GB2473432A

GB2473432A - Improvements in or relating to exfoliation

Info

Publication number: GB2473432A
Application number: GB0915732A
Authority: GB
Inventors: Anton Alexandroff
Original assignee: Individual
Current assignee: Individual
Priority date: 2009-09-09
Filing date: 2009-09-09
Publication date: 2011-03-16
Also published as: WO2011030148A2; WO2011030148A3; GB0915732D0

Abstract

A formulation for use in the exfoliation of a mammalian subject, the formulation comprising:(i) one or more active agents selected from the group, (a), consisting of: Staphylococcus aureus exfoliative toxins A, B or D, or active derivatives, analogues or truncated portions thereof; and/or (ii) one or more active agents selected from the group, (b), consisting of: a binding partner which binds to desmoglein 1, desmoglein 2, desmoglein 3 or desmoglein 4; and/or (iii) one or more active agents selected from the group, (c), consisting of: binding partner which binds to laminin 5, laminin 6, integrin beta 4 subunit, collagen VII, collagen XVII, or Bullous Pemphigoid antigen 230 proteins, and (iv) a pharmaceutically or cosmetically acceptable carrier, diluent or excipient, said active agent(s) being present in the formulation at a known concentration sufficient such that the formulation causes appropriate exfoliation of the subject's skin within a contact time in the range of 2 minutes to 1 hour.

Description

Improvements in or Relating to Exfoliation

Field of the Invention

The present invention relates to a composition for peeling (exfoliation) of mammalian skin. More particularly, it relates to a composition for carrying out peeling by applying an active substance or a mixture of active substances to the skin, and to a method of treatment, especially a cosmetic treatment, of the skin.

Background of the Invention

Peeling refers to resurfacing of skin with the aim to rejuvenate the skin (e.g. by removing wrinkles and improving skin texture), improve the cosmetic appearance of the skin (e.g. by reducing skin discoloration or removing sun-damaged skin such as actinic keratoses andlor improving scar appearances, including scars due to acne, varicella and other inflammatory processes).

Methods of peeling can be generally classified into three groups: chemical peeling, dermabrasion and laser peeling. Chemical peeling is a technique wherein active chemicals such as phenol are applied to the skin surface such that the skin is chemically burned and layers of the skin (i.e. the epidermis and, optionally, dermal layers of the skin) are destroyed, thus causing peeling of the skin. During the process of peeling, a chemical burn produces inflammation. Such inflammation can produce undesirable side effects. Following the destruction of the skin layers, skin regeneration and re-epithelization occur.

Depending on the depth of skin tissue destruction, skin peeling can be generally classified into superficial, medium and deep peeling. The depth of skin peeling depends on several factors including: the choice and concentration of the chemicals used; the duration of the application of chemicals to the skin; and the thickness and other intrinsic characteristics of the skin of an individual. If inadequate superficial chemical peeling is carried out in order to minimize inflammation, this may result in inadequate cosmetic or medical effects. However, excessive deep peeling, e.g. due to one or more of: an excessive concentration of the chemical used; an excessive duration of application of the chemical to the skin; or an inexperienced operator, may lead to excessive inflammation and may result in medical and cosmetic complications including delayed healing, infection (e.g. bacterial, viral or fungal infections), scar formation (atrophic or hypertrophic scars), long lasting dyschromias (e.g. hyperpigmentation or hypopigmentation) (ref. 1). The risk of undesirable dyschromias is especially high in patients with darker, skin e.g. Asian or black skin.

In the process of dermabrasion, fast rotating wire brushes or diamond fraises are used to surgically abrade the skin and remove the layers of the epidermis and, optionally, some of the dermis. Dermabrasion usually requires a general anaesthetic and causes inflammation. If inadequate superficial dennabrasion is carried out to minimize the inflammation, it may produce inadequate cosmetic or medical effects due to under-treatment. In contrast, excessive deep dermabrasion may lead to excessive inflammation which may result in medical and cosmetic complications. It is known that up to 50% of patients undergoing dermabrasion may experience hypopigmentation. Dermabrasion may also be accompanied by milia (i.e. keratin filled cysts) formation (ref 2). The rate of viral infection associated with dermabrasion is sufficiently high to require the use of antiviral prophylaxis when dermabrasion is carried out (ref. 3).

In the process of laser peeling, the skin surface is irradiated with a laser beam which bums off layers of skin. Laser peeling is also accompanied by inflammation.

Furthermore, if inadequate or superficial laser peeling is carried out in order to minimize inflammation, it may produce inadequate cosmetic or medical effects due to under-treatment. In contrast, excessive deep peeling may lead to excessive inflammation which may result in medical and cosmetic complications. The rate of hyperpigmentation and hypopigmentation associated with laser skin peeling can be as high as 36% and 16%, respectively (ref. 2). Laser peeling may also be accompanied by acne and dermatitis (eczema).

In order to minimize complications, any type of skin peeling which produces inflammation will require careful patient selection, excellent surgical technique and fastidious postoperative care (ref. 2). Deeper levels of skin peeling cause more inflammation and are more likely to cause complications. Traditional methods of skin peeling (i.e. chemical, laser peeling and dermabrasion) are very operator and patient dependent, and thus it is more difficult to control the exact depth of skin peeling with the use of these methods. If these procedures are carried out in an inadequate, superficial manner, they are likely to be inefficient and lead to under-treatment.

However, if these procedures peel skin at excessive deep levels, they are likely to be associated with complications. Therefore, there is a need to establish an effective method of skin peeling at a precise level, in order to minimize the rate of complication and, at the same time, maximize the efficiency of skin peeling.

An object of the present invention is to provide an effective alternative skin peeling treatment and composition.

Summary of the Invention

In a first aspect, the invention provides a formulation for use in the exfoliation of a mammalian subject, the formulation comprising: (i) one or more active agents selected from the group, (a), consisting of: Staphylococcus aureus exfoliative toxins A, B or D, or active derivatives, analogues or truncated portions thereof; andlor (ii) one or more active agents selected from the group, (b), consisting of: a binding partner which binds to desmoglein 1, desmoglein 2, desmoglein 3 or desmoglein 4; and/or (iii) one or more active agents selected from the group, (c), consisting of: a binding partner which binds to laminin 5, laminin 6, integrin beta 4 subunit, collagen VII, collagen XVII, or Bullous Pemphigoid antigen 230 proteins; said active agent(s) being present in the formulation at a known concentration sufficient such that the formulation causes appropriate exfoliation of the subject's skin within a contact time in the range of 2 minutes to 1 hour; and (iv) a cosmetically or therapeutically acceptable carrier, diluent or excipient.

In a second aspect, the invention provides a method of exfoliative treatment of a mammalian subject, especially a cosmetic treatment, comprising the step of: applying to one or more selected areas of the subject's skin, in need of such exfoliative treatment, an exfoliative formulation, said formulation being in accordance with the first aspect of the invention defined above; and leaving said formulation in contact with the selected area(s) of the subject's skin for an appropriate duration of time to achieve the desired degree of exfoliation.

In a third aspect, the invention provides a method of making an exfoliative formulation for use on a mammalian subject, the method comprising the step of mixing one or more active agents selected from the groups of substances (a) and/or (b) and/or (c) as aforesaid, with a cosmetically or therapeutically acceptable carrier, diluent or excipient.

In a fourth aspect the invention provides for use of one or more active agents selected from the groups of substances (a) and/or (b) and/or (c) as aforesaid for the manufacture of a therapeutic skin exfoliative formulation.

The subject is preferably human.

In one embodiment, the formulation of the invention comprises at least one active agent selected from group (a) and at least one active agent selected from group (b). In another embodiment the formulation comprises at least one active agent selected from group (a) and at least one active agent selected from group (c). In a further embodiment, the formulation comprises at least one active agent selected from group (b) and one active agent selected from group (c). In yet a further embodiment, the formulation of the invention comprises at least one active agent selected from each of groups (a), (b) and (c).

In one embodiment, the formulation comprises both a type A and a type B exfoliative toxin. In one embodiment the formulation comprises both a type A and a type D exfoliative toxin. In another embodiment the formulation comprises both a type B and a type D exfoliative toxin. In yet another embodiment the formulation comprises a type A, a type B and a type D exfoliative toxin.

By way of explanation, it is known that Staphylococcus aureus exfoliative toxins (including exfoliative toxins A, B, D) can cause blistering of the skin in humans and/or animals during disease, including such diseases as bullous impetigo and staphylococcal scalded-skin syndrome (ref. 4). These diseases usually affect children and neonatal animals (ref. 4). The Staphylococcus aureus exfoliative toxins A, B, and D have been identified and fully sequenced (ref. 4). Other similar exfoliative toxins for example Staphylococcus hyicus exfoliative toxins ExhA, ExhB, ExhC, and ExhD (which can cause similar disease in pigs and other animals), have also been identified (ref. 4). For present purposes, exfoliative toxins of Staphylococcus hyicus (especially Staphylococcus hyicus toxins ExhA, ExhB, EXhC and ExhD) are considered analogues of Staphylococcus aureus exfoliative toxins and hence their use is within the scope of the present invention. The use of Staphylococcus aureus exfoliative toxins in skin peeling, as a cosmetic or in a therapeutic procedure has not been previously reported.

Derivatives of Staphylococcus aureus exfoliative toxins include, for example, point mutations of wild type toxins. Nucleic acid molecules encoding Staphylococcus aureus exfoliative toxins have been cloned (e.g. Jackson & landolo, 1986 J. Bacteriol.

j, 574-580; Yamaguchi et al., 2001 Infection & Immunity, 7760-7771). Thus it is in principle easy to prepare mutant forms of the exfoliative toxins having desirable characteristics. Typically derivatives suitable for use in the present invention will differ by fewer than 20 amino acid residues relative to the respective wild type sequence, preferably differ by fewer than 10 amino acid residues, more preferably by less than 9, 8, 7, 6, 5, 4, 3 or even 2 residues relative to the respective wild type sequence.

Other derivatives which may be useful in the invention include chimeric forms and fusion proteins comprising at least an active portion of the exfoliative toxin. Equally, truncated variants of the exfoliative toxin will comprise at least an active portion thereof. For present purposes, a truncated, mutated or otherwise altered "active portion" of an exfoliative toxin is considered active if it still retains a detectable ability to cleave a desmoglein protein in an appropriate assay. A suitable assay of desmoglein-cleaving activity is disclosed by Nishifuji et al. (ref.4): baculovirus-expressed recombinant extracellular domains of human desmoglein 1, desmoglein 2, desmoglein 3, and desmoglein 4 fused with E tags are incubated in vitro with an exfoliative toxin; the degradation of recombinant human desmogleins is determined by immunoblot analysis with anti-E tag monoclonal antibody. For present purposes, the wild type sequences of Staphylococcus aureus exfoliative toxins are considered to be those disclosed by Jackson & landolo 1986, J. Bact. , 574-580; Gray & Kehoe 1984 Infect. Immun. 4, 615-618; and Yaniaguchi 2002 Infect. Immun. 5835-5845).

When possible, it is preferable to use Staphylococcus aureus exfoliative toxin A rather than toxin B, because toxin A is more stable. For example, Staphylococcus aureus exfoliative toxin A is stable for 12 months at -30°C whereas toxin B is stable for 30 days at -30°C; the difference in stability is even more evident at higher temperatures.

Presently only Staphylococcus aureus exfoliative toxins A, B or D are known to act on human skin. However, Staphylococcus aureus exfoliative toxins A, B or D are also known to act on animal skin, and exfoliative toxins of other strains of Staphylococcus (for example, Staphylococcus hyicus and Staphylococcus chromo genes exfoliative toxins) are known to act on animal skin. Therefore, the present invention includes the use of other Staphylococcus exfoliative toxins, derivatives, truncated products or analogues thereof, and is not restricted to the use of Staphylococcus aureus exfoliative toxins A, B or D only.

Staphylococcus aureus exfoliative toxins cause skin blistering through an enzymatic process by cleaving a cell protein named desmoglein 1 (hereinafter referred to as Dsgl) (ref. 4). There are currently four desmoglein (Dsg) proteins (i.e. Dsgl, Dsg2, Dsg3, Dsg4) that have been identified and are all members of the cadherin cell adhesion molecule superfamily. The Dsg gene family members are located in a cluster on chromosome 18. These Dsg proteins are expressed differently by skin cells in the different layers of skin, and are responsible for keeping skin cells together through formation of cell structures called desmosomes. Desmosomes are cell-cell junctions formed between epithelial, myocardial and certain other cell types.

Cleavage of Dsg I proteins in particular causes the skin cells to fall apart, forming blisters and skin peeling beneath or within the level of Stratum Granulosum of the epidermal layer of skin. Importantly, and in contrast to traditional methods of skin peeling which cause inflammation, Staphylococcus aureus exfoliative toxins peel the skin with minimal or no inflammation as they do not destroy skin cells per se but only proteins which keep skin cells glued' together. (ref. 4). In addition, there is no risk of undesirable deep levels of skin peeling using Staphylococcus aureus exfoliative toxins because Dsg 1 molecules are expressed only at specific levels of the skin (including stratum comeum, stratum granulosum, stratum spinosum, stratum basale), and skin peeling will inevitably stop once all Dsgl molecules are cleaved. Therefore, skin peeling will not progress deeper than the basal layer of epidermis, and will usually stop beneath or within the level of stratum granulo sum of the epidermal layer of skin.

In addition, there is a reduced risk of inefficient skin peeling because Staphylococcus aureus exfoliative toxins, derivatives, truncated products or analogues thereof can be applied to the skin with a relative excess of the product. Furthermore, the time of application of such products to the skin is not limited, due to the reduced complications associated with this technique.

The use of an agent for modulating the activity, stability or expression of a polypeptide derived from the Dsg 1 protein for cosmetic andlor therapeutic use, in particular for preventing and/or treating the signs of skin aging, has been described in W02009081368 (A2). However, it has not been reported previously that Staphylococcus aureus exfoliative toxins A, B or D, derivatives, truncated products or analogues thereof, can be used to cleave the (Dsgl) protein and, in this way, cause skin peeling which is useful for cosmetic and/or medical purposes.

Pemphigus is a rare group of autoimmune diseases that affect the skin and mucous membranes. In pemphigus, autoantibodies form against the Dsg proteins. In patients suffering from the disease pemphigus foliaceus or pemphigus vulgaris, blisters are caused by antibodies directed against the Dsgl and Dsg3 proteins, respectively (refs.

4, 7). In order to confirm that the blistering associated with these diseases is caused by antibodies against the Dsgl and/or Dsg3 proteins, experimental animals were injected with antibodies against Dsg 1 and/or against Dsg3. The animals showed blistering (ref. 7). The combination of antibodies against Dsgl and Dsg3 is known to be more efficient in causing blistering than antibodies against either component separately, and the extent of blistering (for example, generalised or localised) depends on the dose of the antibodies injected (ref. 7). However, it has not previously been suggested that binding partners having binding affinity for the Dsgl protein, Dsg2 protein, the Dsg3 protein or Dsg4 protein, alone, or in combination, or optionally with other components (eg. Staphylococcus aureus exfoliative toxins A, B or D, derivatives or truncated products or analogues thereof) are useful for inclusion in a formulation for skin peeling, as a cosmetic or in a therapeutic procedure.

The binding partner having binding affinity for any one or more of the Dsg 1, 2, 3 or 4 proteins may be any molecule having a suitable binding affinity and, preferably, specificity.

For the purposes of illustration, the binding affinity of the binding partner may be such that the Kd is preferably in the range 1pM to 1OpM, and most preferably in the range 100pM to lOOnM.

Conveniently the binding partner is an immunoglobulin or a derivative thereof. As used herein, the term "immunoglobulin" refers to a family of polypeptides which retain the immunoglobulin fold characteristic of antibody molecules, i.e. containing two f3 sheets and, usually a conserved disuiphide bond. Members of the imrnunoglobulin superfamily are involved in many aspects of cellular and non-cellular interactions in vivo, including widespread roles in the immune system (e.g. antibodies, T-cell receptor molecules and the like), involvement in cell adhesion (e.g. the intracellular adhesion, ICAM, molecules) and intracellular signalling (e.g. receptor molecules, such as the platelet-derived growth factor, PDGF, receptor). The present invention is applicable to all immunoglobulin superfamily molecules which possess binding domains. Preferably, the binding partner is an antibody or derivative thereof, which derivative retains Dsg-binding activity.

Methods of making antibodies to purified proteins are well known to those skilled in the art and are described in detail, for example, in Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990).

Immunoglobulins of use in the invention are preferably an IgG, IgA or 1gM molecules or derivatives thereof They could, however, in principle be an IgE or IgD molecule or a derivative thereof Derivatives of immunoglobulins are well known to those skilled in the art and methods for their isolation, screening, selection and manufacture on an industrial scale are also well known. Examples of derivatives include Fv, Fab, higher order polymers, Fab', single chain Fv fragments and single domain antibodies ("dAbs"). Similarly, methods for the production of these derivatives are well known. Each of these derivatives share the characteristic of retaining the ability to bind to the antigen that is recognised by the parent immunoglobulin from which they may be derived, in the case where they occur naturally and have not been artificially created.

The binding partner is preferably one which can be used several times on the same subject without eliciting an immune response. For example, where the binding partner is an immunoglobulin, the irnmunoglobulin may be "humanised" to reduce its immunogensity. Methods of humanisation include complementarity-determining region (CDR) grafting. Other modifications include adaptation of the immunoglobulin to improve its protease resistance, since human skin secretions include proteases.

In a manner similar to that described for exfoliative toxins, the use of antibodies or other binding partners directed against the Dsgl protein andlor against the Dsg2 protein, Dsg3 protein or Dsg4 protein, or derivatives, truncated products or analogues of such antibodies for skin peeling, is advantageous over traditional methods of skin peeling, as it is self-limiting and will not progress deeper than the stratum basale of the epidermal layer of skin. In addition, these methods are more efficient than traditional methods of skin peeling.

The formulation of the invention, where it comprises a binding partner (especially an antibody or derivative thereof) for a Dsg protein, may conveniently comprise a single molecule which has binding activity for two or more different Dsg proteins (e.g. Dsg 1 and Dsg 3). For example, the binding partner may comprise a bivalent, bispecific antibody which cross-reacts with or binds to both Dsg 1 and Dsg 3. In other embodiments, the formulation of the invention may comprise two or more different binding partners, with different binding specificity, e.g. one antibody which has high binding specificity for Dsg 1, and a second antibody which binds to Dsg 3 with high specificity. The latter approach may be preferable as it allows variation in the relative amounts of Dsg 1 and Dsg 3 binding activity to be provided in the fonnulation.

In preferred embodiments, where the formulation comprises a Dsg protein binding partner, it may comprise one or more molecules which will bind to two or more, or three or more, or even all four types of Dsg protein (Dsg 1-4) known to be present in human skin.

The use of an agent for modulating the activity, stability or expression of a polypeptide derived from the Dsgl protein for cosmetic andlor therapeutic use, and in particular for preventing andlor treating the signs of skin aging has been disclosed in W02009/081368. However, the feature of the present invention wherein antibodies directed against the Dsgl protein and/or against the Dsg2 protein, Dsg3 protein or Dsg4 protein, or derivatives or truncated products or analogues of such antibodies alone or in combination, can be used for preparing a skin peeling formulation has not hitherto been disclosed.

The present inventor believes that there is a cumulative and synergistic effect of skin peeling if antibodies directed against the Dsg 1 protein, Dsg2 protein, Dsg3 protein and/or the Dsg4 protein, or derivatives, truncated products or analogues of such antibodies thereof, are used in combination and/or in combination with other components, such as Staphylococcus aureus exfoliative toxins A, B or D, derivatives, truncated products or analogues thereof.

It is known that antibodies directed against laminin 5, laminin 6, integrin beta 4 subunit, collagen VII, collagen XVII, and Bullous Pemphigoid antigen 230 cause autoimmune blistering diseases, including Bullous pemphigoid, Cicatricial pemphigoid and Epidermolysis bullosa acquisita. It is also known that such antibodies can cause blistering at the level just below the epidermal layer of the skin (refs. 8 & 9). However, it has not previously been suggested that binding partners for laminin 5, laminin 6, integrin beta 4 subunit, collagen VII, collagen XVII, and the Bullous Pemphigoid antigen 230 may be used to peel the skin beneath the epidermal layer for the purposes of cosmetic and/or medical treatment.

As in the case of the Dsg proteins, the binding partner having binding activity for one or more of laminin 5, laminin 6, integrin beta 4 subunity, collagen VII, collagen XVII or Bullous Pemphigoid antigen 230 etc. will conveniently comprise an immunoglobulin or a derivative thereof, specifically an antibody or antigen-binding derivative thereof.

According to the present invention, antibodies against, or other binding partners for, laminin 5, laminin 6, integrin beta 4 subunit, collagen VII, collagen XVII, Bullous Pemphigoid antigen 230 proteins, derivatives truncated products or analogues thereof and the like can be used to peel the skin beneath the epidermal layer of the skin for the purposes of cosmetic and/or medical treatment.

Preferably, the binding partner will have a binding affinity such that that its Kd is in the range 10pM to 1 OjiM, preferably in the range 100pM to 1 j.tM, and most preferably in the range mM to lOOnM.

The method of delivering the composition of the present invention may include the subcutaneous, intradermal, transepidermal, subepidermal, intraepidermal injections, and/or the DERMOJET� intradermal method of drug delivery or similar methods.

According to the present invention, the above compositions for peeling may also be applied to skin as a dust, powder, solution, ointment (including an oily ointment, water-soluble ointment, cream, suspension or hydrogel), dermatological paste, patch, and the like. Examples of methods for delivery of the composition for skin peeling include methods of enhancing percutaneous absorption, including, but not limited to, the use of microneedle units (microneedle technology or microneedle drug delivery, including microneedle units coated with the product, uncoated solid, or hollow microneedle units), and/or the cosmetic ROLLCITTM system (Medical ROLLCITTM, Mediroller), andlor the derivatisation procedures for the product including but not limited to PEGylation' (i.e. the modification of proteins by covalent linking to one or more molecules of polyethylene glycol). The formulation may be applied to the skin as is, and/or using the technique of occlusion. Examples of the additional component include a percutaneous absorption accelerator such as isopropyl myristate, a surfactant, alcohol or the like.

The excipient, diluent or carrier may be any cosmetically suitable substance, whether solid, liquid or gel, depending on the intended nature of the formulation. Suitable excipients, diluents or carriers may include distilled water, undistilled water, 22p.m pore filter-sterilised water or water sterilised by other means, aqueous solutions (such as saline, phosphate -buffered saline, or other buffer solutions), calcium carbonate, kaolin, silica, or any combination of the foregoing. Where the formulation is aqueous, it will be preferred to include at least one buffer in the formulation, at a concentration sufficient to provide adequate stabilisation of p1-I.

The formulation of the present invention is applied to the target skin. The method for applying the composition of the present invention to the skin is not particularly limited, as long as the active ingredient(s) can be absorbed via the skin or delivered to the epidermis and/or dermis layers of skin. Examples of such delivery include injections, spraying, coating, packing, iontophoresis, and other methods outlined above. If the active ingredients are applied to the skin surface, for example as a spray, aqueous solution, ointment or the like, the concentration of active ingredient(s) in the base is generally within the range 0.0 1% to 70% preferably within the range 0.1% to 50% and more preferably within the range 1% to 30%. The duration of application of the composition to the skin is not particularly limited. Preferably, the composition is applied to the skin from about 1 minute to about 12 hours, more preferably from about minutes to about 4 hours and most preferably from about 10 minutes to about 60 minutes.

If exfoliative toxins are applied by injection, using the DERMOJET� intradermal method of drug delivery or using similar methods, the dose of the active ingredient(s) will generally range from about 0.01 ig to about 1 g per 1 cm2, preferably from about 0.ltg to about 100mg per cm2 and more preferably from approximately about lp.g to about 100pg per cm2. If antibodies to Dsg proteins are applied by injections using the DERMOJET� intradermal method of drug delivery or using similar methods, the dose of the active ingredient(s) will generally range from about 1 tg to 1 g per 1 cm2, preferably from about 1 0pg to 100mg per cm2 and more preferably from about 1 00 jig to 5mg per cm2. When the active ingredients are applied by injection, using the DERMOJET� intradermal method of drug delivery or similar methods, it is preferable to adjust the solutions so that pH is between 6.9 and 7.4, preferably between 7.1 and 7.2, and to include any appropriate excipient, including buffers, stabilizers and agent(s) promoting isotonicity, for example sodium chloride andlor sodium citrate.

When Staphylococcus exfoliative toxins and/or antibodies directed against the Dsg proteins are applied to the skin for the purposes of skin peeling, depending on the concentration of active ingredients, skin peeling will occur from between about 1 minute to about 24 hours, preferably from about 10 minutes to about 12 hours, and more preferably from about 30 minutes to about 2 hours following application of the formulation.

Staphylococcus exfoliative toxins and/or antibodies directed against Dsg proteins can be used separately to peel the skin. However, to increase the efficacy of skin peeling it is more preferable to use the combination of antibodies against the Dsgl protein, Dsg2 protein and/or Dsg3 protein for deeper peeling, and to use the combination of antibodies against the Dsgl and/or Dsg4 for more superficial skin peeling.

Staphylococcus exfoliative toxins can be used in the above combinations instead of antibodies against the Dsgl protein, or in combination with antibodies against Dsg proteins in order to improve efficiency of skin peeling.

If antibodies against laminin 5, laminin 6, integrin beta 4 subunit, collagen VII, collagen XVII, Bullous Pemphigoid antigen 230 proteins, derivatives, truncated products or analogues thereof are applied by injection, the DERMOJET� intradermal method of drug delivery or similar methods, the dose of active ingredient(s) is generally from I tg to 1 g per 1 cm2, preferably from 1 0tg to 100mg per cm2 and more preferably from 100tg to 5mg per cm2. When such antibodies are applied to the skin for the purpose of skin peeling, depending of the concentration of active ingredients, skin peeling will occur from about 1 minute to about 24 hours preferably from about minutes to about 12 hours, and more preferably from about 30 minutes to about 2 hours.

According to the present invention, the above exfoliative toxins, antibodies against Dsgl, Dsg2, Dsg3, Dsg4, laminin 5, laminin 6, integrin beta 4 subunit, collagen VII, collagen XVII, Bullous Pemphigoid antigen 230 proteins, derivatives or truncated products or analogues thereof and the like can be used simultaneously, if desired, in order to increase the efficacy of the skin peeling.

Antibodies to many antigens are available commercially. For example, the company Abeam (Cambridge, UK) supplies the following: mouse monoclonal anti-desmoglein 1 antibody (clone No. 3G131); monoclonal anti-desmoglein 2 antibody (clone No. 3G132); anti-desmoglein 3 antibody (Cat. No. ab14416; clone 3G133); mouse monoclonal anti-desmoglein 4 antibody (Cat. No. ab58364); sheep polyclonal anti-Staphylococcus aureus exfoliative toxin A antibody (Cat. No. ab53400); rat monoclonal [A5] anti-laminin antibody (Cat. No. ab2500); rabbit polyclonal anti-laminin antibody (Cat. No. abl 1575); Integrin beta 4 antibodies (Cat. Nos. ab29043; ab63546; ab29044; ab63438; ab77693); mouse monoclonal anti-collagen VII antibody (Cat. No. ab63 12); anti-collagen XVII antibody (Cat. No. ab28440) Antibodies are commercially available from many other sources, including the following: AbD Serotec, Antigenix America Inc., BD Biosciences, Everest Biotech, Lab Vision, Millipore, Novus Biologicals, Santa Cruz Biotechnology Inc., and Sigma-Aldrich.

For the purposes of the present specification invention, the term "cosmetic use" is intended to denote a use intended to provide an aesthetic effect.

For the purposes of the present specification, the term "therapeutic composition" is intended to provide a prophylactic and/or curative effect with respect to epithelial, and in particular epidermal, disorders recognised as reflecting a pathological state.

The methods according to the invention are advantageous as their implementation does not require an invasive procedure.

In general, any composition of the invention may be applied to the skin (on any region of the body) or even to the mucous membranes (buccal, jugal, gingival, genital, conjuctival, etc.). Typically however, the formulation may be applied to the skin of the face, scalp, hands, arms, feet, legs or trunk.

A cosmetic formulation in accordance with the invention may also contain one or more adjuvants which are customary in the field of cosmetics, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic additives, preservatives, antioxidants, solvents, fragrances, fillers, screens, odour absorbers and dyestuffs.

For the avoidance of doubt, it is hereby expressly stated that any feature described as "preferable", "desirable", "convenient", "advantageous", or the like may be present in the invention in isolation or in any combination with any one or more features so described, unless the context dictates otherwise.

The invention will be further described by way of illustrative example and with reference to the accompanying drawing, Figure 1 (adapted from Nishifjui et al., ref. 4) which is a schematic representation of a section through human skin. The skin is formed from two distinct layers: the outermost layer is the epidermis, whilst the underlying layer is the dermis. The epidermis is considered to comprise four layers or strata: the stratum corneum, stratum granulosum, stratum spinosum and stratum basale. As indicated on the right of the figure, Dsgl is distributed throughout the epidermis: Dsg2 and Dsg4 are restricted to the stratum basale and just below the stratum corneum respectively. Dsg3 is present in both the stratum spinosum arid the stratum basale. The proteins laminin 5, laminin 6, integrin beta4, collagen VII, collagen XVII and bulious pemphigoid 230, indicated generally by the arrow, are located primarily in the dermal region of the skin. Accordingly active agents in groups (a) and (b) are likely to cause superficial exfoliation restricted to the epidermis, whilst active agents in group (c) are likely to cause deeper peeling of the dermal layer.

Detailed Description of Embodiments

Example 1

In a specific embodiment, a formulation in accordance with the present invention comprises: (i) Staphylococcus aureus exfoliative toxin A, present in the composition at a concentration of 1 00tg/ml; (ii) an active agent consisting of an immunoglobulin which binds to Dsg3 at a concentration of 1mg/mi; (iii) an active agent consisting of an immunoglobulin which binds to laminin 5 at a concentration of Img/ml; and an aqueous buffer solution adjusted to an appropriate pH (e.g. in the range 6.5-7.5).

The immunoglobulin of part (ii) binds to Dsg with a Kd of mM and the immunoglobulin of part (iii) binds to laminin 5 with a Kd of lOnm.

The formulation is filter-sterilised by passage through a 0.22 tm pore filter and stored frozen at -20°C or refrigerated at 4°C until required.

The formulation of the present invention may be used to treat a subject suffering from scarring due to acne or varicella, or wrinkling due to photodamage. The formulation is administered to the subject using the DERMOJET � intradermal method of drug delivery. Using this technique, approximately 0.1 ml per cm2 of the formulation is administered to the dermal layer of the subject's skin. Prior to administration, the pH of the solution is adjusted such that it has a pH of 7.2. The formulation is left on the subject's skin for 60 minutes, then removed by washing with copious amounts of water (preferably sterile, optionally distilled).

Example 2

In another specific embodiment, a formulation in accordance with the present invention comprises: (i) Staphylococcus aureus exfoliative toxin A, present in the composition at a concentration of 1 Oojig/ml; (ii) an active agent consisting of an immunoglobulin which binds to Dsg3 at a concentration of 1 mg/mi; and an aqueous buffer solution adjusted to an appropriate pH (e.g. in the range 6.5-.7.5).

The immunoglobulin of part (ii) binds to Dsg with a Kd of lnM.

The formulation is filter-sterilised by passage through a 0.22 im pore filter and stored frozen at -20°C or refrigerated at 4°C until required.

The formulation of the present invention may be used to treat a subject suffering from actinic keratoses. The formulation is administered to the subject using the DERMOJET � intradermal method of drug delivery. Using this technique, approximately 1 0tg per cm2 of Staphylococcus aureus exfoliative toxin A of part (i) and approximately 1 OOj.tg per cm2 of anti-Dsg3 immunoglobulin of part (ii) of the formulation is administered to the dermal layer of the subject's skin. Prior to administration, the pH of the solution is adjusted such that it has a pH of 7.2. The formulation is left in contact with the subject's skin for 30 minutes and then any active agent remaining is removed by rinsing with copious amounts of water (preferably sterile and optionally distilled).

References 1. Brody, H.R. 2003. Skin Resurfacing: chemical peels. In Fitzpatrick's dermatology in general medicine. I.M. Freedberg, A.Z.Eisen, K. Wolff, K.F. Austen, L.A. Goldsmith, and S.I. Katz, editors. Mc Graw Hill, New York. 2530-2535.

2. Hruza, G.H., J.S. Dover, and K.A. Amdt. 2003. Skin Resurfacing: laser. in Fitzpatrick's dermatology in general medicine. I.M. Freedberg, A.Z. Eisen, K. Wolff, K.F. Austen, L.A. Goldsmith, and S.I. Katz, editors. Mc Graw Hill, New York. 2538-2544.

3. Harmon, C.B. and J.M. Yarborough. 2003. Skin resurfacing: dermabrasion. In Fitzpatrick's dermatology in general medicine. I.M. Freedberg, A.Z. Eisen, K. Wolff, K.F. Austen, L.A. Goldsmith, and S.!. Katz, editors. Mc Graw Hill, New York. 2536-2538.

4. Nishifuji K, Sugai M, Amagai M. Staphylococcal exfoliative toxins: "molecular scissors" of bacteria that attack the cutaneous defense barrier in mammals. J Dermatol Sd 2008; 49:21-3 1.

5. Kondo I, Sakurai S, Sarai Y. New type of exfoliatin obtained from staphylococcal strains, belonging to phage groups other than group II, isolated from patients with impetigo and Ritter's disease. Infect Immun 1974; 10:851-861.

6. Kapral F.A, Miller MM. Skin lesions produced by Staphylococcus aureus exfoliatin in hairless mice. infect Immun, 1972; 6:877-879.

7. Mahoney M.G, Wang Z, Rothenberger K, Koch P.J, Amagai M, Stanley J.R.

Explanations for the clinical and microscopic localization of lesions in pemphigus foliaceus and vulgaris. JClin Invest 1999; 103:461-468.

8. Liu Z, Diaz L.A, Troy J.L, Taylor A.F, Emery D.J, Fairley J.A. et al. A passive transfer model of the organ-specific autoimmune disease, bullous pemphigoid, using antibodies generated against the hemidesmosomal antigen, BP18O. J Clin Invest 1993; 92:2480-2488.

9. Lazarova Z, Hsu R, Briggaman R.A, Yancey K.B. Fab fragments directed against laminin 5 induce subepidermal blisters in neonatal mice. Clin Immunol 2000; 95:26-32.

Claims

Claims A formulation for use in the exfoliation of a mammalian subject, the formulation comprising: (i) one or more active agents selected from the group, (a), consisting of: Staphylococcus aureus exfoliative toxins A, B or D, or active derivatives, analogues or truncated portions thereof; and/or (ii) one or more active agents selected from the group, (b), consisting of: a binding partner which binds to desmoglein 1, desmoglein 2, desmoglein 3 or desmoglein 4; andlor (iii) one or more active agents selected from the group, (c), consisting of: a binding partner which binds to laminin 5, laminin 6, integrin beta 4 subunit, collagen VII, collagen XVII, or Bullous Pemphigoid antigen 230 proteins; said active agent(s) being present in the formulation at a known concentration sufficient such that the formulation causes appropriate exfoliation of the subject's skin within a contact time in the range of 2 minutes to 1 hour; (iv) a cosmetically acceptable carrier, diluent or excipient.
2. A formulation according to claim 1, comprising at least one active agent from group (a), and at least one active agent from group (b).
3. A formulation according to claim 1, comprising at least one active agent from group (a), and at least one active agent from group (c).
4. A formulation according to any of the preceding claims, comprising at least one active agent from each of groups (a), (b) and (c).
5. A formulation according to claim 1, wherein the binding partner in group (b), andlor the binding partner in group (c), comprises an antibody or an antigen-binding derivative thereof.
6. A formulation according to any of the preceding claims wherein the binding affinity of the binding partner is such that the Kd is in the range 1pM to 1OM.
7. A formulation according to any of the preceding claims wherein the binding affinity of the binding partner is such that the Kd is in the range 100pM to lOOnM.
8. A formulation according to any of the preceding claims, comprising Staphylococcus aureus exfoliative toxin A or an active derivative, analogue or truncated portion thereof.
9. A method of treatment of the skin of a mammalian subject for cosmetic or therapeutic purposes, the method comprising the steps of: applying to selected areas of the subject's skin in need of such treatment an effective amount of a formulation in accordance with any of the preceding claims; and leaving said formulation in contact with the selected areas of the subject's skin for an appropriate amount of time.
10. A method according to claim 9, wherein the formulation is applied to the subject's skin by means of a spray, aqueous solution, cream or ointment.
11. A method according to claim 9, wherein the formulation is applied by injection or intradermal drug delivery system.
12. A method according to any one of claims 9, 10 or 11, wherein the formulation is applied so as to provide a total concentration of active agent in the range 0.01 jg to 1 gm per cm2 of skin.
13. A method of making an exfoliative formulation for use on a mammalian subject, the method comprising the step of mixing one or more active agents selected from the groups of substances (a) andlor (b) and/or (c) as defined in claim 1, with a cosmetically or therapeutically acceptable carrier, diluent or excipient.
14. A method according to claim 13, performance of which results in the manufacture of a formulation in accordance with any of claims 2-8.
15. Use of one or more active agents selected from the groups of substances (a) and/or (b) and/or (c) as defined in claim 1, for the manufacture of a therapeutic skin exfoliation formulation.