Nm ^"2 and a maximum treatment time of 45 s was employed. Significant differences (p < 0.05) in penetrant flux across human epidermal sheets on using brush B relative to brush A was observed for all permeants.

In Figure 2a, which shows the effect of rotating device brush A and B on skin permeation of CF; (■) untreated skin (α) brush A at 300 N m^"2,45s; (+) brush B at 300 N m^"2, 15s; (A) brush B at 300 N m^"2, 25s. Data represents, mean ± s.d. (n=3-6). Device speed maintained at 80 φm. Skin donor used for CF different from that used for MP and BP in Figures 2b-c.

In Figure 2b, which shows the effect of rotating device brush A and B on skin permeation of MP; (■) untreated skin (o) brush A at 300 N m^"2 ,45s; (A) brush B at 300 N m^"2, 25s; (Δ) brush B at 450 N m^"2, 15s. Data represents, mean ± s.d. (n=3-6). Device speed maintained at 80 φm.

In Figure 2c, which shows the effect of rotating device brush A and B on skin permeation of BP; (■) untreated skin (ϋ)brush A at 450 N m^"2, 45s; (+) brush B at 300 N m^"2/15s; (A) brush B at 300 N m^"2, 25s; (Δ)brush B at 450 N m^"2, 15s. Data represents, mean ± s.d. (n=3-6). Device speed maintained at 80 φm.

The higher EF's recorded for brush B are likely to be attributed to the degree of perturbation imposed on the membrane relative to brush A (Tables 5a-c, below) with CF being most markedly affected, with an increase in EF of between 37 and 64.

Table 5a Effect of treatment on in vitro permeation parameters of CF using brush B Penetrant Skin treatment Js K_P T_L EF (IP^"4 μg/cm/s) (10^"8 cm/s) (min)

CF Untreated 1.87 ± 0.26 9.15 ± 1.27 19.69 ± 3.54 450 Nm^"2,45 s^a 8.52 ± 0.29* 41.61 ± 14.19 16.74 ± 3.99 4.55 300 Nm^"2,25 s 120.92 ± 42.55*⁺ 589.93 ± 207.62 - 64.44 300 Nm^"2,15 s 70.19 ± 34. 87*⁺ 342.4 ± 170.1 - 37.41 450 Nm^"2,15 s _. . . . Delipidised 20.43 ± 5.50*⁺ 99.68 ± 26.84 - 10.93 Tapestripped 35.5 ± 29.5*⁺ 173.4 ± 144.0 17.16 ± 9.15 19.01 EtOH/PBS 28. 98 ± 6.39*⁺ 141.4 ± 31.21 26.71 ± 9.26 15.46

Data represents mean ± s.d. (n ≥3). *Flux significantly different from untreated skin (p≤O.05). ^a Represents brush A. Flux significantly different from skin treated with brush A (p≤O.05). ^x Skin donor used for CF different from that used for MP and BP in Tables 4b-c. Device speed maintained at 80 rpm. Table 5b Effect of treatment on in vitro permeation parameters of MP using brush B

Penetrant Skin treatment J_s Kp T_L EF (IP^"3 μg/cm/s) (10^"6cm/s) (min)

MP Untreated 10.90 ±1.28 5.14 ±6.06 15.02 ±3.74 450Nm^"2,45s^a 13.53 ±0.95* 6.38 ±0.45 6.18 ±1.34 1.24 300Nm^"2,25s 50.16 ±9.25^*+ 23.66 ±4.36 3.29 ±2.09 4.60 300Nm^"2,15s - . . . 450Nm^"2,15s 62.34 ± 12.75*⁺ 29.41 ±6.01 6.73 ±3.63 5.72 Delipidised 41.98 ±2.05*⁺ 19.81 ±0.96 - 3.85 Tapestripped 53.86 ± 12.27*⁺ 25.41 ±5.79 2.71 ±1.61 4.94 EtOH/PBS 44.45 ±7.19*⁺ 20.96 ±3.39 7.76 ±5.56 4.08

Data in Table 5b represents mean ± s.d. (n >3). *Flux significantly different from untreated skin (p≤O.05). ^a Represents brush A. Flux significantly different from skin treated with brush A (p≤O.05).

Table 5c Effect of treatment on in vitro permeation parameters of BP using brush B

Penetrant Skin treatment Js Kp T EF (10^"4 μg/cm/s) (10^"5 cm/s) (min)

BP Untreated 4.93 ± 0.22 2.47 ±0.11 35.84 ±1.98 450Nm^"2,45s^a 8.38 ±1.09* 4.19 ±0.54 24.51 ±5.50 1.69 300 Nm^"2' 25 s 10.47 ±0.96* 5.23 ±0.48 23.06 ±2.19 2.12 300 Nm^"2, 15 s 6.16 ±0.89* 3.08 ±0.44 22.65 ±3.22 1.25 450 Nm^"2, 15 s 12.69 ±2.07*⁺ 6.34 ±1.03 14.55 ±5.18 2.57 Delipidised 20.54±2.85*⁺ 10.27±1.43 8.17±5.41 4.16 Tapestripped 15.60 ±3.69*⁺ 7. 80± 1.84 34.78 ±4.44 3.16 EtoH/PBS 18.48 ± 1.61*⁺ 9.24 ±0.80 26.43 ±5.05 3.75

Data in Table 5 c represents mean ± s.d. (n >3). *Flux significantly different from untreated skin (p≤O.05). ^a Represents brush A. Flux significantly different from skin treated with brush A (p≤O.05). The effect of brush B at 300 Nm^"2 was found to increase when treatment duration was increased from 15 s to 25 s. Shorter treatment times and lower pressures were required in the use of brush B, when compared to brush A, to produce significant changes to the barrier nature of the SC and, therefore, drug flux. With CF, significantly higher fluxes were observed with the use of brush B, compared to the other enhancement methods, thus demonstrating the benefit of this device for such molecules. In the case of the parabens, the highest EF recorded on using the novel device was comparable to the established and developmental modes of skin permeation enhancement. Due to the remarkably high fluxes recorded for CF with the use of brush B, the effect of increasing pressure exerted (>300 Nm^"2) on CF flux was not investigated further.

CF Pseudo finite dose study

This part of the study involved the use of the device with brush B and the minimum and ideal conditions of pressure and a variation in treatment time to further evaluate, via the finite dose methodology, the effect of the system using CF (Figure 3). This was performed, in order to investigate the degree of enhancement provided by the device under application conditions more like those that would occur in vivo. CF was selected as a suitable candidate for this part of the study, due to its poor intrinsic permeability across intact skin as well as for the promising results recorded during the optimisation stages. For untreated skin, and chemical enhancement procedures, the presence of CF was only determined after 120 min (limit of detection of analytical method was 0.05 ug/ml).

In Figure 3, which shows the results of the pseudo-finite dose study comparing the effect of the device using brush B (300 Nm^"2/ 25 s) to other established modes of skin permeation enhancement; Mean + s.d. (n=3-6). *Amount of CF in receptor after 120 min. Device speed maintained at 80 φm.

Administered doses were found to dry into a thin film after approximately 1 hr. The amount of CF deposited in the receptor compartment of the Franz cell, with the use of the chemical enhancement method, was found not to be significantly different (p>0.05) from untreated skin. The amount of CF in receptor after 30 min using brush B was found to be significantly higher (p<0.05) and at least double that recorded for the other methods of enhancement.

Permeation of ACV (Pseudo finite dose study)

The poor efficacy of ACN topical preparations have been attributed its poor skin permeability, resulting from its hydrophilicity, which hinders it from reaching the target site of the basal epidermis (Stagni et al, 2004). This, therefore, makes ACN an interesting model candidate for the evaluation of a novel transdermal device. The ability of the novel device to enhance the flux profile of ACV from a commercial cream is demonstrated in Figures 4-5 and in Table 6.

In Figure 4, which shows the effect of treatment time on the skin permeation profile of acyclovir from a topical preparation (Zovirax^®) using a device with brush B; (■) untreated; (•) 10 s; (• with dashed line) 30 s; (A) 60 s. Data represents mean + s.e. (n=4-9). Constant device parameters (speed; 80 φm, pressure; 300 Νm^"2).

In Figure 5, which shows a comparison of the effect of device treatment duration and iontophoretic treatment on skin permeation of radiolabelled H-labelled acyclovir after 60 min. Mean± s.e. (n=4-9). Device speed maintained at 80 φm, pressure; 300

Νm^"2.

A significant increase in ACV flux across the skin was observed as the brush treatment duration was increased, demonstrating the increased benefit of the device on delivering molecules like acyclovir across the skin. At maximum treatment time, the device of the invention resulted in a ca. 600% increase in enhancement factor, when compared to iontophoresis, also operated under optimum conditions (Nolpato et al, 1995, 1998; Morrel et al, 2004). Table 6 Effect of using brush B at different treatment times (10, 30 & 60 s) and iontophoretic treatment on skin absoφtion of radiolabelled acyclovir

Amount in receptor after 60 min ( D g/cm²) EF

Untreated 0.14 ± 0.08

Brush treatment 10 s 5.06 ± 1.88* 36 30 s 12.5 ± 4.02* 89 60 s 30.91 ± 5.45* 220

Iontophoresis⁰ 4.95 ± 2.35* 35

Data represents mean ± s.d. (n=4-9) except otherwise stated. Flux significantly different from that of untreated skin (p≤O.05). Constant device parameters (speed; 80rpm, pressure;300 Nm^"2). ^cCathodal iontophoresis (not post iontophoresis) performed for a period of 10 min, after which the current was switched off.

Permeation of Angiotensin II (Infinite dose study)

The highly significant results in skin permeation enhancement observed for the hydrophilic solutes ACV and CF prompted an investigation into the ability of the device to enhance the permeation of a model peptide, Ang II. The hydrophilic nature and large molecular weight of the peptide makes it an extremely unlikely candidate to be absorbed across the skin.

It can be seen from Figure 6 and Tables 7a and b, below, that the use of all the enhancement techniques, with the exception of post-iontophoresis, was found to significantly increase the permeation of the model peptide, compared to untreated (intact) skin at each defined time period (4 h and 24 h) although the effect was greatest with the device of the invention. In Figure 6, which shows a comparison of the effect of a device fitted with brush B with other enhancement strategies on the skin permeation of Ang II over a (π) 4h and (■) 24 h period. Data represents mean ± s.d. (n=3-6).

The amount of Ang II permeating the skin was generally found to increase with time, however, this increase was found not to be significant for untreated, post- iontophoretic and tape-stripped skin.

Table 7a Effect of using brush B and other treatment methods on the in vitro skin permeation of Ang π after 4 h

Amount in receptor after 4 h (ng/cm²) . EF

Untreated 0.27 ± 0.23

Post-iontophoresis 0.46 ± 0.15⁺ 1.7

Delipidisation 15.71 ± 2.85* 58

Tape-stripping 14.44 ± 4.73* 53

Rotating brush 19.45 ± 0.55* 72

Data represents mean ± s.e. (n=3-6) except otherwise stated. Significantly different from that of untreated skin (p<0.05). ⁺7.53 ± 5.87 ng/cm² was initially recorded (see Figure 3), however this amount was found not to be reproducible on sample re- analysis. Table 7b Effect of using brush B and other treatment methods on the in vitro skin permeation of Ang II after 24 h

Amount in receptor after 24 h (ng/cm²) EF

Untreated 1.69 ± 1.01

Post-iontophoresis 10.02 ± 6.28 6

Delipidisation 83.03 ± 24.28* 49

Tape-stripping 46.93 ± 15.62* 27

Rotating brush 107.49 ± 19.66* 63

Data represents mean ± s.e. (n=3-6) except otherwise stated. Significantly different from that of untreated skin (p<0.05).

Thus, the above Example clearly demonstrates the ability of the oscillating brush device of the invention to enhance in vitro skin permeability and/or reduce lag times of permeants of differing physicochemical properties across the skin, by the disruption of the upper skin layers. The suφrising benefits of this approach when compared to established and developmental methods of permeation enhancement, has also been clearly shown.

REFERENCES

Clemessy M, Courazze G, Bevan B, Puisieux F (1995). Mechanisms involved in iontophoretic transport of angiotensin. Pharm Res. 12, 998-1002

Cormier MJ, Trautman J, Kim HL, Samiee AP, Neukeraians AP, Edwards BP, Lim W, Poutiantine ST (2001). Skin treatment apparatus for sustained transdermal drug delivery. World Patent No. 01/41864 Al. Cullander, C (1992). What are the pathways of iontophoretic current flow through mammalian skin? Adv. Drug Del. Rev. 9, 119-135.

Elias, PM. (1983) Epidermal lipids, barrier function and desquamation. J. Invest. Dermatol. 80, 44-49

Gerstel MS, Place VA (1976). Drug delivery device. US Patent No 3,964, 482.

Guy, R.H., Delgdo-Charro, M.B., Kalia, Y.N (2001). "Iontophoretic transport across the skin", Skin Pharmacol Appl Skin Physiol. 14, SI, 35-40.

Kalia YN, Naik A, Garrison J, Guy RH (2004). Iontophoretic drug delivery. Adv Drug Del Rev. 56, 619-658

Kligmann AM, Christophers, E (1963). Preparation of isolated sheets of human stratum corneum. Arch. Dermatol. 88, 702-708.

Morrel EM, Spruance SL, Lim ST, Brown MB, Goldberg D (2004). A Multicenter, Placebo Controlled, Randomized, Clinic-Initiated Pilot Trial of a Single, Topical, Iontophoretic Application of Acyclovir Cream for the Episodic Treatment of Heφes. J. Invest Dermatol (submitted).

Rastogi SK, Singh J (2001a). Lipid extraction and transport of hydrophilic solutes through porcine epidermis. Int. J. Pharm. 225, 75-82

Rastogi SK, Singh J (2001b). Lipid extraction and iontophoretic transport of leuprolide acetate through porcine epidermis, hit J Pharm, 215, 241-249.

Stagni, G., Ali , ME, Weng D (2004). Pharmacokinetics of acyclovir in rabbit skin after i.v.-bolus, ointment, and iontophoretic administrations. Int J Pharm.15; 274, 201-211

Sugibayashi K, Kagino M, Numajiri S, Inoue N, Kobayashi D, Kimura M, Yamaguchi M, Morimoto Y (2000). Synergistic effects of iontophoresis and jet injector pre- treatment on the in vitro skin permeation of diclofenac and angiotensin II. J Pharm Pharmacol. 52, 1179-1186. Touitou E, Dayan N, Bergelson 1, Godin B, Eliaz M (2000). Ethosomes - novel vesicular carriers for enhanced delivery: characterization and skin penetration properties. J Control Rel. 65, 403-418.

Volpato NM, Santi P, Colombo P (1995). Iontophoresis enhances the transport of acyclovir through nude mouse skin by electrorepulsion and elecfroosmosis. Pharm Res, 12, 1623-1627.

Volpato NM, Nicoli, S., Laureri, C, Colombo, P., Santi P (1998). In vitro acyclovir distribution in human skin layers after transdermal iontophoresis. J Control Release. 50, 291-296.

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Claims

CLAIMS:

1. A mechanised brushing device, wherein the mechanism of the device is adapted to rotate a brush in contact with the skin of a patient, the device having abutment means for contact with the skin, the brush being movably located in relation to the abutment means to allow it to be introduced to the skin, travel of the brush being limited in relation to the abutment means such that pressure on the brush to contact the skin is limited to a predefined range.

2. A device according to claim 1, further comprising pressure limiting means.

3. A device according to claim 1 or 2, adapted to provide a contact pressure of the brush of between about 200 and about 1500 N m^"2 on the skin.

4. A device according to claim 3, wherein the pressure is between about 200 and about 1000 N m^"2,

5. A device according to claim 3 or 4, wherein the pressure is about equal to, or greater than, 300 N m^"2,

6. A device according to claim 3, 4 or 5, wherein the pressure is between about 300 and about 600 N m^"2.

7. A device according to any preceding claim, comprising a timing mechanism to limit the duration of the brushing.

8. A device according to any preceding claim, adapted to provide brushing for between about 10 seconds and 5 minutes.

9. A device according to claim 8, adapted to provide brushing for between about 10 seconds and about two minutes.

10. A device according to claim 8, adapted to provide brushing for between about 20 seconds and one minute.

11. A device according to claim 8, adapted to provide brushing for between about 30 seconds and about 50 seconds.

12. A device according to any preceding claim, wherein the brush is substantially Robertson grade 8, and is adapted to provide brushing for between about 15 seconds to about 40 seconds.

13. A device according to any preceding claim, adapted to provide an oscillatory brushing motion.

14. A device according to claim 13, wherein the oscillatory motion is essentially circular.

15. A device according to claim 13 or 14, wherein the rate of said motion is between 30 and 300 φm.

16. A device according to claim 15, wherein the rate of said motion is between 50 and 200 φm.

17. A device according to claim 15, wherein the rate of said motion is between 60 and 120 φm.

18. A device according to any preceding claim, wherein that part of the brush for contact with the skin consists essentially of bristles, and wherein said bristles have a Robertson number of from about 6 to about 11.

19. A device according to claim 18, wherein the Robertson number is from about 7 to about 10.

20. A device according to claim 18, wherein the Robertson number is from about 8 to about 9.

21. A device according to claim 18, wherein the Robertson number is about 8.

22. A device according to any preceding claim, wherein the cross-sectional area of that part of the brush for contact with the skin is between about 1 mm² and about 10 cm².

23. A device according to claim 22, wherein the cross-sectional area is about 4 mm² to about 5 cm².

24. A device according to claim 22, wherein the cross-sectional area is about 5 mm² to about 2 cm².

25. A method for the conditioning of skin to enhance transdermal delivery of drug, the method comprising continuous brushing for a period sufficient to reduce the barrier qualities of the stratum corneum.

26. A method according to claim 25, comprising, substantially immediately after brushing the area of skin to be treated, applying said drug to the brushed area.

27. A method according to claim 25 or 26, limited in accordance with any of claims 1 to 24.

28. A drug for use with any preceding claim, which is selected from: crotamiton, doxepin hydrochloride, mesulphen, polidocanol, amethocaine, amylocaine, benzocaine, bucricaine, butacaine sulphate, butyl aminobenzoate picrate, cin***e, dimethisoquin hydrochloride, dyclocaine hydrochloride, ethyl chloride, lidocaine, lignocaine, myrtecaine, oxethazaine, prilocaine, propanocaine hydrochloride, tetracaine, antihistamines, antazoline, chlorcyclizine hydrochloride, dimethindene maleate, diphenhydramine, histapyrrodine, isothipendyl hydrochloride, mepyramine, mepyramine maleate, tolpropamine hydrochloride, tripelermamine hydrochloride, triprolidine hydrochloride, corticosteroids, alclometasone dipropionate, beclomethasone dipropionate, betamethasone valerate, clobetasol propionate, clobetasone butyrate, desoximetasone, diflucortolone valerate, fludroxycortide/flurandrenolone, fluocinolone acetonide, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, calcipotriol, coal tar, dithranol, 5-fluouracil, ciclosporin, fumeric acid, lonapalene, methotrexate, methoxsalen, salicylic acid, tacalcitol, tacrolimus, pimecrolimus, tazarotene, azelaic acid, benzoyl peroxide, amorolfine, benzoic acid, bifonazole, bromochlorosalicylanilide, buclosamide, butenafine hydrochloride, chlormidazole hydrochloride, chloφhenesin, ciclopirox olamine, clotrimazole, croconazole hydrochloride, eberconazole, econazole nitrate, fenticlor, fenticonazole nitrate, flutrimazole, haloprogin, ketoconazole, mepartricin, miconazole nitrate, naftifine hydrochloride, natamycin, neticonazole hydrochloride, nystatin, omoconazole nitrate, oxiconazole nitrate, pyrrolnitrin, sertaconazole nitrate, sodium propionate, sulbentine, sulconazole nitrate, sulconazole nitrate, terbinafine, tioconazole, tolciclate, tolnaftate, triacetin, undecenoates/undecanoic acid, 1-docosanol, aciclovir, brivudine, edoxudine, ibacitabine, idoxuridine, idoxuridine in dimethyl sulfoxide, imiquimod, penciclovir, vidarabine, benzyl benzoate, carbaryl, malathion, permethrin, phenothrin, cetrimide, collodion, magnesium sulphate, proflavine, heparinoid, antiperspirants, aluminium chloride, glycopyrroniurn bromide, and mixtures thereof.

29. A drug for use with any of claims 1 to 27, which is selected from non-steroidal anti-inflammatories, actinic keratosis treatments, and capsaicin.

30. A drug for use with any of claims 1 to 29, which is a hydrophilic drug.

31. A drug for use in accordance with claim 30, having a Log P of < 2.

32. A drug for use in accordance with claim 30, having a Log P of < 1.

33. A drug for use in accordance with claim 30, having a Log P of 0 or below.

34. A drug for use with any of claims 1 to 27, which is selected from: methotrexate, aciclovir, dactinomycin, oxytetracychne, 5-fluorouracil, ipatropium bomide, chlortetracycline, ceterizine, carboplatin, aminophylline, ofloxacin, pravastatin sodium, dichloromethotrexate, isoniaziad, theopylline, doxycyline, metronidazole, procaine, 4-aminosalicyclic acid, baclofen, triamcinolone, lidocaine/lignocaine, minoxidil, and combinations thereof.

35. A substance for use with any of claims 1 to 27, which is a protein or peptide, nucleic acid, or a related compound.

36. A substance for use in accordance with claim 35, which is capable of stimulating an immune response when applied to the skin by a device as defined in any of claims 1 to 21.