WO2003047629A2 - Treatment of diseases via the skin - Google Patents

Abstract

A method of delivering a molecule to an individual, the method comprising: (a) providing an agent capable of effecting the degradation of a component of the epidermal barrier of the individual; (b) administering the agent to the individual; and (c) administering the molecule to the individual. Specifically described is the use of proteolysis, and other degradative processes for the reduction of epidermal barrier function, to regulate the passage of agents through the stratum corneum and into the body of an individual. A path comprising one or both agents provides a particularly advantageous way of putting the invention into practice.

Description

TREATMENT OF DISEASES

FIELD

This invention relates to the treatment of diseases. More generally, the invention relates to the delivery of drugs and other agents for various purposes, including topical delivery or delivery of drugs and other agents via the epidermis.

BACKGROUND

Drugs and other agents may be administered to individuals such as patients requiring them in various ways, including oral adm stration, injection, and topical administration.

It is known to incorporate a skin penetration enhancer which is dermatologically acceptable and compatible with the drug into a formulation to increase its penetration from the skin surface into epidermal keratinocytes. A skin enhancer which increases the absorption of the active compound(s) into the skin reduces the amount of agent needed for an effective treatment and provides for a longer lasting effect of the formulation.

Skin penetration enhancers known in the art include dimethyl sulfoxide (U.S. Pat. No.

3,711,602); oleic acid, 1,2-butanediol surfactant (Cooper, J. Pharm. Set, 73: 1153-1156 (1984)); a combination of ethanol and oleic acid or oleyl alcohol (EP 267,617), 2-ethyl-l,3- hexanediol (WO 87/03490); decyl methyl sulphoxide and Azone (Hadgraft, Eur. J. Drug. Metab. Pharmacokinet, 21:165-173 (1996)); alcohols, sulphoxides, fatty acids, esters, Azone, pyrrolidones, urea and polyoles (Kalbitz et al, Pharmazie, 51:619-637 (1996)); terpenes such as 1,8-cineole, menthone, limonene and erolidol (Yamane, J. Pharmacy & Pharmocology, 47:978-989 (1995)); Azone and Transcutol (Harrison et al, Pharmaceutical Res. 13 :542-546 (1996)); and oleic acid, polyethylene glycol and propylene glycol (Singh et al, Pharmazie, 51 :741-744 (1996)). The present invention seeks to solve one or more problems associated with prior art methods of drug delivery.

SUMMARY

We have now discovered that regulation of proteolysis of components of the stratum comeum is a major factor in maintenance of the epidermal or skin barrier function of an animal. In general, therefore, we describe the use of proteolysis and other degradative processes for the reduction of epidermal barrier function, in order to regulate the passage of agents through the stratum comeum and into the body of an individual.. Modulation of endogenous proteolytic and other degradative processes may be employed; alternatively, or in addition, such degradative processes may be set up by the use of external degradative agents.

According to a first aspect of the present invention, we provide use of an agent capable of effecting the degradation of a component of the epidermal barrier of an individual to enable or assist transdermal delivery of a molecule to the individual.

There is provided, according to a second aspect of the present invention, a method of delivering a molecule to an individual, the method comprising: (a) providing an agent capable of effecting the degradation of a component of the epidermal barrier of the individual; (b) administering the agent to the individual; and (c) administering the molecule to the individual.

We provide, according to a third aspect of the present invention, a method of treating a disease in an individual, the method comprising: (a) administration of an agent capable of effecting the degradation of a component of the epidermal barrier of the individual, followed by (b) administration of a drug indicated for the disease.

As a fourth aspect of the present invention, there is provided a composition comprising a molecule to be delivered through an epidermal barrier of an individual together with an agent capable of effecting the degradation of a component of the epidermal barrier of the individual. We provide, according to a fifth aspect of the present invention, an agent capable of effecting the degradation of a component of the epidermal barrier of an individual for use in a method of treatment of a disease in an individual, in which the treatment comprises administration of the agent to enable or assist transdermal delivery of a drug to the individual.

The present invention, in a sixth aspect, provides use of an agent capable of effecting the degradation of a component of the epidermal barrier of an individual in the preparation of a pharmaceutical composition for the treatment of a disease in an individual, in which the treatment comprises administration of the agent or the pharmaceutical composition to enable or assist transdermal delivery of a drug to the individual.

In a seventh aspect of the present invention, there is provided a kit comprising a compound to be delivered through an epidermal barrier of an individual, an agent capable of effecting the degradation of a component of the epidermal barrier of the individual, and instructions for using the agent to effect delivery of the compound.

According to an eighth aspect of the present invention, we provide a method of preparing a composition capable of enhanced transdermal delivery of a compound, the method comprising the steps of providing a composition comprising a compound to be delivered, and adding an agent capable of effecting the degradation of a component of the epidermal barrier of an individual to the composition.

The compound or molecule to be delivered may be administered simultaneously, sequentially or as a mixture with the agent. Alternatively or in addition, the agent is administered to the individual, followed by administration of the compound or molecule to the individual.

In one embodiment, the agent comprises an agent capable of effecting the degradation of a proteinaceous component of the epidermal barrier. Preferably, the proteinaceous component is comprised in the stratum comeum. More preferably, the proteinaceous component comprises an adhesion protein, preferably a desmosomal adhesion protein. In a preferred embodiment, the adhesion protein comprises corneodesmosin.

In a preferred embodiment, the agent comprises a protease. The protease may be selected from the group consisting of: Stratum Comeum Chymotryptic Enzyme (SCC; kallikrein 7, KLK7 ), Stratum Comeum Tryptic Enzyme (SCTE; kallikrein 5, KLK5), aminopeptidase M, carboxypeptidase P, carboxypeptidase Y, caspase 1,4,5, caspase 2,3,7, caspase 6,8,9, chymotrypsin, Factor Xa, pepsin, TEN, thrombin and trypsin.

In an alternative embodiment, the agent comprises an antagonist of a protease inhibitor, such as an antagonist of a antileukoprotease or an antagonist of a elafin protease inhibitor 3 (PI3 or SKALP).

In a further embodiment, the agent is capable of effecting the degradation of a lipidic component of the epidermal barrier. Preferably, the lipidic component is comprised in the lipid lamellae. Most preferably, the agent comprises a lipase.

More than one agent may be administered. Therefore, a particular embodiment of the method according to the first or third aspects of the invention or a use according to the second aspect of the invention further comprises the step of administering a second agent capable of effecting the degradation of a second component of the epidermal barrier of the individual. Furthermore, a composition according to the fourth aspect of the invention may further comprise a second agent capable of effecting the degradation of a second component of the epidermal barrier of the individual.

In a preferred embodiment, the first component comprises a proteinaceous component and the second component comprises a lipidic component. Preferably, the first agent comprises a protease and the second component comprises a lipase. The agent capable of effecting the degradation of a lipidic component and the agent capable of effecting the degradation of the proteinaceous component may be administered in any order. Preferably, the agent capable of effecting the degradation of a lipidic component is administered before the agent capable of effecting the degradation of the proteinaceous component. Furthermore, degradation of the lipidic component of the epidermal barrier preferably enables the agent capable of effecting the degradation of the proteinaceous component to contact the proteinaceous component of the epidermal barrier.

The methods and compositions as described here are useful for enabling or assisting delivery of a molecule to a normal skin. Thus, in a preferred embodiment, the compound or molecule to be delivered is not normally capable of crossing the epidermal barrier of normal human skin.

The methods and compositions as described here may also be used to enable or assist delivery of a molecule to diseased or otherwise abnormal skin. Thus, the compound or molecule to be delivered may be one which is capable of crossing the epidermal barrier of normal human skin but not substantially capable of crossing the epidermal barrier of psoriatic skin.

Any molecule may be delivered according to the methods and compositions as described here. Preferably, the compound or molecule comprises a drug or other therapeutic compound, preferably ascomycin or tacrolimus.

The compound or molecule to be delivered may have a molecular weight of more than about 1000 Daltons. The compound or molecule may have a molecular weight of between about 800 Daltons to about 1000 Daltons.

In a preferred embodiment, the or each agent is administered or applied in the form of a patch comprising the or each agent or both agents. The patch may further comprise the compound or molecule to be delivered in a separate compartment from the or each agent. Alternatively or in addition, the patch comprises a mixture of the or each agent and the compound or molecule to be delivered.

The or each agent may be provided in the form of a cream. The cream may further comprise the molecule or compound to be delivered.

We provide, according to a ninth aspect of the invention, a patch comprising an agent capable of effecting the degradation of a component of the epidermal barrier of an individual. The agent or component in such a patch may be as defined m any of the preceding aspects of the invention.

There is provided, in accordance with a tenth aspect of the present invention, a patch comprising a composition according to the fourth aspect of the invention.

As an eleventh aspect of the invention, we provide a patch comprising a molecule to be delivered to an individual together with an agent capable of effecting the degradation of a component of the epidermal barrier of an individual. The molecule and the agent may be in separate compartments, or they may be in the form of a mixture.

We provide, according to a twelfth aspect of the invention, there is provided the use of a patch according to the twelfth aspect of the invention, for enabling or assisting the delivery of a molecule to an individual.

According to a thirteenth aspect of the present invention, we provide the combined use of an agent capable of effecting the degradation of a proteinaceous component of the epidermal barrier together with an agent capable of effecting the degradation of a lipidic component of the epidermal barrier for assisting the transdermal transport of a therapeutic molecule. There is provided, according to a fourteenth aspect of the present invention, the use of an agent capable of reducing an epidermal barrier ftinction to enable or assist transdermal delivery of a molecule.

We provide, according to a fifteenth aspect of the present invention, use of a protease or a lipase to enable or assist the delivery of a molecule through an epidermal barrier of an individual.

DETAILED DESCRIPTION

In general, the methods and compositions as described here involve the use of one or more agents capable of reducing an epidermal barrier function of an individual, to enable or assist, or otherwise effect, the transdermal deHvery of a molecule (i.e., delivery of a molecule across the epidermal barrier of a patient). Thus, at least a portion of the epidermis of an individual is treated with an agent capable of reducing (preferably degrading) an epidermal barrier function to enable increased epidermal transport of a molecule.

In preferred embodiments, the individual is a patient suffering or likely to suffer from a disease, and the molecule is a drag such as a therapeutic or prophylactic drug. However, it will be appreciated that the methods and compositions described here are suitable for assisting or effecting the delivery of any molecule as desired, not necessary being of medical or therapeutic benefit. Thus, molecules which are for example components of cosmetics may also be delivered by the methods and compositions described here.

The agent capable of reducing an epidermal barrier function preferably comprises an agent capable of effecting the degradation of the epidermal barrier (or a component of the epidermal barrier) associated with the stratum comeum. Preferably, the methods further involve the administration of the molecule, preferably a drug indicated for a disease, to be delivered to an individual (such as a patient). In a preferred embodiment, the methods and compositions as described here involve the use of one or more agents capable of effecting the degradation of a component of an epidermal barrier of an individual, preferably a lipidic or proteinaceous component. Preferably, the agent is one which is capable of effecting the degradation of the epidermal barrier associated with the stratum comeum or the lamellar lipids, preferably the stratum comeum.

The agents as described here are capable of effecting the degradation a component of the epidermal barrier; in other words, the component is degraded in the presence of the agent(s), but not substantially in their absence. The agents may directly degrade the component of the epidermal barrier, or indirectly cause the degradation of the barrier. They may therefore comprise molecules, enzymes or other proteins which act to degrade, destroy, abolish or remove the component.

Where the component comprises a protein or polypeptide, the agent may comprise for example a protease or proteinase or any molecule comprising a proteolytic activity; where the component is lipidic in nature, the agent may comprise a lipase or a molecule capable of degrading, breaking down or digesting a lipid. The agent is preferably an enzyme, or a polypeptide having relevant enzymatic activity, such as a fragment of an enzyme. For example, a polypeptide comprising protease activity, a polypeptide comprising lipase activity, a fragment of a protease, a fragment of a lipase, a polypeptide comprising a protease active site, a polypeptide comprising a lipase active site, etc. Combinations of any of the above may be employed.

The agents may also comprise molecules which act to regulate any of the activities of any of the above.

In preferred embodiments, the protease activity and/or lipase activity comprises an endogenous enzyme activity. Thus, preferably, the protease involved is one which is expressed in the individual concerned, preferably, in the skin of an individual. Similarly, the lipase involved comprises a lipase expressed in the individual, preferably, expressed in his or her skin. Examples of endogenous proteases include S Stratum Comeum Chymotryptic Enzyme (SCCE) and Stratum Comeum Tryptic Enzyme (SCTE).

As noted above, the agent may not itself act to degrade the epidermal barrier, but may act indirectly, for example, by modulating the activity of a protease, lipase or other degrading agent. Thus, the agent may comprise a molecule capable of up-regulating the expression of an endogenous protease, for example one as listed above. Furthermore, the agent may comprise a molecule capable of down-regulating the breakdown or degradation of such a protease. Endogenous protease activity may be regulated by protease inhibitors, for example, endogenous protease inhibitors such as antileukoprotease (skin-derived antileukoprotease) and elafin protease inhibitor 3 (PI3 or SKALP). Thus, the agents may comprise molecules capable of down-regulating the activity of protease inhibitors, preferably endogenous protease inhibitors.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4^th Ed, John Wiley & Sons, Inc. which are incorporated herein by reference), chemical methods, pharmaceutical formulations and delivery and treatment of individuals.

EPIDERMAL BARRIER

The skin is a barrier that retains water within the body and prevents the penetration of environmental agents into the body. The barrier function of the skin is essential to the maintenance of the internal homeostasis (Cork 1997). The epidermis is composed of layers of closely packed keratinocytes that are formed by division in the stratum basale (or germinative layer). As the keratinocytes move up through the prickle and granular layers, they differentiate and a rigid internal structure of keratin, microfilaments and microtubules is formed. The stratum comeum (homy layer) is composed of layers of flattened dead cells that have lost their nucleus, between which is a complex mixture of lipid and proteins.

The "barrier function" of the epidermis, or the "epidermal barrier function", as the terms are used in this document, is therefore intended to refer to the ability of an epidermal layer to resist the penetration of an external agent.

Where a drug is topically applied to the skin of an individual, a sufficient amount of agent must penetrate an individual's skin in order to obtain a desired pharmacological effect. The epidermal permeability barrier provides a barrier to the penetration of drags through the stratum comeum and into the viable part of the epidermis. Once a drag has passed through the stratum comeum it has a passage to the rest of the body. Drags of more than 1000 daltons molecular weight will not normally pass through an intact epidermal permeability barrier. Drags of molecular weight 800-1000 may pass through the normal epidermal barrier but this is variable and influenced by several factors including the structure and lipid solubility. Anything that increases the thickness of the stratum comeum (such as psoriasis) will decrease the penetration of a drug. This problem will be most obvious for drags of molecular weight 800-1000 daltons. Drags this molecular weight range may penetrate through normal skin but not through psoriatic skin (for example Ascomycin and Tacrolimus).

It is generally understood that the absorption of drag into the skin is a function of the nature of the drag, the behaviour of the vehicle, and the skin.

Three major variables account for differences in the rate of absorption or flux of different topical drugs or the same drug in different vehicles; the concentration of drag in the vehicle, the partition coefficient of drag between the stratum comeum and the vehicle and the diffusion coefficient of drug in the stratum comeum. To be effective for treatment, a drag must cross the stratum comeum which contributes to the barrier function of the skin. In general, a topical formulation which exerts a high in vitro skin penetration is effective in vivo. Ostrenga et al (J. Pharm. Set, 60:1175-1179 (1971) demonstrated that in vivo efficacy of topically applied steroids was proportional to the steroid penetration rate into human skin obtained by dermatomed technique in vitro.

The stratum corneum is a barrier that is continually being replaced by proliferation and differentiation of keratinocytes in the viable epidermis. In order to maintain a constant stratum comeum thickness at a given body site superficial parts of the stratum comeum must be continuously shed in the process of desquamation at a rate that balances their production.

REDUCTION OF EPIDERMAL BARRIER FUNCTION

The barrier function or epidermal barrier may comprise one or more components, any number of which are degraded, individually or together, by the methods and compositions as described here. Particularly preferred components of the epidermal barrier comprise proteinaceous components and lipidic components. Such components of the epidermal barrier are described in a separate section below.

An epidermis which has been treated with an agent capable of reducing an epidermal barrier function, for example, an agent capable of effecting the degradation of a component of the epidermal barrier, has an reduced epidermal barrier function. In other words, the molecular weight cut-off of the epidermal barrier is increased, so that molecules of higher molecular weight are able to cross the skin than an untreated skin.

The methods and compositions described here therefore enable the efficient transdermal delivery of a molecule to an individual. As used here, the term "transdermal" is meant to encompass passage of a molecule across the epidermal barrier of an individual. The methods and compositions described here therefore enable the delivery of a molecule to any portion of the body of an individual which is below the epidermal barrier, for example, a subcutaneous portion, a sub-epidermal portion, a muscle portion or a fat portion of the body. An epidermis treated by the methods and compositions as described here is more permeable to an external agent than a corresponding untreated epidermis. The corresponding epidermis is preferably one from the same patient, individual or animal, for example, an epidermis from another part of the patient, individual or animal. Preferably, the corresponding epidermis has the same disease state or normality as the one which is treated. The corresponding epidermis may also be derived from another individual, etc.

For example, where penetration of agent into psoriatic skin is being assessed, a suitable reference material may comprise untreated psoriatic skin from another part of the body of the same individual, or psoriatic skin from another individual.

An epidermis which has been treated by the methods and compositions described here will typically have a reduced or impaired barrier function, and will allow more penetration of an external agent than otherwise. As an extreme example, the untreated sldn may be substantially impermeable to the external agent of interest. Thus, such a skin does not allow substantially any agent to penetrate. Application of the agent capable of effecting the degradation of a component of the epidermal barrier enables an amount of external agent to pass through the epidermal barrier. In this particular extreme example, the treated sldn may have such a degraded epidermal barrier that the skin becomes substantially completely permeable to the external agent. However, it will be appreciated that this is not always the case; all that is necessary is that the epidermal barrier is broken down to such an extent that the amount of agent capable of passing through the barrier is more than in an untreated sldn. Preferably, the epidermal barrier is broken down to such an extent that a desired amount of agent may be delivered, preferably a therapeutically effective amount of agent.

Preferably, a treated epidermis is 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% 90%, 100%, 200%, 300%, 400%, 500%, 10 times or more permeable to an external agent than an untreated epidermis. Increase in penetration or permeability is preferably reflected by increase of mass of agent or drag, activity of an agent or drag (such as a relevant chemical, biological or enzymatic activity) which passes through the epidermal layer. Thus, a treated epidermis will therefore preferably enable penetration of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% 90% or 100% or more agent or drug than an untreated epidermis.

Increase in permeability or penetration may also be reflected by an increased ratio of molecules which pass through compared to those which are retarded, or alternatively, as compared to those which are applied. This ratio, N, may be increased by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% 90% or 100% or more with the methods and compositions as described here.

Measurement of epidermal barrier function may be done in various ways. Similarly, the level or degree of penetration of an agent or composition can be determined by techniques known to those of skill in the art. For example, radiolabeling of an active compound or a tracer compound, followed by measurement of the amount of radiolabeUed compound absorbed by the skin enables one of skill in the art to determine levels of the composition absorbed using any of several methods of determining skin penetration of the test compound. Measurement of the amount of radiolabeUed compound which crosses the sl n layer may also be made.

In a preferred embodiment, a Franz chamber and cadaver system (also known as a Franz chamber penetration system) is used to measure penetration of an agent.. This system measures barrier function by allowing the measurement of amount of radiolabeUed compound which passes through a piece of sldn to a receptacle fluid. The Franz chamber has the advantage in that it is a robust, quick and easy system, and is described in more detail below.

In a preferred embodiment, the methods and compositions described here are used to effect delivery of a drug or agent which is substantiaUy incapable of traversing the epidermal barrier of an untreated epidermis. Such a drug or agent may have a molecular weight, hydrophobicity, conformation or shape etc which is incompatible with movement or diffusion across an untreated epidermal barrier. Use of the methods and compositions described here enables such a drug or agent to cross the epidermal barrier of a treated epidermis. For example, the molecular weight cut-off of normal skin is in the region of about 1000 daltons. The methods and compositions described here preferably enable the passage through the epidermal barrier of molecules of molecular weight of 1000 daltons or higher. Preferably the methods and compositions described here enable the passage through the epidermal barrier, of molecules of 2 kDa or higher, such as molecules of 3 kDa, 4 kDa, 5kDa, lOkDa, 100 l Da, 1000 kDa, 10,000 l Da, 100,000 kDa or higher.

It wiU be appreciated that any molecule of or higher than the relevant modified molecular weight cut-off of the treated skin may be dehvered. The molecular weight of any molecular may readily be determined by means known in the art. Furthermore, searches may be made on suitable databases to identify molecules of specified molecular mass, to ascertain whether they are capable of being delivered to skin treated by the methods and compositions described here.

The epidermal barrier function may be reduced to match the molecular weight of the molecule to be dehvered. In other words, the molecular weight cut-off may be modified according to the molecular weight of the molecule in question. The desired degree of reduction of the epidermal barrier, and therefore the increase in penetration or permeability, may be achieved by various ways. The concentration of the or each degradative agent may be altered, reduced or increased to achieve the desired reduction in epidermal barrier function. The degradative agent may be modified, chemicaUy or by molecular biology techniques to alter the activity of the agent. For example, a protease or lipase may be mutated to modulate the protease or lipase activity. The time of application of the degradative agent may also be changed. Exposure for shorter periods will enable less reduction in epidermal barrier function than exposure for longer periods. The reduction in epidermal barrier ftinction may be monitored to determine the optimal time of contact.

Furthermore, different degradative agents may be chosen based on their ability to cleave or degrade different components, and their efficiency of doing so. Thus, where proteinaceous components of the sldn barrier are being targeted, different proteases may be chosen based on their abiUty to cleave or degrade different components, and their efficiency of doing so. For example, where a higher reduction of the epidermal barrier is desired, two or more proteases may be employed. Each protease may be capable of degrading a different proteinaceous component of the barrier. Thus, a protease capable of degrading corneodesmosin may be combined with a protease capable of degrading desmoglein, for example.

Other combinations are of course possible. For example, combinations of two or more of proteases capable of degrading corneodesmosin, proteases capable of degrading corneodesmosin, proteases capable of degrading desmocollin, proteases capable of degrading desmoplaldn I, proteases capable of degrading desmoplaldn π, proteases capable of degrading plakoglobin (PG) and proteases capable of degrading plakophilin (PP) may be employed. Furthermore, a protease may be combined with a lipase to enable the reduction of both proteinaceous and Upidic components to enable increased reduction in barrier function.

Preferably, the epidermal barrier function is reduced to enable the passage of a molecule of interest, such as a drug, through the epidermal barrier. Preferably, the epidermal barrier function is reduced to enable the accumulation or deUvery of a therapeuticaUy effective amount of a drag to the individual. The epidermal barrier function is preferably reduced only to such an extent as to enable such passage, but not reduced enough to enable passage of molecules of substantiaUy higher molecular weight than the molecule of interest. Thus, in preferred embodiments, the reduced epidermal barrier function does not enable the passage of high molecular weight toxins or microorganisms such as bacterial or viruses.

In preferred embodiments, the breakdown or reduction of the epidermal barrier is not permanent. Thus, preferably, the epidermal barrier is broken down or reduced for long enough for a desired amount of the relevant molecule to be dehvered to pass through for delivery. More preferably, the epidermal barrier is broken down for only such a period, and restored subsequently. Restoration may be achieved by physical removal of the agent, such as by washing with oil or water based solutions, or by physical or chemical inactivation of the agent. Where a protease is involved, for example, a peptide comprising a cleavage site may be used to neutraUse or reduce the activity of the protease. Furthermore, a protease inhibitor may be applied to reduce or remove the protease activity.

It wiU be appreciated that other means of assisting transdermal delivery may be used in conjunction with the methods and compositions described here. For example, the agent or drag may be derivatised to increase or decrease its abiUty to penetrate the epidermal barrier may be employed. For example, derivatisation may alter the hydrophobicity of the drug, its hydrophilicity or both. Other agents or factors which are known to regulate epidermal cell-cell adhesion may be employed in conjunction with the agents described in this document. Such other agents and/or factors include molecules capable of modulating calcium levels, for example, chelators, particularly chelators of divalent ions such as EDTA or EGTA. Glycosidases may also be employed.

Impaired desquamation of corneocytes is characteristic of a number of diseases such as psoriasis, acne vulgaris, ichthyosis and keratinosis pUaris, among others. In psoriasis, impaired desquamation of corneocytes causes an increased thickness of the stratum comeum and the barrier is usuaUy enhanced. Thus, the ability of the a molecule to penetrate the epidermal barrier may differ between individual to individual, depending on their disease state, and the term "epidermal barrier function" should be preferably taken to be refer to the barrier function of the specific individual in question.

Publications relating to skin penetration studies include Reinfenrath, W G and G S

Hawkins. The Weaning Yorkshire Pig as an Animal Model for Measuring Percutaneous Penetration. In: Swine in Biomedical Research (M. E. Tumbleson, Ed.) Plenum, New York, 1986, and Hawkins, G. S. Methodology for the Execution of In Vitro Skin Penetration Determinations. In: Methods for Skin Absorption, B W Kemppainen and W G Reifenrath, Eds., CRC Press, Boca Raton, 1990, pp.67-80; and W. G. Reifenrath, Cosmetics & Toiletries, 110:3-9 (1995). PROTEINACEOUS COMPONENTS OF THE EPIDERMAL BARRIER

The epidermal barrier may comprise for example one or more proteinaceous components; the methods and compositions as described here may therefore involve the degradation of one or more proteinaceous components comprised in the epidermal barrier. Preferably, the proteinaceous component comprises an adhesion protein, as described in further detail below.

By the term "adhesion protein" we mean any protein which is involved in ceU-ceU adhesion. Preferably, the adhesion protein mediates ceU-ceU interaction between epitheUal ceUs, i.e., the adhesion protein is an epitheUal ceU adhesion protein. More preferably, the adhesion protein mediates ceU-ceU adhesion between corneocytes. Most preferably, the adhesion protein is a desmosomal protein or a corneodesmosomal protein. The adhesion protein may suitably comprise any one or more of corneodesmosin, desmoglein, desmocollin, desmoplaldn I, desmoplaldn π, plakoglobin (PG) and plakoph in (PP). Other adhesion proteins suitable for use in the methods and compositions described here are set out in Annex A, Table Al .

Within the epidermis, the barrier function may be performed by the stratum comeum. In this situation, the strength of the barrier function is dependent on strong adhesion between corneocytes (Egelrud, 2000). Accordingly, the effectiveness of the barrier function or epidermal barrier function may be directly proportional to the integrity of the stratum comeum, and in particular, the degree or level of adhesion between corneocytes of the stratum comeum.

The adhesion between corneocytes in the stratum comeum is mediated by comeodesmosomes (Menton and Elisen, 1971; Chapman and Walsh, 1990; North et al, 1999). Accordingly, in a preferred embodiment, the component of an epidermal barrier which is degraded according to the methods and compositions described here comprises a component of a desmosome or comeodesmosome, preferably a proteinaceous component. Preferably, such an agent is capable of decreasing the ceU-ceU adhesion mediated by a comeodesmosome. Comeodesmosomes (also known as desmosomes) are symmetrical structures that form disc-shaped interceUular junctions between epitheUal ceUs. In the epidermis the desmosomes mediate the adhesion between keratinocytes. In psoriasis, various ichtyoses and skin xerosis, the number of comeodesmosomes (desmosomes in upper layers of the epidermis) is increased in the stratum comeum. Immunoelectron microscopy has been used to show the define the interactions within comeodesmosomes between proteins of the extraceUular core domain such as desmoglein and desmocollins and intraceUular comeodesmosomal proteins including desmoplaldns I and π, plakoglobin (PG) and plakophilins (PP) (Cowin and Burke, 1996).

Preferably therefore, the component of an epidermal barrier comprises an intraceUular comeodesmosomal protein, for example, desmoglein, desmocoUin, desmoplaldn I, desmoplaldn π, plakoglobin (PG) and plakophilin (PP). The methods and compositions described here may comprise degradation of one or more of the foregoing, simultaneously or sequentiaUy, by the use of one or more agents.

In a preferred embodiment, the component of an epidermal barrier comprises a glycoprotein. Preferably, the component comprises corneodesmosin. Three forms of the corneodesmosin with different weights 33-36 to 40-46 and 52-56 kDa have been isolated from the epidermis (Simon et al, 1997). The methods and compositions described here therefore include treatment of an epidermis to degrade any one or more of the forms of corneodesmosin, optionaUy in combination with degradation of any one or more components of the epidermal barrier.

The importance of comeodesmosomal proteins in epidermal integrity is demonstrated by inherited disorders such as striate subtype of palmoplantar keratoderma caused by desmoplaldn haploinsufficiency (Armstrong et al, 1999). Mutations in loricrin gene lead to keratoderma of Camisa. LIPID COMPONENTS OF THE EPIDERMAL BARRIER

Another component of the epidermal barrier is the lameUar Upids. The Upid is secreted into lameUar bodies in the stratum granulosum (EUas 1983). At the interface between the stratum granulosum and stratum comeum, the Upids are extracted from the granular ceUs into the intercorneoctye space.

The Upids are then organised into highly organised multimeUar bUayer structures (Landmann 1988). The stratum comeum can be visuaUsed rather tike a brick waU with the corneocytes forming the bricks and the lameUar Upids the mortar (EUas 1983). Corneocytes contain a water retaining substance, natural moisturising factor (NMF), which retains water within them. The high water content causes the corneocytes to sweU, preventing the formation of fissures and cracks between them. The pliabiUty and elasticity of the skin is directly related to its water content. Normal healthy stratum comeum has comparatively high water content.

The epidermal barrier may therefore comprise for example one or more Upid components; the methods and compositions as described here may therefore involve the degradation of one or more tipidic components comprised in the epidermal barrier. Preferably, the methods and compositions described here employ Upases, by which term we mean any agent which is capable of degrading, destroying or cleaving a Upid, in particular, a Upid component of an epidermal barrier.

CORNEODESMOSIN

Corneodesmosin is an adhesion protein which is found in the stratum comeum, particularly in the comeodesmosomes. A corneodesmosin sequence is set out in WO0162788. The term "corneodesmosin" includes any variant or mutation of the corneodesmosin gene or protein, including variants associated with susceptibility to disease (for example as described in Guerrin et al., 2001, Tissue Antigens;57(l):32-8). Examples of corneodesmosin genes, amino acid sequences and nucleotide sequences include NT_007592 Homo sapiens chromosome 6 working draft sequence segment gi|15305200|refjNT_007592.5|Hs6_7749[15305200]; XM_041759 Homo sapiens corneodesmosin (CDSN), mRNA gi|14782117|ref]XM_041759.1|[14782117]; XM_004564 Homo sapiens corneodesmosin (CDSN), mRNA gijl 1419124|reflXM_004564.1|[l 1419124];

AJ238467 Homo sapiens CDSN gene, partial, haplotype HLA B8-Cw*0701; gi|6010136|emb|AJ238467.1|HSA238467[6010136]; AJ238466 Homo sapiens CDSN gene, partial, haplotype HLA B7-Cw*0702; gi|6010134|emb|AJ238466.1|HSA238466[6010134];

AJ238465 Homo sapiens CDSN gene, partial, haplotype HLA B18-Cw*0701 gi|6010132|emb|AJ238465.1|HSA238465[6010132]; AJ238464 Homo sapiens CDSN gene, partial, haplotype HLA B13-Cw*0602 gi|6010130|emb|AJ238464.1|HSA238464[6010130];

AJ238463 Homo sapiens CDSN gene, partial, haplotype HLA B57-Cw*0602,B37-Cw*0602 gi|6010128|emb|AJ238463.1|HSA238463[6010128]; AJ238462 Homo sapiens CDSN gene, partial, haplotype HLA B62-Cw*0304 gi|6010126|emb|AJ238462.1|HSA238462[6010126]; AJ238461 Homo sapiens CDSN gene, partial, haplotype HLA B62-Cw*0304 gi|6010124|emb|AJ238461.1|HSA238461[6010124]; AF286165 Homo sapiens corneodesmosin (CDSN) gene, CDSN- 1.43 allele, partial cds gi|9937326|gb|AF286165.1|AF286165[9937326]; AF224752 Homo sapiens corneodesmosin

(CDSN) gene, CDSN- 1.42 allele, partial cds gi|9027570|gb|AF224752.2|AF224752[9027570]; AF224758 Homo sapiens corneodesmosin

(CDSN) gene, CDSN-2.23 aUele, partial cds gi|8699045|gb|AF224758.1|AF224758[8699045]; AF224757 Homo sapiens corneodesmosin

(CDSN) gene, CDSN-2.22 allele, partial cds gi|8699043|gb|AF224757.1|AF224757[8699043]; AF224756 Homo sapiens corneodesmosin (CDSN) gene, CDSN-2.21 allele, partial cds gi|8699041|gb|AF224756.1|AF224756[8699041]; AF224755 Homo sapiens corneodesmosin

(CDSN) gene, CDSN-2.11 allele, partial cds gi|8699039|gb|AF224755.1|AF224755 [8699039]; AF224754 Homo sapiens corneodesmosin

(CDSN) gene, CDSN- 1.52 allele, partial cds gi|8699037|gb|AF224754.1|AF224754[8699037]; AF224753 Homo sapiens corneodesmosin (CDSN) gene, CDSN- 1.51 allele, partial cds gi|8699035|gb|AF224753.1|AF224753[8699035]; AF224751 Homo sapiens corneodesmosin

(CDSN) gene, CDSN- 1.41 allele, partial cds gi|8699031|gb|AF224751.1|AF224751[8699031]; AF224750 Homo sapiens corneodesmosin (CDSN) gene, CDSN- 1.32 allele, partial cds , gi|8699029|gb|AF224750.1|AF224750[8699029]; AF224749 Homo sapiens corneodesmosin

(CDSN) gene, CDSN- 1.31 allele, partial cds gi|8699027|gb|AF224749.1|AF224749[8699027]; AF224748 Homo sapiens corneodesmosin

(CDSN) gene, CDSN- 1.21 allele, partial cds gi|8699025|gb|AF224748.1|AF224748[8699025]; AF224747 Homo sapiens corneodesmosin

(CDSN) gene, CDSN- 1.11 allele, partial cds gi|8699023|gb|AF224747.1|AF224747[8699023]; NM_001264 Homo sapiens corneodesmosin (CDSN), mRNA gi|4502758|reflNM_001264.1|[4502758]; AF030130

Homo sapiens corneodesmosin mRNA, complete cds gi|2921269|gb|AF030130.1|AF030130[2921269]; AI930552 ul66bl2.yl Sugano mouse embryo mewa Mus musculus cDNA clone IMAGE:2135519 5' similar to SW:CDSN_HUMAN

Q15517 CORNEODESMOSIN PRECURSOR ;, mRNA sequence gi|5666516|gb|AI930552.1|AI930552[5666516]; AI787690 ul21d03.yl Sugano mouse embryo mewa Mus musculus cDNA clone MAGE:2088197 5' simUar to SW:CDSN_HUMAN Ql 5517 CORNEODESMOSIN PRECURSOR ;, mRNA sequence gi|5335397|gb|AI787690.1|AI787690[5335397]; AI768204 wg82d08.xl

Soares_NSF_F8_9W_OT_PA_P_Sl Homo sapiens cDNA clone AGE:23715993' similar to SW:CDSN_HUMAN Q15517 CORNEODESMOSIN PRECURSOR ;, mRNA sequence gi|5234713|gb|AI768204.1|AI768204[5234713]; AI746833 ul08a01.yl Sugano mouse embryo mewa Mus musculus cDNA clone MAGE:2076168 5' simUar to SW:CDSN_HUMAN

Q15517 CORNEODESMOSIN PRECURSOR ;, mRNA sequence gi|5125097|gb|AI746833.1|AI746833[5125097] REGULAΉON OF PROTEOLYSIS

LI a preferred aspect, the methods and compositions described here involve the regulation of proteolysis of an adhesion protein comprised in the epidermal barrier, to enable transdermal deUvery of a molecule.

Proteases suitable for use in the methods and compositions described here are set out in

Annex A, Table A2.

In one aspect, the methods and compositions described here involve the regulating (preferably up-regulating) the activity of an endogenous protease which degrades a component of the epidermal barrier of an individual. Examples of endogenous proteases include Stratum Comeum Chymotryptic Enzyme (SCCE; kallikrein 7, KLK7), Stratum Comeum Tryptic Enzyme (SCTE; kallUcrein 5, KLK5), KLKl, KLK2, KLK3 and KLK4. Furthermore, such endogenous proteases may be applied to the sldn using the methods and compositions described here, to supplement the activity of he protease in the skin. Endogenous proteases are referred to here as "primary" proteases.

An advantage of using endogenous proteases relates to their allogenicity. Use of endogenous proteases is less likely to provoke an inappropriate immune response to cause for example inflammation. Endogenous proteases are also generaUy more specific in their activity, in that they are only capable of degrading certain substrates. Accordingly, use of endogenous proteases wiU avoid degradation of other components of the skin, for example, components of the sldn that do not contribute to the strength or integrity of the epidermal barrier.

Preferably, an agent is employed which is capable of achieving this. Such an agent may comprise a molecule, such as a transcription factor, capable of increasing the quantity, activity and/or bio-avaUabiUty of a protease responsible for break down of an adhesion protein. The methods and compositions as described here may further involve up-regulating the expression and/or activity of a protease responsible for breaking down an adhesion protein. For example, the transcription and/or translation of such a protease may be up-regulated as a means to enable breakdown of a proteinaceous component of the epidermal barrier to enable transdermal deUvery of a molecule. Suitable proteases the expression of which may be regulated include SCCE and SCTE, described in detaU elsewhere. Thus, an agent comprising a molecule which up-regulates protease activity may be used in the methods and compositions described here.

Furthermore, the protease whose activity, expression, bioava abitity, etc is upregulated need not be one which is normaUy involved in the proteolysis of the epidermal barrier component. Thus, the methods and compositions described here include the administration of any protease to the individual, so long as such administration is capable of degrading a component of an epidermal barrier to enable transdermal deUvery of a drag or agent. Such a protease is referred to here as a "secondary protease".

Examples of suitable secondary proteases include aminopeptidase M, carboxypeptidase

P, carboxypeptidase Y, caspase 1,4,5, caspase 2,3,7, caspase 6,8,9, chymotrypsin, Factor Xa, pepsin, TEN, thrombin or trypsin may therefore be employed.

Regulation of proteolysis may also be achieved through regulation of activities of molecules involved in regulating protease activity.

Accordingly, the expression and/or activity of a protease inhibitor which is capable of inhibiting a protease activity capable of breakdown of an adhesion protein may be down- regulated as a means to effect transdermal deUvery of an agent. The protease inhibitor whose expression and/or activity is down-regulated preferably comprises a natural protease inhibitor which physiologicaUy inhibits the protease in question (i.e., the protease responsible for proteolysis of the adhesion protein). Down-regulation of protease inhibitor may be achieved by deactivation or repression of transcription and/or translation of the protease inhibitor to increase the activity of proteases. Preferred protease inhibitors include for example, SKALP and SLPI, wh e preferred proteases include SCCE and SCTE.

Expression of the protease inhibitor may be effected at the transcriptional and/or the translational level.

It wiU be appreciated that the methods described above need not be carried out independently, and may be combined with each other. For example, a protease may be administered in combination with an agent which down-regulates a protease inhibitor.

In a specific example, an agent such as a protease may be formulated in an emoUient base. The protease or other agent wUl effect the degradation of an epidermal barrier component (such as an adhesion protein, for example corneodesmosin). As a result, the epidermal barrier is compromised; in particular, the cohesion between corneocytes is reduced, allowing transport of a relevant agent or drug across the epidermal barrier.

Expression of one or more genes coding for a protease, whether a primary protease or a secondary protease, may be used to increase protease activity. For example, we provide for the subcutaneous (or otherwise) injection of expression vectors which express, for example, SCCE, SCTE, aminopeptidase M, carboxypeptidase P, carboxypeptidase Y, caspase 1,4,5, caspase 2,3,7, caspase 6,8,9, chymotrypsin, Factor Xa, pepsin, TEN, thrombin or trypsin, or other proteases in order to increase protease activity to effect proteolysis of the adhesion protein. Furthermore, as mentioned above, endogenous production of primary proteases may be enhanced by up-regulating transcription and/or translation of the relevant protease genes, by means known in the art. PROTEASES AND PROTEASE INHIBITORS

As noted above, a preferred agent for use in the methods and compositions described here comprises any protease capable of effecting the degradation of a component of an epidermal barrier, preferably an adhesion protein.

Particular examples of proteases which may be used in the methods and compositions described here include the foUowing:

Stratum Comeum Chymotryptic Enzyme (SCCE) Stratum Comeum Tryptic Enzyme (SCTE)

Stratum comeum chymotryptic enzyme (SCCE) or kallikrein 7 (KLK7) as well as stratum comeum tryptic enzyme (SCTE) or kallikrein 5 (KLK5) are members of large serine protease famUy. Analysis of their expression profiles suggests their skin specificity. Both enzymes are expressed in high suprabasal keratinocytes and interfoUicular epidermis and have maximum activity at the physiologic pH of stratum comeum (Elcholm et al, 2000; Eugelrud, 1992, Elcholm and Eugelrud, 1998). SCCE and SCTE are transported to the stratum comeum extraceUular space during comification. SCCE is produced as an inactive precursor and there is a need for an activating enzyme to confer to SCCE its proteolytic activity. SCTE is thought to be the enzyme involved in SCCE activation. A cluster of kallikrein genes including SCCE and SCTE has been mapped in chromosome 19ql3.3-13.4. This includes at least 6 structuraUy and evolutionary- related members (KLKl, KLK2, KLK3, KLK4, SCCE and SCTE). Of those SCCE (KLK7) and SCTE (KLK-L2) are mainly expressed in skin. They are also expressed in other tissues such as brain, kidney, mammary and saUvary glands (Yousef et al, 2000; Yousef and Diamandis, 1999).

Also useful for the methods and compositions described here are homologues of adhesion proteins, proteases and protease inhibitors. For example, skin specific proteases such as SCCE may be identified by conventional screening or database homology searching. Such SCCE homologues may also be used in the methods and compositions described here.

Protease inhibitors whose activity may be down-regulated include any of the protease inhibitors set out in Annex A, Table A3. Particularly preferred examples of protease inhibitors include the Mowing:

Antileukoprotease andElafin Protease Inhibitor 3

Several serine protease inhibitors are present in human epidermis including antileukoprotease (skin-derived antileukoprotease) and elafin. Antileukoprotease is a powerful inhibitor of SCCE. Elafin protease inhibitor 3 (PI3 or SKALP) is another serine protease inhibitor produced by keratinocytes. It is over-expressed in the subcomeal layers of lesional psoriatic sldn and in other skin disorders such as Behcet's syndrome, Sweet's syndrome, pyoderma gangrenosum, urticaria and cutaneous aUergic vasculitis (Tanaka et al. 2000). Changes of SKALP expression are capable of affecting SCCE and SCTE activities and hence disturb the structure of superficial layers of the epidermis in skin disorders such as psoriasis and eczema.

Mutants, variants, etc of the adhesion proteins, proteases, and protease inhibitors etc are encompassed by the invention. Such mutants include deletions, transversions, transitions, nucleic acid substitutions, amino acid substitutions (preferably with conserved residues), insertions, etc.

AGONISTS AND ANTAGONISTS

The methods and compositions of our invention rely, in some embodiments, on blocking the activity of protease inhibitors, or blocldng the activity of inhibitors of Upase activity, or both. They may further employ in some embodiments agents capable of increasing the activity of a protease and/or Upase. Agents which are capable of increasing the activity of a protease are referred to as agonists of that activity. Similarly, antagonists reduce the activity of a protease inhibitor. The term "antagonist", as used in the art, is generaUy taken to refer to a compound which binds to an enzyme and inhibits the activity of the enzyme. The term as used here, however, is intended to refer broadly to any agent which inhibits the activity of a molecule, not necessarily by binding to it. Accordingly, it includes agents which affect the expression of a protein such as a protease inhibitor, or the biosynthesis of a molecule such as a protease inhibitor, or the expression of modulators of the activity of the protease or protease inhibitor. The specific activity which is inhibited may be any activity which is characteristic of the enzyme or molecule, for example, protease activity or protease inhibitor activity. Assays for protease activity and protease inhibitor activity are known in the art.

The antagonist may bind to and compete for one or more sites on the relevant molecule, for example, a protease enzyme, preferably, the catalytic site of the protease enzyme. Preferably, such binding blocks the interaction between the molecule and another entity (for example, the interaction between a protease enzyme and its substrate, for example, an adhesion molecule). However, the antagonist need not necessarily bind directly to a catalytic site, and may bind for example to an adjacent site, another protein (for example, a protein which is complexed with the enzyme) or other entity on or in the ceU, so long as its binding reduces the activity of the enzyme or molecule.

Where antagonists of a enzyme such as a protease enzyme are concerned, an antagonist may include a substrate of the enzyme, or a fragment of this which is capable of binding to the enzyme. In addition, whole or fragments of a substrate generated natively or by peptide synthesis may be used to compete with the substrate for binding sites on the enzyme. Alternatively, or in addition, an immunoglobulin (for example, a monoclonal or polyclonal antibody) capable of binding to the protease inhibitor may be used. The antagonist may also include a peptide or other smaU molecule which is capable of interfering with the binding interaction. Other examples of antagonists are set forth in greater detaU below, and wiU also be apparent to the skilled person.

Blocking the activity of a protease or protease inhibitor may also be achieved by reducing the level of expression of the protease or inhibitor in the ceU. For example, the cell may be treated with antisense compounds, for example oligonucleotides having sequences specific to the protease or protease inhibitor mRNA.

The antagonist or agent may itself be a protease which cleaves the protease or protease inhibitor. Examples of proteases include aminopeptidase M, carboxypeptidase P, carboxypeptidase Y, caspase 1,4,5, caspase 2,3,7, caspase 6,8,9, chymotrypsin, Factor Xa, pepsin, TEN, thrombin, trypsin etc.

AGENTS AND MOLECULES

An agent of use in the methods and compositions described here is one which is capable of reducing epidermal barrier function, for example, by degrading a component of the epidermal barrier.

As used herein, in general, the term "agenf (including agonists and antagonists) includes but is not limited to agents such as an atom or molecule, wherein a molecule may be inorganic or organic, a biological effector molecule and/or a nucleic acid encoding an agent such as a biological effector molecule, a protein, a polypeptide, a peptide, a nucleic acid, a peptide nucleic acid (PNA), a virus, a virus-tike particle, a nucleotide, a ribonucleotide, a synthetic analogue of a nucleotide, a synthetic analogue of a ribonucleotide, a modified nucleotide, a modified ribonucleotide, an amino acid, an amino acid analogue, a modified amino acid, a modified amino acid analogue, a steroid, a proteoglycan, a Upid, a fatty acid and a carbohydrate. An agent may be in solution or in suspension (e.g., in crystalline, coUoidal or other particulate form). The agent may be in the form of a monomer, dimer, oUgomer, etc, or otherwise in a complex.

The terms "antagonist" and "agent" are also intended to include, a protein, polypeptide or peptide including, but not limited to, a structural protein, an enzyme, a cytokine (such as an interferon and/or an interleuldn) an antibiotic, a polyclonal or monoclonal antibody, or an effective part thereof, such as an Fv fragment, which antibody or part thereof may be natural, synthetic or humanised, a peptide hormone, a receptor, a signalling molecule or other protein; a nucleic acid, as defined below, including, but not limited to, an oUgonucleotide or modified oUgonucleotide, an antisense oUgonucleotide or modified antisense oUgonucleotide, cDNA, genomic DNA, an artificial or natural chromosome (e.g. a yeast artificial chromosome) or a part thereof, RNA, including mRNA, tRNA, rRNA or a ribozyme, or a peptide nucleic acid (PNA); a virus or viras- like particles; a nucleotide or ribonucleotide or synthetic analogue thereof, which may be modified or unmodified; an amino acid or analogue thereof, which may be modified or unmodified; a non-peptide (e.g., steroid) hormone; a proteoglycan; a lipid; or a carbohydrate. SmaU molecules, including inorganic and organic chemicals, which bind to and occupy the active site of the polypeptide thereby making the catalytic site inaccessible to substrate such that normal biological activity is prevented, are also included. Examples of small molecules include but are not limited to small peptides or peptide-tike molecules.

Furthermore, as noted above, any molecule may be delivered according to the methods and compositions described here. Such a molecule to be deUvered may take the form of any of the entities as specified above for agents. In particular, the molecules to be deUvered preferably comprise therapeutic compounds such as drugs. However, the methods and compositions described here are also suitable for the deUvery of compounds having a cosmetic function.

ANTISENSE COMPOUNDS

As described above, the antagonist may comprise one or more antisense compounds, including antisense RNA and antisense DNA, which are capable of reducing the level of expression of a protease inhibitor and thereby increasing protease activity, preferably endogenous protease activity. Preferably, the antisense compounds comprise sequences complementary to the mRNA encoding the protease inhibitor.

Preferably, the antisense compounds are otigomeric antisense compounds, particularly oUgonucleotides. The antisense compounds preferably specifically hybridize with one or more nucleic acids encoding the protease inhibitor. As used herein, the term "nucleic acid encoding protease" or encompasses DNA encoding the protease, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an otigomeric compound with its target nucleic acid interferes with the normal ftinction of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generaUy referred to as "antisense". The functions of DNA to be interfered with may include repUcation and transcription. The functions of RNA to be interfered with may include aU vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, spUcing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or faciUtated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of the protease inhibitor.

Antisense constructs are described in detail in US 6,100,090 (Monia et al), and Neckers et al, 1992, Crit Rev Oncog 3(1-2):175-231, the teachings of which document are specifically incorporated by reference.

In the context of the present invention, "modulation" means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. For example, the expression of a gene encoding a protease may be increased, or the expression of a gene encoding an inhibitor of protease inhibitor activity may be decreased, or both. However, preferably, inhibition of expression, in particular, inhibition of expression of protease inhibitor, is the preferred form of modulation of gene expression and mRNA is a preferred target.

DISEASES ASSOCIATED WITH INCREASED ADHESION

In a particular embodiment, the methods and compositions described here are useful for enabling transdermal deUvery of a molecule across the skin of an individual suffering from a disease associated with increased ceU-ceU adhesion of epithelial ceUs, in particular corneocytes. Increased adhesion results in an increased thickness of the stratum comeum, and increased barrier function. Such diseases are referred to in this document as Group 2 diseases. Examples of Group 2 diseases include psoriasis, the ichtyoses, acne vulgaris and keratoses pilaris.

For example, in psoriasis and the ichthyoses, there is a generaUsed thickening of the stratum comeum. In acne there is a locaUsed focal thickening of the stratum comeum at the entrance to the pilosebaceous duct.

Accordingly, the skin of such diseased individuals provides more resistance to drug penetration than normal undiseased skin. The methods and compositions described here enable the epidermal barrier of such diseased sldn to be reduced to allow passage and delivery of a molecule such as a drag. The molecule or drug may be one which is intended to reUeve or treat the condition leading to the increased barrier ftinction, i.e., the Group 2 disease, but need not be.

As noted above, the sldn of a Group 2 patient such as a psoriatic individual, is more impermeable than a normal undiseased skin. Whereas the normal molecular weight cut-off for an undiseased skin is typically in the region of 1000 Daltons a psoriatic sldn is substantiaUy incapable of aUowing passage of molecules above about 800 Da. Therefore, in a particular embodiment, the methods and compositions described here are used to enable delivery of molecules such as drags above about 800 Da in molecular weight across the sldn of a Group 2 individual. Preferably, the molecule to be deUvered comprises ascomycin or tacrolimus.

It wiU be appreciated that, as ceU-ceU adhesion in Group 2 sldn is stronger than in normal sldn, the amount or activity of agent appUed to effect degradation of an adhesion protein or lipid may need to be increased.

PSORIASIS

The compositions and methods of our invention are suitable for assisting or enabling the deUvery of drugs or other agents across psoriatic sldn. They are therefore useful for drug deUvery to psoriatic individuals. Psoriasis manifests itself as inflamed swoUen skin lesions covered with silvery white scale. Characteristics of psoriasis include pus-like bUsters (pustular psoriasis), severe sloughing of the skin (erythrodermic psoriasis), drop-tike dots (guttate psoriasis) and smooth inflamed lesions (inverse psoriasis).

The causes of psoriasis are currently unknown, although it has been estabUshed as an autoimmune sldn disorder with a genetic component. One in three people report a family history of psoriasis, but there is no pattern of inheritance. However, there are many cases in which children with no apparent famUy history of the disease wiU develop psoriasis. Whether a person actuaUy develops psoriasis may depend on "trigger factors" which include systemic infections such as strep throat, injury to the sldn (the Koebner phenomenon), vaccinations, certain medications, and intramuscular injections or oral steroid medications. Once something triggers a person's genetic tendency to develop psoriasis, it is thought that in turn, the immune system triggers the excessive skin ceU reproduction.

Sldn cells are programmed to foUow two possible programs: normal growth or wound healing. In a normal growth pattern, sldn ceUs are created in the basal ceU layer, and then move up through the epidermis to the stratum comeum, the outermost layer of the sldn. This normal process takes about 28 days from cell birth to death. When skin is wounded, a wound healing program (regenerative maturation) is triggered, in which ceUs are produced at a much faster rate, the blood supply increases and localized inflammation occurs. Lesional psoriasis is characterized by ceU growth in the alternate growth program. Sldn cells (Iceratinocytes) switch from the normal growth program to regenerative maturation, ceUs are created and pushed to the surface in as Uttle as 2-4 days, and the sldn cannot shed the ceUs fast enough. The excessive skin ceUs butid up and form elevated, scaly lesions. The white scale ("plaque") that usuaUy covers the lesion is composed of dead skin cells, and the redness of the lesion is caused by increased blood supply to the area of rapidly dividing slcin ceUs.

Psoriasis is a geneticaUy determined disease of the skin characterized by two biological hallmarks. First, there is a profound epidermal hyperproliferation related to accelerated and incomplete differentiation. Second, there is a marked inflammation of both epidermis and dermis with an increased recruitment of T lymphocytes, and in some cases, formation of neutrophU microabcesses. Many pathologic features of psoriasis can be attributed to alterations in the growth and maturation of epidermal keratinocytes, with increased proUferation of epidermal ceUs, occurring within 0.2 mm of the skin's surface. Traditional investigations into the pathogenesis of psoriasis have focused on the increased proUferation and hyperplasia of the epidermis. In normal skin, the time for a ceU to move from the basal layer through the granular layer is 4 to 5 weeks. In psoriatic lesions, the time is decreased sevenfold to tenfold because of a shortened ceU cycle time, an increase in the absolute number of ceUs capable of proUferating, and an increased proportion of ceUs that are actuaUy dividing. The hyperproUferative phenomenon is also expressed, although to a substantiaUy smaUer degree, in the cUnicaUy uninvolved skin of psoriatic individuals.

A common form of psoriasis, psoriasis vulgaris, is characterized by weU-demarcated erythematous plaques covered by thick, silvery scales. A characteristic finding is the isomorphic response (Koebner phenomenon), in which new psoriatic lesions arise at sites of cutaneous trauma.

Lesions are often localized to the extensor surfaces of the extremities, and the nails and scalp are also commonly involved. Much less common forms include guttate psoriasis, a form of the disease that often erupts foUowing streptococcal pharyngitis, and pustular psoriasis, which is characterized by numerous sterile pustules, often 2 to 5 mm in diameter, on the palms and soles or distributed over the body.

Objective methods which are employed for establishing the effect of treatment of psoriasis individuals include the resolution of plaques by visual monitoring and with photography. The visual scoring is done using PASI (Psoriasis Area and Severity Index) score (see Fredericksson, A J, Peterssonn B C Dermatologies 157:238-244 (1978)). Psoriasis affects approximately 2% of the UK population and may be associated with arthritis (in 7-25%) and Crohn's disease. The effect upon the individual self confidence and social activity can be catastrophic. Currently therapies for psoriasis are only suppressive and systemic treatments have significant adverse effects. Psoriasis is a multifactorial disease and genome wide scans have demonstrated a significant linlcage to several chromosomes including 6p21, lq21, 4q, 19p and 17q. In 6p21 the strongest association is with the major histocompatibiUty region (MHC). The MHC S gene (corneodesmosin) is located 160kb telomeric of HLA-C and is expressed in keratinocyte differentiation as a component of the cormeodesmosomes.

DISEASES ASSOCIATED WITH DECREASED CEUL-CELL ADHESION

The methods and compositions described here are also suitable for effecting, assisting or enabling the deUvery of drugs or other agents Compositions described here are useful for enabling transdermal deUvery of a molecule across the skin of an individual suffering from a disease associated with decreased ceU-ceU adhesion of epitheUal ceUs, in particular corneocytes. Such diseases are referred to in this document as Group 1 diseases. Decreased adhesion results in a decreased thickness of the stratum comeum.

Group 1 diseases are characterised by impaired barrier ftinction. It wUl be appreciated that within such diseases, penetration of molecules or drags to be delivered is already greater compared to normal, undiseased, skin. The methods and compositions described here are however useful if such impaired barrier function associated with the Group I disease is insufficient to enable deUvery of a desired amount of drug to the individual. Accordingly, the methods and compositions described here are particularly useful for deUvery of drugs and other therapeutic molecules in for example mUd forms of Group I diseases, such as atopic eczema.

Within the Group 1 diseases, three sub-groups may be identified.

A first subgroup of Group 1 diseases (Subgroup 1.1) is characterised by an impaired barrier which permits more penetration of an irritant, aUergen or other xenobiotic compared to normal undiseased skin. Examples of such diseases affecting the skin include atopic eczema, sebarrhoeic eczema, irritant contact dermatitis, and aUergic contact dermatitis. A second subgroup of Group 1 diseases (Subgroup 1.2) is characterised by an impaired barrier that permits more penetration of bacteria, virus, other micro-organisms or micro-organism products e.g. superantigenic exotoxin, compared to normal undiseased skin. Examples of such diseases include atopic eczema, contact dermatits, impetigo, viral warts, MoUuslum Contagiosum, secondary bacterial and/or viral infections such as meningitis and peptic ulceration caused by or associated with penetration of Helicobacteria pylori. A third subgroup of Group 1 diseases (Subgroup 1.3) is characterised by an impaired barrier that permits more penetration of a carcinogen compared to normal undiseased sldn. Examples of such diseases affecting the skin include melanoma, squamous ceU carcinoma, basal cell carcinoma, cutaneous lymphoma, and other skin cancers. Examples of such diseases affecting the bowel include matignancies of the entire gastrointestinal tract. Lung malignancies are examples of diseases affecting the lung.

DESMOSOMES AND CORNEODESMOSOMES

Desmosomes are symmetrical structures that form disc-shaped interceUular junctions between epitheUal ceUs. In the epidermis the desmosomes mediate the adhesion between keratinocytes. In psoriasis, various ichtyoses and sldn xerosis, the number of comeodesmosomes (desmosomes in upper layers of the epidermis) is increased in the stratum comeum. Immunoelectron microscopy has been used to show the define the interactions within comeodesmosomes between proteins of the extraceUular core domain such as desmoglein and desmocoUins and intraceUular comeodesmosomal proteins including desmoplakins I and JJ, plakoglobin (PG) and plakophilins (PP) (Cowin and Burke, 1996). The importance of comeodesmosomal proteins in epidermal integrity is demonstrated by inherited disorders such as striate subtype of palmoplantar keratoderma caused by desmoplaldn haploinsuffieiency (Armstrong et al, 1999). Mutations in loricrin gene lead to keratoderma of Camisa.

Corneodesmosin is a glycoprotein of comeodesmosomes. Three forms of the corneodesmosin with different weights 33-36 to 40-46 and 52-56 kDa have been isolated from the epidermis (Simon et al, 1997). Mutations within the corneodesmosin/S gene and related genes within the MHC epidermal gene cluster (chromosome 6p21) result in a reduced cohesion between corneocytes.

ANΠBODJES

The methods and compositions described here may employ an antibody against a protease inhibitor, to down-regulate its activity, and to enable degradation of a component of an epidermal barrier. The antibody may be polyclonal or monoclonal, and may be capable of specifically binding to epitopes on the protease inhibitor.

In preparing the antibody, the protease inhibitor, or a fragment of it, may be used as a source of the immunogen. Peptide amino acid sequences isolated from the amino acid sequence of the protease inhibitor may also be used as an immunogen. Thus, for example, antibodies may be generated against the protease inhibitors Antileukoprotease and Elafin Protease Inhibitor 3, by immunisation with the whole proteins, a fragment of the protein, or a peptide derived or synthesised from the protein.

Conveniently, the antibodies may be prepared against a synthetic peptide based on the sequence, or prepared recombinantly by cloning techniques or the natural gene product and/or portions thereof may be isolated and used as the immunogen. Such proteins or peptides may be used to produce antibodies by standard antibody production technology weU known to those sldUed in the art as described generaUy in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988.

For producing polyclonal antibodies a host, such as a rabbit or goat, is immunized with the protein or peptide, generaUy with an adjuvant and, if necessary, coupled to a carrier; antibodies to the protein are coUected from the sera.

For producing monoclonal antibodies, the technique involves hyperimmunization of an appropriate donor, generaUy a mouse, -with the protein or peptide fragment and isolation of splenic antibody producing ceUs. These ceUs are fused to a ceU having immortaUty, such as a myeloma ceU, to provide a fused ceU hybrid which has immortaUty and secretes the required antibody. The ceUs are then cultured, in bulk, and the monoclonal antibodies harvested from the culture media for use.

The antibody may be bound to a soUd support substrate or conjugated with a detectable moiety or be both bound and conjugated as is well known in the art. (For a general discussion of conjugation of fluorescent or enzymatic moieties see Johnstone and Thorpe, Immunochemistry in Practice, BlackweU Scientific Publications, Oxford, 1982.) The binding of antibodies to a solid support substrate is also well known in the art. (see for a general discussion Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Publications, New York, 1988) The detectable moieties contemplated with the present invention may include, but are not limited to, fluorescent, metalUc, enzymatic and radioactive markers such as biotin, gold, ferritin, alkaline phosphatase, β-galactosidase, peroxidase, urease, fluorescein, rhodamine, tritium, ¹⁴ C and iodination.

NUCLEIC ACID

Nucleic acids useful in the methods and compositions described here include antisense molecules which are capable of binding to and down-regulating the level or expression or activity of a protease inhibitor or lipase inhibitor. Furthermore, expression of the adhesion protein may also be downregulated by the use of an appropriate antisense nucleic acid against an adhesion protein, for example, an antisense RNA to corneodesmosin, or a fragment of corneodesmosin.

Protease activity or Upase activity may be upregulated by increasing the expression of protease or Upase. For example, a nucleic acid sequence encoding the protease and/or lipase, suitably linked to a promoter, may be introduced to the skin of an individual; expression of the protease or Upase enables or assists degradation of the appropriate components of the epidermal barrier. As used herein, the term "nucleic acid" is defined to encompass DNA and RNA or both synthetic and natural origin which DNA or RNA may contain modified or unmodified deoxy- or dideoxy- nucleotides or ribonucleotides or analogues thereof. The nucleic acid may exist as single- or double-stranded DNA or RNA, an RNA/DNA heteroduplex or an RNA/DNA copolymer, wherein the term "copolymer" refers to a single nucleic acid strand that comprises both ribonucleotides and deoxyribonucleotides.

The term "synthetic", as used herein, is defined as that which is produced by in vitro chemical or enzymatic synthesis.

Nucleic acid sequences useful according to the methods and compositions described her include those encoding adhesion proteins, as well as proteases, protease inhibitors, etc. They furthermore include sequences encoding nuclear proteins, cytoplasmic proteins, rnitochondrial proteins, secreted proteins, plasmalemma-associated proteins, serum proteins, viral antigens, bacterial antigens, protozoal antigens and parasitic antigens. Also included are sequences encoding proteins, lipoproteins, glycoproteins, phosphoproteins and nucleic acids (e.g., RNAs such as ribozymes or antisense nucleic acids). Ribozymes of the hammerhead class are the smaUest known, and lend themselves both to in vitro synthesis and deUvery to cells (summarised by SuUivan, 1994, J. Invest. Dermatol., 103: 85S-98S; Usman et al., 1996, Curr. Opin. Struct. Biol., 6: 527-533).

One sldUed in the art wiU readtiy recognise that as more proteins and polypeptides become identified, their corresponding genes can be cloned into the gene expression vector(s) of choice, administered to a tissue of a recipient or other vertebrate, and expressed in that tissue.

EXPRESSION VECTORS

We further provide vectors comprising an expression control sequence operatively linked to the nucleic acid sequence, for example, a nucleic acid sequence encoding a protease such as Stratum Comeum Chymotryptic Enzyme and Stratum Comeum Tryptic Enzyme, as weU as host ceUs, selected from suitable eukaryotic and prokaryotic ceUs, which are transformed with these vectors.

It is possible to transform host ceUs, including E. coli, using the appropriate vectors so that they carry recombinant DNA sequences derived from the protease transcript or containing the entire protease transcript in its normal form or a mutated sequence containing point mutations, deletions, insertions, or rearrangements of DNA. Such transformed cells may be used as "factories" to express protease, which (with optional processing) may be administered to an individual to enable transdermal deUvery of a molecule.

Vectors are known or may be constructed by those sktiled in the art and should contain all expression elements necessary to achieve the desired transcription of the sequences. Other beneficial characteristics may also be contained within the vectors such as mechanisms for recovery of the nucleic acids in a different form. Phagemids are a specific example of such beneficial vectors because they may be used either as plasmids or as bacteriophage vectors. Examples of other vectors include viruses such as bacteriophages, baculoviruses and retrovirases, DNA viruses, cosmids, plasmids and other recombination vectors. The vectors may also contain elements for use in either procaryotic or eucaryotic host systems. One of ordinary slciU in the art wiU know which host systems are compatible with a particular vector.

The vectors may be introduced into ceUs or tissues by any one of a variety of known methods within the art. Such methods may be found generaUy described in Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1992), in Ausubel et al., Current Protocols in Molecular Biology, John WUey and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor, Mich. (1995) and GUboa et al (1986) and include, for example, stable or transient transfection, Upofection, electroporation and infection with recombinant viral vectors. Introduction of nucleic acids by infection offers several advantages over the other Usted methods. Higher efficiency may be obtained due to their infectious nature. See also U.S. Pat. Nos. 5,487,992 and 5,464,764. Moreover, viruses are very specialized and typicaUy infect and propagate in specific ceU types. Thus, their natural specificity may be used to target the vectors to specific ceU types in vivo or within a tissue or mixed culture of ceUs. Viral vectors may also be modified with specific receptors or tigands to alter target specificity through receptor mediated events.

Recombinant methods known in the art may also be used to achieve the sense, antisense or triplex inhibition of a target nucleic acid. For example, vectors containing antisense nucleic acids may be employed to express protein or antisense message to reduce the expression of the target nucleic acid and therefore its activity.

A specific example of DNA viral vector for introducing and expressing antisense nucleic acids is the adenovirus derived vector Adenop53TK. This vector expresses a herpes virus thymidine lcinase (TK) gene for either positive or negative selection and an expression cassette for desired recombinant sequences such as antisense sequences. This vector may be used to infect ceUs that have an adenovirus receptor. This vector as weU as others that exhibit similar desired functions may be used to treat a mixed population of ceUs include, for example, an in vitro or ex vivo culture of ceUs, a tissue or a human subject.

Additional features may be added to the vector to ensure its safety and/or enhance its therapeutic efficacy. Such features include, for example, markers that may be used to negatively select against ceUs infected with the recombinant virus. An example of such a negative selection marker is the TK gene described above that confers sensitivity to the anti-viral gancyclovir. Negative selection is therefore a means by which infection may be confroUed because it provides inducible suicide through the addition of antibiotic. Such protection ensures that if, for example, mutations arise that produce altered forms of the viral vector or sequence, cellular transformation wiU not occur. Features that limit expression to particular cell types may also be included. Such features include, for example, promoter and regulatory elements that are specific for the desired ceU type. Recombinant viral vectors are another example of vectors useful for in vivo expression of a desired nucleic acid because they offer advantages such as lateral infection and targeting specificity. Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected ceU produces many progeny virions that bud off and infect neighbouring ceUs. The result is that a large area becomes rapidly infected, most of which was not initiaUy infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny. Viral vectors may also be produced that are unable to spread laterally. This characteristic may be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.

As described above, viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. TypicaUy, viruses infect and propagate in specific ceU types. The targeting specificity of viral vectors utilizes its natural specificity to specificaUy target predetermined cell types and thereby introduce a recombinant gene into the infected ceU. The vector to be used in the methods of the invention wiU depend on desired ceU type to be targeted. For example, a vector specific for epitheUal ceUs may be used for treatment of skin diseases. Likewise, if diseases or pathological conditions of the lung are to be treated, then a viral vector that is specific for lung ceUs and their precursors, preferably for the specific type of lung cell, should be used.

Retroviral vectors may be constructed to function either as infectious particles or to undergo only a single initial round of infection. In the former case, the genome of the vims is modified so that it maintains ati the necessary genes, regulatory sequences and packaging signals to synthesize new viral proteins and RNA. Once these molecules are synthesized, the host ceU packages the RNA into new viral particles which are capable of undergoing further rounds of infection. The vector's genome is also engineered to encode and express the desired recombinant gene. In the case of non-infectious viral vectors, the vector genome is usuaUy mutated to destroy the viral packaging signal that is required to encapsulate the RNA into viral particles. Without such a signal, any particles that are formed wiU not contain a genome and therefore cannot proceed through subsequent rounds of infection. The specific type of vector will depend upon the intended application. The actual vectors are also known and readUy avaUable within the art or may be constructed by one skilled in the art using weU-known methodology.

If viral vectors are used, for example, the procedure may take advantage of their target specificity and consequently, do not have to be administered locaUy at the diseased site. However, local administration may provide a quicker and more effective treatment, adiriinistration may also be performed by, for example, intravenous or subcutaneous injection into the subject. Injection of the viral vectors into a spinal fluid may also be used as a mode of administration, especiaUy in the case of neuro-degenerative diseases. FoUowing injection, the viral vectors wiU circulate until they recognize host ceUs with the appropriate target specificity for infection.

Transfection vehicles such as liposomes may also be used to introduce the non-viral vectors described above into recipient ceUs within the inoculated area. Such transfection vehicles are known by one skiUed within the art.

ADMINISTRATION OF AGENT

The agent capable of reducing an epidermal barrier function may be administered to an individual in a number of ways.

In a preferred embodiment, the agent is appUed to an individual in the form of a cream or emoUient. In a further preferred embodiment, the agent is administered or applied in the form of a patch comprising the agent. It wUl be appreciated that in each of these cases more than one agent may be used, for example, a protease and a lipase, and that therefore the cream or emoUient or patch may comprise more than one agent capable of reducing an epidermal barrier function. Furthermore, it wiU be appreciated that the creams may be used in conjunction with the patches. For example a cream comprising an agent capable of reducing an epidermal barrier function may be appUed to the skin, foUowed by a patch containing the drug (optionaUy together with an agent capable of reducing an epidermal barrier function, which may be the same agent as in the cream, or a different agent).

For some applications, it is preferable to administer a long acting form of agent or composition using formulations known in the arts, such as polymers. The agent can be incorporated into a dermal patch (Junginger, H. E., in Ada Pharmaceutica Nordica 4:117 (1992); Thacharodi et al, in Biomaterials 16:145-148 (1995); Niedner R., in Hautarzt 39:761- 766 (1988)) or a patch according to methods known in the art, to increase the efficiency of delivery of the drug to the areas to be treated.

CREAMS

A cream comprising the agent may be used together with a separate cream comprising the drug or molecule to be deUvered. Thus, for example, a cream may be supptied containing the agent or agents, together with one containing the drag or drugs. In the simplest formulation, a cream comprising an agent capable of reducing an epidermal barrier function, for example a cream comprising a protease capable of degrading corneodesmosin, is suppUed for use with, or together with a cream comprising a drag or other molecule to be deUvered.

Two creams comprising two different agents (for example, a cream comprising a protease and a cream comprising a Upase) may be suppUed with one containing the drag. However, it will be appreciated that the cream containing the agent may be suppUed on its own, for use with pre-existing creams or other formulations comprising the drag to be deUvered, to enhance their deUvery. TypicaUy, the cream containing the agent wiU be administered to the individual and aUowed to act before administration of the cream containing the drug to be delivered.

The creams may be appUed simultaneously or sequentiaUy, or as mixtures with each other. Preferably, a first cream comprising an agent capable of reducing an epidermal barrier function is appUed to the individual before a cream comprising a drag or molecule to be deUvered. Preferably, the agent capable of reducing an epidermal barrier function is appUed for sufficient time to substantiaUy reduce epidermal barrier ftinction before the molecule or drug to be deUvered. Thus, in a particular embodiment, a first cream comprising the agent capable of reducing an epidermal barrier ftinction is administered substantially before a second cream comprising a molecule or drug to be delivered.

The cream or emoUient comprising the agent may further comprise the drug or molecule to be deUvered. Thus, a single cream may be produced comprising one or more agents with one or more drugs.

The term "cream" should be taken to include reference to a Uquid, semi-Uquid, gel or sol preparation comprising the agent, optionaUy with other components, for example, an emoUient, a lotion, an ointment, a tincture, etc. The cream may comprise semi-soUd and gel-Ulce vehicles that include a polymer thickener, water, preservatives, active surfactants or emulsifiers, antioxidants, sun screens, and a solvent or mixed solvent system. U.S. Pat. No. 5,863,560 (Osbome) discusses a number of different combinations which can aid in the exposure of the sldn to a medicament.

The cream may comprise one or more carriers, which may include in any number or combination an aqueous Uquid, an alcohol base liquid, a water soluble gel, a lotion, an ointment, a nonaqueous Uquid base, a mineral oU base, a blend of mineral oil and petrolatum, lanolin, Uposomes, protein carriers such as serum albumin or gelatin, powdered cellulose carmel, and combinations thereof.

The agent may also be incorporated into and administered in the form of a smear, spray, a Uquid absorbed wipe, and combinations thereof.

The cream may comprise a polymer thickener. Polymer thickeners that may be used include those known to one skiUed in the art, such as hydrophiUc and hydroalcohoUc gelling agents frequently used in the cosmetic and pharmaceutical industries. Preferably, the hydrophiUc or hydroalcohoUc gelling agent comprises "CARBOPOL" (B. F. Goodrich, Cleveland, Ohio), "HYPAN" (Kingston Technologies, Dayton, N.J.), "NATROSOL" (Aqualon, Wilmington, Del.), "KLUCEL" (Aqualon, Wilmington, Del.), or "STABILEZE" (ISP Technologies, Wayne, N.J.). Preferably, the geUing agent comprises between about 0.2% to about 4% by weight of the composition. More particularly, the preferred compositional weight percent range for "CARBOPOL" is between about 0.5% to about 2%, while the preferred weight percent range for "NATROSOL" and "KLUCEL" is between about 0.5% to about 4%. The preferred compositional weight percent range for both "HYPAN" and "STABILEZE" is between about 0.5% to about 4%.

"CARBOPOL" is one of numerous cross-linked acrytic acid polymers that are given the general adopted name carbomer. These polymers dissolve in water and form a clear or slightly hazy gel upon neutralization with a caustic material such as sodium hydroxide, potassium hydroxide, triethanolamine, or other amine bases. "KLUCEL" is a ceUulose polymer that is dispersed in water and forms a uniform gel upon complete hydration. Other preferred gelling polymers include hydroxyethylceUulose, ceUulose gum, MVEMA decadiene crosspolymer, PVM7MA copolymer, or a combination thereof.

Preservatives may also be used in this invention and preferably comprise about 0.05% to

0.5%) by weight of the total composition. The use of preservatives assures that if the product is microbiaUy contaminated, the formulation wiU prevent or diminish microorganism growth. Some preservatives useful in this invention include methylparaben, propylparaben, butylparaben, chloroxylenol, sodium benzoate, DMDM Hydantoin, 3-Iodo-2-Propylbutyl carbamate, potassium sorbate, chlorhexidine digluconate, or a combination thereof. Titanium dioxide may be used as a sunscreen to serve as prophylaxis against photosensitization. Alternative sun screens include methyl cinnamate. Moreover, BHA may be used as an antioxidant, as weU as to protect ethoxydiglycol and/or dapsone from discoloration due to oxidation. An alternate antioxidant is BHT.

Pharmaceuticals for use in aU embodiments of the invention include antimicrobial agents, anti-inflammatory agents, antiviral agents, local anesthetic agents, corticosteroids, destructive therapy agents, antifungals, and antiandrogens. In the treatment of acne, active pharmaceuticals that may be used include antimicrobial agents, especiaUy those having anti-inflammatory properties such as dapsone, erythromycin, minocycUne, tetracycline, clindamycin, and other antimicrobials. The preferred weight percentages for the antimicrobials are 0.5% to 10%.

Local anesthetics include tetracaine, tetracaine hydrochloride, Udocaine, Udocaine hydrochloride, dyclonine, dyclonine hydrochloride, dimethisoquin hydrochloride, dibucaine, dibucaine hydrochloride, butambenpicrate, and pramoxine hydrochloride. A preferred concentration for local anesthetics is about 0.025% to 5% by weight of the total composition. Anesthetics such as benzocaine may also be used at a preferred concentration of about 2% to 25% by weight.

Corticosteroids that may be used include betamethasone dipropionate, fluocinolone actinide, betamethasone valerate, triamcinolone actinide, clobetasol propionate, desoximetasone, diflorasone diacetate, amcinonide, flurandrenolide, hydrocortisone valerate, hydrocortisone butyrate, and desonide are recommended at concentrations of about 0.01% to 1.0% by weight. Preferred concentrations for corticosteroids such as hydrocortisone or methylprednisolone acetate are from about 0.2%> to about 5.0%> by weight.

Destructive therapy agents such as saUcyUc acid or lactic acid may also be used. A concentration of about 2% to about 40% by weight is preferred. Cantharidin is preferably utilized in a concentration of about 5% to about 30% by weight. Typical antifungals that may be used in this invention and their preferred weight concentrations include: oxiconazole nitrate (0.1 % to 5.0%)), ciclopirox olamine (0.1% to 5.0%>), ketoconazole (0.1%> to 5.0%>), miconazole nitrate (0.1%) to 5.0%), and butoconazole nitrate (0.1% to 5.0%>). For the topical treatment of seborrheic dermatitis, hirsutism, acne, and alopecia, the active pharmaceutical may include an antiandrogen such as flutamide or finasteride in preferred weight percentages of about 0.5% to 10%.

TypicaUy, treatments using a combination of drugs include antibiotics in combination with local anesthetics such as polymycin B sulfate and neomycin sulfate in combination with tetracaine for topical antibiotic gels to provide prophylaxis against infection and reUef of pain. Another example is the use of minoxidil in combination with a corticosteroid such as betamethasone diproprionate for the treatment of alopecia ereata. The combination of an anti-inflammatory such as cortisone with an antifungal such as ketoconazole for the treatment of tinea infections is also an example.

Methods of producing such creams are known in the art. Indeed, such creams may be considered pharmaceutical compositions, and the detailed description of pharmaceutical compositions wiU be taken to be equaUy applicable to the creams containing the agents.

PATCHES

In a preferred embodiment, agent is administered or applied in the form of a patch comprising the agent or agents. We therefore provide a patch comprising an agent capable of effecting the degradation of a component of the epidermal barrier of an individual. We further provide a patch comprising a molecule to be deUvered to an individual together with an agent capable of effecting the degradation of a component of the epidermal barrier. The use of such a patch for enabling or assisting the deUvery of a molecule to an individual is also provided.

The patch may comprise a cream (comprising an agent capable of reducing an epidermal barrier function, optionaUy together with a molecule or drug to be deUvered) as described above. Thus, we envisage the appUcation of such a cream to a conventional patch or bandage, as sold for example in a pharmacy, and appUcation of such a bandage or patch to the skin

Where more than one agent is employed, a single patch comprising each agent may be used, or multiple patches each comprising a separate agent. An embodiment in which the agent capable of reducing an epidermal barrier function is appUed in one patch, and the drug or molecule to be deUvered is applied in another patch. The or each patch may comprise the compound or molecule to be deUvered, as a mixture with the agent, or in a separate compartment from the agent. Where the term "patch" is used in this document, this term should be taken to include reference to any preferably planar material capable of maintaining contact between a molecule retained therewith (such as an agent capable of degrading the epidermal barrier, or a drug to be deUvered) and the sldn of an individual. Preferably, such a patch is capable of occluding the agent, molecule, etc. The patch may be permeable to other molecules such as gas, such as air, or water or other Uquid, or it may be substantiaUy impermeable to any or aU of these. In a particular embodiment, the patch is occlusive to gas or other molecules.

The term "patch" should be taken to include a simple bandage comprising for example cotton wool taped to an individual's skin, but preferably should be taken to include a simple patch, a Band- Aid, a surgical dressing, plasters, etc.

In a highly preferred embodiment of the invention, the patch comprises a transdermal patch. Transdermal patches are known in the art.

Contact between the molecule and the sldn may be maintained by the bandage comprising an adhesive, as in the case of a Band- Aid or by any other means, such as an elastic strap. The bandage may comprise a compartment made of for example an absorbent material to retain the molecule to be administered. The compartment is generally centrally positioned within the bandage, and may suitably be made of cotton wool or gauze.

Bandages come in a variety of shapes and sizes. They are used to cover and protect wounds such as cuts, abrasions, punctures, and other forms of wounds inflicted on human tissue such as the skin of the arms, legs, hands, and other parts of the human body. TypicaUy, a bandage comprises a flexible backing with an adhesive surface for attaching the bandage to the sldn. The bandage may have a patch of gauze or absorbent material disposed at the centre of the adhesive surface. Such adhesive bandages are weU-known and staple items in a first-aid kit or medicine cabinet. The agent may be appUed to a bandage either directly or in one of the other carriers. The bandages may be sold damp or dry, wherein the agent is in a lyophilized form on the bandage. The bandage may be a time-release bandage.

A description of a specific example of a bandage foUows. A adhesive bandage includes a flexible backing, such as a strip of plastic material or woven or non-woven fabric or the like capable of receiving and retaining adhesive of the type normaUy employed for adhesive bandages. The backing may be made of a material that is breathable, gas-permeable, and/or hydrophobic. The backing may also contain one or more designs and colours and could also be made to look translucent, transparent, or opaque. These materials, their dimensions, modifications, etc., are weU known in the industry. The backing may be in the shape of a strip having end portions extending from a central portion. A skin-contacting or bottom surface of the end portions is coated with an adhesive for attaching the bandage to the skin. GeneraUy disposed at the central portion of the bottom surface of the flexible backing may be an absorbent pad, made of gauze, cotton, or some other absorbent material, that is meant for direct contact with the area of the skin where administration of the agent and/or molecule/compound is to be deUvered. This area is therefore void of adhesive on the skin-facing surface. The agent is preferably held in the absorbent pad, optionaUy together with the compound, molecule or drug to be delivered.

Thus, in one embodiment, the bandage comprises a mixture of the or each agent and the compound or molecule to be delivered.

In another embodiment, the bandage comprises the compound or molecule to be deUvered in a separate compartment from the or each agent. The separate compartments may comprise portions of an absorbent pad, or more than one absorbent pad. In one embodiment, the absorbent pad may comprise a first portion comprising the agent capable of reducing an epidermal barrier function, and a second portion comprising the molecule to be deUvered. Preferably, the first and second portion are such that the agent and the molecule/drag are substantiaUy separated, i.e., there is limited or preferably substantiaUy no diffusion or passage of molecules between the first portion and the second portion. This may be achieved by the presence of a physical barrier, such as a non-permeable membrane between the first and second portion.

The first portion and/or the second portion may be in the form of layers, which are disposed in the plane of the bandage. For example, the second portion may be disposed on the bandage so that it is sandwiched between the first portion and the backing of the bandage, i.e., disposed as follows: skin-first portion-second portion-backing. This is advantageous for deUvery as the agent capable of reducing an epidermal barrier function is contacted with the sldn before the molecule or drug to be deUvered; the epidermal barrier is thus reduced to faciUtate passage of the drug or molecule.

Modifications of the above embodiment, in which the agent capable of reducing an epidermal barrier function and the drug or molecule are comprised in separate absorbent pads, instead of portions of a single pad, are possible. Thus, in another embodiment, two absorbent pads are disposed in the bottom surface of the flexible backing. A first absorbent pad comprises the agent capable of reducing an epidermal barrier function, while a second absorbent pad comprises the molecule to be deUvered.

PHARMACEUTICAL COMPOSITIONS

The invention also relates to pharmaceutical compositions comprising one or more agents capable of reducing an epidermal barrier ftinction. Such agents include agents capable of degrading a proteinaceous component of the epidermal barrier, and/or agents capable of degrading a lipidic component of the epidermal barrier, as described above

While it is possible for the composition comprising the agent or agents to be administered alone, it is preferable to formulate the active ingredient(s) as a pharmaceutical formulation. The composition may include the agent(s), a structuraUy related compound, or an acidic salt thereof. The pharmaceutical formulations described here comprise an effective amount of agent together with one or more pharmaceuticaUy-acceptable carriers. An "effective amounf ' of an agent is the amount sufficient to enable or assist transdermal deUvery of a relevant molecule, compound or drag, as the case may be. The drag, etc is one which is suitable for administration to an individual suffering from a disease. The effective amount of the agent may therefore vary between individuals, and may need to be taUored for different individuals.

Factors influencing the effective amount of the agent include in particular the degree of permeabiUty of the individual's skin to the drug, etc. Thus, where an individual is already suffering from a Group I disease as described above (whether or not this is a disease which is to be treated by administration of the drug, etc), his sldn may exhibit decreased ceU-ceU adhesion and hence greater permeabiUty (lower degree of barrier function). In such a case, the effective amount of agent capable of reducing an epidermal barrier ftinction may be lower than for "normal" skin. Conversely, an individual suffering from a Group II disease wiU have enhanced barrier function, and the amount of agent capable of reducing an epidermal barrier ftinction wtil need to be increased for effectiveness. A guide to an effective amount of agent is the amount needed for permeabiUty of the relevant drag, etc, in a "normal" (undiseased) skin; this amount may be increased or decreased according to the disease state of the individual.

Another factor which is important for determining the effective amount of agent capable of reducing an epidermal barrier function wiU be the molecular weight of the drug or molecule to be deUvered. A higher molecular weight may mean that the effective amount of agent wiU be higher than normal, and vice versa.

Other factors which may play a part include the particular disease or syndrome to be treated or aUeviated, as weU as other factors including the age and weight of the individual, how advanced the disease etc state is, the general health of the individual, the severity of the symptoms, and whether the agent is being administered alone or in combination with other therapies. Suitable pharmaceuticaUy acceptable carriers are weU known in the art and vary with the desired form and mode of administration of the pharmaceutical formulation. For example, they can include effluents or excipients such as fiUers, binders, wetting agents, disintegrators, surface- active agents, lubricants and the like. TypicaUy, the carrier is a soUd, a Uquid or a vaporizable carrier, or a combination thereof. Each carrier should be "acceptable" in the sense of being compatible with the other ingredients in the formulation and not injurious to the individual. The carrier should be biologicaUy acceptable without eUciting an adverse reaction (e.g. immune response) when administered to the host.

Pharmaceutical compositions include those suitable for topical and oral administration, with topical formulations being preferred where the tissue affected is primarily the sldn or epidermis (for example, psoriasis and other epidermal diseases). The topical formulations include those pharmaceutical forms in which the composition is applied extemaUy by direct contact with the skin surface to be treated. A conventional pharmaceutical form for topical application includes a soak, an ointment, a cream, a lotion, a paste, a gel, a stick, a spray, an aerosol, a bath oil, a solution and the Uke. Topical therapy is deUvered by various vehicles, the choice of vehicle can be important and generally is related to whether an acute or chronic disease is to be treated. As an example, an acute skin proUferation disease generally is treated with aqueous drying preparations, whereas chronic skin proliferation disease is treated with hydrating preparations. Soaks are the easiest method of drying acute moist eruptions. Lotions (powder in water suspension) and solutions (medications dissolved in a solvent) are ideal for hairy and intertriginous areas. Ointments or water-in-oU emulsions, are the most effective hydrating agents, appropriate for dry scaly eruptions, but are greasy and depending upon the site of the lesion sometimes undesirable. As appropriate, they can be appUed in combination with a bandage, particularly when it is desirable to increase penetration of the agent composition into a lesion. Creams or oU- in-water emulsions and gels are absorbable and are the most cosmeticaUy acceptable to the individual. (Guzzo et al, in Goodman & Gilman's Pharmacological Basis of Therapeutics, 9th Ed., p. 1593-15950 (1996)). Cream formulations generaUy include components such as petroleum, lanolin, polyethylene glycols, mineral oU, glycerin, isopropyl palmitate, glyceryl stearate, cetearyl alcohol, tocopheryl acetate, isopropyl myristate, lanolin alcohol, simethicone, carbomen, methylchlorisothiazolinone, methylisothiazolinone, cyclomethicone and hydroxypropyl methylceUulose, as weU as mixtures thereof.

Other formulations for topical application include shampoos, soaps, shake lotions, and the Uke, particularly those formulated to leave a residue on the underlying sldn, such as the scalp (Arndt et al, in Dermatology In General Medicine 2:2838 (1993)).

In general, the concentration of the agent composition in the topical formulation is in an amount of about 0.5 to 50% by weight of the composition, preferably about 1 to 30%, more preferably about 2-20%), and most preferably about 5-10%. The concentration used can be in the upper portion of the range initially, as treatment continues, the concentration can be lowered or the appUcation of the formulation may be less frequent. Topical applications are often appUed twice daily. However, once-daUy application of a larger dose or more frequent appUcations of a smaller dose may be effective. The stratum comeum may act as a reservoir and allow gradual penetration of a drag into the viable sldn layers over a prolonged period of time.

For some appUcations, it is preferable to administer a long acting form of agent or composition using formulations Icnown in the art, such as polymers.

The pharmaceutical composition may further include, together with the agent capable of reducing an epidermal barrier function, a Icnown skin penetration enhancer. Such sldn penetration enhancers include dimethyl sulfoxide (U.S. Pat. No. 3,711,602); oleic acid, 1,2-butanediol surfactant (Cooper, J. Pharm. Sci., 73:1153-1156 (1984)); a combination of ethanol and oleic acid or oleyl alcohol (EP 267,617), 2-ethyl-l,3-hexanediol (WO 87/03490); decyl methyl sulphoxide and Azone (Hadgraft, Eur. J. Drug. Metab. Pharmacokinet, 21 : 165-173 (1996)); alcohols, sulphoxides, fatty acids, esters, Azone, pyrrolidones, urea and polyoles (Kalbitz et al, Pharmazie, 51 :619-637 (1996)); terpenes such as 1,8-cineole, menthone, limonene and neroUdol (Yamane, J. Pharmacy & Pharmocology, 47:978-989 (1995)); Azone and Transcutol (Harrison et al, Pharmaceutical Res. 13:542-546 (1996)); and oleic acid, polyethylene glycol and propylene glycol (Singh et al, Pharmazie, 51:741-744 (1996)). The pharmaceutical composition may also include the drug to be deUvered, in combination with the agent capable of reducing an epidermal barrier function and optionaUy a known skin penetration enhancer. Alternatively, the agent capable of reducing an epidermal barrier function may be suppUed in the form of a separate pharmaceutical composition from the drug. Such a kit may further include instructions for administering the agent and the drug, according to the methods already described, for efficient penetration of the drug.

OptionaUy, the topical formulations of this invention can have additional excipients for example; preservatives such as methylparaben, benzyl alcohol, sorbic acid or quaternary ammonium compound; stabilizers such as EDTA, antioxidants such as butylated hydroxytoluene or butylated hydroxanisole, and buffers such as citrate and phosphate.

EXAMPLES

Example 1. In vitro Assay for Penetration

The penetration of radiolabeUed molecules may be used to measure penetration through the composites. However this system is not quantitative and a Franz chamber method is preferable.

Use of Franz chamber and cadaver system alca Franz chamber penetration system

The Franz chamber is a standard method used to measure the penetration of drags and other xenobiotics through skin. This system may be modified by tape stripping the skin to reduce the integrity of the epidermal barrier.

In our experiments we take normal sldn and measure the penetration of a group of radiolabeUed drugs with different molecular weights. In dupUcate Franz chambers the normal skin are treated with either SCCE or SCTE for two hours. The penetration of the same group of drags is measured in these pre-treated Franz chambers and compared with the untreated control skin. In the Franz chambers containing skin pre-tieated with either SSCE or SCTE, drags of a higher molecular weight are able to pass into the receiving chamber.

REFERENCES

Chapman and Walsh (1991). Desmosomes, comeosomes and desquamation. An ultrastractural study of adult pig epidermis. Arch Dermatol Res 282; 304-310.

Coleman, R., Trembath, R.C., Harper, J.I. (1997) Genetic studies of atopic and atopic deπnatitis. Rr. J. Dermatol. 136: 1-5

Cork, M.J. (1997) The importance of skin barrier function. J. Derm Treatment. 8, S7- S13.

Daser A, Koez K, Batjer N, Jung M, Ruschendorf F, Goltz M, EUerbrok H, Renz H

Walter J, Paulsen M. (2000). Genetics of atopy in a mouse model: polymorphism of the IL-5 receptor α chain. Immunogenetics 51: 632-638.

Egelrud, T. (1993). Purification ang preliminary characterization of stratum corneoum chymotryptic enzyme: a proteinase that may be involved in desquamation. 101; 200-204.

Egelrud, T. (2000) Desquamation in the stratum comeum. Ada. Derm. Venerol. Supp.

208, 44-45.

Elcholm, E. and Egelrud, T. (1998) The expression of stratum comeum chymotrytic enzyme in human anagen hair follicles: further evidence for its involvement in desquamation-Uke processes. Brit. J. Derm. 139, 585-590.

EUas, P.M. (1983) Epidermal Upids, barrier function and desquamation. J.Invest.

Dermatol. 80,(6) 44-49. Guerrin, M., Simon, M., Montezin, M., Haftek, M., Vincent, C. and Serre, G. (1998) Expression cloning of human corneodesmosin proves its identity with product of the S gene and aUows improved characterisation of its processing during keratinocyte differentiation. J. Biol. Chem., 273 22640-22647.

M. Guerrin, C. Vincent, M. Simon, R. Tazi Ahnini, M. Fort, and G. Serre. (2000).

Identification of six novel polymoφhisms in the human corneodesmosin gene. Tissue Antigens, in press.

Jenisch S, Koch S, Henseler T, Nair RP, Elder JT, Watts CE, Westphal E, Voorhees JJ, Christophers E, Kronlce M. (1999). Corneodesmosin gene polymoφhism demonstrates strong Unlcage disequilibrium with HLA and association with psoriasis. Tissue Antigens, 54: 439-449.

Landmann, L.(1998) The epidermal permeability barrier. A nat. Embryol. (Berl.). 172, 1-13.

Li, A., Hopkin, M. (1997) Atopic phenotype in subjects with variants of the β subunit of the high affinity IgE receptor. Thor 52: 654-655

Menton and Eisen (1971). Structure and organisation of mammatian stratum comeum. J

Utrastruct. Res 35: 247-264

Muφhy R, Wiltiams HC, Duff GW, Cork MJ. Total and specific IGE in atopic dermatitis. Some considerations for linlcage analysis (submitted, 2000 ).

North AJ, Bardsley WG, Hyam J, Bornslaeger EA, Cordingley HC, Trinnaman B,

Halzfeld M, Green KJ, Magee Al, Garrod DR. Molecular map of the desmosomal plaque. Journal of Cell Science 112, 4325-4336 (1999). Serre, G., Mils, V., Hafϊtek, M., Vincent, C, Croute, F., Reano, A., Ouhayoun,J-P., Bettinger, S., Soleilhavoup, J-P. (1991) J.Invest. Dermatol. 97: 1061-1072

Shirakawa, T., Li, A., Dubowitz, M., et al., (1994) Association between atopic and variants of the β subunit of the high afinity immunoglobuUn E receptor. Nat. Genet. 7: 125-130

Simon, M., Montezin, M., Guerrin, M., Durieux, J.J. and Serre, G. (1997)

Characterisation and purification of human corneodesmosin, an epidermal basic glycoprotein associated with corneocyte-specific modified desmosomes. J. Biol. Chem., 272, 31770-31776.

Sundberg JP, France M, Boggess D, Sυndberg BA, Jenson AB, Beamer WG, Shultz LD. (1997). Development of and progression of psoriasiform dermatitis and systemic lesions in the flaky skin (fsn) mouse mutant. Pathobiology, 65: 271-286.

Wiltiams HC, Burney PG, Hay RJ et al. The LK working party's diagnostic criteria for atopic dermatitis I: derivation of a minimum set of discrimination for atopic dermatitis. Br J Dermatol 1994; 131:383-396.

Zhou, Y. and Chaplin, D.D. (1993) Identification in the HLA class I region of a gene expressed late in keratinocyte differentiation. Proc. Natl. Acad. Sci. USA, 90,9470-9473.

Each of the appUcations and patents mentioned above, and each document cited or referenced in each of the foregoing appUcations and patents, including during the prosecution of each of the foregoing appUcations and patents ("appUcation cited documents") and any manufacturer's instructions or catalogues for any products cited or mentioned in each of the foregoing appUcations and patents and in any of the appUcation cited documents, are hereby incorporated herein by reference. Furthermore, aU documents cited in this text, and aU documents cited or referenced in documents cited in this text, and any manufacturer's instructions or catalogues for any products cited or mentioned in this text, are hereby incorporated herein by reference. Narious modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the mvention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the invention.

The invention includes within its claims a method to allow the admimstration of vaccines via the transcutaneous route. Vaccines have previously only been administered by injection below the skin eg. subdermal subcutaneous or intramuscular. This invention transiently breaks down the skin barrier which permits the penetration of the vaccine into the systematic circulation. This then allows immunity to develop to the particular disease.

A further claim of the invention is a method to allow the admimstration of allergens via the transcutaneous route, in order to desensitise the individual to the allergens. Allergens have previously been administered via the subcutaneous route. This carries a significant risk of inducing an anaphylactic response. This invention transiently breaks down the skin barrier which permits the penetration of the allergen.

ANNEX A: ADHESION PROTEINS, PROTEASES AND PROTEASE INHIBITORS

Tables Al, A2 and A2 detail adhesion proteins, proteases and protease inhibitors, with their GenBank accession numbers. Each of these adhesion proteins, proteases and protease inhibitors may be used in the methods and compositions described in this document.

Thus, where the term "adhesion protein" is used in this document, this should be talcen to include reference to each, a combination or aU of the entities in Table Al. Sirmlarly, where the term "protease" is used in this document, this should be talcen to include each, a combination or aU of the entities in Table A2. Where the term "protease inhibitor" is used in this document, this should be talcen to include each, a combination or all of the entities in Table A2.

TABLE Al : ADHESION PROTEINS

Table Al. Adhesion proteins suitable for use in the methods and compositions described here

TABLE A2: PROTEASES

Table A2. Proteases suitable for use in the methods and compositions described here

TABLEA3: PROTEASE INHIBITORS

Table A3. Protease inhibitors suitable for use in the methods and compositions described here

Claims

1. Use of an agent capable of effecting the degradation of a component of the epidermal barrier of an individual to enable or assist transdermal deUveryof a molecule to the individual.

2. A method of deUvering a molecule to an individual, the method comprising:

(a) providing an agent capable of effecting the degradation of a component of the epidermal barrier of the individual;

(b) administering the agent to the individual; and

(c) administering the molecule to the individual.

3. A method of treating a disease in an individual, the method comprising:

(a) administration of an agent capable of effecting the degradation of a component of the epidermal barrier of the individual, foUowed by

(b) administration of a drug indicated for the disease.

4. A composition comprising a molecule to be deUvered through an epidermal barrier of an individual together with an agent capable of effecting the degradation of a component of the epidermal barrier of the individual.

5. An agent capable of effecting the degradation of a component of the epidermal barrier of an individual for use in a method of treatment of a disease in an individual, in which the treatment comprises administration of the agent to enable or assist transdermal deUvery of a drag to the individual.

6. Use of an agent capable of effecting the degradation of a component of the epidermal barrier of an individual in the preparation of a pharmaceutical composition for the treatment of a disease in an individual, in which the treatment comprises administration of the agent or the pharmaceutical composition to enable or assist transdermal deUvery of a drug to the individual.

7. A ldt comprising a compound to be deUvered through an epidermal barrier of an individual, an agent capable of effecting the degradation of a component of the epidermal barrier of the individual, and instructions for using the agent to effect delivery of the compound.

8. A method of preparing a composition capable of enhanced transdermal delivery of a compound, the method comprising the steps of providing a composition comprising a compound to be deUvered, and adding an agent capable of effecting the degradation of a component of the epidermal barrier of an individual to the composition.

9. A method, use, composition, agent or ldt according to any preceding claim, in which a compound or molecule to be deUvered is administered simultaneously, sequentiaUy or as a mixture with the agent.

10. A method, use, composition, agent or kit according to any preceding claim, in which the agent is administered to the individual, foUowed by administration of the compound or molecule to the individual.

11. A method, use, composition, agent or ldt according to any preceding claim, in which the agent comprises an agent capable of effecting the degradation of a proteinaceous component of the epidermal barrier.

12. A method, use, composition, agent or ldt according to Claim 11, in which the proteinaceous component is comprised in the stratum comeum.

13. A method, use, composition, agent or kit according to Claim 11 or 12, in which the proteinaceous component comprises an adhesion protein, preferably a desmosomal adhesion protein.

14. A method, use, composition, agent or kit according to Claim 13, in which the adhesion protein comprises corneodesmosin.

15. A method, use, composition, agent or kit according to any of Claims 11 to 14, in which the agent comprises a protease.

16. A method, use, composition, agent or ldt according to Claim 15, in which the protease is selected from the group consisting of: Stratum Comeum Chymotryptic Enzyme (SCC; kallikrein 7, KLK7 ), Stratum Comeum Tryptic Enzyme (SCTE; kalUlcrein 5, KLK5), aminopeptidase M, carboxypeptidase P, carboxypeptidase Y, caspase 1,4,5, caspase 2,3,7, caspase 6,8,9, chymotrypsin, Factor Xa, pepsin, TEV, thrombin and trypsin.

17. A method, use, composition, agent or kit according to any of Claims 11 to 14, in which the agent comprises an antagonist of antileukoprotease or an antagonist of elafin protease inhibitor 3 (PI3 or SKALP).

18. A method, use, composition, agent or kit according to any of Claims 1 to 10, in which the agent is capable of effecting the degradation of a Upidic component of the epidermal barrier.

19. A method, use, composition, agent or kit according to Claim 18, in which the Upidic component is comprised in the Upid lameUae.

20. A method, use, composition, agent or kit according to Claim 18 or 19, in which the agent comprises a lipase.

21. A method according to Claim 1 or 3, a use according to Claim 2, or a method or use according to any of 8 to 20 as dependent thereon, in which the method or use further comprises the step of administering a second agent capable of effecting the degradation of a second component of the epidermal barrier of the individual.

22. A composition according to Claim 4 or any of 8 to 20 as dependent thereon, in which the composition further comprises a second agent capable of effecting the degradation of a second component of the epidermal barrier of the individual.

23. A method, use or composition according to Claim 21 or 22, in which the first component comprises a proteinaceous component and the second component comprises a lipidic component.

24. A method, use or composition according to Claim 21 , 22 or 23, in which the first agent comprises a protease and the second component comprises a lipase.

25. A method, use or composition according to Claim 22, 23, or 24, in which the agent capable of effecting the degradation of a Upidic component is administered before the agent capable of effecting the degradation of the proteinaceous component.

26. A method, use or composition according to Claim 25 , in which degradation of the Upidic component of the epidermal barrier enables the agent capable of effecting the degradation of the proteinaceous component to contact the proteinaceous component of the epidermal barrier.

27. A method, use, composition, agent or kit according to any of Claims 1 to 26, in which the compound or molecule to be delivered is capable of crossing the epidermal barrier of normal human skin but not substantiaUy capable of crossing the epidermal barrier of psoriatic skin.

28. A method, use, composition, agent or ldt according to any preceding claim, in which the compound or molecule comprises ascomycin or tacrolimus.

29. A method, use, composition, agent or kit according to any preceding claim, in which the compound or molecule has a molecular weight of more than about 1000 Daltons.

30. A method, use composition, agent or kit according to any of Claims 1 to 28, in which the compound or molecule has a molecular weight of more than about 800 Daltons.

31. A method, use, composition, agent or ldt according to any preceding claim, in which the or each agent is administered or applied in the form of a bandage comprising the or each agent or both agents.

32. A method, use, composition, agent or ldt according to Claim 31 , in which the bandage further comprises the compound or molecule to be delivered in a separate compartment from the or each agent.

33. A method, use, composition, agent or kit according to Claim 31, in which the bandage comprises a mixture of the or each agent and the compound or molecule to be delivered.

34. A method, use, composition, agent or ldt according to any preceding claim, in which the or each agent is provided in the form of a gel, cream, lotion, ointment or tincture.

35. A method, use, composition, agent or ldt according to Claim 34, claim, in which the cream further comprises the molecule or compound to be delivered.

36. A bandage comprising an agent capable of effecting the degradation of a component of the epidermal barrier of an individual.

37. A bandage according to Claim 36, in which the agent or component is as defined in any of Claims 8 to 35.

38. A bandage comprising a composition according to Claim 4 or any of Claims 9 to 35 as dependent thereon.

39. A bandage comprising a molecule to be deUvered to an individual together with an agent capable of effecting the degradation of a component of the epidermal barrier.

40. A bandage according to Claim 39, in which the molecule and the agent are in separate compartments.

41. A bandage according to Claim 39, comprising a mixture of an agent capable of effecting the degradation of a component of the epidermal barrier together with a molecule to be deUvered.

42. Use of a bandage according to any of Claims 36 to 41 , for enabling or assisting the deUvery of a molecule to an individual.

43. The combined use of an agent capable of effecting the degradation of a proteinaceous component of the epidermal barrier together with an agent capable of effecting the degradation of a lipidic component of the epidermal barrier for assisting the transdermal transport of a therapeutic molecule.

44. The use of an agent capable of reducing an epidermal barrier function to enable or assist transdermal deUvery of a molecule.

45. Use of a protease or a lipase to enable or assist the delivery of a molecule through an epidermal barrier of an individual.

46. A method, use, composition, agent or kit according to any of claims 1 to 26 in which the compound or molecule to be delivered is not normally capable of crossing the epidermal barrier of normal human sldn.