WO2010046706A1

WO2010046706A1 - Treatment of inflammatory disorders

Info

Publication number: WO2010046706A1
Application number: PCT/GB2009/051423
Authority: WO
Inventors: Thomas Walther
Original assignee: The University Of Hull
Priority date: 2008-10-23
Filing date: 2009-10-22
Publication date: 2010-04-29
Also published as: GB0819446D0

Abstract

The present disclosure relates to the treatment of inflammatory disorders. In particular, although not exclusively the present disclosure relates to agents which inhibit a Mas related gene (Mrg) receptor for use in the treatment or prevention of a disorder associated with inflammation. Also included in the present disclosure are methods of treatment, assays and other subject matter.

Description

TREATMENT OF INFLAMMATORY DISORDERS

The present invention relates to agents and methods for use in the treatment of inflammation. The agents of the invention are inhibitors of the activation of NF-κB via a Mas related gene family receptor. Also included in the present invention are assays and methods for screening for such agents for use in the treatment of inflammatory diseases, as well as other subject matter.

BACKGROUND Inflammation is the complex biological response to harmful stimuli such as pathogens, damaged cells or irritants. Chemicals such as bradykinin, histamine, serotonin and others are released, attracting tissue macrophages and white blood cells to localise in an area to engulf and destroy foreign substances. During this process, chemical mediators such as TNFα are released, giving rise to inflammation. However, sustained or chronic inflammation can lead to a variety of disorders. Examples of disorders which are associated with inflammation include e.g. asthma, autoimmune diseases, chronic inflammation, inflammatory bowel disease, rheumatoid arthritis and pelvic inflammatory disease. These disorders are characterised by dysregulation of the immune system and inappropriate mobilisation of the body's defences against its own healthy tissue. A number of treatments are available for inflammatory conditions. For example, the treatment of rheumatoid arthritis typically includes the administration of (a) non-steroidal anti-inflammatory drugs (NSAIDS e.g. ibuprofen, ketoprofen, indomethacin, diclofenac, aspirin and fenoprofen) (b) steroid (e.g. cortisone, hydrocortisone, prednisone, triamcinolone and dexamethasone) (c) disease modifying antirheumatic drugs (DMARDS e.g. cyclosporine, azathioprine, methotrexate, leflunomide and sulfasalazine) or (d) recombinant proteins e.g. etanercept, a soluble TNF receptor fusion protein and infliximab, a chimeric monoclonal anti-TNF antibody. Despite the variety of treatments available, there remains a need for new improved medicaments for the treatment of inflammatory disorders.

G protein-coupled receptors (GPCRs) are a large family of receptor proteins which all have a common structural motif of seven alpha helices formed from seven sequences of between 22 to 24 hydrophobic amino acids, each of which spans the cell membrane. The transmembrane helices are joined by strands of amino acids having a larger loop between the fourth and fifth transmembrane helix on the extracellular side of the membrane. Another larger loop, composed primarily of hydrophilic amino acids, joins transmembrane helices five and six on the intracellular side of the membrane. The carboxy terminus of the receptor lies intracellular^ with the amino terminus residing in the extracellular space. It is thought that the loop joining helices five and six, as well as the carboxy terminus, interact with the G protein. Currently, the G proteins that have been identified are Gq, Gs, Gi, and Go.

Under physiological conditions, GPCRs exist in the cell membrane in equilibrium between two different states or conformations: an "inactive" state and an "active" state. A receptor in an inactive state is unable to link to the intracellular transduction pathway to produce a biological response. A receptor in the active state allows linkage to the transduction pathway to produce a biological response. Physiologically, conformational change between the inactive and active state are induced in response to binding of a molecule to the receptor. Several types of biological molecules can bind to specific receptors, such as peptides, hormones or lipids, and can cause a cellular response.

One member of the GPCR superfamily is the Mas receptor which was first detected in vivo by its tumorogenic properties which originate from rearrangement of its 5' flanking region (Young, D. et al., Cell 45:71 1-719 (1996)). Subsequent studies have indicated that the tumorogenic properties of Mas appear to be negligible. Mas is constitutively activated.

A family of GPCRs which share sequence identity to Mas has been identified recently (Dong et al, Cell, 2001 , Sept. 7, 106(5), 619-32). Members of this large family are known as Mas-related genes (Mrgs) or alternatively Mas-related G-protein coupled receptor members. Most are orphan receptors and have short (3-21 amino acid) N termini, with no apparent signal peptide, which are predicted to be located extracellularly. The transmembrane domains and intracellular domains are highly conserved, suggesting that the receptors have a shared function. Under normal physiological conditions, members of the Mrg family are expressed in small-diameter sensory neurons of dorsal root ganglia. However, they may be expressed in other tissues under abnormal and/or pathological conditions.

BRIEF SUMMARY OF THE DISCLOSURE

The present invention describes for the first time the relationship between the Mrg receptors, including the Mas receptor, and the upregulation of pro-inflammatory cytokines via NF-κB, a transcription factor. The present invention has identified agents which act to inhibit e.g. block activation of NF-kappaB (NF-κB) via this Mrg receptor pathway and which may be used in the treatment of inflammation and disorders associated with an inflammatory response. Furthermore, the present invention shows for the first time that several members of the Mrg family are constitutively active. Thus, blockade of these receptors may result in a reduction of intracellular events which are controlled by the constitutive activity of these receptors.

Thus, the present invention is based, at least in part, on the findings that activation of NF-κB can be controlled via control of a Mas-related gene family receptor (Mrg receptor). NF-κB is responsible, amongst other functions, for initiating the up-regulation and expression of pro-inflammatory cytokines. Thus, the present invention includes methods of controlling inflammation by administering agents which control an Mrg receptor activity. In one embodiment, the agent blocks the constitutive activity of an Mrg receptor, thus decreasing NF-κB activity in a cell which expresses the Mrg receptor. The level of NF-κB activity can be determined by calculating the level of NF-κB activity within a cell, determining the level of NF-κB proteins in a cell or by determining the level of NF-KB mRNA in the cell.

Embodiments of the present invention relate to the treatment of disorders caused by or associated with an inappropriate inflammatory response. Agents of the invention may have utility in the treatment of inflammatory disorders by inhibition e.g. blockade of the Mrg receptor-NF-κB pathway.

In one embodiment, the agent is an inhibitor of a Mas receptor. Thus, in one embodiment, the agent is capable of blocking the Mas receptor, thus reducing the constitutive activity of the receptor. The term "Mas" as used herein includes the human sequences found in GeneBank Accession No. CR542261 , naturally occurring allelic variants, mammalian orthologs and recombinant mutants thereof. In one embodiment, the Mas receptor is a human Mas receptor protein.

In one embodiment, the agent is an inhibitor of MrgD (Mas-related gene D). Thus, in one embodiment, the agent is capable of blocking the MrgD receptor, thus reducing the constitutive activity of the receptor. MrgD is expressed across species from rodents to nonhuman primates and humans and under normal physiological conditions its expression is believed to be restricted to dorsal root ganglion (DRG) neurons. The term "MrgD" as used herein includes the sequences found in Accession No. NP944605 and Swiss-Prot Accession No. Q8TDS7-1 , naturally occurring allelic variants, mammalian orthologs and recombinant mutants thereof. In one embodiment, the MrgD receptor is a human MrgD receptor protein.

In one embodiment, the agent is an inhibitor of MrgX4 (Mas-related gene X4). Thus, in one embodiment, the agent is capable of blocking the MrgX4 receptor, thus reducing the constitutive activity of the receptor. The term "MrgX4" as used herein includes the sequences found in Swiss Prot Accession No. Q96LA9, naturally occurring allelic variants, mammalian orthologs and recombinant mutants thereof. In one embodiment, the MrgX4 receptor is a human MrgX4 receptor protein.

In an alternative embodiment, the agent is an inhibitor of an Mrg receptor selected from MRG, MrgE, MrgG, MrgH, MrgX1 , MrgX2 and MrgX3. The Mrg receptor may be for example a human Mrg receptor protein.

In one embodiment, the agent is an inhibitor of Mas receptor induced NF-κB activation. In one embodiment, the agent is an inhibitor of MrgD induced NF-κB activation. In one embodiment, the agent is an inhibitor of MrgX4 receptor induced NF-κB activation.

Thus, the present invention also provides methods and assays for determining whether a compound is suitable for treating an inflammatory disorder by inhibiting the activation of NF-κB via an Mrg receptor. Several of the Mrg receptors are constitutively active and therefore in one embodiment, the agent blocks the constitutive activity of an Mrg receptor e.g. Mas, MrgD or MrgX4.

The present invention indicates that NF-κB is activated via different members of the Mrg receptor family and therefore inhibitors of these members may be used to treat inflammation and disorders associated therewith. Thus, the present invention provides agents which inhibit NF-κB activation via constitutive activation of a Mrg receptor.

In one aspect of the present invention, there is provided an agent which blocks a Mas related gene (Mrg) receptor for the treatment or prevention of a disorder associated with inflammation. In one embodiment, the agent inhibits NF-κB activation. In one embodiment, the agent blocks a receptor selected from Mas, MrgD and MrgX4, wherein optionally the agent blocks constitutive activity of the receptor. In one embodiment, the agent blocks a Mas receptor so as to inhibit NF-κB activation.

In one embodiment, the agent is selected from an antibody, a peptide, a polypeptide, a fusion protein, a compound and a nucleic acid.

In one embodiment, the agent is a compound of Formula (Ia):

(Ia)

or a pharmaceutically acceptable salt, solvate or hydrate thereof; wherein: G is C(=O) or S(=O)₂; R₁ is n-propyl optionally substituted with 1 , 2, 3, 4, 5, 6, or 7 fluorine atoms;

R₂, Rβ, R₄, and R₅ are each selected independently from the group consisting of H, Ci-6 acyl, Ci_-6 acyloxy, C_2-6 alkenyl, Ci_-6 alkoxy, Ci_-6 alkyl, Ci_-6 alkylamino, Ci_-6 alkylcarboxamide, C_2-6 alkynyl, Ci_-6 alkylsulfonamide, Ci_-6 alkylsulfinyl, Ci_-6 alkylsulfonyl, Ci_-6 alkylthio, Ci_-6 alkylthiocarboxamide, Ci_-6 alkylthioureyl, Ci_-6 alkylureyl, amino, di-Ci_-6- alkylamino, Ci_-6 alkoxycarbonyl, carboxamide, carboxy, cyano, C_3-6 cycloalkyl, di-Ci_-6- alkylcarboxamide, di-Ci_-6-alkylsulfonamide, di-Ci_-6- alkylthiocarboxamido, Ci_-6 haloalkoxy, Ci_-6 haloalkyl, halogen, Ci_-6 haloalkylsulfinyl, Ci_-6 haloalkylsulfonyl, Ci_-6 haloalkylthio, heterocyclic, hydroxyl, nitro, sulfonamide, and thiol; and

Ar is aryl or heteroaryl each optionally substituted with 1 , 2, 3, 4, 5, 6, or 7 substituents selected independently from the group consisting of Ci_-6 acyl, Ci_-6 acyloxy, C_2-6 alkenyl, Ci_-6 alkoxy, Ci_-6 alkyl, Ci_-6 alkylamino, Ci_-6 alkylcarboxamide, C_2-6 alkynyl, Ci_-6 alkylsulfonamide, Ci_-6 alkylsulfinyl, Ci_-6 alkylsulfonyl, Ci_-6 alkylthio, Ci_-6 alkylthiocarboxamide, Ci_-6 alkylthioureyl, Ci_-6 alkylureyl, amino, di-Ci_-6-alkylamino, Ci_-6 alkoxycarbonyl, carboxamide, carboxy, cyano, C_3-6 cycloalkyl, di-Ci_-6-alkylcarboxamide, di-Ci_-6-alkylsulfonamide, di-Ci_-6-alkylthiocarboxamido, Ci_-6 haloalkoxy, Ci_-6 haloalkyl, halogen, Ci_-6 haloalkylsulfinyl, Ci_-6 haloalkylsulfonyl, Ci_-6 haloalkylthio, heterocyclic, hydroxyl, nitro, sulfonamide, and thiol; provided that said compound is not of the group consisting of: 1 '-(propyl)-l,2-dihydro-5,7-dimethyl-l-(2-chloro-benzenesulfonyl)-spiro[3H- indole- 3,4'-piperidine];

1 '-(propyl)-l,2-dihydro-5-methyl-l-(2,3-difluoro-benzoyl)-spiro[3H-indole-3,4'- piperidine]; and

1 '-(propyl)-l ,2-dihydro-5-methyl-1 -(2,6-difluoro-benzoyl)-spiro[3H-indole-3,4'- piperidine].

Other small molecules which may have utility in the invention are described in more detail under the heading "Exemplary agents".

In one embodiment, the agent is for use in the treatment of a chronic inflammatory disorder. In one embodiment, the agent is for the treatment of an inflammatory disorder selected from arthritis, scleroderma, systemic lupus erythematosis, vasculitis, multiple sclerosis, autoimmune thyroiditis, dermatitis, autoimmune skin diseases, myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, colitis, ulcerative colitis, diabetes mellitus (type I); inflammatory conditions of, e.g., the skin, nervous system, liver, kidney (e.g., nephritis) and pancreas (e.g., pancreatitis); cholesterol metabolic disorders, oxygen free radical injury; disorders associated with wound healing; respiratory disorders, acute inflammatory conditions, transplant rejection and allergy. In one embodiment, the agent is for use in the treatment of a kidney disorder. In one embodiment, the agent is for use in the treatment of chronic kidney inflammation.

In one embodiment, the agent is for the treatment of inflammation of the sensory neurons and/ or the brain. In one embodiment, the agent is for the treatment of renal inflammation e.g. nephritis. In one embodiment, the agent is for the treatment of arthritis e.g. rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, lupus-associated arthritis and ankylosing spondylitis. In one embodiment, the agent is for the treatment of dermatitis, e.g. atopic dermatitis and eczematous dermatitis. In one embodiment, the agent is for the treatment of an inflammatory disorder of the skin, e.g. psoriasis, acute and chronic urticaria (hives). In one embodiment, the agent is for the treatment of an inflammatory disorder of the nervous system, e.g. Alzheimer's disease and amyotrophic lateral sclerosis. In one embodiment, the agent is for the treatment of an inflammatory disorder of the liver e.g. hepatitis. In one embodiment, the agent for the treatment of an inflammatory respiratory disorder e.g. asthma and COPD.

In one embodiment, the agent is for the treatment of inflammation of the cardiovascular system e.g. myocarditis.

In one embodiment, the agent is for the treatment of an acute inflammatory disorder e.g. endotoxemia, septicemia, septic shock, toxic shock syndrome and infectious disease. In one embodiment, the agent is for the treatment of allergy e.g. anaphylaxis, angioedema, atopy, insect sting allergies and allergic rhinitis. In one embodiment, the agent is for the treatment of pain associated with an inflammatory disorder.

In a further aspect of the invention, there is provided use of an agent which blocks a Mas related gene (Mrg) receptor for the manufacture of a medicament for the treatment or prevention of a disorder associated with inflammation. In one embodiment, the agent inhibits NF-κB activation. In one embodiment, the agent blocks a receptor selected from Mas, MrgD and MrgX4. Other features of the agent are described above and in the following sections.

In one embodiment, the inflammatory disorder is selected from arthritis, scleroderma, systemic lupus erythematosis, vasculitis, multiple sclerosis, autoimmune thyroiditis, dermatitis, autoimmune skin diseases, myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, colitis, ulcerative colitis, diabetes mellitus (type I); inflammatory conditions of, e.g., the skin, nervous system, liver, kidney (e.g., nephritis) and pancreas (e.g., pancreatitis); cholesterol metabolic disorders, oxygen free radical injury; disorders associated with wound healing; respiratory disorders, acute inflammatory conditions, transplant rejection and allergy.

In a further aspect of the present invention, there is provided a method for modulating an inflammatory process in a subject, wherein the method involves administering an effective amount of an agent which blocks a Mas related gene (Mrg) receptor to a subject in need thereof. In one embodiment, the method comprises modulating an amount of pro-inflammatory cytokines induced during the inflammatory process. In one embodiment, the pro-inflammatory cytokines are selected from IL-6, TNF-α, MCP-1 and combinations thereof. In one embodiment, the method is for inhibiting an inflammatory process.

In a further aspect of the present invention, there is provided a method of treating or preventing a disorder associated with inflammation comprising administering a therapeutically effective amount of an agent which inhibits (e.g. blocks) a Mas related gene (Mrg) receptor to a subject. In one embodiment, the agent inhibits NF-κB activation via an Mrg receptor. The subject may be for example a human subject.

In one embodiment, the method is for treating of a disorder selected from the group consisting of arthritis, scleroderma, systemic lupus erythematosis, vasculitis, multiple sclerosis, autoimmune thyroiditis, dermatitis, autoimmune skin diseases, myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, colitis, ulcerative colitis, diabetes mellitus (type I); inflammatory conditions of, e.g., the skin, nervous system, liver, kidney (e.g., nephritis) and pancreas (e.g., pancreatitis); cholesterol metabolic disorders, oxygen free radical injury; disorders associated with wound healing; respiratory disorders, acute inflammatory conditions, transplant rejection and allergy.

In one embodiment, the method includes the use of an agent is selected from a polypeptide, an antibody, a compound, a peptide and a nucleic acid. In one embodiment, the method comprises administering a 1 ,2-dihydro-spiro[3H-indole-3,4'- piperidine compound to the subject.

In a further aspect of the invention, there is provided an assay or method for identifying an agent which prevents or reduces inflammation comprising: a) contacting a cell expressing a Mrg receptor with a test compound and b) determining the level of constitutive activity of the Mrg receptor, wherein the level of constitutive activity will be reduced if the test compound is an agent which prevents or reduced inflammation.

In one embodiment, the assay includes determining the effect of the test compound on NF-κB in the cell. In one embodiment, the assay includes determining the concentration of (1 ) NF-κB protein; (2) mRNA or (3) NF-κB activity in the cell. In one embodiment, the assay includes comparing NF-κB activity with a basal NF-κB in the absence of the test compound, wherein reduction of NF-κB activity, mRNA or protein indicates an agent which prevents or reduces inflammation. In one embodiment, step (b) comprises carrying out an electrophoretic mobility shift assay to determine NF-κB activity.

In one embodiment, the assay comprises detecting expression levels of pro- inflammatory cytokines, wherein optionally the pro-inflammatory cytokines are selected from IL-6 and MCP-1.

In one embodiment, the assay and/or method is automated for high content screening (HCS) or medium through-put screening (MTS).

Other details of the present invention are described in more detail below.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is described in more detail with reference to the following diagrams:

FIGURE 1

Unilateral ureteral obstruction (UUO) was set in Mas-deficient (Mas-/-) mice and their wild-type controls (Mas+/+), and animals were studied after 2, 5 and 7 days; Figure 1A shows Masson staining and Figure 1 B shows Azan blue staining after 2 days, Figure 1C shows Masson staining after 7 days; magnification: 10Ox, 20Ox and 10Ox, respectively.

Figure 1 D demonstrates lower concentrations of blood urea nitrogen in Mas-deficient mice 2 days after UUO.

Figure 1 E shows evaluation of apoptosis-related proteins at 2 days of UUO. Data of renal Bax/Bcl-xl ratio expressed as n-fold of increase vs. contralateral kidneys of each genotype, expressed as mean ± SEM of 6-8 animals per group. Open and black bars represent Mas+/+ or Mas-/- respectively; C: contralateral, O: obstructed kidneys; ^* P<0.05 vs. contralateral kidney of its own genotype; # P<0.05 vs. Mas+/+ obstructed kidney.

Figure 1 F shows the computer analysis of moncytes/macrophages scoring 2, 5 and 7 days of UUO. The presence of inflammatory cell infiltration was determined by immunohistochemistry with anti-F4/80 antibody (specific for monocytes/macrophages; brown staining); magnification: 20Ox.

Results are expressed as F4/80 positive cells/mm² as mean ± SEM of 6-10 animals per group. Open bars show data of Mas +/+ and black bars of Mas -/- kidneys, ^* P<0.05 vs. contralateral kidney of the same genotype; # P<0.05 vs. Mas +/+ obstructed kidneys. (G) illustrates inflammatory cell infiltration in obstructed kidneys (cortex) of Mas+/+ and Mas-/- mice (representative kidneys of 6-10 studied in each genotype, each time point).

FIGURE 2

Figure 2A shows renal NF-κB activation measured in Mas deficient (Mas-/-) mice at 2 and 5 days of UUO using an electrophoretic mobility shift assay (EMSA) experiment. Competition assay with a 100-fold excess of unlabelled NF-KB shows the specificity of the binding (marked by arrows). The position of free oligonucleotides is indicated.

Figure 2B shows data of gene expression of proinflammatory factors (MCP-1 and IL-6) obtained by real-time PCR experiments and expressed as n-fold increase vs. contralateral kidney as mean +/- SEM of 8-10 animals per group; C- contralateral, O: obstructed kidneys. ^*P< 0.05 vs. contralateral kidney of the same genotype; # P<0.05 vs. Mas +/+ obstructed kidney.

FIGURE 3 Ischemia reperfusion (I/R) was performed in mice deficient for Mas (I/R Mas -/-) and their wild-type controls (I/R Mas +/+). Animals were studied 3 days after I/R. Figures 3A and 3B show Ladewig and PAS staining respectively of group-representing renal sections. Magnification: 40Ox. Figure 3C shows renal NF-κB activity after I/R as measured by EMSA

Figure 3D shows renal NF-κB activity after I/R as calculated as renal NF-κB activity expressed as n-fold increase vs. wild-type controls and shown as mean ± SEM of 6-10 animals per group analyzed in duplicate. ¥ P<0.05 vs. saline-infused mice; # P<0.05 vs. Mas+/+ mice. Figure 3E shows mRNA levels of MCP-1 and IL-6 expressed as n-fold increase vs. wild-type controls and expressed as mean ± SEM of 6-8 animals per group analyzed in duplicate. ¥ P<0.05 vs. saline-infused mice; # P<0.05 vs. Mas +/+ mice.

Figure 4 shows the constitutive activity of Mrg receptors in HEK cells as a function of luciferase production. Cells expressing Mas receptors, MrgD and MrgX4 show constitutive activity.

Figure 5 shows NF-κB activity in HEK cells expressing different Mrg receptors as a function of luciferase production. Cells expressing Mas receptors, MrgD and MrgX4 show NF-κB activation.

Figure 6 shows the inhibitory effect on NF-κB activation by a compound which inhibits Mas receptor activity.

DETAILED DESCRIPTION

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19- 854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd. ,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed. ), Molecular Biology and Biotechnology : a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). Definitions and additional information known to one of skill in the art in immunology can be found, for example, in Fundamental Immunology, W. E. Paul, ed., fourth edition, Lippincott-Raven Publishers, 1999. As stated above, the present invention relates to agents that can be used in the reduction, prevention or treatment of inflammation. In one embodiment the agent is for use in alleviating the symptoms associated with inflammation and/or inflammatory disorders including for example pain. Exemplary disorders which may be treated by the agents disclosed herein are described in more detail later.

The agents of the present invention are inhibitors of Mrg receptor activity and typically of NF-κB activation via activation of an Mrg receptor, which activation may be constitutive activation.

NF-κB is a protein complex which acts as a transcription factor. It is found in almost all animal cells types and is involved in cellular responses to stimuli such as stress, cytokines, free radicals, ultraviolet irradiation and bacterial and viral antigens. It plays a key role in regulating the immune response to infection. NF-κB has been implicated in inflammatory responses.

NF-κB is activated via a pathway which is initiated by activation of a membrane receptor. There are five NF-κB proteins in mammals (ReIA, ReIB, c-Rel, NF-κB1 , and NF-κB2), and they form a variety of homodimers and heterodimers, each of which activates its own characteristic set of genes. The inhibitory protein, IKB, binds to the dimers and holds them in an inactive state within large protein complexes in the cytoplasm. Signals activate the dimers by triggering a signaling pathway that leads to the phosphorylation, ubiquitylation, and consequent degradation of IKB. The degradation of IKB exposes a nuclear localization signal on the NF-κB proteins, which moves into the nucleus and stimulate the transcription of specific genes. The phosphorylation of IKB is performed by a serine/threonine kinase, IKB kinase (IKK).

In the present invention, the membrane receptor is a member of the Mas-related gene (Mrg) family. In one embodiment, the Mrg receptor is Mas. In one embodiment, the agent is an inhibitor of a receptor other than Mas, e.g. MrgD and/or MrgX4. In one embodiment, the agent is an agonist of a Mrg receptor ligand. Thus, embodiments of the present invention may have utility in the treatment of inflammatory disorders as a result of inhibiting activation of the NF-κB pathway via a Mas-related gene family receptor.

In some embodiments, the Mrg receptor is constitutively active and therefore the agent may reduce the level of constitutive Mrg receptor activity. In one embodiment, the agent binds directly to the Mrg receptor in an antagonistic manner. Thus, in one embodiment the agent is an Mrg receptor antagonist. The agent may act to reduce the baseline intracellular, e.g. NF-κB, response of the receptor in comparison to the intracellular, e.g. NF-κB, response in the absence of the agent.

As used herein, the terms "'modulatory, modulation," "modulator," "inhibitory,"

"inhibiting," "inhibitors, activating," and "activators," including their various grammatical forms, are used interchangeably to refer to modulating, inhibiting and/or activating a Mrg receptor. "Modulatory effect" refers to up-regulation, induction, stimulation, potentiation, attenuation, and/or relief of inhibition, as well as inhibition and/or down- regulation or suppression. Inhibitors are compounds that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate Mrg receptor genes or proteins, e.g., antagonists. Activators are compounds that, e.g., bind to, stimulate, increase, open, activate, facilitate, enhance activation, sensitize, or up regulate Mrg receptor genes or proteins, e.g., agonists. As used herein, the term "modulates the activity of the target protein" refers to any change in the activity of the target protein, such as a decrease or an increase in the activity.

EXEMPLARY AGENTS

Exemplary agents of the invention include, but are not limited to, proteins, peptides, antibodies, peptibodies, carbohydrates or small organic molecules. Further details of suitable agents are provided below:

In one embodiment, the agent is a small molecule. Exemplary agents include e.g. certain 1 ,2-dihydro-spiro[3H-indole-3,4'-piperidine] compounds and pharmaceutically acceptable salts, free bases, solvates, hydrates, stereoisomer and prodrugs thereof. A class of compounds suitable for use in the present invention is disclosed in WO2007/0021 14, the entire contents of which are incorporated herein by reference. In one embodiment, the agent is a compound of Formula (Ia):

(Ia)

or a pharmaceutically acceptable salt, solvate or hydrate thereof; wherein: G is C(=O) or S(=O)₂;

Ri is n-propyl optionally substituted with 1 , 2, 3, 4, 5, 6, or 7 fluorine atoms; R₂, R3, R₄, and R₅ are each selected independently from the group consisting of H, Ci-6 acyl, Ci_-6 acyloxy, C_2-6 alkenyl, Ci_-6 alkoxy, Ci_-6 alkyl, Ci_-6 alkylamino, Ci_-6 alkylcarboxamide, C_2-6 alkynyl, Ci_-6 alkylsulfonamide, Ci_-6 alkylsulfinyl, Ci_-6 alkylsulfonyl, Ci_-6 alkylthio, Ci_-6 alkylthiocarboxamide, Ci_-6 alkylthioureyl, Ci_-6 alkylureyl, amino, di-Ci_-6- alkylamino, Ci_-6 alkoxycarbonyl, carboxamide, carboxy, cyano, C_3-6 cycloalkyl, di-Ci_-6- alkylcarboxamide, di-Ci_-6-alkylsulfonamide, di-Ci_-6- alkylthiocarboxamido, Ci_-6 haloalkoxy, Ci_-6 haloalkyl, halogen, Ci_-6 haloalkylsulfinyl, Ci_-6 haloalkylsulfonyl, Ci_-6 haloalkylthio, heterocyclic, hydroxyl, nitro, sulfonamide, and thiol; and

Ar is aryl or heteroaryl each optionally substituted with 1 , 2, 3, 4, 5, 6, or 7 substituents selected independently from the group consisting of Ci_-6 acyl, Ci_-6 acyloxy,

C_2-6 alkenyl, Ci_-6 alkoxy, Ci_-6 alkyl, Ci_-6 alkylamino, Ci_-6 alkylcarboxamide, C_2-6 alkynyl, Ci_-6 alkylsulfonamide, Ci_-6 alkylsulfinyl, Ci_-6 alkylsulfonyl, Ci_-6 alkylthio, Ci_-6 alkylthiocarboxamide, Ci_-6 alkylthioureyl, Ci_-6 alkylureyl, amino, di-Ci_-6-alkylamino, Ci_-6 alkoxycarbonyl, carboxamide, carboxy, cyano, C_3-6 cycloalkyl, di-Ci_-6-alkylcarboxamide, di-Ci_-6-alkylsulfonamide, di-Ci_-6-alkylthiocarboxamido, Ci_-6 haloalkoxy, Ci_-6 haloalkyl, halogen, Ci_-6 haloalkylsulfinyl, Ci_-6 haloalkylsulfonyl, Ci_-6 haloalkylthio, heterocyclic, hydroxyl, nitro, sulfonamide, and thiol; provided that said compound is not of the group consisting of: 1 '-(propyl)-l,2-dihydro-5,7-dimethyl-l-(2-chloro-benzenesulfonyl)-spiro[3H- indole- 3,4'-piperidine];

1 '-(propyl)-l,2-dihydro-5-methyl-l-(2,3-difluoro-benzoyl)-spiro[3H-indole-3,4'- piperidine]; and 1 '-(propyl)-l,2-dihydro-5-methyl-1-(2,6-difluoro-benzoyl)-spiro[3H-indole-3,4'- piperidine].

In one embodiment, the agent is a compound having a structure according to Formula (Na):

(Ha) or a pharmaceutically acceptable salt, solvate or hydrate thereof; wherein: G is C(=O) or S(=O)₂;

R₂, R₄, and R₅ are each independently H, -CH₃, or F;

R₃ is selected from the group consisting of H, -OCH₃, -CH₃, -CH₂CH₃, -CH(CH₃)₂, -C(CH₃)₃, -S(=O)₂NH₂, -S(=O)₂NHCH₃, -S(=O)CH₃, -S(=O)₂CH₃, -SCH₃, -C(=O)NH₂, - C≡N, -C(=O)N(CH₃)₂, -OCF₃, -CF₃, -CF₂CF₃, F, Cl, Br, I, -S(=O)CF₃, -S(=O)₂CF₃, -SCF₃, -OH, and -S(=O)₂NH₂; and

Ar is selected from the group consisting of 2-chlorophenyl, 4-methoxyphenyl, 4- nitrophenyl, 3,4-difluorophenyl, 2-fluorophenyl, 4-chlorophenyl, 3-dichloromethylphenyl, 3,5-bis-trifluoromethylphenyl, 2,4-difluorophenyl, 2-methoxyphenyl, 3,5- dimethoxyphenyl, 3,4,5-trimethoxyphenyl, 3-fluorophenyl, 4-fluorophenyl, 2,3- difluorophenyl, 2,5-difluorophenyl, 3-trifluoromethyl-4-fluorophenyl, 3- trifluoromethylphenyl, phenyl, 3-nitrophenyl, 3-nitro-4-methylphenyl, naphthalen-1-yl, naphthalen-2-yl, 4-trifluoromethylphenyl, 3-chlorophenyl, 3,5-dichlorophenyl, 2,4- dichlorophenyl, 2,6-difluorophenyl, 2,6-dichlorophenyl, 2,6-bis-trifluoromethylphenyl, 2- chloro-6-fluoro-phenyl, and 2,3-dichlorophenyl.

In one embodiment, the agent is a compound having a structure according to In one embodiment, the compound has a structure according to Formula (Ilia):

(Ilia)

or a pharmaceutically acceptable salt, solvate or hydrate thereof; wherein: Ri is n-propyl optionally substituted with fluorine;

G is C(=O) or S(=O)₂; R₃ is -CH(CHs)₂ or -C(CH₃)₃; and

In one embodiment, the agent is a compound having a structure according to Formula (IVa):

(IVa)

or a pharmaceutically acceptable salt, solvate or hydrate thereof;

Ri is n-propyl optionally substituted with fluorine; R₃ is -CH(CHs)₂ or -C(CH₃)₃; and Ar is selected from the group consisting of 2-chlorophenyl, 4-methoxyphenyl, 4- nitrophenyl, 3,4-difluorophenyl, 2-fluorophenyl, 4-chlorophenyl, 3-dichloromethylphenyl,

3,5-bis-trifluoromethylphenyl, 2,4-difluorophenyl, 2-methoxyphenyl, 3,5- dimethoxyphenyl, 3,4,5-trimethoxyphenyl, 3-fluorophenyl, 4-fluorophenyl, 2,3- difluorophenyl, 2,5-difluorophenyl, 3-trifluoromethyl-4-fluorophenyl, 3- trifluoromethylphenyl, phenyl, 3-nitrophenyl, 3-nitro-4-methylphenyl, naphthalen-1-yl, naphthalen-2-yl, 4-trifluoromethylphenyl, 3-chlorophenyl, 3,5-dichlorophenyl, 2,4- dichlorophenyl, 2,6-difluorophenyl, 2,6-dichlorophenyl, 2,6-bis-trifluoromethylphenyl, 2- chloro-6-fluoro-phenyl, and 2,3-dichlorophenyl.

In one embodiment, the agent is a compound having a structure according to Formula (Va):

(Va) or a pharmaceutically acceptable salt, solvate or hydrate thereof; wherein:

R₃ is -CH(CHa)₂, or -C(CHa)₃; and

Ar is phenyl or naphthyl each optionally substituted with 1 , 2, 3, 4, 5, 6, or 7 substituents selected independently from the group consisting of F, Cl, Br, -OCH₃, - OCH₂CH₃, -OCH(CH₃)₂, NO₂, -CH₃, -S(=O)₂CH₃, -S(=O)₂NH₂, -C≡N, -CF₃, and -OCF₃.

(Va)

or a pharmaceutically acceptable salt, solvate or hydrate thereof; wherein:

R₃ is -CH(CHs)₂, -C(CHa)₃, CF₃, or Cl; and Ar is selected from the group consisting of 2-chlorophenyl, 2-fluorophenyl, 2,4- difluorophenyl, 2-methoxyphenyl, 2,3-difluorophenyl, 2,5-difluorophenyl, naphthalen-1- yl, 2,4-dichlorophenyl, 2,6-difluorophenyl, 2,6-dichlorophenyl, 2,6-bis- trifluoromethylphenyl, 2-chloro-6-fluoro-phenyl, and 2,3-dichlorophenyl.

In one embodiment, the agent is a compound having a structure according to (Va):

(Va) or a pharmaceutically acceptable salt, solvate or hydrate thereof; wherein: R₃ is -CH(CH₃)₂, -C(CH₃)₃, CF₃, or Cl; and

Ar is thienyl, pyridinyl, furanyl, pyrazolyl, benzofuranyl, benzothiophenyl, or naphthyridinyl each optionally substituted with 1 , 2, 3, 4, or 5 substituents selected independently from the group consisting of F, Cl, Br, -OCH₃, -OCH₂CH₃, -OCH(CH₃)₂, - NO₂, -CH₃, and -CF₃.

In one embodiment, the agent is 1 '-(allyl)-1 ,2-dihydro-5-fluoro-1-(2,3-difluorobenzoyl)- spiro[3H]-indole-3,4'piperidine] or a pharmaceutically acceptable salt or free base thereof. This compound is referred to in WO2007/002114 on page 4, lines 25 to 30 as "Compound S75".

The 1 ,2-dihydro-spiro[3H-indole-3,4'-piperidine] compound as indicated above may be synthesised using the methods disclosed in WO2007/0021 14 (US Publication No. US2008200491 ) on for example page 31 , line 18 to page 41 , line 34 of the PCT application and in Schemes 1 to 13. In one embodiment, the agent is a protein, peptide, antibody, antibody fragment or fusion protein e.g. an isolated protein, peptide, antibody, antibody fragment or fusion protein. An "isolated" or "purified" protein or biologically active fragment thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of the protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.

In all of the embodiments of the invention described herein in which the agent is a polypeptide, the amino acid sequence of the agent may be modified by one or more changes in sequence which do not eliminate the underlying biological function and utility of the agents as described herein. Modifications may include substitution of individual amino acids with other naturally occurring or non-naturally occurring amino acids.

The agents of the invention may be, for example, an antibody or fragment thereof, e.g. a Fab fragment. An antibody and immunologically active portions thereof, for instance, are typically molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen. The antibody may be for example an antibody which binds to a Mrg receptor. In one embodiment, the agent is an antibody which binds to Mas. In one embodiment, the agent is an antibody which binds to MRG and e.g. inhibits the activation of the NF-κB pathway via this receptor.. In one embodiment, the agent is an antibody which binds to MrgD and e.g. inhibits the activation of the NF-κB pathway via this receptor. In one embodiment, the agent is an antibody which binds to MrgX4 and e.g. inhibits the activation of the NF-κB pathway via this receptor.

Preferred antibodies and fragments are Fab fragments or scFv. Naturally within the scope of the agents of the invention are antibodies or fragments which are monoclonal, polyclonal, chimeric, human, or humanized.

A naturally occurring antibody (for example, IgG) includes four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (λ) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Full-length immunoglobulin light chains are generally about 25 Kd or 214 amino acids in length. Full-length immunoglobulin heavy chains are generally about 50 Kd or 446 amino acid in length. Light chains are encoded by a variable region gene at the NH2-terminus (about 1 10 amino acids in length) and a kappa or lambda constant region gene at the COOH- terminus. Heavy chains are similarly encoded by a variable region gene (about 1 16 amino acids in length) and one of the other constant region genes.

The basic structural unit of an antibody is generally a tetramer that consists of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions bind to an antigen, and the constant regions mediate effector functions. Immunoglobulins also exist in a variety of other forms including, for example, Fv, Fab, and (Fab')₂, as well as bifunctional hybrid antibodies and single chains (e.g., Lanzavecchia et al., Eur. J. Immunol. 17:105, 1987; Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85:5879-5883, 1988; Bird et al., Science 242:423-426, 1988; Hood et al., Immunology, Benjamin, N.Y., 2nd ed., 1984; Hunkapiller and Hood, Nature 323:15-16, 1986).

Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1 , CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to Fc receptors. An immunoglobulin light or heavy chain variable region includes a framework region interrupted by three hypervariable regions, also called complementarity determining regions (CDR's) (see, Sequences of Proteins of Immunological Interest, E. Kabat et al., U.S. Department of Health and Human Services, 1983). As noted above, the CDRs are primarily responsible for binding to an epitope of an antigen. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. In one embodiment, the antibody is a monoclonal antibody. A monoclonal antibody is produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. Generally, a monoclonal antibody is produced by a specific hybridoma cell, or a progeny of the hybridoma cell propagated in culture. A hybridoma or other cell producing an antibody may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.

A suitable class of agents may be chimeric antibodies which bind to an Mrg receptor e.g. Mas, MrgD and/or MrgX4. Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant region genes belonging to different species. For example, the variable segments of the genes from a mouse monoclonal antibody can be joined to human constant segments, such as kappa and gamma 1 or gamma 3. In one example, a therapeutic chimeric antibody is thus a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody, although other mammalian species can be used, or the variable region can be produced by molecular techniques. Methods of making chimeric antibodies are well known in the art, e.g., see U.S. Patent No. 5,807,715, which is herein incorporated by reference.

In one embodiment, the agent may be a humanized antibody or fragment thereof. A "humanized" immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a

"donor" and the human immunoglobulin providing the framework is termed an

"acceptor." In one embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Exemplary conservative substitutions are those such as gly, ala; val, ile, leu; asp, glu; asn, gin; ser, thr; lys, arg; and phe, tyr (see U.S. Patent No. 5,585,089, which is incorporated herein by reference). Humanized immunoglobulins can be constructed by means of genetic engineering, e.g., see U.S. Patent No. 5,225,539 and U.S. Patent No. 5,585,089, which are herein incorporated by reference.

In one embodiment, the agent is a human antibody. A human antibody is an antibody wherein the light and heavy chain genes are of human origin. Human antibodies can be generated using methods known in the art. Human antibodies can be produced by immortalizing a human B cell secreting the antibody of interest. Immortalization can be accomplished, for example, by EBV infection or by fusing a human B cell with a myeloma or hybridoma cell to produce a trioma cell. Human antibodies can also be produced by phage display methods (see, e.g., Dower et al., PCT Publication No. WO91/17271 ; McCafferty et al., PCT Publication No. WO92/001047; and Winter, PCT Publication No. WO92/20791 , which are herein incorporated by reference), or selected from a human combinatorial monoclonal antibody library (see the Morphosys website). Human antibodies can also be prepared by using transgenic animals carrying a human immunoglobulin gene (e.g., see Lonberg et al., PCT Publication No. WO93/12227; and Kucherlapati, PCT Publication No. WO91/10741 , which are herein incorporated by reference).

Antibodies may also be obtained using phage display technology. Phage display technology is known in the art for example Marks et al J. MoI. Biol. 222: 581 -597 and Ckackson et al, Nature 352: 624-628, both incorporated herein by reference. Phage display technology can also be used to increase the affinity of an antibody. To increase antibody affinity, the antibody sequence is diversified, a phage antibody library is constructed, and a higher affinity binders are selected on antigen (see for example Marks et al Bio/ Technology 10:779-783, Barbas et al Proc. Natl. Acad. Sci USA 91 :3809-3813 and Schier et al J. MoI. Biol. 263: 551-567, all incorporated herein by reference.) In one embodiment, the agent is an antibody fragment. Various fragments of antibodies have been defined, including Fab, (Fab')₂, Fv, dsFV and single-chain Fv (scFv) which have specific antigen binding. These antibody fragments are defined as follows: (1 ) Fab, the fragment that contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain or equivalent^ by genetic engineering; (2) Fab', the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')₂, the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction or equivalently by genetic engineering; (4) F(Ab')₂, a dimer of two FAb' fragments held together by disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; dsFV, which is the variable region of the light chain and the variable region of the heavy chain linked by disulfide bonds and (6) single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Single chain antibodies may also be referred to as single chain variable fragments (scFv). Methods of making these fragments are routine in the art.

Reference is made to the numbering scheme from Kabat, E. A., et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987) and (1991 ). In these compendiums, Kabat lists many amino acid sequences for antibodies for each subclass, and lists the most commonly occurring amino acid for each residue position in that subclass. Kabat uses a method for assigning a residue number to each amino acid in a listed sequence, and this method for assigning residue numbers has become standard in the field. For purposes of this invention, to assign residue numbers to a candidate antibody amino acid sequence which is not included in the Kabat compendium, one follows the following steps. Generally, the candidate sequence is aligned with any immunoglobulin sequence or any consensus sequence in Kabat. Alignment may be done by hand, or by computer using commonly accepted computer programs; an example of such a program is the Align 2 program discussed in this description. Alignment may be facilitated by using some amino acid residues which are common to most Fab sequences. For example, the light and heavy chains each typically have two cysteines which have the same residue numbers; in VL domain the two cysteines are typically at residue numbers 23 and 88, and in the VH domain the two cysteine residues are typically numbered 22 and 92. Framework residues generally, but not always, have approximately the same number of residues, however the CDRs will vary in size. For example, in the case of a CDR from a candidate sequence which is longer than the CDR in the sequence in Kabat to which it is aligned, typically suffixes are added to the residue number to indicate the insertion of additional residues (see, e.g. residues I OOabcde in fig. 5). For candidate sequences which, for example, align with a Kabat sequence for residues 34 and 36 but have no residue between them to align with residue 35, the number 35 is simply not assigned to a residue.

CDR and FR residues are also determined according to a structural definition (as in Chothia and Lesk, J. MoI. Biol. 196:901-917 (1987). Where these two methods result in slightly different identifications of a CDR, the structural definition is preferred, but the residues identified by the sequence definition method are considered important FR residues for determination of which framework residues to import into a consensus sequence.

Aptamers

A further class of agents which may be useful in the present invention are aptamers. Aptamers have been defined as artificial nucleic acid ligands that can be generated against amino acids, drugs, proteins and other molecules. They are isolated from complex libraries of synthetic nucleic acids by an iterative process of adsorption, recovery and re-amplification.

RNA aptamers are nucleic acid molecules with affinities for specific target molecules. They have been likened to antibodies because of their ligand binding properties. They may be considered as useful agents for a variety of reasons. Specifically, they are soluble in a wide variety of solution conditions and concentrations, and their binding specificities are largely undisturbed by reagents such as detergents and other mild denaturants. Moreover, they are relatively cheap to isolate and produce. They may also readily be modified to generate species with improved properties. Extensive studies show that nucleic acids are largely non-toxic and non-immunogenic and aptamers have already found clinical application. Furthermore, it is known how to modulate the activities of aptamers in biological samples by the production of inactive dsRNA molecules in the presence of complementary RNA single strands (Rusconi et ai, 2002). It is known from the prior art how to isolate aptamers from degenerate sequence pools by repeated cycles of binding, sieving and amplification. Such methods are described in US 5,475,096, US 5,270,163 and EP0533 38 and typically are referred to as SELEX (Systematic Evolution of Ligands by EX-ponential Enrichment). The basic SELEX system has been modified for example by using Photo-SELEX where aptamers contain photo-reactive groups capable of binding and/or photo cross-linking to and/or photo- activating or inactivating a target molecule. Other modifications include Chimeric- SELEX, Blended-SELEX, Counter-SELEX, Solution-SELEX, Chemi-SELEX, Tissue- SELEX and Transcription-free SELEX which describes a method for ligating random fragments of RNA bound to a DNA template to form the oligonucleotide library. However, these methods even though producing enriched ligand-binding nucleic acid molecules, still produce unstable products. In order to overcome the problem of stability it is known to create enantiomeric "spiegelmers" (WO 01/92566). The process involves initially creating a chemical mirror image of the target, then selecting aptamers to this mirror image and finally creating a chemical mirror image of the SELEX selected aptamer. By selecting natural RNAs, based on D-ribose sugar units, against the non- natural enantiomer of the eventual target molecule, for example a peptide made of D- amino acids, a spiegelmer directed against the natural L-amino acid target can be created. Once tight binding aptamers to the non-natural enantiomer target are isolated and sequenced, the Laws of Molecular Symmetry mean that RNAs synthesised chemically based on L-ribose sugars will bind the natural target, that is to say the mirror image of the selection target. This process is conveniently referred to as reflection- selection or mirror selection and the L-ribose species produced are significantly more stable in biological environments because they are less susceptible to normal enzymatic cleavage, i.e. they are nuclease resistant.

Thus, in one embodiment, the agent is an aptamer which binds to an Mrg receptor, e.g.

Mas, MrgD, MrgX4 and/or other Mrg receptors and e.g. has an inhibitory effect on the activation of the NF-κB pathway via the receptor.

RNAi molecules

In one embodiment, the agent is an inhibitory RNA molecule which inhibits expression of a Mrg receptor. In one embodiment, the agent is an interfering RNA molecule which blocks gene expression of an Mrg receptor e.g. an Mrg receptor selected from Mas, MrgX4 and MrgD. RNA interference or "RNAi" is a term initially coined by Fire and co-workers to describe the observation that double-stranded RNA (dsRNA) can block gene expression when it is introduced into worms (Fire etal., Nature 391 : 806-81 1 ,1998). Short dsRNA directs gene specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. RNAi has been suggested as a method of developing a new class of therapeutic agents.

The agent may be for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA) and short hairpin RNA (shRNA) molecules, which collectively fall under the general term of iRNA agents. The iRNA agents can be unmodified or chemically-modified nucleic acid molecules. The iRNA agents can be chemically synthesized or expressed from a vector or enzymatically synthesized. The invention provides various chemically-modified synthetic iRNA agents capable of modulating gene expression or activity in cells and in a mammal by RNAi. The use of a chemically-modified iRNA agent can improve one or more properties of an iRNA agent through increased resistance to degradation, increased specificity to target moieties, improved cellular uptake, and the like.

An "iRNA agent" as used herein, is an RNA agent which can, or which can be cleaved into an RNA agent which can, down regulate the expression of a target gene i.e. a Mrg gene. While not wishing to be bound by theory, an iRNA agent may act by one or more of a number of mechanisms, including post-transcriptional cleavage of a target mRNA sometimes referred to in the art as RNAi, or pre-transcriptional or pre-translational mechanisms. An iRNA agent can include a single strand or can include more than one strands, e. g. it can be a double stranded iRNA agent. If the iRNA agent is a single strand it is particularly preferred that it include a 5'modification which includes one or more phosphate groups or one or more analogs of a phosphate group.

The iRNA agent should include a region of sufficient homology to the target gene e.g. the Mas gene, the MrgD gene or the MrgX4 gene, and be of sufficient length in terms of nucleotides, such that the iRNA agent, or a fragment thereof, can mediate down regulation of the target gene. (For ease of exposition the term nucleotide or ribonucleotide is sometimes used herein in reference to one or more monomeric subunits of an RNA agent. It will be understood herein that the usage of the term "ribonucleotide" or "nucleotide" herein can, in the case of a modified RNA or nucleotide surrogate, also refer to a modified nucleotide, or surrogate replacement moiety at one or more positions. ) Thus, the iRNA agent is or includes a region which is at least partially, and in some embodiments fully, complementary to the target RNA. It is not necessary that there be perfect complementarity between the iRNA agent and the target, but the correspondence must be sufficient to enable the iRNA agent, or a cleavage product thereof, to direct sequence specific silencing, e. g. by inhibitory RNA cleavage of the target RNA, e. g. mRNA.

Complementarity, or degree of homology with the target strand, is most critical in the antisense strand. While perfect complementarity, particularly in the antisense strand, is often desired some embodiments can include, particularly in the antisense strand, one or more but preferably 6, 5, 4,3, 2, or fewer mismatches (with respect to the target RNA).

Proteins and Peptides

In one embodiment, the agent is a peptide or polypeptide. In one embodiment, the agent is a peptibody. The term "peptibody" refers to a molecule comprising an antibody Fc domain attached to at least one peptide. The production of peptibodies is generally described in PCT publication WO 00/24782, published May 4, 2000.

In one embodiment, the agent is a fusion protein i.e. a protein comprising at least two heterologous peptide sequences. The fusion protein may comprise a linker between the at least two peptide sequences. In one embodiment, the fusion protein is an antibody fusion protein. Examples of antibody fusion proteins are detailed in "Antibody Fusion Proteins" (Chamow and Ashenazi, Wiley-Liss 1999). In one embodiment, the agent may be an Fc fusion protein i.e. comprises an Fc portion of an antibody.

The agents of the present invention, if comprising a peptide sequence, for example an antibody, a fusion protein, a peptide or a protein, may be encoded by a nucleic acid sequence. The present invention includes any nucleic acid sequence which encodes an agent as defined herein. The present invention also includes a nucleic acid sequence which encodes the agent of the invention but which differs from the wild-type nucleic acid as a result of the degeneracy of the genetic code.

The present invention also includes nucleic acids that share at least 80% homology with a nucleic acid sequence which encodes an agent of the present invention. In particular, the nucleic acid may have 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to a nucleic acid which encodes an agent of the present invention.

Calculations of sequence homology or identity (the terms are used interchangeably herein) between sequences are performed as follows.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the percent identity between two amino acid sequences is determined using the Needleman et al. (1970) J. MoI. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. In one embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of the invention) are a BLOSUM 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers et al. (1989) CABIOS 4:1 1-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

In one aspect of the invention, there is provided a nucleic acid molecule which hybridises under stringent conditions to a nucleic acid molecule which encodes an agent of the present invention. Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other. The stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001 ); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology — Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993). The T_m is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand. The following have been found as exemplary for hybridization conditions but without limitation:

Very High Stringency (allows sequences that share at least 90% identity to hybridize)

Hybridization: 5x SSC at 65⁰C for 16 hours Wash twice: 2x SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5x SSC at 65⁰C for 20 minutes each

High Stringency (allows sequences that share at least 80% identity to hybridize)

Hybridization: 5x-6x SSC at 65°C-70°C for 16-20 hours Wash twice: 2x SSC at RT for 5-20 minutes each Wash twice: 1x SSC at 55°C-70°C for 30 minutes each

Low Stringency (allows sequences that share at least 50% identity to hybridize)

Hybridization: 6x SSC at RT to 55⁰C for 16-20 hours Wash at least twice: 2x-3x SSC at RT to 55⁰C for 20-30 minutes each.

In one embodiment, the nucleic acids hybridize over substantially their entire length.

USES OF AGENTS

The agents of the present invention are for use in treating inflammatory disorders. The term "treating" can be taken to include preventing, reducing or alleviating the symptoms of a disorder. As used herein, the terms "subject" and "patient" refers to any human or nonhuman mammal.

In another aspect, the invention relates to a method of treating, preventing or alleviating an inflammatory disease, such as, for example, chronic inflammatory disease (e.g., RA) or respiratory disorder/disease (e.g., asthma), in a subject (e.g., a human subject) comprising administering an agent which is an inhibitor of an Mrg receptor to a subject. The agent typically inhibits NF-κB activation by an Mrg receptor.

Inflammatory disorders which may be treated in the present invention include, for example, arthritis. In one embodiment, the treatment of arthritis includes for example rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, lupus- associated arthritis and ankylosing spondylitis.

In one embodiment, the inflammatory disorder is for example selected from scleroderma, systemic lupus erythematosis, vasculitis, multiple sclerosis and autoimmune thyroiditis.

In one embodiment, the inflammatory disorder is dermatitis, including for example atopic dermatitis and eczematous dermatitis.

In one embodiment, the inflammatory disorder is an autoimmune skin disease, myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, colitis, ulcerative colitis or diabetes mellitus (type I). In one embodiment, the inflammatory disorder is an inflammatory condition of, e.g., the skin (e.g., psoriasis, acute and chronic urticaria (hives)).

In one embodiment, the inflammatory disorder is an inflammatory condition of the nervous system (e.g., Alzheimer's disease, amyotrophic lateral sclerosis). In one embodiment, the agent is for the treatment of a disorder associated with inflammation of the brain. In one embodiment, the agent is for the treatment of a disorder caused by neuronal inflammation.

In one embodiment, the inflammatory disorder is an inflammatory condition of the liver (e.g., hepatitis).

In one embodiment, the inflammatory disorder is an inflammatory condition of the kidney (e.g., nephritis) or pancreas (e.g., pancreatitis). In one embodiment, the agent is for the treatment of renal inflammation and/or disorders associated with renal inflammation.

In one embodiment, the inflammatory disorder is an inflammatory condition of the cardiovascular system e.g., myocarditis, cholesterol metabolic disorders and oxygen free radical injury. In one embodiment, the inflammatory disorder is a disorder associated with wound healing or a respiratory disorder, e.g., asthma and COPD (e.g., cystic fibrosis). In one embodiment, the inflammatory disorder is an; acute inflammatory conditions (e.g., endotoxemia, septicemia, septic shock, toxic shock syndrome and infectious disease). In one embodiment, the inflammatory disorder is transplant rejection. In one embodiment, the inflammatory disorder is allergy (e.g., anaphylaxis, angioedema, atopy, insect sting allergies, allergic rhinitis).

In one embodiment, the agent is for the treatment of a symptom associated with an inflammatory disorder. In one embodiment, the agent is for the treatment of pain associated with an inflammatory disorder.

In one embodiment, the agent is comprised in a pharmaceutical composition. Actual dosage levels of the agent in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active agent(s) that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration (referred to herein as a "therapeutically effective amount"). The selected dosage level will depend upon the activity of the particular agent, the severity of the condition being treated and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound at levels lower than required for to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

Also included in the present invention is a pharmaceutical formulation comprising an agent as described herein; in embodiments the formulation is a composition comprising the agent and a pharmaceutically acceptable diluent, carrier or excipient. Such formulations may further routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents.

The formulations may also include antioxidants and/or preservatives. As antioxidants may be mentioned tocopherols, butylated hydroxyanisole, butylated hydroxytoluene, sulfurous acid salts (e.g. sodium sulfate, sodium bisulfite, acetone sodium bisulfite, sodium metabisulfite, sodium sulfite, sodium formaldehyde sulfoxylate, sodium thiosulfate) and nordihydroguaiareticacid. Suitable preservatives may for instance be phenol, chlorobutanol, benzylalcohol, methyl paraben, propyl paraben, benzalkonium chloride and cetylpyridinium chloride.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The pharmaceutically acceptable carriers useful in the methods disclosed herein are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co, Easton,

PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the agents herein disclosed.

Delivery of active agents The agent of the present invention may be delivered to the subject by any suitable means. The skilled reader will appreciate that the administration may take place periodically throughout the term of the treatment, e.g. at periods of twice a day, once a day or longer. Substantially continuous administration by, for example, infusion is not excluded. In one embodiment, the mode of administration of the agent of the invention may be intravenous, inter-arterial or subcutaneous injection or infusion, or by oral administration.

In one embodiment, the agent is for oral administration. According to a further aspect of the disclosure there is provided an oral pharmaceutical formulation including an agent of the disclosure, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.

The oral pharmaceutical formulation may be for repeated administration e.g. one a day, two a day or greater frequency. Solid dosage forms for oral administration include capsules, tablets (also called pills), powders and granules. In such solid dosage forms, the active compound is typically mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or one or more fillers, extenders, humectants, dissolution aids, ionic surface active agents. The active compounds may also be in micro-encapsulated form, if appropriate, with one or more of excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as water or other solvents, solubilizing agents and emulsifiers.

The agent may be for administration via parental route. Parenteral preparations can be administered by one or more routes, such as intravenous, subcutaneous, intradermal and infusion; a particular example is intravenous. A formulation disclosed herein may be administered using a syringe, injector, plunger for solid formulations, pump, or any other device recognized in the art for parenteral administration.

The agent of the present invention may be for administration in combination with a second active ingredient either separately, simultaneously or sequentially. Thus, the agent may be for administration with a second anti-inflammation agent. For example, the agent may be for administration, with (a) a non-steroidal anti-inflammatory drug e.g. ibuprofen, ketoprofen, indomethacin, diclofenac, aspirin or fenoprofen, (b) a steroid (e.g. cortisone, hydrocortisone, prednisone, triamcinolone and dexamethasone); (c) disease modifying antirheumatic drugs (DMARDS e.g. cyclosporine, azathioprine, methotrexate, leflunomide and sulfasalazine) or (d) recombinant proteins e.g. etanercept, a soluble TNF receptor fusion protein and infliximab, a chimeric monoclonal anti-TNF antibody.

Screening Assays and Methods

As described above, the present invention is also concerned with an assay for screening for agents which can be used to treat inflammation and disorders associated with inflammation. The assays involve cell-free and cell-based assays that identify compounds (modulators) which bind to and/or inhibit the activity of an Mrg receptor and also typically cause inhibition of NF-κB activity.

In one embodiment, the assay determines binding of a test compound to an Mrg receptor. Determining the ability of the test compound to bind to an Mrg receptor can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the Mrg receptor-expressing cell can be measured by detecting the labelled compound in a complex. The radioisotope can be detected by direct counting of radioemmission or by scintillation counting. Alternatively, the test compound can be enzymatically labelled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

In another embodiment, the assay is a cell-based assay comprising contacting a cell expressing an Mrg receptor or biologically active fragment thereof, or a fusion protein which includes all or a portion of the Mrg receptor expressed on the cell surface with a test compound and determining the ability of the test compound to inhibit the activity of the Mrg receptor, in particular, on NF-κB activity. The effect of a test compound on NF-

KB activity can be determined e.g. by monitoring NF-κB expression or by determining expression levels of pro-inflammatory cytokines which are regulated by NF-κB e.g. IL-6 and MCP-1. A test compound may be considered an agent which is suitable for treating inflammatory disorders if the level of NF-κB activity or expression of pro-inflammatory cytokines is reduced compared to the level of NF-κB activity or expression of proinflammatory cytokines in absence of the test compound.

Suitable test compounds for use in the screening assays of the invention can be obtained from any suitable source, e.g., conventional compound libraries. The test compounds can also be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91 :1 1422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261 :1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061 ; and Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten (1992)

Bio/Techniques 13:412-421 ), or on beads (Lam (1991 ) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos.

5,571 ,698; 5,403,484; and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad.

Sci. USA 89:1865-1869) or phage (Scott and Smith (1990) Science 249:386-390;

Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA

87:6378-6382; and Felici (1991 ) J. MoI. Biol. 222:301-310).

Thus, in one embodiment the invention comprises a high-throughput screening method to identify compounds which inhibit Mrg receptor activity and e.g. inhibit NF-κB pathway activation via a Mrg receptor pathway.

So-called high throughput screening methods typically involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (e.g., ligand or modulator compounds). Such combinatorial chemical libraries or ligand libraries are then screened in one or more assays to identify those library members (e.g., particular chemical species or subclasses) that display a desired characteristic activity. The compounds so identified can serve as conventional lead compounds, or can themselves be used as potential or actual therapeutics. A combinatorial chemical library is a collection of diverse chemical compounds generated either by chemical synthesis or biological synthesis, by combining a number of chemical building blocks (i.e., reagents such as amino acids). As an example, a linear combinatorial library, e.g., a polypeptide or peptide library, is formed by combining a set of chemical building blocks in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide or peptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.

The preparation and screening of combinatorial chemical libraries is well known to those having skill in the pertinent art. Combinatorial libraries include, without limitation, peptide libraries (e.g. U.S. Pat. No. 5,010,175; Furka, 1991 , Int. J. Pept. Prot. Res., 37:487-493; and Houghton et al., 1991 , Nature, 354:84-88). Other chemistries for generating chemical diversity libraries can also be used. Nonlimiting examples of chemical diversity library chemistries include, peptides (PCT Publication No. WO 91/019735), encoded peptides (PCT Publication No. WO 93/20242), random bio-oligomers (PCT Publication No. WO 92/00091 ), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993, Proc. Natl. Acad. Sci. USA, 90:6909-6913), vinylogous polypeptides (Hagihara et al., 1992, J. Amer. Chem. Soc, 1 14:6568), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., 1992, J. Amer. Chem. Soc, 1 14:9217-9218), analogous organic synthesis of small compound libraries (Chen et al., 1994, J. Amer. Chem. Soc, 116:2661 ), oligocarbamates (Cho et al., 1993, Science, 261 :1303), and/or peptidyl phosphonates (Campbell et al., 1994, J. Org. Chem., 59:658), nucleic acid libraries (see Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (U.S. Pat. No. 5,539,083), antibody libraries (e.g., Vaughn et al., 1996, Nature Biotechnology, 14(3):309-314) and PCT/US96/10287), carbohydrate libraries (e.g., Liang et al., 1996, Science, 274-1520-1522) and U.S. Pat. No. 5,593,853), small organic molecule libraries (e.g., benzodiazepines, Baum C&EN, Jan. 18, 1993, page 33; and U.S. Pat. No. 5,288,514; isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337; and the like).

Devices for the preparation of combinatorial libraries are commercially available (e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky.; Symphony, Rainin, Woburn,

Mass.; 433A Applied Biosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford,

Mass.). In addition, a large number of combinatorial libraries are commercially available

(e.g., ComGenex, Princeton, N. J.; Asinex, Moscow, Russia; Tripos, Inc., St. Louis, Mo.;

ChemStar, Ltd., Moscow, Russia; 3D Pharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md., and the like).

A "library" according to the present invention relates to a (mostly large) collection of (numerous) different chemical entities that are provided in a sorted manner that enables both a fast functional analysis (screening) of the different individual entities, and at the same time provide for a rapid identification of the individual entities that form the library. Examples are collections of tubes or wells or spots on surfaces that contain chemical compounds that can be added into reactions with one or more defined potentially interacting partners in a high-throughput fashion. After the identification of a desired "positive" interaction of both partners, the respective compound can be rapidly identified due to the library construction. Libraries of synthetic and natural origins can either be purchased or designed by the skilled artisan.

Examples of the construction of libraries are provided in, for example, Breinbauer R, Manger M, Scheck M, Waldmann H. Natural product guided compound library development. Curr Med. Chem. 2002 December; 9(23):2129-45, wherein natural products are described that are biologically validated starting points for the design of combinatorial libraries, as they have a proven record of biological relevance. This special role of natural products in medicinal chemistry and chemical biology can be interpreted in the light of new insights about the domain architecture of proteins gained by structural biology and bioinformatics. In order to fulfil the specific requirements of the individual binding pocket within a domain family it may be necessary to optimise the natural product structure by chemical variation. Solid-phase chemistry is said to become an efficient tool for this optimisation process, and recent advances in this field are highlighted in this review article. Other related references include Edwards P J, Morrell A I. Solid-phase compound library synthesis in drug design and development. Curr Opin Drug Discov Devel. 2002 July; 5(4):594-605; Merlot C, Domine D, Church D J. Fragment analysis in small molecule discovery. Curr Opin Drug Discov Devel. 2002 May; 5(3):391-9. Review; Goodnow R A Jr. Current practices in generation of small molecule new leads. J Cell Biochem Suppl. 2001 ; Suppl 37:13-21.

The term "polypeptide library" or "library of protein sequences" is used herein to indicate a variegated ensemble of polypeptide sequences, where the diversity of the library may result from cloning, mutagenesis, or random or semi-random synthesis of nucleic acid sequences. In an embodiment, the polypeptide library is a variegated ensemble of prey proteins. The term "gene library" has a similar meaning, indicating a variegated ensemble of nucleic acid molecules.

Thus in one embodiment the invention comprises a method of screening a combinatorial chemical library to identify a compound which has an inhibitory effect on a Mrg receptor and/or NF-κB activity.

In another aspect, the present invention encompasses screening and small molecule (e.g., drug) detection assays which involve the detection or identification of small molecules that can bind to a Mrg receptor, e.g., Mas, MrgD and/or MrgX4.

The present invention is described in more detail below with reference to the following non-limiting examples.

EXAMPLES

MATERIALS AND METHODS ANIMALS

Three-month old Mas-deficient mice (Walther et al, J. Biol. Chem 283: 11867-1 1873) and their wild-type controls, both on a C57B1/6 background, were used in the experiments. Furthermore, mice deficient in all three Angiotensin (Ang) Il receptors (AT1A, AT1 B, AT2; triple knockouts) and their own wild-type controls were used from the breeding colony of Thomas Walther at the FEM, Berlin, Germany. All animals were maintained under standardized conditions with an artificial 12-h dark-light cycle, with free access to food and water. Animals were killed by cervical dislocation, and kidneys were immediately removed and further processed for histological studies or frozen in liquid nitrogen for evaluation of RNA, protein and transcription factor activity. Control (saline-infused) animals and control animals without surgery (sham) of the same age were used as internal controls in all experimental settings. All animal studies were performed according to national guidelines and approved by the institutional animal care studies in Spain and Germany. This research was in compliance with the Guide for the Care and the Use of Laboratory Animals published by the OPRR (Office for Protection against Research risks) of the US National Institutes of Health, Washington, D.C. (NIH Publication No. 85-23, revised 1985).

UNILATERAL URETERAL OBSTRUCTION (UUO) Mas-deficient mice (Mas-/-) and their age-matched wild-type controls (Mas+/+) underwent UUO. Mice were anesthetized by pentobarbital injection. The left ureter was ligated with silk (4/0) at two locations and cut between ligatures (obstructed kidney), as described before (Esteban et al, 2004 J. Am. Soc. Nephrol 15:1514-1529). Contralateral kidneys served as controls. Animals were studied for 2, 5 and 7 days (n=6-10 animals per genotype, per time point). Renal function was characterized as described before (Taneda et al J. Am. Soc. Nephrol 14: 968-980).

RENAL ISCHEMIA/REPERFUSION (I/R)

Mice were anesthetized by isoflurane inhalation. Anesthesia was maintained using a mixture of N₂O/O₂/isofluane. Normal body temperature was maintained by placing the animals on heating pads until recovery from anesthesia. Following a midline abdominal incision, the left renal pedicle was localized and the renal artery and vein were dissected. An atraumatic micro-vascular clamp was placed, and the left kidney was occluded during 25 minutes. After inspection for signs of ischemia, the wound was covered with PBS soaked cotton and the animal was covered with tin foil insulation sheet. After release of the clamp, restoration of blood-flow was inspected visually and a contra-lateral nephrectomy was performed. The excised right kidney was snap frozen and stored at -80⁰C for further analysis. The abdominal wound was closed in two layers using 5/0 sutures (B. Braun, Melsungen, Germany). The animals were given 0.5 ml PBS subcutaneously and placed under a heating lamp to recover from surgery.

RENAL HISTOLOGY AND INFLAMMATORY CELL INFILTRATION Paraffin sections of mouse tissues were prepared and stained using standard histology procedures, including hematoxylin/eosin (HE), Azan blue, Masson, Ladewig and van Gieson, as described before (Esteban et al (2004) Am Soc Nephrol 15: 1514-1529). The protocol for periodic acid-Schiff (PAS) staining was adapted from Padi & Chopra (Padi & Chopra (2002) Pharmacol. Res. 45:413-420). The slides were deparafinized through zylene, and hydrated through graded ethanol. Finally, they were examined by light microscopy.

Inflammatory cell infiltration was determined by monoclonal antibodies against F4/80 antigen (Serotec, Oxford, UK), present in murine monocytes/macrophages. Briefly, paraffin-embedded sections were rehydrated, their endogenous peroxidase blocked, and incubated for 1 hour at 25°C with 8% bovine serum albumin (BSA)/5% goat serum in phosphate-buffered saline (PBS) to eliminate non-specific protein binding sites. The slides were then exposed (overnight, 4°C) to the monoclonal F4/80 antibody (dilution 1/50). After removing excess antibody, slides were treated with the corresponding anti- IgG biotinylated-conjugated antibody followed by the avidin-biotin-peroxidase complex (Dako, Dako Diagnόsticos S.A, Barcelona, Spain), and 3,3'-diaminobenzidine as chromogen. Some tissue samples were incubated without the primary antibody or unrelated IgG, as negative controls (data not shown).

Infiltrating cells were quantified by image analysis using a KZ 300 imaging system 3.0 (Zeiss, Munchen-Hallbergmoos, Germany). Briefly, the percentage of the stained area was calculated as the ratio of stained area and the total filed area. For each sample, the mean staining area was obtained by analysis of 10 different fields (x200). The staining score is expressed as F4/80-positive cells/mm². The immunohistochemistry experiments were performed in two kidney sections per experimental animal to obtain a mean score for each of them. In all cases, evaluations were performed by two independent observers in a blinded fashion and the mans core value calculated for each mouse.

QUANTITATIVE REAL-TIME PCR

Total RNA was isolated with Trizol (Gibco BRL, Paisley, Scotland, UK) with subsequent chloroform-isopropanol extraction according to the manufacturer's instructions. Two μg of RNA underwent random primed reverse transcription using a modified Maloney murine leukaemia virus tanscriptase (Superscript II; Life Technologies, Faithersburg, MD, USA) for 10 minutes at 25°C and 37°C for 2 hours. Proinflammatory gene expression was analyzed by real-time PCR, performed on an ABI Prism 7500 sequence detection PCR system (Applied Biosystems, Foster City, CA, USA) according to manufacturer's protocol. After an initial hold of 2 minutes at 50⁰C and 10 minutes at 95°C, the samples were cycled 40 times at 95°C for 15 seconds and 60⁰C for 60 seconds. For all quantitative cDNA analysis, the ΔCt technique was applied. Assay IDs used were MCP-1 , Mm00441242_m1 and IL-6, Mm00446190_m1. To normalize data different approaches were done using several housekeeping genes, including CAPDH, Histone-3 and 18s ribosomal RNA expression (assay IDs: Mm99999915_g1 and Hs99999901_s). All primers, probes, and reagents were obtained from Applied Biosystems (Foster City, CA, USA). All measurements were performed in duplicate. Controls consisting of ddH₂O were negative in all runs.

DETERMINATION OF NF-KB ACTIVITY WITH ELECTROPHORETIC MOBILITY SHIFT ASSAY (EMSA)

A. Protein extraction:

For protein extraction from tissues, frozen kidney pieces were pulverized in a metallic chamber and resuspended in a cold extraction buffer [20 mmol/L HEPES-NaOH (pH 7.6), 20% (vol-vol) glycerol, 0.35 mol/L NaCI, 5 mmol/L MgCI₂, 0.1 mmol/L EDTA, 1 mmol/L DTT, 0.5 mmol/L PMSF]. The homogenate was vigorously shaken for 30 minutes, and the insoluble materials precipitated by centrifugation at 40,000 g for 30 minutes at 4°C. For protein extraction from cultured cells, cells were resuspended in extractioin buffer (10 mmol/L HEPES, pH 7.8, 15 mmol/L KCI, 2 mmol/L MgCI2, 0.1 mmol/L EDTA, 1 mmol/L dithiothreitol, 1 mmol/L PMSF) and homogenized. Nuclei and cytosolic fractions were separated by centrifugation at 1 ,000 x g for 10 minutes. The nuclei were resuspended in extraction buffer to a final concentration of 0.39 mol/L KCI and centrifuged at 100,000 x g for 30 minutes. Supernatants dialyzed overnight against a binding buffer containing 20 mmol/L HEPES-NaOH (pH 7.6), 20% (v/v) glycerol, 0.1 mmol/L NaCI, 5 mmol/ MgCI2, 0.1 mmol/L EDTA, 1 mmol/L dithiothreitol, and 0.5 mmol/L PMSF. The dialysates were cleared by centrifugation at 10,000 x g for 15 minutes at 4°C and frozen at -80⁰C. Protein concentration was quantified by the bicinchoninic acid method (Pierce, Rockford, IL, USA).

B. Electrophoretic mobility shift assay: NF-κB activity was evaluated by binding of 60μg of tissue extracts of tissue of 8-10 μg of nuclear extracts from cells, as described (Esteban et al 2002 Am. Soc. Nephrol 15: 1514-1529). NF-KB consensus oligonucleotide (δ'-AGTTGAGGGGACTTTCCCAGGC- 3') was end-labeled with [Y-³²P]-ATP (Amersham, Buckinghamshire, UK) and T4 polynucleotide kinase (Promega, Madison, Wl, USA). Samples were equilibrated for 10 minutes in a binding buffer [4% glycerol, 1 mmol/L MgCI2, 05 mmol/L EDTA, 0.5 mmol/L dithiothreitol, 50 mmol/L NaCI, 10 mmol/L Tris-HCI, pH 7.5, and 50 mg/ml of poly(dl-dC)] (Pharmacia LKB, Uppsala Sweden), then the NF-κB consensus oligonucleotide labelled [Y- P]-ATP (0.35 pmol) was added and incubated for 20 minutes at room temperature.

Negative controls without cellular extracts, and competition assays with a 100-fold excess of unlabeled NF-κB, mutant NF-κB and AP-1 (unrelated) oligonucleotides, were performed to establish the specificity of the reaction (not shown). When competition assays were done, the unlabeled probe was added to this buffer 10 minutes prior to the addition of the labelled probe. The results of the EMSA experiments were analyzed using a Densitometer (GS-800, Biorad, Alcobendas, Madrid, Spain). The specificity of the antibodies was confirmed by Western blot (data not shown). Oligonucleotides were from Pomega Corp. (Madison, Wl, USA). The reaction was stopped by adding gel- loading buffer (250 mmol/L Tris-HCL, 0.2% bromophneol blue, 0.2% xylene cyanol, and 40% glycerol) and protein-DNA complexes were separated on a nondenaturing, 4% acrylamide gel in Tris-borate. The gels were dried and exposed to X-ray film.

WESTERN BLOT ANALYSIS

Protein levels were assessed by Western blotting. Total proteins were resolved on 12% sodium dodecyl sulphate-polyacrylamide gels, electrophoretically transferred to polyvinylidene difluoride membranes, blocked (in buffer containing 0.01 mM Tris, pH 7.5, 0.4 M NaCI, 0.1 % Tween-20, 1% bonie serum albumin, and 5% milk), and incubated for 18 h at 4°C with Bax and Bcl-xL antibodies (1 :1000 and 1 :500, respectively) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Detection was performed with peozidase-conjugated secondary antibody, using an ECL chemiluminescence kit (Amersham, Arlington Heights, IL, USA).

STATISTICAL ANALYSIS

Since all investigated parameters were normally distributed having continuous variables with equal variances, we analyzed with student t test for ANOVA. For UUO, all parameters investigate did not differ between contralateral kidneys and kidneys from control mice (without surgery) in each genotype studied (data not shown). Data are expressed as mean ± SEM. A P<0.05 was considered significant. Tests were done using the SPSS 1 1.5 software package.

Example 1 Evolution of renal lesions in the model of unilateral ureteral obstruction (UUO) in Mas- deficient and wild-type mice.

The unilateral ureteral obstruction (UUO) model is characterized by interstitial inflammatory cell infiltration, NF-κB activation, apoptosis and fibrosis (Klahr, Morrissey (2002) Am J Physiol Renal Physiol 283: F861-875). Studies have demonstrated that Ang II, via AT₁ Or AT₂ receptors, contributes to renal damage following UUO (Esteban et al (2004) Am Soc Nephrol 15: 1514-1529).

The effects of deficiency in the Mas receptor associated to Ang-(1-7) signalling in the UUO model were investigated. Thus, UUO was performed in Mas-knockout mice (Mas- /-) and their wild-type controls (Mas+/+) and kidneys were studied after 2, 5 and 7 days.

Masson staining of kidney sections two days after UUO showed occasional perivascular mononuclear cell infiltrates and tubular damage in wild-type kidneys, while obstructed kidneys lacking Mas showed no inflammatory infiltrates and tubular lesions (Figure 1A, lower panel).

Wild-type UUO kidneys after two days revealed prominent perivascular, interstitial matrix deposition that was less pronounced in Mas -/- mice (Figure 1 B, Azan blue staining after 2 days.)

This difference further increased at the endpoint of 7 days, when wild-type kidneys showed pronounced infiltration of cells in the interstitium and fibrosis while much less stained cells in the knockout kidneys were detectable (Figure 1 C, Masson staining). Notably, the UUO kidneys with Mas receptors showed occasional glomerular collapse (arrowheads) that was not observed in kidneys lacking the receptor (Figure 1 C).

Importantly, the significantly less renal damage after UUO in Mas-deficient mice led also to a preserved renal function compared to wild-type mice with UUO. Blood urea nitrogen (BUN) (Figure 1 D) and urea (data not shown) were significantly less in mice with Mas deficiency two days after UUO. Notably, under sham conditions both strains did not differ in renal function (data not shown).

These results suggest that the absence of the receptor Mas prevents renal disease progression in the UUO model. Example 2

Mas deficiency and apoptosis

The expression of apoptosis-related proteins that promote (Bax) or protect (Bcl-xL) from cell death was examined. Two days after UUO, the Bax/Bcl-xL ratio increased in the obstructed kidneys of both genotypes in comparison to the contra-lateral control kidneys but significantly less in Mas-deficient mice. This less pronounced increase in the Bax/Bcl-xL ratio in UUO-kidneys of Mas-/- mice implied the induction of reduced apoptosis (Figure 1 E).

Example 3

The inflammatory response was explored by examining inflammatory cell infiltration by immunohistochemistry with a specific anti-F4/80 antibody that recognizes murine monocytes/macrophages. In control samples and in non-injured kidneys of both genotypes, only few cells were positive for F4/80 (data not shown). Two and five days after UUO, obstructed kidneys of Mas+/+ mice showed scattered infiltrates of mononuclear cells within interstitial spaces focally distributed mainly at juxtamedular level, compared to contralateral kidneys (Figure 1 F; Figure 1 G, upper panels). Obstructed kidneys of Mas-deficient mice lacked this pronounced increase in infiltrating cells (Figure 1 F; Figure 1 G, lower panels). After 7 days of UUO, the presence of monocytes/macrophages infiltrates in Mas-deficient kidneys was significantly than in obstructed wild-type kidneys (Figure 1 F; Figure 1 G, right panels) showing that renal Mas deficiency retards UUO damage progression by reducing inflammation.

Example 4

NF-KB activation

UUO significantly increased renal NF-κB activity in saline-treated and Mas-wild type mice (obstructed vs. contralateral kidney). Confirming the immunohistological data, this activation was significantly reduced in mice lacking the Mas receptor (Figure 2A)

NF-κB is a pivotal transcription factor in chronic immune and inflammatory disease. In human kidney diseases, elevated renal NF-κB activity correlates with upregulation of proinflammatory parameters. Experimental models have shown that NF-κB blockade attenuates renal inflammation (Ruiz-Ortega et al 2006, Curr Opin Nephrol Hypertens 15: 159-166). It was examined whether NF-κB downstream signalling was influenced by Mas deficiency. Obstructed kidneys of wild type mice showed increased mRNA expression of proinflammatory cytokines (TNF-α, IL-6), and chemokines (MCP-1 ) compared to the contralateral ones (Figure 2E) as previously described, while in Mas-deficient mice, obstructed kidneys presented significant less gene activation compared to Mas-wild type mice, both at 2 and 5 days (Figure 2B)

Example 5

Impact of Mas on renal ischemia/reperfusion injury The effects on NF-κB regulation of the Mas receptor in other models of renal inflammation was determined using a second experimental kidney model that is characterized by an inflammatory component. This model can be applicable to other forms of inflammation in tissues other than the kidney. The renal ischemia/reperfusion (I/R) injury was chosen to evaluate whether the receptor was involved in the primary mechanisms determining ischemia-mediated renal failure, due to the significance of renal ischemia as a cause of serious morbidity and mortality.

I/R was performed in mice deficient for Mas (I/R Mas -/-) and their wild-type controls (I/R Mas +/+). Animals were studied 3 days after I/R. In wild-type animals (I/R Mas +/+), renal ischemia/reperfusion led to inflammatory infiltrates in interstitial spaces (data not shown) and diffuse matrix deposition as well as partial glomerular collapse 3 days after reperfusion (Figure 3A and B, first panels)

Furthermore, reduced glomerular perfusion was detected (Figure 3B, first panel). Mas- deficient kidneys showed no evidence of matrix deposition in the renal parenchyma 3 days post ischemia. Notably, there were also no significant structural changes in the glomeruli (Figure 3A and B, second panels).

As in the UUO model, the strong beneficial impact of the lack of the Mas receptor on l/R-induced pathomorphological changes was congruent with less NF-κB activation and significant lower renal mRNA of cytokines with proinflammatory properties (Figure 3C-E)

These examples identify the Mas receptor to have a significant impact on inflammation. The models used were renal inflammation models and the findings may be generally applicable to inflammation in other tissues. In the context of renal inflammation, the recruitment of immune cells into the damaged kidney is a main feature of many renal diseases. These infiltrating inflammatory cells (monocytes/macrophages, T cells, and neutrophils) mediate the initiation and progression of damage by direct cytotoxicity, the secretion of soluble factors, such as proinflammatory cytokines, metalloproteinases, and growth factors, which modulate the local response and increase inflammation within the damaged kidney. However, regulation renal inflammation is complex, involving activation of transcription factors and induction of chemokines and other proinflammatory mediators.

The present invention indicates that the proinflammatory properties of the Mas receptor in the kidney are primarily stimulated by a local activation of the NF-κB pathway, and the consequent upregulation of proinflammatory genes under its control, like MCP-1 and IL-6, rather than by systemic effects as stimulating bone-marrow based progenitors of inflammatory cells. Thus, the blockade of constitutive Mas activity significantly reduces stimulation of NF-κB and downstream factors.

In addition to the Mas receptor, the present invention is also concerned with the treatment of inflammation by inhibition of NF-κB stimulation by other members of the Mrg family, as indicated by Example 6.

Example 6 Mrg receptors

The Mas receptor is a constitutively active receptor. Experiments were carried out to determine whether Mrg receptors other than Mas are constitutively active. The materials and methods were similar to those described in Gembardt et al, MoI. Cell.

Biochem, 2008 JuI 18., published. In short, luciferase reporter gene assays were performed with the Dual Luciferase Reporter Assay System (Promega GmbH,

Mannheim, Germany) according to manufacturer's protocol. Briefly, HEK293 cells were seeded into 48-well plates and co-transfected with the rising concentrations of Mrg vectors (hMas, hMRG, hMrgD, hMrgE, ratMrgE, hMrg G, rat MrgG, mouseMrgH, hMrgXI , hMrgX2, hMrgX3, hMrgX4 all cloned in pcDNA3.1 ) and pSRE.L encoding firefly luciferase reporter plasmid and pRL-TK encoding renilla luciferase control vector.

To represent unstimulated cells, the HEK cells were transfected with a control vector, pcDNA3.1. The transfected cells were maintained in DMEM with 0.5% FCS for 18 hours and then stimulated as indicated. Afterwards the cells were lysed and luciferase activities were determined with a microplate luminometer (Orion, Berthold Detection Systems GmbH, Pforzheim, Germany) in a white 96-well plate. The given values are firefly/renilla luciferase ratios as a percentage of the luciferase ratios in unstimulated cells.

As shown in Figure 4, at least hMrgD and hMrgX4 were also constitutively active.

Example 7

Stimulation of NFKB via Mrg receptor.

Experiments were carried out to determine whether NFKB is activated by Mrg receptors other than Mas. To do this, a method similar to that described in Gembardt et al, MoI. Cell. Biochem, 2008 JuI 18., epublished was used. However, in place of measuring serum response factor, NFKB was measured. Furthermore, it was determined that Mrg receptors other than Mas are also constitutively active.

In short, luciferase reporter gene assays were performed with the Dual Luciferase Reporter Assay System (Promega GmbH, Mannheim, Germany) according to manufacturer's protocol. Briefly, HEK293 cells were seeded into 48-well plates and co- transfected with a pNFkB-Luc vector or pSRE.L vector encoding firefly luciferase reporter and pRL-TK encoding renilla luciferase control vector. The transfected cells were maintained in DMEM with 0.5% FCS for 18 hours and then stimulated as indicated. Afterwards the cells were lysed and luciferase activities were determined with a microplate luminometer (Orion, Berthold Detection Systems GmbH, Pforzheim, Germany) in a white 96-well plate. The given values are firefly/renilla luciferase ratios as a percentage of the luciferase ratios in unstimulated cells. To represent unstimulated cells, the HEK cells were transfected with a control vector, pcDNA3.1. Other HEK cells were transfected with rising concentrations of an Mrg vector (HumanMas, hMrgD, MrgX4, MrgE and MrgG)

The results shown that there is a significant increase in luciferase production in cells transfected with hMas, hMrgD and hMrgX4. The two negative controls, HSMrgE and HSMrgG, showed no significant increase in luciferase production.

Thus, the results shown in Figure 5 indicate that constitutive activity of at least Mas, MrgD and MrgX₄ stimulates activation of NF-KB. Example 8

Blockade of the constitutive Mas receptor activity results in inhibition of N FKB

To exemplify that agents could be used to inhibit the constitutive activity of the Mas receptor to results in inhibition of NF-KB, a compound with the formula 1 '-(allyl)-1 ,2- dihydro-5-fluoro-1-(2,3-difluorobenzoyl)-spiro[3H]-indole-3,4'piperidine] was applied to transfected HEK cells. Luciferase reporter gene assays were performed with the Dual Luciferase Reporter Assay System (Promega GmbH, Mannheim, Germany) as described above. Figure 6 shows that increasing concentrations of the compound result in increased inhibition of NF-KB activation. Thus, the present invention shows that a blockade of the constitutive activity of an Mrg receptor ,e.g. Mas, can be used to inhibit NF-KB activation and therefore inhibit NF-KB mediated inflammatory responses.

Claims

1. An agent which inhibits a Mas related gene (Mrg) receptor for use in the treatment or prevention of a disorder associated with inflammation.

2. An agent according to claim 1 which inhibits NF-κB activation.

3. An agent according to claim 1 or claim 2, which blocks a receptor selected from Mas, MrgD and MrgX4, wherein optionally the agent blocks constitutive activity of the receptor.

4. An agent according to claim 3, which blocks a Mas receptor so as to inhibit NF- KB activation.

5. An agent according to claim 3, which blocks an MrgD receptor so as to inhibit NF-κB activation.

6. An agent according to claim 3, which blocks an MrgX4 receptor so as to inhibit NF-κB activation.

7. An agent according to any preceding claim, which is selected from an antibody, a peptide, a polypeptide, a fusion protein, a compound and a nucleic acid.

8. An agent according to claim 7, which is a compound of Formula (Ia):

(Ia)

Ri is n-propyl optionally substituted with 1 , 2, 3, 4, 5, 6, or 7 fluorine atoms; R₂, R₃, R₄, and R₅ are each selected independently from the group consisting of H, Ci-6 acyl, Ci_-6 acyloxy, C_2-6 alkenyl, Ci_-6 alkoxy, Ci_-6 alkyl, Ci_-6 alkylamino, Ci_-6 alkylcarboxamide, C_2-6 alkynyl, Ci_-6 alkylsulfonamide, Ci_-6 alkylsulfinyl, Ci_-6 alkylsulfonyl, Ci_-6 alkylthio, Ci_-6 alkylthiocarboxamide, Ci_-6 alkylthioureyl, Ci_-6 alkylureyl, amino, di-Ci_-6- alkylamino, Ci_-6 alkoxycarbonyl, carboxamide, carboxy, cyano, C_3-6 cycloalkyl, di-Ci_-6- alkylcarboxamide, di-Ci_-6-alkylsulfonamide, di-Ci_-6- alkylthiocarboxamido, Ci_-6 haloalkoxy, Ci_-6 haloalkyl, halogen, Ci_-6 haloalkylsulfinyl, Ci_-6 haloalkylsulfonyl, Ci_-6 haloalkylthio, heterocyclic, hydroxyl, nitro, sulfonamide, and thiol; and

C_2-6 alkenyl, Ci_-6 alkoxy, Ci_-6 alkyl, Ci_-6 alkylamino, Ci_-6 alkylcarboxamide, C_2-6 alkynyl,

Ci_-6 alkylsulfonamide, Ci_-6 alkylsulfinyl, Ci_-6 alkylsulfonyl, Ci_-6 alkylthio, Ci_-6 alkylthiocarboxamide, Ci_-6 alkylthioureyl, Ci_-6 alkylureyl, amino, di-Ci_-6-alkylamino, Ci_-6 alkoxycarbonyl, carboxamide, carboxy, cyano, C_3-6 cycloalkyl, di-Ci_-6-alkylcarboxamide, di-Ci_-6-alkylsulfonamide, di-Ci_-6-alkylthiocarboxamido, Ci_-6 haloalkoxy, Ci_-6 haloalkyl, halogen, Ci_-6 haloalkylsulfinyl, Ci_-6 haloalkylsulfonyl, Ci_-6 haloalkylthio, heterocyclic, hydroxyl, nitro, sulfonamide, and thiol; provided that said compound is not of the group consisting of: 1 '-(propyl)-l,2-dihydro-5,7-dimethyl-l-(2-chloro-benzenesulfonyl)-spiro[3H- indole- 3,4'-piperidine];

9. An agent according to claim 7, which is a compound having a structure according to Formula (Na):

(Ha) or a pharmaceutically acceptable salt, solvate or hydrate thereof; wherein:

G is C(=O) or S(=O)₂; R₂, R₄, and R₅ are each independently H, -CH₃, or F;

Ar is selected from the group consisting of 2-chlorophenyl, 4-methoxyphenyl, 4- nitrophenyl, 3,4-difluorophenyl, 2-fluorophenyl, 4-chlorophenyl, 3-dichloromethylphenyl,

10. An agent according to claim 7, which is a compound having a structure according to Formula (Ilia):

(Ilia)

or a pharmaceutically acceptable salt, solvate or hydrate thereof; wherein: Ri is n-propyl optionally substituted with fluorine; G is C(=O) or S(=O)₂; R₃ is -CH(CH₃)₂ or -C(CH₃)₃; and

1 1. An agent according to claim 7, which is a compound having a structure according to Formula (IVa):

(IVa)

or a pharmaceutically acceptable salt, solvate or hydrate thereof; Ri is n-propyl optionally substituted with fluorine; R₃ is -CH(CHa)₂ or -C(CH₃)₃; and

12. An agent according to claim 7, which is a compound having a structure according to Formula (Va):

R₃ is -CH(CHa)₂, or -C(CHa)₃; and

13. An agent according to claim 7, which is a compound having a structure according to Formula (Va):

(Va)

or a pharmaceutically acceptable salt, solvate or hydrate thereof; wherein: R₃ is -CH(CH₃)₂, -C(CHa)₃, CF₃, or Cl; and

Ar is selected from the group consisting of 2-chlorophenyl, 2-fluorophenyl, 2,4- difluorophenyl, 2-methoxyphenyl, 2,3-difluorophenyl, 2,5-difluorophenyl, naphthalen-1- yl, 2,4-dichlorophenyl, 2,6-difluorophenyl, 2,6-dichlorophenyl, 2,6-bis- trifluoromethylphenyl, 2-chloro-6-fluoro-phenyl, and 2,3-dichlorophenyl.

14. An agent according to claim 7, which is a compound having a structure according to Formula (Va):

(Va) or a pharmaceutically acceptable salt, solvate or hydrate thereof; wherein: R₃ is -CH(CHs)₂, -C(CHa)₃, CF₃, or Cl; and

15. An agent according to any preceding claim, which is for use in the treatment of a chronic inflammatory disorder.

16. An agent according to any preceding claim, which is for use in the treatment of an inflammatory disorder selected from arthritis, scleroderma, systemic lupus erythematosis, vasculitis, multiple sclerosis, autoimmune thyroiditis, dermatitis, autoimmune skin diseases, myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, colitis, ulcerative colitis, diabetes mellitus (type I); inflammatory conditions of, e.g., the skin, nervous system, liver, kidney (e.g., nephritis) and pancreas (e.g., pancreatitis); cholesterol metabolic disorders, oxygen free radical injury; disorders associated with wound healing; respiratory disorders, acute inflammatory conditions, transplant rejection and allergy.

17. An agent according to claim 16, which is for the treatment of inflammation of the sensory neurons and/ or the brain.

18. An agent according to claim 16, which is for use in the treatment of renal inflammation and is optionally for the treatment of nephritis.

19. An agent according to claim 16, which is for use in the treatment of arthritis wherein optionally said arthritis is selected from rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, lupus-associated arthritis and ankylosing spondylitis.

20. An agent according to claim 16, which is for use in the treatment of dermatitis, wherein optionally the dermatitis is selected from atopic dermatitis and eczematous dermatitis.

21. An agent according to claim 16, which is for use in the treatment of an inflammatory disorder of the skin, wherein optionally said condition is selected from psoriasis, acute and chronic urticaria (hives).

22. An agent according to claim 16, which is for use in the treatment of an inflammatory disorder of the nervous system, wherein optionally said condition is selected from Alzheimer's disease and amyotrophic lateral sclerosis.

23. An agent according to claim 16, which is for use in the treatment of an inflammatory disorder of the liver wherein optionally said disorder is hepatitis.

24. An agent according to claim 16, which is for use in the treatment of an inflammatory respiratory disorder which is optionally selected from asthma and COPD.

25. An agent according to claim 16, which is for use in the treatment of an acute inflammatory disorder which is optionally selected from endotoxemia, septicemia, septic shock, toxic shock syndrome and infectious disease.

26. An agent according to claim 16, which is for use in the treatment of allergy and is optionally for the treatment of anaphylaxis, angioedema, atopy, insect sting allergies and allergic rhinitis.

27. An agent according to any preceding claim, which is for use in the treatment of pain associated with an inflammation disorder.

28. Use of an agent which blocks a Mas related gene (Mrg) receptor for the manufacture of a medicament for the treatment or prevention of a disorder associated with inflammation.

29. Use according to claim 28, wherein the agent inhibits NF-κB activation.

30. Use according to claim 28 or claim 29, wherein the agent blocks a receptor selected from Mas, MrgD and MrgX4.

31. Use according to any of claims 28 to 30, wherein the inflammatory disorder is selected from arthritis, scleroderma, systemic lupus erythematosis, vasculitis, multiple sclerosis, autoimmune thyroiditis, dermatitis, autoimmune skin diseases, myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, colitis, ulcerative colitis, diabetes mellitus (type I); inflammatory conditions of, e.g., the skin, nervous system, liver, kidney (e.g., nephritis) and pancreas (e.g., pancreatitis); cholesterol metabolic disorders, oxygen free radical injury; disorders associated with wound healing; respiratory disorders, acute inflammatory conditions, transplant rejection and allergy.

32. Use according to any of claims 28 to 31 , wherein the agent is as recited in any of claims 2 to 14.

33. A method for modulating an inflammatory process in a subject, said method comprising administering an effective amount of an agent which blocks a Mas related gene (Mrg) receptor to a subject in need thereof.

34. A method according to claim 33, comprising modulating an amount of proinflammatory cytokines induced during the inflammatory process.

35. A method according to claim 34, wherein said pro-inflammatory cytokines are selected from IL-6, TNF-α, MCP-1 and combinations thereof.

36. A method according to any of claim 33 to 35, which is for inhibiting an inflammatory process.

37. A method of treating or preventing a disorder associated with inflammation comprising administering a therapeutically effective amount of an agent which inhibits a

Mas related gene (Mrg) receptor to a subject in need thereof.

38. A method according to claim 37, wherein the agent inhibits NF-κB activation via an Mrg receptor.

39. A method according to claim 37 or claim 38, which is for the treatment of a disorder selected from the group consisting of arthritis, scleroderma, systemic lupus erythematosis, vasculitis, multiple sclerosis, autoimmune thyroiditis, dermatitis, autoimmune skin diseases, myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, colitis, ulcerative colitis, diabetes mellitus (type I); inflammatory conditions of, e.g., the skin, nervous system, liver, kidney (e.g., nephritis) and pancreas (e.g., pancreatitis); cholesterol metabolic disorders, oxygen free radical injury; disorders associated with wound healing; respiratory disorders, acute inflammatory conditions, transplant rejection and allergy.

40. A method according to any of claim 37 to 39, wherein the agent is selected from a polypeptide, an antibody, a compound, a peptide and a nucleic acid.

41. A method according to claim 40, wherein the agent is a 1 ,2-dihydro-spiro[3H- indole-3,4'-piperidine compound.

42. An assay for identifying an agent which prevents or reduces inflammation comprising: a) contacting a cell expressing a Mrg receptor with a test compound and b) determining the level of constitutive activity of the Mrg receptor, wherein the level of constitutive activity will be reduced if the test compound is an agent which prevents or reduced inflammation.

43. An assay according to claim 42, which further comprises determining the effect of the test compound on NF-κB in the cell.

44. An assay according to claim 43, which further comprises determining the concentration of (1 ) NF-κB protein; (2) mRNA or (3) NF-κB activity in the cell.

45. The assay of claim 43 or claim 44, which comprises comparing NF-κB activity with a basal NF-κB in the absence of the test compound, wherein reduction of NF-κB activity, mRNA or protein indicates an agent which prevents or reduces inflammation.

46. The assay of any of claims 42 to 45, wherein step (b) comprises carrying out an electrophoretic mobility shift assay to determine NF-κB activity.

47. The assay of any of claims 42 to 46, which comprises detecting expression levels of pro-inflammatory cytokines, wherein optionally the pro-inflammatory cytokines are selected from IL-6 and MCP-1.