WO2008037266A1

WO2008037266A1 - Inhibitors of conventional protein kinase c isozymes and use thereof for treating inflammatory diseases

Info

Publication number: WO2008037266A1
Application number: PCT/EP2006/009085
Authority: WO
Inventors: Cécile SPRINGAEL; Séverine THOMAS; Michel Goldman; Esra Aksoy; Fabienne Alice Raisa Willems
Original assignee: Universite Libre De Bruxelles
Priority date: 2006-09-25
Filing date: 2006-09-25
Publication date: 2008-04-03

Abstract

The present invention relates to the use of an inhibitor that inhibits a conventional protein kinase C alpha isozyme and/or of an inhibitor that inhibits a conventional protein kinase C alpha isozyme and that inhibits a conventional protein kinase C gamma isozyme as a medicament or for the preparation of a medicament for treating inflammatory diseases, in particular inflammatory diseases that are mediated by a Toll-like receptor 2 (TLR-2) and/or an Interleukin-1 receptor (IL-1R). The present invention further provides suitable inhibitors for use in the present invention; pharmaceutical compositions and kits for treating such TLR-2 and/or IL-1R mediated inflammatory diseases.

Description

Inhibitors of conventional protein kinase C isozymes and use thereof for treating inflammatory diseases

Field of the invention The present invention relates to the pharmaceutical and medical field. The invention provides specific inhibitors of conventional protein kinase C isozymes, in particular isozymes having alpha or gamma activity, which are useful in the treatment of inflammatory diseases. The present invention further provides pharmaceutical compositions, kits and methods for treating inflammatory diseases, preferably inflammatory diseases that are mediated by receptors of the TLR and/or IL-1 R family. The present invention further relates to methods for modulating the signal transduction pathway mediated by receptors of the TLR and/or IL-1 R family, and methods for screening conventional protein kinase C isozyme-specific inhibitors that are useful for treating inflammatory diseases, as defined herein.

Background

Protein kinase C (PKC) consists of a family of closely related enzymes that function as serine/threonine kinases. Protein kinase C plays an important role in cell-cell signaling, gene expression, and in the control of cell differentiation and growth. At present, there are currently at least ten known isozymes of PKC that differ in their tissue distribution, enzymatic specificity, and regulation. They are divided into three subfamilies: conventional (or classical), novel, and atypical based on their second messenger requirements. Conventional (c)PKCs contain the isoforms a, β_u β_n, and y. Novel (n)PKCs include the δ, e, η, and θ isoforms. On the other hand, Atypical (a)PKCs (including ζ and 11 λ isoforms).

Protein kinase C isozymes are single polypeptide chains ranging from 592 to 737 amino acids in length. The isozymes contain a regulatory domain and a catalytic domain connected by a linker peptide. The regulatory and catalytic domains can be further subdivided into constant and variable regions. The catalytic domain of protein kinase C is very similar to that seen in other protein kinases while the regulatory domain is unique to the PKC isozymes.

Protein kinase C is a membrane-associated enzyme that is allosterically regulated by a number of factors, including membrane phospholipids, calcium, and certain membrane lipids such as diacylglycerols that are liberated in response to the activities of phospholipases. The protein kinase C isozymes, alpha, beta-1 , beta-2 and gamma, require membrane phospholipid, calcium and diacylglycerol/phorbol esters for full activation. The delta, epsilon, eta, and theta forms of PKC require DAG but are calcium-independent in their mode of activation. The zeta and lambda forms of PKC are independent of both calcium and diacylglycerol and are believed to require only membrane phospholipid for their activation.

Summary The present invention is directed to the use of inhibitors that inhibit the activity of conventional protein kinase C (cPKC) isozymes, in particular inhibitors that are capable of inhibiting the activity of conventional protein kinase C alpha isozymes (cPKCα-inhibitors) and inhibitors that are capable of inhibiting the activity of conventional protein kinase C alpha and gamma isozymes (cPKCα/^inhibitors), for the treatment of inflammatory diseases. More in particular, the present invention is at least in part based on the Applicants' findings that inhibition of conventional PKC isozymes alpha and combined inhibition of conventional PKC isozymes alpha and gamma by means of pharmacological inhibitors significantly reduces inflammatory responses that are mediated by receptors of the TLR and/or IL-1 R family, and preferably by Toll-like receptor 2 (TLR-2) and interleukin-1 receptor (IL-1 R). The Applicant demonstrated that such specific inhibitors work by selectively targeting p38 MAPKs and c-jun N-terminal kinase (JNK)1/2 mitogen-activated protein kinases (MAPKs). In addition, the Applicant has shown that inhibitors that inhibit the activity of conventional protein kinase C alpha and that inhibit activity of conventional protein kinase C isozymes alpha and gamma can selectively inhibit TLR-2 and IL- 1 R signaling without effecting the tumor necrosis factor (TNF)-α signaling pathway. The Applicant has compared the role of cPKCα inhibitors or cPKCα/γ inhibitors within TLR2, IL-1 R and TNF-α signaling pathways in terms of inflammatory responses and showed that TNFα is not effected. As far as TLR2 signaling is concerned, MAPK pathway is inhibited while NF-κB pathway is intact within TLR2 signaling. Hence, the Applicant showed that the present inhibitors specifically target MAPK pathway (but not NF-κB) within TLR2 and/or IL1R signaling pathways but not the (TNF)-α signaling.

Increasing prevalence of autoimmune and inflammatory disorders as well as cancer in the industrialized countries generates current requirements for new drug development and therapies. In accordance with the present invention small molecule inhibitors are therefore provided and employed in therapies, which are aimed to inhibit specific conventional PKC isoforms involved in inflammatory processes. The present invention thus relates to methods, compositions and kits for treating inflammatory diseases, which are effective, specific and which have limited side-effects.

To this end, the present invention relates in a first aspect, to the use as a medicament of an inhibitor that inhibits a conventional protein kinase C alpha isozyme and/or of an inhibitor that inhibits a conventional protein kinase C alpha isozyme and that inhibits a conventional protein kinase C gamma isozyme.

In a second aspect, the present invention relates to the use of an inhibitor that inhibits a conventional protein kinase C alpha isozyme and/or of an inhibitor that inhibits a conventional protein kinase C alpha isozyme and that inhibits a conventional protein kinase C gamma isozyme for the preparation of a medicament for treating inflammatory diseases, preferably inflammatory diseases that are mediated by receptors of the TLR and/or IL-1 R family, and preferably by a Toll-like receptor 2 (TLR-2) and/or an interleukin-1 receptor (IL-1 R).

In a third aspect the invention relates to a pharmaceutical composition for treating inflammatory diseases, preferably inflammatory diseases that are mediated by receptors of the TLR and/or IL-

1 R family, and preferably by a Toll-like receptor 2 (TLR-2) and/or an interleukin-1 receptor (IL-

1 R), comprising a therapeutically effective amount of an inhibitor that inhibits a conventional protein kinase C alpha isozyme and/or an inhibitor that inhibits a conventional protein kinase C alpha isozyme and that inhibits a conventional protein kinase C gamma isozyme and further comprising a pharmaceutically acceptable excipient.

The present invention further relates to kits and methods for treating inflammatory diseases, preferably inflammatory diseases that are mediated by receptors of the TLR and/or IL-1 R family, and preferably by a Toll-like receptor 2 (TLR-2) and/or an interleukin-1 receptor (IL-1R). Also provided are methods for modulating the signal transduction pathway mediated by receptors of the TLR and/or IL-1 R family, and methods for indentifying and/or screening cPKCα inhibitor and/or cPKCα/γ inhibitors that are useful for treating inflammatory diseases, as defined herein.

Those skilled in the art will immediate recognize the many other effects and advantages of the present methods and compositions or kits and the numerous possibilities for end uses of the present invention from the detailed description and examples provided below.

Description of the figures

Fig. 1a illustrates effects of conventional PKC inhibitor Go 6976 on TLR2/TLR6-induced IL- 12p40 or TNF-α expression by human monnocyte-derived dendritic cells (DCs Fig. 1b illustrates effects of conventional PKC inhibitor Go 6976 on TLR2/TLR1 -induced IL- 12p40 or TNF-α expression by human monnocyte-derived dendritic cells (DCs). Fig. 1c illustrates that a PKC-α specific inhibitor, HBDDE abolishes TLR2-mediated inflammatory responses similar to conventional PKC inhibitor, Gό6976 in human monnocyte- derived dendritic cells (DCs). Fig. 1d shows a kinetic study of the effects of conventional PKC inhibitor Go 6976 on IL-12p40 and IL-12p19 mRNA induction mediated by FSL-1.

Fig. 1e shows that inhibition of conventional PKC activity by Go 6976 diminishes TLR2- mediated induction of IL-12p40 mRNA levels. Fig. 1f illustrates that inhibition of PKC-α diminishes IL-1-but not TNF-α-mediated inflammatory cytokine expression in human mo-DCs.

Fig. 2 illustrates that PKCα deficient BMDCs or wild type (WT) BMDCs treatment with conventional PKC inhibitor Go 6976 display defective responses following TLR2 activation.

Fig. 3a shows that conventional PKC inhibition abolishes TLR2-mediated JNK-1/-2 and p38 MAPK phosphorylation.

Fig. 3b shows the inhibition of basal and TLR2-mediated c-jun DNA-binding activity.

Fig. 3c shows that Inhibition of PKCα kinase activity results in the loss of TLR2-mediated MKK4 activation.

Fig. 4a-b illustrate that pharmacological inhibitors of conventional PKC isoforms do not target NF-/fB pathway activation in response to TLR2 stimulation.

Fig. 5 shows that dominant negative (DN) PKCα overexpression inhibits AP1 reporter activity in

HEK 293T cells stably expressing TLR2.

Fig. 6 is a scheme of primary structures the protein kinase C family members showing domain composition. The regulatory domain is located in the N-terminal region containing different domains, depending on the isoenzyme. Pseudosubstrate; the C1 domain; the C2 domain.

According to the orientation of the eight ^-strands that form the domain, the cPKCs exhibit a type I topology C2 domain with the arrow pointing to the left, and nPKCs exhibit a type Il topology C2 domain with the arrow pointing to the right. The PB1 domain contains the OPCA motif represented by a blue box. All kinases have a conserved kinase core that contains the ATP binding, the substrate binding and the phosphotransfer sites. This family of kinases can be divided into three subfamilies depending on their primary structure and biochemical properties.

The cPKCs are regulated by Ca²⁺, phosphatidylserine and diacylglycerol. The nPKCs are regulated by diacylglycerol and acidic phospholipids independently of Ca²⁺. Atypical PKCs are regulated by acidic phospholipids, ceramides and protein-protein interactions.

Detailed description of the invention

This invention is at least in part based on the demonstration of an interplay between two key groups of molecules involved in innate immunity and inflammation. The first group includes the protein kinase C (PKC), which are serine/threonine kinases. The second group is the Toll-like receptor (TLR) family of proteins, which are amongst the most ancient and evolutionarily conserved pathogen recognition receptor families and serve as the earliest surveillance system for infections (Akira et al.; Janeway et al.).

The Applicant has demonstrated that inhibition of cPKC having alpha activity and/or that inhibition of cPKC having alpha activity and cPKC having gamma activity, by means of isozyme- selective pharmacological inhibitors that inhibit cPKC having α activity and/or isozyme-selective pharmacological inhibitors that inhibit cPKC having α activity and that inhibit conventional PKC having γ activity, substantially reduces inflammatory cytokine production mediated by MyD88- dependent signalling receptors including TLR and interleukin-1 receptor (IL-1R) family in innate immune cells such as dendritic cells (DCs) and in epithelial cells in mammals. The current invention is designed at inhibiting conventional PKCα/γ kinase activities as therapy to control inflammation-induced pathologies particularly mediated by TLR and/or IL-1 R family pathway.

Definitions

Conventional PKC (cPKC) belong to the group of the "protein kinase C", family of enzymes that are capable of modifying other proteins by chemically adding phosphate groups to them (phosphorylation), (see futher below).

Toll-like receptors (TLRs) are a family of receptors involved in the reconitaion of a wide range of microbial molecules, e.g. lipopolysaccharides (LPS) from G negative (-) bacteria and lipoproteins from G (positive) (+) bacteria. The receptors are designated TLR-2, TLR-4, TLR-5 etc. and each receptor recognizes a small range of conserved molecules from a group of pathogens. Binding of TLR leads to the production of inflammatory cytokines.

Interleukin-1 (IL-1) is a cytokine that is secreted by macrophages, monocytes and dendritic cells. It is an important part of the inflammatory response of the body against infection. There are a few molecules of the IL-1 family. The two most studied molecular forms of interleukin-1 , are: IL-1σ IL-1£. For the most part, these two forms of IL-1 bind to the same cellular receptor, the interleukin-1 receptor (IL-1 R).

JNK (= Jun kinase; c-jun N-terminal kinase; stress-activated protein kinase; SAPK) refers to a family of kinases involved in intracellular signaling cascades. p38 is a mitogen-activated protein kinase (MAPK) that is regulated by stress and cytokines. NF-KB (Nuclear Factor-KappaB) is a heterodimeric protein composed of different combinations of members of the ReI family of transcription factors. The ReI/ NF-κB family of transcription factors are involved mainly in immune and inflammatory responses. Introduction

Structure and function of protein kinase C isoforms

PKCs are categorized into three major subtypes according to the absence or presence of motifs dictating their cofactor requirements for their optimal catalytic activity (Fig. 6). Conventional PKCs (cPKC: α, βl-II (splice variants) and γ) and the subtype novel PKCs (nPKC: δl-III, ε, η and ΘI— II) bind to diacylglycerol (DAG), which stimulate their catalytic activities, whereas atypical PKCs (aPKC: ζ and PKMζ (catalytic fragment of PKCζ) and i/λ) do not interact with DAG. Furthermore, cPKCs, but not other PKC subtypes, require Ca²⁺ for their functions. In resting cells or in absence of lipid hydrolysis, PKCs are localized primarily in the cytosol. Cellular activation by various stimuli leads to the hydrolysis of phosphoinositol 4,5-bisphosphate (PIP₂). generating DAG and inositol 1 ,4,5-triphosphate (IP₃). DAG binds to C1 domain of conventional and novel PKCs and increases their affinity for membrane phospholipids. Consequently, not only the PKC levels on the membrane are increased but also DAG-binding to PKCs confers conformational changes on PKC molecule to phosphorylate its substrates. Overall, the diverse and distinct roles of individual PKC isoforms can be attributed to the differences in their structural features and the mechanisms that modulate their activation.

The role of PKC in the immune system

Originally identified in 1977, the protein kinase C (PKC) molecules comprise a subfamily of the protein kinase A, protein kinase G and protein kinase C (AGC) serine/threonine protein kinases.

PKCs display broad tissue distribution and cellular function. PKC isoforms are known to regulate a wide variety of cellular processes that influence cell growth, differentiation, cytoskeletal remodeling, and gene expression in response to diverse stimuli. PKC enzymes regulate both positive and negative signal transduction pathways essential for initiation and homeostasis of immune responses. PKCs mediate an evolutionarily conserved function in host defense from primitive organisms up to mammals in resistance against fungal and bacterial infections.

The role of protein kinase C in adaptive immunity

PKC is central to the signal transduction pathways involved in adaptive immunity. In lymphocytes, biochemical and genetic studies have positioned PKC as an important component of T and B cell antigen receptor pathways.

The novel PKC member, PKCΘ plays an important role in TCR signaling, since its expression is restricted to T cells (and the skeletal muscle) and co-localizes with TCR in the immunological synapses. Furthermore, overexpression or inhibition of PKCΘ, but not other isoforms, have shown that PKCΘ mediates the activation of NF-κB and AP-1 by TCR/CD28 co-stimulation in T cells. The essential role of PKCΘ in TCR is demonstrated using T cells from two independently generated PKCΘ^"'^" mice strains. In either mice strain, their peripheral T cells exhibit reduced proliferation and IL-2 production due to impaired TCR/CD28-mediated AP-1 and NF-κB activation. Although, each study supports the role of PKCΘ in TCR signaling, there is a notable difference in TCR/CD28-induced activation of NFAT (nuclear factor of activated T cells) as it is abrogated in only one strain of mice. Albeit this discrepancy needs to be clarified, in vitro studies implicate in the role of PKCΘ in the synergistic activation NFAT and calcineurin. The normal thymocyte differentiation in PKCΘ^"'^" mice suggests that PKCΘ function is developmental stage specific and may be compensated by other PKCs such as PKCα. The cPKC member PKCα is also expressed at high levels in thymocytes and PKCα transgenic mice exhibit massive thymocyte proliferation and IL-2 production in response to TCR stimulation. More recently, it was demonstrated that PKCα but not PKCβ is required for NF-κB activation following TCR/CD28-induced T cell activation. Furthermore, transfection of foetal thymic T cells using either a constitutively active or a dominant negative form of PKCα impairs allelic exclusion and differentiation during thymocyte development. In the B cell compartment, distinct PKC isoforms are essentially involved in the regulation of humoral responses and NF-κB-mediated transcription. Mice deficient for PKCβl gene display B cell dysfunction and impaired humoral responses to T cell-independent antigens. PKCβ^"'^" B cells show defective activation of NF-κB and induction of NF-κB-responsive survival genes (i.e. Bcl-2 and BCI-X_L) Indeed, PKCβ is an important component of Bruton's tyrosine kinase (Btk) signaling to NF-κB in B cells. Other studies have shown that PKCζ^"'^" B cells also fail to induce NF-κB- dependent transcription, since PKCζ is required for the direct phosphorylation of p65/RelA subunit, thereby increasing NF-κB transactivation potential. In addition, PKCδ controls B cell tolerance that was established using mice deficient in PKCδ gene. PKCδ^"'^" mice have severely deregulated immune system characterized by lymphoadenopathy and autoimmune disease potentially due to breakdown of B cell tolerance leading to premature death.

The role of protein kinase C in innate immunity

PKCs play a critical role in integrating several pathways mediated by LPS and influence innate immune responses. Gram negative LPS activate several PKC isoforms including conventional, novel and atypical PKCs in various cell types. MΦs from C3H/HeJ strain (carrying a point mutation in the intracytoplasmic domain of TLR4) or monocytes activated in presence of blocking antibodies directed against CD14 exhibit defective translocation of novel and conventional PKCs upon LPS activation. LPS inducible IRAKI activation downstream of TLR4 can be inhibited by protein kinase C inhibitor, calphostin C. Furthermore, PKC activation is implicated in PMA-mediated differentiation of CD34⁺ hematopoietic progenitors into DCs and can be inhibited by PKC inhibitors. Previous studies have shown the important role of novel PKC member PKCε as a mediator of MΦ function in vitro and in vivo. LPS triggering of mouse MΦs was observed to activate PKCε. Cellular inhibition studies using conventional or novel PKC specific antisense oligonucleotides have demonstrated that PKCα, βl and δ are important regulators of iNOS expression and participate in NF-κB transcriptional activity in RAW 264.7 MΦs. Overexpression of PKCε is sufficient to induce NO production in RAW 264.7 MΦ cell line. An important study performed using mice with a deletion in the gene encoding PKCε established that PKCε is required for resistance and survival against Gram (-) and Gram (+) bacterial infections. In addition, PKCε^"'^" mice do not display defects in the differentiation of monocytes and MΦs from bone marrow precursors. Most importantly, PKCε^''^" MΦs exhibit defective generation of inflammatory mediators including TNF-α, IL-1β and nitric oxide synthase 2 (NOS2) in responses to LPS and/or IFN-γ stimulation due to a defect in activating NF-κB. However, loss of PKCε expression in MΦs only partially inhibits LPS-mediated activation of MAPKs. PKCε^"'^" MEFs are also shown to display reduced responsiveness to IFN-γ, originating from an impairment of IFN-γ-induced Tyr phosphorylation of STAT1. PKCδ is an important component of type I IFNR independently of TLR activation. Notably, pan-PKC inhibitors, H7 and retinal are shown to inhibit IFN-β- but not IFN-γ-mediated cytotoxic functions of MΦs. Other studies have demonstrated that atypical PKCζ is implicated in TNF-α and LT-βR responses of MEFs. PKCβ^"7" mice display defective IgE- antigen-induced mast cell degranulation and IL-6 production. In contrast, PKCδ is implicated in the negative regulation of antigen-induced mast cell degranulation, since PKCδ^"'^" mast cells exhibit more sustained Ca⁺² mobilization and antigen-induced degranulation. Atypical PKCζ is important for chemoattractant-induced integrin-dependent neutrophil adhesion and chemotaxis.

The role of protein kinase C in TLR signaling

PKC isoforms are essential signaling components that contribute to innate immune defense against microbial and viral infections and regulate adaptive immune responses. The applicant has demonstrated specific roles of conventional PKC isoforms in TLR signalling, controlling c- jun-N-terminal kinase (JNK) and p38 MAPK pathways and the downstream inflammatory gene regulation. In addition, the applicant showed which conventional PKC isoforms are involved in the regulation of innate immunity in mammals. Up to now such effects and mode of action of specific PKC isoform were unknown.

1^s' and 2^nd medical use

The present invention relates in a first aspect to the use as a medicament of: an inhibitor that inhibits a conventional protein kinase C alpha isozyme and/or of - an inhibitor that inhibits a conventional protein kinase C alpha isozyme and that inhibits a conventional protein kinase C gamma isozyme.

The term "inhibitor" as used herein refers to a compound that is capable of binding a conventional PKC and decreases or inhibits its biological activity. The terms "isoform", "isozyme", "isotype", and "isoenzyme" are used herein as synonyms and refer to enzymes that differ in amino acid sequence but that can catalyze the same chemical reaction. These enzymes usually display different kinetic parameters (i.e. different K_M values), or different regulatory properties.

Inhibitors used in accordance with the present invention are isozyme selective inhibitors. The term "isozyme selective" means the preferential inhibition of one protein kinase C isozyme (or subgroup) over the other isozymes. For example a compound may selectively inhibit the alpha or gamma isozymes over protein kinase C isozymes beta-1 or beta-2, delta, epsilon, zeta, and eta. In general, isozyme selective compounds demonstrate a minimum of a eightfold differential

(preferably a ten fold differential) in the dosage required to inhibit one subtype compared to the dosage required for equal inhibition of a different subtype (for example, inhibition of PKC alpha or gamma isozyme as compared to the beta-1 or beta-2 protein kinase C isozyme) as measured in a PKC assay. The compounds demonstrate this differential across the range of inhibition and are exemplified at the IC50, i.e., a 50% inhibition. Thus, for example, an alpha and/or alpha+gamma isozyme-selective compound inhibits the alpha and/or alpha+ gamma isozymes of protein kinase C at much lower concentrations with lower toxicity by virtue of their minimal inhibition of the other PKC isozymes.

The present invention relates to the use of an "isozyme selective" inhibitor for the inhibition of conventional protein kinase C alpha. For this, in one embodiment, cPKCa inhibitors are applied which are selective for alpha PKC isozyme. The terms "cPKCα inhibitor" or "PKCα inhibitor" or the like are used herein as synonyms and refer to an inhibitor which is capable of selectively inhibiting a protein kinase C alpha isozyme over other protein kinase C isozymes.

Alternatively or in combination therewith, the present invention also relates to the use of an isozyme inhibitor that can inhibit a conventional protein kinase C alpha isozyme and that can inhibiti a conventional protein kinase C gamma isozyme. The terms "cPKCa/y inhibitor" or "PKCaJy inhibitor" or the like are used herein as synonyms and refer to an inhibitor which is capable of selectively inhibiting a protein kinase C alpha isozyme and a protein kinase C gamma isozyme over other protein kinase C isozymes. The Applicant has shown that combined inhibition of conventional protein kinase C alpha and gamma isozymes provides a cumulative and stronger effect compared to inhibition of conventional protein kinase C alpha isozyme. In another embodiment, the invention relates to the use of

- an inhibitor that inhibits a conventional protein kinase C alpha isozyme, and/or of

- an inhibitor that inhibits a conventional protein kinase C alpha isozyme and that inhibits a conventional protein kinase C gamma isozyme for the preparation of a medicament for treating inflammatory diseases.

The term "treating," as used herein, describes the management and care of a patient for the purpose of combating the disease, condition, or disorder and includes the administration of an inhibitor according to the present invention to prevent the onset of the symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition, or disorder.

The term "inflammatory diseases" as used herein refers to any diseases involving any type of inflammation. Inflammation is the first response of the immune system to infection or irritation and may be referred to as the innate cascade. Inflammation is characterised by the following quintet: redness (rubor), heat (calor), swelling (tumor), pain (dolor) and dysfunction of the organs involved (functio laesa). Inflammation is the reaction to the tissue damage or to an extrenal microbial (e.g. from bactierla, viral, fungal origin, a chemical (e.g. irritating stubstances), fysical (heat, radioactive or UV radiation) stimulus, but may also be a consequence is of an auto-immune response of the body like. Inflammation aims to remove the detrimental agent and repairing "damage". Inflammatory diseases which may be treated with inhibitors as defined herein include but are not limited to acute or chronic inflammatory diseases or disorders or autoimmune diseases e. g. rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, diabetes type I or Il and the disorders associated therewith, respiratory diseases such as asthma or inflammatory lung injury, inflammatory liver injury, inflammatory glomerular injury, cutaneous manifestations of immunologically-mediated disorders or illnesses, inflammatory and hyperproliferative skin diseases (such as psoriasis, atopic dermatitis, allergic contact dermatitis, irritant contact dermatitis and further eczematous dermatitises, seborrhoeic dermatitis), inflammatory eye diseases, e. g. Sjoegren's syndrome, keratoconjunctivitis or uveitis, (chronic) inflammatory bowel disease, Crohn's disease or ulcerative colitis, Scleroderma, Autoimmune Thyroid Diseases such as Hashimoto's thyroidis, Graves' disease (GD), primary myxedema or autoimmune thyroid failure, Hashitoxicosis; inflammatory heart diseases, e.g. myocarditis, pericarditis, systemic inflammatory response syndrome (SIRS) or sepsis, cancers that are associated with inflammation including but not limited to and inflammation-associated cancers such as hepatocellular carcinoma, colon carcinoma, esophageal adenocarcinoma, liver cancer, bladder and colon carcinomas, cancer of the stomach, lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, and the like.

In a preferred embodiment, the inflammatory disease which may be treated with inhibitors as defined herein is an inflammatory disease that is mediated by receptors of the TLR and/or IL-1 R family, and preferably by a Toll-like receptor 2 (TLR-2) and/or an lnterleukin-1 receptor (IL-1 R). Preferred examples of such diseases include but are not limited to sepsis, myocarditis, arthritis, chronic inflammatory bowel diseases (e.g. Crohn's disease).

Inhibitors cPKCa inhibitors

In one embodiment the present invention relates to the use of an inhibitor that inhibits a conventional protein kinase C alpha isozyme (i.e. a cPKCα-inhibitor) that is a bisindolemaleimide or a derivative thereof.

By the term "derivatives" as used herein is meant N-oxides, salts, solvates, hydrates, stereoisomeric forms, racemic mixtures, tautomeric forms, optical isomers, esters, and the like.

In a preferred embodiment, an inhibitor that inhibits a conventional protein kinase C alpha isozyme (cPKCα-inhibitor) for use according to the present invention is represented by formula IA or formula IB or a pharmaceutically acceptable salt thereof,

Formula IA Formula IB wherein R¹ is independently hydrogen or selected from the group comprising halogen, hydroxyl, alkyl, alkoxy, -NHCO(alkyl), and -NR⁸R⁹, wherein each group may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, preferably C_L10 alkyl, C_1-6 alkyl, or C_1-4 alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl; wherein R² and R³ are each independently hydrogen or oxo (=0); wherein R⁴ and R⁵ are each independently hydrogen or selected from the group comprising alkyl, aryl, cyanoalkyl, thioalkyl, alkylthioalkyl, aminoalkyl, alkylaminoalkyl, heteroaryl, heterocyclyl, cycloalkyl, heterocyclylalkyl, and aminoiminothioalkyl, wherein each group may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, preferably C_1-10 alkyl, C_1-6 alkyl, or C_1-4 alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl; wherein R⁶ and R⁷ are each independently hydrogen or selected from the group comprising halogen, hydroxyl, alkyl, alkoxy, -NHCO(alkyl), and -NR⁸R⁹, benzyloxy, hydroxy, and aminoalkoxy, wherein each group may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, preferably C_1-10 alkyl, C_1-6 alkyl, or C_1-4 alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl; wherein R⁸ and R⁹ are independently hydrogen or selected from the group comprising alkyl, alkanoyl or halo(alkanoyl) or wherein R⁸ and R⁹ taken together with the N atom to which they are bound form a 5 or 6-membered ring; wherein each group may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, preferably C_1-10 alkyl, C_1-6 alkyl, or C_1-4 alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl; and wherein R¹⁰ and R¹¹ are each independently hydrogen or selected from the group comprising alkyl, aryl, cyanoalkyl, thioalkyl, alkylthioalkyl, aminoalkyl, alkylaminoalkyl, heteroaryl, heterocyclyl, cycloalkyl, heterocyclylalkyl, and aminoiminothioalkyl, wherein each group may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, preferably C_1-10 alkyl, C_1-6 alkyl, or C₁^ alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl; or wherein R⁴ and R¹⁰ together form a heterocyclyl or a heteroaryl ring, wherein said ring may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, preferably C_1-10 alkyl, C_1-6 alkyl, or C₁^ alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl; and/or wherein R⁵ and R¹¹ together form a heterocyclyl or a heteroaryl ring wherein said ring may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, preferably C_1-10 alkyl, C_1-6 alkyl, or C_1-4 alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl.

In a preferred embodiment, the inhibitor that inhibits a conventional protein kinase C alpha isozyme for use according to the present invention is represented by formula MA or HB, or a pharmaceutically acceptable salt thereof,

formula MA Formula MB wherein R², R³, R⁴, R¹⁰, and R¹¹ are as defined above.

In a particularly preferred embodiment, the inhibitor is selected from the group comprising bisindolylmaleimide I, bisindolylmaleimide II, bisindolylmaleimide III, bisindolylmaleimide IV, bisindolylmaleimide V, bisindolylmaleimide Vl, bisindolylmaleimide VII, bisindolylmaleimide VIII, bisindolylmaleimide IX, bisindolylmaleimide X, bisindolylmaleimide Xl hydrochloride salt, 2-[1-(3- Dimethylaminopropyl)-1 H-indol-3-yl]-3-(1 H-indol-3-yl)-maleimide, 2-[1-[2-(1-

Methylpyrrolidino)ethyl]-1 H-indol-3-yl]-3-(1 H-indol-3-yl)maleimide, 2-[1-(3-Aminopropyl)-1 H- indol-3-yl]-3-(1H-indol-3-yl)maleimide, 2,3-bis(1H-lndol-3-yl)maleimide, 12-(2-cynaoethyl)- 6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo [2, 3-a] pyrrollo [3, 4-c] carbazole, 2-[1-(3- dimethylaminopropyl)-5-methoxyinol-3-yo]-3-(1 H-indol-3-yl) maleimide and 2,3-bis(1H-lndol-3- yl)-N-methylmaleimide.

A particularly preferred example of the inhibitor is 12-(2-cynaoethyl)-6,7,12,13-tetrahydro-13- methyl-5-oxo-5H-indolo [2, 3-a] pyrrollo [3, 4-c] carbazole, herein also denoted as G06976. This inhibitor has the following specificity: Go 6976 inhibits PKCαr (IC₅₀=2.3 nM) and PKQ01 (IC₅₀=6.2 nM) but has no effect on δ, e or ζ isotypes.

Another particularly preferred example of the inhibitor is (2-{8-[(Dimethylamino)methyl]-6,7,8,9- tetrahydropyrido[1 ,2-a]indol-3-yl)-3-(1-methylindol-3-yl)maleimide hydrochloride, herein also denoted as bisindolylmaleimide Xl hydrochloride salt or Ro 32-0432.

In yet another preferred example of the inhibitor is 2-[1-(3-dimethylaminopropyl)-5-methoxyinol- 3-yo]-3-(1 H-indol-3-yl) maleimide, herein also denoted as Gό6983.

In another embodiment the present invention relates to the use of an inhibitor that inhibits a conventional protein kinase C alpha isozyme that is a substituted biphenyl, or a derivative thereof.

By the term "derivative" as used herein is meant N-oxides, salts, solvates, hydrates, stereoisomeric forms, racemic mixtures, tautomeric forms, optical isomers, esters, and the like. In a preferred embodiment, an inhibitor that inhibits a conventional protein kinase C alpha isozyme for use according to the present invention is represented by formula III, or a pharmaceutically acceptable salt thereof,

Formula III wherein R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, and R²¹ are each independently hydrogen or selected from the group comprising alkyl, aryl, het, nitro, amino, cyano, halogen, formyl, hydroxyl, haloalkyl, hydroxyalkyl, cyanoalkyl, alkoxy, oxyalkyl, alkyloxyalkyl, carboxy, alkylcarboxy, alkylcarbonyl, arylcarbonyl, alkylthio, alkylsulfonyl, alkylsulfoxide, alkylamino, arylamino, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, aryloxy, arylamino, arylalkyloxy, aryloxyalkyl, arylalkylamino, arylcarboxy, arylalkylcarboxy, arylaminocarbonyl, alkylcarbonylamino, alkylcarbonylalkylamino, arylcarbonylamino, arylcarbonylalkylamino, alkylsulfonylamino, arylsulfonylamino, alkylsulfonylalkylamino, and arylsulfonylalkylamino, wherein each group may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, preferably C_1-10 alkyl, C_1-6 alkyl, or C_1-4 alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl.

In a preferred embodiment, the invention provides an inhibitor that inhibits a conventional protein kinase C alpha isozyme (cPKCα) for use according to the present invention represented by formula III, wherein R¹² is independently hydrogen or selected from the group comprising alkyl, aryl, nitro, amino, cyano, halogen, haloalkyl, formyl, hydroxyl, alkoxy, oxyalkyl, alkyloxyalkyl, carboxy, alkylcarboxy, alkylcarbonyl, arylcarbonyl, aryloxy, arylalkyloxy, aryloxyalkyl, and arylcarboxy, and preferably from the group comprising hydrogen, alkyl, hydroxyl, alkoxy, oxyalkyl, and alkyloxyalkyl, wherein each group may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, preferably Ci_-10 alkyl, C₁^ alkyl, or C_1-4 alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl; wherein R¹³ is independently hydrogen or selected from the group comprising alkyl, aryl, nitro, amino, cyano, halogen, haloalkyl, formyl, hydroxyl, alkoxy, oxyalkyl, alkyloxyalkyl, carboxy, alkylcarboxy, alkylcarbonyl, arylcarbonyl, aryloxy, arylalkyloxy, aryloxyalkyl, and arylcarboxy, and preferably from the group comprising hydrogen, alkyl, hydroxyl, alkoxy, oxyalkyl, and alkyloxyalkyl, wherein each group may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazine cyano, alkyl, preferably d.i₀ alkyl, C_1-6 alkyl, or C_1-4 alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoaikyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl; wherein R¹⁴ is independently hydrogen or selected from the group comprising alkyl, aryl, nitro, amino, cyano, halogen, haloalkyl, formyl, hydroxyl, alkoxy, oxyalkyl, alkyloxyalkyl, carboxy, alkylcarboxy, alkylcarbonyl, arylcarbonyl, aryloxy, arylalkyloxy, aryloxyalkyl, and arylcarboxy, and preferably from the group comprising hydrogen, alkyl, hydroxyl, alkoxy, oxyalkyl, and alkyloxyalkyl, wherein each group may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, preferably C_1-10 alkyl, C_1-6 alkyl, or C₁^ alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl; wherein R¹⁵ is independently hydrogen or selected from the group comprising alkyl, aryl, nitro, amino, cyano, halogen, haloalkyl, formyl, hydroxyl, alkoxy, oxyalkyl, alkyloxyalkyl, carboxy, alkylcarboxy, alkylcarbonyl, arylcarbonyl, aryloxy, arylalkyloxy, aryloxyalkyl, and arylcarboxy, and preferably from the group comprising hydrogen, alkyl, hydroxyl, alkoxy, oxyalkyl, and alkyloxyalkyl, wherein each group may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazine cyano, alkyl, preferably Ci_-10 alkyl, Ci_-6 alkyl, or C_1-4 alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl; wherein R¹⁶ is independently hydrogen or selected from the group comprising alkyl, aryl, nitro, amino, cyano, halogen, haloalkyl, formyl, hydroxyl, alkoxy, oxyalkyl, alkyloxyalkyl, carboxy, alkylcarboxy, alkylcarbonyl, arylcarbonyl, aryloxy, arylalkyloxy, aryloxyalkyl, and arylcarboxy, and preferably from the group comprising hydrogen, alkyl, hydroxyl, alkoxy, oxyalkyl, and alkyloxyalkyl, wherein each group may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, preferably C_1-10 alkyl, C_1-6 alkyl, or C_1-4 alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl; wherein R¹⁷ is independently hydrogen or selected from the group comprising alkyl, aryl, nitro, amino, cyano, halogen, haloalkyl, formyl, hydroxyl, alkoxy, oxyalkyl, alkyloxyalkyl, carboxy, alkylcarboxy, alkylcarbonyl, arylcarbonyl, aryloxy, arylalkyloxy, aryloxyalkyl, and arylcarboxy, and preferably from the group comprising hydrogen, alkyl, hydroxyl, alkoxy, oxyalkyl, and alkyloxyalkyl, wherein each group may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, preferably C_1-10 alkyl, C_1-6 alkyl, or C_1-4 alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl; wherein R¹⁸ is independently hydrogen or selected from the group comprising alkyl, aryl, nitro, amino, cyano, halogen, haloalkyl, formyl, hydroxyl, alkoxy, oxyalkyl, alkyloxyalkyl, carboxy, alkylcarboxy, alkylcarbonyl, arylcarbonyl, aryloxy, arylalkyloxy, aryloxyalkyl, and arylcarboxy, and preferably from the group comprising hydrogen, alkyl, hydroxyl, alkoxy, oxyalkyl, and alkyloxyalkyl, wherein each group may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, preferably C_1-10 alkyl, C_1-6 alkyl, or C_1-4 alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl; wherein R¹⁹ is independently hydrogen or selected from the group comprising alkyl, aryl, nitro, amino, cyano, halogen, haloalkyl, formyl, hydroxyl, alkoxy, oxyalkyl, alkyloxyalkyl, carboxy, alkylcarboxy, alkylcarbonyl, arylcarbonyl, aryloxy, arylalkyloxy, aryloxyalkyl, and arylcarboxy, and preferably from the group comprising hydrogen, alkyl, hydroxyl, alkoxy, oxyalkyl, and alkyloxyalkyl, wherein each group may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, preferably C_1-10 alkyl, C_1-6 alkyl, or C_1-4 alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl; wherein R²⁰ is independently hydrogen or selected from the group comprising alkyl, aryl, nitro, amino, cyano, halogen, haloalkyl, formyl, hydroxyl, alkoxy, oxyalkyl, alkyloxyalkyl, carboxy, alkylcarboxy, alkylcarbonyl, arylcarbonyl, aryloxy, arylalkyloxy, aryloxyalkyl, and arylcarboxy, and preferably from the group comprising hydrogen, alkyl, hydroxyl, alkoxy, oxyalkyl, and alkyloxyalkyl, wherein each group may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, preferably C_1-10 alkyl, C_1-6 alkyl, or C_1-4 alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl; wherein R²¹ is independently hydrogen or selected from the group comprising alkyl, aryl, nitro, amino, cyano, halogen, haloalkyl, formyl, hydroxyl, alkoxy, oxyalkyl, alkyloxyalkyl, carboxy, alkylcarboxy, alkylcarbonyl, arylcarbonyl, aryloxy, arylalkyloxy, aryloxyalkyl, and arylcarboxy, and preferably from the group comprising hydrogen, alkyl, hydroxyl, alkoxy, oxyalkyl, and alkyloxyalkyl, wherein each group may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, preferably C_1-10 alkyl, C_1-6 alkyl, or C_1-4 alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl; In a particularly preferred embodiment, the inhibitor is 2,2',3,3^l,4₎4^l-Hexahydroxy-1 ,1^l-biphenyl- 6,6'-dimethanol dimethyl ether, herein also denoted as HBDDE. This inhibitor has the following specificity: HBDDE inhibits PKCσ (IC₅₀=43 μM) and PKCK (IC₅₀=50 μM) without inhibiting the δ, β\ and yff 11 isotypes.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art. When describing the compounds of the invention, the terms used are to be construed in accordance with the following definitions, unless a context dictates otherwise.

As used herein the terms such as "alkyl, aryl, or heteroaryl, each being optionally substituted with" or "alkyl, aryl, or heteroaryl, optionally substituted with" refers to optionally substituted alkyl, optionally substituted aryl and optionally substituted heteroaryl.

The term "substituted" is used in the present invention, it is meant to indicate that one or more hydrogens on the atom indicated in the expression using "substituted" is replaced with a selection from the indicated groups, provided that the indicated atom's normal valency is not exceeded, and that the substitution results in a chemically stable compound, i.e. a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into a therapeutic agent.

The term "alkyl" by itself or as part of another substituent, refers to a straight or branched saturated hydrocarbon group joined by single carbon-carbon bonds having 1 to 20 carbon atoms, for example 1 to 10 carbon atoms, for example 1 to 8 carbon atoms, preferably 1 to 6 carbon atoms, more preferably 1 , 2, 3 or 4 carbon atoms. When a subscript is used herein following a carbon atom, the subscript refers to the number of carbon atoms that the named group may contain. Thus, for example, C₁^ alkyl means an alkyl of one to four carbon atoms. Examples of alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, 2-methylbutyl, pentyl iso-amyl and its isomers, hexyl and its isomers, heptyl and its isomers and octyl and its isomer. When the term "alkyl" is used as a suffix following another term, as in "aminoalkyl," this is intended to refer to an alkyl group, as defined above, being substituted with one or two (preferably one) substituent(s) selected from the other, specifically-named group, also as defined herein. As used herein, the term Cr₂₀ alkyl refers to an alkyl of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. The term "optionally substituted alkyl" refers to an alkyl group optionally substituted with one or more substituents, for example 1 to 4 substituents, for example 1 , 2, 3 or 4 substituents, at any available point of attachment. Non-limiting examples of such substituents include halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, preferably C_1-I0 alkyl, C_1-6 alkyl, or C_1-4 alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, alkyloxy, thiol, alkylthio, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, aminosulfonyl, and the like.

Where alkyl groups as defined are divalent, i.e., with two single bonds for attachment to two other groups, they are termed "alkylene" groups. Non-limiting examples of alkylene groups includes methylene, ethylene, methylmethylene, trimethylene, propylene, tetramethylene, ethylethylene, 1 ,2-dimethylethylene, pentamethylene and hexamethylene. Alkylene group may be optionally substituted according to the present invention with one or more substituents, for example 1 to 4 substituents, for example 1 , 2, 3 or 4 substituents, at any available point of attachment. Non-limiting examples of such substituents include halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, preferably C_1-10 alkyl, C_1-6 alkyl, or C_1-4 alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, alkyloxy, thiol, alkylthio, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, aminosulfonyl, and the like. The term "cycloalkyl group" as used herein by itself or as part of another group is a cyclic alkyl group, that is to say, a monovalent, saturated, or unsaturated hydrocarbyl group having 1 , 2 or 3 cyclic structure. Cycloalkyl includes all saturated or partially saturated (containing 1 or 2 double bonds) hydrocarbon groups containing 1 to 3 rings, including monocyclic, bicyclic or polycyclic alkyl groups. Cycloalkyl groups may comprise 3 or more carbon atoms in the ring and generally, according to this invention comprise from 3 to 10, more preferably from 3 to 8 carbon atoms still more preferably from 3 to 6 carbon atoms. The further rings of multi-ring cycloalkyls may be either fused, bridged and/or joined through one or more spiro atoms. Cycloalkyl groups may also be considered to be a subset of homocyclic rings discussed hereinafter. Examples of cycloalkyl groups, are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl and cyclodecyl with cyclopropyl being particularly preferred. An "optionally substituted cycloalkyl" refers to a cycloalkyl having optionally one or more substituents (for example 1 to 3 substituents, for example 1 , 2, 3 or 4 substituents), selected from those defined above for substituted alkyl. When the suffix "ene" is used in conjunction with a cyclic group, this is intended to mean the cyclic group as defined herein having two single bonds as points of attachment to other groups. An "optionally substituted cycloalkyl" refers to a cycloalkyl having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4 substituents), selected from those defined above for substituted alkyl.

The term "aryl" as used herein by itself or as part of another group refers but is not limited to 5 to 24 carbon-atom homocyclic (i.e., hydrocarbon) monocyclic, bicyclic or tricyclic aromatic rings or ring systems containing 1 to 4 rings which are fused together or linked covalently, typically containing 5 to 8 atoms; at least one of which is aromatic. The aromatic ring may optionally include one to three additional rings (either cycloalkyl, heterocyclyl or heteroaryl) fused thereto. Non-limiting examples of aryl comprise phenyl, biphenylyl, biphenylenyl, 5- or 6-tetralinyl, 1-, 2-, 3-, A-, 5-, 6-, 7- or 8-azulenyl, 1- or 2-naphthyl, 1-, 2- or 3-indenyl, 1-, 2- or 9-anthryl, 1- 2-, 3-, A- or 5-acenaphtylenyl, 3-, 4- or 5-acenaphtenyl, 1-, 2-, 3-, 4- or 10-phenanthryl, 1- or 2-pentalenyl, 1 , 2-, 3- or 4-fluorenyl, 4- or 5-indanyl, 5-, 6-, 7- or 8-tetrahydronaphthyl, 1 ,2,3,4- tetrahydronaphthyl, 1 ,4-dihydronaphthyl, dibenzo[a,d]cylcoheptenyl, 1-, 2-, 3-, 4- or 5-pyrenyl. As used herein, the term C₅-C₂₄ aryl refers to an aryl of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24 carbon atoms. An "optionally substituted aryl" refers to an aryl having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4 substituents), selected from those defined above for substituted alkyl.

The term "heteroaryl" as used herein by itself or as part of another group refers but is not limited to 5 to 12 carbon-atom aromatic rings or ring systems containing 1 to 3 rings which are fused together or linked covalently, typically containing 5 to 8 atoms; at least one of which is aromatic in which one or more carbon atoms in one or more of these rings can be replaced by oxygen, nitrogen or sulfur atoms where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quatemized. Such rings may be fused to an aryl, cycloalkyl, heteroaryl or heterocyclyl ring. Non-limiting examples of heteroaryl can be 2- or 3-furyl, 2- or 3-thienyl, 1-, 2- or 3-pyrrolyl, 1-, 2-, 4- or 5-imidazolyl, 1-, 3-, 4- or 5-pyrazolyl, 3-, A- or 5-isoxazolyl, 2-, 4- or 5-oxazolyl, 3-, 4- or 5-isothiazolyl, 2-, 4- or 5-thiazolyl, 1 ,2,3-triazol-1-, - 2-, -4- or -5-yl, 1 ,2,4-triazol-1-, -3-, -A- or -5-yl, 1 ,2,3-oxadiazol-4- or -5-yl, 1 ,2,4-oxadiazol-3- or - 5-yl, 1 ,2,5-oxadiazolyl, 1 ,3,4-oxadiazolyl, 1 ,2,3-thiadiazol-4- or -5-yl, 1 ,2,4-thiadiazol-3- or -5-yl, 1 ,2,5-thiadiazol-3- or -4-yl, 1 ,3,4-thiadiazolyl, and the like. An "optionally substituted heteroaryl" refers to a heteroaryl having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted aryl. The terms "heterocyclyl" or "heterocyclo" as used herein by itself or as part of another group refer to non-aromatic, fully saturated or partially unsaturated cyclic groups (for example, 3 to 13 member monocyclic, 7 to 17 member bicyclic, or 10 to 20 member tricyclic ring systems, or containing a total of 3 to 10 ring atoms) which have at least one heteroatom in at least one carbon atom-containing ring. Each ring of the heterocyclic group containing a heteroatom may have 1 , 2, 3 or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized. The heterocyclic group may be attached at any heteroatom or carbon atom of the ring or ring system, where valence allows. The rings of multi- ring heterocycles may be fused, bridged and/or joined through one or more spiro atoms. An "optionally substituted heterocyclic" refers to a heterocyclic having optionally one or more substituents (for example 1 to 4 substituents, or for example 1 , 2, 3 or 4), selected from those defined above for substituted aryl. Exemplary heterocyclic groups include piperidinyl, azetidinyl, imidazolinyl, imidazolidinyl, isoxazolinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, piperidyl, succinimidyl, 3H-indolyl, indolinyl, isoindolinyl, chromenyl, isochromanyl, xanthenyl, 2H-pyrrolyl, 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, pyrrolidinyl, 4H-quinolizinyl, 4aH-carbazolyl, 2- oxopiperazinyl, piperazinyl, homopiperazinyl, 2-pyrazolinyl, 3-pyrazolinyl, pyranyl, dihydro-2H- pyranyl, 4H-pyranyl, 3,4-dihydro-2H-pyranyl, triazinyl, cinnolinyl, phthalazinyl, oxetanyl, thietanyl, 3-dioxolanyl, 1 ,4-dioxanyl, 2,5-dioximidazolidinyl, 2,2,4-piperidonyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, indolinyl, tetrahydropyranyl, tetrahydrofuranyl, tetrehydrothienyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1 , 3-dioxolanyl, 1 ,4-oxathianyl, 1 ,4-dithianyl, 1 ,3,5-trioxanyl, 6H-1 ,2,5-thiadiazinyl, 2H-1 ,5,2-dithiazinyl, 2H-oxocinyl, I H-pyrrolizinyl, tetra hydro- 1,1 - dioxothienyl, N- formylpiperazinyl, and morpholinyl.

The term "hydroxyalkyl" by itself or as part of another substituent refers to a -R^b-OH group wherein R^b is alkylene as defined herein. An "optionally substituted hydroxyalkyl" refers to a hydroxyalkyl having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl.

The term "alkoxy" or "alkyloxy" by itself or as part of another substituent refers to the group -O- R^a wherein R^a is alkyl as defined herein. An "optionally substituted alkoxy" refers to an alkoxy having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl.

The term "aryloxy" by itself or as part of another substituent refers to the group -O-R^c wherein R^c is aryl as defined herein. An "optionally substituted aryloxy" refers to an aryloxy having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl. The term "alkoxyalkyl" or "alkyloxyalkyl" by itself or as part of another substituent refers to the group -R^b-O-R^a wherein R^a is alkyl as defined herein and R^b is alkylene as defined herein. An "optionally substituted alkoxyalkyl" refers to an alkoxyalkyl having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl.

The term "arylalkyloxy" by itself or as part of another substituent refers to the group -O-R^b-R^c wherein R^c is aryl as defined herein and R^b is alkylene as defined herein. An "optionally substituted arylalkyloxy" refers to an arylalkyloxy having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl.

The term "aryloxyalkyl" by itself or as part of another substituent refers to the group -R^b-O-R^c wherein R^c is aryl as defined herein and R^b is alkylene as defined herein. An "optionally substituted aryloxyalkyl" refers to an aryloxyalkyl having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl.

The term "nitro" refers to the group -NO₂. The term "amino" refers to the group -NH₂.

The term "aminoalkyl" by itself or as part of another substituent refers to the group -R^b-NH₂ wherein R^b is alkylene as defined herein. An "optionally substituted aminoalkyl" refers to an aminoalkyl having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl. For instance, in the aminoalkyl group the alkyl may be unsubstituted or substituted by hydroxy or C_1-4 alkoxy, and the nitrogen may be unsubstituted, mono- or disubstituted by C_1-4 alkyl, and/or the substituents, together with the nitrogen to which they are attached, may form a C₃-C₆ heterocyclic ring wherein the alkyl chain is unsubstituted or substituted by a C₁^ alkyl

The term "alkylamino" by itself or as part of another substituent refers to the group -NH-R^a wherein R^a is alkyl as defined herein. An "optionally substituted alkylamino" refers to an alkylamino having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl. The term "alkylaminoalkyl" alone or in combination refers to the group — R^b-NH-R^a wherein R^a is alkyl as defined herein, and wherein R^b is alkylene as defined herein. An "optionally substituted alkylaminoalkyl" refers to an alkylaminoalkyl having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl. The term "arylamino" by itself or as part of another substituent refers to the group -NH-R^C wherein R^c is aryl as defined herein. An "optionally substituted arylamino" refers to an arylamino having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl. The term "arylalkylamino" by itself or as part of another substituent refers to the group -NH-R^b- R^c wherein R^c is aryl as defined herein, and R^b is alkylene as defined herein. An "optionally substituted arylalkylamino" refers to an arylalkylamino having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl. The term "aminoalkoxy" or "aminoalkyloxy" by itself or as part of another substituent refers to the group -O-R^a-NH₂ wherein R^a is alkyl as defined herein. An "optionally substituted aminoalkyloxy" refers to an aminoalkyloxy having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl. For instance, in the aminoalkoxy group the alkyl may be unsubstituted or substituted by hydroxy or Ci-C₄ alkoxy, and the nitrogen may be unsubstituted, mono- or disubstituted by C₁-C₄ alkyl, and/or the substituents, together with the nitrogen to which they are attached, may form a C₃-C₆ heterocyclic ring wherein the alkyl chain is unsubstituted or substituted by a Ci-C₄ alkyl.

The term "cyano" refers to the group -CN. The term "cyanoalkyl" by itself or as part of another substituent refers to an alkyl radical having the meaning as defined above wherein one or more hydrogens are replaced with a cyano as defined above. An "optionally substituted cyanoalkyl" refers to a cyanoalkyl having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl.

The term "thiol" or ""sulfhydryl" refers to the group -SH. The term "alkylthio" by itself or as part of another substituent refers to the group -SR^a group wherein R^a is alkyl as defined herein. This term refers to a group consisting of a sulfur atom attached to an alkyl group. An "optionally substituted alkylthio" refers to an alkylthio having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl. Non-limiting examples of alkylthio groups include methylthio (SCH₃), ethylthio (SCH₂CH₃), n-propylthio, isopropylthio, n-butylthio, isobutylthio, sec-butylthio, tert-butylthio, n-hexylthio, and the like.

The term "thioalkyl" by itself or as part of another substituent refers to the group -R^b-SH wherein

R^b is alkylene as defined herein. An "optionally substituted thioalkyl" refers to a thioalkyl having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl. Non-limiting examples of thioalkyl groups include thiomethyl, thioethyl, thiopropyl, thiobutyl, thiopentyl, thiohexyl, thioheptyl, thiooctyl, thiooctadecyl, and the like.

The term "alkylthioalkyl" by itself or as part of another substituent refers to the group -R^b-S-R^a wherein R^b is alkylene as defined herein, and R^a is alkyl as defined herein. An "optionally substituted alkylthioalkyl" refers to an alkylthioalkyl having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl.

The term "aminoiminothioalkyl" by itself or as part of another substituent refers to the group -R^b- S-C(=NH)-NH₂, wherein R^b is alkylene as defined herein. An "optionally substituted aminoiminothioalkyl" refers to an aminoiminothioalkyl having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl.

The term "alkylsulfonyl" by itself or as part of another substituent refers to the group -SO₂-R^a wherein R^a is alkyl as defined herein. An "optionally substituted alkylsulfonyl" refers to an alkylsulfonyl having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl.

The term "alkylsulfonylamino" by itself or as part of another substituent refers to the group - NR^d-SO₂-R^a wherein R^a is alkyl as defined herein, and wherein R^d is hydrogen or alkyl which is optionally substituted with one or more substituents selected from the list given above. An "optionally substituted alkylsulfonylamino" refers to an alkylsulfonylamino having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl.

The term "arylsulfonylamino" by itself or as part of another substituent refers to the group -NR^d- SO₂-R⁰ wherein R^c is aryl as defined herein, and wherein R^d is hydrogen or alkyl which is optionally substituted with one or more substituents selected from the list given above. An "optionally substituted arylsulfonylamino" refers to an arylsulfonylamino having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl.

The term "alkylsulfoxide" by itself or as part of another substituent refers to the group -S(=O)-R^a wherein R^a is alkyl as defined herein. An "optionally substituted alkylsulfoxide" refers to an arylsulfonylamino having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl.

The term "halo" or "halogen" as a group or part of a group is generic for fluoro, chloro, bromo or iodo. The term "haloalkyl" by itself or as part of another substituent refers to an alkyl radical having the meaning as defined above wherein one or more hydrogens are replaced with a halogen as defined above. Non-limiting examples of such haloalkyl radicals include chloromethyl, 1- bromoethyl, fluoromethyl, difluoromethyl, trifluoromethyl, 1 ,1 ,1 -trifluoroethyl and the like. An "optionally substituted "haloalkyl" refers to a haloalkyl having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl.

As used herein, the term "oxo" or "=O" forms a carbonyl moiety with the carbon atom to which it is attached. The term "alkanoyl" or "alkylcarbonyl", by itself or as part of another substituent refers to the group-C(=O)R^a, wherein R^a is alkyl as defined herein. An "optionally substituted" alkylcarbonyl" refers to an alkylcarbonyl having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl. Said alkylcarbonyl can be exemplified by acetyl, propionyl, butyryl, valeryl and pivaloyl. The term "aminocarbonyl" refers to the group -C(=O)-NH₂.

The term "alkylaminocarbonyl", by itself or as part of another substituent refers to the group - C(=O)-NR^d-R^a, wherein R^a is alkyl as defined herein, and wherein R^d is hydrogen or alkyl which is optionally substituted with one or more substituents selected from the list given above for substituted alkyl. An "optionally substituted "alkylaminocarbonyl" refers to an alkylaminocarbonyl having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl.

The term "arylaminocarbonyl" by itself or as part of another substituent refers to the group - C(=O)-NR^d-R^c, wherein R^c is aryl as defined herein, and wherein R^d is hydrogen or alkyl which is optionally substituted with one or more substituents selected from the list given above for substituted alkyl. An "optionally substituted "arylaminocarbonyl" refers to an arylaminocarbonyl having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl.

The term "alkylcarbonylamino", by itself or as part of another substituent refers to the group - NR^d-C(=O)-R^a, wherein R^a is aryl as defined herein, and wherein R^d is hydrogen or alkyl which is optionally substituted with one or more substituents selected from the list given above for substituted alkyl. An "optionally substituted "alkylcarbonylamino" refers to an alkylcarbonylamino having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl. The term "arylcarbonylamino", by itself or as part of another substituent refers to the group - NR^d-C(=O)-R^c, wherein R^c is aryl as defined herein, and wherein R^d is hydrogen or alkyl which is optionally substituted with one or more substituents selected from the list given above for substituted alkyl. An "optionally substituted "arylcarbonylamino" refers to an arylcarbonylamino having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl.

The term "carboxy" is equivalent to "hydroxycarbonyl" and refers to the group -CO₂H.

The term "alkylcarboxy" is equivalent to "alkyloxycarbonyl" and refers by itself or as part of another substituent to the group -CO₂-R^a, wherein R^a is alkyl as defined herein. An "optionally substituted "alkylcarboxy " refers to an alkylcarboxy having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl.

The term "arylcarboxy" refers by itself or as part of another substituent to the group -CO₂-R⁰, wherein R^c is aryl as defined herein. An "optionally substituted "arylcarboxy" refers to an arylcarboxy having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl.

The term "arylalkylcarboxy" by itself or as part of another substituent refers to the group -CO₂- R^b-R°, wherein R^c is aryl as defined herein, and where is R^b alkylene as defined herein. An "optionally substituted "arylalkylcarboxy" refers to an arylalkylcarboxy having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl.

The term carboxaldehyde or formyl refers to the group -C(=O)H.

The term "alkylcarbonyl" by itself or as part of another substituent refers to the group-C(=O)R^a, wherein R^a is alkyl as defined herein. An "optionally substituted "alkylcarbonyl" refers to an alkylcarbonyl having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl. Said alkylcarbonyl can be exemplified by acetyl, propionyl, butyryl, valeryl and pivaloyl.

The term "arylcarbonyl" by itself or as part of another substituent refers to the group-C(=O)R^c, wherein R^c is aryl as defined herein, and wherein R^b is alkylene as defined herein. An "optionally substituted "arylcarbonyl" refers to an arylcarbonyl having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl. Said alkylcarbonyl can be exemplified by acetyl, propionyl, butyryl, valeryl and pivaloyl. For therapeutic use, the salts of the inhibitors according to the invention are those wherein the counter-ion is pharmaceutically or physiologically acceptable. The pharmaceutically acceptable salts of the inhibitors according to the invention, i.e. in the form of water-, oil-soluble, or dispersible products, include the conventional non-toxic salts or the quaternary ammonium salts which are formed, e.g., from inorganic or organic acids or bases. Examples of such acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate. Base salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicydohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such a sarginine, lysine, and so forth. Also, the basic nitrogen-containing groups may be quatemized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl- bromides and others. Other pharmaceutically acceptable salts include the sulfate salt ethanolate and sulfate salts. cPKCa/y inhibitors

In another embodiment the present invention relates to the use of an inhibitor that is capable of inhibiting conventional protein kinase C alpha isozyme and that is apable of inhibiting conventional protein kinase C gamma isozyme that is a bisindolemaleimide or a derivative thereof. In a preferred embodiment, such cPKCα/κ inhibitor that inhibits a conventional protein kinase C alpha isozyme and a gamma isozyme for use according to the present invention is represented by formula IA, IB, HA, or MB as indicated above.

In a preferred embodiment, the inhibitor that inhibits a conventional protein kinase C alpha isozyme and a conventional protein kinase C gamma isozyme is selected from the group comprising bisindolylmaleimide I, bisindolylmaleimide II, bisindolylmaleimide III, bisindolylmaleimide IV, bisindolylmaleimide V, bisindolylmaleimide Vl, bisindolylmaleimide VII, bisindolylmaleimide VIII, bisindolylmaleimide IX, bisindolylmaleimide X, bisindolylmaleimide Xl hydrochloride salt, 2-[1 -(3-Dimethylaminopropyl)-1 H-indol-3-yl]-3-(1 H-indol-3-yl)-maleimide, 2- [1-[2-(1-Methylpyrrolidino)ethyl]-1 H-indol-3-yl]-3-(1 H-indol-3-yl)maleimide, 2-[1-(3-Aminopropyl)-

1 H-indol-3-yl]-3-(1 H-indol-3-yl)maleimide, 2,3-bis(1 H-lndol-3-yl)maleimide, 12-(2-cynaoethyl)- 6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo [2, 3-a] pyrrollo [3, 4-c] carbazole, 2-[1-(3- dimethylaminopropyl)-5-methoxyinol-3-yo]-3-(1 H-indol-3-yl) maleimide and 2,3-bis(1 H-lndol-3- yl)-N-methylmaleimide.

In another embodiment the present invention relates to the use of an inhibitor that inhibits a conventional protein kinase C alpha isozyme and a conventional protein kinase C gamma isozyme that is a substituted biphenyl, or a derivative thereof. In a preferred embodiment, an inhibitor that inhibits a conventional protein kinase C alpha isozyme and a conventional protein kinase C gamma isozyme for use according to the present invention is represented by formula III as indicated above. In a particularly preferred embodiment, the cPKC alγ inhibitor is 2,2^l,3,3',4,4'-Hexahydroxy-1,1'- biphenyl-6,6'-dimethanol dimethyl ether, herein also denoted as HBDDE.

In accordance with the present invention, the applicant has shown that inhibitory compounds that are selective to one or two protein kinase C isozymes, in particular to alpha and gamma isozymes, relative to the other PKC isozymes, are superior therapeutic agents in the treatment of inflammatory diseases as defined herein. Such inhibitors demonstrate greater efficacy and lower toxicity by virtue of their specificity.

Inhibitor specificity can be represented by means of the inhibitors' IC₅₀ values. The "IC₅₀" value as used herein represents the concentration of an inhibitor as defined herein that is required for 50% inhibition of a cPKC isozyme as defined herein. In a preferred embodiment, the invention provides an inhibitor that inhibits a conventional protein kinase C alpha isozyme (cPKCα inhibitor) having an IC₅₀ value for alpha activity lower than 10OnM and preferably lower than 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50 nM. In another embodiment, the invention provides an inhibitor that inhibits a conventional protein kinase C alpha and gamma isozyme (cPKCα/p inhibitor) having an IC₅₀ value for alpha activity lower than 50 μM and preferably lower than 48, 46, or 45 μM and an IC₅₀ value for gamma activity lower than 70 μM and preferably lower than 68, 66, or 60 μM.

Pharmaceutical compositions

In another embodiment, the invention relates to a pharmaceutical composition for treating inflammatory diseases as defined herein, comprising a therapeutically effective amount of: - an inhibitor that inhibits a conventional protein kinase C alpha isozyme and/or

- an inhibitor that inhibits a conventional protein kinase C alpha isozyme and a conventional protein kinase C that inhibits gamma isozyme and further comprising a pharmaceutically acceptable excipient. Suitable inhibitors for use in a composition include inhibitors as defined above and represented by the formula IA, IB, MA, NB or III given above.

In one embodiment, the invention provides a pharmaceutical composition for treating inflammatory diseases as defined herein, comprising an inhibitor that is represented by formula IA, IB, HA, or MB as defined herein, and a pharmaceutically acceptable excipient. In a preferred embodiment, the invention provides a pharmaceutical composition for treating inflammatory diseases as defined herein, comprising an inhibitor that is selected from the group comprising bisindolylmaleimide I₁ bisindolylmaleimide II, bisindolylmaleimide III, bisindolylmaleimide IV, bisindolylmaleimide V, bisindolylmaleimide Vl, bisindolylmaleimide VII, bisindolylmaleimide VIII, bisindolylmaleimide IX, bisindolylmaleimide X, bisindolylmaleimide Xl hydrochloride salt, 2-[1-(3- Dimethylaminopropyl)-1 H-indol-3-yl]-3-(1 H-indol-3-yl)-maleimide, 2-[1-[2-(1-

Methylpyrrolidino)ethyl]-1 H-indol-3-yl]-3-(1 H-indol-3-yl)maleimide, 2-[1 -(3-Aminopropyl)-1 H- indol-3-yl]-3-(1 H-indol-3-yl)maleimide, 2,3-bis(1 H-lndol-3-yl)maleimide, 12-(2-cynaoethyl)- 6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo [2, 3-a] pyrrollo [3, 4-c] carbazole, 2-[1-(3- dimethylaminopropyl)-5-methoxyinol-3-yo]-3-(1 H-indol-3-yl) maleimide (Go 6983) and 2,3- bis(1 H-lndol-3-yl)-N-methylmaleimide, and a pharmaceutically acceptable excipient.

In another embodiment, the invention provides a pharmaceutical composition for treating inflammatory diseases as defined herein, comprising an inhibitor that is represented by formula III as defined herein, and a pharmaceutically acceptable excipient. In a preferred embodiment, the invention provides a pharmaceutical composition for treating inflammatory diseases as defined herein, comprising an inhibitor that is 2,2^l,3,3',4,4^l-Hexahydroxy-1 ,1^l-biphenyl-6,6'- dimethanol dimethyl ether and a pharmaceutically acceptable excipient.

The inhibitors as defined herein may be administered in free form or in pharmaceutically acceptable salt form e.g. as indicated above. Such salts may be prepared in conventional manner and exhibit the same order of activity as the free compounds.

The term "therapeutically effective amount" as used herein means that amount of inhibitor that elicits the biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease being treated. The particular dose of the inhibitor administered according to this invention will, of course, be determined by the particular circumstances surrounding the case, including the inhibitor administered, the route of administration, the particular condition being treated, and similar considerations. The inhibitors can be administered by a variety of routes including the oral, rectal, transdermal, subcutaneous, topical, intravenous, intramuscular or intranasal routes. For all indications, a typical daily dose will contain from about 0.01 mg/kg to about 20 mg/kg of the active inhibitor of this invention. Preferred daily doses will be about 0.05 to about 10 mg/kg, for instance about 0.1 to about 5 mg/kg. For instance, for topical administration a typical dosage may be about 1 to about 500 μg inhibitor per cm² of an affected tissue, for instance about 30 to about 300 μg/cm², or for instance from about 50 to about 200 μg/cm², or for instance from about 60 to about 100 μg/cm².

Pharmaceutical compositions comprising one or more inhibitors as defined herein in free form or in pharmaceutical acceptable salt form in association with at least one pharmaceutical acceptable excipient, e.g. carrier or diluent may be manufactured in conventional manner by mixing with a pharmaceutically acceptable carrier or diluent. For instance, at least one inhibitor formula I, II, III or IV, in free form or in pharmaceutical acceptable salt form, one or more solid or liquid pharmaceutical excipients and, if desired, in combination with other pharmaceutical active compounds, are brought into a suitable administration form or dosage form which can then be used as a pharmaceutical in human medicine or veterinary medicine.

Particular forms of the pharmaceutical composition may be, for example, solutions, suspensions, emulsions, creams, tablets, capsules, nasal sprays, liposomes or micro- reservoirs, especially compositions in orally ingestible or sterile injectable form, for example, as sterile injectable aqueous or oleaginous suspensions or suppositories. The preferred form of composition contemplated is the dry solid form, which includes capsules, granules, tablets, pills, boluses and powders. The solid carrier may comprise one or more excipients, e.g. lactose, fillers, disintegrating agents, binders, e.g. cellulose, carboxymethylcellulose or starch or anti- stick agents, e.g. magnesium stearate, to prevent tablets from adhering to tabletting equipment. Tablets, pills and boluses may be formed so as to disintegrate rapidly or to provide slow release of the active ingredient.

In order to enhance the solubility and/or the stability of the inhibitors of a pharmaceutical composition according to the invention, it can be advantageous to employ a-, β- or y- cyclodextrins or their derivatives. In addition, co-solvents such as alcohols may improve the solubility and/or the stability of the inhibitors. In the preparation of aqueous compositions, addition of salts of the inhibitors of the invention are obviously more suitable due to their increased water solubility. Appropriate cyclodextrins are a-, β- or y-cyclodextrins (CDs) or ethers and mixed ethers thereof wherein one or more of the hydroxy groups of the anhydroglucose units of the cyclodextrin are substituted with alkyl, particularly methyl, ethyl or isopropyl, e.g. randomly methylated /?-CD; hydroxyalkyl, particularly hydroxyethyl, hydroxypropyl or hydroxybutyl; carboxyalkyl, particularly carboxymethyl or carboxyethyl; alkylcarbonyl, particularly acetyl; alkyloxycarbonylalkyl or carboxyalkyloxyalkyl, particularly carboxymethoxypropyl or carboxyethoxypropyl; alkylcarbonyloxyalkyl, particularly 2-acetyloxypropyl. Especially noteworthy as complexants and/or solubilizers are β-CO, randomly methylated β-CD, 2,6-dimethyl- jff-CD, 2-hydroxyethyl-£- CD, 2-hydroxyethyl-κ-CD, 2-hydroxypropyl-κ-CD and (2-carboxymethoxy)propyl- /?-CD, and in particular 2-hydroxypropyl- yff-CD (2-HP- /?-CD). The term mixed ether denotes cyclodextrin derivatives wherein at least two cyclodextrin hydroxy groups are etherified with different groups such as, for example, hydroxypropyl and hydroxyethyl. An interesting way of formulating the analogues in combination with a cyclodextrin or a derivative thereof has been described in EP- A-721 ,331. Although the formulations described therein are with antifungal active ingredients, they are equally interesting for formulating the analogues. Said formulations may also be rendered more palatable by adding pharmaceutically acceptable sweeteners and/or flavors.

More in particular, the compositions may be formulated in a pharmaceutical formulation comprising a therapeutically effective amount of particles consisting of a solid dispersion of the inhibitors of the invention and one or more pharmaceutically acceptable water-soluble polymers.

The term "a solid dispersion" defines a system in a solid state (as opposed to a liquid or gaseous state) comprising at least two components, wherein one component is dispersed more or less evenly throughout the other component or components. When said dispersion of the components is such that the system is chemically and physically uniform or homogenous throughout or consists of one phase as defined in thermodynamics, such a solid dispersion is referred to as "a solid solution". Solid solutions are preferred physical systems because the components therein are usually readily bioavailable to the organisms to which they are administered. The term "a solid dispersion" also comprises dispersions that are less homogenous throughout than solid solutions. Such dispersions are not chemically and physically uniform throughout or comprise more than one phase.

The water-soluble polymer is conveniently a polymer that has an apparent viscosity of 1 to 100 mPa.s when dissolved in a 2 % aqueous solution at 20⁰C solution. Preferred water-soluble polymers are hydroxypropyl methylcelluloses or HPMC. HPMC having a methoxy degree of substitution from about 0.8 to about 2.5 and a hydroxypropyl molar substitution from about 0.05 to about 3.0 are generally water soluble. Methoxy degree of substitution refers to the average number of methyl ether groups present per anhydroglucose unit of the cellulose molecule. Hydroxy-propyl molar substitution refers to the average number of moles of propylene oxide which have reacted with each anhydroglucose unit of the cellulose molecule. Various techniques exist for preparing solid dispersions including melt-extrusion, spray-drying and solution-evaporation, melt-extrusion being preferred.

It may further be convenient to formulate the analogues in the form of nanoparticles which have a surface modifier adsorbed on the surface thereof in an amount sufficient to maintain an effective average particle size of less than 1000 nm. Suitable surface modifiers can preferably be selected from known organic and inorganic pharmaceutical excipients. Such excipients include various polymers, low molecular weight oligomers, natural products and surfactants. Preferred surface modifiers include nonionic and anionic surfactants. Yet another interesting way of formulating the inhibitors according to the invention involves a pharmaceutical composition whereby the inhibitors are incorporated in hydrophilic polymers and applying this mixture as a coat film over many small beads, thus yielding a composition with good bio-availability which can conveniently be manufactured and which is suitable for preparing pharmaceutical dosage forms for oral administration. Said beads comprise (a) a central, rounded or spherical core, (b) a coating film of a hydrophilic polymer and an antiretroviral agent and (c) a seal-coating polymer layer. Materials suitable for use as cores in the beads are manifold, provided that said materials are pharmaceutically acceptable and have appropriate dimensions and firmness. Examples of such materials are polymers, inorganic substances, organic substances, and saccharides and derivatives thereof. KTT

In an embodiment, the invention relates to a kit comprising i) an inhibitor represented by formula IA, IB, MA, and/or MB according to the invention, and ii) an anti-inflammatory compound as a combined preparation for simultaneous, separate or sequential use in the treatment of inflammatory diseases, as defined herein. In another embodiment, the invention relates to a kit comprising i) an inhibitor represented by formula III according to the invention, and ii) an anti-inflammatory compound as a combined preparation for simultaneous, separate or sequential use in the treatment of inflammatory diseases, as defined herein.

In yet another embodiment, the invention relates to a kit comprising i) an inhibitor represented by formula IA, IB, MA, and/or MB and an inhibitor represented by formula III according to the invention, and ii) an anti-inflammatory compound as a combined preparation for simultaneous, separate or sequential use in the treatment of inflammatory diseases, as defined herein.

Preferably, said inhibitor of formula IA, IB, MA, or MB is selected from the group comprising comprising bisindolylmaleimide I, bisindolylmaleimide II, bisindolylmaleimide Ml, bisindolylmaleimide IV, bisindolylmaleimide V, bisindolylmaleimide Vl₁ bisindolylmaleimide VII, bisindolylmaleimide VIII, bisindolylmaleimide IX, bisindolylmaleimide X, bisindolylmaleimide Xl hydrochloride salt, 2-[1 -(3-Dimethylaminopropyl)-1 H-indol-3-yl]-3-(1 H-indol-3-yl)-maleimide, 2- [1-[2-(1-Methylpyrrolidino)ethyl]-1 H-indol-3-yl]-3-(1 H-indol-3-yl)maleimide, 2-[1-(3-Aminopropyl)- 1 H-indol-3-yl]-3-(1 H-indol-3-yl)maleimide, 2,3-bis(1 H-lndol-3-yl)maleimide, 12-(2-cynaoethyl)- 6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo [2, 3-a] pyrrollo [3, 4-c] carbazole, 2-[1-(3- dimethylaminopropyl)-5-methoxyinol-3-yo]-3-(1H-indol-3-yl) maleimide (Go 6983) and 2,3- bis(1 H-lndol-3-yl)-N-methylmaleimide.

The inhibitor of formula III preferably is 2,2^l,3_l3',4,4'-Hexahydroxy-1 ,1'-biphenyl-6,6'-dimethanol dimethyl ether. Preferably the anti-inflammatory compound which can be used in combination with an inhibitor according to the invention for treating inflammatory diseases is selected from the group comprising but not limited to glucocorticoids such as prednisonem dexamethasone, hydrocortisone, or non-steroidal anti-inflammatory drugs, usually abbreviated to NSAIDs, including salicylates such as aspirin, Methyl salicylate, Diflunisal.Benorylate, Faislamine, or Amoxiprin; Arylalkanoic acids such as Diclofenac, Indomethacin, or Sulindac; 2-Arylpropionic acids (profens) such as Carprofen, Fenoprofen, Flurbiprofen, Ibuprofen, Ketoprofen, Ketorolac, Loxoprofen, Naproxen, or Tiaprofenic acid; N-Arylanthranilic acids (fenamic acids) such as Mefenamic acid or Meclofenamic acid; Pyrazolidine derivatives such as Phenylbutazone or Oxyphenylbutazone; Oxicams such as Piroxicam or Meloxicam, coxibs such as Parecoxib or Etoricoxib; sulphonanilides such as Nimesulide, or Omega-3 Fatty Acids.

In yet another embodiment, the invention relates to a kit comprising i) an inhibitor that inhibits a conventional protein kinase C alpha isozyme and ii) an inhibitor that inhibits a conventional protein kinase C alpha isozyme and that inhibits a conventional protein kinase C gamma isozyme as a combined preparation for simultaneous, separate or sequential use in the treatment of inflammatory diseases, as defined herein.

In another embodiment, the invention relates to a kit comprising i) an inhibitor that inhibits a conventional protein kinase C alpha isozyme and ii) an anti-inflammatory compound, as defined herein, as a combined preparation for simultaneous, separate or sequential use in the treatment of inflammatory diseases, as defined herein. In still another embodiment, the invention relates to a kit comprising i) an inhibitor that inhibits a conventional protein kinase C alpha isozyme and that inhibits a conventional protein kinase C gamma isozyme and ii) an anti-inflammatory compound as defined herein, as a combined preparation for simultaneous, separate or sequential use in the treatment of inflammatory diseases, as defined herein.

Methods of treatment

In another embodiment, the invention relates to a method for treating inflammatory diseases as defined herein, and preferably inflammatory diseases that are mediated by receptors of the TLR and/or IL-1 R family, and preferably by a Toll-like receptor 2 (TLR-2) and/or an interleukin-1 receptor (IL-1 R), in a mammal, e.g. a human or an animal, in need thereof comprising administering a therapeutically effective amount of an inhibitor that inhibits a conventional protein kinase C alpha isozyme and/or of an inhibitor that inhibits a conventional protein kinase C alpha isozyme and that inhibits a conventional protein kinase C gamma isozyme optionally in combination with one or more other anti-inflammatory compounds as defined herein.

In another embodiment, the invention relates to a method for treating inflammatory diseases as defined herein, and preferably inflammatory diseases that are mediated by receptors of the TLR and/or IL-1 R family, and preferably by a Toll-like receptor 2 (TLR-2) and/or an interleukin-1 receptor (IL-1 R), in a mammal, e.g. a human or an animal, in need thereof comprising administering a therapeutically effective amount of an inhibitor of formula IA, IB, MA, MB and/or III as disclosed herein, optionally in combination with one or more other anti-inflammatory compounds as defined herein.

In yet another embodiment, the invention relates to a method for treating inflammatory diseases as defined herein, and preferably inflammatory diseases that are by receptors of the TLR and/or IL-1 R family, and preferably by a Toll-like receptor 2 (TLR-2) and/or an Interleukin-1 receptor (IL-1 R), in a mammal, e.g. a human or an animal, in need thereof comprising administering a therapeutically effective amount of a pharmaceutical composition as disclosed herein.

The present treatment methods comprise methods for treatment of inflammation-mediated diseases including autoimmune syndromes, myocarditis, arthritis and certain cancers, as disclosed herein, which depend on TLR and/or IL-1 R family signaling. In another embodiment of the invention there is provided a method for inhibiting TLR and/or IL-1 R family mediated inflammation, e.g. TLR-2-mediated septic shock, which comprises administering to a mammal in need of such treatment a therapeutically effective amount of a PKCα inhibitor and/or of a PKCα/γ inhibitor. In yet another embodiment of the invention there is provided a method for inhibiting TLR- and/or IL-1 R family-mediated inflammatory diseases which comprises administering to a mammal in need of such treatment a therapeutically effective amount of a PKCα inhibitor and/or of a PKCα/γ inhibitor.

As indicated above, due to their favourable selective inhibitory properties the inhibitors according to the present invention are particularly useful in the treatment of individuals suffering from inflammatory disease as disclosed herein. For these purposes, an inhibitor and/or pharmaceutical composition of the present invention may be administered orally, parenterally, i.e. including subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques, by inhalation spray, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. In accordance with the method of the present invention, said inhibitor and/or pharmaceutical composition can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms. The present invention is therefore to be understood as embracing all such regimes of simultaneous or alternating treatment and the term "administering" is to be interpreted accordingly.

In another embodiment of the method of the invention, the administration may be performed with food, e.g., a high-fat meal. The term 'with food' means the consumption of a meal either during or no more than about one hour before or after administration of an inhibitor and/or a pharmaceutical composition according to the invention. For an oral administration form, the inhibitor and/or composition of the present invention can be mixed with suitable additives, such as excipients, and brought by means of the customary methods into the suitable administration forms, such as tablets, coated tablets, hard capsules, aqueous, alcoholic, or oily solutions. Examples of suitable excipients are inert carriers such as gum arabic, magnesia, magnesium carbonate, potassium phosphate, lactose, glucose, or starch, in particular, corn starch. In this case, the preparation can be carried out both as dry and as moist granules. Suitable oily excipients or solvents are vegetable or animal oils, such as sunflower oil or cod liver oil. Suitable solvents for aqueous or alcoholic solutions are water, ethanol, sugar solutions, or mixtures thereof. Polyethylene glycols and polypropylene glycols are also useful as further auxiliaries for other administration forms. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.

The oral administration of a inhibitor or a pharmaceutically acceptable salt or ester thereof and/or pharmaceutical composition according to the invention, is suitably accomplished by uniformly and intimately blending together a suitable amount of the steroid compound in the form of a powder, optionally also including a finely divided solid carrier, and encapsulating the blend in, for example, a hard gelatin capsule. The solid carrier can include one or more substances, which act as binders, lubricants, disintegrating agents, coloring agents, and the like. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.

Oral administration of a inhibitor or a pharmaceutically acceptable salt or ester thereof and/or pharmaceutical composition according to the invention can also be accomplished by preparing capsules or tablets containing the desired amount of the inhibitor, optionally blended with a solid carrier as described above. Compressed tablets containing the pharmaceutical composition of the invention can be prepared by uniformly and intimately mixing the active ingredient with a solid carrier such as described above to provide a mixture having the necessary compression properties, and then compacting the mixture in a suitable machine to the shape and size desired. Molded tablets maybe made by molding in a suitable machine, a mixture of powdered steroid compound moistened with an inert liquid diluent.

When administered by nasal aerosol or inhalation, an inhibitor or a pharmaceutically acceptable salt or ester thereof and/or pharmaceutical composition according to the invention may be prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. Suitable pharmaceutical formulations for administration in the form of aerosols or sprays are, for example, solutions, suspensions or emulsions of the compounds of the invention or their physiologically tolerable salts in a pharmaceutically acceptable solvent, such as ethanol or water, or a mixture of such solvents. If required, the formulation can also additionally contain other pharmaceutical auxiliaries such as surfactants, emulsifiers and stabilizers as well as a propellant.

For subcutaneous or intravenous administration, the active analogue, if desired with the substances customary therefor such as solubilizers, emulsifiers or further auxiliaries, are brought into solution, suspension, or emulsion. The inhibitors of the invention can also be lyophilized and the lyophilizates obtained used, for example, for the production of injection or infusion preparations. Suitable solvents are, for example, water, physiological saline solution or alcohols, e.g. ethanol, propanol, glycerol, in addition also sugar solutions such as glucose or mannitol solutions, or alternatively mixtures of the various solvents mentioned. The injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1 ,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.

When rectally administered in the form of suppositories, these formulations may be prepared by mixing the inhibitors according to the invention with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidify and/or dissolve in the rectal cavity to release the drug. Methods for modulating the signal transduction pathway mediated by receptors of the TLR and/or IL-1 R family

In a further embodiment, the invention relates to a method for modulating in an eukaryotic cell the signal transduction pathway mediated by a receptor of the TLR and/or IL-1 R family, and preferably by a Toll-like receptor 2 (TLR-2) and/or an interleukin-1 receptor (IL-1 R).

In particular, the invention provides a method for inhibiting in an eukaryotic cell a signaling pathway mediated by a receptor of the TLR and/or IL-1 R family, and preferably by a Toll-like receptor 2 (TLR-2) and/or an interleukin-1 receptor (IL-1 R) comprising administering to said cell in vitro an effective amount of : - an inhibitor that inhibits a conventional protein kinase C alpha isozyme and/or of

- an inhibitor that inhibits a conventional protein kinase C alpha isozyme and that inhibits a conventional protein kinase C gamma isozyme.

Preferably the method for inhibiting a signaling pathway mediated by a receptor of the TLR and/or IL-1 R family, and preferably by a Toll-like receptor 2 (TLR-2) and/or an Interleukin-1 receptor (IL-1 R) in an eukaryotic cell comprises administering to said cell in vitro an effective amount of an inhibitor represented by formula IA, IB, MA, MB and/or III as defined herein, and preferably selected from the group comprising bisindolylmaleimide I, bisindolylmaleimide II, bisindolylmaleimide III, bisindolylmaleimide IV, bisindolylmaleimide V, bisindolylmaleimide Vl, bisindolylmaleimide VII, bisindolylmaleimide VIII, bisindolylmaleimide IX, bisindolylmaleimide X, bisindolylmaleimide Xl hydrochloride salt, 2-[1-(3-Dimethylaminopropyl)-1 H-indol-3-yl]-3-(1 H- indol-3-yl)-maleimide, 2-[1 -[2-(1 -Methylpyrrolidino)ethyl]-1 H-indol-3-yl]-3-(1 H-indol-3- yl)maleimide, 2-[1-(3-Aminopropyl)-1 H-indol-3-yl]-3-(1 H-indol-3-yl)maleimide, 2,3-bis(1 H-lndol- 3-yl)maleimide, 12-(2-cynaoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo [2, 3-a] pyrrollo [3, 4-c] carbazole, 2-[1-(3-dimethylaminopropyl)-5-methoxyinol-3-yo]-3-(1 H-indol-3-yl) maleimide (Go 6983) and 2,3-bis(1 H-lndol-3-yl)-N-methylmaleimide, or from the group comprising 2,2',3,3',4,4^I-Hexahydroxy-1 ,1^l-biphenyl-6,6'-dimethanol dimethyl ether

In a further embodiment, the invention provides a method for inhibiting TLR- or IL-1 R-family mediated inflammation by blocking PKC isozymes with α activity, optionally in combination with PKC isozymes with gamma activity. The invention thus provides a method for inhibiting TLR and/or IL-1 R-mediated signaling, which comprises in vitro administration of an effective amount of an indicated type of an PKCα inhibitor and/or of a PKCα/γ inhibitor to eukaryotic cells such as mammalian immune cells or cells of epithelial origin, for instance from animal (mice, rat, etc..) or human origin. In particular, the invention provides a method for inhibiting TLR and/or IL-1 R- family-mediated signaling pathways by targeting p38 MAPK and c-jun N-terminal kinase without effecting nuclear-factor (NF)-kB activation. In another embodiment, the invention provides a method for inhibiting expression, in an eukaryotic cell, e.g. mammalian cells, preferably immune cells or cells of epithelial origin, for instance from animal (mice, rat, etc..) or human origin, of a gene whose transcription is regulated by a receptor of the TLR and/or IL-1 R family, and preferably by a Toll-like receptor 2 (TLR-2) and/or an interleukin-1 receptor (IL-1 R). The mehod involves administering to said cell:

- an inhibitor that inhibits a conventional protein kinase C alpha isozyme and/or

In a particularly preferred embodiment, the gene whose transcription is regulated by a receptor of the TLR and/or IL-1 R family is not a gene encoding NF-/cB. In a preferred embodiment, said gene is an inflammatory gene, preferably selected from the group comprising but not limited to genes encoding TNF-α, IL-12 (e.g. IL12p40, IL12p19), IL-6, IL-8, IL-18, IL-1.

In another preferred embodiment, said gene is regulated by a MAP kinase and even more preferably said gene is regulated by either a p38 MAP kinase and/or JNK kinases such as e.g. c-jun N-terminal kinase

In another preferred embodiment, said gene is a target or a potential target of AP1 transcription factor.

The above-given methods for modulating the signal transduction pathway mediated by receptors of the TLR and/or IL-1 R family comprises inhibiting the activity of a conventional protein kinase C isozyme alpha and/or inhibiting the activity of a conventional protein kinase C alpha isozyme and gamma isozyme. Inhibition can be effected by using suitable cPCK inhibitors as defined herein.

Methods of screening In yet another embodiment, the invention provides a highly-specific screening protocol to define the specific actions of cPKCα and/or cPKCα/γ inhibitors that are useful for treating inflammatory diseases, as defined herein in mammals. The method comprises the steps of: a) evaluating whether an inhibitor: a1) inhibits the activity of a conventional protein kinase C alpha isozyme or a2) inhibits the activity of a conventional protein kinase C alpha isozyme and the activity of a conventional protein kinase C gamma isozyme, b) evaluating whether the inhibitor inhibits expression of a gene whose transcription is regulated by a receptor of the TLR and/or IL-1 R family, and preferably by a Toll-like receptor 2 (TLR-2) and/or an interleukin-1 receptor (IL-1 R), c) evaluating whether the inhibitor influences the transcriptional activity (i.e. DNA-binding activity) of a gene encoding NF-KB, and d) selecting an inhibitor that d1 ) inhibits the activity of a conventional protein kinase C alpha isozyme and/or that inhibits the activity of a conventional protein kinase C alpha isozyme and gamma isozyme, d2) that inhibits expression of a gene as defined under step b), and d3) that does not effect the transcriptional activity (i.e. DNA-binding activity) of a gene as defined under step c). The present method can be applied in vitro by using suitable eukaryotic cells, e.g. mammalian cells, preferably immune cells or cells of epithelial origin. In a preferred embodiment, the gene whose transcription is regulated by a receptor of the TLR and/or IL-1 R family, and preferably by a Toll-like receptor 2 (TLR-2) and/or an interleukin-1 receptor (IL-1 R), is an inflammatory gene, preferably selected from the group comprising but not limited to genes encoding TNF-α, IL-12 (e.g. IL12p40, IL12p19), IL-6, IL-8, IL-18, IL-1. In another preferred embodiment, the gene whose transcription is regulated by a receptor of the TLR and/or IL-1 R family, and preferably by a Toll-like receptor 2 (TLR-2) and/or an interleukin-1 receptor (IL-1 R), is regulated by a gene encoding a MAP kinase and even more preferably by a gene encoding a p38 MAP kinase and/or a gene encoding a JNK kinase such as e.g. c-jun N-terminal MAP kinase. In yet another embodiment, the gene whose transcription is regulated by a receptor of the TLR and/or IL-1 R family, and preferably by a Toll-like receptor 2 (TLR-2) and/or an interleukin-1 receptor (IL-1 R), is a gene whose transcription is regulated by AP1.

Inhibitors, as determined by methods as disclosed herein, that exhibit cPKC inhibiting activity selectively for alpha and/or alpha+gamme isozyme, an ability to inhibit p38 and/or JNK kinase activity, an ability to inhibit TNF-α, IL-12 (e.g. IL12p40, IL12p19), IL-6, IL-8, IL-18, IL-1 activity, and/or that does not have any effect on the activation of NF-/cB and its DNA-binding activity are particularly suitable for being used as a medicament or for the preparation of medicaments of the treatment of inflammatory diseases, in particular inflammatory diseases that are mediated by a Toll-like receptor (TLR) and/or an interleukin-1 receptor (IL-1 R).

Examples

The following examples are meant to illustrate the invention. In the examples following methods were applied. MATERIAL AND METHODS

Generation of monocyte-derived DC

DCs were generated from peripheral blood mononuclear cells (PBMCs) of healthy human subjects as described by Romani et al. (Romani et al.). After six days, non-adherent cells that corresponds to the DC-enriched fraction, routinely contained more than 95% of DC as assessed by morphologic and fluorescence-activated cell-sorter (FACS) analysis was described earlier (Goriely et al.).

Mice and generation of bone-marrow-derived DC

PKCα knockout (-/-) mice were obtained from M. Leitges, Max-Planck Institute for Experimental Immunology (Hannover, Germany) and were generated as described previously (Letiges, M molecular endocrinology 2002). Wild-type 129/SV mice were purchased from Harlan. Bone marrow (BM)-DCs were generated from the BMs of wild-type or PKCa^''^" 129/SV mice as described previously (Lutz et al.). Cells were cultured in RPM1 1640 medium supplemented with

10% FCS and GM-CSF. The DCs obtained on day 14 of culture, routinely contained 95% of DC as assessed by FACS analysis.

Stable cell lines and reagents

Human embryonic kidney (HEK) 293 parental cells and cells stably expressing GFP-TLR2 and GFP-TLR4 (TLR3-293) were kindly provided by D. Golenbock (see MTA). The AP1 reporter construct was purchased from Stratagene. Gό6976 and 2,2',3,3',4,4'-Hexahydroxy-1-1'- biphenyl-6-6'-dimethanol-dimethyl ether (HBDDE) were from Biomol (Boechout, Belgium). The TLR-2 ligands FSL-1 and Pam3CysK4 were purchased from EMC microcollections (Tuebingen, Germany).

Determination of cytokine levels

DCs or HEK 293 cells (5 x 10⁵/ml) were stimulated with FSL-1 (0.5μg/ml), or Pam3CysK4 (0.5μg/ml) for 20 h followed by quantification of cytokines in culture supernatants. Quantification of human IL-12p40 and TNF-α were performed using antibodies from Biosource and for mouse from R&D Systems.

RNA purification and quantitative RT-PCR

Total cellular mRNA from DC (1 x 10⁶/ml) was extracted using a MagnaPure LC mRNA isolation Kit (Roche) according to the manufacturer's specification. Reverse transcription and quantitative RT-PCR were performed using Light-Cycler-RNA Master Hybridization probes (one-step procedure) on a Lightcycler^® apparatus (Roche). The conditions were previously described (Goriely et al.). lmmunoblotting

DC (1 x 10⁵/ml) were collected and directly lysed in Laemmli buffer. Equal volumes of whole cell lysate from each condition were resolved by 8% SDS-polyacrylamide gel electrophoresis (PAGE), and analyzed by western blotting, lmmunoblots were probed with polyclonal antibodies directed against phospho-JNK1/2, phospho-p38, phospho-c-Jun all obtained from Cell Signalling Technologies (Bioke, The Netherlands). Equal loading was verified either by anti- JNK1 , -p38 or anti-c-Jun antibodies all purchased from Santa Cruz. The immunoreactive bands were revealed using the ECL^® detection kit (Amersham Biotechnologies).

Determination ofAP-1 and NF-κB DNA binding activities Nuclear extracts were prepared as described earlier (Osborn et al.). AP-1 or NF-/cB DNA- binding activities in nuclear extracts were measured by Trans-AM c-Jun or p65 transcription factor assay kits (Active Motif Europe, Rixensart, Belgium) according to the manufacturer's protocols. Briefly, 5 μg of each nuclear extract was incubated in plates-coated with consensus AP-1 and NF-/cB oligonucleotides, respectively. Plates were washed and anti-c-Jun or p65 antibodies were added to the wells. Antibody binding was detected with a secondary HRP- conjugated antibody and developed with TMB substrate. The intensity of the reactions was measured at 450nm.

Innate inflammatory response to endotoxin challenge in vivo.

For lethality tests, use is made of PKC alpha knockout mice and wild-type (wt) counterparts, which receive various doses of salmonella LPS (0.5-1000 μg) and the resulting lethality can be observed until 48h after challenge as previously described.

Example 1: Conventional PKC inhibition abolishes TLR2- or IL-1-β-mediated inflammatory cytokine gene expression. Fig. 1a-c illustrate that inhibition of PKC αlγ or only PKC-α activity by conventional PKC inhibitor Gό6976 or PKC-α-specific inhibitor HBDDE abolishes inflammatory cytokine expression mediated by TLR2/6 and TLR2/1 ligands in monnocyte-derived dendritic cells (DCs). These figures illustrate that inhibition of PKC-α or PKC-α// activity abolishes FSL-1 -mediated inflammatory cytokine production. Immature mo-DCs were incubated in vehicle (DMSO) or indicated concentrations of G06976 for 1 h then were either left in medium or activated by FSL- 1 (a) or PAM₃CSK₄ (b). IL-12p40 (upper) or TNF-α (lower) concentrations in culture supernatants were analyzed by ELISA. (c) Comparison of the effects of PKCa inhibitor HBDDE to Gό6976 on FSL-1 -mediated IL-12p40 (upper) or TNF-α (lower) production in DCs. Data represent means ± SEM of 3-5 independent experiments from different blood donors. Fig. 1d-e illustrate the effects of conventional PKC inhibitor Go 6976 on IL-12p40 and IL-12p19 mRNA transcription mediated by TLR2 ligands in human mo-DCs. These figures illustrate that conventional PKC inhibitor, Go 6976 represses IL-12p40 and IL-12p19 mRNA transcription, (d) IL-12p40 (upper) and IL-12p19 (lower) mRNA accumulation at indicated time intervals following FSL-1 (0.5 μg/ml) stimulation was quantified by quantitative RT-PCR. The mRNA levels were normalized to β-actin mRNA levels and depicted as fold index compared to unstimulated samples. Data represent of one independent experiment out of 3 performed from different donors, (e) Quantification of IL-12p40 mRNA accumulation at 6 h after FSL-1 stimulation was quantified by quantitative RT-PCR. Data represent means ± SEM of 5-6 independent experiments performed from different donors.

Fig. 1f illustrates that inhibition of PKC-α diminishes IL-1-but not TNF-α-mediated inflammatory cytokine expression in human mo-DCs. Inhibition of PKCα or PKCα/κ-activity abolishes IL-1yff- mediated inflammatory cytokine production. Immature mo-DCs were incubated in vehicle (DMSO) or HBDDE (40 μM) or indicated concentrations of G56976 for 1 h then were either left in medium or activated by IL-1/9 (upper) or TNF-oc (lower). IL-12p40 concentration in culture supematants were analyzed by ELISA. Data represent means ± SEM of 3-5 independent experiments from different blood donors.

Example 2: Bone marrow-derived DCs (BMDCs) from PKCα deficient mice or treatment of Go 6976 results in the inhibition of inflammatory cytokine production in response to TLR2 engagement.

Fig. 2 illustrates that PKCα deficient BMDCs or wild type (WT) BMDCs treatment with conventional PKC inhibitor Go 6976 display defective responses following TLR2 activation. BMDCs from WT or PKCα deficient mice were either incubated in vehicle (DMSO) or Go 6976 for 1 h and then stimulated with PaITi₃CSK₄ (0.5 μg/ml). After 20 h, IL-12p40 (upper) and TNF-α (lower) concentrations in culture supematants were analyzed by ELISA. Data represent means ± SEM of 5 independent experiments.

Example 3: Pharmacological inhibitors of conventional PKC isoforms selectively target and inhibit JNK1/2 and p38 MAPK pathways leading to depression of AP-I transcriptional activity.

FIG. 3a-c illustrates that Conventional PKC inhibitor Go 6976 abolishes TLR-2-mediated p38 MAPK and JNK1/2 activation and results in the inhibition of AP-1 DNA binding activity, (a) Immature mo-DCs were incubated with vehicle (DMSO) or Go 6976 (1 μM) for 1 h prior to activation by FSL-1 (0.5 μg/ml). At indicated time intervals, cells were harvested, lysed and the protein extracts were analyzed by direct western blotting using an antibodies directed against phospho-JNK1/2 (upper) or phospho-p38 (lower). Protein loading was controlled by probing with an anti-pJNK1 (upper) or anti-p38 (lower) antibody. One representative out of 3-4 independent experiments is shown, (b) Immature DCs were treated with vehicle (DMSO) or G56976 (1 μM) for 1 h and then were activated by FSL-1 (0.5 μg/ml) (upper) or Pam3CSK4 (0.5 μg/ml) (lower). Cells were harvested after 2 h following cellular activation, nuclear extracts were prepared and analyzed for c-jun DNA-binding activity using the TransAM transcription factor assay kits. Data are means ± SEM of 5-6 independent experiments, (c) Immature DCs were treated with vehicle (DMSO), Go 6976 (1 μM) or HBDDE (40 μM) for 1 h and then activated as in (a). Protein extracts were analyzed by direct western blotting using an anti-phospho MKK4 antibody. Protein loading was controlled using MKK4 antibody. One representative out of 3 independent experiments is shown.

Example 4: Pharmacological inhibitors of conventional PKC isoforms do not target NF-κB pathway activation in response to TLR2 stimulation.

Fig. 4 illustrates that pharmacological inhibitors of conventional PKC isoforms do not target NF- KB pathway activation in response to TLR2 stimulation. In Fig. 4a immature mo-DCs were incubated with vehicle (DMSO) or Go 6976 (1 μM) for 1 h prior to activation by FSL-1 (0.5 μg/ml). At indicated time intervals, cells were harvested, lysed and the protein extracts were analyzed by direct western blotting using an antibodies directed against phospho-IKK-α/β (upper) or kB-α (lower). Protein loading was controlled by probing with an anti-IKK-b (upper) or anti-Gapdh (lower) antibody. One representative out of 3 independent experiments is shown. In Fig. 4b immature DCs were treated with vehicle (DMSO) or Gό6976 (1 μM) for 1 h and then were activated by FSL-1 (0.5 μg/ml) (left) or PaIT)₃CSK₄ (0.5 μg/ml) (right). Cells were harvested after 2 h following cellular activation; nuclear extracts were prepared and analyzed for p65/Rel- A DNA-binding activity using the TransAM transcription factor assay kits. Data are means ± SEM of 5-6 independent experiments.

Example 5: Dominant negative (DN) PKCα overexpression inhibits AP1 reporter activity in HEK 293T cells stably expressing TLR2.

In the experiment illustrated in Fig. 5, TLR2 stably expressing HEK 293 cells were transiently co-transfected with an AP-1 reporter plasmid (1 μg/ml) or with an empty vector (vehicle, 160 ng). The following day, cells were stimulated by FSL-1 (0.5 μg/ml) or left un-stimulated. After 18 h, whole cell lysate were harvested and luciferase reporter gene activity was measured and normalized using Renilla luciferase activities.

In addition, physical interactions of PKCα with the molecular components of MAPK pathway downstream of TLR2 and IL-1 R signaling are studied. Also studied are i) impact of inactivating PKCα or PKCα/κ activity or loss of PKCα expression on TLR-2-ligand mediated shock in vivo; and ii) impact of inactivating PKCα or PKCα/κ or loss of PKCα expression on TLR-2-mediated inflammation in vivo.

In summary, the present invention is directed to the specific inhibition of conventional PKC having alpha (α) or α/γ activity and thus to selectively target the inflammatory MAPK pathways involving p38 and c-jun N terminal kinases (JNKs) in response to TLR-IL-1 R signaling which abolishes the inflammatory cytokine network. The present methods, compositions, and kits are highly selective to inflammatory signaling pathways which use MyD88 adapter molecule in mammals.

References

Akira.S. and K.Takeda. 2004. Toll-like receptor signalling. Nat.Rev. Immunol. 4:499-511.

Janeway.C.A., Jr. and R.Medzhitov. 2002. Innate immune recognition. Annu.Rev.lmmυnol. 20:197-216.

Romani.N., D.Reider, M.Heuer, S.Ebner, E.Kampgen, B.Eibl, D.Niederwieser, and G.Schuler.

1996. Generation of mature dendritic cells from human blood. An improved method with special regard to clinical applicability. J. Immunol. Methods 196:137-151.

Goriely.S., B.Vincart, P.Stordeur, J.Vekemans, F.Willems, M.Goldman, and D.De Wit. 2001. Deficient IL-12(p35) gene expression by dendritic cells derived from neonatal monocytes.

J.Immunol. 166:2141-2146.

Osbom.L, S.Kunkel, and G.J.Nabel. 1989. Tumor necrosis factor alpha and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor kappa

B. Proc.Natl.Acad.Sci.U.S.A 86:2336-2340. Lutz , M. B., Kukutsch, N., Ogilvie, A. L., Rossner, S., Koch, F., Romani, N., and Schuler, G.

(1999) J.lmmunol.Methods 223, 77-92.

Claims

1. Use as a medicament of: an inhibitor that inhibits a conventional protein kinase C alpha isozyme and/or of an inhibitor that inhibits a conventional protein kinase C alpha isozyme and that inhibits a conventional protein kinase C gamma isozyme.

2. Use of an inhibitor that inhibits a conventional protein kinase C alpha isozyme and/or of an inhibitor that inhibits a conventional protein kinase C alpha isozyme and that inhibits a conventional protein kinase C gamma isozyme for the preparation of a medicament for treating inflammatory diseases.

3. Use according to claim 2, wherein said inflammatory disease is an inflammatory disease that is mediated by receptors of the TLR and/or IL-1 R family, and preferably by a Toll-like receptor 2 (TLR-2) and/or an interleukin-1 receptor (IL-1 R).

4. Use according to claim 2 or 3, wherein said inflammatory disease is selected from the group comprising sepsis, myocarditis, arthritis, and chronic inflammatory bowel diseases.

5. Use according to any of claims 1 to 4, wherein the inhibitor that inhibits a conventional protein kinase C alpha isozyme is represented by formula IA or formula IB or a pharmaceutically acceptable salt thereof,

Formula IA Formula IB wherein R¹ is independently hydrogen or selected from the group comprising halogen, hydroxyl, alkyl, alkoxy, -NHCO(alkyl), and -NR⁸R⁹, wherein each group may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl; wherein R² and R³ are each independently hydrogen or oxo (=0); wherein R⁴ and R⁵ are each independently hydrogen or selected from the group comprising alkyl, aryl, cyanoalkyl, thioalkyl, alkylthioalkyl, aminoalkyl, alkylaminoalkyl, heteroaryl, heterocyclyl, cycloalkyl, heterocyclylalkyl, and aminoiminothioalkyl, wherein each group may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl; wherein R⁶ and R⁷ are each independently hydrogen or selected from the group comprising halogen, hydroxyl, alkyl, alkoxy, -NHCO(alkyl), and -NR⁸R⁹, benzyloxy, hydroxy, and aminoalkoxy, wherein each group may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl; wherein R⁸ and R⁹ are independently hydrogen or selected from the group comprising alkyl, alkanoyl or halo(alkanoyl) or wherein R⁸ and R⁹ taken together with the N atom to which they are bound form a 5 or 6-membered ring; wherein each group may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl; wherein R¹⁰ and R¹¹ are each independently hydrogen or selected from the group comprising alkyl, aryl, cyanoalkyl, thioalkyl, alkylthioalkyl, aminoalkyl, alkylaminoalkyl, heteroaryl, heterocyclyl, cycloalkyl, heterocyclylalkyl, and aminoiminothioalkyl, wherein each group may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl; or wherein R⁴ and R¹⁰ together form a heterocyclyl or a heteroaryl ring, wherein said ring may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl; and/or wherein R⁵ and R¹¹ together form a heterocyclyl or a heteroaryl ring wherein said ring may be optionally substituted with one or more substituents selected from the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazino, cyano, alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl.

6. Use according to any of claims 1 to 4, wherein the inhibitor that inhibits a conventional protein kinase C alpha isozyme and that inhibits a conventional protein kinase C gamma isozyme is represented by formula IA or IB as defined in claim 5.

7. Use according to any of claims 1 to 4, wherein the inhibitor that inhibits a conventional protein kinase C alpha isozyme is represented by formula III or a pharmaceutically acceptable salt thereof,

Formula III wherein R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, and R²¹ are each independently hydrogen or selected from the group comprising alkyl, aryl, het, nitro, amino, cyano, halogen, formyl, hydroxyl, haloalkyl, hydroxyalkyl, cyanoalkyl, alkoxy, oxyalkyl, alkyloxyalkyl, carboxy, alkylcarboxy, alkylcarbonyl, arylcarbonyl, alkylthio, alkylsulfonyl, alkylsulfoxide, alkylamino, arylamino, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, aryloxy, arylamino, arylalkyloxy, aryloxyalkyl, arylalkylamino, arylcarboxy, arylalkylcarboxy, arylaminocarbonyl, alkylcarbonylamino, alkylcarbonylalkylamino, arylcarbonylamino, arylcarbonylalkylamino, alkylsulfonylamino, arylsulfonylamino, alkylsulfonylalkylamino, and arylsulfonylalkylamino, wherein each group may be optionally substituted with one or more substituents selected the group comprising halogen, haloalkyl, hydroxyl, carbonyl, halocarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, nitro, amino, formyl, imines, azido, hydrazine cyano, alkyl, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkyloxy, thiol, alkylthio, thioalkyl, carboxyl, alkylcarboxy, acylamino, alkyl esters, carbamates, thioamides, urea, sulphonamides, alkylsulfonyl, alkylsulfoxide, and aminosulfonyl.

8. Use according to any of claims 1 to 4, wherein the inhibitor that inhibits a conventional protein kinase C alpha isozyme and that inhibits a conventional protein kinase C gamma isozyme is represented by formula III as defined in claim 7.

9. Pharmaceutical composition for treating inflammatory diseases comprising a therapeutically effective amount of: - an inhibitor that inhibits a conventional protein kinase C alpha isozyme and/or

- an inhibitor that inhibits a conventional protein kinase C alpha isozyme and that inhibits a conventional protein kinase C gamma isozyme, and further comprising a pharmaceutically acceptable excipient.

10. Composition according to claim 9, wherein said inhibitor is as defined in any of claims 5 to 8.

11. Composition according to claim 9 or 10, wherein said inflammatory diseases are as defined in claim 3 or 4.

12. Method of inhibiting a signaling pathway mediated by receptors of the TLR and/or IL-1 R family, and preferably by a Toll-like receptor 2 (TLR-2) and/or an interleukin-1 receptor (IL-

1 R) in an eukaryotic cell comprising administering to said cell in vitro an effective amount of

13. Method according to claim 12, wherein said inhibitor is as defined in any of claims 5 to 8.

14. Method for inhibiting expression, in a eukaryotic cell, of a gene whose transcription is regulated by receptors of the TLR and/or IL-1 R family, and preferably by a Toll-like receptor

2 (TLR-2) and/or an interleukin-1 receptor (IL-1 R), the method comprising administering to said cell:

- of an inhibitor that inhibits a conventional protein kinase C alpha isozyme and that inhibits a conventional protein kinase C gamma isozyme.

15. Method according to any of claims 12 to 14, wherein said method is carried out on mammalian cells, preferably immune cells or cells of epithelial origin.