WO2015169884A2 - Compounds and methods for the treatment of itch - Google Patents

Compounds and methods for the treatment of itch Download PDF

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WO2015169884A2
WO2015169884A2 PCT/EP2015/060019 EP2015060019W WO2015169884A2 WO 2015169884 A2 WO2015169884 A2 WO 2015169884A2 EP 2015060019 W EP2015060019 W EP 2015060019W WO 2015169884 A2 WO2015169884 A2 WO 2015169884A2
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compound
etar
itch
endothelin
fluoro
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WO2015169884A3 (en
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Joerg BUDDENKOTTE
Martin Steinhoff
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Westfaelische Wilhelms-Universitaet Muenster
The Regents Of The University Of California
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    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
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    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
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    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
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    • C12Y304/24071—Endothelin-converting enzyme 1 (3.4.24.71)

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Abstract

The present application provides a compound for use in the treatment of a subject suffering from itch, wherein said compound is endothelin-converting enzyme (ECE-1), a MEK1 inhibitor, or an ETAR inhibitor. Similarly, these compounds can be used in a topic pharmaceutical composition and for the preparation of a medicament. Also, the present invention provides a method for the screening of a compound useful for the treatment of histamine-independent, endothelin-1 (ET-1)/endothelin A receptor (ETAR) induced itch and a method for diagnosing a subject suffering from a histamine-independent, ET-1/ETAR induced itch.

Description

COMPOUNDS AND METHODS FOR THE TREATMENT OF ITCH
FIELD OF THE INVENTION
[0001] The present invention relates to the field of itch treatment. In particular, the present invention is related to compounds and methods of treating itch and preparing pharmaceutical compositions for the itch treatment. In another aspect, the invention relates to diagnosing subjects suffering from itch and treating such subjects.
BACKGROUND OF THE INVENTION
[0002] The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
[0003] Itch or pruritus is a common dermatological symptom of numerous skin and systemic diseases in humans and animals such as dermatologic, allergic, autoimmune diseases and tumors, but its molecular and cellular basis is still poorly understood. Itch is an unpleasant sensation that elicits the desire to scratch, and - similar to chronic pain - can be deleterious. Since all human species experience pruritus in the course of their life time, one can distinguish between acute itch, which is of a limited time period ranging from seconds to weeks and serves as an important protective function by warning against harmful agents in the environment such as insects, toxic plants or other irritants, and chronic pruritus, which lasts for greater than 6 weeks. Chronic pruritus (itch) is a common symptom that relates to many different diseases, e.g. prurigo nodularis, atopic dermatitis, allergies, psoriasis, urticaria, uremic pruritus of dialysis patients, diabetes mellitus or leukemia.
[0004] Generalized pruritus may be classified in different categories on the basis of the underlying causative disease. However, due to the poorly understood pathophysiology and neurological basis of pruritus, the development of effective treatment modalities for pruritus has proven to be particularly difficult. The therapy of pruritus is challenging and currently takes on an individualistic approach. At present, there is no universally accepted, well established therapy regime for pruritogenic skin disease, and pruritus therapy varies depending on the underlying etiology (1 ). New therapies are based on advances in the understanding of the mechanisms that cause pruritus. Histamine is the best known endogenous agent that evokes itch. Even though histamine-induced itch has been studied for some time, the underlying mechanism of itch is just beginning to emerge (2). Previous treatments for pruritus including the use of antihistamines and corticosteroids offer marginal benefit and have undesired side effects, e.g. result in allergic contact dermatitis, but are tried because of their low cost and potential for providing relief.
[0005] Itch can be triggered by exogenous (e.g. allergens, toxins, medication, illegal drugs, microbes) or endogenous (e.g. amines, proteases, neuropeptides, cytokines, prostanoids) stimuli that transmit signals via C-fibers to the central nervous system. While the existence of itch-selective C-fibers in the sensory nervous system appears to be generally acknowledged, the neuronal regulatory circuits and the intracellular cell signaling cascade during pruritus in both the peripheral and central nervous system are still not well understood, including potential endogenous anti-pruritic mechanisms.
[0006] Recently, the existence of at least three distinct molecular pathways that contribute to the transduction of itch responses to different pruritogens have been described (3). Thus, it has been discovered that the function of phospholipase C33 (ΡΙ β3), a component of a canonical signal transduction cascade, is critical for serotonin- and histamine-induced scratching in mice. It has also been reported that histamine requires a functional transient receptor potential cation channel V1 (TRPV1 ) for mediating itch transmission, whereas serotonin elicits itch independently of TRPV1 . Another TRP-family member, TRP ankyrin A1 (TRPA1 ), is necessary for histamine-independent itch that is induced by the Mas-related G protein-coupled receptor-mediated or endothelin-1 (ET-l )-induced itch. TRPAq is required not only for transduction of chronic itch to the central nervous system (CNS), but also for the changes in gene expression that was observed in both neurons and skin. However, ET-1 requires neither histamine-1 receptor (H1 R), TRPV1 nor ΡΙ β3 function for itch induction. ET-1 is a 21 -amino acid peptide and is expressed by a variety of cell types, including immune cells, endothelial cells, neurons and glial cells of the central and peripheral nervous system (4-10). ET-1 is a potent vasoconstrictor that can also evoke pain sensations in rodents and humans (3, 6, 1 1 -20). ET-1 is upregulated in response to hypoxia, sheer stress and a range of inflammatory cytokines. ET-1 is further involved in cell signaling and inflammatory processes, which are important in chronic itchy skin disorders such as atopic dermatitis (22).
[0007] The biological effects of ET-1 are mediated by two distinct G protein-coupled receptors (GPCRs): endothelin A receptor (ETAR) and endothelin B receptor (ETBR) (21 ). The pruritogenic effect of ET-1 in rodents is mediated in large part by ETAR, although expression of ETBR has been detected in satellite glial cells and non-myelinated Schwann cells of dorsal root ganglia (DRG) (17, 23). When activated, ETAR internalizes and recycles back to the plasma membrane, whereas ETBR internalizes but apparently does not recycle (27). Although different endothelin A B receptor inhibitors have been characterized in the past which could be useful to regulate the physiological or pathophysiological role of the endothelin receptors (24), the use of ETAR inhibitors is up to now directed to diverse therapeutic approaches associated with blood pressure regulation, in particular treatment of pulmonary arterial hypertension, and cancer diseases (25, 26). However, the use of ETAR inhibitors in therapeutic treatment of pruritogenic skin diseases such as prurigo nodularis and atopic dermatitis has not been described so far.
[0008] It was previously reported that the zinc metalloendopeptidase endothelin-converting enzyme-1 (ECE-1 ) is present in acidified endosomes and degrades neuropeptides to promote receptor recycling and resensitization of GPCRs that induce neurogenic inflammation (28-30) and to terminate endosomal neuropeptide signaling (28, 29, 31 ). Although ET-1 is known to induce itch and pain in mouse models (33), and the meaning of ECE-1 in neuropeptide receptor internalization, recycling and cell signaling has been reported (28-31 ), a fundamental role of the intracellular neural peptidase ECE-1 in regulating the intracellular cell signaling in DRG neurons and the involvement of ECE-1 in the pathophysiology of ET-1 -induced pruritic disease has not been acknowledged so far. Moreover, a therapeutic treatment of itchy skin diseases using ECE-1 endopeptidase has not been discovered until now.
[0009] The mitogen-activated protein kinase (MAPK) pathway has emerged as the central piece of a signaling network regulating cell growth and survival, and the MAPK pathway has been widely observed to be dysregulated in various human malignancies. Thus, a wide spectrum of inhibitors against components of this pathway has demonstrated anti-cancer effects by suppressing tumor growth. Some time ago, the mitogen-activated protein kinase kinase (MEK) as downstream protein of the MAPK pathway has been described as target for inhibitors in cancer therapy, and MEK inhibitors were the first selective inhibitors of MAPK pathway activation to enter clinical trials (32). However, any importance of MEK inhibitors for use in the treatment of itch and pruritic skin disease has not been demonstrated so far.
[0010] With regard to the state of the art, there is a demand for new, specific inhibitors useful to treat deliterious itch in the course of pruritic disease. In particular there is a need for compounds useful to treat pathophysiologies which are ET-1 -induced, but histamine- independent. Such compounds can advantageously be utilized to design new medicaments for treatment of patients suffering from itch. Moreover, such medicaments would be particular useful to improve situation of patients suffering from chronic itchy diseases and to adjust the treatment regimen of said patients. Additionally, diagnosis of new molecular markers involved in the pathophysiology of pruritic diseases as well as cellular mechanisms underlying the development of histamine-independent, ET-1 -induced itch would be of great relevance in order to understand the underlying mechanism of itch in more details. [0011] In sum, there is an urgent need to provide new, alternative means and methods that help to treat patients suffering from itch. Further technical problems solved by the present invention will become apparent from the descriptions, examples and figures that follow.
SUMMARY OF THE INVENTION
[0012] The present invention is, at least partly, based on the surprising finding of several compounds to treat itch. In one aspect, the inventors discovered ECE-1 can be used to ET-1 induced pruritus. It is found for the first time that the ET-1/ETAR/ECE-1/MEK/ERK axis plays a central role in the pathophysiology of pruritic skin diseases, and that ECE-1 plays an central role in regulating the pathophysiology of ET-1 induced pruritic diseases. As shown in the examples of the present application, the cutaneous expression of ET-1 , ETAR and ECE- 1 was determined in mice in skin, cutaneous nerve fibers and DRG neurons. As disclosed by the present invention, mice with pruritic chronic dermatitis exhibited an increased ET-1 level in epidermal layers compared to normal skin, and it was demonstrated that ET-1 exerts its action on small- to medium-sized sensory nerves by binding to ETAR. Additionally, activation and trafficking of ETAR by ET-1 stimulation and modulation of ET-1 /ETAR induced itch by endosomal ECE-1 in peripheral sensory neurons was demonstrated by subcellular localization of an ET-1/ETAR-complex in early endosomes of DRG neurons, which are also ECE-1 positive.
[0013] The inventors also discovers that endosomal ECE-1 modulates itch by regulating ET- 1/ETAR signaling in mice and is the first identified endogenous negative regulator of itch signaling in sensory nerves. Moreover, it was shown that ETAR, ET-1 and ECE-1 are expressed and co-localized in murine DRG neurons and human skin nerves. In neurons of murine dorsal root ganglia neurons, ET-1 induced internalization of ETAR to ECE-1 - containing endosomes. ET-1 -induced scratching behavior in mice was significantly augmented by ECE-1 inhibition and abrogated by MEK1/ERK1/2 inhibition in vivo. Using iontophoresis, ET-1 was shown to be a potent histamine-independent pruritogen in humans.
[0014] The results of the present invention demonstrate for the first time that ECE-1 can be used as an active ingredient to treat subjects suffering from itch, in particular from histamine- independent, ET-1/ETAR induced itch.
[0015] The invention is also based at least partly on the discovery that ETAR inhibitors were locally injected into mice prior to ET-1 injection, showing a significantly reduced scratching behavior in a chronic pruritic dermatitis mouse model. Moreover, the present inventors showed that ETAR inhibition ameliorates itch in mouse model. The present invention contributes to the state of the art with the finding that the ET-1 /ETAR induced pathway plays an important role in controlling chronic itch in mice and identified ETAR inhibitors as useful compounds to treat itch, in particular histamine-independent, ET-1/ETAR induced itch.
[0016] Furthermore, the present inventors also discovered that the MAPK signaling (MEK- ERK pathway) is a key-mechanism involved in ET-1 /ETAR induced itch in DRG neurons. Treatment of DRG neurons with ERK1/2 and MEK1 inhibitors, respectively, prior to ET-1 application to mice successfully abolished of ET-1 -evoked scratching behavior in mouse models. MEK-ERK inhibition has now been discovered to suppress the effects of all tested pruritogens. Accordingly, the present invention identifies that the ET-1/ETAR/ECE- 1/MEK/ERK pathway serves as an important target for treating and regulate pruritus in humans. MEK1 inhibitors blocking the ET-1/ETAR induced pathway is now shown to be a compound useful for the treatment of a subject suffering from ET-1 /ETAR induced itch.
[0017] Moreover, the present discovery in rodents can be translated into the human system, which is of high relevance because the impact of ET-1 induced pruritus in humans is still poorly understood. The inventors compared the immunoreactivity of ETAR and ET-1 in skin biopsies from patients with atopic dermatitis and prurigo nodularis, and demonstrated that ET-1 also account for itch symptoms involved in human skin diseases. Immunoreactivity of ET-1 was significantly increased in all layers of the epidermis, but ETAR was not elevated, while no or very weak staining for ETAR and ET-1 was observed in healthy skin controls. Thus, the present invention provides the first evidence that human PN patients exhibit a high upregulation of ET-1. Further it was found that ET-1 elicited an itch response in human subjects that was mainly independent from histamine-1 receptor (H 1 R) function. Together, in vivo and ex vivo findings in human demonstrates that ET-1 is increased in chronic pruritic skin diseases, is released by C-fibers, keratinocytes and endothelial cells in prurigo, and activates ETAR on myelinated nerve fibers.
[0018] In sum, the inventors successfully identified several compounds that can be advantageously used for the treatment of a subject suffering from itch. Since at present no universally accepted, well established therapy regime for pruritic skin diseases is available and treatment of itch is unspecific, the present invention solves the need to of providing reliable therapeutic approaches in the treatment of pruritus.
[0019] The inventors of the present application innovatively suggest the treatment of pruritic skin diseases with the use of ECE1 , MEK-1 inhibitors, or ETAR inhibitors. ECE1 , MEK-1 inhibitors, and ETAR inhibitors are particularly useful to treat subjects suffering from a histamine-independent, ET-1 /ETAR induced itch and represent new tools for the treatment of pruritic skin disease.
[0020] In one aspect, the present invention provides a method of treating itch by compounds described in this patent. In another aspect the present invention provides endothelin- converting enzyme-1 (ECE-1 ) for use in the treatment of a subject suffering from itch. In another aspect, the present invention provides MEK1 inhibitors for use in the treatment of a subject suffering from itch. In a further aspect, the present invention provides endothelin A receptor (ETAR) inhibitors for use in the treatment of a subject suffering from itch.
[0021] Preferably, the itch to be treated is a histamine-independent, endothelin-1 (ET- 1 )/ETAR induced itch. In one embodiment, the subject of treatment has a chronic pruritic skin disease. In another embodiment, the chronic pruritic skin disease is atopic dermatitis. In another embodiment, the chronic pruritic skin disease is prurigo nodularis. In other embodiments, the chronic pruritic skin disease is a skin eczema. In a further embodiment, the chronic pruritic skin disease is xerosis cutis. In another embodiment, the chronic pruritic skin disease is Graver's disease. In another embodiment, the chronic pruritic skin disease is a cutaneous post herpetic itch.
[0022] In one embodiment, the MEK1 inhibitor of the present invention is N-[(2R)-2,3- dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide. In one embodiment, the MEK1 inhibitor of the present invention is 6-(4-bromo-2-fluoroanilino)-7-fluoro-N-(2- hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide. In one embodiment, the MEK1 inhibitor of the present invention is N-[(2S)-2,3-dihydroxypropyl]-3-(2-fluoro-4- iodoanilino)pyridine-4-carboxamide. In one embodiment, the MEK1 inhibitor of the present invention is 6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3- methylbenzimidazole-5-carboxamide. In one embodiment, the MEK1 inhibitor of the present invention is 2-(2-fluoro-4-iodoanilino)-N-(2-hydroxyethoxy)-1 ,5-dimethyl-6-oxopyridine-3- carboxamide. In one embodiment, the MEK1 inhibitor of the present invention is N-[3,4- difluoro-2-(2-fluoro-4-iodoanilino)-6-methoxyphenyl]-1 -[(2S)-2,3- dihydroxypropyl]cyclopropane-1 -sulfonamide. In one embodiment, the MEK1 inhibitor of the present invention is 2-(2-chloro-4-iodoanilino)-N-(cyclopropylmethoxy)-3,4- difluorobenzamide. In one embodiment, the MEK1 inhibitor of the present invention is N-[3- [3-cyclopropyl-5-(2-fluoro-4-iodoanilino)-6,8-dimethyl-2,4,7-trioxopyrido[4,3-d]pyrimidin-1 - yl]phenyl]acetamide. In one embodiment, the MEK1 inhibitor of the present invention is N-[3- [5-(2-aminopyrimidin-4-yl)-2-tert-butyl-1 ,3-thiazol-4-yl]-2-fluorophenyl]-2,6- difluorobenzenesulfonamide; methanesulfonic acid. In one embodiment, the MEK1 inhibitor of the present invention is N-[3-[5-(2-aminopyrimidin-4-yl)-2-tert-butyl-1 ,3-thiazol-4-yl]-2- fluorophenyl]-2,6-difluorobenzenesulfonamide. In one embodiment, the MEK1 inhibitor of the present invention is N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4- iodoanilino)benzamide. In one embodiment, the MEK1 inhibitor of the present invention is 3,4-difluoro-2-(2-fluoro-4-iodoanilino)-N-(2-hydroxyethoxy)-5-[(3-oxooxazinan-2- yl)methyl]benzamide. In one embodiment, the MEK1 inhibitor of the present invention is (2S,3S)-2-[(4R)-4-[4-[(2R)-2,3-dihydroxypropoxy]phenyl]-2,5-dioxoimidazolidin-1 -yl]-N-(2- fluoro-4-iodophenyl)-3-phenylbutanamide. In one embodiment, the MEK1 inhibitor of the present invention is 3-[(2R)-2,3-dihydroxypropyl]-6-fluoro-5-(2-fluoro-4-iodoanilino)-8- methylpyrido[2,3-d]pyrimidine-4,7-dione. In one embodiment, the MEK1 inhibitor of the present invention is [3,4-difluoro-2-(2-fluoro-4-iodoanilino)phenyl]-[3-hydroxy-3-[(2S)- piperidin-2-yl]azetidin-1 -yl]methanone. In one embodiment the MEK1 inhibitor of the present invention is 5-bromo-N-(2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4- iodoanilino)benzamide. In one embodiment, the MEK1 inhibitor of the present invention is 2- [4-[(2-butyl-4-oxo-1 ,3-diazaspiro[4.4]non-1 -en-3-yl)methyl]-2-(ethoxymethyl)phenyl]-N-(4,5- dimethyl-1 ,2-oxazol-3-yl)benzenesulfonamide. In one embodiment, the MEK1 inhibitor of the present invention is 2-[4-[(2-butyl-4-oxo-1 ,3-diazaspiro[4.4]non-1 -en-3-yl)methyl]-2- (ethoxymethyl)phenyl]-N-(3,4-dimethyl-1 ,2-oxazol-5-yl)benzenesulfonamide. In one embodiment, the MEK1 inhibitor of the present invention is 2-[4-[(2-butyl-4-oxo-1 ,3- diazaspiro[4.4]non-1 -en-3-yl)methyl]-2-propylphenyl]-N-(4,5-dimethyl-1 ,2-oxazol-3- yl)benzenesulfonamide.
[0023] In one embodiment, the ETAR inhibitor of the present invention is 4-tert-butyl-N-[6-(2- hydroxyethoxy)-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl)pyrimidin-4-yl]benzenesulfonamide. In one embodiment, the ETAR inhibitor of the present invention is (5-(4-bromophenyl)-6-[2- (5-bromopyrimidin-2-yl)oxyethoxy]-N-(propylsulfamoyl)pyrimidin-4-amine. In one embodiment, the ETAR inhibitor of the present invention is (2S)-2-[(4,6-dimethylpyrimidin-2- yl)oxy]-3-methoxy-3,3-diphenylpropanoic acid. In one embodiment, the ETAR inhibitor of the present invention is N-[6-(2-Hydroxyethoxy)-5-(2-methoxyphenoxy)-2-[2-(2H-tetrazol-5- yl)pyridin-4-yl]pyrimidin-4-yl]-5-propan-2-ylpyridine-2-sulfonamide. In one embodiment the ETAR inhibitor of the present invention is N-(4-chloro-3-methyl-1 ,2-oxazol-5-yl)-2-[2-(6- methyl-2H-1 ,3-benzodioxol-5-yl)acetyl]thiophene-3-sulfonamide. In one embodiment, the ETAR inhibitor of the present invention is N-(3-Methoxy-5-methylpyrazin-2-yl)-2-[4-(1 ,3,4- oxadiazol-2-yl)phenyl]pyridine-3-sulfonamide. In one embodiment, the ETAR inhibitor of the present invention is 2-[(3R,6R,9S,12R,15S)-6-(1 H-indol-3-ylmethyl)-9-(2-methylpropyl)- 2,5,8,1 1 ,14-pentaoxo-12-propan-2-yl-1 ,4,7,10,13-pentazabicyclo[13.3.0]octadecan-3- yl]acetic acid. In one embodiment the ETAR inhibitor of the present invention is 3- benzodioxol-5-yl)-1 -[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3- carboxylic acid. In one embodiment, the ETAR inhibitor of the present invention is (2R,3R,4S)-4-(1 ,3-benzodioxol-5-yl)-1 -[2-(dibutylamino)-2-oxoethyl]-2-(4- methoxyphenyl)pyrrolidine-3-carboxylic acid. In one embodiment, the ETAR inhibitor of the present invention is (2R,3R,4S)-4-(1 ,3-benzodioxol-5-yl)-1 -[2-(dibutylamino)-2-oxoethyl]-2-(2- methoxyphenyl)pyrrolidine-3-carboxylic acid. In one embodiment, the ETAR inhibitor of the present invention is 3-(1 ,3-benzodioxol-5-yl)-5-hydroxy-5-(4-methoxyphenyl)-4-[(3,4,5- trimethoxyphenyl)methyl]furan-2-one. In one embodiment, the ETAR inhibitor of the present invention is (2R)-2-[[(2R)-2-[[(2S)-2-(azepane-1 -carbonylamino)-4-methylpentanoyl]amino]-3- (1 -formylindol-3-yl)propanoyl]amino]-3-(1 H-indol-3-yl)propanoic acid. In one embodiment, the ETAR inhibitor of the present invention is 2-(1 ,3-benzodioxol-5-yl)-4-(4-methoxyphenyl)-4- oxo-3-[(3,4,5-trimethoxyphenyl)methyl]but-2-enoate. In one embodiment, the ETAR inhibitor of the present invention is (2S)-2-(4,6-dimethoxypyrimidin-2-yl)oxy-3-methoxy-3,3- diphenylpropanoic acid. In one embodiment, the ETAR inhibitor of the present invention is N- [6-methoxy-5-(2-methoxyphenoxy)-2-pyridin-4-ylpyrimidin-4-yl]-5-methylpyridine-2- sulfonamide. In one embodiment, the ETAR inhibitor of the present invention is N-[5-(2- methoxyphenoxy)-2-pyridin-4-yl-6-(trideuteriomethoxy)pyrimidin-4-yl]-5-methylpyridine-2- sulfonamide. In one embodiment, the ETAR inhibitor of the present invention is N-[6-(2- hydroxyethoxy)-5-(2-methoxyphenoxy)-2-[2-(2H-tetrazol-5-yl)pyridin-4-yl]pyrimidin-4-yl]-5- methylpyridine-2-sulfonamide. In one embodiment, the ETAR inhibitor of the present invention is [6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-[2-(1 ,2,3-triaza-4- azanidacyclopenta-2,5-dien-5-yl)pyridin-4-yl]pyrimidin-4-yl]-(5-methylpyridin-2- yl)sulfonylazanide. In one embodiment, the ETAR inhibitor of the present invention is 4- amino-N-(3,4-dimethylisoxazol-5-yl)benzenesulfonamide. In one embodiment, the ETAR inhibitor of the present invention is [(7R)-5-chloro-3-[(1 E,3E,5S)-3,5-dimethylhepta-1 ,3- dienyl]-7-methyl-6,8-dioxoisochromen-7-yl] acetate.
[0024] According to the present invention, the subject suffering from itch is a mammal, preferably human. In some embodiments the mammal is a mouse, guinea pig, rabbit, cat, dog, monkey, horse, cow, or other mammals.
[0025] Preferably, the compound for use in the treatment of a subject suffering from itch is administered in a therapeutically effective amount to said subject. This therapeutically effective amount inhibits or alleviates said histamine-independent, ET-1/ETAR induced itch. The therapeutically effective amount is preferably between about 0.01 mg per kg body weight and about 1 g per kg body weight. More preferably, the therapeutically effective amount is between 0.01 mg per kg body weight and 10 mg per kg body weight.
[0026] The administration of the compounds for use in the treatment of a subject suffering from itch according to the present invention can be carried out in method known in the art. In some embodiments, the administration is carried out orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, transdermally, or by application to mucous membranes, or combinations thereof. Preferably, the administration is carried out epicutaneously.
[0027] In some embodiments, the compound for use in the treatment of a subject suffering from itch is administered together with a pharmaceutically acceptable excipient. In some embodiments the compound for use in the treatment of a subject suffering from itch is administered together with a pharmaceutically acceptable carrier. In some embodiments the compound for use in the treatment of a subject suffering from itch is administered in a combination with another anti-pruritic drug. In some embodiments the compound for use in the treatment of a subject suffering from itch is administered in combination with a compound used to treat skin diseases. In some embodiments the combination is administered as a combined formulation. In some embodiments the combination is administered separate from each other. In some embodiments the compound for use in the treatment of a subject suffering from itch is administered in combination with ultraviolett radiation therapy.
[0028] In a further aspect, the present invention provides a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w ECE-1 and one or more pharmaceutically acceptable excipients. Another aspect of the present invention provides a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w MEK1 inhibitor and one or more pharmaceutically acceptable excipients. Still another aspect of the present invention provides a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w endothelin A receptor (ETAR) inhibitor and one or more pharmaceutically acceptable excipients.
[0029] In some embodiment, the topical pharmaceutical composition of the present invention is in the form of an ointment. In some embodiment, the topical pharmaceutical composition of the present invention is in the form of a cream. In some embodiment, the topical pharmaceutical composition of the present invention is in the form of a lotion. In some embodiment, the topical pharmaceutical composition of the present invention is in the form of a gel. In some embodiment, the topical pharmaceutical composition of the present invention is in the form of a balm-stick. In some embodiment, the topical pharmaceutical composition of the present invention is in the form of a spray. In some embodiment, the topical pharmaceutical composition of the present invention is in the form of foam.
[0030] The topical pharmaceutical composition of the present invention can further comprise a cooling agent. In some embodiments the cooling agent is menthol. In some embodiments the cooling agent is an isomer of menthol. In some embodiments the cooling agent is a menthol derivative. In some embodiments the cooling agent is 4-Methyl-3-(1 -pyrrolidinyl)- 2[5H]-furanone. In some embodiments the cooling agent is WS-23. In some embodiments the cooling agent is, lcilin. In some embodiments the cooling agent is lcilin Unilever Analog. In some embodiments the cooling agent is 5-methyl-4-(1 -pyrrolidinyl)-3-[2H]-furanone; 4,5- dimethyl-3-(1 -pyrrolidinyl)-2[5H]-furanone. In some embodiments the cooling agent is isopulegol, 3-(l-menthoxy)propane-1 ,2-diol. In some embodiments the cooling agent is 3-(l- menthoxy)-2-methylpropane-1 ,2-diol. In some embodiments the cooling agent is p- menthane-2,3-diol. In some embodiments the cooling agent is p-menthane-3,8-diol. In some embodiments the cooling agent is 6-isopropyl-9-methyl-1 ,4-dioxas-piro[4,5]decane-2- methanol. In some embodiments the cooling agent is menthyl succinate and its alkaline earth metal salts. In some embodiments the cooling agent is trimethylcyclohexanol. In some embodiments the cooling agent is N-ethyl-2-isopropyl-5-methylcyclohexanecarb-oxamide. In some embodiments the cooling agent is Japanese mint (Mentha arvensis) oil. In some embodiments the cooling agent is peppermint oil. In some embodiments the cooling agent is menthone. In some embodiments the cooling agent is menthone glycerol ketal. In some embodiments the cooling agent is menthyl lactate. In some embodiments the cooling agent is 3-(1 -menthoxy)ethan-1 -ol. In some embodiments the cooling agent is 3-(l-menthoxy)propan- 1 -ol, 3-(l-menthoxy)butan-1 -ol, 1 -menthylacetic acid N-ethylamide. In some embodiments the cooling agent is l-menthyl-4-hydroxypentanoate. In some embodiments the cooling agent is l-menthyl-3-hydroxybutyrate. In some embodiments the cooling agent is N,2,3-trimethyl-2-(1 - methylethyl)-butanamide. In some embodiments the cooling agent is spearmint oil. In some embodiments the cooling agent is camphor. In some embodiments the cooling agent is a camphor derivate.
[0031] In a further aspect, the present invention provides a method for the treatment of a subject suffering from itch, said method comprising administering a therapeutically effective amount of ECE-1 to a subject in need thereof. In another aspect, the present invention provides a method for the treatment of a subject suffering from itch, said method comprising administering a therapeutically effective amount of a MEK1 inhibitor to a subject in need thereof. In still another aspect, the present invention provides a method for the treatment of a subject suffering from itch, said method comprising administering a therapeutically effective amount of an endothelin A receptor (ETAR) inhibitor to a subject in need thereof.
[0032] Another aspect of the present invention provides a method for screening a compound useful for the treatment of a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch, the method comprising assaying said compound for the activity in inhibiting the ET-1/ETAR induced signaling cascade. The method for screening a compound useful for the treatment of a histamine-independent, endothelin-1 (ET- 1 )/endothelin A receptor (ETAR) induced itch comprises administering the compound suspected as to be useful for the treatment of a histamine-independent ET-1/ETAR induced itch to an animal suffering from an acute or chronic pruritic skin disease, wherein said animal is a histamine-impervious animal. The method for screening a compound useful for the treatment of a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch further comprises measuring the scratching movements of said animal within a defined time interval, and comparing the scratching movements of said animal to the scratching movements of a reference animal to which the suspected compound has not been administered, the scratching movements of said reference animal measured within the same defined time interval. A lower amount of scratching movements of the animal under administration of the suspected compound in comparison to the reference animal shows that said suspected compound is useful for the treatment of a histamine-independent, endothelin- 1 (ET-1 )/endothelin A receptor (ETAR) induced itch. An approximate or higher amount of scratching movements of the animal under administration of the suspected compound in comparison to the reference animal indicates that said suspected compound is not useful for the treatment of a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch.
[0033] In some embodiments, the histamine impervious animal of the present invention is a histamine-1 receptor (H1 R) knockout animal. In some embodiments, the histamine impervious animal of the present invention is a histamine-4 receptor (H4R) knockout animal. In some embodiments, the histamine impervious animal of the present invention is a histamine-1 receptor (H1 R) and histamine-4 receptor (H4R) double knockout animal. In some embodiments, the histamine impervious animal of the present invention is mast cell deficient animal. In some embodiments, the histamine impervious animal of the present invention is a mouse or a rat. In some embodiments...
[0034] Another aspect of the present invention provides a method for diagnosing histamine- independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch in a subject, the method comprising determining the amount of endothelin-1 in a skin sample of said subject, and comparing the amount of endothelin-1 determined in a) to a reference skin sample. In some embodiments the skin sample comprises keratinocytes. In some embodiments the skin sample comprises endothelial cells. In some embodiments the skin sample comprises C- fibres. In some embodiments the determination of ET-1 in a skin sample includes measuring the level of ET-1 mRNA in said skin sample. A significantly increased amount of ET-1 in all layers of the epidermis as compared to said reference sample demonstrates a histamine- independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch. No significantly increased amount of ET-1 in all layers of the epidermis as compared to said reference sample demonstrates that said subject does not suffer from a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch.
[0035] In a further aspect, the present invention provides a diagnostic kit for detecting a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch, comprising a binding molecule which specifically binds to endothelin-1 (ET-1 ). In some embodiment the binding molecule comprised by a diagnostic kit for detecting a histamine- independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch is an antibody. In some embodiments the antibody comprised by a diagnositic kit for detecting a histamine- independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch is a polyclonal antibody. In some embodiments the antibody comprised by a diagnositic kit for detecting a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch is a monoclonal antibody. In some embodiments the antibody comprised by a diagnositic kit for detecting a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch is a chimeric antibody. In some embodiments the antibody comprised by a diagnositic kit for detecting a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch is a human or humanized antibody. Said antibody can be present in bound or soluble form. In some embodiments the binding molecule comprised by a diagnostic kit for detecting a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch is a FISH probe.
[0036] In another aspect, the present invention provides a compound for the preparation of a medicament for treatment of a human subject suffering from histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch, wherein said compound is selected from the group consisting of endothelin-converting enzyme-1 (ECE-1 ), a MEK1 inhibitor, and an ETAR inhibitor.
* * *
[0037] Unless otherwise stated, the following terms used in this document, including the description and claims, have the definitions given below.
[0038] Those skilled in the art will recognize, or be able to ascertain, using not more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.
[0039] It is to be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.
[0040] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the methods and uses described herein. Such equivalents are intended to be encompassed by the present invention.
[0041] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or sometimes when used herein with the term "having". [0042] When used herein "consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein any of the terms "consisting", "consisting of" and "consisting essentially of may be replaced with either of the other two terms.
[0043] As used herein, the conjunctive term "and/or" between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by "and/or", a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term "and/or" as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term "and/or" as used herein.
[0044] As described herein, "preferred embodiment" means "preferred embodiment of the present invention". Likewise, as described herein, "various embodiments" and "another embodiment" means "various embodiments of the present invention" and "another embodiment of the present invention".
[0045] The word "about" as used herein refers to a value being within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. The term "about" is also used to indicate that the amount or value in question may be the value designated or some other value that is approximately the same. The phrase is intended to convey that similar values promote equivalent results or effects according to the invention. In this context "about" may refer to a range above and/or below of up to 10%. The word "about" refers in some embodiments to a range above and below a certain value that is up to 5%, such as up to up to 2%, up to 1 %, or up to 0.5 % above or below that value. In one embodiment "about" refers to a range up to 0.1 % above and below a given value.
[0046] Several documents are cited throughout the text of this disclosure. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. [0047] The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.
DETAILED DESCRIPTION OF THE INVENTION
[0048] The present invention provides, inter alia, new compounds for the treatment of a subject suffering from itch of pathophysiologies independent of histamine. Such compounds are preferably ECE-1 , MEK1 inhibitors, or ETAR inhibitors, which can further be used in a topic pharmaceutical composition and for the preparation of a medicament to treat pruritic skin diseases. Moreover, a method for the screening of a compound useful for the treatment of histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch and a method for diagnosing a subject suffering from a histamine-independent, ET-1/ETAR induced itch are provided herein.
[0049] The present inventors found that ET-1 elicits an itch response in human subjects mainly independent from histamine-1 receptor (H1 R) function and identified neural endothelin-converting enzyme-1 (ECE-1 ) as first intracellular, negative regulator of itch on sensory nerves. Moreover, the inventors showed that a neural peptidase, ECE-1 , directly regulates ET-1 -induced, histamine-independent pruritus in humans and mice, and implicate the ET-1/ECE-1/MEK ERK pathway as an important target to treat pruritus in humans. In this regard, the present inventors found that MEK1 inhibitors and ETAR inhibitors, only known to treat pulmonary arterial hypertension and cancer, can be used to treat a subject suffering from itch. This disclosure was unexpected as ECE-1 , MEK1 inhibitors and ETAR inhibitors have not been discovered so far as to treat subjects suffering from itch. Moreover, these compounds have not been described as to be particularly useful to treat a subject suffering from a histamine-independent, ET-1 /ETAR induced itch.
[0050] Moreover, to identify new compounds useful to selectively treat deliterious itch in the course of pruritic disease, the present invention also provides a method for screening a compound useful for the treatment of a histamine-independent, ET-1 -induced itch using a histamine-impervious animal model. The method comprises assaying said compound for the activity in inhibiting the ET-1/ETAR induced signaling cascade.
[0051] The findings of the present invention allow for the diagnosis of pruritic diseases. The present invention further provides methods for the diagnosis of a histamine-independent, ET- 1/ETAR induced itch and diagnostic kits for the detection of a histamine-independent, ET- 1/ETAR induced itch, which determines the amounts of ET-1 in a skin sample of a subject suspected as to suffer from said histamine-independent, ET-1/ETAR induced itch. The analysis of ET-1 quantities in the skin sample of a patient can be used to evaluate the progression of an anti-pruritic therapy.
[0052] Prior to the present invention, the central role of ECE-1 and the involvement of the MEK/ERK signaling cascade in ET-1/ETAR induced itch and pathophysiology of pruritic skin diseases was unknown. The disclosure of the present invention contributes to the understanding of the cellular mechanisms underlying the development of histamine- independent, ET-1 -induced itch. The present inventors emphasize, that the use of ECE-1 , MEK1 inhibitors, and ETAR inhibitors to specifically treat subjects suffering from deliterious itch should prompt serious consideration in the therapy of pruritic skin diseases. These compounds can further be utilized to design new medicaments for patients suffering from chronic itchy diseases such as atopic dermatitis, prurigo nodularis, neurodermatitis, urticaria, allergies, psoriasis, or uremic pruritusa.
[0053] The present invention provides, amongst others, methods for the treatment of a subject suffering from itch, wherein said method comprises the administration of a therapeutically effective amount of ECE-1 , a MEK1 inhibitor, or an ETAR inhibitor to a subject in need thereof. These therapeutic approaches will sustainably improve the physical conditions of a subject suffering in particular from a histamine independent, ET-1/ETAR induced pruritic skin disease.
Compounds for itch treatment
[0054] The present invention provides compounds for use in the treatment of a subject suffering from itch, including ECE-1 , MEK-1 inhibitors, ETAR inhibitors, ERK1/2 inhibitors and TRPA1 inhibitors.
[0055] The therapeutic use of ECE-1 in the treatment of subjects suffering from itch has never recognized before. Although ETAR inhibitors have been reported as to be useful in ETAR inhibition as directed to diverse therapeutic approaches associated with blood pressure regulation, in particular treatment of pulmonary arterial hypertension, and cancer diseases (25, 26), use of ETAR inhibitors in therapeutic treatment of pruritogenic skin diseases has not been described so far. Also the therapeutic use of MEK1 inhibitors as selective inhibitors of MAPK pathway in cancer therapy is acknowledged (32). It is surprising that MEK1 inhibitors can be used in the treatment of a subject suffering from itch.
[0056] As confirmed by the data disclosed herein, ECE-1 is important for the recycling of ETAR in DRG neurons (Figure 10) and ECE-1 inhibition prolongs ET-1 -induced phosphorylation of ERK1/2 in DRG neurons (Figure 13). In one aspect of the present invention, endothelin converting enzyme-1 (ECE-1 ) can be used in the treatment of a subject suffering from itch.
[0057] The terms "treat", "treating", or "treatment" as used herein means to reduce, stabilize, or inhibit the progression of itch and/or symptoms associated therewith. Said symptoms may be hallmarks of human pruritus such as epidermal hyperplasia, acanthosis, fibrosis, collagenosis, and/or an increased infiltration of lymphocyte like T-cells, mast cells, or eosinophiles into the dermis of said subject. Those in need of treatment include those already with the disorder as well as those prone to having the disorder. Preferably, a treatment reduces, stabilizes, or inhibits progression of a symptom that is associated with the presence and/or progression of a disease or pathological condition.
[0058] The term "subject" as used herein, also addressed as an individual, refers to a mammal including a human or a non-human mammal such as cattle. The methods, uses and compositions described in this document are generally applicable to both human and non- human mammals.
[0059] In various embodiments, the subject of the present invention treated with a compound as described elsewhere herein is a mammal. In some embodiment the mammal of the present invention is a mouse. In some embodiment the mammal of the present invention is a rat. In some embodiment the mammal of the present invention is a guinea pig. In some embodiment the mammal of the present invention is a rabbit. In some embodiment the mammal of the present invention is a cat. In some embodiment the mammal of the present invention is a dog. In some embodiment the mammal of the present invention is a monkey. In some embodiment the mammal of the present invention is a horse. In a preferred embodiment the mammal of the present invention is a human.
[0060] In the context of the present invention, the subject treated with a compound as disclosed elsewhere herein suffers from itch. The terms "itch" or "itchy" as used herein means the sensation that elicits the desire or reflex for scratching movements. According to the present invention, the terms "itch" and "itchy" can be equivalently replaced herein by the terms "pruritus" and "pruritic".
[0061] According to the present disclosure, the subject suffering from itch has an acute or chronic pruritic skin disease. Typical examples of disease causing pruritus (itch) and which are embodiments of the present invention are diabetes, hyperthyroidism, disorders of the parathyroid gland, carcinoid syndrome, hepatic disease, pregnancy, intrahepatic cholestasis, obstructive jaundice (in biliary tract or extrahepatic), primary biliary cirrhosis, drug induced cholestasis, chronic renal failure, uraemia, polycythaemia vera, iron deficiency, Hodgkin's Disease, Mycrosis fungoides, Lymphosarcoma, Chronic leukemia, Myleomatosis, Paraproteinaemia, Mast cell disease, HIV, Sezary's syndrome (T-cell lymphoma), leukaemia, multiple myeloma, Waldenstrom's macroglobinaemia, mycosis fungoides, benign gammopathy, systemic mastocytosis, haematological and lymphoproliferative disorders, carcinomatosis, adenocarcinoma and squamous cell carcinoma of various organ, multiple sclerosis and brain tumors.
[0062] In a preferred embodiment of the present invention, the pruritic skin disease is atopic dermatitis. In another preferred embodiments, the pruritic skin disease is prurigo nodularis/prurigo simplex subacuta/Lichen simplex chronicus. In still another preferred embodiments, the pruritic skin disease is a skin eczema. In still another preferred embodiments, the pruritic skin disease is xerosis cutis. In still another preferred embodiments, the pruritic skin disease is Graver's disease. In still another preferred embodiments, the pruritic skin disease is neuropathic postherpetic itch. In still another preferred embodiments, the pruritic skin disease is post zoster neuralgia. In still another preferred embodiments, the pruritic skin disease is acne vulgaris. In still another preferred embodiments, the pruritic skin disease is urticaria. In still another preferred embodiments, the pruritic skin disease is psoriasis. In still another preferred embodiments, the pruritic skin disease is uremic pruritus. In other embodiments, the pruritic skin disease is a skin disorder associated with chronic renal diseases, including, but not limited to, perpigmentation, pallor xerosis, ichthyosis, pruritus, prurigo nodularis, Kyrle disease, bacterial, fungal and viral infections (e.g. with Streptococcus species, Staphylococcus aureus, Tinea infections, herpes zoster), purpura, porphyria cutanea tarda, pseudoporphyria, calciphylaxis, benign nodular calcification, half-and-half nails, koilonychias, transverse leukonychia, onychomycosis, onycholysis, splinter hemorrhages, subungual hyperkeratosis, brittle hair, sparse body and scalp hair, alopecia, pingueculitis, angular cheilitis, uremic frost, and nephrogenic fibrosing dermopathy. In other embodiments, the pruritic skin disease is notalgia paresthetica. In other embodiments, the pruritic skin disease is lichen planus. In other embodiments, the pruritic skin disease is Chronic allergic and toxic contact dermatitis. In other embodiments, the pruritic skin disease is mastocytosis. In other embodiments, the pruritic skin disease is seborrhoic dermatitis. In other embodiments, the pruritic skin disease is neoplasms. In other embodiments, the pruritic skin disease is mycosis fungoides. In other embodiments, the pruritic skin disease is cutaneous T cell lymphoma. In other embodiments, the pruritic skin disease is Hodgkin lymphoma. In other embodiments, the pruritic skin disease is liver- disease associated pruritus. In other embodiments, the subject suffering from itch is has anorexia. Preferably, the pruritic skin disease is atopic dermatitis. Preferably, the pruritic skin disease is prurigo nodularis. Preferably, the pruritic skin disease is skin eczema. Preferably, the pruritic skin disease is xerosis cutis.
[0063] In some embodiment of the invention, the use of a compound for the treatment of a subject suffering from itch is associated with or caused by insect bites, such as insect bite inflammation, or is associated or caused by allergic or toxic reaction to insects or parasites, such as cinguantera toxin from fishes. In another embodiment, the use of a compound for the treatment of a subject suffering from itch is associated with insect stings or parasite bites. In another embodiment, the use of a compound for the treatment of a subject suffering from itch is associated with sunburn or any form of chronic pruritic photodermatitis. In another embodiment, the use of a compound for the treatment of a subject suffering from itch is associated with scabies. In another embodiment, the use of a compound for the treatment of a subject suffering from itch is associated with lichen simplex chronicus or lichen planus.
[0064] In some embodiment, the itch of the present invention is a histamine-independent itch. The term histamine-independent as used herein refers to itch induced independently from the action of histamine. As disclosed by the present invention, in human chronic pruritic diseases the pruritogenic effect is mediated in large part by ET-1/ETAR induction and the further activated intracellular signaling cascade. In fact, ET-1 elicited an itch response in human subjects that was mainly independent from histamine-1 receptor (H1 R) function (Figure 15C). Therefore, histamine and serotonin are not involved in itch induction via the ET-1/ETAR/ECE-1/MEK/ERK pathway. In a preferred embodiment of the present invention is particularly useful for histamine-independent, ET-1/ETAR induced itch.
[0065] The MAPK signaling (MEK-ERK pathway) is a key-mechanism involved in ET- 1/ETAR induced itch in DRG neurons and treatment of DRG neurons with ERK1/2 and MEK1 inhibitors, respectively, prior to ET-1 application to mice abolished of ET-1 -evoked scratching behavior in mouse models (Figure 14). Accordingly, the present invention demonstrates that blocking the ET-1/ETAR/ECE-1/MEK/ERK pathway using a MEK1 inhibitors are particularly useful for the treatment of a subject suffering from itch, eminently histamine-independent, ET-1/ETAR induced itch. Therefore, in another aspect of the present invention, the compound for use in the treatment of a subject suffering from itch is a mitogen- activated protein kinase kinase 1 (MEK1 ) inhibitor.
[0066] Any MEK1 inhibitors can be used within the scope of the present invention. Preferred MEK1 inhibitors for use in the treatment of a subject suffering from itch according to the present invention are depicted in Figure 3. In some embodiments, the MEK1 inhibitor according to the present invention is N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-
4- iodoanilino)benzamide. In some embodiments, the MEK1 inhibitor according to the present invention is 6-(4-bromo-2-fluoroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-
5- carboxamide. In some embodiments, the MEK1 inhibitor according to the present invention is N-[(2S)-2,3-dihydroxypropyl]-3-(2-fluoro-4-iodoanilino)pyridine-4-carboxamide. In some embodiments, the MEK1 inhibitor according to the present invention is 6-(4-bromo-2- chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide. In some embodiments, the MEK1 inhibitor according to the present invention is 2-(2-fluoro-4- iodoanilino)-N-(2-hydroxyethoxy)-1 ,5-dimethyl-6-oxopyridine-3-carboxamide. In some embodiments, the MEK1 inhibitor according to the present invention is N-[3,4-difluoro-2-(2- fluoro-4-iodoanilino)-6-methoxyphenyl]-1 -[(2S)-2,3-dihydroxypropyl]cyclopropane-1 - sulfonamide. In some embodiments, the MEK1 inhibitor according to the present invention is 2-(2-chloro-4-iodoanilino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide. In some embodiments, the MEK1 inhibitor according to the present invention is N-[3-[3-cyclopropyl-5- (2-fluoro-4-iodoanilino)-6,8-dimethyl-2,4,7-trioxopyrido[4,3-d]pyrimidin-1 -yl]phenyl]acetamide. In some embodiments, the MEK1 inhibitor according to the present invention is N-[3-[5-(2- aminopyrimidin-4-yl)-2-tert-butyl-1 ,3-thiazol-4-yl]-2-fluorophenyl]-2,6- difluorobenzenesulfonamide;methanesulfonic acid. In some embodiments, the MEK1 inhibitor according to the present invention is N-[3-[5-(2-aminopyrimidin-4-yl)-2-tert-butyl-1 ,3- thiazol-4-yl]-2-fluorophenyl]-2,6-difluorobenzenesulfonamide. In some embodiments, the MEK1 inhibitor according to the present invention is N-[(2R)-2,3-dihydroxypropoxy]-3,4- difluoro-2-(2-fluoro-4-iodoanilino)benzamide. In some embodiments, the MEK1 inhibitor according to the present invention is 3,4-difluoro-2-(2-fluoro-4-iodoanilino)-N-(2- hydroxyethoxy)-5-[(3-oxooxazinan-2-yl)methyl]benzamide. In some embodiments, the MEK1 inhibitor according to the present invention is (2S,3S)-2-[(4R)-4-[4-[(2R)-2,3- dihydroxypropoxy]phenyl]-2,5-dioxoimidazolidin-1 -yl]-N-(2-fluoro-4-iodophenyl)-3- phenylbutanamide. In some embodiments, the MEK1 inhibitor according to the present invention is 3-[(2R)-2,3-dihydroxypropyl]-6-fluoro-5-(2-fluoro-4-iodoanilino)-8- methylpyrido[2,3-d]pyrimidine-4,7-dione. In some embodiments, the MEK1 inhibitor according to the present invention is [3,4-difluoro-2-(2-fluoro-4-iodoanilino)phenyl]-[3- hydroxy-3-[(2S)-piperidin-2-yl]azetidin-1 -yl]methanone. In some embodiments, the MEK1 inhibitor according to the present invention is 5-bromo-N-(2,3-dihydroxypropoxy)-3,4-difluoro- 2-(2-fluoro-4-iodoanilino)benzamide. In some embodiments, the MEK1 inhibitor according to the present invention is 2-[4-[(2-butyl-4-oxo-1 ,3-diazaspiro[4.4]non-1 -en-3-yl)methyl]-2- (ethoxymethyl)phenyl]-N-(4,5-dimethyl-1 ,2-oxazol-3-yl)benzenesulfonamide. In some embodiments, the MEK1 inhibitor according to the present invention is 2-[4-[(2-butyl-4-oxo- 1 ,3-diazaspiro[4.4]non-1 -en-3-yl)methyl]-2-(ethoxymethyl)phenyl]-N-(3,4-dimethyl-1 ,2-oxazol- 5-yl)benzenesulfonamide. In some embodiments, the MEK1 inhibitor according to the present invention is 2-[4-[(2-butyl-4-oxo-1 ,3-diazaspiro[4.4]non-1 -en-3-yl)methyl]-2- propylphenyl]-N-(4,5-dimethyl-1 ,2-oxazol-3-yl)benzenesulfonamide.
[0067] As further indicated by the data disclosed herein, ET-1 , ETAR and ECE-1 are co- expressed by small to medium-sized sensory nerves projecting into skin (Figure 5) and ETAR inhibition alleviates pruritic responses in chronic pruritic mouse models (Figure 9), suggesting a central role of ETAR in ET-1 induced itch. Therefore, in still another aspect of the present invention, the compound for use in the treatment of a subject suffering from itch is an endothelin A receptor (ETAR) inhibitor. Any ETAR inhibitors may be used within the scope of the present invention. Preferred ETAR inhibitors for use in the treatment of a subject suffering from itch according to the present invention are depicted in Figure 2. In some embodiments, the ETAR inhibitor of the present invention is 4-tert-butyl-N-[6-(2- hydroxyethoxy)-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl)pyrimidin-4-yl]benzenesulfonamide. In some embodiments, the ETAR inhibitor of the present invention is (5-(4-bromophenyl)-6- [2-(5-bromopyrimidin-2-yl)oxyethoxy]-N-(propylsulfamoyl)pyrimidin-4-amine. In some embodiments, the ETAR inhibitor of the present invention is (2S)-2-[(4,6-dimethylpyrimidin-2- yl)oxy]-3-methoxy-3,3-diphenylpropanoic acid. In some embodiments, the ETAR inhibitor of the present invention is N-[6-(2-Hydroxyethoxy)-5-(2-methoxyphenoxy)-2-[2-(2H-tetrazol-5- yl)pyridin-4-yl]pyrimidin-4-yl]-5-propan-2-ylpyridine-2-sulfonamide. In some embodiments the ETAR inhibitor of the present invention is N-(4-chloro-3-methyl-1 ,2-oxazol-5-yl)-2-[2-(6- methyl-2H-1 ,3-benzodioxol-5-yl)acetyl]thiophene-3-sulfonamide. In some embodiments, the ETAR inhibitor of the present invention is N-(3-Methoxy-5-methylpyrazin-2-yl)-2-[4-(1 ,3,4- oxadiazol-2-yl)phenyl]pyridine-3-sulfonamide. In some embodiments, the ETAR inhibitor of the present invention is 2-[(3R,6R,9S,12R,15S)-6-(1 H-indol-3-ylmethyl)-9-(2-methylpropyl)- 2,5,8,1 1 ,14-pentaoxo-12-propan-2-yl-1 ,4,7,10,13-pentazabicyclo[13.3.0]octadecan-3- yl]acetic acid. In some embodiments the ETAR inhibitor of the present invention is 3- benzodioxol-5-yl)-1 -[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3- carboxylic acid. In some embodiments, the ETAR inhibitor of the present invention is (2R,3R,4S)-4-(1 ,3-benzodioxol-5-yl)-1 -[2-(dibutylamino)-2-oxoethyl]-2-(4- methoxyphenyl)pyrrolidine-3-carboxylic acid. In some embodiments, the ETAR inhibitor of the present invention is (2R,3R,4S)-4-(1 ,3-benzodioxol-5-yl)-1 -[2-(dibutylamino)-2-oxoethyl]- 2-(2-methoxyphenyl)pyrrolidine-3-carboxylic acid. In some embodiments, the ETAR inhibitor of the present invention is 3-(1 ,3-benzodioxol-5-yl)-5-hydroxy-5-(4-methoxyphenyl)-4-[(3,4,5- trimethoxyphenyl)methyl]furan-2-one. In some embodiments, the ETAR inhibitor of the present invention is (2R)-2-[[(2R)-2-[[(2S)-2-(azepane-1 -carbonylamino)-4- methylpentanoyl]amino]-3-(1 -formylindol-3-yl)propanoyl]amino]-3-(1 H-indol-3-yl)propanoic acid. In some embodiments, the ETAR inhibitor of the present invention is 2-(1 ,3- benzodioxol-5-yl)-4-(4-methoxyphenyl)-4-oxo-3-[(3,4,5-trimethoxyphenyl)methyl]but-2- enoate. In some embodiments, the ETAR inhibitor of the present invention is (2S)-2-(4,6- dimethoxypyrimidin-2-yl)oxy-3-methoxy-3,3-diphenylpropanoic acid. In some embodiments, the ETAR inhibitor of the present invention is N-[6-methoxy-5-(2-methoxyphenoxy)-2-pyridin- 4-ylpyrimidin-4-yl]-5-methylpyridine-2-sulfonamide. In some embodiments, the ETAR inhibitor of the present invention is N-[5-(2-methoxyphenoxy)-2-pyridin-4-yl-6- (trideuteriomethoxy)pyrimidin-4-yl]-5-methylpyridine-2-sulfonamide. In some embodiments, the ETAR inhibitor of the present invention is N-[6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)- 2-[2-(2H-tetrazol-5-yl)pyridin-4-yl]pyrimidin-4-yl]-5-methylpyridine-2-sulfonamide. In some embodiments, the ETAR inhibitor of the present invention is [6-(2-hydroxyethoxy)-5-(2- methoxyphenoxy)-2-[2-(1 ,2,3-triaza-4-azanidacyclopenta-2,5-dien-5-yl)pyridin-4-yl]pyrimidin- 4-yl]-(5-methylpyridin-2-yl)sulfonylazanide. In some embodiments, the ETAR inhibitor of the present invention is 4-amino-N-(3,4-dimethylisoxazol-5-yl)benzenesulfonamide. In some embodiments, the ETAR inhibitor of the present invention is [(7R)-5-chloro-3-[(1 E,3E,5S)- 3,5-dimethylhepta-1 ,3-dienyl]-7-methyl-6,8-dioxoisochromen-7-yl] acetate.
[0068] As suggested by the data of the present invention with TRPA"'" and TRPV"'" neurons (Figure 7A), ET-1 does not activate a subset of transient receptor potential cation channel V1 (TRPV1 ), but acts via TRP ankyrin A1 (TRPAI )-positive sensory neurons, indicating an important role of TRPA1 for ET-induced itch. This can be underlined by the use of TRPA1 inhibitor HC-030331 in vivo (Figure 9). Thus, ET-1 induced pruritus is not linked to a function TRPV1 channel as described for histamine and serotonin (3, 34), but an operating TRPA1 channel and contradicts earlier results where TRP-inhibitors were deployed. According to another aspect of the present invention, the compound for use in the treatment of a subject suffering from itch is a TRPA1 inhibitor.
[0069] The inventors discovered that ET-1 mediates activation of ERK1/2 in DRG neurons and ECE-1 modulates the status of ET-1 triggered ERK1/2 phosphorylation (Figure 13, Figure 14). As demonstrated herein, pre-treatment of mice with ERK inhibitor calbiochem reduced or abolished ET-1 or chloroquine evoked scratching behavior and pruritogenic actions. According to a further aspect of the present invention, the compound for use in the treatment of a subject suffering from itch is an ERK1/2 inhibitor. In some embodiments, the ERK1/2 inhibitor is 5,6,15-trihydroxy-17-methoxy-1 1 -methyl-12-oxacicyclo[12.4.0]octadeca- 1 (14),2,8,15,17-pentaene-7,13-dione. In some embodiments, the ERK1/2 inhibitor is 5-(2- phenylpyrazolo[1 ,5-a]pyridine-3-yl)2H-pyrazolo[3,4-c]pyridazin-3-amine. In some embodiments, the ERK1/2 inhibitor is 3-(2-Aminoethyl)-5-((4-ethoxyphenyl)methylene)-2,4- thiazolidinedione. In some embodiments, the ERK1/2 inhibitor is 3-(2-aminoethyl)-5-(4- ethoxybenzylidene)-1 ,3-thiazolidine-2,4-dione.
[0070] Figure 15 shows skin biopsies from patients with atopic dermatitis and prurigo nodularis indicate a significantly increased immunoreactivity of ET-1 in all layers of the epidermis and ETAR, ET-1 and ECE-1 expression was confirmed for peripheral nerves, indicating that ET-1 /ETAR signaling is contributing to pathophysiology of pruritic skin diseases in human subjects. Therefore, as disclosed by the present invention, compound such as ECE-1 , MEK1 inhibitors, and ETAR inhibitors as described herein represent suitable new tools for use in the treatment of pruritic skin disease and might lead to specific, beneficial new therapies. Accordingly, a method for the treatment of a subject suffering from itch is envisaged herein as well, said method comprising administering a therapeutically effective amount of ECE-1 to a subject in need thereof. Further, the invention provides a method for the treatment of a subject suffering from itch, said method comprising administering a therapeutically effective amount of a MEK1 inhibitor to a subject in need thereof. Additionally, the present invention provides a method for the treatment of a subject suffering from itch, said method comprising administering a therapeutically effective amount of an endothelin A receptor (ETAR) inhibitor to a subject in need thereof.
[0071] Moreover, the present inventors investigated the effect of several ETAR blocking compounds (40 mg/kg body-weight) in a chronic oxazolone-induced, atopic-dermatitis-like itch model in mice (Figure 18). The experimental results reveal that under the application of all tested ETAR-inhibitors the scratching behavior was significantly reduced as compared to control mice. Accordingly, the data underline that ETAR-inhibitors are well suited for the treatment of a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch.
[0072] The compounds of the present invention significantly contribute to the regulation of histamine-independent pruritus and reduce hallmarks of pruritic skin diseases when administered to a subject in need thereof. Because of the described involvement of the ET- 1/ETAR/ECE-1/MEK/ERK pathway in induction of pruritus, which is independent of histamine impact as described elsewhere herein, theses compound can be useful for the treatment of a subject suffering from a histamine-independent, ET-1 induced itch. Therefore, the compounds of the present invention typically block ET-1/ETAR induced signaling.
The compounds for use in the treatment of a subject suffering from itch according to the present invention are generally administered to the subject in a therapeutically effective amount. Said therapeutically effective amount is sufficient to inhibit or alleviate the itch. By "therapeutic effect" or "therapeutically effective" is meant that the compound for use will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term "therapeutically effective" further refers to the inhibition of factors causing or contributing to the disease. The term "therapeutically effective amount" includes that the amount of the compound when administered is sufficient to significantly improve the progression of the disease being treated or to prevent development of said disease. The therapeutically effective amount will vary depending on the compound, the disease and its severity and on individual factor of the subject such. Therefore, the compound of the present invention will not in all cases turn out to be therapeutically effective, because the method disclosed herein cannot provide a 100% safe prediction whether or not a subject may be responsive to said compound, since individual factors are involved as well. It is to expect that age, body weight, general health, sex, diet, drug interaction and the like may have a general influence as to whether or not the compound for use in the treatment of a subject suffering from itch will be therapeutically effective. In the scope of the present invention, it is for example envisaged that the therapeutic effect is detected by way of surgical resection or biopsy o the affected skin or the effected tissue, which is subsequently analyzed by way of, for example immunological techniques. Additionally or alternatively, it is also possible to evaluate the general appearance of the respective patient, which will also aid the skilled practitioner to evaluate whether the therapy is effective. Those skilled in the art are aware of numerous other ways which will enable a practitioner to observe a therapeutic effect of the compound for use in the treatment of itch as disclosed herein in the context of a method or use of the present invention.
[0073] It is envisaged that the therapeutic effect is detected by way of surgical resection or biopsy of the affected skin or the effected tissue, which is subsequently analyzed by way of, for example immunological techniques. Alternatively, it is also envisaged that biomarkers in the skin of the patient are detected in order to diagnose whether or not the therapeutic approach is effective. Additionally or alternatively, it is also possible to evaluate the general appearance of the respective patient which will also aid the skilled practitioner to evaluate whether the therapy is effective. Those skilled in the art are aware of numerous other ways which will enable a practitioner to observe a therapeutic effect of a compound used for the treatment of itch in the context of a method or use of the present disclosure.
[0074] Generally, the therapeutically effective amount of the compound used to treat a subject suffering from itch is sufficient to inhibit or alleviate histamine-independent, ET- 1/ETAR induced itch. Preferably, the therapeutically effective amount of the compound used to treat a subject suffering from itch is between about 0.01 mg per kg body weight and about 1 g per kg body weight, such as about 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 200, 300, 400, 500, 600, 700, 800, or about 900 mg per kg body weight. Even more preferably, the therapeutically effective amount of the compound used to treat a subject suffering from itch is between about 0.01 mg per kg body weight and about 100 mg per kg body weight, such as between about 0.1 mg per kg body weight and about 10 mg per kg body weight. The therapeutic effective amount of the compound will vary with regard to the weight of active compound contained therein, depending on the species of subject to be treated.
[0075] The present invention further provides a method for screening a compound useful for the treatment of a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch, the method comprising assaying said compound for the activity in inhibiting the ET-1/ETAR induced signaling cascade. This method comprises a) administering the compound suspected as to be useful for the treatment of a histamine-independent ET- 1/ETAR induced itch to an animal suffering from an acute or chronic pruritic skin disease, wherein said animal is a histamine-impervious animal, b) measuring the scratching movements of said animal within a defined time interval, c) comparing the scratching movements of said animal to the scratching movements of a reference animal to which the suspected compound has not been administered, the scratching movements of said reference animal measured within the same defined time interval.
[0076] The term "assaying the activity" of a compound as used in the present disclosure refers to examining the ability of said compound to significantly inhibit ET-1/ETAR induced signaling. It will be understood by those skilled in the art, that such an assessment is usually not intended to be correct for 100% of the compound to be investigated. The term, however, requires that a prediction can be made for a significant inhibition of ET-1/ETAR signaling by the compound suspected as to be useful to treat endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch in a proper and correct manner. Whether a compound is statistically significant in inhibiting ET-1/ETAR induced signaling can be determined by those skilled in the art using various well known statistic evaluation tools, e.g. determination of confidence intervals, p-value determination, Student's t-test, and Mann-Whitney test. Suitable confidence intervals are, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%. Suitable p-values are preferably 0.1 , 0.05, 0.01 , 0.005, or 0.0001 .
[0077] In a preferred embodiment, assaying a compound useful for the treatment of a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch in inhibiting the ET-1/ETAR induced signaling comprises measuring the scratching movements of an animal suffering from an acute or chronic pruritic skin disease within a defined time interval, and comparing the scratching movements of said animal to the scratching movements of a reference animal to which the suspected compound has not been administered, the scratching movements of said reference animal measured within the same defined time interval. In this regard the reference animal is an animal not suffering from an acute or chronic pruritic skin disease. A lower amount of scratching movements of the animal under administration of the suspected compound in comparison to the reference animal indicates that said suspected compound is useful for the treatment of a histamine- independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch. An approximate or higher amount of scratching movements of the animal under administration of the suspected compound in comparison to the reference animal indicates that said suspected compound is not useful for the treatment of a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch.
[0078] In another embodiment, assaying a compound useful for the treatment of a histamine- independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch in inhibiting the ET-1/ETAR induced signaling comprises in the administration of the compound suspected as to be useful to treat endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch to a cell line overexpressing ET-1 . In this regard, the change of the expression level of a biomarker by said cell line under administration of the suspected compound will be measured or detected.
[0079] The term "biomarker" is defined as a physical sign or laboratory measurement that occurs in association with a natural or pathological process, and that has putative diagnostic and/or prognostic utility. More precisely, the term "biomarker" may comprise a protein or a gene encoding a protein/peptide, which is expressed at a lower or higher level by a cell under different cellular conditions. In the present disclosure, said biomarker is preferably expressed and released by a subject under native and/or pathological conditions, such as pruritus-like skin diseases. Preferably, the biomarker disclosed herein is ET-1 expressed in one or more or all layers of the epidermis or endothelial cells of said subject. Further biomarker described herein are cytokines expressed and released by an immune cell, in particular activated T-cells, eosinophiles and/or a mast cells in the lesional skin of a subject. Said biomarker is preferably one of IL-2, IL-4, IL-10, IL-13 IL-17, IL-31 , and IFNy. In the present invention, measuring the change of the expression level of one or more of the biomarkers disclosed herein can be used for screening a compound useful for treating and/or preventing pruritic skin diseases. It is also possible to measure the membrane ETAR and/or ECE-1 level by immunohistochemistry or FACS of cells as biomarkers.
[0080] The term "detect" or "detecting" when used in the context of a biomarker refers to any method that can be used to identify the presence of a protein/polypeptide, such as ET-1 and cytokines, released or expressed by a cell. When used herein in combination with the words "level", "amount" or "value", the words "detect" and "detecting" are understood to refer to a quantitative as well as a qualitative level.
[0081] Said biomarker is preferably ET-1 . In some embodiments said biomarker is a cytokine and will be selected from the group consisting of IL-31 , IFNy, IL-4, IL-2, IL-10, IL-13, and IL- 17. The expression level of one or more of said biomarker will be compared to a reference expression level of said biomarker released by the same cell line which instead was not treated with the compound suspected as to be useful to treat endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch. The expression level of one or more biomarker is measured in a sample obtained from the cell line overexpressing ET-1 and quantified by methods known to those skilled in the art, e.g. by PCR or ELISA techniques. Preferably, quantifying the expression level of a biomarker includes measuring the level of the biomarkers mRNA in the sample. In such a case, biomarker mRNA is extracted from the cell, reverse transcribed into cDNA and quantified by real-time PCR as described elsewhere herein.
[0082] In some embodiments, assaying said compound for the activity in inhibiting the ET- 1/ETAR induced signaling cascade comprises administering the compound suspected as to be useful for the treatment of a histamine-independent ET-1/ETAR induced itch to an animal suffering from an acute or chronic pruritic skin disease, wherein said animal is a histamine- impervious animal. In this regard, in some embodiments, the histamine-impervious animal suffering from an acute or chronic pruritus skin disease used within the method for screening a compound useful for the treatment of a histamine-independent, endothelin-1 (ET- 1 )/endothelin A receptor (ETAR) induced itch as described herein, is a histamine-1 receptor (H1 R) knockout animal. In another embodiment, the histamine-impervious animal suffering from an acute or chronic pruritus skin disease used within the method for screening a compound useful for the treatment of a histamine-independent, endothelin-1 (ET- 1 )/endothelin A receptor (ETAR) induced itch is a histamine-4 receptor (H4R) knockout animal. In still another embodiment the histamine-impervious animal suffering from an acute or chronic pruritus skin disease used within the method for screening a compound useful for the treatment of a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch is a histamine-1 receptor (H1 R) and histamine-4 receptor (H4R) double knockout animal. In some embodiments, the histamine-impervious knockout animal suffering from an acute or chronic pruritus skin disease and used within the scope of the present invention is a heterozygous knockout animal. In some embodiments, the histamine- impervious knockout animal suffering from an acute or chronic pruritus skin disease and used within the scope of the present invention is a homozygous knockout animal.
[0083] Techniques for generating genetically altered animal models have been perfected over the years and the skilled artisan is aware of the variety of techniques used to knockout gene in laboratory animals to study the function or pathology involved with that particular gene. Such techniques of gene targeting allow for the introduction of engineered genetic mutations into an animal at a determined genomic locus and were described in detail in the state of the art (35).
[0084] In yet another embodiment, the histamine-impervious animal suffering from an acute or chronic pruritus skin disease used within the method for screening a compound useful for the treatment of a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch is a mast cell-deficient animal.
[0085] In the context of the present invention, the change of the expression level of the biomarker by said histamine-impervious animal suffering from an acute or chronic pruritus skin disease will be measured. Said measured biomarker is preferably ET-1 . In some embodiments the biomarker will be one or more cytokines selected from the group consisting of IL-31 , IFNy, IL-4, IL-2, IL-10, and IL-17. The expression level of one or more of said biomarkers can be compared to a reference expression level of said biomarker released by the histamine-impervious animal which instead was not treated with the compound suspected as to be useful for the treatment of a histamine-independent ET-1/ETAR induced itch. According to the present invention, the suspected compound is useful for treating itch or preventing histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch, if the expression of said biomarker under administration of said suspected compound is significant lower than the reference expression level for said biomarker. The suspected compound is not useful for treating histamine-independent, endothelin-1 (ET- 1 )/endothelin A receptor (ETAR) induced itch or preventing histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch, if the expression of said biomarker under administration of a 4-1 BB/4-1 BBL antagonist is approximate or higher than the reference expression level of said biomarker.
[0086] The expression level of the biomarker is measured in a sample obtained from the non-human transgenic animal overexpressing 4-1 BB in basal keratinocytes and quantified by methods known to those skilled in the art, e.g. by PCR or ELISA techniques. The sample is preferably a tissue sample of said transgenic animal, preferably a sample taken from the skin of said animal. In some embodiments the sample is taken from lesional skin of said transgenic animal. Measuring the expression level of one or more biomarkers by the non- human transgenic animal preferably includes measuring the level of biomarkers mRNA in the sample. In such a case, biomarker mRNA is extracted from the tissue sample, reverse transcribed into cDNA and quantified by real-time PCR as described elsewhere herein.
[0087] The compound for use in the treatment of a subject suffering from itch according to the present invention can be administered to the subject described herein. In some embodiments the administration is carried out orally. In some embodiments the administration is carried out parenterally. In some embodiments the administration is carried out subcutaneously. In some embodiments the administration is carried out intravenously. In some embodiments the administration is carried out intramuscularly. In some embodiments the administration is carried out intraperitoneally. In some embodiments the administration is carried out by intranasal instillation. In some embodiments the administration is carried out by implantation. In some embodiments the administration is carried out by intracavitary instillation. In some embodiments the administration is carried out by intravesical instillation. In some embodiments the administration is carried out intraocularly. In some embodiments the administration is carried out intraarterially. In some embodiments the administration is carried out intralesionally. In some embodiments the administration is carried out transdermally. In some embodiments the administration is carried out by application to mucous membranes. The compound for use in the treatment of a subject suffering from itch will be administered in substantielly non-toxic quantities.
The present invention further provides the use of a compound for the preparation of a medicament for treatment of a human subject suffering from histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch, wherein said compound is selected from the group consisting of endothelin-converting enzyme-1 (ECE-1 ), a MEK1 ECE-1 . The present invention further provides the use of a compound for the preparation of a medicament for treatment of a human subject suffering from histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch, wherein said compound is a MEK1 inhibitor. The present invention further provides the use of a compound for the preparation of a medicament for treatment of a human subject suffering from histamine- independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch, wherein said compound is an ETAR inhibitor. The present invention further provides the use of a compound for the preparation of a medicament for treatment of a human subject suffering from histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch, wherein said compound is an ERK1/2 inhibitor. The present invention further provides the use of a compound for the preparation of a medicament for treatment of a human subject suffering from histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch, wherein said compound is a TRPA1 inhibitor. In this context, the compounds of the present invention are typically provided as components of a pharmaceutical composition, optionally including at least one further active ingredient, preferably together with pharmaceutically acceptable excipients and/or additives as described in detail elsewhere herein.
[0088] In another aspect of the present invention, the compounds described herein for use in the treatment of a subject suffering from itch might be used to prevent a human subject from developing a pruritic skin disease. In this context, the human subject to whom a compound suspected as to be useful to treat itch has been administered, will not exhibit characteristic hallmarks of pruritus. In some embodiments said compound will be used to prevent a human subject from developing epidermal hyperplasia. In some embodiments the said compound will be used to prevent a human subject from developing irregular acanthosis. In some embodiments said compound will be used to prevent a human subject from developing fibrosis. In some embodiments said compound will be used to prevent a human subject from developing collagenosis. In some embodiments said compound will be used to prevent a human subject from an increased infiltration of lymphocytes, mast cells and/or eosinophiles into the dermis of said human subject. Preferably, the human subject who can be prevented from developing pruritic skin diseases is a healthy human subject not exhibiting any hallmarks of human pruritus.
Pharmaceutical compositions [0089] While it is possible to administer a compound of the present invention directly without any formulation, in another aspect of the present invention the compounds are preferably employed in the form of a pharmaceutical or veterinary formulation composition, comprising a pharmaceutically or veterinarily acceptable carrier, diluent or excipient and a compound of the present invention. The carrier used in combination with the compound of the present invention is water-based and forms an aqueous solution. An oil-based carrier solution containing the compound of the present invention is an alternative to the aqueous carrier solution. Either aqueous or oil-based solutions further contain thickening agents to provide the composition with the viscosity of a liniment, cream, ointment, gel, or the like. Suitable thickening agents are well known to those skilled in the art.
[0090] Alternative embodiments of the present invention can also use a solid carrier containing the compound for use in the treatment of itch as disclosed elsewhere herein. This enables the alternative embodiment to be applied via a stick applicator, patch, or suppository. The solid carrier further contains thickening agents to provide the composition with the consistency of wax or paraffin.
[0091] Alternatively, the compound for us in the treatment of a subject suffering from itch can be administered in a combination with another anti-pruritic drug and/or a compound used to treat skin disease. Many compounds are known to have anti-pruritic effects and are therapeutically used to treat skin disease. In some embodiments the anti-pruritic drug and/or a compound used to treat skin disease is an antihistamine. In some embodiments the antipruritic drug and/or a compound used to treat skin disease is a glucocorticosteroid. In some embodiments the anti-pruritic drug and/or a compound used to treat skin disease is calcineurin inhibitor. In some embodiments the anti-pruritic drug and/or a compound used to treat skin disease is local anesthetic. In some embodiments the anti-pruritic drug and/or a compound used to treat skin disease is serotonin-reuptake inhibitors (SSRI). In some embodiments the anti-pruritic drug and/or a compound used to treat skin disease is menthol. In some embodiments the anti-pruritic drug and/or a compound used to treat skin disease is camphor. In some embodiments the anti-pruritic drug and/or a compound used to treat skin disease is a neuroleptic. In some embodiments the anti-pruritic drug and/or a compound used to treat skin disease is a topical antidepressant In some embodiments the anti-pruritic drug and/or a compound used to treat skin disease is a tetracyclic. In some embodiments the anti-pruritic drug and/or a compound used to treat skin disease is neurokinin-1 receptor antagonist. In some embodiments the anti-pruritic drug and/or a compound used to treat skin disease is a mu-opioid receptor antagonists. In some embodiments the anti-pruritic drug and/or a compound used to treat skin disease is a kappa opioid receptor antagonist. In some embodiments the anti-pruritic drug and/or a compound used to treat skin disease is a protease inhibitor. In some embodiments the anti-pruritic drug and/or a compound used to treat skin disease is a protease-activated receptor antagonist. In some embodiments the anti-pruritic drug and/or a compound used to treat skin disease is a gastrin-realizing peptide. In some embodiments the anti-pruritic drug and/or a compound used to treat skin disease is a gastrin-releasing peptide receptor antagonist. In some embodiments the anti-pruritic drug and/or a compound used to treat skin disease is a brain-derived natriuretic peptide (BNP). In some embodiments the anti-pruritic drug and/or a compound used to treat skin disease is a brain-derived natriuretic peptide (BNP) receptor antagonist. In some embodiments the antipruritic drug and/or a compound used to treat skin disease is a dynorhin receptor antagonist. In some embodiments the anti-pruritic drug and/or a compound used to treat skin disease is a cytokine. In some embodiments the anti-pruritic drug and/or a compound used to treat skin disease is a chemokine receptor antagonist. In some embodiments the anti-pruritic drug and/or a compound used to treat skin disease is a botulinum toxin. Said combination according to the present invention can be administered as a combined formulation or separate from each other.
[0092] Moreover, the compounds for use in the treatment of a subject suffering from itch according to the present invention can be combined with ultraviolet radiation therapy. In some embodiments the ultraviolet radiation therapy is UVA radiation. In some embodiments the ultraviolet radiation therapy is UVB radiation. In some embodiments the ultraviolet radiation therapy is PUVA radiation.
[0093] In one aspect, the present invention particularly provides a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w ECE-1 and one or more pharmaceutically acceptable excipients, such as 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8 or 9% w/w ECE-1 . In another aspect, the present disclosure provides a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w MEK1 inhibitor and one or more pharmaceutically acceptable excipients, such as 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8 or 9% w/w MEK1 inhibitor. In still another aspect, the present invention provides a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w endothelin A receptor (ETAR) inhibitor and one or more pharmaceutically acceptable excipients, such as 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8 or 9% w/w endothelin A receptor (ETAR) inhibitor. Pharmaceutical excipients according to the present invention include, by the way of illustration and not limitation, diluent, disintegrants, binding agents, adhesives, wetting agents, polymers, lubricants, gliands, substances added to mask or counteract a disagreeable texture, taste or odor, flavors, dyes, fragrances, and substances added to improve appearance of the composition. Acceptable excipients include lactose, sucrose, starch powder, maize starch or derivatives thereof, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate, polyvinyl-pyrrolidone, and/or polyvinyl alcohol, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, and the like. Examples of suitable excipients for soft gelatin capsules include vegetable oils, waxes, fats, semisolid and liquid polyols. Suitable excipients for the preparation of solutions and syrups include, without limitation, water, polyols, sucrose, invert sugar and glucose. Suitable excipients for injectable solutions include, without limitation, water, alcohols, polyols, glycerol, and vegetable oils. The pharmaceutical compositions can additionally include preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorings, buffers, coating agents, or antioxidants.
[0094] In preferred embodiments, the present invention provides a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2- (2-fluoro-4-iodoanilino)benzamide, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w 6-(4-bromo-2-fluoroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3- methylbenzimidazole-5-carboxamide, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w N-[(2S)-2,3-dihydroxypropyl]-3-(2-fluoro-4-iodoanilino)pyridine-4- carboxamide, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w 6-(4- bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w 2-(2-fluoro-4- iodoanilino)-N-(2-hydroxyethoxy)-1 ,5-dimethyl-6-oxopyridine-3-carboxamide, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w N-[3,4-difluoro-2-(2-fluoro-4- iodoanilino)-6-methoxyphenyl]-1 -[(2S)-2,3-dihydroxypropyl]cyclopropane-1 -sulfonamide, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w 2-(2-chloro-4- iodoanilino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w N-[3-[3-cyclopropyl-5-(2-fluoro-4-iodoanilino)- 6,8-dimethyl-2,4,7-trioxopyrido[4,3-d]pyrimidin-1 -yl]phenyl]acetamide, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w N-[3-[5-(2-aminopyrimidin-4- yl)-2-tert-butyl-1 ,3-thiazol-4-yl]-2-fluorophenyl]-2,6- difluorobenzenesulfonamide;methanesulfonic acid, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w N-[3-[5-(2-aminopyrimidin-4-yl)-2-tert-butyl-1 ,3-thiazol-4- yl]-2-fluorophenyl]-2,6-difluorobenzenesulfonamide, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4- iodoanilino)benzamide, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w 3,4-difluoro-2-(2-fluoro-4-iodoanilino)-N-(2-hydroxyethoxy)-5-[(3-oxooxazinan-2- yl)methyl]benzamide, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w (2S,3S)-2-[(4R)-4-[4-[(2R)-2,3-dihydroxypropoxy]phenyl]-2,5-dioxoimidazolidin-1 -yl]-N-(2- fluoro-4-iodophenyl)-3-phenylbutanamide, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w 3-[(2R)-2,3-dihydroxypropyl]-6-fluoro-5-(2-fluoro-4-iodoanilino)-8- methylpyrido[2,3-d]pyrimidine-4,7-dione, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w [3,4-difluoro-2-(2-fluoro-4-iodoanilino)phenyl]-[3-hydroxy-3-[(2S)- piperidin-2-yl]azetidin-1 -yl]methanone, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w 5-bromo-N-(2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4- iodoanilino)benzamide, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w 2-[4-[(2-butyl-4-oxo-1 ,3-diazaspiro[4.4]non-1 -en-3-yl)methyl]-2-(ethoxymethyl)phenyl]-N- (4,5-dimethyl-1 ,2-oxazol-3-yl)benzenesulfonamide, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w 2-[4-[(2-butyl-4-oxo-1 ,3-diazaspiro[4.4]non-1 -en-3- yl)methyl]-2-(ethoxymethyl)phenyl]-N-(3,4-dimethyl-1 ,2-oxazol-5-yl)benzenesulfonamide, or a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w 2-[4-[(2-butyl-4-oxo- 1 ,3-diazaspiro[4.4]non-1 -en-3-yl)methyl]-2-propylphenyl]-N-(4,5-dimethyl-1 ,2-oxazol-3- yl)benzenesulfonamide.
[0095] In preferred embodiments, the present invention provides a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w 4-tert-butyl-N-[6-(2-hydroxyethoxy)-5-(2- methoxyphenoxy)-2-(pyrimidin-2-yl)pyrimidin-4-yl]benzenesulfonamide, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w (5-(4-bromophenyl)-6-[2-(5- bromopyrimidin-2-yl)oxyethoxy]-N-(propylsulfamoyl)pyrimidin-4-amine, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w (2S)-2-[(4,6- dimethylpyrimidin-2-yl)oxy]-3-methoxy-3,3-diphenylpropanoic acid, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w N-[6-(2-Hydroxyethoxy)-5-(2- methoxyphenoxy)-2-[2-(2H-tetrazol-5-yl)pyridin-4-yl]pyrimidin-4-yl]-5-propan-2-ylpyridine-2- sulfonamide, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w N-(4- chloro-3-methyl-1 ,2-oxazol-5-yl)-2-[2-(6-methyl-2H-1 ,3-benzodioxol-5-yl)acetyl]thiophene-3- sulfonamide, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w N-(3- Methoxy-5-methylpyrazin-2-yl)-2-[4-(1 ,3,4-oxadiazol-2-yl)phenyl]pyridine-3-sulfonamide, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w 2- [(3R,6R,9S, 12R, 15S)-6-(1 H-indol-3-ylmethyl)-9-(2-methylpropyl)-2,5,8, 1 1 , 14-pentaoxo-12- propan-2-yl-1 ,4,7, 10,13-pentazabicyclo[13.3.0]octadecan-3-yl]acetic acid, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w 3-benzodioxol-5-yl)-1 -[2- (dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w (2R,3R,4S)-4-(1 ,3- benzodioxol-5-yl)-1 -[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3- carboxylic acid, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w (2R,3R,4S)-4-(1 ,3-benzodioxol-5-yl)-1 -[2-(dibutylamino)-2-oxoethyl]-2-(2- methoxyphenyl)pyrrolidine-3-carboxylic acid, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w 3-(1 ,3-benzodioxol-5-yl)-5-hydroxy-5-(4-methoxyphenyl)-4- [(3,4,5-trimethoxyphenyl)methyl]furan-2-one, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w (2R)-2-[[(2R)-2-[[(2S)-2-(azepane-1 -carbonylamino)-4- methylpentanoyl]amino]-3-(1 -formylindol-3-yl)propanoyl]amino]-3-(1 H-indol-3-yl)propanoic acid, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w 2-(1 ,3- benzodioxol-5-yl)-4-(4-methoxyphenyl)-4-oxo-3-[(3,4,5-trimethoxyphenyl)methyl]but-2- enoate, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w (2S)-2-(4,6- dimethoxypyrimidin-2-yl)oxy-3-methoxy-3,3-diphenylpropanoic acid, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w N-[6-methoxy-5-(2-methoxyphenoxy)-2- pyridin-4-ylpyrimidin-4-yl]-5-methylpyridine-2-sulfonamide, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w N-[5-(2-methoxyphenoxy)-2-pyridin-4-yl-6- (trideuteriomethoxy)pyrimidin-4-yl]-5-methylpyridine-2-sulfonamide, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w N-[6-(2-hydroxyethoxy)-5-(2- methoxyphenoxy)-2-[2-(2H-tetrazol-5-yl)pyridin-4-yl]pyrimidin-4-yl]-5-methylpyridine-2- sulfonamide, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w [6-(2- hydroxyethoxy)-5-(2-methoxyphenoxy)-2-[2-(1 ,2,3-triaza-4-azanidacyclopenta-2,5-dien-5- yl)pyridin-4-yl]pyrimidin-4-yl]-(5-methylpyridin-2-yl)sulfonylazanide, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w 4-amino-N-(3,4-dimethylisoxazol-5- yl)benzenesulfonamide, a topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w [(7R)-5-chloro-3-[(1 E,3E,5S)-3,5-dimethylhepta-1 ,3-dienyl]-7-methyl-6,8- dioxoisochromen-7-yl] acetate.
[0096] A variety of techniques for the formulation of pharmaceutical compositions and administration are known in the art and can be found for example in Remington: The Science and Practice of Pharmacy (20th ed, Lippincott Williams & Wilkens Publishers (2003)), which is incorporated herein by reference in its entirety.
[0097] The form of the topical pharmaceutical composition comprising a compound for use in the treatment of a subject suffering from itch will vary depending on the respective therapeutic approach. In some embodiments the topical pharmaceutical composition according to the present invention is in the form of an ointment. In some embodiments the topical pharmaceutical composition according to the present invention is in the form of a cream. In some embodiments the topical pharmaceutical composition according to the present invention is in the form of a lotion. In some embodiments the topical pharmaceutical composition according to the present invention is in the form of a gel. In some embodiments the topical pharmaceutical composition according to the present invention is in the form of a balm-stick. In some embodiments the topical pharmaceutical composition according to the present invention is in the form of a spray. In some embodiments the topical pharmaceutical composition according to the present invention is in the form of foam. Such formulations are prepared in a conventional manner in accordance with standard practice known to those skilled in the art. The formulation will vary with regard to the weight of active compound contained therein, depending on the species of subject to be treated.
[0098] In some embodiment, the pharmaceutical composition of the present invention further comprises a keratolytic agent. In this regard, a keratolytic agent is a compound supporting or elevating the absorption of the pharmaceutical composition into a skin layer deeper than the exterior layer of the epidermis. Various keratolytic agents are comprised within the scope of the present invention. In some embodiments the keratolytic agent is salicylic acid. In some embodiments the keratolytic agent is acetyl salicylic acid. In some embodiments the keratolytic agent is magnesium salicylate. In some embodiments the keratolytic agent is sodium salicylate. In some embodiments the keratolytic agent is benzoic acid. In some embodiments the keratolytic agent is urea. In some embodiments the keratolytic agent is sulfur. In some embodiments the keratolytic agent is colloidal sulfer. In some embodiments the keratolytic agent is podophyllum resin. In some embodiments the keratolytic agent is benzoyl peroxide. In some embodiments the keratolytic agent is resorcinol. In some embodiments the keratolytic agent is selenium disulphide. In some embodiments the keratolytic agent is ohydroxy acid. In some embodiments the keratolytic agent is hydroxybenzoic acids In some embodiments the keratolytic agent is alpha-hydroxycarboxylic acid.
[0099] In some embodiments, the pharmaceutical composition of the present invention further comprises a cooling agent, including, but not limited to menthol, an isomer of menthol, a menthol derivative, 4-Methyl-3-(1 -pyrrolidinyl)-2[5H]-furanone, WS-23, Icilin, Icilin Unilever Analog, 5-methyl-4-(1 -pyrrolidinyl)-3-[2H]-furanone; 4,5-dimethyl-3-(1 -pyrrolidinyl)-2[5H]- furanone; isopulegol, 3-(l-menthoxy)propane-1 ,2-diol, 3-(l-menthoxy)-2-methylpropane-1 ,2- diol, p-menthane-2,3-diol, p-menthane-3,8-diol, 6-isopropyl-9-methyl-1 ,4-dioxas- piro[4,5]decane-2-methanol, menthyl succinate and its alkaline earth metal salts, trimethylcyclohexanol, N-ethyl-2-isopropyl-5-methylcyclohexanecarb-oxamide, Japanese mint (Mentha arvensis) oil, peppermint oil, menthone, menthone glycerol ketal, menthyl lactate, 3-(1 -menthoxy)ethan-1 -ol, 3-(l-menthoxy)propan-1 -ol, 3-(l-menthoxy)butan-1 -ol, 1 - menthylacetic acid N-ethylamide, l-menthyl-4-hydroxypentanoate, l-menthyl-3- hydroxybutyrate, N,2,3-trimethyl-2-(1 -methylethyl)-butanamide, spearmint oil, camphor, or camphor derivates.
[0100] The compositions according to the present invention are preferably formulated in a unit dosage form, each dosage containing about 1 to about 500 mg, more usually about 5 to about 300 mg, of the active ingredient. The term "unit dosage form" as used herein refers to physically discreet units suitable as unitary dosages for human subjects or other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical carrier. As used herein, a "dosage" refers to an amount of therapeutic agent administered to a patient. As used herein, a "daily dosage" refers to the total amount of therapeutic agent administered to a patient in a day.
[0101] The compounds disclosed herein are also useful for treating pruritus in domestic animals such as cats, dogs, rabbits, guinea pigs, cows, sheeps, and horses. Thus, the invention also provides a veterinary formulation comprising a compound for use in the treatment of a subject suffering from itch as defined elsewhere herein together with a veterinary acceptable diluents or carrier. Such formulations include in particular ointments, pour-on formulations, spot-on formulations, dips, sprays, mousses, shampoos, collar, and powder formulations.
Diagnosis and diagnostic kits
[0102] As disclosed by the present invention (Figure 15), skin biopsies from patients with atopic dermatitis and prurigo nodularis, diseases characterized by chronic, intractable pruritus indicate a significantly increased immunoreactivity of ET-1 in all layers of the epidermis, while ETAR was not elevated. The results further confirm that ET-1 elicited an itch response in human subjects mainly independent from histamine-1 receptor (H1 R) function. Together the human findings in vivo and ex vivo indicate that ET-1 is increased in chronic prurigo skin diseases, is released by C-fibers, keratinocytes and endothelial cells in prurigo, and activates ETAR on unmyelinated nerve fibers, showing that ET-1 plays an important role in human pruritus pathology.
[0103] Therefore, another aspect of the present invention provides a method for diagnosing a histamine-independent, endothelin-1 (ET-1 )/en doth el in A receptor (ETAR) induced itch in a subject, the method comprising determining the amount of endothelin-1 in a skin sample of said subject, and comparing the amount of endothelin-1 determined in a) to a reference skin sample. In this regard, a significantly increased amount of ET-1 in all layers of the epidermis as compared to said reference sample demonstrates a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch. No significantly increased amount of ET-1 in all layers of the epidermis as compared to said reference sample demonstrates that said subject does not suffer from a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch. The reference skin sample can be obtained from the same patient from an area which does not suffer from itch. The reference skin sample can also be obtained from a different patient who does not suffer from itch or a different patient with histamine-dependent urticarial that responds to antihistamines.
[0104] The term ""determine" or determining" as used herein refers to any method that can be used to identify the presence of ET-1 , released or expressed in a skin sample of the subject according to the present invention. When used herein in combination with the words "amount" or "level", the terms "determine" or "determining" are understood to refer to a quantitative as well as a qualitative level.
[0105] The sample of the present invention is preferably a sample taken from the skin of the subject of the present invention. In some embodiments, the sample is taken from the lesional skin of said subject. The term "skin sample" is meant to include skin of the entire embody including scalp, the forehead, the head, arms, legs, breast, and so forth. The term "skin" is also meant to include various layers of skin, such as stratum corneum, epidermis and dermis.
[0106] According to the present invention, the skin sample may comprise C-fibers, keratinocytes, and endothelial cells. Frequently the sample will be a "clinical sample" which is as sample derived from a patient. Such samples include skin tissue samples or cells taken therefrom. Such skin tissue samples can be analyzed directly or they may be subject to some preparation prior to use in the methods or assays of the present invention. Such preparation can include, but is not limited to, suspension/dilution of the sample in water or an appropriate buffer or removal of cellular debris, e.g. by centrifugation, or selection of particular fractions of the samples before analysis.
[0107] In some embodiments, determining the amount of ET-1 in a skin sample includes measuring the level of ET-1 mRNA in said sample. The ET-1 mRNA expression level in a skin sample of a subject may be determined using any desired techniques known to those skilled in the art and methods disclosed herein. Methods to measure the expression level of ET-1 mRNA may be carried by any methods known in the art and include but are not limited to RT-PCR, Microarrays, and the like.
[0108] According to another aspect, the present invention provides diagnostic kits for detecting a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch, comprising a binding molecule which specifically binds to endothelin-1 (ET-1 ). In some embodiments the binding molecule as used within the scope of the present invention is an antibody. Antibodies for use herein may be specifically directed against ET-1 . The term "antibody" as used herein comprises polyclonal as well as monoclonal antibodies, chimeric antibodies, human or humanized antibodies, which may be present in bound or soluble form. Said term also includes fragments or derivatives of an antibody, such as Fab, F(ab)2, Fv, scFv, nanobodies, domain antibodies and the like. Polyclonal antibodies are heterogeneous mixtures of antibody molecules being produced from sera of animals which have been immunized with the antigen. The present invention comprises also polyclonal monospecific antibodies which are obtained by purification of the antibody mixture (e.g. via chromatography over a column carrying peptides of the specific epitope). A monoclonal antibody represents a homogenous population of antibodies specific for a single epitope of the antigen. Monoclonal antibodies can be prepared according to methods described in the prior art (Kohler & Milstein, Nature (1975) 256: 495-497; US-Patent 4,376,1 10; Ausubel et al., Current Protocols in Molecular Biology (1998) John Wiley & Sons, New York). The antibody comprised by the diagnostic kit according to the present invention specifically recognizes endothelin-1 as an antigen. The diagnosis kit of the invention comprising an antibody specifically directed against ET-1 is preferably carried out by an ELISA or immunochromatography technique.
[0109] In some embodiment, the antibody used within the scope of the present invention is a labeled antibody, preferably a dye-labeled antibody. Dyes used according to the present invention are known in the art.
[0110] In another embodiment, the binding molecules used in a diagnostic kit for detecting a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch as provided within the scope of the present invention are probes used for fluorescence in situ hybridization (FISH). In some embodiments such FISH probes comprise RNA probes designed for visualization of ET-1 mRNA, ET-1 IncRNA and ET-1 miRNA in tissues and cells derived from the subject suspected as to suffer from a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch.
[0111] The diagnosis kits of the invention are preferably used for diagnosing whether the subject is suffering from a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch. The diagnostic kits of the present invention can used to diagnose endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch infection in a subject. In this context, skin sample of a subject suspected as to suffer from a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch is analyzed using the diagnostic kits according to the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0112] Figure 1 : Overview of the signal transduction pathway for the stimulation of histamine-independent, ET-1/ETAR induced itch/pruritus in DRG neurons following the ET- 1/ETAR/ECE-17/MEK/ERK signaling cascade.
[0113] Figure 2: Nonexclusive list of ETAR inhibitors that can be used within the scope of the present invention.
[0114] Figure 3: Nonexclusive list of MEK-1 inhibitors that can be used within the scope of the present invention.
[0115] Figure 4: Nonexclusive list of ERK1/2 inhibitors that can be used within the scope of the present invention.
[0116] Figure 5: ETAR, ET-1 and ECE-1 are expressed in adult mouse DRG neurons and skin. (A) ETAR, ET-1 and ECE-1 mRNA was detectable in mouse DRG neurons and skin. (B) Expression of ETAR and ECE-1 protein was confirmed for mouse DRG neurons and skin by Western-blot analysis. (C) ETAR, ET-1 and ECE-1 were detected in small- to medium- sized DRG neurons by IHC (scale bar = 50 μηη) and co-localized to some extent with CGRP, IB4 and TRPV1 (arrows, see table 1 ). (D) ETAR, ET-1 and ECE-1 co-localize with PGP9.5+ peripheral sensory nerves (arrows) of skin, as examined by IHC using serial sections of regional skin obtained from mice suffering from chronic pruritic dermatitis, (scale bar = 50 μηη). (E) Expression of ET-1 in epidermal layers of biopsies obtained from WT mice (NMS = normal mouse skin) or mice with AD-like phenotype visualized with IHC.
[0117] Figure 6: ET-1 induces marked Ca2+ response in murine DRG neurons. DRG neurons treated in vitro with 100 nM ET-1 at indicated time point (arrow) release intracellular Ca2+ suggesting intact ETAR function in neuronal cells (n=7).
[0118] Figure 7: ET-1 induced scratching behavior in mice is dependent on TRPA1 and ECE-1 , but not histamine-1 receptor activation. (A) Scratching elicited by ET-1 (100 pmol/10 μΙ into the cheek) is eliminated in TRPA1 -deficient mice but not TRPV1 -, TRPV4- or TRPM8- deficient mice. Total number of scratching bouts/30 min (n = 4-10 mice/group). (B) ET-1 - induced calcium mobilization. Percentages of ET-1 -responsive neurons which also respond to other compounds (Mann Whitney U test, n=409-1525 cells/group). For quantification, 15- 68 dishes/group were used, and approximately 40 cells/ dish were counted. (C) Characterization of ET-1 -responsive DRG neurons. Percentages of ET-1 -responsive neurons in different KO mice (Mann Whitney U test, n=526-1057 cells/group. *p<0.05) compared with either His (histamine), Cap (capsaicin), or AITC (mustard oil) group. Error bars indicated as SEM. (D) Venn diagrams for DRG neurons in percentages. (E) ECE-1 modulates itch behavior responses to ET-1 . Total bouts of scratching over the 30 min observation period show substantial enhancement in behavioral response to intradermally administered ET-1 (nape of neck; n = 7, 100 pmol/site) when mice are pre-treated with ECE-1 inhibitor SM- 19712 30 min prior to agonist (n = 7; 25 mg/kg,). At this concentration, no pain behaviour was observed. (F) ET-1 elicited scratching behavior is independent of functional H1 R. Diphenhydramine hydrochloride (anti-histamine, AH; 3 mg/kg in H20) was applied orally 30 min prior to intradermal ET-1 injection (100 pmol/100 μΙ; nape of neck) (n = 4-5) (***; p< 0.001 ; *, p < 0.05).
[0119] Figure 8: Dose dependency of ET-1 activated itch in the mouse cheek model.
[0120] Figure 9: ET-1 induced scratching behavior in mice is dependent on TRPA1 and ETAR. Scratching elicited by ET-1 (100 pmol/10 μΙ into the cheek) is eliminated in WT mice pre-treated (30 min before agonist) intraperitoneally with TRPA1 antagonist (HC-030031 , 30 mg/kg) or with ETAR inhibitor BQ-123 (1 nmol) locally into the cheek of ET-1 injection.
[0121] Figure 10: ECE-1 is important for the recycling of ETAR in DRG neurons. DRG neurons were cultured with ET-1 in presence or absence of ECE-1 inhibitor. (A) Membrane bound ETAR (arrows) internalized and co-localized with ECE-1 (arrowheads) in cytosolic vesicles of DRG neurons within 10 min after ET-1 stimulation. Recovery of the receptor to the plasma membrane is observed 60 min after ET-1 application (arrow). (B) ECE-1 inhibitor SM-19712 does not affect initial internalization of ETAR (arrows, arrowheads), but prevents recycling of ETAR to the plasma membrane. (C) Time-dependence of ETAR translocation in DRG neurons in response to ET-1 (100 nM). In unstimulated cells, ETAR is mainly located at the plasma membrane. ET-1 stimulation for 10 min induced translocation of ETAR from plasma membrane to cytosol. Immunofluorescence for ETAR reappeared at plasma membrane 60 min after stimulation (black bars). Co-stimulation with the ECE-1 inhibitor SM- 19712 (10 μΜ) inhibited re-location of ETAR immunofluorescence to the plasma membrane (n = 10 cells/group, error bars are indicated as +/- SEM, Students i-test, ** p<0.01 ;*** p< 0.001 ). Subcellular distribution was analyzed from captured images using Image J (http://rsbweb.nih.gov/ij/) (D) ECE-1 degrades ET-1 at endosomal pH 5.5, but not at pH 7.4. Degradation of ET-1 by rhECE-1 at pH 5.5 and 7.4 was assessed by HPLC. (E) ET-1 was hydrolyzed by ECE-1 at Asp18-lle19 (scissor).
[0122] Figure 11 : ET-1 activated ETAR localizes in EEA-1 + acidic endosomes of murine DRG neurons. After ET-1 stimulation, ETAR trafficked from plasma membrane (arrows) to endosomes containing the endosomal marker EEA-1 (arrowheads). Recovery of ETAR to plasma membrane was observed 60 min after stimulation (arrows). Scale bar 30μΜ.
[0123] Figure 12: ET-1 trafficks to ECE-1 containing vesicles in cultured DRG neurons. Murine DRG neurons were treated with 100nM ET-1 . Upon ETAR activation by ET-1 , ET-1 trafficked to intracellular vesicles where it co-localized with ECE-1 (arrowheads). Scale bar 30μΜ.
[0124] Figure 13: Raw MALDI-TOFF data of (rh)ET-1 degradation by ECE-1 in (A) absence or (B) presence of the converting enzyme at pH 5.5. (C) Tabular summarization of two representative traces.
[0125] Figure 14: ECE-1 inhibition prolongs ET-1 -induced phosphorylation of ERK1/2 in DRG neurons. DRG cells were pre-treated with SM19712 (10μΜ) or vehicle for 60 min and stimulated with ET-1 (100nM). The cellular lysates were subjected to Western blot analysis for ERK1/2, and p38. ET-1 caused ERK activation in DRG neurons with maximum after 5 min, and ERK activation was sustained by pre-treatment with SM19712. ET-1 did not activate p38, in DRG neurons. Data are representative of three independent experiments.
[0126] Figure 15: ERK1/2 plays a central role in ET-1 -induced itch in vivo. (A) ET-1 (10pmol/site) was intradermal^ injected to the nape of neck 30 min after intraperitoneal injection of an ERK1/2 inhibitor (30 mg/kg) and/ or ECE-1 inhibitor SM-19712 (25 mg/kg) (n= 9 mice/ group). (B) Scratching elicited by ET-1 , IL-31 and CQ is eliminated or significantly decreased in ERK inhibitor (30 mg/kg bw, targets ERK1/2)- or PD031250 (10 mg/kg bw, targets ERK1/2phosphorylation)-treated mice. Total number of scratching bouts per 30 min in response to intradermal injection to the cheek of mice (n=4) of a pruritic compound panel was monitored. Compound concentrations: ET-1 100 pmol/10 μΙ, histamine 100 μg/10 μΙ, IL- 31 5 nmol/10 μΙ, CQ 200 pg/10 μΙ (one-way ANOVA with Dunnetts post-hoc test; (*, p < 0.05; ***; p < 0.001 ). (C) Amelioration of itch by ETAR blockage in mice with AD-like phenotype. Cohorts of WT mice were treated with multiple challenges of oxazolone onto the nape of neck to induce a chronic pruritus. Oxazolone challenging induced a robust and persistent scratching behavior in mice (baseline was evaluated on days 12-18). Intradermal injection of BQ-123 (10 nmol/100 μΙ or 25 nmol/100 μΙ) into the nape of neck was performed on day 18. With 30 min after BQ-123 injection, total numbers of scratching bouts were determined for 30 min.
[0127] Figure 16: Increase of ET-1 and ETAR in pruritic human skin and independence of ET-1 -evoked itch from H 1 receptor function in humans. (A) Localization of ET-1 , ETAR and ECE-1 in peripheral nerves of skin from patients with prurigo nodularis. Immunoreactivity was examined with IHC using serial sections. Positive staining for ETAR and ET-1 and (arrows) was detected for PGP9.5+ cutaneous sensory nerve fibers (scale bar = 50 μηη). (B) Staining for ET-1 is significantly enhanced in the epidermis of prurigo nodularis affected skin compared to healthy skin (NHS) and atopic dermatitis (AD). Samples from atopic dermatitis patients do not show enhanced ET-1 staining in cells per keratinocyte, but due to acanthosis the harboured total ET-1 may be increased when compared to healthy skin. Keratinocytes stain positively but fibroblasts were negative for ET-1 . (C) Time course of itch intensity (0: no itch, 10: worst imaginable itch) for ten minutes after the termination of a 60-second iontophoresis with histamine (open circles) and ET-1 (black circles) on the forearm skin of healthy human subjects (n = 6) who orally took a fexofenadine 60 mg tablet (anti-histamine H-i receptor blocker) 3 hours before. ET-1 induced itch independent of histamine release in humans though the anti-histamine H 1 receptor blocker significantly ameliorates the itch perception of volunteers from minute 4 until end of monitoring. Controls of treatment only with agonist are shown for ET-1 (black squares) and histamine (open squares), respectively. The asterisk indicates a statistically significant difference (P < 0.05) and error bars show SEM.
[0128] Figure 17: The time course of pain, pricking and burning intensities (0: no sensation, 10: worst imaginable pain, pricking or burning) for ten minutes after the termination of a 60- second iontophoresis with endothelin-1 on the forearm skin of healthy human subjects (n = 4), ET-1 significantly induced instant pain, pricking or burning (pain-related sensations) that were only described for seconds after agonist application. Error bars as SEM.
[0129] Figure 18: Amelioration of itch by ETAR blockage in mice with an AD-like phenotype. Cohorts of WT mice were treated with challenges of oxazolone (OXA) to the nape of neck to induce chronic pruritus. Oxazolone challenge induced a robust and persistent scratching behavior in mice (baseline was evaluated on days 18). On day 18, mice were i.p. injected into the peritoneum with Ambrisentan, Bosentan, Macitentan, Sitaxentan or Zibotentan (40 mg/kg body-weight). Within 30 minutes after BETAR inhibition, the total number of scratching bouts over a 30-minute period was determined. *P < 0.05; **P < 0.01 ; ***P < 0.001 . One-way ANOVA with Dunnett's post-hoc test.
EXAMPLES
[0130] The following examples illustrate the invention. These examples should not be construed as to limit the scope of this invention. The examples are included for purposes of illustration and the present invention is limited only by the claims. It will be clear to a skilled person in the art that the invention may be practiced in other ways than as particularly described in the present description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.
[0131] Mice
Adult C57bl/s mice (TRPV1 TRPV4"'", TRPA1"'", and TRPM8"'") were maintained under specific pathogen-free conditions with free access to water and food. All experiments were performed according to institutional regulations and in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. For the isolation of dorsal root ganglia neurons and dorsal skin, mice were deeply anesthetized by intraperitoneal injection of a mixture of 0.2% Xylazine/ 1 % Ketamine/0.9% NaCI (Ceva Sante Animale), and then perfused transcardially with Ca2+-free and Mg2+-free phosphate buffered saline (PBS). When samples were dedicated to immunohistochemical or immunofluorescence analysis, transcardial perfusion was performed using PBS containing 4% paraformaldehyde. To establish a primary DRG culture, harvested DRG neurons were incubated with Hanks BSS containing 1 .3 mg/ml papain and 0.65 mg/ml L-cysteine for 10min at 37°C, and then incubated with Hanks BSS containing 3 mg/ml collagenase for 10 min at 37°C. Digests were washed with complete medium consisting of MEM Eagle's with Earles's BSS medium supplemented with 10% (v/v) Horse serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomysin, 1x GIBCO MEM Vitamin Solution and 1x N1 medium supplement. Strain cell suspension was filtered through a 100 μηη cell strainer and cultured in dishes with or without poly-D-Lysine and Laminin coated glass cover slips containing complete media overnight. Cells were preincubated with vehicle or SM-19712 (10 μΜ) 2 hours before agonist stimulation. Inhibitor incubation was performed throughout the experiments. Cells were stimulated with vehicle or ET-1 (100 nM). For Calcium imaging upper- to mid-cervical mouse DRGs were enzymatically digested and processed.
[0132] RT-PCR
Total RNA was extracted from homogenized samples of DRG neurons or skin using TRIzol reagent. To avoid DNA contamination, total RNA was treated with 10U of RQ1 RNase-Free DNase. A quantity of 1 μg whole RNA was reverse-transcribed with the RevertAid First Strand cDNA Synthesis Kit. Subsequent RT-PCRs were carried out using the GoTaq DNA polymerase. The primers for mouse ET-1 (235 bp), ECE-1 (54 bp), ETAR (129 bp), ETBR (240 bp) and β-actin (147 bp) were used as previously reported (36).
The sequences of the primers were as follows:
ET-1 (mouse, NM 010104.3) forward 5'-GGAAACTACGAAGGTTGGAGGC-3' (SEQ ID NO: 1 ), reverse 5'-CTGTAGAAGCCACACAGATGGTCT-3' (SEQ ID NO: 2);
ECE-1 (mouse, NM 199307.2) forward 5'-GTGGCATTGGTGTCGTAGTG-3' (SEQ ID NO: 3), reverse 5'-CTTGATCATCGAAAGCGTGA-3' (SEQ ID NO: 4);
ETAR (mouse, NM 010332.2) forward 5'-GCTGGTTCCCTCTTCACTTAAGC-3' (SEQ ID NO: 5), reverse 5'-TCATGGTTGCCAGGTTAATGC-3' (SEQ ID NO: 6); β-actin (mouse, NM 007392.3), forward 5'-TTG CTGACAG G ATG CAG AAG-3 ' (SEQ ID NO: 7), reverse 5'-TGATCCACATCTGCTGGAAG-3' (SEQ ID NO: 8).
PCR conditions: annealing temperature 60°C, 35 cycles. PCR products were separated with a 2 % (w/v) agarose gel.
[0133] SDS-PAGE and Western blotting
Lysates of homogenized samples of DRG neurons, skin or whole cell lysates were homogenized in protein lysis buffer containing a protease inhibitor mixture and sonicated; cell debris was removed by centrifugation (14000 x g, 4°C, 10min). Samples were boiled in sample buffer (50 mM Tris-HCL, pH 6.8; 2% (w/v) SDS; 0.1 % (w/v) bromophenol blue; 10% (v/v) glycerol; and 2.5% (v/v) 2-mercaptoethanol) for 5 minutes, separated by SDS-PAGE (7 or 10% acrylamide) and blotted onto a nitrocellulose membrane. Membrane blocking was performed with milk powder (5% w/v) in Tris-buffered saline with 0.05% (v/v) Tween 20 for 1 hour. Primary antibodies were applied overnight at 4°C. After 5 washes with PBS/Tween, the membrane was incubated with secondary antibody for 2 hours at room temperature (RT). Immunoreactive polypeptides were visualized using the ECL Plus Western Blotting Detection Kit.
[0134] Immunofluorescence of tissues Tissue samples (10 μηη sections of DRG neurons) were postfixed in 4% paraformaldehyde solution for 2 hours at 4°C, transferred to 30% sucrose solution, embedded in Neg-50 for 24 h at 4°C and kept at -80°C until use. Sections were washed in PBS and incubated with 2% (w/v) bovine serum albumin, 5% (v/v) Horse serum and 0.3% (v/v) Triton X-100 in PBS for 1 hour. Sections were incubated with primary antibodies overnight at 4°C, then washed with PBS (37). Second primary antibodies were applied overnight at 4°C. Sections were washed with PBS and incubated with secondary antibodies at RT for 2 hours.
[0135] Confocal Microscopy
Tissue stainings were taken with x40, 20x or x10 magnification objectives in the inverted configuration. Confocal images for primary cultured DRG cells were obtained with x63 magnification objective in the inverted configuration. For all confocal images a regular phase transmission image was obtained. In DRGs, the subcellular distribution of ETAR, ECE-1 and ET-1 were analysed from captured images using Image J (http://rsbweb.nih.gov/ij/) as described previously (38, 39). To analyze the subcellular distribution of ETAR, plasma membrane fluorescence was calculated by subtracting cytoplasmic pixel counts from total cell pixel counts. To analyze fluorescence intensity of ET-1 and ECE-1 over time, total cell pixel counts were calculated.
[0136] Immunohistochemistry
6 μηη paraffin sections were de-paraffinized, rehydrated, and heated in a steamer (MultiGourmet plus FS20) for 25 minutes in Target Retrieval Solution. Sections were allowed to cool down in Target Retrieval Solution for 20 minutes. Endogenous peroxidase activity was quenched with 100 mM NaN 3/0. 1 % (w/v) H202 in PBS for 20 minutes at RT. After washing with PBS, sections were blocked with 2% (w/v) bovine serum albumin, 5% (w/v) Horse serum and 0.3% (v/v) Triton-X 100 for 1 hour at RT. Sections were incubated with primary antibodies overnight at 4°C in a humid chamber. After being rinsed with PBS, sections were incubated with secondary antibodies for 1 hour at RT (EnVision + System Labelled Polymer-HRP, anti-rabbit, anti-goat or anti-sheep). Immunoreactivity was detected by using the Liquid DAB + Substrate Chromogen System. Nuclei were counterstained with Hematoxylin QS and mounted with Aquamount. Stained sections were examined using the Axiophot microscope, equipped with a DP70 CCD camera. Morphometric analysis of images was performed using the DP-Soft 3.2 software (Olympus).
[0137] Scratching behavior
Mice were placed individually in a glass cage (13x13x20 cm) for at least 30 min before behavioral studies. ET-1 (1 pmol-1 nmol/site in 0.9% NaCI) or vehicle was administered intradermally to the nape or cheek of the mouse. Scratching and wiping behavior was videotaped for 30 min with personal kept out of the observation room and analyzed double- blinded. Analyzing the video-material for scratching bouts, only scratching with the hind paw toward the injected site and wiping of the right cheek with the front paw were counted. A series of scratching movements within 1 second was counted as one bout of scratching. SM- 19712 (25 mg/kg body weight in 0.9% NaCI), ERK1/2 inhibitor (30 mg/kg body weight in 0.9% NaCI, 10%DMSO), HC-030031 (30 mg/kg body weight in 0.9% NaCI), PD0325901 (in 0.9% NaCI, 10%DMSO) or vehicle were injected i.p. 30 min before ET-1 injection. The H1 R inhibitor Diphenhydramine hydrochloride (3 mg/kg in H20) was applied orally 30 min before ET-1 injection. BQ-123 (1 -25 nmol in 0.9% NaCI) was injected intradermally 30 min before ET-1 injection.
[0138] Chronic contact dermatitis
Mice were sensitized by topical treatment with 5% oxazolone (10 μΙ) applied to left ear. After 7 days oxazolone-sensitized mice were topically treated with 0.2% OXA (60 μΙ) on the flank every second day for a total of 2 weeks. Skin biopsies of mice were taken on last day of oxazolone treatment.
[0139] Chronic pruritus model
WT mice were treated one time with 10% oxazolone in acetone/olive oil (4:1 v/v) on the shaved nape of the neck (100 μΙ). After a resting periode of 7 days, mice were treated with 1 % oxazolone in acetone/olive oil (4:1 v/v) on the nape of the neck (100 μΙ) every other day for additional 10 days. Baseline scratching behavior was evaluated for 30 min on days 0, 12, 14, 16 and 18.
[0140] Immunofluorescence of cultured cells
Cultured and stimulated DRG cells were fixed in 4% paraformaldehyde, 100mM PBS, pH7.4, washed and incubated in PBS containing 1 % fetal calf serum and 0.1 % saponin for 30 minutes, and incubated with primary antibodies overnight at 4°C. Cells were washed, incubated with secondary antibodies for 2 hours at RT, washed and mounted.
[0141] Peptide degradation by ECE-1
ET-1 (250 μΜ) was incubated with rhECE-1 (100 nM) in 50mM Mes/KOH (pH 5.5) or 50mM TrisHCI (pH 7.4) for 0-90 min at 37°C. Reactions were stopped by adding TFA. Samples were run on a reversed-phase HPLC and analyzed using an ABI 4700 MALDI TOF/TOF mass spectrometer. The predicted masses of the peptides were calculated using the MS- Product feature of the Protein Prospector program. This program can be accessed at the following website: http://prospector.ucsf.edu/prospector/mshome.htm
[0142] Measurement of single cell [Ca2+li
DRG cells were seeded on glass cover slips (18mm) coated with Poly-D-Lysine and Laminin overnight. 2 hours before experiments, neurons were washed and kept in a medium containing 1 % Horse serum. Coverslips were washed with HEPES buffer and subsequently incubated with complete medium containing 2.5μΜ Fluo-3AM for 30 minutes at 37°C. Medium was removed and extracellular contamination with Fluo-3AM was eliminated by rinsing with HEPES buffer. Glass cover slips were transferred to a perfusion chamber and the cells were perfused with medium for 10 minutes to allow complete de-esterification of cytosolic dye. ET-1 at a concentration of 100 nM was washed-in. Fluorescent signals were measured with a confocal laser scanning microscope using an excitation wavelength of 488 nm. Emission signals between 505 nm and 530 were recorded. Two-dimensional images were recorded every 0.78 s. Relative [Ca2+], levels are shown as pseudo ratios (F/F0).
[0143] Calcium imaging
Upper- to mid-cervical DRGs removed from mice were enzymatically digested as mentioned above. Cells were plated on poly-D-lysine-coated glass coverslips, and cultured for 16-24 h. Cells were incubated in Ringer's solution (pH 7.4; 140 mM NaCI, 4 mM KCI, 2 mM CaCI2, 1 mM MgCI2, 10 mM Hepes and 4.54 mM NaOH) with 10 187 μΜ of Fura-2 AM and 0.05% of Pluronic F-127. Coverslips were mounted on a custom-made aluminum perfusion block and viewed through an inverted fluorescence microscope. Fluorescence was excited by UV light at 340 nm and 380 nm alternately and the emitted light was collected via a CoolSnap camera attached to a Lambda LS lamp and a Lambda optical filter changer. Ratiometric measurements were made using computer software Simple PCI (Compix Inc., Cranberry Township, PA) every 3 sec. Approximately 40 cells were observed per dish and subjected to the identical stimulus sequence. Solutions were delivered by a solenoid-controlled 8-channel perfusion system at a flow rate at 6 ml/min. ET-1 (1 μΜ) and histamine (100 μΜ), SLIGRL- NH2 (100 μΜ) or chloroquine (300 μΜ) were delivered, usually in this order. After applications of pruritogens, 1 μΜ capsaicin or 100 μΜ allyl isothiocyanate (AITC) and 144 mM potassium were applied in this order. Stimulus duration was 30 s (10 s for capsaicin and AITC). Ratios were normalized to prestimulus baseline. Cells were judged to be responsive if the ratio value increased by more than 10% of the resting level following chemical application. Stimulus duration was 30 s (10 s for capsaicin). Ratios were normalized to prestimulus baseline. Cells were judged to be responsive if the ratio following chemical application increased by more than 10% of the resting level. [0144] Iontophoresis in human subjects
Six healthy adult human subjects (4 women and 2 men aged 30.2 years) participated in the experiment. Three hours after a tablet of fexofenadine 60 mg, a commercially available antihistamine H-i receptor blocker, was orally administered to the subjects, histamine dehydrochloride (10 mg/ml in distilled water) and endothelin-1 (10 μg ml in distilled water) were iontophoretically applied to the volar side of their left forearm as has been described before (40). Histamine and endothelin-1 were applied in a random order with a 10-minute interval, in which both the subjects and the examiner were blind to the information which substance was either histamine or endothelin-1. For iontophoresis of each substance, 0.2 mA constant current was applied for 60 seconds to the skin through the iontophoresis and counter electrodes in a round shape of a 5 mm and a 15 mm diameter, respectively, which were placed with a 5 cm distance. The subjects were asked to describe itch intensity on a 0 (no itch)-to-10 (worst itch imaginable) scale every 10 seconds for 10 minutes after the termination of iontophoresis.
[0145] Statistical analysis
Data are presented as mean ± SEM. Analyses were performed using the GraphPad Prism 4.0 software (GraphPad, LaJolla, USA). Between-group comparisons were performed by two-tailed Student's t test. Multiple comparisons within groups were performed by repeated- measures one-way ANOVA, followed by Bonferoni post hoc test or Dunnett's post hoc test if not indicated otherwise. Statistical significance was set at p< 0.05.
[0146] Study approval and human subjects
Written and verbal informed consent was obtained prior to enrollment in the study, according to the Declaration of Helsinki. Permission for human studies regarding obtaining tissue and immunostaining were given by the Ethical Committee of the University of Mijnster, Germany and University of California San Francisco. Patients did not receive topical medications within 2 weeks before obtaining biopsies. Skin samples included 4 lesional AD (2 males and 2 females, age 20-60 years, mean 30.8 years), 4 PN (2 males and 2 females, age 25-82 years, mean 47.4 years) and 7 normal skin, obtained from postoperative material (4 males and 3 females, age 20-86 years, mean 45.6 years), lontophorese is a non-invasive, relatively harmless method. Study was performed by volunteering authors of this manuscript (2 males and 4 females, age 25-40, mean 30.2 years) and results were analyzed by a third party to guarantee independence of the obtained results. None of the participants had neither skin lesions nor history of atopic diseases including allergic rhinitis and asthma, both personally and in the family. Written informed consent was obtained from all human subjects in this study. [0147] ETAR, ET-1 and ECE-1 co-localization in murine dorsal root ganglion neurons and skin
The inventors found that ECE-1 contributes to ET-1 -mediated itch behavior in mice. First, the expression of ET-1 , ETAR and ECE-1 in skin, cutaneous nerve fibers and DRG neurons has been tested and RT-PCR confirmed presence of mRNA for ET-1 , ETAR and ECE-1 in skin and the peripheral nervous system (Figure 5A). Protein expression of ETAR and ECE-1 by DRG neurons and skin was also confirmed by Western blot analysis (Figure 5B). Immunoreactive ET-1 , ETAR and ECE-1 were mainly localized in a subset of small to medium-sized DRG neurons, where 95.0 ± 1.4% of ETAR-positive neurons express ECE-1 (Figure 5C). Further, the present inventors classified these neurons on the basis of size (Table 1 ). Of the ETAR-expressing neurons, 69.2 ± 2.0% were small-sized (diameter <25μη"ΐ) and 30.8 ± 2.0% were medium-sized (25-40μη-ι). Of ET-1 -positive DRG neurons, 82.0 ± 2.8% were small-sized DRG neurons and 18.0 ± 2.5% were medium-sized. From all ECE-1 - positive neurons, 70.1 ± 3.6% were small-sized, 28.7 ± 3.4% were medium-sized and 1 .2 ± 0.2% were large-sized (diameter >40μη-ι) (Table 1 ). ETAR, ET-1 and ECE-1 co-localized with the peptidergic marker calcitonin gene-related peptide (CGRP) (ETAR, 37.5 ± 0.7%; ET-1 , 40.6 ±5.1 %; ECE-1 , 39.0 ± 4.7%) and to a lesser extent with the nonpeptidergic marker isolectin B4 (IB4) (ETAR, 7.9 ± 3.5%; ET-1 , 42.1 ±1 .1 %; ECE-1 , 26.1 ± 5.3%) (Figure 1 C, Table 1 ). Furthermore, 33% of ETAR- and ET-1-positive neurons also expressed TRPV1 , which is expressed by sensory nerves conveying itch as well as pain signals (3, 41 , 42). Further, the cutaneous expression of ET-1 , ETAR and ECE-1 in mice with pruritic chronic dermatitis was examined. Staining for ET-1 , ETAR and ECE-1 was observed in keratinocytes, inflammatory cells and endothelial cells; no staining was discernable in fibroblasts (data not shown) in any analysed skin samples. ET-1 , ETAR and ECE-1 also co- localized with the neuronal marker protein gene product 9.5 (PGP9.5) in subepidermal cutaneous nerve fibers (Figure 5D). Induction of an atopic-like phenotype induced increased ET-1 staining in epidermal layer compared to normal murine skin (Figure 5E). The present inventors further demonstrate, that ET-1 induces a strong Ca2+-signals in neurons (43) and epithelial cells (44). To confirm the functional expression of ET-1 , ETAR and ECE-1 in sensory nerves, the effects of ET-1 on the modulation of [Ca2+], in murine DRG neurons in vitro was examined. Stimulation with 100 nM ET-1 100 nM increased [Ca2+], in DRG neurons (Figure 6), which indicates that ET-1 activates intracellular signaling cascades in murine DRG neurons. Based on the expression pattern of ETAR and ETBR in murine DRG neurons, ET-1 exerts its actions on small- to medium-sized sensory nerves by binding to ETAR. Similar to the percentage of neurons that respond to other endogenous pruritogens, such as bovine adrenal medulla 8-22 peptide (BAM8-22) or thymic stromal lymphopoietin (45, 46), only a small proportion of these small- to medium sized DRG neurons (3.0%; 78/2589) exhibited a transient increase in [Ca2+], in response to ET-1 application (Figure 7B). It was found that 69.2% of 13 ET-1 -responsive DRG neurons responded to histamine, 24.0% of 25 cells responded to chloroquine (CQ), 14.8% of 27 cells responded to SLIGRL-NH2, 85.5 of 29 cells responded to capsaicin, and 73.5% of 49 cells responded to allyl isothiocyanate (AITC, mustard oil, MO) (Figure 7C, D). To further examine the role of TRPV1 and TRPA1 for ET-1 evoked [Ca2+]i, TRPA1 and TRPV1 "'" DRG neurons were additionally tested. In 526 tested TRPV1"'" DRG neurons, the percentage of ET-1 -responsive was comparable to WT DRG neurons (Figure 7B). The percentage of ET-1 -responsive cells TRPA1 "'" DRG neurons trended to be reduced slightly, but was not significant in 901 tested cells (Mann Whitney U test, p=0.595; Figure 2B). These data suggest that ET-1 does not activate a subset of TRPV1 -, but could act partly via TRPA1 -positive sensory neurons. Our results indicate that ET-1 , ETAR and ECE-1 are expressed by small- to medium-sized sensory nerves projecting into the skin, and suggest that ET-1/ETAR-induced itch may be modulated by endosomal ECE-1 in peripheral sensory neurons in mice.
[0148] Inhibition of ECE-1 enhances ET-1 -induced scratching behavior in vivo
ET-1 evokes itch sensation in BALB/c and male Swiss mice (1 1 , 17). Intradermal injection of ET-1 (100 pmol) to the cheek evoked a marked scratching response also in C57BL/6 wild type (WT) mice (Figure 7A, E) (47, 48). Gomes et al. (47) reported that ET-1 also acts as a nociceptive agonist, thus the inventors took advantage of the cheek model to distinguish between ET-1 induced scratching behavior or pain behavior (49). In the present experimental setting, ET-1 induced a concentration-dependent scratching response (from 1 pmol to 1 nmol/site, Figure 8). However, in contrast to the published observation, any wiping movements in any treated mouse using those concentrations were not observed (data not shown). ET-1 -induced itch was independent of a contribution by a functional H1 R (Figure 7F). Also TRPV1 "'", TRPV4"'" and TRPM8"'" mice show no differences in ET-1 -induced scratching responses (Figure 7A), whereas TRPA1 "'" mice showed a marked reduction in ET- 1 triggered itch when compared to WT mice (Figure 7A). Therefore a TRPA1 inhibitor was injected intraperitonally (i.p.; 30 mg/kg, HC-030031 ) 30 min before ET-1 injection (100 pmol/site) into the cheek and a reduced scratching behavior as compared to vehicle control could be observed (Figure 9). The present inventors found that ECE-1 controls ET-1 -evoked itch by the following. Mice were pre-treated with SM-19712, a specific ECE-1 inhibitor, followed by intradermal injection of ET-1 (100 pmol) to the nape of the neck and measured scratching behavior. A significant increase of scratching bouts was found when ET-1 was applied after SM-19712 pre-treatment (ECE-1 blockage). In contrast, SM-19712 pre- treatment alone did not change scratching behavior in mice in vivo (data not shown).
[0149] ET-1 induces internalization of ETAR in DRG neurons To determine if ET-1 stimulates activation and trafficking of ETAR, the subcellular localization of immunoreactive ETAR and ECE-1 in DRG neurons was examined by confocal microscopy. In unstimulated small diameter DRG neurons, ETAR was prominently localized to the cell surface, whereas ECE-1 was mainly localized in vesicles (Figure 10A). It has been previously shown in KNRK and HEK cells that ECE-1 b and ECE-1 d isoforms are co-localized with early endosomal antigen-1 (EEA-1 ) in endosomes (28). Previous studies with Chinese Hamster ovary (CHO) cells demonstrated that ET-1 co-localizes with ETAR in internalized vesicular structures (27, 50). When DRG neurons were stimulated with ET-1 (100 nM) for 10 min, ETAR redistributed to early endosomes containing ECE-1 (Figure 10A), and EEA-1 (Figure 1 1 ). By using Image J quantification, it could be demonstrated that immunofluorescence for ETAR decreased from the cell membrane and increased in the cytosol after ET-1 stimulation (Figure 10C). ETAR reappears on the cell membrane 60 min after ET-1 stimulation whereas ECE-1 re-localized in ETAR-negative endosomes, indicating receptor recycling (Figure 10A, Figure 10C for statistical analysis) (28). It could be further confirmed that internalized ET-1 co-localized with ECE-1 (Figure 12). Immunofluorescence for ET-1 increased after ET-1 stimulation but disappeared over time whereas ECE-1 immunofluorescence was constant over a 120 min time course. Therefore, stimulation of ETAR on DRG neurons by ET-1 induces co-localization of the ligand/receptor-complex in early endosomes, which are also ECE-1 positive. The present results indicate that ETAR recycles back to the cell membrane, while ET-1 gets degraded. The role of ECE-1 in the ETAR recycling was determined. Pre-treatment of cultured primary DRG neurons with ECE-1 inhibitor SM-19712 (10 μΜ) did not influence ET-1 evoked internalization and co-localization of ETAR with ECE-1 but prolonged co-localization of ETAR with ECE-1 in endosomes and prevented recycling of the receptor back to the cell surface even after 60 min (Figure 10B, 10C). Previous studies in KNRK and HEK model cell lines have shown that ECE-1 degrades neuropeptides in acidified early endosomes and allow the receptor, freed from agonist and β- arrestins to recycle back to the plasma membrane (28, 30). Here, it is show for the first time that ECE-1 has a similar role in DRG neurons, which naturally express ET-1 and ETAR.
[0150] Internalized ET-1 is degraded by endosomal ECE-1
It is well known that extracellular localized ECE-1 hydrolyzes big endothelin-1 at neutral pH and thereby generates bioactive ET-1 (33). However, endosomal ECE-1 isoforms can degrade neuropeptides at acidic milieu with an optimum of pH 5.6 - 5.8 (33). The present results show that ECE-1 co-localized with ET-1 and ETAR in endosomes of DRG neurons (Figure 10 and Figure 1 1 , 12). This demonstrates that ECE-1 degrades ET-1 in acidified endosomes (lysosomes) of DRG neurons to regulate ETAR recycling and endosomal signaling, confirmed by the observation of ECE-1 mediated degradation of ET-1 at acidic endosomal pH using an in vitro approach: ET-1 was incubated with recombinant human (rh) ECE-1 at endosomal (pH 5.5) and extracellular (pH 7.4) acidities for 90 min. The degradation products were analyzed by HPLC and MALDI-Toff spectrometry. Intact ET-1 control peptide that was not incubated with ECE-1 eluted from HPLC at 28 min. After incubation with rhECE- 1 at pH 5.5, two degradation products were identified that eluted at 24 and 26 min from HPLC. During incubation for 90 min, intact ET-1 decreased to 48% of the initial value, and the two degradation products increased (Figure 10D, Figure 13). Mass spectrometry analysis indicated that ET-1 was hydrolyzed by ECE-1 between amino acid residues Asp18-lle19 (Figure 10E). ET-1 degradation was pH-dependent, since there was minimal degradation at pH 7.4 (99% intact ET-1 after 90 min incubation with rhECE-1 ). Together, these results indicate that internalized ET-1 could be degraded by endosomal ECE-1 at acidic pH in DRG neurons in vivo.
[0151] ECE-1 inhibitor prolongs ET-1 -induced phosphorylation of ERK1/2 in DRG neurons in vitro
The present results clearly demonstrate that ECE-1 promotes ETAR recycling and thus resensitization in DRG neurons in vitro, and that inhibition of ECE-1 intensifies ET-1 -evoked scratching behavior in mice in vivo. These observations indicate a fundamental contribution of ligand (ET-1 ) dissociation and degradation by ECE-1 in the pathophysiology of itch. The inventors demonstrated that ET-1 mediates activation of ERK1/2 in DRG neurons and that ECE-1 modulates the status of ET-1 -triggered ERK1/2 phosphorylation, confirmed by pre- treating DRG neurons with SM19712 (10 μΜ) or vehicle for 60 minutes and subsequently stimulated cultured DRG neurons with ET-1 (100 nM). Cellular lysates were analyzed by western blotting to detect the phosphorylation status of ERK1/2 and p38. ET-1 rapidly activated of ERK1/2 which peaked at 5 min, and ERK1/2 phosphorylation subsequently declined, and was not observable 120 min after agonist application (Figure 14). No phosphorylation of the pain-modulator p38 (51 , 52) was induced by ET-1 . This data confirms a recent publication by Cevikbas et al. showing that the pruritogenic cytokine IL-31 induce phosphorylation of ERK1/2 but not of p38 (53). In contrast, the time-course of ET-1 induced ERK1/2 activation in DRG neurons pre-treated with the ECE-1 inhibitor SM-19712 resembled that of vehicle-treated probes (Figure 14). As in DRG neurons challenged with ET-1 alone, those co-treated with ET-1 and SM-19712 demonstrated a rapid activation of ERK1/2 (within 5 min after agonist application). However, in marked contrast to neurons treated with ET-1 alone, ECE-1 inhibition evoked a remarkably sustained ET-1 -stimulated ERK1/2 activation, that remained elevated over the monitored time-course of 120 min (Figure 14).
[0152] Inhibition of ERK1/2 abolishes ET-1 induced pruritus in mice in vivo The present in vitro data indicate a potential role of ERK1/2 signaling in ET-1 -induced pruritus in vivo. Consequently, a pharmacological approach in a mouse model of itch to determine the importance of ERK1/2 activation for ET-1 -triggered pruritus in vivo was used. An ERK1/2 inhibitor (ERK inhibitor, Calbiochem, 30 mg/kg) was administered i.p. 30 min prior to ET-1 application either in the nape of neck (Figure 15A) or cheek (Figure 15B) of WT mice. This pre-treatment abolished of ET-1 -evoked scratching behaviour in both mouse models (Figure 15A, 15B). Importantly, mice co-treated with ET-1 plus ERK inhibitor or ERK inhibitor alone showed no signs of sedation, pain or inactivity. Administration of a MEK1/ERK1/2 inhibitor (PD0325901 , 10 mg/kg) recapitulated the effect of ERK1/2 inhibitor treatment (Figure 15B). To study a more fundamental and central role of ERK1/2 in conveying itch signals, a panel of multiple pruritogens were investigated in their dependency on the MEK-ERK pathway. With the exception of histamine-induced (100 μg/10 μΙ) itch, MEK-ERK inhibition suppressed the effects of all tested pruritogens (Figure 15B). Thus, in addition to ET-1 , MEK1 or ERK1/2 inhibition significantly reduced or abolished the pruritogenic actions of chloroquine (200 μg/10 μΙ) and IL-31 (5 nmol/10 μΙ).
[0153] ETAR inhibition alleviates pruritic responses in chronic pruritic mouse models
To test whether ET-1 /ETAR might be integral to pathophysiology of pruritic diseases, ETAR signaling in WT mice and in a chronic, oxazolone-driven pruritic mouse model (54) was inhibited. Local injection of BQ-123 (1 nmol/site) 30 min prior ET-1 injection into the cheek (100 pmol) was sufficient to inhibit scratching behavior (Figure 9). Chronic treatment with oxazolone for several days induced a chronic pruritic skin disorder characterized by eczema and intolerable itch. Chronification of the mouse model was established starting from day 12 and identified by chronic and stable scratching behavior. Concurrent local injection into oxazolone-applied skin with the ETAR inhibitor BQ-123 (10 nmol or 25 nmol/100 μΙ) significantly reduced the scratching behavior in chronic pruritic dermatitis (Figure 15C). The present inventors conclude that ET-1/ETAR/ECE-1 pathway plays an important role in controlling chronic itch in mice.
[0154] ET-1 is upregulated in human chronic pruritic diseases
In order to develop beneficial new therapies in humans, it is critical to translate findings from rodents into the human system. Although ET-1 is as a potent pruritogen in mice acting at a picomolar range (1 1 ), the mechanism and impact of ET-1 -induced pruritus in humans is poorly understood. To test whether ET-1 may also account for itch symptoms involved in human skin diseases, the immunoreactivity of ETAR and ET-1 in skin biopsies from patients with atopic dermatitis (AD, n=4) and prurigo nodularis (PN, n = 4), a disease characterized by chronic, intractable pruritus, were compared to healthy skin controls (n = 7). In PN (Figure 16B), the immunoreactivity of ET-1 was significantly increased in all layers of the epidermis but ETAR was not elevated while no or very weak staining for ETAR and ET-1 was observed in healthy skin controls (Figure 16B). In AD, the amount of total ET-1 in the epidermis might be enhanced due to acanthosis albeit increased immunoreactivity on the level of a single keratinocyte was not observed (Figure 16C). Likewise, ETAR immunostaining was not enhanced on keratinocytes or sensory nerves in human AD skin (data not shown). ETAR, ET-1 and ECE-1 expression was also confirmed for peripheral nerves in skin biopsies taken from PN patients (Figure 16A).
[0155] ET-1 induces histamine-independent pruritus in humans
To correlate the increased distribution of ET-1 and ETAR in prurigo with ET-1 -induced effects in humans, ET-1 was applied into human skin forearm by iontophoresis. Corresponding to the results observed in mice (48) and our own, ET-1 elicited an itch response in human subjects that was mainly independent from histamine-1 receptor (H1 R) function (Figure 16C). Pre-application of a H 1 R antagonist did not significantly diminish the ET-1 induced effects in human skin for the first 5 minutes after agonist application but alleviated from itch in the following monitored time interval (Figure 16C). Of note, while ET-1 induced a long-lasting itch in human subjects treated with an anti-H1 R antagonist, histamine failed to produce itch in the setting. The ET-1 -induced local erythema was comparable to the histamine-induced erythema after iontophoresis but no significant axon-reflex flare or a wheal was observed for ET-1 (data not shown), which is usually observed after histamine application. This demonstrates a different mechanism of ET-1 - to histamine-induced pruritus in humans. Because ET-1 has been linked to pain perception, the inventors asked the human subjects also for non-itch responses after ET-1 application. Almost all human subjects had described a stinging sensation during the first 2-3 min after ET-1 stimulation but a pain-related sensation was only described for seconds after ET-1 application (Figure 17). Together, in vivo and ex vivo findings in human demonstrates that ET-1 is increased in chronic pruritic skin diseases, is released by C-fibers, keratinocytes and endothelial cells in prurigo, and activates ETAR on myelinated nerve fibers.
[0156] The cellular mechanisms that regulate histamine-independent pruritus and itch- associated cell signaling in neurons are still poorly understood. Here, it is described for the first time that an intracellular neural peptidase, ECE-1 , regulates ET-1 -induced pruritus and intracellular cell signaling in DRG neurons. This is based on the following evidence: ET-1 , ETAR and ECE-1 are expressed on mouse DRG neurons at RNA- and protein level, and can be co-localized on small- to medium-size diameter neurons, predominantly of the peptidergic (CGRP-positive) type. ET-1 , ETAR and ECE-1 are co-localized in endosomes of murine DRG neurons after ETAR stimulation, suggesting that ECE-1 is important for hydrolyzing of ET-1 and recycling of the internalized ET-1/ETAR complex. The inventors discovered that ECE-1 hydrolyses ET-1 in acidified endosomes (lysosomes) with the observation that ECE-1 cleaves ET-1 at a pH of 5.5, but not 7.4, indicating that ECE-1 cleaves ET-1 in lysosomes. ET-1 induces Ca2+-mobilization and ERK1/2 phosphorylation in DRG neurons. Application of ET-1 into the murine skin results in marked scratching behaviour in vivo. ECE-1 inhibition markedly enhances ET-1 -induced scratching behavior in vivo. The itch response that was induced by ET-1 and augmented by ECE-1 in mice in vivo is associated with a sustained ERK1/2 induction in DRG neurons, but not p38. ERK1/2 inhibition abrogates ET-1 -induced scratching behavior in vivo. Finally, in humans ET-1 induces a pruritic response, independent of H1 R and its expression is upregulated in pruritic skin of patients with chronic prurigo suggesting a role of this pathway in human chronic pruritic disease. Based on the translational data, it is shown that ECE-1 is an important regulator of ET-1 -induced pruritus in mice and humans.
[0157] The results shown herein demonstrate that inhibition of ECE-1 lead to a sustain activation of ERK1/2. The inventors successfully identified the role of ECE-1 in the mechanism of itch. Here, there is a first evidence that ET-1 -induced pruritus is also histamine-independent in humans, and that ET-1 is upregulated in patients with chronic itch (PN) suggesting a role of this neural peptide and its receptor ETAR in human pruritic disease, and inaugurates the peptidase ECE-1 as a regulator of ET-1 -induced signaling. Of note, this is the first description establishing an endogenous negative regulator of itch transmission in mice and humans. This represents new strategies to treat pruritus and various pruritic diseases.
[0158] ETAR blocking compounds (40 mg/kg body-weight) in chronic oxazolone-induced, atopic-dermatitis-like itch model in mice
Reagents. ETAR/ETBR inhibitors Ambrisentan, Bosentan, Macitentan, Sitaxentan and Zibotentan were purchased from Selleck Chemicals (Headquaters Boston USA; Munich, Germany).
Chronic atopic-like pruritus model. WT mice were treated one time with 10% oxazolone in acetone/olive oil (4:1 v/v) on the shaved nape of the neck (100μΙ). After a resting period of 7 days, mice were treated with 1 % oxazolone in acetone/ olive oil (4:1 v/v) on the nape of the neck (100 Dl) every other day for an additional 10 days. Scratching behavior was evaluated for 30 minutes on days 18. For measurement, mice were placed individually in a glass cage for at least 30 minutes before behavioral studies. Scratching and wiping behavior was videotaped for 30 minutes with personnel kept out of the observation room and was analyzed in a double-blinded fashion. Analysis of the video footage to determine the number of scratching bouts included only scratching with the hind paw toward the chronically treated sit. A series of scratching movements within 1 second was counted as 1 bout of scratching. Ambrisentan, Bosentan, Macitentan, Sitaxentan and Zibotentan (all 40 mg/kg BW), or vehicle was injected i.p. 30 minutes before taping for scratching behavior.
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Claims

1 . A compound for use in the treatment of a subject suffering from itch, wherein said compound is endothelin-converting enzyme-1 (ECE-1 ).
2. A compound for use in the treatment of a subject suffering from itch, wherein said compound is a MEK1 inhibitor.
3. A compound for use in the treatment of a subject suffering from itch, wherein said compound is an endothelin A receptor (ETAR) inhibitor.
4. A compound for use according to any one of claim 1 to 3, wherein said itch is a histamine-independent, endothelin-1 (ET-1 )/ETAR induced itch.
5. The compound for use according to claim 4, wherein said subject has an acute or chronic pruritic skin disease.
6. The compound for use according to claim 5, wherein said acute or chronic pruritic skin disease is idiopathic chronic pruritus, inflammatory skin diseases, atopic dermatitis, skin eczemas, psoriasis, Lichen planus, neuropathic itch, hepatic disease, chronic renal failure, uraemia, HIV, Sezary's syndrome, T-cell lymphoma, leukemias, lymphoproliferative disorders, chronic urticaria, chronic renal diseases, mycosis fungoides, mastocytosis, contact dermatitis, nostalgia paresthetica, herpes zoster, pruritus, ichthyosis, pallor xerosis, prurigo nodularis, skin eczema, xerosis cutis, Graver's disease, or cutaneous post herpetic itch.
7. The compound for use according to any one of claim 2, 4 to 6, wherein said MEK1 inhibitor is selected from the group consisting of:
N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide,
6-(4-bromo-2-fluoroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5- carboxamide,
N-[(2S)-2,3-dihydroxypropyl]-3-(2-fluoro-4-iodoanilino)pyridine-4-carboxamide,
6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5- carboxamide, 2-(2-fluoro-4-iodoanilino)-N-(2-hydroxyethoxy)-1 ,5-dimethyl-6-oxopyridine-3- carboxamide,
N-[3,4-difluoro-2-(2-fluoro-4-iodoanilino)-6-methoxyphenyl]-1 -[(2S)-2,3- dihydroxypropyl]cyclopropane-1 -sulfonamide,
2- (2-chloro-4-iodoanilino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide,
N-[3-[3-cyclopropyl-5-(2-fluoro-4-iodoanilino)-6,8-dimethyl-2,4,7-trioxopyrido[4,3- d]pyrimidin-1 -yl]phenyl]acetamide,
N-[3-[5-(2-aminopyrimidin-4-yl)-2-tert-butyl-1 !3-thiazol-4-yl]-2-fluorophenyl]-2,6- difluorobenzenesulfonamide;methanesulfonic acid,
N-[3-[5-(2-aminopyrimidin-4-yl)-2-tert-butyl-1 !3-thiazol-4-yl]-2-fluorophenyl]-2,6- difluorobenzenesulfonamide,
N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide;
3,4-difluoro-2-(2-fluoro-4-iodoanilino)-N-(2-hydroxyethoxy)-5-[(3-oxooxazinan-2- yl)methyl]benzamide,
(2S,3S)-2-[(4R)-4-[4-[(2R)-2!3-dihydroxypropoxy]phenyl]-2,5-dioxoimidazolidin-1 -yl]-N-(2- fluoro-4-iodophenyl)-3-phenylbutanamide,
3- [(2R)-2,3-dihydroxypropyl]-6-fluoro-5-(2-fluoro-4-iodoanilino)-8-methylpyrido[2,3- d]pyrimidine-4,7-dione,
[3,4-difluoro-2-(2-fluoro-4-iodoanilino)phenyl]-[3-hydroxy-3-[(2S)-piperidin-2-yl]azetidin-1 - yl]methanone,
5-bromo-N-(2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide,
2-[4-[(2-butyl-4-oxo-1 ,3-diazaspiro[4 ]non-1 -en-3-yl)methyl]-2-(ethoxymethyl)phenyl]-N- (4,5-dimethyl-1 ,2-oxazol-3-yl)benzenesulfonamide,
2-[4-[(2-butyl-4-oxo-1 ,3-diazaspiro[4 ]non-1 -en-3-yl)methyl]-2-(ethoxymethyl)phenyl]-N- (3,4-dimethyl-1 ,2-oxazol-5-yl)benzenesulfonamide, 2-[4-[(2-butyl-4-oxo-1 ,3-diazaspiro[4 ]non-1 -en-3-yl)methyl]-2-propylphenyl]-N-(4,5- dimethyl-1 ,2-oxazol-3-yl)benzenesulfonamide.
8. The compound for use according any one of claim 3 to 6, wherein said ETAR inhibitor is selected from the group consisting of:
4-tert-butyl-N-[6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl)pyrimidin-4- yl]benzenesulfonamide,
(5-(4-bromophenyl)-6-[2-(5-bromopyrimidin-2-yl)oxyethoxy]-N-(propylsulfamoyl)pyrimidin- 4-amine,
(2S)-2-[(4,6-dimethylpyrimidin-2-yl)oxy]-3-methoxy-3,3-diphenylpropanoic acid,
N-[6-(2-Hydroxyethoxy)-5-(2-methoxyphenoxy)-2-[2-(2H-tetrazol-5-yl)pyridin-4- yl]pyrimidin-4-yl]-5-propan-2-ylpyridine-2-sulfonamide,
N-(4-chloro-3-methyl-1 ,2-oxazol-5-yl)-2-[2-(6-methyl-2H-1 ,3-benzodioxol-5- yl)acetyl]thiophene-3-sulfonamide,
N-(3-Methoxy-5-methylpyrazin-2-yl)-2-[4-(1 ,3,4-oxadiazol-2-yl)phenyl]pyridine-3- sulfonamide,
2- [(3R!6R!9S!12R!15S)-6-(1 H-indol-3-ylmethyl)-9-(2-methylpropyl)-2!5!8!1 1 ,14-pentaoxo- 12-propan-2-yl-1 ,4,7,10,13-pentazabicyclo[13.3.0]octadecan-3-yl]acetic acid,
3- benzodioxol-5-yl)-1 -[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3- carboxylic acid,
(2R,3R,4S)-4-(1 ,3-benzodioxol-5-yl)-1 -[2-(dibutylamino)-2-oxoethyl]-2-(4- methoxyphenyl)pyrrolidine-3-carboxylic acid,
(2R,3R,4S)-4-(1 ,3-benzodioxol-5-yl)-1 -[2-(dibutylamino)-2-oxoethyl]-2-(2- methoxyphenyl)pyrrolidine-3-carboxylic acid,
3-(1 ,3-benzodioxol-5-yl)-5-hydroxy-5-(4-methoxyphenyl)-4-[(3,4,5- trimethoxyphenyl)methyl]furan-2-one, (2R)-2-[[(2R)-2-[[(2S)-2-(azepane-1 -carbonylamino)-4-methylpentanoyl]amino]-3-(1 - formylindol-3-yl)propanoyl]amino]-3-(1 H-indol-3-yl)propanoic acid,
2-(1 ,3-benzodioxol-5-yl)-4-(4-methoxyphenyl)-4-oxo-3-[(3,4,5- trimethoxyphenyl)methyl]but-2-enoate,
(2S)-2-(4,6-dimethoxypyrimidin-2-yl)oxy-3-methoxy-3,3-diphenylpropanoic acid,
N-[6-methoxy-5-(2-methoxyphenoxy)-2-pyridin-4-ylpyrimidin-4-yl]-5-methylpyridi sulfonamide,
N-[5-(2-methoxyphenoxy)-2-pyridin-4-yl-6-(trideuteriomethoxy)pyrimidin-4-yl]-5- methylpyridine-2-sulfonamide,
N-[6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-[2-(2H-tetrazol-5-yl)pyridin-4- yl]pyrimidin-4-yl]-5-methylpyridine-2-sulfonamide,
[6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-[2-(1 ,2,3-triaza-4-azanidacyclopenta-2,5- dien-5-yl)pyridin-4-yl]pyrimidin-4-yl]-(5-methylpyridin-2-yl)sulfonylazanide,
4-amino-N-(3,4-dimethylisoxazol-5-yl)benzenesulfonamide, and
[(7R)-5-chloro-3-[(1 E,3E,5S)-3,5-dimethylhepta-1 ,3-dienyl]-7-methyl-6,8- dioxoisochromen-7-yl] acetate.
9. The compound for use according to any of the preceding claims, wherein said subject is a mammal.
10. The compound for use according to claim 9, wherein said mammal is mouse, rat, guinea pig, rabbit, cat, dog, monkey, horse or human.
1 1 . The compound for use according to any of the preceding claims, wherein the use comprises administering a therapeutically effective amount of said compound to the subject.
12. The compound for use according to claim 1 1 , wherein said therapeutically effective amount is sufficient to inhibit or alleviate said histamine-independent, ET-1/ETAR induced itch.
13. The compound for use according to claim 12, wherein the amount is between 0.01 mg per kg body weight and 1 g per kg body weight, preferably between 0.01 mg per kg body weight and 10 mg per kg body weight.
14. The compound for use according to any one of claim 1 1 to 13, wherein said administration is carried out orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, transdermally, or by application to mucous membranes.
15. The compound for use according to any one of claim 1 1 to 14, wherein said compound is administered together with a pharmaceutically acceptable excipient or carrier.
16. The compound for use according to any one of claim 1 1 to 14, wherein said compound is administered in a combination with another anti-pruritic drug and/or a compound used to treat skin diseases.
17. The compound for use according to claim 16, wherein said anti-pruritic drug and/or compound used to treat skin diseases is selected from the group consisting of antihistamines, glucocorticosteroids, calcineurin inhibitors, local anaesthetics, serotonin- reuptake inhibitors (SSRI), menthol, campher, neuroleptics, topical antidepressants, tetracyclics, neurokinin-1 receptor antagonists, mu-opioid receptor antagonists, kappa opioid receptor antagonists, protease inhibitors, protease-activated receptor antagonists, gastrin-realising peptide and gastrin-releasing peptide receptor antagonists, brain-derived natriuretic peptide (BNP) and its receptor antagonists, dynorhin receptor antagonists, cytokine and chemokine receptor antagonists, botulinum toxin.
18. The compound for use according to claim 16 or 17, wherein said combination is administered as a combined formulation or separate from each other.
19. A topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w ECE-1 and one or more pharmaceutically acceptable excipients.
20. A topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w MEK1 inhibitor and one or more pharmaceutically acceptable excipients.
21 . A topical pharmaceutical composition comprising 0.1 % w/w to 10% w/w endothelin A receptor (ETAR) inhibitor and one or more pharmaceutically acceptable excipients.
22. The compound for use according to any one of claim 1 1 to 14, wherein said compound is administered in combination with ultraviolet radiation therapy.
23. The composition according to claim 20, wherein said MEK1 inhibitor is selected from the group consisting of:
N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide
(compound XXVIII),
6-(4-bromo-2-fluoroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5- carboxamide,
N-[(2S)-2,3-dihydroxypropyl]-3-(2-fluoro-4-iodoanilino)pyridine-4-carboxamide,
6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5- carboxamide,
2-(2-fluoro-4-iodoanilino)-N-(2-hydroxyethoxy)-1 ,5-dimethyl-6-oxopyridine-3- carboxamide,
N-[3,4-difluoro-2-(2-fluoro-4-iodoanilino)-6-methoxyphenyl]-1 -[(2S)-2,3- dihydroxypropyl]cyclopropane-1 -sulfonamide,
2-(2-chloro-4-iodoanilino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide,
N-[3-[3-cyclopropyl-5-(2-fluoro-4-iodoanilino)-6,8-dimethyl-2,4,7-trioxopyrido[4,3- d]pyrimidin-1 -yl]phenyl]acetamide,
N-[3-[5-(2-aminopyrimidin-4-yl)-2-tert-butyl-1 ,3-thiazol-4-yl]-2-fluorophenyl]-2,6- difluorobenzenesulfonamide;methanesulfonic acid,
N-[3-[5-(2-aminopyrimidin-4-yl)-2-tert-butyl-1 ,3-thiazol-4-yl]-2-fluorophenyl]-2,6- difluorobenzenesulfonamide,
N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide;
3,4-difluoro-2-(2-fluoro-4-iodoanilino)-N-(2-hydroxyethoxy)-5-[(3-oxooxazinan-2- yl)methyl]benzamide, (2S!3S)-2-[(4R)-4-[4-[(2R)-2!3-dihydroxypropoxy]phenyl]-2,5-dioxoimidazolidin-1 -yl]-N-(2- fluoro-4-iodophenyl)-3-phenylbutanamide,
3- [(2R)-2,3-dihydroxypropyl]-6-fluoro-5-(2-fluoro-4-iodoanilino)-8-methylpyrido[2,3- d]pyrimidine-4,7-dione,
[3,4-difluoro-2-(2-fluoro-4-iodoanilino)phenyl]-[3-hydroxy-3-[(2S)-piperidin-2-yl]azetidin yl]methanone,
5-bromo-N-(2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide,
2-[4-[(2-butyl-4-oxo-1 ,3-diazaspiro[4.4]non-1 -en-3-yl)methyl]-2-(ethoxymethyl)phenyl]-N- (4,5-dimethyl-1 ,2-oxazol-3-yl)benzenesulfonamide,
2-[4-[(2-butyl-4-oxo-1 ,3-diazaspiro[4.4]non-1 -en-3-yl)methyl]-2-(ethoxymethyl)phenyl]-N- (3,4-dimethyl-1 ,2-oxazol-5-yl)benzenesulfonamide,
2-[4-[(2-butyl-4-oxo-1 ,3-diazaspiro[4.4]non-1 -en-3-yl)methyl]-2-propylphenyl]-N-(4,5- dimethyl-1 ,2-oxazol-3-yl)benzenesulfonamide.
24. The composition according to claim 21 , wherein said ETAR inhibitor is selected from the group consisting of:
4- tert-butyl-N-[6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl)pyrimidin-4- yl]benzenesulfonamide,
(5-(4-bromophenyl)-6-[2-(5-bromopyrimidin-2-yl)oxyethoxy]-N-(propylsulfamoyl)pyrimidin- 4-amine,
(2S)-2-[(4,6-dimethylpyrimidin-2-yl)oxy]-3-methoxy-3,3-diphenylpropanoic acid,
N-[6-(2-Hydroxyethoxy)-5-(2-methoxyphenoxy)-2-[2-(2H-tetrazol-5-yl)pyridin-4- yl]pyrimidin-4-yl]-5-propan-2-ylpyridine-2-sulfonamide,
N-(4-chloro-3-methyl-1 ,2-oxazol-5-yl)-2-[2-(6-methyl-2H-1 ,3-benzodioxol-5- yl)acetyl]thiophene-3-sulfonamide, N-(3-Methoxy-5-methylpyrazin-2-yl)-2-[4-(1 ,3,4-oxadiazol-2-yl)phenyl]pyridine-3- sulfonamide,
2- [(3R!6R!9S 2R 5S)-6-(1 H-indol-3-ylmethyl)-9-(2-methylpropyl)-2!5!8!1 1 ,14-pentaoxo- 12-propan-2-yl-1 ,4,7,10,13-pentazabicyclo[13.3.0]octadecan-3-yl]acetic acid,
3- benzodioxol-5-yl)-1 -[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3- carboxylic acid,
(2R,3R,4S)-4-(1 ,3-benzodioxol-5-yl)-1 -[2-(dibutylamino)-2-oxoethyl]-2-(4- methoxyphenyl)pyrrolidine-3-carboxylic acid,
(2R,3R,4S)-4-(1 ,3-benzodioxol-5-yl)-1 -[2-(dibutylamino)-2-oxoethyl]-2-(2- methoxyphenyl)pyrrolidine-3-carboxylic acid,
3- (1 ,3-benzodioxol-5-yl)-5-hydroxy-5-(4-methoxyphenyl)-4-[(3,4,5- trimethoxyphenyl)methyl]furan-2-one,
(2R)-2-[[(2R)-2-[[(2S)-2-(azepane-1 -carbonylamino)-4-methylpentanoyl]amino]-3-(1 - formylindol-3-yl)propanoyl]amino]-3-(1 H-indol-3-yl)propanoic acid,
2-(1 ,3-benzodioxol-5-yl)-4-(4-methoxyphenyl)-4-oxo-3-[(3,4,5- trimethoxyphenyl)methyl]but-2-enoate,
(2S)-2-(4,6-dimethoxypyrimidin-2-yl)oxy-3-methoxy-3,3-diphenylpropanoic acid,
N-[6-methoxy-5-(2-methoxyphenoxy)-2-pyridin-4-ylpyrimidin-4-yl]-5-methylpyridine-2- sulfonamide,
N-[5-(2-methoxyphenoxy)-2-pyridin-4-yl-6-(trideuteriomethoxy)pyrimidin-4-yl]-5- methylpyridine-2-sulfonamide,
N-[6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-[2-(2H-tetrazol-5-yl)pyridin-4- yl]pyrimidin-4-yl]-5-methylpyridine-2-sulfonamide,
[6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-[2-(1 ,2,3-triaza-4-azanidacyclopenta-2,5- dien-5-yl)pyridin-4-yl]pyrimidin-4-yl]-(5-methylpyridin-2-yl)sulfonylazanide,
4- amino-N-(3,4-dimethylisoxazol-5-yl)benzenesulfonamide, and [(7R)-5-chloro-3-[(1 E,3E,5S)-3,5-dimethylhepta-1 ,3-dienyl]-7-methyl-6,8- dioxoisochromen-7-yl] acetate.
25. The topical pharmaceutical composition according to any one of claim 19 to 23 in the form of ointment, cream, lotion, gel, balm-stick, spray or foam.
26. The topical pharmaceutical composition according to any one of claim 19 to 24, wherein said compound further comprises a keratolytic agent.
27. The topical pharmaceutical composition according to claim 25, wherein said keratolytic agent is one selected from the group consisting of salicylic acid, acetyl salicylic acid, magnesium salicylate, sodium salicylate, benzoic acid, urea, sulfur, colloidal sulfer; podophyllum resin, benzoyl peroxide, resorcinol, selenium disulphide, a-hydroxy acids, hydroxybenzoic acids, or alpha-hydroxycarboxylic acids.
28. The topical pharmaceutical composition according to any one of claim 19 to 26, further comprising a cooling agent.
29. The topical pharmaceutical composition according to claim 27, wherein said cooling agent is selected from the group consisting of menthol, an isomer of menthol, a menthol derivative, 4-Methyl-3-(1 -pyrrolidinyl)-2[5H]-furanone, WS-23, lcilin, lcilin Unilever Analog, 5-methyl-4-(1 -pyrrolidinyl)-3-[2H]-furanone; 4,5-dimethyl-3-(1 -pyrrolidinyl)-2[5H]-furanone; isopulegol, 3-(l-menthoxy)propane-1 ,2-diol, 3-(l-menthoxy)-2-methylpropane-1 ,2-diol, p- menthane-2,3-diol, p-menthane-3,8-diol, 6-isopropyl-9-methyl-1 ,4-dioxas- piro[4,5]decane-2-methanol, menthyl succinate and its alkaline earth metal salts, trimethylcyclohexanol, N-ethyl-2-isopropyl-5-methylcyclohexanecarb-oxamide, Japanese mint (Mentha arvensis) oil, peppermint oil, menthone, menthone glycerol ketal, menthyl lactate, 3-(1 -menthoxy)ethan-1 -ol, 3-(l-menthoxy)propan-1 -ol, 3-(l-menthoxy)butan-1 -ol, 1 -menthylacetic acid N-ethylamide, l-menthyl-4-hydroxypentanoate, l-menthyl-3- hydroxybutyrate, N,2,3-trimethyl-2-(1 -methylethyl)-butanamide, spearmint oil, camphor, or camphor derivates.
30. A method for the treatment of a subject suffering from itch, said method comprising administering a therapeutically effective amount of ECE-1 to a subject in need thereof.
31 . A method for the treatment of a subject suffering from itch, said method comprising administering a therapeutically effective amount of a MEK1 inhibitor to a subject in need thereof.
32. A method for the treatment of a subject suffering from itch, said method comprising administering a therapeutically effective amount of an endothelin A receptor (ETAR) inhibitor to a subject in need thereof.
33. The method according to any one of claim 29 to 31 , wherein said itch is a histamine- independent, endothelin-1 (ET-1 )/ETAR induced itch.
34. The method according to claim 32, wherein said subject has an acute or chronic pruritic skin disease.
35. The method according to claim 33, wherein said acute or chronic pruritic skin disease is idiopathic chronic pruritus, inflammatory skin diseases, atopic dermatitis, skin eczemas, psoriasis, Lichen planus, neuropathic itch, hepatic disease, chronic renal failure, uraemia, HIV, Sezary's syndrome, T-cell lymphoma, leukemias, lymphoproliferative disorders, chronic urticaria, chronic renal diseases, mycosis fungoides, mastocytosis, contact dermatitis, nostalgia paresthetica, herpes zoster, pruritus, ichthyosis, pallor xerosis, prurigo nodularis, skin eczema, xerosis cutis, Graver's disease, or cutaneous post herpetic itch.
36. The method according to any one of claim 29 to 34, wherein said therapeutically effective amount is sufficient to inhibit or alleviate itch.
37. The method according to claim 35, wherein the amount is between 0.01 mg per kg body weight and about 1 g per kg body weight, preferably between 0.1 and 10 mg per kg body weight.
38. The method according to any one of claim 30, 32 to 36, wherein said MEK1 inhibitor is selected from the group consisting of:
N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide,
6-(4-bromo-2-fluoroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5- carboxamide,
N-[(2S)-2,3-dihydroxypropyl]-3-(2-fluoro-4-iodoanilino)pyridine-4-carboxamide,
6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5- carboxamide, 2-(2-fluoro-4-iodoanilino)-N-(2-hydroxyethoxy)-1 ,5-dimethyl-6-oxopyridine-3- carboxamide,
N-[3,4-difluoro-2-(2-fluoro-4-iodoanilino)-6-methoxyphenyl]-1 -[(2S)-2,3- dihydroxypropyl]cyclopropane-1 -sulfonamide,
2- (2-chloro-4-iodoanilino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide,
N-[3-[3-cyclopropyl-5-(2-fluoro-4-iodoanilino)-6,8-dimethyl-2,4,7-trioxopyrido[4,3- d]pyrimidin-1 -yl]phenyl]acetamide,
N-[3-[5-(2-aminopyrimidin-4-yl)-2-tert-butyl-1 !3-thiazol-4-yl]-2-fluorophenyl]-2,6- difluorobenzenesulfonamide;methanesulfonic acid,
N-[3-[5-(2-aminopyrimidin-4-yl)-2-tert-butyl-1 !3-thiazol-4-yl]-2-fluorophenyl]-2,6- difluorobenzenesulfonamide,
N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide;
3,4-difluoro-2-(2-fluoro-4-iodoanilino)-N-(2-hydroxyethoxy)-5-[(3-oxooxazinan-2- yl)methyl]benzamide,
(2S,3S)-2-[(4R)-4-[4-[(2R)-2!3-dihydroxypropoxy]phenyl]-2,5-dioxoimidazolidin-1 -yl]-N-(2- fluoro-4-iodophenyl)-3-phenylbutanamide,
3- [(2R)-2,3-dihydroxypropyl]-6-fluoro-5-(2-fluoro-4-iodoanilino)-8-methylpyrido[2,3- d]pyrimidine-4,7-dione,
[3,4-difluoro-2-(2-fluoro-4-iodoanilino)phenyl]-[3-hydroxy-3-[(2S)-piperidin-2-yl]azetidin-1 - yl]methanone,
5-bromo-N-(2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide,
2-[4-[(2-butyl-4-oxo-1 ,3-diazaspiro[4 ]non-1 -en-3-yl)methyl]-2-(ethoxymethyl)phenyl]-N- (4,5-dimethyl-1 ,2-oxazol-3-yl)benzenesulfonamide,
2-[4-[(2-butyl-4-oxo-1 ,3-diazaspiro[4 ]non-1 -en-3-yl)methyl]-2-(ethoxymethyl)phenyl]-N- (3,4-dimethyl-1 ,2-oxazol-5-yl)benzenesulfonamide, 2-[4-[(2-butyl-4-oxo-1 ,3-diazaspiro[4.4]non-1 -en-3-yl)methyl]-2-propylphenyl]-N-(4,5- dimethyl-1 ,2-oxazol-3-yl)benzenesulfonamide.
39. The method according to any one of claim 31 to 36, wherein said ETAR inhibitor is selected from the group consisting of:
4-tert-butyl-N-[6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl)pyrimidin-4- yl]benzenesulfonamide,
(5-(4-bromophenyl)-6-[2-(5-bromopyrimidin-2-yl)oxyethoxy]-N-(propylsulfamoyl)pyrimidin- 4-amine,
(2S)-2-[(4,6-dimethylpyrimidin-2-yl)oxy]-3-methoxy-3,3-diphenylpropanoic acid,
N-[6-(2-Hydroxyethoxy)-5-(2-methoxyphenoxy)-2-[2-(2H-tetrazol-5-yl)pyridin-4- yl]pyrimidin-4-yl]-5-propan-2-ylpyridine-2-sulfonamide,
N-(4-chloro-3-methyl-1 ,2-oxazol-5-yl)-2-[2-(6-methyl-2H-1 ,3-benzodioxol-5- yl)acetyl]thiophene-3-sulfonamide,
N-(3-Methoxy-5-methylpyrazin-2-yl)-2-[4-(1 ,3,4-oxadiazol-2-yl)phenyl]pyridine-3- sulfonamide,
2- [(3R,6R,9S,12R,15S)-6-(1 H-indol-3-ylmethyl)-9-(2-methylpropyl)-2,5,8,1 1 ,14-pentaoxo- 12-propan-2-yl-1 ,4,7,10,13-pentazabicyclo[13.3.0]octadecan-3-yl]acetic acid,
3- benzodioxol-5-yl)-1 -[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3- carboxylic acid,
(2R,3R,4S)-4-(1 ,3-benzodioxol-5-yl)-1 -[2-(dibutylamino)-2-oxoethyl]-2-(4- methoxyphenyl)pyrrolidine-3-carboxylic acid,
(2R,3R,4S)-4-(1 ,3-benzodioxol-5-yl)-1 -[2-(dibutylamino)-2-oxoethyl]-2-(2- methoxyphenyl)pyrrolidine-3-carboxylic acid,
3-(1 ,3-benzodioxol-5-yl)-5-hydroxy-5-(4-methoxyphenyl)-4-[(3,4,5- trimethoxyphenyl)methyl]furan-2-one, (2R)-2-[[(2R)-2-[[(2S)-2-(azepane-1 -carbonylamino)-4-methylpentanoyl]amino]-3-(1 - formylindol-3-yl)propanoyl]amino]-3-(1 H-indol-3-yl)propanoic acid,
2-(1 ,3-benzodioxol-5-yl)-4-(4-methoxyphenyl)-4-oxo-3-[(3,4,5- trimethoxyphenyl)methyl]but-2-enoate,
(2S)-2-(4,6-dimethoxypyrimidin-2-yl)oxy-3-methoxy-3,3-diphenylpropanoic acid,
N-[6-methoxy-5-(2-methoxyphenoxy)-2-pyridin-4-ylpyrimidin-4-yl]-5-methylpyridi sulfonamide,
N-[5-(2-methoxyphenoxy)-2-pyridin-4-yl-6-(trideuteriomethoxy)pyrimidin-4-yl]-5- methylpyridine-2-sulfonamide,
N-[6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-[2-(2H-tetrazol-5-yl)pyridin-4- yl]pyrimidin-4-yl]-5-methylpyridine-2-sulfonamide,
[6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-[2-(1 ,2,3-triaza-4-azanidacyclopenta-2,5- dien-5-yl)pyridin-4-yl]pyrimidin-4-yl]-(5-methylpyridin-2-yl)sulfonylazanide,
4-amino-N-(3,4-dimethylisoxazol-5-yl)benzenesulfonamide, and
[(7R)-5-chloro-3-[(1 E,3E,5S)-3,5-dimethylhepta-1 ,3-dienyl]-7-methyl-6,8- dioxoisochromen-7-yl] acetate.
40. The method according to any one of claim 29 to 38, wherein said subject is a mammal.
41 . The method according to claim 39, wherein said mammal is mouse, rat, guinea pig, rabbit, cat, dog, monkey or human.
42. The method according to any one of claim 29 to 40, wherein said administration is carried out epicutaneously, orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, transdermally, or by application to mucous membranes.
43. The method according to any one of claim 29 to 41 , wherein said compound is administered together with a pharmaceutically acceptable excipient or carrier.
44. The method according to any one of claim 29 to 41 , wherein said compound is administered in a combination with another anti-pruritic drug and/or a compound used to treat skin diseases.
45. The method according to claim 43, wherein said anti-pruritic drug and/or compound used to treat skin diseases is selected from the group consisting of antihistamines, glucocorticosteroids, calcineurin inhibitors, local anaesthetics, serotonin-reuptake inhibitors (SSRI), menthol, campher, neuroleptics, topical antidepressants, tetracyclics, neurokinin-1 receptor antagonists, mu-opioid receptor antagonists, kappa opioid receptor antagonists, protease inhibitors, protease-activated receptor antagonists, gastrin-realising peptide and gastrin-releasing peptide receptor antagonists, brain-derived natriuretic peptide (BNP) and its receptor antagonists, dynorhin receptor antagonists, cytokine and chemokine receptor antagonists, botulinum toxin, or ultraviolett therapy.
46. The method according to claim 44, wherein said combination is administered as a combined formulation or separate from each other.
47. The method according to any one of claim 29 to 41 , wherein said compound is administered in combination with ultraviolet radiation therapy.
48. A method for screening a compound useful for the treatment of a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch, the method comprising assaying said compound for the activity in inhibiting the ET-1/ETAR induced signaling cascade.
49. The method according to claim 46, the method comprising:
a) administering the compound suspected as to be useful for the treatment of a histamine-independent ET-1/ETAR induced itch to an animal suffering from an acute or chronic pruritic skin disease, wherein said animal is a histamine-impervious animal, b) measuring the scratching movements of said animal within a defined time interval, c) comparing the scratching movements of said animal to the scratching movements of a reference animal to which the suspected compound has not been administered, the scratching movements of said reference animal measured within the same defined time interval.
50. The method according to claim 47, wherein a lower amount of scratching movements of the animal under administration of the suspected compound in comparison to the reference animal indicates that said suspected compound is useful for the treatment of a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch.
51 . The method according to claim 47, wherein an approximate or higher amount of scratching movements of the animal under administration of the suspected compound in comparison to the reference animal indicates that said suspected compound is not useful for the treatment of a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch.
52. The method according to any one of claim 47 to 49, wherein said histamine- impervious animal suffering from an acute or chronic pruritus skin disease is a histamine-1 receptor (H1 R) knockout animal.
53. The method according to any one of claim 47 to 49, wherein said histamine- impervious animal suffering from an acute or chronic pruritus skin disease is a histamine-4 receptor (H4R) knockout animal.
54. The method according to any one of claim 47 to 49, wherein said histamine- impervious animal suffering from an acute or chronic pruritus skin disease is a histamine-1 receptor (H1 R) and histamine-4 receptor (H4R) double knockout animal.
55. The method according to any one of claim 50 to 52, wherein said knockout animal is a heterozygous or homozygous knockout animal.
56. The method according to any one of claim 47 to 49, wherein said histamine- impervious animal suffering from an acute or chronic pruritus skin disease is a mast cell-deficient animal.
57. A method for diagnosing histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch in a subject, the method comprising:
a) determining the amount of endothelin-1 in a skin sample of said subject, and b) comparing the amount of endothelin-1 determined in a) to a reference skin sample.
58. The method according to claim 54, wherein a significantly increased amount of ET-1 in all layers of the epidermis as compared to said reference sample indicates a histamine- independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch.
59. The method according to claim 54, wherein no significantly increased amount of ET-1 in all layers of the epidermis as compared to said reference sample indicates that said subject does not suffer from a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch.
60. The method according to any one of claim 54 to 56, wherein determining the amount of ET-1 in a skin sample includes measuring the level of ET-1 mRNA in said sample.
61 . A diagnostic kit for detecting a histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch, comprising a binding molecule which specifically binds to endothelin-1 (ET-1 ).
62. The binding molecule according to claim 58, wherein said binding molecule is an antibody.
63. The binding molecule according to claim 59, wherein said antibody is one of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, and a human or humanized antibody present in bound or soluble form.
64. The binding molecule according to claim 58, wherein said binding molecule is a FISH probe.
65. Use of a compound for the preparation of a medicament for treatment of a human subject suffering from histamine-independent, endothelin-1 (ET-1 )/endothelin A receptor (ETAR) induced itch, wherein said compound is selected from the group consisting of endothelin- converting enzyme-1 (ECE-1 ), a MEK1 inhibitor, and an ETAR inhibitor.
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