# Patent

## Title
Use of NRF2 inducers to treat epidermolysis bullosa simplex and related diseases

## Abstract

The present invention relates to methods and compositions for the prevention and treatment of keratin-based skin diseases. In particular, the application describes compositions and methods of treating a patient suffering from skin blistering comprising the use of phase II enzyme inducers.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation application of U.S.S.N.12/690,494, filed Jan. 20, 2010, which is a national stage application filed under 35 U.S.C. §of international application no. PCT/US2008/08694, filed Jul. 17, 2008, which claims the benefit of U.S. Provisional Application No. 60/929,985, filed Jul. 20, 2007, the contents of each of which are incorporated by reference in their entireties.
CROSS- REFERENCE TO RELATED APPLICATIONS
This application claims the benefit U.S. Provisional Application No. 60/929,985.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 3, 2013, is named 85531CON(71699)_SL.txt and is 16,083 bytes in size.
BACKGROUND OF THE INVENTION
The skin is continuously exposed to changes in the external environment, including oxidative insults, heat, cold, UV radiation, injury, and mechanical stresses. The stratum corneum, composed of terminally differentiated keratinocytes, constitutes the natural barrier that prevents loss of water and prevents entry of infectious agents (e.g., bacteria, viruses), small objects (e.g., particles), and a broad variety of water-soluble chemicals.
Intermediate filaments (IFs), microtubules (MTs) and microfilaments (MFs) constitute the cytoskeleton and play important roles in the organization and mechanical Integrity of skin keratinocytes (Fuchs and Cleveland, 1998). Keratins are a large family of proteins that form the intermediate filament cytokeleton in epithelial cells. Keratins are encoded by two groups of genes, type I and II, which are distinct at the level of genomic structure and nucleotide sequence. Each type of keratin gene is clustered within separate loci in the mouse and human genomes (Fuchs, 1995; Schweizer at al., 2006). Type II keratin proteins, which include K1-K8/K71-K74 in soft epithelia and K81-K86 in hard epithelia, such as hair, nail, and oral papilla, are larger (52 to 70 kDa) and basic-neutral in charge; type I keratins, which comprise K9-K28 in soft epithelia and K31-K40 in hard epithelia, are smaller (40 to 64 kDa) and acidic (Schweizer et al., 2006). The physical proximity, identical substructure and transcriptional orientation, and high sequence homology of type II keratin genes K5, K6α, K6β and K6hf, and type I keratin genes K14, K16, K17 and K17n, strongly suggest that each subset was generated through successive duplications from a common ancestral gone (Wong at al., 2005). Type I epidermal keratin genes K14, K16 and K17 share high amino acid sequence identity (Troyanovsky et al., 1992; McGowan and Coulombe, 1998; 1998b), and are structurally and functionally related (Paladini and Coulombe, 1999; Coulombe et al., 2004; Tong and Coulombe, 2006).
In most epithelial cells the keratin filament network spans the entire cytoplasm, from the surface of the nucleus to the cell periphery, where it contacts cell-matrix (hemidesmosomes) and cell-cell (desmosomes) adhesive sites (e.g., Fuchs, 1995; Gu and Coulombe, 2007). Keratin Intermediate filaments provide cells and tissues with mechanical resilience and protects them against physical stress. Disruption of the keratin scaffold leads to tissue and cell fragility in the skin and its appendages (hair, nail, glands), oral mucosa, and cornea. Several genetic diseases are caused by dominantly-acting mutations altering the coding sequence of keratin proteins (Fuchs and Cleveland, 1998; On and Coulombe, 2007; Irvine and McLean, 1999; Omary et al., 2004). Most of these mutations are missense or small in-frame insertions or deletions affecting the central rod domain of keratin proteins, and interfering with their structural support function (Cassidy et al. 2002; Gu and Coulombe 2007).
Epidermolysis bullosa simplex (BBS) is a rare autosomal dominant disease in which the epidermis loses its integrity following trivial mechanical trauma (Fine at al., 1991; 2000). The disease is characterized by extreme fragility of the keratinocytes, and skin blistering, resulting from missense mutations in the gene that encodes keratin 5 (K5) or keratin 14 (K14) (Fuchs and Cleveland, 1998; Cassidy at al. 2002; Gu and Coulombs 2007; Omary et al., 2004). K5 and K14, which are abundant cellular proteins, normally co-polymerize to form an intricate network of 10-12 nm-wide, “intermediate-sized” filaments in basal keratinocytes of epidermis and related epithelia (Nelson and Sun, 1983; Fuchs, 1995). EBS may manifest itself as a relatively mild blistering condition involving the hands and feet (EBS, Weber-Cockayne type), or as a generalized blistering condition, sometimes associated with mucosal blistering that involves the oropharynx, the esophagus and ocular mucosa, and which can be fatal (e.g., EBS, Dowling-Meara type). In individuals affected by EBS Weber-Cockayne (EBS-WC), blisters are rarely present at birth and may occur on the knees and shins with crawling, or on the feet in late infancy or later, during adolescence or early adulthood. Neonates affected by EBS, Koebner type (EBS-K), present blisters at birth or develop blisters within the first few months of life (Fine at al., 1991; 2000). In individuals suffering from EBS with mottled pigmentation (EBS-MP), skin fragility is evident at birth and children develop progressive brown pigmentation over time, interspersed with depigmented spots on the trunk and extremities, which disappears in adult life (see Gu and Coulombs, 2007, and refs. therein). Individuals affected by BBS-DM develop widespread and severe blistering and/or multiple grouped clumps of small blisters at birth, with hyperkeratosis of the palms and soles, that improves during mid to late childhood. The blistering in EBS-DM can be severe enough to result in neonatal or infant death (Fine at al., 2000).
Management of all types of EBS consists of supportive care to protect the skin from blistering, dressings that promotes healing, and prevention and treatment of secondary infection. These treatment options are therefore palliative and have limited success. Furthermore, EBS is representative of a large number of tissue fragility conditions caused by inherited mutations in intermediate filament protein-encoding genes (sue Fuchs and Cleveland, 1998; Cassidy at al., 2002; Omary et al., 2004; Gu and Coulombs, 2007).
Accordingly, there is a need in the art for improved treatment options for EBS and the present invention satisfies that need.
SUMMARY OF THE INVENTION
It is, therefore, an object of the invention to provide solutions to the aforementioned deficiencies in the art.
Further to this object, the invention provides a method to ameliorate the compromised state of mechanical resilience of skin in a patient comprising administering to the patient a composition comprising a therapeutically effective amount of an Nrf2 inducer. The patient to be treated may suffer from skin blistering. In one aspect of the invention, the patient may be affected by Epidermolysis bullosa simplex. In a preferred embodiment of the invention, the patient suffers from Epidermolysis bullosa simplex-Weber-Cockayne type, Epidermolysis bullosa simplex-Koebner type, Epidermolysis bullosa simplex with mottled pigmentation, Epidermolysis bullosa simplex-Dowling-Meara type, or Epidermolysis bullosa simplex with muscular dystrophy.
In a further embodiment, the method to ameliorate the mechanical resistance of the skin comprises administering a phase II enzyme inducer. In one embodiment, the phase II inducer is an isothiocyanate. In a preferred embodiment the phase II enzyme inducer is sulforaphane. In another preferred embodiment, the phase II enzyme inducer is a sulforaphane synthetic analogue. In yet another embodiment, the Nrf2 inducer is keratinocyte growth factor, also known as fibroblast growth actor 7. In an additional embodiment, the Nrf2 inducer is oltipraz. In a further embodiment, the Nrf2 inducer is ethacrynic acid. In still another preferred embodiment, the Nrf2 inducer causes the selective induction of K6, K16 or K17 in the keratinocytes in the skin of the patient.
In an additional embodiment, the present invention provides a method for treating or preventing skin blistering in a patient comprising administering to the patient a composition comprising a therapeutically effective amount of an Nrf2 inducer. In one aspect of the invention, the patient may be affected by Epidermolysis bullosa simplex. In a preferred embodiment of the invention, the patient suffers from Epidermolysis bullosa simplex-Weber-Cockayne type, Epidermolysis bullosa simplex-Koebner type, Epidermolysis bullosa simplex with mottled pigmentation, Epidermolysis bullosa simplex-Dowling-Meara type, or Epidermolysis bullosa simplex with muscular dystrophy.
In a further embodiment, the method for treating or preventing skin blistering comprises administering a phase II enzyme inducer. In one embodiment, the phase II inducer is an isothiocyanate. In a preferred embodiment the phase II enzyme inducer is sulforaphane. In another preferred embodiment, the phase II enzyme inducer is a sulforaphane synthetic analogue. In yet another embodiment, the Nrf2 inducer is keratinocyte growth factor. In an additional embodiment, the Nrf2 inducer is oltipraz. In a further embodiment, the Nrf2 inducer is ethacrynic acid. In a further embodiment, the Nrf2 inducer is a Michael reaction acceptor, such as triterpenoids or cyclic/acyclic bis-benzylidene-alkalones. In still another preferred embodiment, the Nrf2 inducer causes the selective induction of K6, K16 or K17 in the keratinocytes in the skin of the patient.
In yet another embodiment, the present invention provides a method for treating or preventing a keratin-based skin disease in a patient comprising administering to the patient a composition comprising a therapeutically effective amount of an Nrf2 inducer. The patient to be treated may be affected by an epidermolytic or non-epidermolytic keratin-based skin disease. Exemplary types of keratin-based skin diseases (Cassidy at al., 2002) to be treated include, but are not limited to, epidermolytic hyperkeratosis, ichtyosis bullosa of Siemens, pachyonychia congenita, epidermolytic or non-epidermolytic palmoplantar keratoderma (diffuse or focal) steatocystoma multiplex, Naegeli-Franceschetti-Jadassohn syndrome and dermatopathia pigmentosa reticularis. In one embodiment, the keratin-based skin disease is caused by a mutation at the K14 locus.
In a further embodiment, the method for treating or preventing a keratin-based skin disease comprises administering a phase II enzyme inducer. In one embodiment, the phase II inducer is an isothiocyanate. In a preferred embodiment the phase II enzyme inducer is sulforaphane. In another preferred embodiment, the phase II enzyme inducer is a sulforaphane synthetic analogue. In yet another embodiment, the Nrf2 inducer is keratinocyte growth factor. In an additional embodiment, the Nrf2 inducer is oltipraz. In a further embodiment, the Nrf2 inducer is ethacrynic acid. In a further embodiment, the Nrf2 inducer is a Michael reaction acceptor, such as triterpenoids or cyclic/acyclic bis-benzylidene-alkalones. In still another preferred embodiment, the Nrf2 inducer causes the selective induction of K6, K16 or K17 in the keratinocytes in the skin of the patient.
In an additional embodiment, the present invention provides a composition for topical application to the skin comprising a therapeutically effective amount of an Nrf2 inducer and a vehicle suitable for delivery. Topical compositions may be in several forms, such as solutions, oils, creams, ointments, gels, lotions, or pastes, and include, for instance, the penetration enhancer “transcutanol” (diethylene glycol monoethylether), or other excipients well known in the art.
Preferably, the Nrf2 inducer in the composition is a phase II enzyme inducer. More preferably, the phase II inducer is an isothiocyanate. Even more preferably, the phase II enzyme inducer is sulforaphane or a sulforaphane synthetic analogue. In another embodiment, the Nrf2 inducer is keratinocyte growth factor. In yet another embodiment, the Nrf2 inducer is oltipraz. In a further embodiment, the Nrf2 inducer is ethacrynic acid. In a further embodiment, the Nrf2 inducer is a Michael reaction acceptor, such as triterpenoids or cyclic/acyclic bis-benzylidene-alkalones. In a preferred embodiment, the Nrf2 inducer causes the selective induction of K6, K16 or K17 in the keratinocytes in the skin of the patient.
The Nrf2 inducer in the composition of the invention may be administered alone or in combination with additional active agents, including pharmaceutical, biological and/or molecular biological active agents in the context of combination or adjuvant therapy.
The foregoing general description and following brief description of the drawings and the detailed description are exemplary and explanatory and, along with the manuscript appended, are intended to provide further explanation of the invention as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of the invention.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the clinical features of Epidermolysis bullosa simplex in a 2-month old baby girl born prematurely. The patient had recurrent bullae from shortly after birth, particularly in trauma-prone areas. The blisters typically healed with hypopigmentation as new lesions formed. The patient's father bad similar lesions as a newborn and complained of recurrent bullae on his hands and feet. FIG. 1A shows the presence of several skin erosions at various stages of healing in the abdomen and upper thighs (the source of frictional trauma in the upper thighs is the diaper). FIG. 1B details the presence of several large fluid-filled blisters on the dorsal and lateral sides of toes and heel (depicted by single arrows and double arrows, respectively). Some of the toenails are also affected. The prominent soft bandage wrapped around the distal portion of the right foot is an attempt to prevent further trauma to the toes.
FIG. 2 illustrates the alignment of the predicted amino acid sequences for mouse K17 (SEQ ID NO: 1) and human K17(SEQ ID NO: 2), K14 (SEQ ID NO: 3)and K16(SEQ ID NO: 4). This alignment was produced using the DNASIS v.3.5 software (Hitachi Software Engineering Inc., Japan). Default parameters were applied. The boundaries of the major domains recognized in all IF proteins (Fuchs and Weber, 1994) are depicted with brackets: the non-helical head domain at the N-terminus; the α-helical subdomains 1A, 1B, 2A, and 2B characteristic of the central rod domain; and the non-helical tail domain at the C-terminus. Nonsense stop codons are depicted by asterisks. Both symbols “+” and “#” underneath the sequences identify residues that are different between mouse and human K17. The symbol “#” marks the subset for which mouse K17 is identical to either K14 or K16; the symbol “^” identifies residues that are conserved between mouse and human K17, but different from K14 and K16.



DETAILED DESCRIPTION OF THE INVENTION
Transcription factor NF-E2-related factor 2 (Nrf2) belongs to the CNC (Cap-N-Collar) family of transcription factors and possesses a highly conserved basic region-leucine zipper (bZip) structure. Nrf2 plays a critical role in the constitutive and inducible expression of anti-oxidant and detoxification genes, commonly known as phase II genes, that encode defensive enzymes, including drug metabolizing enzymes, such as glutathione S-transferase, NADP(H):quinone oxidoreductase and UDP-glucuronosyltransferase, and anti-oxidant enzymes, such as heme oxygenase-1-(HO-1)1 and γ-glutamylcysteine synthetase (GCS), in response to oxidative and xenobiotic stress (Braun et al., 2002; Fahey at al., 1997; Fahey and Talalay, 1999; Holtzclaw et al., 2004; Motohashi and Yamamoto, 2004). These enzymes are regulated through a promoter called anti-oxidant responsive element (ARE) or electrophile response element (EpRE). Phase II genes are responsible for cellular defense mechanisms that include the scavenging of reactive oxygen or nitrogen species (ROS or RNS), detoxification of electrophiles and maintenance of intracellular reducing potential (Holtzclaw et al., 2004; Motohashi and Yamamoto, 2004).
Nrf2 is normally sequestered in the cytoplasm of the cells by an actin-bound regulatory protein called Keap1. When cells are exposed to oxidative or electrophilic stress, the Keap1-Nrf2 complex undergoes a conformational change, and Nrf2 is liberated from the complex and released into the nucleus. The active Nrf2 dimerizes with small Maf proteins, binds to ARE and activates phase II gene transcription (Braun et al., 2002; Motohashi and Yamamoto, 2004).
There is increasing evidence that the induction of phase II enzymes protects from carcinogenesis and mutagenesis and enhances the antioxidant capability of the cells (Fahey and Talalay, 1999; lida et al., 2004). To date, nine classes of phase II enzyme inducers have been identified: 1) diphenols, phenylene diamines and quinones; 2) Michael acceptors; 3) isothiocyanates; 4) hydroperoxides and hydrogen peroxide; 5) 1,2-dithiole-3-thiones; 6) dimercaptans; 7) trivalent arsenicals; 8) divalent heavy metals; and 9) carotenoids, curcumins and related polyenes (Fahey and Talalay, 1999). These phase II enzyme inducers are considered very efficient antioxidants because unlike direct antioxidants, they are not consumed stoichiometrically during oxide-reduction reactions, have long duration of action, support the function of direct antioxidants, such as tocopherols and CoQ, and enhance the synthesis of glutathione, a strong antioxidant (Fahey and Talalay, 1999).
The diuretic ethacrynic acid (EA), an electrophilic Michael acceptor, oltipraz, and the isothiocyanate sulforaphane have been shown to inhibit lipopolysaccharide (LPS)-induced secretion of high-mobility group box 1 (HMGB1), a proinflammatory protein implicated in the pathogenesis of inflammatory diseases, from immunostimulated macrophages (Killeen at al., 2006). Oltipraz prevents carcinogenesis in liver and urinary bladder by enhancing carcinogen detoxification (Iida at al., 2004). The cytoprotective effect of keratinocyte growth factor (KGF) against oxidative stress in injured and inflamed tissues, including wounded skin, has been related to KGF's stimulation of Nrf2 during cutaneous wound repair (Braun et al., 2002).
Isothiocyanates, which are primarily derived from in cruciferous vegetables, are potent antioxidants and effective agents in the chemoprevention of tumors via the activation of phase II enzymes, inhibition of carcinogen-activating phase I enzymes and induction of apoptosis (Hecht, 1995; Zhang and Talalay, 1994; Zhang et al., 1994). Isothiocyanates are formed in plants from the hydrolysis of glucosinolates, which are β-thioglucoside-N-hydroxysulfates, when maceration of the vegetables by predators, food preparation or chewing causes disruption of the calls with consequent activation and release of the enzyme myrosinase. The resultant aglycones undergo non-enzymatic intramolecular rearrangement to yield isothiocyanates, nitriles and epithionitriles.
Sulforaphane is the aglycone breakdown product of the glucosinolate glucoraphanin, also known as sulforaphane glucosinolate (SOS). The molecular formula of sulforaphane is C6H11NOS2, and its molecular weight is 177.29 daltons. Sulforaphane is also known as 4-methylsulfinylbutyl isothiocyanate and (−)-1-isothiocyanato-4(R)-(methylsulfinyl)butane. The structural formula of sulforaphane is:




Sulforaphane was first synthesized (Schmid and Karrer, 1945), and then isolated from the weed hoary cress (Cardaria draba), savoy and red cabbage (Prochazka, 1959). More recently, sulforaphane was identified in broccoli and shown to be a potent phase II enzyme inducer in isolated murine hepatoma cells (Zhang et al., 1992), block the formation of mammary tumors in Sprague-Dawley rats (Zhang at al.; 1994), prevent promotion of mouse skin tumorigenesis (Gills at al., 2006; Xu et al., 2006) and increase heme oxygenase-1 (HO-1) expression in human hepatoma HepC2 cells (Keum et al., 2006). Sulforaphane was also shown to inhibit ultraviolet (UV) light-induced activation of the activator protein-1 (AP-1), a promoter of skin carcinogenesis, in human keratinocytes (Zhu et al., 2004), and there is evidence that topical application of sulforaphane extract increases the level of phase II enzymes NAD(P)H:quinone oxidoreductase I (NQO1), glutathione S-transferase A1 and heme oxygenase 1 in mouse skin epidermis (Dinkova-Kostova et al., 2007). Moreover, sulforaphane protects human epidermal keratinocytes against sulfur mustard, a potent cytotoxic agent and powerful mutagen and carcinogen (Gross of al., 2006), and inhibits cell growth, activates apoptosis, inhibits histone deacetylase (HDAC) activity and decreases the expression of estrogen receptor-α, epidermal growth factor receptor and human epidermal growth factor receptor-2, which are key proteins involved in breast cancer proliferation, in human breast cancer cells (Pledgie-Tracy et al., 2007). Further, sulforaphane was showed to eradicate Helicobacter pylori from human gastric xenografts (Haristoy et al., 2003).
The present inventors discovered that topical application of Nrf2 inducers to the skin markedly improve the mechanical resilience of skin and prevents or reduce skin blistering in mammals, and specifically in human subjects suffering from a keratin-based skin disease, particularly a skin disease caused by a mutation at the K14 locus, thanks to their ability to trigger ectopic expression of structurally- and functionally-related keratins in basal layer keratinocytes.
Type 1 epidermal keratins K14, K16, and K17 are remarkably similar at the primary sequence level (see FIG. 2). There is direct and indirect evidence in the literature indicating that K14, K16, and K17 are redundant to a significant extent in their ability to foster structural support in stratified epithelia such as epidermis. There also is evidence that K17 fulfills two additional functions, likely in a context-dependent fashion, in skin epithelia. The first function is the protection against TNFalpha-induced programmed cell death, which is shared with K14 and K16 (Tong and Coulombe, 2006). The second function is stimulation of protein synthesis and epithelial cell growth, which is so far unique to K17 and restricted to the wound repair response (Kim et al., 2006). These two roles would be expected to be either neutral, or beneficial, in the context of EBS treatment. It has also been proposed that K16 plays a role in the process of keratinocyte activation that occurs after acute injury to the stratified epithelia (Paladini et al., 1996; Paladini and Coulombe, 1998; 1999). Since the structural support function of keratins is defective in EBBS and related conditions, accumulation of “surrogate” keratins (e.g., K16, K17) can “dilute away”, or attenuate, the dominant negative impact of the mutant protein (e.g., K14) responsible for the disease (Cao et al., 2001; Kerns et al.).
The inventors of the present application have made the discovery that treatment with sulforaphane (SF), a chemical naturally present in the diet, significantly decreases the massive skin blistering seen in a mouse model of EBS, thanks to sulforaphane's ability to selectively induce keratin genes whose structural support function is markedly redundant with K14. These genes are K16 and K17 (see Paladini and Coulombe, 1999; McGowan et al., 2002; Coulombe et al., 2004; Tong and Coulombe, 2006) (FIG. 2). In contrast to its clear impact on K16 and K17 expression, SF is not effective at inducing other relevant keratins in the epidermis, including K5, or K14, and has a weaker impact on K6 expression (Kerns et al.). The key aspect of SF's efficacy in treating EBS consists in its ability to cause activation of K16 and K17 expression in basal keratinocytes of epidermis. However, the mechanism by which sulforaphane causes induction of select keratins in treated skins is not known.
Our studies have shown that sulforaphane's effect does not reach the basal layer of epidermis, a key requirement for EBS therapy, when dissolved in an organic solvent like acetonitrile or acetone (date not shown). In contrast, sulforaphane impacts gene expression in the desired manner in basal keratinocytes of mouse epidermis when topically administered in select formulations that comprise a carrier, such as, but not limited to, jojoba oil and evening primrose oil, that allows sulforaphane to reach basal keratinocytes in the epidermis. These formulations may be modified according to various factors affecting human akin, including the age of the subject being treated and the site of treatment in the body.
The terms “subject” and “patient” are used interchangeably and are meant to refer to an animal. In a preferred aspect of the invention, the patient is a mammal. In the most preferred aspect of the Invention, the mammal is a human. Other suitable subjects or patients include, but are not limited to, laboratory animals, such as mouse, rat, rabbit or guinea pigs, farm animals and domestic animals or pets.
An epidermolytic or a non-epidermolytic keratin-based skin disease, as used in the current context, should be obvious to the person skilled in the art, and is meant to include any abnormality in the skin, where a keratin gene mutation is involved in the etiology of the disorder or is affected by the disorder. Examples of epidermolytic or a non-epidermolytic keratin-based skin diseases for which the current invention could be used preferably include, but are not limited to, epidermolysis bullosa simplex, epidermolytic hyperkeratosis, ichtyosis bullosa of Siemens, pachyonychia congenita, epidermolytic or non-epidermolytic palmoplantar keratoderma (diffuse or focal) steatocystoma multiplex, Naegeli-Franceschetti-Jadassohn syndrome and dermatopathia pigmentosa reticularis.
The treatment envisioned by the invention can be used for patients with a pre-existing condition, or for patients pre-disposed to a keratin-based skin disease. Additionally, the methods of the invention can be used to alleviate symptoms of keratin-based skin disease in patients, or as a preventative measure in patients. Finally, the treatment envisioned could also be used as a complement to other agents in the context of a combination or adjuvant therapy for administration to a subject that is being treated with one or more conventional drugs. Such drugs can be administered concurrently with, prior to or sequentially with Nrf2 inducer or phase II enzyme inducer treatment.
As used herein, “a pharmaceutically effective amount” is intended to mean an amount effective to elicit a cellular response that is clinically significant.
The present invention relates to methods of preventing or treating EBS and other keratin-based skin diseases using phase II enzyme inducers as described above.
Isothiocyanates are compounds containing the isothiocyanate (NCS) moiety and are easily identifiable by one of ordinary skill in the art. An example of an isothiocyanate includes, but is not limited to sulforaphane or its analogs. The description and preparation of isothiocyanate analogs is described in U.S. Reissue Pat. No. 36,784, and is hereby incorporated by reference in its entirety. The sulforaphane analogs used in the present invention include 6-isothiocyanato-2-hexanone, exo-2-acetyl-6-isothiocyanatonorbornane, exo-2-isothiocyanato-6-methylsulfonyinorbornane, 6-isothiocyanato-2-hexanol, 1-isothiocyanato-4-dimethylphosphonylbutane, exo-2-(1′-hydroxyethyl)-5-isothiocyanatonorbornane, exo-2-acetyl-5-isothiocyanatonorbornane, 1-isothiocyanato-5-methylsulfonylpentane, cis-3-(methylsulfonyl)cyclohexylmethylisothiocyanate and trans-3-(methylsulfonyl)cyclohexylmethylisothiocyanate.
Other compounds contemplated by the present invention include keratinocyte growth factor (KGF), oltipraz, ethacrynic acid, and analogs thereof, as well a additional Michael reaction acceptors, such as triterpenoids or cyclic/acyclic bis-benzylidene-alkalenes.
The compounds used in the methods of the present invention can be formulated into pharmaceutical compositions with suitable, pharmaceutically acceptable excipients for topical administration to mammals. Such excipients are well known in the art. Topical administration includes administration to the skin or mucosa, including surfaces of the lung and eye.
Dosage forms for topical administration include, but are not limited to, ointments, creams, emulsions, lotions and gels and agents that favor penetration within the epidermis. In a preferred embodiment, the composition is in the form of topical ointment.
The compounds of the invention may be administered alone or in combination with additional active agents, including pharmaceutical, biological and/or molecular biological active agents in the context of combination or adjuvant therapy. The compositions can also contain adjuvants such as, but not limited to, solubilizers, skin permeation enhancers, preservatives, wetting agents, moisturizers, gelling agents, buffering agents, surfactants, emulsifying agents, emollients, thickening agents, stabilizers, humectants and dispersing agents.
Moisturizers include carriers that allow the Nrf2 inducer or phase II enzyme inducer to reach basal keratinocytes in the epidermis. This may be achieved by varying the formulation according to several factors affecting human skin, including the age of the subject being treated and the body site. Examples of moisturizers include, but are not limited to, jojoba oil and evening primrose oil.
Suitable skin permeation enhancers are well known in the art and include lower alkanols, such as methanol ethanol and 2-propanol; alkyl methyl sulfoxides such as dimethylsulfoxide (DMSO), decylmethylsulfoxide (C10 MSO) and tetradecylmethyl sulfoxide; pyrrolidones, urea; N,N-diethyl-m-toluamide; C2-C6 alkanediols; dimethyl formamide (DMF), N,N-dimethylacetamide (DMA) and tetrahydrofurfuryl alcohol.
Examples of solubilizers include, but are not limited to, hydrophilic ethers such as diethylene glycol monoethyl ether (ethoxydiglycol, available commercially as Transcutol®) and diethylene glycol monoethyl ether oleate (available commercially as Softcutol®); polyoxy 35 castor oil, polyoxy 40 hydrogenated castor oil, polyethylene glycol (PEG), particularly low molecular weight PEGs, such as PEG 300 and PEG 400, and polyethylene glycol derivatives such as PEG-8 caprylic/capric glycerides (available commercially as Labrasol®); alkyl methyl sulfoxides, such as DMSO; pyrrolidones, DMA, and mixtures thereof.
Prevention and/or treatment of infections can be achieved by the inclusion of antibiotics, as well as various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like, in the compositions of the invention.
One of ordinary skill will appreciate that effective amounts of the agents in the compositions used in the methods of the Invention can be determined empirically. It will be understood that, when administered to a human patient, the total daily usage of the composition of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors: the type and degree of the response to be achieved; the activity of the specific composition employed; the age, body weight, general health, sex and diet of the patient; the duration of the treatment; drugs used in combination or coincidental with the method of the invention; and like factors well known in the medical arts.
Typically, the amount of Nrf2 inducer in the composition topically administered to the patient will be from about 100 nmol to about 1 μmol/cm2, and the composition will be applied directly on the skin over relevant portions of the body of the patient two or three times a week, so as to prevent or minimize blistering resulting from frictional trauma.
EXAMPLES
Source of Sulforaphane
Pure sulforaphane (SF) was used in our studies (Zhang et al., 1992).
Example 1
K14 Null and K5 Null Mouse Strains as Models for Very Severe EBS Disease
Introduction of null mutations at the K14 locus (Lloyd et al., 1995) and K5 locus (Peters et al., 2001) in mice essentially abrogate the keratin filament network in basal keratinocytes in the epidermis, and renders the keratinocytes acutely fragile in the face of physiological levels of mechanical trauma. The presence of small amounts of K15, a type I keratin related to K14, leaves a residual but wispy keratin filament network in basal keratinocytes of K14 null epidermis. Accordingly, K5 null mice show more extensive skin blistering and die sooner (before P0.5) than K14 null mice (P2-P3 in K14 null mice). Thus, these two mouse models represent very severe forms of the disease.
The K14 null mouse strain (Lloyd at al., 1995) has proven to be the more useful model for these studies. The selective fragility of epidermal basal cells and the associated trauma-induced skin blistering seen in K14-null mice mimics EBS as seen in humans. This said, this mouse model presents unique challenges that are relevant only to a small subset of human EBS patients (FIG. 1). The main challenge is premature death, which is largely due to extensive oral blistering and its acute impact on the feeding, stamina, and growth of newborn pups. Beginning shortly after birth, K14 null mice become fragile, lethargic, and can be distinguished from their “normal” littermates (K14+/+ or K14+/−). By P2, K14 null pups are significantly smaller, lack milk in their bellies, and are unable to close their mouths, correlating with local swelling. At P0 and P2, their lips and tongue exhibit very severe epithelial blistering. In our hands, the mean survival of the K14-null mice is 2.5±0.35 days (n=14, p<0.01). Oral lesions are only seen occasionally in newborns afflicted with EBS (Fine et al., 1991; see also FIG. 1B). The K14 null mouse strain thus provides a very stringent test for the notion that treatment with SF could be effective in the therapeutic management of skin blistering in EBS patients.
Example 2
Ectopic Expression of GB2 Rescues Skin Blistering in K14 Null Mice but not in K5 Null Mice
Keratin 17 is a direct target for the transcription factor Gli2, a powerful terminal effector of hedgehog signaling pathways (Bianchi et al., 2005). In Gli2TG transgenic mice (Grachtehouk et al., 2000), expression of the Gli2 coding sequence is controlled by the K5 gene promoter, thereby causing its accumulation in the basal layer of epidermis. Gli2TG mice appear normal at birth and in the days thereafter, but they develop epidermal hyperplasia as young adults, which progresses to basal cell carcinoma by 2-3 months of age. Availability of Gli2TG transgenic mice provided an opportunity to conduct a “proof of principle” experiment, whereby constitutive expression of Gli2TG transgene in the setting of K14−/− mice should cause a stable upregulation of K17 in basal keratinocytes of epidermis, and hence, rescue their oral and skin blistering. Conversely, the Gli2TG transgene should not rescue the phenotype arising in K5 null mice, given that Gli2 has a very modest impact on type II keratin gene regulation in epidermis (see Kerns et al.).
Gli2TG transgenic mice were thus mated with K14 null mice, and the resulting offsprings analyzed for readouts relevant to the K14 null mutation. Unlike their K14−/− littermates, the K14−/− Gli2TG mice were initially viable and showed normal skin, correlating with the presence of K17 in basal cells. This was in contrast to K5−/− Gli2TG mice, which died shortly after birth, exactly as straight K5 null mice did. These findings strongly suggest that rescue of an EBS-like condition can be achieved by exploiting functional redundancy within the keratin multigene family, and the presence or activation of a relevant transcription factor in basal keratinocytes can ectopically induce a keratin gene without affecting epidermal physiology.
Example 3
Sulforaphane Selectively Induces K16 and K17 in Skin Keratinocytes In Vitro and In Vivo
To evaluate the effect of sulforaphane (SF) on keratin gene transcription in vitro, a mouse keratinocyte line (308 cells) was exposed to 1 μM SF in acetonitrile vehicle, and mRNA levels were measured at 12, 24, and 48 hours after treatment. Relative to vehicle treatment, SF-treated keratinocytes showed a significant increase in the mRNA levels of NQO1, a well-established SF target, at all time points as expected (Dinkova-Kostova, et al., 2006). Similarly to NQO1, K17 and K16 mRNAs were each elevated  ̃2.5 fold at 12 h after SF treatment, but their induction was shorter-lived and levels returned to baseline by 24 hours. No significant change was measured for K5, K6a, K6b, K14 and K15 mRNA levels. Indirect immunofluorescence revealed an obvious induction of K17, but not K14, at the protein level.
At higher doses and in some specific contexts, SF induces apoptosis, or programmed cell death (Gamet-Payrestre et al., 2000; Fimongnari et al., 2002; Misiewicz et al., 2003; Gingras, et al., 2004). However, in the experiments described above, SF did not alter the intrinsically low rate of apoptosis seen in primary cultures of mouse keratinocytes, when present at a 1 μM concentration in the culture medium. At higher concentrations (5 μM), SF did alter the rate of apoptosis (16% in control treated-cells versus 94% in SF-treated cells). Thus, at a dose sufficient to alter keratin gene expression in a selective fashion (1 μM), SF does not cause apoptosis in cultured primary mouse keratinocytes.
To uncover whether SF had a similar effect on keratin expression in vivo and after sustained treatment, SKH-1 hairless mice were topically treated with 1 μmole SF in jojoba oil, a vehicle that readily penetrates the skin (El Laithy and El-Shaboury, 2002), twice a week for four weeks. Compared to vehicle-treated skin, SF-treated skin showed markedly increased K17 immunoreactivity that extended to the basal layer of the epidermis. K16 immunoreactivity was also increased, while K14 showed no change. Total protein extracts were prepared from the dorsal skin of these mice and analyzed to confirm these alterations in keratin expression. Relative to vehicle-treated samples, SF-treated samples exhibited increased levels of K17 and K16, whereas the level of K14 was unaltered. This sustained treatment regimen did not appear to affect skin morphology or alter the rate of apoptosis, in agreement with a previous study involving topical SF treatment at a higher frequency over an 11-week period (Dinkova-Kostova et al., 2006).
To ascertain whether chronic topical application can result in systemic exposure, blood and liver samples were tested for levels of SF and its metabolites, collectively known as dithio-carbamates (DTCs) (Zhang et al., 1996; Ye et al., 2002). The levels of SF and DTCs in blood and liver homogenates were below the sensitivity of the assay. Liver homogenates were also tested for NQO1 activity, and there was no significant difference between control groups (1150 mOD/min/mg in untreated mice; 1053 mOD/min/mg in vehicle-treated mice) and SF-treated mice (1161 mOD/min/mg). Taken together, these results suggest that systemic exposure is unlikely to occur in the context of the treatment regimen used.
Example 4
Optimization of Sulforaphane Treatment Regime for K14 Null Mice
The effectiveness of treatment with sulforaphane (SF) in preventing or reducing skin blistering was tested in K14 null mice. The treatment regimen initially used entailed topical application of 1 μmol SF in jojoba oil (100 μl volume) at P0, P1, and P3. This postnatal treatment regimen reduced cutaneous blistering in several K14 null pups, but the “clinical success” achieved proved variable. Histological analyses revealed that K14-null mice already exhibited a significant amount of “sub-clinical blistering” at birth, that is, prior to the first SF application. In other instances where K14 null mice were successfully rescued, expression of the “rescue” keratin (K16, K17) began prior to birth in basal keratinocytes of the epidermis (Paladini and Coulombe, 1999). SF administered to pregnant mice crosses the placental barrier (Coulombe and Kerns, unpublished data; Noyan-Ashraf et al., 2006), and could conceivably cause an induction of K16/K17 in fetal epidermis at a prenatal stage. Pregnant female mice were given an intraperitoneal (IP) injection of 5 μmol SF, and the embryos were retrieved and their skin and body were separately assayed for levels of SF and DTCs. These analyses provided evidence that K17 is indeed induced in fetal epidermis. Based on these results, the treatment regimen was set as consisting of 3 IP injections of 5 μmol SF administered to the mother every other day during the week prior to delivery, followed by topical application of 1 μmol SF (in 100 μl of jojoba oil) at P0, P1, P3 and P5 post-birth.
These findings established that the SF treatment regimen can be modified to meet the “clinical demands” of the overall phenotype.
Example 5
Sulforaphane Treatment Reduces Cutaneous Blistering in K14 Null Mice
The revised sulforaphane (SF) treatment regimen had a dramatic impact on the appearance and integrity of K14 null mouse skin. K14 null pups could no longer be identified based on their appearance and behavior at P0.5 and even P2.5 At P4.5, many of the K14 null pups showed limited blistering restricted to the front paws. During this early postnatal period, the difference between untreated and SF-treated K14 null pups was indeed dramatic. Whereas nearly all of the untreated K14 null pupa had died by P3 (n=14), 90% of the SF-treated K14 null pups (n=26) were alive and thriving at P4.
Histological analyses were conducted with a special focus on forepaw, which consistently shows very severe skin blistering in K14 null mice. At P2.5, the skin was significantly protected in SF-treated mice relative to untreated K14 null controls. K14 null pups had markedly less sub-clinical blistering of their forepaws, back skin, and snout than control at P0. These data were confirmed by quantification of the surface area of forepaw skin showing blistering in untreated and SF-treated K14 null mice.
Virtually all of the SF-treated K14-null mice developed progressive wasting beyond P4, and most of them died within a day or two. Unlike skin, which remained largely blister-free, the lips and oral mucosa showed the telltale signs of severe blistering. The topical mode of SF delivery during the postnatal phase of the treatment regimen was not effective for maintenance of K16/K17 expression in the oral mucosa, and accordingly this component of the K14 null phenotype was likely responsible for the demise of the mice beyond P4.
Example 6
The Therapeutic Benefit of Sulforaphane Correlates with K17 Induction in Epidermis
Sulforaphane (SF) treatment regimen had no obvious impact on mouse epidermal architecture. To further probe into this issue, intact back skin tissue from P2 mice, SF-treated and untreated, was subjected to ultrastructural and immunohistochemical analyses. Phenotypic rescue correlated with the presence of K17 antigens in the basal layer of SF-treated epidermis. These results contrasted with untreated K14 null skin, which showed a spotty distribution of K17 (and K16) restricted to the suprabasal compartment. Early and late differentiation markers, such as K1 and filaggrin, were completely normal in SF-treated K14-null mouse epidermis.
The K14, K16 and K17 genes and proteins are highly conserved between human and mouse at the level of sequence (FIG. 2), tissue distribution, and regulation (McGowan et al., 1998; Coulombe et al., 2004). In particular, these keratins' ability to provide structural support in the epidermis are similar (Paladini and Coulombe, 1999; Kerns et al.). Thus, the data collected from the K14 null mouse model for EBS are directly applicable to the skin of patients suffering from EBS as a result of mutations at the K14 locus. While there are only a few reports describing the equivalent of a K14 null mutation in the human population (Chan et al., 1994; Rugg et al., 1994; Jonkman et al., 1996; El-Ghalbzouri et al., 2003), the present inventors have provided evidence that the SF-dependent induction of K16 and K17 in the basal keratinocytes of human epidermis alleviates the dominant negative impact of missense K14 alleles.
Accordingly, topical application of SF or an Nrf2 inducer is effective in preventing skin blistering in the relevant subset of EBS patients. Studies conducted in our laboratory have shown that SF-induced K17 protein is very long-lived in newborn as well as adult mouse epidermis (Bernot et al., 2005). The data provided above show that, in the mouse, there is no systemic exposure to SF or a pharmacologically active metabolite in the context of the topical treatment regimen devised. These findings indicate that, beyond infancy, EBS patients will achieve a significant preventive benefit from topical application of SF or an Nrf2 inducer twice a week for most body sites and under most conditions. More frequent applications may be needed during the first few months after birth and especially during the neonatal period, when for unknown reasons the EBS clinical symptoms are most pronounced (Fine et al., 1991; 2000).
SF-mediated Induction of K16 and K17, along with its impact on the expression or metabolic and anti-oxidant enzymes and proteins, is also beneficial in the treatment of other conditions in which the skin exhibits fragility as a result of a mutation in a gene encoding a key cytoskeletal component.
It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover the modification and variations of the invention provided they come within the scope of the appended claims and their equivalents.
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## Claims

1. A method for treating or preventing skin blistering in a patient in need thereof or at risk of developing skin blistering, comprising administering to the patient a composition comprising a therapeutically effective amount of an Nrf2 inducer.

2. The method of claim 1, wherein the patient suffers from skin blistering.

3. The method of claim 1, wherein the Nrf2 inducer is a phase II enzyme inducer.

4. The method of claim 3, wherein the phase II inducer is an isothiocyanate.

5. The method of claim 4, wherein the phase II enzyme inducer is sulforaphane.

6. The method of claim 4, wherein the phase II enzyme inducer is a sulforaphane synthetic analogue.

7. The method of claim 6, wherein the sulforaphane synthetic analogue is exo-2-acetyl-6-isothiocyanatonorbomane.

8. The method of claim 1, wherein the Nrf2 inducer is keratinocyte growth factor.

9. The method of claim 1, wherein the Nrf2 inducer is oltipraz.

10. The method of claim 1, wherein the Nrf2 inducer is ethacrynic acid.

11. The method of claim 1, wherein the Nrf2 inducer causes the selective induction of K6, K16 or K17 in the keratinocytes in the skin of the patient.

12. The method of claim 1, wherein the composition comprising the NRF2 inducer is topically administered to the patient.

13. The method of claim 12, wherein the amount of Nrf2 inducer in the composition topically administered to the patient is from about 100 nmol/cm2 to about 1 μmol/cm2.

14. The method of claim 12, wherein the composition comprising the Nrf2 inducer is a topical preparation selected from the group consisting of ointment, cream, emulsion, lotion and gel.

15. The method of claim 13, wherein the composition father comprises one or more pharmaceutical, biological or molecular biological active agents.

16. The method of claim 1, wherein the patient is a mammal.

17. The method of claim 16, wherein the mammal is a human.

18. The method of claim 1, wherein the patient suffers from Epidermolysis bullosa simplex.

19. The method of claim 1, wherein the patient suffers from Epidermolysis bullosa simplex-Weber-Cockayne type, Epidermolysis bullosa simplex-Koebner type, Epidermolysis bullosa simplex with mottled pigmentation, Epidermolysis bullosa simplex-Dowling-Meara type, or Epidermolysis bullosa simplex with muscular dystrophy.

20. The method of claim 19, wherein the Epidermolysis bullosa simplex is caused by a mutation at the K14 locus.

21. The method of claim 1, wherein the Nrf2 inducer is a phase II enzyme inducer.

22. The method of claim 21, wherein the phase II inducer is an isothiocyanate.

23. The method of claim 21, wherein the phase II enzyme inducer is sulforaphane.

24. The method of claim 21, wherein the phase II enzyme inducer is a sulforaphane synthetic analogue.

25. The method of claim 24, wherein the sulforaphane synthetic analogue is exo-2-acetyl-6-isothiocyanatonorbornane.

26. The method of claim 1, wherein said skin blistering is a keratin-based skin disease.

27. The method of claim 26, wherein the keratin-based skin disease is selected from the group consisting of epidermolytic hyperkeratosis, ichtyosis bullosa of Siemens, pachyonychia congenita, epidermolytic or non-epidermolytic palmoplantar keratoderma (diffuse or focal) steatocystoma multiplex, Naegeli-Franceschetti4adessohn syndrome and dermatopathia pigmentosa reticularis.

28. The method of claim 26, wherein the disease is caused by a mutation at the K14 locus.

29. The method of claim 26, wherein the Nrf2 inducer is a phase II enzyme inducer.

30. The method of claim 29, wherein the phase II inducer is an isothiocyanate.

31. The method of claim 29, wherein the phase II enzyme inducer is sulforaphane.

32. The method of claim 29, wherein the phase II enzyme inducer is a sulforaphane synthetic analogue.

33. The method of claim 32, wherein the sulforaphane synthetic analogue is exo-2-acetyl-6-isothiocyanatonorbornane.

34. The method of claim 26, wherein the Nrf2 inducer is keratinocyte growth factor.

35. The method of claim 26, wherein the Nrf2 inducer is oltipraz.

36. The method of claim 26, wherein the Nrf2 inducer is ethacrynic acid.

37. The method of claim 26, wherein the Nrf2 inducer causes the selective induction of K6, K16 or K17 in the keratinocytes in the skin of the patient.

38. The method of claim 26, wherein the composition comprising the Nrf2 inducer is topically administered to the patient.

39. The method of claim 38, wherein the amount of Nrf2 inducer in the composition topically administered to the patient is from about 100 nmol to about 1 μmol/cm2.

40. The method of claim 38, wherein the composition comprising the Nrf2 inducer is a topical preparation selected from the group consisting of ointment, cream, emulsion, lotion and gel.

41. The method of claim 39, wherein the composition further comprises one or more pharmaceutical, biological or molecular biological active agents.

42. The method of claim 26, wherein the patient is a mammal.

43. The method of claim 42, wherein the mammal is a human.

44. The method of claim 15, wherein the one or more pharmaceutical, biological or molecular biological active agents comprises jojoba oil.

45. The method of claim 41, wherein the one or more pharmaceutical, biological or molecular biological active agents comprises jojoba oil.

46. The method of claim 1, further comprising identifying a patient with skin blistering or pre-disposed to developing skin blistering.

47. The method of claim 1, wherein the method comprises treating skin blistering in a patient in need thereof comprising administering to the patient a composition comprising a therapeutically effective amount of an Nrf2 inducer.