U.S. patent application number 12/690494 was filed with the patent office on 2011-01-06 for use of nrf2 inducers to treat epidermolysis bullosa simplex and related diseases.
Invention is credited to Pierre A. Coloumbe, Michelle L. Kerns.
Application Number | 20110003747 12/690494 |
Document ID | / |
Family ID | 39765040 |
Filed Date | 2011-01-06 |
United States Patent
Application |
20110003747 |
Kind Code |
A1 |
Coloumbe; Pierre A. ; et
al. |
January 6, 2011 |
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.
Inventors: |
Coloumbe; Pierre A.;
(Laurel, MD) ; Kerns; Michelle L.; (Baltimore,
MD) |
Correspondence
Address: |
EDWARDS ANGELL PALMER & DODGE LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Family ID: |
39765040 |
Appl. No.: |
12/690494 |
Filed: |
July 17, 2008 |
PCT Filed: |
July 17, 2008 |
PCT NO: |
PCT/US08/08694 |
371 Date: |
September 3, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60929985 |
Jul 20, 2007 |
|
|
|
Current U.S.
Class: |
514/9.2 ;
514/514 |
Current CPC
Class: |
A61K 31/497 20130101;
A61K 9/0014 20130101; A61K 38/1825 20130101; A61K 31/192 20130101;
A61K 31/00 20130101; A61P 17/00 20180101; A61K 31/26 20130101; A61K
31/275 20130101 |
Class at
Publication: |
514/9.2 ;
514/514 |
International
Class: |
A61K 31/26 20060101
A61K031/26; A61K 38/18 20060101 A61K038/18; A61P 17/00 20060101
A61P017/00 |
Claims
1. A method to ameliorate the mechanical resilience of skin in a
patient in need thereof 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 IT enzyme inducer is
sulforaphane.
6. The method of claim 4, wherein the phase II enzyme inducer is a
sulforaphane synthetic analogue.
7. (canceled)
8. The method of claim 1, wherein the Nrf2 inducer is keratinocyte
growth factor.
9-11. (canceled)
12. The method of claim 1, wherein the composition comprising the
Nrf2 inducer is topically administered to the patient.
13-15. (canceled)
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. 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.
19. The method of claim 18, wherein the patient suffers from
Epidermolysis bullosa simplex.
20. The method of claim 18, 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.
21. The method of claim 20, wherein the Epidermolysis bullosa
simplex is caused by a mutation at the K14 locus.
22. The method of claim 18, wherein the Nrf2 inducer is a phase II
enzyme inducer.
23-36. (canceled)
37. 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.
38. The method of claim 37, 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,
Nacgeli-Franceschetti-Jadassohn syndrome and dermatopathia
pigmentosa reticularis.
39-54. (canceled)
55. A composition for topical application to the skin comprising a
therapeutically effective amount of an Nrf2 inducer and a carrier
that allows the Nrf2 inducer to reach basal keratinocytes in the
epidermis.
56. (canceled)
57. The composition of claim 55, wherein the Nrf2 inducer is a
phase II enzyme inducer.
58-67. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/929,985.
BACKGROUND OF THE INVENTION
[0002] 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.
[0003] 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 cytoskeleton
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 et 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, K6.alpha., K6.beta.
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 gene (Wong et al., 2005). Type
I epidermal keratin genes K17, K16 and K17 share high amino acid
sequence identity (Troyanovsky et al., 1992; McGowan and Coulombe,
1998a; 1998b), and are structurally and functionally related
(Paladini and Coulombe, 1999; Coulombe et al., 2004; Tong and
Coulombe, 2006).
[0004] 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; Gu and Coulomb;
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).
[0005] Epidermolysis bullosa simplex (EBS) is a rare autosomal
dominant disease in which the epidermis loses its integrity
following trivial mechanical trauma (Fine et 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 et al. 2002; Gu and Coulombe 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 et 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 Coulombe, 2007, and refs. therein). Individuals affected by
EBS-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 et al., 2000).
[0006] 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 (see Fuchs and
Cleveland, 1998; Cassidy et al., 2002; Omary et al., 2004; Gu and
Coulombe, 2007).
[0007] 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
[0008] It is, therefore, an object of the invention to provide
solutions to the aforementioned deficiencies in the art.
[0009] 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.
[0010] 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 TI 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 factor 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.
[0011] 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.
[0012] 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.
[0013] 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 et 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,
[0014] 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.
[0015] 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.
[0016] 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.
[0017] 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.
[0018] 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 TYKE DRAWINGS
[0019] 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,
[0020] 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).
[0021] 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.
[0022] FIG. 2 illustrates the alignment of the predicted amino acid
sequences for mouse K17 and human K17, K17 and K16. 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 .alpha.-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
[0023] Transcription factor NF-E-related factor 2 (Nrf) belongs to
the CNC (Cap-N-Collar) family of transcription factors and
possesses a highly conserved basic region-leucine zipper (blip)
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 .gamma.-glutamylcysteine synthetase (GCS),
in response to oxidative and xenobiotic stress (Braun et al., 2002;
Fahey et 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).
[0024] 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).
[0025] 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;
Iida et al., 2004). To date, nine classes of phase II enzyme
inducers have been identified: 1) diphenols, phenylene diamincs 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 oxido-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).
[0026] 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 et al., 2006). Oltipraz
prevents carcinogenesis in liver and urinary bladder by enhancing
carcinogen detoxification (Iida et 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).
[0027] Isothiocyanates, which are primarily derived from in
calciferous 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
.beta.-thioglucoside-N-hydroxysulfates, when maceration of the
vegetables by predators, food preparation or chewing causes
disruption of the cells with consequent activation and release of
the enzyme myrosinase. The resultant aglycones undergo
non-enzymatic intramolecular rearrangement to yield
isothiocyanates, nitriles and epithionitriles.
[0028] Sulforaphane is the aglycone breakdown product of the
glucosinolate glucoraphanin, also known as sulforaphane
glucosinolate (SGS). The molecular formula of sulforaphane is
C.sub.6H.sub.11NOS.sub.2, 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:
##STR00001##
[0029] Sulforaphane was first synthesized (Schmid and Karrer,
1948), 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 et al., 1994), prevent promotion of mouse skin
tumorigenesis (Gills et al., 2006; Xu et al., 2006) and increase
heme oxygenase-1 (HO-1) expression in human hepatoma HepG2 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 1 (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
et al., 2006), and inhibits cell growth, activates apoptosis,
inhibits histone deacetylase (HDAC) activity and decreases the
expression of estrogen receptor-.alpha., 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 (Pledgic-Tracy et al., 2007). Further,
sulforaphane was showed to eradicate Helicobacter pylori from human
gastric xenografts (Haristoy et al., 2003).
[0030] 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.
[0031] Type I 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 EBS 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.).
[0032] 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 at.). 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.
[0033] 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 skin, including the
age of the subject being treated and the site of treatment in the
body.
[0034] 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.
[0035] 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.
[0036] 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 a
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.
[0037] As used herein, "a pharmaceutically effective amount" is
intended to mean an amount effective to elicit a cellular response
that is clinically significant.
[0038] 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.
[0039] 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 United States
Reissue Patent 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-methylsulfonylnorbornane,
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.
[0040] 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-alkaloses.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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 (C.sub.10 MSO) and tetradecylmethyl
sulfoxide; pyrrolidones, urea; N,N-diethyl-m-toluamide;
C.sub.2-C.sub.6 alkanediols; dimethyl formamide (DMF),
N,N-dimethylacetamide (DMA) and tetrahydrofurfuryl alcohol.
[0046] Examples of solubilizers include, but are not limited to,
hydrophilic ethers such as diethylene glycol monoethyl ether
(ethoxydiglycol, available commercially as Transcutol.RTM.) and
diethylene glycol monoethyl ether oleate (available commercially as
Softcutol.RTM.); 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.RTM.); alkyl methyl sulfoxides, such as
DMSO; pyrrolidones, DMA, and mixtures thereof.
[0047] 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.
[0048] 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.
[0049] Typically, the amount of Nrf2 inducer in the composition
topically administered to the patient will be from about 100 nmol
to about 1 .mu.mol/cm.sup.2, 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
[0050] 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
[0051] 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.
[0052] The K14 null mouse strain (Lloyd et 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 axe 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.sup.+/+ or K14.sup.+/-). By P, 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 P, 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 Gli2 Rescues Skin Blistering in K14 Null Mice
but not in K5 Null Mice
[0053] Keratin 17 is a direct target for the transcription factor
Gli, a powerful terminal effector of hedgehog signaling pathways
(Bianchi et al., 2005). In Gli2.sup.TG transgenic mice (Grachtchouk
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. Gli2.sup.TG 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 Gli2.sup.TG transgenic mice provided
an opportunity to conduct a "proof of principle" experiment,
whereby constitutive expression of Gli2.sup.TG transgene in the
setting of K14.sup.-/- mice should cause a stable upregulation of
K17 in basal keratinocytes of epidermis, and hence, rescue their
oral and skin blistering. Conversely, the Gli2.sup.TG 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 ee al).
[0054] Gli2.sup.TG 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.sup.-/- littermates, the
K14.sup.-/- Gli2.sup.TG mice were initially viable and showed
normal skin, correlating with the presence of K17 in basal cells.
This was in contrast to K5.sup.-/- Gli2.sup.TG 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
[0055] To evaluate the effect of sulforaphane (SF) on keratin gene
transcription in vitro, a mouse keratinocyte line (308 cells) was
exposed to 1 .mu.M 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 .about.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.
[0056] 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 .mu.M
concentration in the culture medium. At higher concentrations (5
.mu.M), 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 .mu.M), SF does not cause apoptosis in cultured primary mouse
keratinocytes.
[0057] 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 .mu.mole 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).
[0058] 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 (OTCs) (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
[0059] 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 .mu.mol SF in jojoba oil (100 .mu.l 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 (1016, 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 .mu.mol 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
.mu.mol SF administered to the mother every other day during the
week prior to delivery, followed by topical application of 1
.mu.mol SF (in 100 .mu.l of jojoba oil) at P0, P1, P3 and P5
post-birth.
[0060] 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
[0061] 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 pups had died by P3 (n=14),
90% of the SF-treated K14 null pups (n=26) were alive and thriving
at P4.
[0062] 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.
[0063] 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
[0064] 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.
[0065] 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.
[0066] 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).
[0067] 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.
[0068] 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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