HK1179162A - Method for treatment of inflammatory disease and disorder - Google Patents

Description

Methods for treating inflammatory diseases and disorders

Background of the disclosure

Technical Field

The present disclosure relates generally to methods of treating diseases and more particularly to the treatment of inflammatory diseases and conditions.

Background

The initiation of inflammation begins with an inflammatory response and results in the activation of neutrophils, granulocytes, monocytes, macrophages and other immune cells. This can lead to local or systemic inflammatory cascades involving inflammatory cytokines and mediators (e.g., interleukins, TNF α, and prostaglandins). This complex inflammatory-mediated cascade triggers an overall range of responses such as cell chemotaxis and endothelial injury and results in the recruitment of additional cells from the natural and acquired immune system.

The skin acts as an important boundary between the body and the environment, preventing contact with potentially harmful pathogens. In the case of antigen/pathogen invasion, an inflammatory response is often induced to remove the antigen. This response results in dermal infiltration consisting primarily of T cells, polymorphonuclear cells and macrophages.

The inflammatory response is not necessarily associated with external stimuli or may be caused by harmless environmental substances (if allergy occurs). In both cases, the over-expression of inflammatory cytokines, which is not properly controlled, leads to inflammation, which is often a hallmark of local or systemic inflammation, as well as various inflammatory diseases and disorders. Inflammation is associated with a variety of conditions such as eczema and dermatitis, including for example atopic dermatitis, seborrheic dermatitis, dyshidrotic eczema, nummular eczema, stasis dermatitis, allergic dermatitis, psoriasis, pruritus, multiple sclerosis, skin inflammation, cicatricial pemphigoid, scleroderma, hidradenitis suppurativa, toxic epidermal necrolysis, acne, osteomyelitis, Graft Versus Host Disease (GVHD), pyoderma gangrenosum (pyroproderma gangrenosum) and behcet's syndrome.

Not surprisingly, overproduction of proinflammatory cytokines has been implicated in many inflammatory and autoimmune diseases. For example, the secretion of cytokines such as TNF α and Interleukin (IL) -23 that promote the survival and proliferation of Th17 cells is highly correlated with psoriasis, while IL-6 is required for development of Th17 in addition to its general role as an inflammatory cytokine. Other cytokines like IL-12 and IP-10 are initiating agents and are involved in the Th1 pathway representative of psoriasis and other autoimmune diseases. Interleukin 5(IL-5), a cytokine that increases eosinophil production, is overexpressed in asthma, leading to the accumulation of eosinophils in the bronchial mucosa of asthma, a hallmark of allergic inflammation. Interleukin 4(IL-4) and interleukin 13(IL-13) are known mediators of smooth muscle over-contraction present in inflammatory bowel disease and asthma. In addition, as discussed further below, inflammatory cytokines have been shown, by way of example, to be associated with psoriasis, multiple sclerosis, arthritis, ischemia, septic shock, and organ transplant rejection.

Similarly, granulocyte macrophage colony-stimulating factor (GM-CSF) is a regulator of maturation of the granulocyte and macrophage lineage population and has been implicated as a key factor in many inflammatory and autoimmune diseases. For example, antibodies that inhibit secretion of GM-CSF have been shown to ameliorate autoimmune diseases.

Thus, as discussed herein, the development of therapies that reduce the secretion of proinflammatory cytokines and modulate immune modulators is generally beneficial in mitigating local and systemic inflammation as well as in many inflammatory and/or autoimmune diseases. Various lines of evidence indicate that modulators of the PKC isoforms are useful in achieving these results.

Several in vivo studies, with skin-associated cells (e.g., keratinocytes, dendritic cells and T helper cells) as key players in the development of inflammatory responses involved in the pathogenesis of psoriasis and other autoimmune inflammatory diseases, have shown involvement of T helper (Th)17 cells and secretion of cytokines such as interleukins and TNF α. As used herein, in vivo ("in vivo" latin) is an experiment using whole, living organisms as opposed to partial or dead organisms or in vitro ("in glass" e.g. in test tubes or petri dishes) controlled environments. Secretion of cytokines that promote Th17 cell survival and proliferation (e.g., TNF α and Interleukin (IL) -23) also function as key major cytokine modulators of these diseases (Fitch et al (2007) Curr Rheumatol rep.9: 461-7). In turn, Th17 cells in the dermis decrease IL-17A and IL-22 secretion. IL-22, in particular, causes keratinocyte hyperproliferation and increases inflammatory response in psoriasis (Fitch et al (2007) Currrheumatol Rep 9: 461-7).

The protein kinase c (pkc) family represents a group of phospholipid-dependent enzymes that catalyze the covalent transfer of phosphate from ATP to serine and threonine residues of proteins. This family is currently believed to consist of at least 12 individual isoforms belonging to 3 distinct classes based on their activation via calcium ions and other factors. The PKC family consists of at least ten members, usually divided into three subgroups: traditional, new and atypical PKCs (fig. 1). Specific cofactor requirements, tissue distribution and cellular compartmentalization suggest distinct functions of each isoform and the tuning of specific signaling. Thus, specific stimuli can lead to different responses through isoform-specific PKC signaling regulated by their factors, such as: expression, localization and/or phosphorylation status in a particular biological context. PKC isoforms are activated by various extracellular signals and, in turn, modulate the activity of cellular proteins, including receptors, enzymes, cytoskeletal proteins, and transcription factors. Thus, the PKC family plays a central role in cell signaling (including regulation of cell proliferation, differentiation, survival, and death).

The highly abundant PKC α in skin is the major conventional Ca2+ -responsive PKC isoform in the epidermis and only cPKCK was originally detected in keratinocytes in vitro and in vivo (Dlugosz et al (1992) Biomed Pharmacother 46: 304; Wang et al (1993) J Cancer Res Clin Oncol 119: 279-287). Thus, PKC α has been proposed as a key player in Ca2+ -induced differentiation (Denning et al (1995) Cell Growth Differ 6: 149-157; Dlugosz et al (1992) Biomed Pharmacother 46: 304). Present in the epidermis and confined mainly to the basal lamina (Denning et al (2004) Int J Biochem Cell Biol 36: 1141-1146), PKC α is involved in the arrest of the Cell cycle and is mainly associated with the Cell-Cell junctions of the keratin cytoskeleton and desmosome (Jansen et al (2001) IntJ Cancer 93: 635-643; Tibudan et al (2002) J Invest Dermatol.119: 1282-1289). Thus, the labeling of spinous processes is inhibited when exposed to the traditional PKC activator TPA (12-O-tetradecanoyl phorbol-13-acetate), PKC α is thought to largely result in the transformation of differentiation from spinous processes to granular as a result of TPA activation (Dlugosz and Yuspa (1993) J Cell Biol 120: 217-225; Lee et al (1998) J Invest Dermatol 111: 762 766; Matsui et al (1992) J Invest Dermatol 99: 565-571; Punnonen et al (1993) J Invest Dermatol 101: 719-726). Indeed, blocking PKC α activity by antisense oligonucleotides or their synthesis appears to eliminate the granular marker and restore spinous process markers like K1 and K10. Similarly, the achievement of dominant negative PKC α appears to restore (late) the spinous process marker involucrin (Deucher et al (2002) J Bio1 Chem 277: 17032-17040). Thus, incomplete differentiation in skin Cancer (Tennenbaum et al (1993) Cancer Res 3: 4803-4810; Tomakidi et al (2003) J Pathol 200: 298-307) is associated with increased PKC α activity, as also observed in vitro tumor cells (Dlugosz et al (1992) Biomed Phaother 46: 304; Yang et al (2003) J Cell physiology 195: 249-259). However, overexpression of PKC α in normal human keratinocytes did not appear to alter their differentiation pattern (Deucher et al (2002) J Biol Chem 277: 17032-17040). The effect of PKC α on cell trafficking and membrane recruitment of β 1 integrins during migration (Ng et al (1999) EMBO J18: 3909-.

Overexpression of PKC α in transgenic mice has been shown to reduce significant inflammatory responses, increase epidermal thickening and edema associated with neutrophil infiltration, mini-multiple abscesses, and significant increases in inflammatory cytokines and chemokines (e.g., TNF α, MIP-2, COX-2, or Macrophage Inflammatory Protein (MIP)). These results implicate PKC α in epidermal inflammatory responses (Wang and Smart (1999) J Cell Sci 112: 3497-. Treatment with TPA, a PKC α activator, appears to cause epidermal hyperplasia, inflammation of the inner epidermis and massive apoptosis (Cataisson et al (2003) J Immunol 171: 2703-. Furthermore, recent in vivo studies in PKC isozyme selective knockouts and transgenic mice show that individual PKCs have distinct functions in the immune system. These gene analyses, together with biochemical studies, appear to suggest that PKC-regulated signaling pathways play an important role in many aspects of immune response. For example, members of the PKC family appear to be critical in T cell signaling pathways. In particular, PKC α, isoform appears to determine a characteristic property of lymphocytes in vivo effectors. PKC α has also been discussed as being involved in macrophage activation and appears to be involved in mast cell signaling (Cataisson et al (2005) J Immunol 174: 1686-1692). Thus, PKC isoforms are potent drug targets in acquired immunity.

An example of an inflammatory disease is psoriasis. There are two main hypotheses about the basic pathology leading to the development of psoriasis. The first is that psoriasis is primarily a condition of overgrowth and regeneration of skin cells. The second hypothesis considers psoriasis as an immune-mediated disorder in which excessive regeneration of skin cells is secondary to factors produced by the immune system. Thus, most drugs of psoriasis target one element of the disease, the hyperproliferative state of skin cells, or the inflammatory response of the skin present in psoriatic plaques.

Recent data supports the idea that both pathways underlie the pathology of a disease through interactions between skin cells and the immune environment (surrounding, location, and/or background). Traditional genomic linkage analysis has identified nine loci (loci)) named psoriasis susceptibility 1 to 9(PSORS 1 to PSORS 9) loci on different chromosomes associated with a trend to develop psoriasis. In these loci, several genes are characterized and found to encode proteins expressed in epidermal cells, such as corneal-linked proteins, which are expressed in the granular and keratinized layers of the epidermis and are upregulated in psoriasis. In other aspects, other psoriasis-associated genes encode proteins involved in the regulation of the immune system, wherein, for example, IL12B on chromosome 5q expressing interleukin-12B is characterized (Frank et al (2009) NEngl J Med 361: 496-.

Another example of an inflammatory disease is Multiple Sclerosis (MS). MS is a chronic and unpredictable inflammatory disease of the CNS that can affect the brain and spinal cord, which commonly affects young adults (Hafler et al (2005) Immunol Rev 204: 208-31). It is currently considered to be the most common neurological disorder in young adults, and usually attacks between the ages of 20 and 40, with a tendency to appear in women with almost twice the likelihood compared to men.

In MS, myelin, the material that surrounds and protects nerve cells, and/or its ability to be used for production is compromised, which is called "demyelination". This damage has the effect of slowing or blocking the information between the brain and body, resulting in symptoms observed with MS. Demyelination and scarring or other lesions in diffuse areas of the brain and/or spinal cord are considered to be characteristic of the disease (Beeton et al (2007) Journal of visualized experiments 594-604). These lesions appear to alter neurotransmission in the CNS and induce disabling neurological deficits that vary with the location of the demyelinated plaque (Beeton et al (2007) Journal of visualized Experiments 594-. Its clinical signs and symptoms are variable and depend on the part of the CNS that it affects, and may include motor, sensory, autonomic, and cognitive disorders (Noseworthy et al (2000) N Engl J Med 343: 938-52).

Some common symptoms of MS include: 1) irresistible feelings of fatigue; 2) balance-walking and coordination difficulties; 3) visual problems-double vision and blindness; 4) numbness and tingling in the hands and feet; 5) pain-both mild and severe; loss of muscle strength; 6) muscle stiffness and spasm; 7) mood swings-depression and anxiety; 8) dysmnesia and uneasiness of concentration; language barrier (The National MS Society Web Site).

Progressive disability is the fate of most patients with MS, especially when 25 years of vision is involved. Half of the MS patients need crutches to walk within 15 years of disease onset. MS is the leading cause of neurological dysfunction in young and middle-aged adults, and until the last decade, there has been no known beneficial treatment. MS is difficult to diagnose because of non-specific clinical manifestations, which has led to the development of highly structured diagnostic criteria, including several technological advances consisting of MRI scans, evoked potentials, and cerebrospinal fluid (CSF) studies. Diagnostic criteria generally rely on scattered lesions (scattered lesions) in the central white matter occurring at different times and without general principles explained by other etiologies such as infection, vascular disorders or autoimmune disorders.

MS is widely recognized as an autoimmune disease whereby unknown agent or agents trigger T cell-mediated inflammatory attacks leading to demyelination of CNS tissue (Weiner et al (2004) Arch Neurol 61: 1613-1615). Evidence that the autoimmune response targets myelin is strong but not conclusive. Primary oligodendrocyte apoptosis, which is accompanied by microglial activation, is described, for example, in early multiple sclerosis lesions in the absence of lymphocyte or myelin phagocytosis (Manuel et al (2006) Brain).

MS is typically reported as having four disease modes: relapsing-remitting ms (rrms), primary progressive ms (ppms), Progressive Relapsing Multiple Sclerosis (PRMS), and secondary progressive ms (spms). It is estimated that 50% of patients with RRMS will develop SPMS within 10 years, while up to 90% of RRMS patients will eventually develop SPMS. Each pattern of disease may be characterized as mild, moderate or severe. Persons with RRMS exhibit established episodes of worsening neurological function. These episodes are followed by a partial or complete recovery period (remission) during which no disease progression occurs, (approximately 85% of people are initially diagnosed with RRMS). PPMS is characterized by a slowly worsening neurological function from the onset, with no apparent relapse or remission (approximately 10% of people are diagnosed as PPMS). In SPMS, after the initial phase of RRMS, many people develop secondary progressive disease processes in which the disease worsens more steadily (about 50% of people with RRMS develop this form of disease within 10 years). In PRMS, one experiences stable worsening disease symptoms from The beginning, but with a clear onset of worsening neurological function in The process, while The disease appears to progress without remission (5%) (The National MS Society web site).

Although several treatments are available that attempt to reduce disease activity and disease progression, there is currently no cure for MS. Six of the four classes of drugs are approved in the united states for the treatment of MS. FDA approved disease treatments include the following: interferons, IIFN-beta-1 a (And) And IIFN-beta-1 bGlatiramer acetate(a polypeptide), natalizumabAnd mitoxantrone(a cytotoxic agent). Other drugs have been used with varying degrees of success, including glucocorticoids, methotrexate, cyclophosphamide, azathioprine, Intravenous (IV) immunoglobulins. The benefits of the currently approved therapies are relative mitigation of relapse rates and prevention of disability in MS.

(interferon beta 1a) is a drug manufactured by biotechnological processes, which produce the same interferon beta as present in the human body.Three subcutaneous administrations per week (from FDA approved) are reportedPrescription information of).

(interferon beta 1a) is a drug manufactured by biotechnological processes, which produce the same interferon beta as present in the human body.Weekly intramuscular injections are reportedOne shot (from FDA approved)Prescription information of).

(interferon beta 1b) is a drug manufactured by biotechnological processes, which complement the same interferon beta present in the human body.Subcutaneous injections (from FDA approved) are reported every other dayPrescription information of).

(glatiramer acetate) is a synthetic protein that mimics myelin basic protein. Although the mechanism is not fully understood, this drug appears to prevent myelin from damaging T cells by acting as a myelin bait.Once daily subcutaneous injections (from FDA approved) are reportedPrescription information of).

(natalizumab) is a laboratory-produced monoclonal antibody. It is designed to block potentially harmful immune cells from the blood stream passing through itThe "blood brain barrier" enters the locomotion of the brain and spinal cord.Administration by intravenous infusion once every four weeks is reported (from FDA approvedPrescription order information).

(mitoxantrone) belongs to the general group of drugs known as antineoplastic agents. It has been used to treat certain forms of cancer. It has been reported to play a role in MS therapy by inhibiting the activity of T cells, B cells and macrophages, which are presumed to cause attacks on myelin. (from FDA approvalPrescription information of).

Existing treatments for combined inflammatory diseases often fail to provide a multi-element approach that targets multiple elements of the pathogenesis. For example, many treatments for autoimmune diseases involve targeting a single element of the disease, either by preventing cell proliferation or by suppressing the immune response to prevent inflammation. Thus, there is a strong need to provide effective treatments that target multiple elements of the pathogenesis of inflammatory diseases by targeting and modulating PCK isoform activity. A specifically targeted therapy capable of selectively inhibiting or activating specific PKC isoforms is necessary and would provide a therapeutic approach that targets multiple elements of the inflammatory disease pathogenesis while maintaining low levels of side effects, such as when administered topically. Thus, as discussed herein, the development of treatments that reduce the secretion of inflammatory cytokines and/or control immune modulators through PKC isoform modulators would be beneficial in mitigating general local and systemic inflammation as well as many inflammatory and/or autoimmune diseases.

Disclosure of Invention

The present disclosure relates to treatment of inflammatory diseases and disorders by administering to a subject a modulator of PKC, e.g., an inhibitor of PKC epsilon or PKC eta or an activator of PKC delta.

Accordingly, in one aspect, the present disclosure provides a method of treating an inflammatory disease or disorder in a subject. The methods comprise administering to the subject an inhibitor of PKC, thereby treating an inflammatory disease or disorder in the subject. In exemplary embodiments, the inhibitor is a polypeptide that selectively inhibits PKC α, PKC epsilon, or PKC η, such as SEQ ID NO: 1-29.

In another aspect, the present disclosure provides a method of treating an inflammatory disease or disorder in a subject. The methods comprise administering to the subject an activator of PKC δ, thereby treating the inflammatory disease or disorder in the subject. In various embodiments, the activator is a polypeptide that selectively activates PKC δ, such as SEQ ID NO: 30-37.

In another aspect, the disclosure provides a method of treating pruritus in a subject. The method comprises administering to the subject an inhibitor of PKC, thereby treating pruritus in the subject. In various embodiments, the inhibitor is an inhibitor of PKC α, PKC epsilon, or PKC η. In exemplary embodiments, the inhibitor is a polypeptide that selectively inhibits PKC α, PKC epsilon, or PKC η, such as SEQ ID NO: 1-29.

In another aspect, the disclosure provides a method of treating pruritus in a subject. The method comprises administering to the subject an activator of PKC δ, thereby treating pruritus in the subject. In various embodiments, the activator is a polypeptide that selectively activates PKC δ, such as SEQ ID NO: 30-37.

In another aspect, the present disclosure provides a method of treating multiple sclerosis in a subject. The methods comprise administering to the subject an inhibitor of PKC α, PKC η, PKC epsilon, or PKC epsilon, thereby treating multiple sclerosis in the subject. In exemplary embodiments, the inhibitor is a polypeptide that selectively inhibits PKC α or PKC η, such as SEQ ID NO: 1-13 and 26-29.

In various aspects, the present disclosure provides kits for performing the methods of the present disclosure. In one embodiment, the kit comprises an inhibitor of PKC, e.g., an inhibitor of PKC α, PKC epsilon, or PKC η, or an activator of PKC δ, and instructions for administering the inhibitor or activator.

In another aspect, the disclosure provides SEQ ID NO: 3 or a physiologically acceptable salt thereof, wherein the polypeptide is N-myristoylated. In an exemplary embodiment, the polypeptide is SEQ ID NO: 12.

the present disclosure also provides a polypeptide comprising SEQ ID NO: 3 or a physiologically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein the polypeptide is N-myristoylated.

In another aspect, the disclosure provides a polypeptide comprising SEQ ID NO: 4 or a physiologically acceptable salt thereof. In exemplary embodiments, the isolated polypeptide is SEQ ID NO: 10 or SEQ ID NO: 13.

The present disclosure also provides pharmaceutical compositions comprising an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or a physiologically acceptable salt thereof.

In another aspect, the present disclosure provides a polypeptide selected from SEQ ID NOs: 30-33 or a physiologically acceptable salt thereof. In exemplary embodiments, the isolated polypeptide is SEQ ID NO: 34-37.

The present disclosure also provides pharmaceutical compositions comprising an isolated polypeptide comprising the amino acid sequence of SEQ ID NOs: 30-33, or a physiologically acceptable salt thereof.

Drawings

Figure 1 is a pictorial representation depicting the different members of the PKC family of isoforms.

Figure 2 is a series of graphical representations depicting the modulation of the inhibition of PKC α which characterizes the structural integrity of keratinocytes in psoriasis. Skin tissue was paraffin embedded and stained with hematoxylin and eosin (H & E) general histological dyes or different markers for various skin layers, including keratin 14(K14) for the basal layer, keratin 1(K1) for the spinous layer, keratin 6(K6) for keratinocyte migration, and PCNA for keratinocyte proliferation. The results demonstrate normalization of skin properties after PKC α inhibition (left column is wild-type (WT), right column is PKC α knockout).

Figure 3 is a histogram comparing the severity of scaling in different knockout mice after treatment with IMQ with controls.

Figure 4 is a series of image representations showing scaling in knockout mice compared to controls following treatment with IMQ.

FIG. 5 is a series of graphical representations showing the expression of filaggrin (Fil), loricrin (Lor), and keratin 1 (K1).

FIGS. 6A-B are a series of images and graphical representations of in vitro and in vivo assessment of keratinocyte proliferation. Figure 6A is a graphical representation showing PCNA expression. FIG. 6B is a histogram comparing the percentage of PCNA positive cells treated with HO/02/10 to controls.

FIG. 7 is a series of graphical representations showing the expression of filaggrin (Fil), loricrin (Lor), keratin 1(K1), PCNA, and keratin 14 (K14).

Fig. 8 is a graphical representation showing a summary of protein expression data in keratinocytes for different peptide PKC α inhibitors.

FIG. 9 is a histogram comparing the burst pressure of skin samples treated with HO/02/10 to controls.

FIG. 10 is a histogram comparing the anti-inflammatory effects of HO/02/10 on skin wounds in B57BL/6J mice 4 and 9 days post-injury.

FIG. 11 is a histogram comparing cytokine secretion in splenocytes treated with HO/02/10.

Fig. 12 is a series of image representations showing ICAM expression in basal keratinocytes and epithelial cells in the blood vessels of the skin.

Fig. 13 is a series of image representations showing ICAM expression in basal keratinocytes and epithelial cells in the blood vessels of the skin.

FIG. 14 is a histogram comparing the percentage of mice showing positive ICAM-1 staining at the wound margins.

FIG. 15 is a histogram comparing the number of cells in each domain of Iba-1 positively stained cells.

FIGS. 16A-B are a series of images and graphical representations showing MAC-2 expression in keratinocytes. FIG. 16A is a series of stains showing MAC-2 expression. Fig. 16B is a histogram comparing the number of cells in each domain of MAC-2 positively stained cells to controls, 1, 10, and 100 micrograms of pcka inhibitor per mL (from the left).

FIGS. 17A-D are a series of histograms comparing cytokine secretion in LPS-activated keratinocytes treated with HO/02/10. FIG. 17A compares secretion of IL-6, IL-1 α, and GM-CSF. FIG. 17B compares secretion of G-CSF. FIG. 17C compares secretion of MIP-2. FIG. 17D compares KC secretion.

FIGS. 18A-C are a series of histograms comparing cytokine secretion in LPS-activated macrophages treated with HO/02/10. FIG. 18A compares the secretion of G-CSF, KC and MIP-2. FIG. 18B compares secretion of IL1 α (left of histogram pair) and TNF α (right of histogram pair). Fig. 18C compares secretion of IL1 β (left of histogram pair) and IL12 (right of histogram pair).

Fig. 19 is a histogram comparing cytokine secretion in LPS-activated keratinocytes treated with peptide PKC α inhibitors.

Fig. 20 is a histogram comparing cytokine secretion in LPS-activated keratinocytes treated with peptide PKC α inhibitors.

FIGS. 21A-B are histograms comparing cytokine secretion in TNF α -activated keratinocytes treated with peptide PKC α inhibitors. FIG. 21A compares the secretion of ILIA. FIG. 21B compares IL-6 secretion.

FIGS. 22A-B are histograms comparing cytokine secretion in TNF α -activated keratinocytes treated with peptide PKC α inhibitors. FIG. 22A compares secretion of G-CSF. FIG. 22B compares secretion of GM-CSF.

FIGS. 23A-B are histograms comparing cytokine secretion in TNF α -activated keratinocytes treated with peptide PKC α inhibitors. FIG. 23A compares the secretion of MIP-2. FIG. 23B compares the secretion of IP-10.

FIGS. 24A-B are histograms comparing cytokine secretion in IL-17A activated keratinocytes treated with peptide PKC α inhibitors. FIG. 24A compares IL-1A secretion. FIG. 24B compares IL-6 secretion.

FIGS. 25A-B are histograms comparing cytokine secretion in IL-17A activated keratinocytes treated with peptide PKC α inhibitors. Figure 25A compares TNF α secretion. FIG. 25B compares the secretion of IP-10.

FIGS. 26A-B are histograms comparing cytokine secretion in IL-17A activated keratinocytes treated with peptide PKC α inhibitors. FIG. 26A compares secretion of G-CSF. FIG. 26B compares secretion of GM-CSF.

FIGS. 27A-B are histograms comparing cytokine secretion in IL-17A activated keratinocytes treated with peptide PKC α inhibitors. FIG. 27A compares KC secretion. FIG. 27B compares secretion of MIP-2.

Figure 28 is a series of images and graphical representations showing down-regulation of T cell infiltration into the dermis and epidermis during the inflammatory phase following treatment with HO/02/10. Fig. 28A is a series of stains with anti-CD 3 antibody.

Fig. 28B is a histogram comparing the number of cells in each domain of CD3 positive stained cells.

FIG. 29 is a graphical representation showing an overview of the effect of treatment with the peptide PKC α inhibitor MPDY-1 on different cell types.

FIG. 30 is a graphical representation showing an overview of the overall effect of HO/02/10 on psoriasis-associated pathways.

Fig. 31A-B are a series of images and graphical representations showing down-regulation of neutrophil infiltration into the dermis and epidermis during the inflammatory phase following treatment with HO/02/10. Fig. 31A is staining with neutrophil-specific antibodies. Fig. 31B is a histogram comparing the number of cells in each domain of neutrophils specifically positively stained cells.

FIG. 32 is a graphic representation of SDS PAGE staining with Ser176/180 antibody.

FIG. 33 is a graphical representation showing EAE scores over time course of processing.

FIG. 34 is a graphical representation showing EAE scores over time course of processing.

FIG. 35 is a graphical representation showing EAE scores over time course of processing.

FIG. 36 is a graphical representation showing EAE scores over time course of processing.

FIG. 37 is a graphical representation showing EAE scores over time course of processing.

FIG. 38 is an image representation of the mechanism of action of histamine used in the prick test model to assess the effect of MPDY-1 on pruritus.

FIG. 39 is an image representation showing forearms of subjects injected with histamine and treated with or without MPDY-1.

FIG. 40 is an image representation showing forearms of subjects injected with histamine and treated with or without MPDY-1.

FIG. 41 is an image representation showing forearms of subjects injected with histamine and treated with or without MPDY-1.

FIG. 42 is an image representation showing forearms of subjects injected with histamine and treated with or without MPDY-1.

FIG. 43 is a table of data collected in vitro immunoassays for the PKC α inhibitor MPDY-1 and the PKC δ activator DAP-1(SEQ ID NO: 34) (not all data shown).

FIG. 44 is a tabular summary of the results for the cytokine secretion of the PKC delta activator DAP-1(SEQ ID NO: 34) in keratinocytes treated with TNF α and an inhibitor.

Fig. 45 is a histogram showing comparison of cytokine secretion in keratinocytes treated with LPS or TNF α and different PKC epsilon inhibitors.

FIG. 46 is a histogram showing comparison of cytokine secretion in keratinocytes treated with LPS or TNF α and different PKC epsilon inhibitors.

FIG. 47 is a histogram showing comparison of cytokine secretion in keratinocytes treated with LPS or TNF α and different PKC epsilon inhibitors.

Fig. 48 is a histogram showing comparison of cytokine secretion in keratinocytes treated with LPS or TNF α and different PKC epsilon inhibitors.

Figure 49 is a tabular summary of the results of various PKC epsilon inhibitors of cytokine secretion in keratinocytes treated with LPS or TNF α and inhibitor.

FIG. 50 is a histogram comparing cytokine secretion in LPS-activated keratinocytes treated with peptide PKC α inhibitors including MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7), AIP-2(SEQ ID NO: 8), AIP-1(SEQ ID NO: 9), and PPDY (SEQ ID NO: 10).

FIG. 51 is a histogram comparing cytokine secretion in LPS-activated keratinocytes treated with peptide PKC α inhibitors including MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7), AIP-2(SEQ ID NO: 8), AIP-1(SEQ ID NO: 9), and PPDY (SEQ ID NO: 10).

FIG. 52 is a histogram comparing cytokine secretion in LPS-activated keratinocytes treated with peptide PKC α inhibitors including MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7), AIP-2(SEQ ID NO: 8), AIP-1(SEQ ID NO: 9), and PPDY (SEQ ID NO: 10).

FIG. 53 is a histogram comparing cytokine secretion in LPS-activated keratinocytes treated with peptide PKC α inhibitors including MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7), AIP-2(SEQ ID NO: 8), AIP-1(SEQ ID NO: 9), and PPDY (SEQ ID NO: 10).

FIG. 54 is a histogram comparing cytokine secretion in LPS-activated keratinocytes treated with peptide PKC α inhibitors including MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7), AIP-2(SEQ ID NO: 8), AIP-1(SEQ ID NO: 9), and PPDY (SEQ ID NO: 10).

FIG. 55 is a histogram comparing cytokine secretion in TNF α -activated keratinocytes treated with peptide PKC α inhibitors including MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7), AIP-2(SEQ ID NO: 8), AIP-1(SEQ ID NO: 9), and PPDY (SEQ ID NO: 10).

FIG. 56 is a histogram comparing cytokine secretion in TNF α -activated keratinocytes treated with peptide PKC α inhibitors including MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7), AIP-2(SEQ ID NO: 8), AIP-1(SEQ ID NO: 9), and PPDY (SEQ ID NO: 10).

FIG. 57 is a histogram comparing cytokine secretion in TNF α -activated keratinocytes treated with peptide PKC α inhibitors including MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7), AIP-2(SEQ ID NO: 8), AIP-1(SEQ ID NO: 9), and PPDY (SEQ ID NO: 10).

FIG. 58 is a histogram comparing cytokine secretion in LPS-activated keratinocytes treated with peptide PKC α inhibitors including MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7), AIP-2(SEQ ID NO: 8), AIP-1(SEQ ID NO: 9), and PPDY (SEQ ID NO: 10).

FIG. 59 is a histogram comparing cytokine secretion in LPS-activated keratinocytes treated with the peptide PKC α inhibitor MPDY-1(SEQ ID NO: 6).

FIG. 60 is a histogram comparing cytokine secretion in LPS-activated keratinocytes treated with the peptide PKC α inhibitor MPDY-1(SEQ ID NO: 6).

FIG. 61 is a histogram comparing cytokine secretion in LPS-activated keratinocytes treated with the peptide PKC α inhibitor MPDY-1(SEQ ID NO: 6).

FIG. 62 is a histogram comparing cytokine secretion in LPS-activated keratinocytes treated with the peptide PKC α inhibitor AWOT-1(SEQ ID NO: 7).

FIG. 63 is a histogram comparing cytokine secretion in LPS-activated keratinocytes treated with the peptide PKC α inhibitors MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7) and AIP-2(SEQ ID NO: 8).

FIG. 64 is a histogram comparing cytokine secretion in LPS-activated keratinocytes treated with the peptide PKC α inhibitors MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7) and AIP-2(SEQ ID NO: 8).

FIG. 65 is a histogram comparing cytokine secretion in IL-17A activated keratinocytes treated with the peptide PKC α inhibitors MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7), and AIP-2(SEQ ID NO: 8).

FIG. 66 is a histogram comparing cytokine secretion in TNF α -activated keratinocytes treated with the peptide PKC α inhibitors MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7) and AIP-2(SEQ ID NO: 8).

FIG. 67 is a histogram comparing cytokine secretion in TNF α -activated keratinocytes treated with the peptide PKC α inhibitors MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7), and AIP-2(SEQ ID NO: 8).

FIG. 68 is a histogram comparing cytokine secretion in TNF α -activated keratinocytes treated with the peptide PKC α inhibitors MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7) and AIP-2(SEQ ID NO: 8).

FIG. 69 is a histogram comparing cytokine secretion in TNF α -activated keratinocytes treated with the peptide PKC α inhibitors MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7) and AIP-2(SEQ ID NO: 8).

FIG. 70 is a histogram comparing cytokine secretion in TNF α -activated keratinocytes treated with the peptide PKC α inhibitors MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7) and AIP-2(SEQ ID NO: 8).

FIG. 71 is a histogram comparing cytokine secretion in IL-17A activated keratinocytes treated with the peptide PKC α inhibitors MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7) and AIP-2(SEQ ID NO: 8).

FIG. 72 is a histogram comparing cytokine secretion in IL-17A activated keratinocytes treated with the peptide PKC α inhibitors MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7), and AIP-2(SEQ ID NO: 8).

FIG. 73 is a histogram comparing cytokine secretion in IL-17A activated keratinocytes treated with the peptide PKC α inhibitors MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7) and AIP-2(SEQ ID NO: 8).

FIG. 74 is a histogram comparing cytokine secretion in IL-17A activated keratinocytes treated with the peptide PKC α inhibitors MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7) and AIP-2(SEQ ID NO: 8).

FIG. 75 is a histogram comparing cytokine secretion in IL-17A activated keratinocytes treated with the peptide PKC α inhibitors MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7), and AIP-2(SEQ ID NO: 8).

FIG. 76 is a histogram comparing cytokine secretion in IL-17A activated keratinocytes treated with the peptide PKC α inhibitors MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7) and AIP-2(SEQ ID NO: 8).

FIG. 77 is a histogram comparing cytokine secretion in IL-17A activated keratinocytes treated with the peptide PKC α inhibitors MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7) and AIP-2(SEQ ID NO: 8).

FIG. 78 is a histogram comparing cytokine secretion in IL-17A activated keratinocytes treated with the peptide PKC α inhibitors MPDY-1(SEQ ID NO: 6), AWOT-1(SEQ ID NO: 7) and AIP-2(SEQ ID NO: 8).

FIG. 79 is a histogram comparing cytokine secretion in LPS-activated keratinocytes treated with the peptide PKC α inhibitor MPDY-1(SEQ ID NO: 6) and PDY-1(SEQ ID NO: 13).

FIG. 80 is a tabular summary of results for different PKC α inhibitors of cytokine secretion in keratinocytes treated with LPS, TNF α or IL-17A and inhibitors.

Detailed Description

The present disclosure is based on the original discovery that modulators of the PKC isoforms can be administered as effective treatments for inflammatory diseases and conditions. The involvement of the PKC isoform in skin cells and the major cellular processes of many components of the immune system makes it a possible target for the treatment of inflammatory pathologies. The data presented herein demonstrate that PKC family isoforms modulate activation processes in skin and immune cells associated with inflammation and inflammatory diseases.

It is to be understood that this disclosure is not limited to the particular compositions, methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

The principles and operation of a method according to the present disclosure may be better understood with reference to the drawings and the accompanying description.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods and/or steps of the type described herein that will be apparent to those skilled in the art upon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, some preferred methods and materials are now described.

As used herein, the term "subject" refers to a mammalian subject. In this connection, treatment of any animal of the mammalian order is envisioned. These animals include, but are not limited to, horses, cats, dogs, rabbits, mice, goats, sheep, non-human primates, and humans. The methods of the present disclosure are therefore contemplated for use in veterinary applications as well as human use.

"treatment" of a subject herein refers to both therapeutic treatment and prophylactic (preventative) or preventative measures. Those in need of treatment include those already with an inflammatory disease or condition and those in which an inflammatory disease or condition is to be prevented. Thus, the subject may have been diagnosed with an inflammatory disease or disorder or may be susceptible or sensitive to an inflammatory disease or disorder.

As used herein, "inflammatory disease or disorder" is intended to include any disease or disorder having an etiology associated with the modulation of PKC family isoforms. These diseases include, but are not limited to, pruritus, dermatitis, psoriasis, multiple sclerosis, rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, hashimoto's thyroiditis, myasthenia gravis, diabetes type I or II, asthma, inflammatory lung injury, inflammatory liver injury, inflammatory glomerular injury, atopic dermatitis, allergic contact dermatitis, irritant contact dermatitis, seborrheic dermatitis, sjogren's syndrome, keratoconjunctivitis, uveitis, inflammatory bowel disease, crohn's disease, ulcerative colitis, inflammatory diseases of the joints, skin or muscles, acute or chronic idiopathic inflammatory arthritis, myositis, demyelinating disease, chronic obstructive pulmonary disease, interstitial lung disease, interstitial nephritis and chronic active hepatitis.

A "symptom" of an inflammatory disease or condition is a phenomenon or deviation from normal in structure, function, or sensation experienced by the subject and indicative of any pathological condition of the inflammatory disease or condition.

The expression "effective amount" refers to an amount of an inhibitor or activator of PKC isoforms (e.g., polypeptides of SEQ ID NOs: 1-37) that is effective for preventing, ameliorating or treating an inflammatory disease or condition. These effective amounts will generally result in an improvement in the signs, symptoms, and/or other indications of the inflammatory disease or condition. For example, in skin inflammation, an effective amount results in reduction of swelling and/or inflammation and/or clearance of redness. For pruritus, an effective amount may result in the removal of redness and/or pruritus. For MS, an effective amount may result in reduced recurrence rates, prevention of disability, reduction in the number and/or volume of brain MRI lesions, improved walking at 25 steps timing, extended duration of disease progression, and the like.

As used herein, the term "PKC isoform" as used herein encompasses all PKC isoforms, including PKC α, PKC β, PKC δ, PKC ε, PKC η, PKCC ζ, PKC γ, PKC θ, and PKC λ.

The phrase "modulating the expression and/or activity of PKC isoforms" relates to increasing or decreasing the expression and/or activity of PKC isoforms. Increased expression results in increased production of PKC isoforms.

The term "activator" is used herein to describe a molecule that increases the expression and/or activity of an isoform of PKC. The term "inhibitor" is used herein to describe a molecule that inhibits the expression and/or activity of PKC isoforms. The phosphoryl transfer region, the pseudo-substrate domain, the phorbol ester binding sequence, and the phosphorylation site may be targets for, among other things, modulating isoenzyme-specific PKC activity.

The "pseudosubstrate region" or auto-inhibitory domain of PKC isoforms is defined herein as a consensus sequence of the substrate of the kinase that has substantially no phosphorylatable residues. The pseudo substrate domain is located in a regulatory region, very similar to the substrate recognition motif, which blocks the recognition site and prevents phosphorylation. Thus, inhibitory peptides of PKC isoforms (e.g., polypeptides of the disclosure) are obtained by replacing a phosphorylatable residue of serine (S) or threonine (T) with alanine (a). PKC δ is the only PKC isoform known to have an additional binding site that enables isoform activation at the C2 domain, conserved domain 2 of PKC δ.

PKC is the primary signaling pathway that regulates keratinocyte proliferation, migration, and differentiation. Many PKC isoforms are known to be expressed in skin tissue and their expression/activity appears to play a role in cell proliferation and/or cell migration and/or cell differentiation. However, specifically modulating their expression and activity to effect treatment of inflammatory diseases was previously unknown and demonstrated in this disclosure.

In summary, the results presented herein demonstrate that modulating the expression and/or activity of different PKC isoforms is effective in the treatment of inflammation and inflammatory diseases.

Accordingly, in one aspect, the present disclosure provides a method of treating an inflammatory disease or disorder in a subject. The methods comprise administering to the subject an inhibitor of PKC, thereby treating an inflammatory disease or disorder in the subject. In exemplary embodiments, the inhibitor is a polypeptide that selectively inhibits PKC α, PKC epsilon, or PKC η, such as SEQ ID NO: 1-29.

As disclosed in the examples, administration of PKC isoform inhibitors has been shown to reduce the secretion of proinflammatory cytokines, chemokines and Th1 cytokines in a variety of different skin cell types (not just skin cells, i.e. macrophages present and active in other tissues). In addition, administration of PKC isoforms reduces the expression of activated factors (e.g., ICAM-1 on keratinocytes and endothelial cells and mac-2 on macrophages). Furthermore, PKC α inhibitors have been found to be effective in the treatment of skin inflammation and to attenuate inflammatory symptoms in an inflammatory skin model of psoriasis. As discussed further in the examples, the mechanism of action of inhibitors of the PCK isoforms has been elucidated, demonstrating their utility as effective treatments for inflammatory diseases and conditions. For example, peptide inhibitors of the PCK isoform have been shown to: 1) normalizing epidermal differentiation marker expression by reducing terminal differentiation; 2) attenuation of abnormal hyperproliferation; 3) regulating skin structure and increasing skin strength; and/or 4) as outlined, down-regulating inflammation by differentially affecting recruitment and activation of different cell types in different steps of the inflammatory process, e.g., in fig. 30.

Also, as disclosed in the examples, activators of PKC δ have also been shown to reduce the secretion of proinflammatory cytokines in a variety of different skin cell types. Thus, in another aspect, the present disclosure provides methods of treating an inflammatory disease or disorder in a subject by administering to the subject an activator of PKC δ, thereby treating the inflammatory disease or disorder in the subject. In various embodiments, the activator is a polypeptide that selectively activates PKC δ, such as SEQ ID NO: 30-37.

Furthermore, as disclosed in the examples, administration of PKC α inhibitors and PKC η inhibitors has been found to attenuate symptoms of MS. Likewise, in another aspect, the present disclosure provides a method of treating multiple sclerosis in a subject. The methods comprise administering to the subject an inhibitor of PKC α or PKC η, thereby treating multiple sclerosis in the subject.

Furthermore, administration of PKC isoform inhibitors has been found to be effective in the treatment of pruritus. Likewise, in another aspect, the disclosure provides a method of treating pruritus in a subject. The method comprises administering to the subject an inhibitor of PKC, thereby treating pruritus in the subject.

The examples and figures show data demonstrating the ability of activators of PKC δ to inhibit secretion of major inflammatory cytokines (e.g., IL-1, IL-6, and TNF α). A variety of PKC isoform inhibitors show similar data, including PKC α, PKC epsilon, and PKC η. As shown in the examples, formulations comprising PKC inhibitors and active agents of the present disclosure have been shown to inhibit secretion of major inflammatory cytokines. In the case of pruritus, without being bound by a particular theory, it is believed that reducing the level of the inflammatory agent prevents activation of endothelial cells in nearby blood vessels and thus replenishes the psoriatic plaque with neutrophils, macrophages, and T cells. Furthermore, TH1 and TH17 cells have been shown to be involved in the pathogenesis of psoriasis through secretion of specific cytokines, which appear to enhance inflammation or promote excessive proliferation of keratinocytes, respectively. The pro-inflammatory cytokines mentioned above are essential for the development of these Th17 cells (Mangan et al (2006) Nature 441: 231-234; Bettelli et al (2006) Nature 441: 235-238) and Th1 cell activities. Decreasing their secretion via PKC inhibitors and activators shows their use in the effective treatment of inflammatory disorders and psoriasis.

In various embodiments, the inhibitor of an isoform of PKC is an inhibitor of the pseudosubstrate region of PKC and is a polypeptide, and the activator of an isoform of PKC is likewise a polypeptide. The terms "polypeptide", "peptide" or "protein" are used interchangeably herein to designate a series of linear amino acid residues linked to one another by peptide bonds between the alpha amino and carboxyl groups of adjacent residues.

In various embodiments, examples of peptide PKC activators and inhibitors that may be used include, but are not limited to, the peptide PKC activator and inhibitor shown in table 1 as SEQ ID NO: 1-5, 14-19, 26, 27 and 30-33, or a physiologically acceptable salt thereof, and SEQ ID NOs: 6-13, 20-25, 28, 29, 34-37.

Table 1: PKC isoform inhibitors and activator peptides

In various embodiments, the peptide PKC inhibitors or activators typically contain between 6 and 12 amino acids, but may be longer or shorter in length. In various embodiments, the peptide PKC inhibitor or activator may range from 6 to 45, 6 to 40, 6 to 35, 6 to 30, 6 to 25, 6 to 20, 6 to 15, or 6 to 10 amino acids in length. In one embodiment, the peptide comprises 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

In various embodiments, the peptide PKC inhibitor activator may be N-acylated, preferably acylated with an acyl group from a C12-C20 fatty acid, such as a C14 acyl group (myristoyl group) or a C16 acyl group (palmitoyl group).

Typically, peptide PKC α inhibitors include the common motif sequence Phe-Ala-Arg-Lys-Gly-Ala (SEQ ID NO: 1). Alternatively, in another embodiment, the PKC α inhibitor comprises the common motif sequence Thr-Leu-Asn-Pro-Gln-Trp-Glu-Ser (SEQ ID NO: 5).

When the peptide PKC inhibitors and activators are defined by exact sequences or motif sequences, one of skill in the art will appreciate that peptides having similar sequences may have similar functions. Thus, peptides having substantially the same sequence or having substantially the same or similar sequence as the PKC inhibitors or activators of table 1 are contemplated. As used herein, the term "substantially identical sequence" includes a peptide comprising an amino acid sequence identical to SEQ ID NO: 1-37 has a sequence identity of at least 60 +% (meaning a percentage of sixty or more), preferably 70 +%, more preferably 80 +% and most preferably 90 +%, 95 +% or 98 +% and inhibits or activates PKC isoform activity.

A further indication that two polypeptides are substantially identical is that one peptide is immunologically cross-linked with a second peptide. Thus, a polypeptide is typically substantially identical to a second polypeptide when, for example, the two peptides differ only by conservative substitutions.

In the case of proteins or peptides, the term "conservative substitutions" is used to reflect amino acid substitutions that do not substantially alter the activity (e.g., antimicrobial activity) of the molecule. Typically conservative amino acid substitutions include the substitution of one amino acid for another with similar chemical properties (e.g., charge or hydrophobicity). The following six groups each contain amino acids that are typically conservative substitutions for each other: 1) alanine (a), serine (S) threonine (T); 2) aspartic acid (D) glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y) and tryptophan (W).

The term "amino acid" is used in its broadest sense to include naturally occurring amino acids as well as non-naturally occurring amino acids comprising amino acid analogs. In this broad sense, those skilled in the art will know that reference herein to amino acids includes, for example, naturally occurring proteinogenic (L) -amino acids, (D) -amino acids, chemically modified amino acids such as amino acid analogs, naturally occurring non-proteinogenic amino acids such as norleucine, and chemically synthesized compounds having properties known in the art to be characteristic of amino acids. As used herein, the term "proteinogenic" indicates that amino acids can be incorporated into proteins in cells via metabolic pathways.

The term "identity" or percent "identity" in the context of two polypeptide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using a sequence alignment algorithm or by visual inspection.

The phrase "substantially identical" in the context of two polypeptides refers to two or more sequences that have at least 60 +%, preferably 80 +%, most preferably 90-95 +% amino acid sequence identity when compared and aligned for maximum identity, as measured using a sequence alignment algorithm or by visual inspection.

As is well known in the art, optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman ((1981) Adv Appl Math 2: 482), by the homology alignment algorithm of Needleman & Wunsch ((1970) J Mol Biol 48: 443), by the exploration of similarity methods by Pearson & Lipman ((1988) Proc Natl Acad Sci USA 85: 2444), by computer implementation of these algorithms via visual inspection, or other effective methods.

The peptide PKC inhibitors or activators may have modified amino acid sequences or non-naturally occurring terminal modifications. Modifications to the peptide sequence may include, for example, additions, deletions or substitutions of amino acids, provided that the peptide produced by such modifications retains PKC α inhibitory activity. In addition, the peptide may be present in a free terminal or structural formula having an amino-protected (e.g., N-protected) and/or carboxy-protected (e.g., C-protected) terminal. The protecting group includes: (a) aromatic polyurethane-based protecting groups including benzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, isoniazidonyloxycarbonyl and 4-methoxybenzyloxycarbonyl; (b) aliphatic polyurethanes protecting groups including t-butoxycarbonyl, t-pentyloxycarbonyl, isopropoxycarbonyl, 2- (4-biphenyl) -2-propoxycarbonyl, allyloxycarbonyl, and methylsulfonylethoxycarbonyl; (c) cycloalkyl polyurethane-based protecting groups including adamantyloxycarbonyl, cyclopentyloxycarbonyl, cyclohexyloxycarbonyl, isobornyloxycarbonyl; (d) an acyl protecting group or a sulfonyl protecting group. Additional protecting groups include benzyloxycarbonyl, t-butoxycarbonyl, acetyl, 2-propylvaleryl, 4-methylpentanoyl, t-butylacetyl, 3-cyclohexylpropionyl, n-butanesulfonyl, benzylsulfonyl, 4-methylbenzenesulfonyl, 2-naphthalenesulfonyl, 3-naphthalenesulfonyl and 1-camphorsulfonyl.

In various embodiments, the peptide PKC isoform inhibitors and activators may be administered by any suitable means, including topical, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, intravenous, and/or intralesional administration, in order to treat a subject. However, in exemplary embodiments, the peptides are formulated for topical administration, e.g., as liquids, creams, gels, ointments, foam sprays, and the like.

Therapeutic formulations of PKC isoform inhibitors or activators for use in accordance with the present disclosure are prepared, for example, by mixing a PKC isoform inhibitor or activator having the desired purity with, optionally, pharmaceutically acceptable carriers, excipients, and/or stabilizers (see, e.g., Remington's pharmaceutical sciences, 16 th edition, Osol, editors a (1980)). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphate, citrate, and other organic acids; antioxidants include ascorbic acid and methionine; preservatives (for example octadecyl dimethyl benzyl ammonium chloride, chlorhexidine, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine(ii) an amino acid; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g. zinc-protein complexes) and/or nonionic surfactants, e.g. TWEEN^TM、PLURONICS^TMOr polyethylene glycol (PEG).

In an exemplary embodiment, the PKC isoform inhibitor or activator is formulated in a cream. Because of the activity of the PKC enzyme, inhibitors and activators of the PKC isoform are ideal for topical treatment of skin inflammation and other inflammatory diseases, and may be specifically targeted. Inhibition or activation of specific PKC enzymes is achieved by selectively modulating PKC isoforms at lower concentrations without affecting the ability of other PKC isoforms.

An exemplary formulation for topical application is disclosed in example 4, wherein the peptide MPDY-1(SEQ ID NO: 6) is formulated as a cream for topical application. However, it will be appreciated by those skilled in the art that variations in formulation may be made while retaining the necessary characteristics of the cream, such as viscosity, stability, non-toxicity, and the like. Likewise, one skilled in the art will recognize that the formulations may be used as carriers for any of the peptide PKC inhibitors or activators of the present disclosure.

In another embodiment, an article of manufacture, such as a kit, is provided that includes materials useful for practicing the therapeutic methods of the present disclosure. In various embodiments, the kit comprises a PKC isoform activator or inhibitor, i.e., a peptide PKC isoform inhibitor or activator as disclosed herein, and instructions for administering the active agent or inhibitor to a subject.

The term "instructions" or "package insert" is used to mean instructions typically included in commercial packages of therapeutic products, which contain information about indications, usage, dosage, administration, contraindications, other therapeutic products combined with the packaged products and/or warnings regarding the use of these therapeutic products, and the like.

As disclosed herein, inhibitors of PKC α can be formulated for specific routes of administration. As such, the kit may include a formulation containing an inhibitor of PKC α contained in a suitable container (e.g., such as a tube, bottle, vial, syringe, etc.). The container may be formed of various materials such as glass or plastic. The container contains or contains a composition effective for treating inflammatory diseases and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one component of the formulation is an inhibitor or activator of the PKC isoform. The label or package insert indicates that the composition provides a formulation comprising an inhibitor or active agent of the PKC isoform in accordance with the specific instructions for treating an inflammatory disease in a subject suffering from the inflammatory disease with respect to the amounts and intervals of administration. The article may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

It will be understood that the specific dose level and frequency of dosage administered to any particular subject in need of treatment may be varied and will depend upon a variety of factors including the activity of the PKC isoform inhibitor or activator employed, the metabolic stability and length of action of the compound, the age, body weight, health, sex, diet, mode and time of administration, the severity of the particular condition, and the host undergoing therapy. In general, however, the dosage will be about typical for known methods of administration of specific PKC isoform inhibitors or activators. Optimal dosages, methods of administration, and repetition rates can be readily determined by those skilled in the art. The exact formulation and dosage can be selected by The individual physician taking into account The condition of The patient ((Fingl et al "The Pharmacological Basis of therapy)", Chapter 1, page 1 (1975)).

Thus, depending on the severity and responsiveness of the condition to be treated, administration may be a single or repeated administration, with the course of treatment lasting from several days to several weeks or until a cure is achieved or a reduction in the disorder is obtained.

In various embodiments, when the PKC isoform inhibitor or activator is a peptide, the peptide is provided at a concentration of between 0.001 μ g/ml and 100 μ g/ml in the composition. For example, the concentration may be between 0.001. mu.g/ml and 100. mu.g/ml, 0.01. mu.g/ml and 50. mu.g/ml, 0.01. mu.g/ml and 10. mu.g/ml, 0.01. mu.g/ml and 1. mu.g/ml, and 0.01 and 0.5. mu.g/ml.

In one dosing procedure, the method comprises topically administering to the subject an inhibitor or activator of the peptide PKC isoform, e.g., as a cream. The peptide is topically applied at a concentration of from about 1 μ g/ml to about 1000 μ g/ml, 1 μ g/ml to about 500 μ g/ml, 1 μ g/ml to about 100 μ g/ml, 1 μ g/ml to about 10 μ g/ml, or 10 μ g/ml to about 100 μ g/ml. The peptide is administered at least once daily until the condition is treated.

In another dosing procedure, the method comprises parenterally, subcutaneously or intravenously administering to the subject an inhibitor or activator of the peptide PKC isoform. The peptides are applied at a concentration of from about 1 μ g/ml to about 1000 μ g/ml, 1 μ g/ml to about 500 μ g/ml, 1 μ g/ml to about 100 μ g/ml, 1 μ g/ml to about 10 μ g/ml, or 10 μ g/m1 to about 100 μ g/ml. The peptide is administered at least once daily, weekly, biweekly, or monthly until the condition is treated.

The following examples are provided to further illustrate embodiments of the present disclosure and are not intended to limit the scope. Although they are typical of those that may be used, other operations, methods, or techniques known to those skilled in the art may alternatively be used.

Example 1

Modulation of inhibition of PKC α characterizes the keratinocyte structural integrity of inflammatory skin disorders psoriasis

Inhibition of PKC α was shown to modulate the structural integrity of keratinocytes that characterize psoriasis. Skin tissue was paraffin embedded and stained with H & E (hematoxylin and eosin) general histological dyes or different markers for various skin layers, including keratin 14(K14) for the basal layer, keratin 1(K1) for the spinous layer, keratin 6(K6) for keratinocyte migration, and PCNA for keratinocyte proliferation. The results demonstrate normalization of skin properties after PKC α inhibition (fig. 2).

Example 2

Model for assessing in vivo and ex vivo treatment of inflammation via psoriasis model

Psoriasis has been previously studied using a variety of animal models, but none of these models adequately mimic the human disease pathology characterized by excessive skin production, neovascularization, and severe immune dysfunction. In general, to be considered a useful model of psoriasis, the model must share certain histopathological features with psoriasis, exhibit similar pathogenesis and/or disease mechanisms, and respond similarly to therapeutic agents used to treat the disease. The existing models exhibit several characteristics, including acanthosis hypertrophy, altered epidermal differentiation, increased vascularization, and leukocyte/T cell infiltration. However, in existing mouse models, there is little response to existing drugs and treatments. As such, existing models were used to develop new in vitro, ex vivo, and in vivo models to evaluate the psoriasis treatment used in the following examples.

In vitro model

The models developed included cell culture studies using cell lines and primary cultures of skin-derived cells as well as immune cells, using constructs and tools to overexpress and inactivate STAT3 and PKC α -mediated signaling pathways. A variety of techniques for the study of skin cell proliferation, migration, differentiation, inflammation and signaling are used and prove useful in studying the mechanisms of psoriasis development and in studying the therapeutic effects of PKC α inhibition in psoriasis.

In vivo model

PKC α overexpression and knock-out mouse models were used. Overexpression of PKC α in keratinocytes of mice transgenic with K5-PKC α was shown to exhibit severe intraepidermal neutrophil infiltration and disruption of the epidermis mimicking conditions such as pustular psoriasis. Transgenic mice were established in both forms of PKC α and DN by in vivo studies using subcutaneous application. In addition, PKC α knockout mice have also been used to study the effects of PKC α inactivation on skin structure and function.

A STAT3 overexpression mouse model was used. The main mouse model for psoriasis, in terms of similarity to human psoriasis, is a transgenic mouse in which Stat3 is overexpressed in epidermal keratinocytes. These mice develop a psoriasis condition with epidermal acanthosis hypertrophia and have an epidermal lymphocytic infiltration primarily of CD4+ in the dermis and CD8+ in the epidermis, all with features similar to psoriasis in humans.

Wounds as a model for skin inflammation and hyperproliferation. A screening methodology was developed to detect and quantitatively assess inflammation within skin lesions in a traumatic background that allows tracking of epidermal inflammatory responses in different skin compartments and identification of agents affecting this response.

In vitro model

Psoriatic skin transplanted on chick embryo chorioallantoic membrane (CAM). For the purpose of testing ex vivo therapeutic applications, techniques were developed to implant psoriatic skin on chick embryo chorioallantoic membrane (CAM). Although this technique is generally used for skin tumor studies and angiogenesis experiments, it is employed and used for psoriasis studies. This original approach allows new drugs to be applied directly to human psoriasis skin, thus leading to more clinically relevant studies of new drugs for the treatment of psoriasis. After transplantation, psoriatic human skin is used to establish the utility and timing of different treatments in different formulations using morphological, histological and biochemical analyses.

Example 3

PKC alpha knockReduced scaling in mice

PKC α knockout mouse models were developed and used to study the effects of PKC α inactivation on skin structure and function. As shown in fig. 3 and 4, a reduction in scaling was observed in PKC α knockout mice. Figure 3 is a histogram showing a more than 50% reduction in mean scaling severity in PKC α knockout mice compared to controls, demonstrating that inhibition of PKC α is a key requirement for the treatment of psoriasis. This is also shown in fig. 4, which is a series of photographs comparing scaling in different mice.

Example 4

Topical PKC alpha inhibitor formulations

Topical PKC α inhibitor formulations were developed and evaluated for efficacy in the treatment of psoriasis. The peptide PKC α inhibitor MPDY-1(SEQ ID NO: 6) (also referred to herein as HO/02/10), the components of which are shown in Table 2, was formulated in a cream.

Table 2: MPDY-1 cream base preparation

Component (A)
	Water (W)
Glycerol
	Propylene glycol
P-hydroxybenzoic acid methyl ester
	Phenoxyethanol
Stearic acid glyceride SE
	Cetyl alcohol
Squalane (coscol)
	PEG-40Stearath
Sucrose distearate
	Isopropyl myristate
Butylated hydroxytoluene
	Paraffin oil
Capric/caprylic triglyceride
	Vaseline
Propyl p-hydroxybenzoate
	MPDY-1

Example 5

Effect of PKC alpha inhibitors on epidermal differentiation in vitro

The formulation of example 4 (HO/02/10) was determined to control epidermal differentiation in vitro. Basal keratinocytes differentiate to form the stratum spinosum characterized by K1/K10 keratin, the stratum granulosum characterized by paphiopediin/filaggrin, and the stratum corneum. Defects in the expression and binding of loricrin and filaggrin filaments are associated with various immune skin diseases including psoriasis. Therefore, the effect of HO/02/10 on skin differentiation and proliferation was evaluated. As shown in fig. 5 and 6, HO/02/10 normalized skin Proliferation (PCNA) by reducing expression of loricrin and filaggrin (fig. 6) and regulated skin differentiation while the stratum spinosum remained unaffected (fig. 5). Since psoriatic skin keratinocytes differentiate rapidly to produce granular and predominantly abundant corneal cells (scales) while the stratum spinosum is thinned, HO/02/10 functions to normalize psoriatic skin by improving skin characteristics toward a normal phenotype.

FIG. 5 shows HO/02/10 controls granular differentiation of epidermis in vitro. Keratinocyte derived from C57BL/6J mouse is cultured in the presence of Ca²⁺To induce keratinocyte differentiation. The cells were then cultured in the presence of HO/02/10 (1. mu.g/ml). Cells were harvested, expanded on SDS PAGE gels and immunoblotted with antibodies against filaggrin (Fil), anti-loricrin (Lor) and anti-keratin 1 (K1).

FIG. 6 shows that HO/02/10 reduces keratinocyte proliferation in vitro and in vivo. Primary mouse keratinocytes from 2 day Balb/c mice at 0.05mM Ca²⁺Growth in MEM medium was carried out for 5 days to reach complete confluence (confluency). Treatment with HO/02/10 prior to inducing differentiation (10)^-6M and 10^-5M) for 6 hours. Cells were harvested, expanded on SDS PAGE gels and immunoblotted with anti-PCNA antibodies. The results are shown in fig. 6A. 8-10 week old C57B1ack mice were subjected to full-thickness trauma in the upper dorsal region to induce epidermal remodeling and differentiation. After wounding, mice were treated with HO/02/10 (in the range of 40-4000 mg/kg/day) daily for 7 days. At the end point, mice were euthanized and upper back skin samples were fixed in 4% paraformaldehyde solution before paraffin embedding and preparation of sections. The skin samples were then subjected to immunohistochemical staining using PCNA antibodies. (n ═ 18). The results are shown in FIG. 6B.

FIGS. 7 and 8 show additional expression data in keratinocytes using MPDY-1(SEQ ID NO: 6) and data for peptide PKC α inhibitors AIP-1(SEQ ID NO: 9), AIP-2(SEQ ID NO: 8), AWOT-1(SEQ ID NO: 7) and PPDY-1(SEQ ID NO: 10). Figure 7 shows immunohistochemical staining with anti-PCNA, anti-filaggrin (Fil), anti-loricrin (Lor), anti-keratin 1(K1) and anti-keratin 14(K14) antibodies in keratinocytes treated with different peptide PKC α inhibitors. Figure 8 represents a summary of expression data in keratinocytes for different peptide PKC α inhibitors.

To test skin strength and elasticity, the rupture chamber is used to measure the pressure required to rupture the skin sample (a measurable indicator of skin elasticity and durability). The results in figure 9 demonstrate that HO/02/10 treated skin exhibits enhanced skin strength. Thus, inhibition of PKC α may be beneficial to psoriatic skin, as it appears to enhance skin integrity and prevent rupture of psoriatic plaques.

FIG. 9 shows that HO/02/10 significantly enhances skin strength. The skin of the mice was treated with HO/02/10 for 14 days and subsequently subjected to a burst pressure analysis. The rupture chamber device is closed by one end and is connected with high pressure CO via a control valve and a pressure gauge²A fixed volume metal cylinder connected to the container. At the other end of the chamber, an adjustable holder is mounted to hold and contain the skin tissue under test in place. Gas is gradually released into the chamber and the internal pressure is continuously monitored until rupture of the tested skin occurs.

Example 6

Effect of PKC isoform inhibitors and activators on skin inflammation

A screening method was developed to detect and quantitatively assess inflammation of skin lesions in a traumatic background, which allows one to track epidermal inflammatory responses in different skin compartments and identify agents affecting this response (as a preliminary screen). The inflammatory response is considered severe when two of the following three conditions are evident: (1) abscess formation; (2) an increase in excess leukocytes (> 100 cells in fixed domain x 200); (3) high WBC/RBC ratio in the vessel, with > 20% WBC content in the vessel shown in the fixed domain x 200. The mechanistic characteristics of the immune response were studied using markers to identify infiltration and activation of specific immune cells. Examples of such markers are: ICAM-1 (as a marker for activated basal keratinocytes and endothelial cells), MAC-2 (as a marker for activated macrophages), and CD3(T cell marker). Using this quantitative approach, strong anti-inflammatory effects of HO/02/10 and other peptide PCK inhibitors in intact skin and skin lesions of different cell types and processes in several animal models were demonstrated to be possible.

The anti-inflammatory effects of HO/02/10 on skin wounds in B57BL/6J mice 4 and 9 days post-trauma were demonstrated by representative results below (FIG. 10). FIG. 10 shows the effect of dose response of HO/02/10 on inflammation in C57BL/6J mice. The skin of C57BL/6J mice was treated by daily application of HO/02/10 (4. mu.g/kg/day) or (40. mu.g/kg/day) (6 mice/group). Treatment is a local application. Biopsies were collected 4 and 9 days after trauma. Tissues were extracted from euthanized animals to assess inflammation by histology and immunohistochemistry.

HO/02/10 was also shown to reduce proinflammatory cytokine secretion from LPS-activated splenocytes. To assess overall anti-inflammatory effects in vitro, primary splenocytes of mouse origin were used as an immune model. Splenocytes were derived from C57BL/6J mice, red blood cells were lysed and cells were cultured in 96-well plates at 500,000 per well. LPS (1. mu.g/ml for the IL-1 and TNF. alpha. tests, and 0.2ng/ml for the IL-6 tests) was added and the cells were treated with MPDY-1 (1. mu.g/ml) or PBS. No LPS was added to the negative control sample. The medium was collected after 2 days and the amount of secreted cytokines was quantified using ELISA.

FIG. 11, as well as FIGS. 17-27, 43 and 50 demonstrate the ability of HO/02/10 to significantly reduce secretion of major inflammatory cytokines (e.g., TNF α, IL-1, and IL-6) from activated keratinocytes. In particular, IL-6 was shown to be essential for the development of TH17 cells involved in the pathogenesis of psoriasis, and to potentiate the effects that have been demonstrated on IL-1 and TNF α. TNF alpha and IL-6 are known targets for the treatment of psoriasis. FIG. 11 demonstrates the effect of 1. mu.g/ml HO/02/10.

HO/02/10 has also been shown to inhibit basal keratinocyte and endothelial cell immune activity in vivo. ICAM is an adhesion molecule that allows infiltration of granulocytes into inflammatory lesions. In particular, in the skin, basal keratinocytes express ICAM-1 upon immune activation, which increases infiltration of neutrophils and CD8-T cells into the epidermis, one of the hallmarks of psoriasis. Thus, the effect of HO/02/10 on ICAM expression in skin was examined by immunohistochemistry in an in vivo wound inflammatory background.

Downregulation of activated keratinocytes and endothelial cells (ICAM-1 staining) in skin inflammation was observed. A2 cm longitudinal cut was made in the upper back of C57BL/6J mice. After trauma, the sterile pad was sutured to the skin of the mice. Animals were treated daily with HO/02/10 (n-12). Five days after trauma, when the inflammatory phase reached its peak, mice were sacrificed, skin tissue was embedded in paraffin and immunohistochemical staining was performed with anti-ICAM-1 antibody.

As shown in fig. 12, HO/02/10 significantly reduced ICAM expression in basal keratinocytes and endothelium in the blood vessels of the skin. This effect appears to be dose dependent with the greatest effect at 10 μ g/ml administration.

Figure 13 shows additional staining showing down-regulation of activated keratinocytes and endothelial cells (ICAM-1 staining) in skin inflammation. A2 cm longitudinal cut was made in the upper back of C57BL/6J mice as described above. After trauma, the sterile pad was sutured to the skin of the mice. Animals were treated daily with MPDY-1 (n ═ 6). Five days after trauma, when the inflammatory phase reached its peak, mice were sacrificed, skin tissue was embedded in paraffin and immunohistochemical staining was performed with anti-ICAM-1 antibody.

FIG. 14 is a histogram comparing the percentage of mice showing positive ICAM-1 staining at both wound margins.

The effect of MPDY-1 on macrophage infiltration was also shown by Iba-1 staining. Iba-1 is a general marker for macrophages. FIG. 15 is a histogram comparing the number of cells in each domain showing Iba-1 positive staining. A2 cm longitudinal cut was made in the upper back of C57BL/6J mice as described above. After trauma, the sterile pad was sutured to the skin of the mice. Animals were treated daily with MPDY-1 (n ═ 6). Five days after trauma, when the inflammatory phase reached its peak, the mice were sacrificed, skin tissue embedded in paraffin and immunohistochemically stained with anti-Iba-1 antibody. A dose-dependent effect of MPDY-1 on macrophage infiltration was observed.

The effect of MPDY-1 on macrophage activation is also shown by MAC-2 staining. MAC-2 is a specific marker for activated macrophages. FIG. 16 shows a series of MAC-2 stains and histograms comparing the number of cells in each domain that will show a positive MAC-2 stain. A 2cm slit was made as described above. Daily use of specific concentrations of DPBS^-/-(control) or MPDY-1 treated animals (n ═ 6). After 5 days, immunohistochemical staining was performed with anti-MAC-2 antibody. Bar1 μm (/ p (control comparison MPDY-110 μ g) ═ 0.0028). Macrophage activation was significantly inhibited after MPDY-1 treatment.

MPDY-1 as shown in figure 32 also showed a significant reduction in TNF α -induced IKK activation in keratinocytes in a dose-dependent manner. Primary keratinocytes in mice were grown for 4 days to be low in Ca⁺²Fully confluent in MEM. Cells were pretreated for 1 hour with the indicated concentrations of MPDY-1 as depicted in the figure prior to TNF α induction. Following MPDY-1 pretreatment, cells were incubated with TNF α 35 ng/ml for 15 minutes. The reaction was stopped by adding frozen dPBS-/-and the keratinocytes were homogenized in RIPA buffer. Using phosphorylated IKKA/b (Serl76/180 antibody), samples were subjected to SDS PAGE Western blot analysis. Pretreatment with MPDY-1 significantly reduced TNF α -induced IKK activation in keratinocytes in a dose-dependent manner, with the lowest MPDY-1 concentration (0.1mg/ml) showing the strongest inhibition, thereby inhibiting NFKB activation.

As discussed above, HO/02/10 was also shown to reduce cytokine secretion from activated keratinocytes and macrophages. In recent years, both immune and skin components have been found to contribute equally to cycles based on the pathogenesis of psoriasis. Self skin cells and immune cells (both self and infiltrating cells) interact through cell-cell interactions and cytokine secretion in the inflammatory psoriasis process. Thus, the direct effect of HO/02/10 on the secretion of inflammatory, chemotactic and immune pathway-related cytokines that form both keratinocytes and immune cells (e.g., macrophages and dendritic cells) was examined. The results depicted in FIGS. 17 and 18 demonstrate that HO/02/10 down-regulates the secretion of immune-related cytokines such as IL-6, IL-1a, GM-CSF, MIP-2, and KC from keratinocytes and macrophages.

The results in FIG. 17 show the effect of HO/02/10 on cytokine secretion in keratinocytes. Keratinocytes were derived from neonatal C57BL/6J mouse skin. Cells were cultured in 24-well plates for 5 days. Cells were then treated with DPBS-/-, LPS (100ng/ml) or HO/02/10 (1. mu.g/ml) + LPS (100 ng/ml). The medium containing the secreted cytokines was collected after 48 hours and analyzed using the Luminex system.

The results of FIG. 18 show that HO/02/10 down-regulates cytokine secretion in macrophages. Bone marrow cells were derived from B6 mice. Cells were cultured for 6 days in the presence of GM-CSF (20ng/ml) and then treated with DPBS-/-, LPS (100ng/ml) or HO/02/10+ LPS (1. mu.g/ml and 100ng/ml, respectively).

Other peptide PKC α inhibitors have also been shown to decrease cytokine secretion from activated keratinocytes and macrophages. FIGS. 19 to 23 show that the peptide inhibitors MPDY-1(SEQ ID NO: 6), MPDY-1 sh (SEQ ID NO: 12) and PDY-1(SEQ ID NO: 13) reduce cytokine secretion from LPS and TNF α -activated keratinocytes. FIGS. 24 to 27 show that the peptide inhibitors MPDY-1(SEQ ID NO: 6), MPDY-1 sh (SEQ ID NO: 12) and PDY-1(SEQ ID NO: 13) reduce cytokine secretion from IL-17A-activated keratinocytes.

Table 3 summarizes the results of cytokine action and source according to HO/02/10.

Table 3: effect of HO/02/10 on stimulated mouse-derived cells

Various other PKC α inhibitors have also been shown to decrease cytokine secretion in activated keratinocytes. To determine their effect, keratinocytes were derived from neonatal BALB/C mouse skin. Cells were cultured on 24-well plates for 5 days. The cells were then cultured with PBS-/-as a control or stimulated with LPS, TNF α or IL-17. PKC α inhibitors were added as indicated. Media containing secreted cytokines were collected after 48 hours and analyzed using ELISA. Figure 50 is a tabular overview of cytokine secretion. The PKC α inhibitors MPDY-1(SEQ ID NO: 6), AIP-2(SEQ ID NO: 8), AIP-1(SEQ ID NO: 9), AWOT (SEQ ID NO: 7) and PPDY-1(SEQ ID NO: 10) have all been shown to be effective in reducing cytokine secretion in keratinocytes.

HO/02/10 was also shown to attenuate T cell infiltration into the skin. The effect of HO/02/10 on T cell infiltration was studied in vivo using anti-CD 3 specific staining.

As can be seen in figure 28, HO/02/10 down-regulated T cell infiltration into the dermis and epidermis during the inflammatory phase. In particular, HO/02/10 inhibited the infiltration of T cells into the epidermis, which suggests additional anti-inflammatory properties that also characterize psoriatic plaques. A 2cm slit was made as described above. Animals were treated daily with HO/02/10 (n-12). Immunohistochemical staining was performed after 9 days using anti-CD 3 antibody. Fig. 28B is a histogram comparing the number of cells per domain of CD3 positive staining. The effect was statistically significant at concentrations of 1. mu.g/ml and 10. mu.g/ml, with 1. mu.g/ml treatment showing a stronger effect than 10. mu.g/ml.

HO/02/10 was also shown to attenuate neutrophil infiltration into the skin (FIG. 31). The effect of HO/02/10 on neutrophil infiltration was studied in vivo using neutrophil specific staining. A 2cm slit was made as described above. Animals were treated daily (n ═ 6) with DPBS-/- (control) or PKC α inhibitors at specific concentrations. Five days later, the mice were sacrificed and skin tissues were embedded in paraffin and immunohistochemical staining for neutrophils was performed. Although a dose-dependent trend was observed, the results were not statistically significant.

PKC δ activators have also been shown to have anti-inflammatory effects on keratinocytes and splenocytes. Keratinocytes were derived from neonatal BALB/C mouse skin. Cells were cultured on 24-well plates for 5 days. Cells were then cultured with PBS-/-as a control or stimulated with LPS or TNF α. The PKC delta inhibitor DAP-1(SEQ ID NO: 34) was added. Media containing secreted cytokines were collected after 48 hours and analyzed using ELISA. Figure 43 is a tabular summary showing cytokine secretion in splenocytes stimulated with LPS. Figure 44 is a tabular summary of cytokine secretion in keratinocytes stimulated with TNF α. DAP-1 was shown to significantly reduce inflammatory cytokine secretion in both keratinocytes and splenocytes.

PKC epsilon inhibitors have also been shown to have anti-inflammatory effects on keratinocytes. Keratinocytes were derived from neonatal BALB/C mouse skin. Cells were cultured on 24-well plates for 5 days. Cells were then cultured with PBS-/-as a control or stimulated with LPS or TNF α. The PKC epsilon inhibitors EPIP-1(SEQ ID NO: 20), EPIP-2(SEQ ID NO: 21) or EPIP-4(SEQ ID NO: 23) were added. Media containing secreted cytokines were collected after 48 hours and analyzed using ELISA. Fig. 45-48 show the results of specific cytokine secretion and fig. 49 is a tabular summary of cytokine secretion for different PKC epsilon inhibitors. Several PKC epsilon inhibitors have been shown to significantly reduce inflammatory cytokine secretion in keratinocytes.

In summary, the mechanism of action of PKC isoform inhibitors and activators has been identified, suggesting their use as effective treatments for inflammation and inflammatory diseases. These peptides show 1) normalization of epidermal differentiation marker expression by reducing terminal differentiation; 2) attenuation of abnormal hyperproliferation; 3) regulating skin structure and increasing skin strength; and 4) downregulation of inflammation by differentially affecting different cell types recruitment and activation at different steps of the inflammatory process.

Figure 30 shows a summary depicting the overall effect of PKC isoform inhibitors and activators of the present disclosure on relevant pathways of skin inflammation and psoriasis. The summary outlines the inhibitory effect of the inhibitors and activators on various cell types and inflammatory stages in the skin. PKC isoform inhibitors and activators inhibit the secretion of proinflammatory cytokines (e.g., IL-1, IL-6, and TNF α) by own skin immune cells. Thus, a decrease in endothelial cell and keratinocyte activation is obtained, resulting in a significant decrease in ICAM-1 expression, chemokine secretion, and a decrease in infiltration of granulocytes into sites of inflammation, including neutrophils, macrophages, and T cells. Cytokines involved in the development and progression of the Th1 and Th17 pathways, two important pathways in psoriasis, are also down-regulated.

Example 7

Treatment of multiple sclerosis using PKC α and PKC η inhibitors.

Animal models of multiple sclerosis: since CNS tissue is not easily sampled, a large number of models are developed and commonly used in the literature in order to obtain information about the disease mechanism. These models reportedly include myelin mutants, chemically-induced lesions, viruses, and autoimmune models that mimic the clinical state and pathology of MS (Baker et al (2007) ACNR6 (6): 10-12). Among these models, Experimental Allergic Encephalomyelitis (EAE) is reported to be the most commonly used model of MS (Baker et al (2007) ACNR6 (6): 10-12).

Autoimmune models of multiple sclerosis: experimental Allergic Encephalomyelitis (EAE) apparently has received the most attention as a model of MS disease and progression, and is among the therapeutic strategies commonly used to detect MS (Baker et al (2007) ACNR6 (6): 10-12).

This disease model shows many clinical and histological features of MS and is reported to be caused by induction of autoimmunity to antigens naturally or artificially expressed in the CNS (Lavi et al (2005) ISBN0-387-25517-6 and Owens et al (2006) Adv Neurol 98: 77-89). Following sensitization to myelin antigens, the animals appeared to be ill, characterized by paralysis of the limbs. This is associated with blood brain barrier dysfunction, infiltration of monocytes into the CNS and block of conduction leading to impaired neurotransmission.

EAE is polygenic, with susceptibility and clinical course appearing to vary depending on the immunizing antigen and the strain/species of the animal being studied. MOG, small myelin protein, appears to induce chronic paralytic EAE in C57BL/6 mice. EAE is not a single model, but a number of models of varying degrees of pathology with similar changes to MS (Lavi et al (2005) ISBN 0-387-.

To test the effect of several PKC inhibitors on development and clinical condition of EAE mice, various studies were performed using the following experimental procedure. In particular, the peptide PKC α inhibitor MPDY-1(SEQ ID NO: 6) and the peptide PKC η inhibitor MPE-1(SEQ ID NO: 28) were evaluated.

Female 8-10 week old C57BL/6J mice were used for the study. The total number of groups is 7 (n-7 per group); the total number of animals was 49. After anesthesia, mice were immunized with MOG 35-55/CFA. Mice were immunized subcutaneously (s.c.) in the lateral abdomen with 200g of MOG35-55/CFA supplemented with 300 μ g of tuberculosis (Mt) H37RA (Difco). Pertussis Toxin (PTX) was injected intravenously (i.v.) at immunization time and 48 hours later. The need for secondary immunizations was determined based on a preliminary calibration experiment (20 mice) using synthetic MOG35-55 peptide (performed as a calibration procedure prior to treatment experiments).

The treatment was administered as follows. Mice were treated three times a week by i.p (intraperitoneal) injection (200 μ l/injection) starting on the day of immunization.

Clinical observations and scoring were performed at an observation period of 6 days/week for 49 days. Body weight was measured before immunization and twice weekly thereafter. As shown in table 4 below, active EAE was scored on a scale of 0-6 depending on the clinical performance that the mice could quantify.

Table 4: EAE score

Score of	Damage or injury
		0	Non-damage
1	Weakness of the tail
		2	Weakness of the tail and paresis of the hind limbs
3	Greater than or equal to 1 hind limb paresis
		4	Whole hind limb and hind body paresis
5	Posterior paresis and anterior paresis
		6	Death was caused by death

Groups of mice were treated as shown in table 5.

Table 5: treatment regimens

Group of	Treatment of
		1	DPBS-/-(control)
2	CRAMP2mg/kg(CRAMP)
		3	CRAMP0.2mg/kg(CRAMP)
4	MPDY-10.1mg/kg
		5	MPDY-11mg/kg
6	MPE-10.1mg/kg
		7	MPE-1mg/kg

The results are shown and summarized in FIGS. 33-37.

As can be seen from FIG. 33, mice treated with MPDY-10.1mg/kg showed signs of disease beginning on day 13 of the experiment two days later than the control group. In most of the days of the experiment, mice in the MPDY-10.1mg/kg group showed lower scores compared to mice in the control group. Moreover, none of the mice in the MPDY-10.1mg/kg and MPE-10.1 mg/kg groups died in the experiment (score 6), while in all other groups the mice died over the course of time (control group-first mouse died on day 34 of the experiment and the other on day 35). A summary of all mouse deaths is provided in table 6 below.

Table 6: overview of mouse death during treatment

Group of	Number of mice dead during 49 days	Details (days)
			1	2	34，35
2	2	23，44
			3	2	27，38
4	0
			5	2	16，38
6	0
			7	4	20，33，37，35

A summary of the scores from figures 33-37 at particular time points is shown in table 7.

Table 7: overview of specific time Point scores

Group of	The initial day	Score at day 18	Score on day 29	Score at day 37
					Control	11(0.285)	3.000	2.357	2.857
CRAMP0.2mg/kg	10(0.571)	3.214	2.000	2.571
					CRAMP2mg/kg	11(0.143)	2.643	2.643	2.143
MPDY-10.1mg/kg	13(1.000)	2.571	2.000	1.500
					MPDY-11mg/kg	11(0.214)	3.143	3.286	3.071
MPE-10.1mg/kg	11(0.429)	2.857	1.929	1.429
					MPE-11mg/kg	10(0.429)	3.071	4.286	4.286

The treated groups 4 and 6 showed resistance to the development of clinical symptoms of the disease, including no mortality until day 49 of the experiment. MPDY-1 and MPE treatment at a concentration of 0.1 μ g/ml (group 4) reduced EAE severity and protected mice from lethal EAE observed later in disease in control animals, thus resulting in a reduction of the mean group score (1.93 for MPDY-1; 2.00 for MPE-1) to below the mean of the control group scores (2.86) at the end of the experiment (day 42). MPDY-1 treatment at a concentration of 0.1 μ g resulted in a two-day delay in the development of clinical conditions compared to EAE in the control group. Thus, MPDY-1 and MPE appear to be effective agents for the treatment of MS.

Example 8

In vivo assessment of pruritus treatment

A prick test model using histamine to assess pruritus as shown in figure 8 was developed. The forearms of each subject were injected with histamine solution and placebo. The formulation of example 4 was applied topically with different concentrations of MPDY-1 and evaluated for pruritus over the course of time as shown in fig. 39-42.

The test is performed by placing a drop of a solution containing the possible allergen of the skin on the skin and a series of abrasions or needle punctures to allow the solution to enter the skin. The extract is passed into the outer layer of the skin (epidermis) using a fine needle (e.g., a 26G disposable needle). This test is painless and usually does not cause bleeding because the needle merely scrapes the surface of the skin. If the skin appears red, swollen, itchy areas (called wheal), it is the result of an allergic reaction to allergens. This is called a positive reaction. A drop of the extract is introduced through a fine needle (e.g., a 26G disposable needle). The test resulted in no discomfort and minimal trauma so that control and negative experiments only showed the site of puncture (if any).

A26G needle was used to introduce a stock of histamine (Histamrol positive control histamine, 1mg/ml, code # HIST14999V, Trupham). In a double-blind randomized trial, the test was performed on healthy volunteers. The formulation was applied to the forearm. Three treatment areas were selected and marked (the surface of the forearm from elbow to wrist divided laterally into three sections, proximal, middle and distal); the area is punctured prior to the following treatment.

One area was treated with the active formulation in a double-blind fashion for 10 minutes-labeled a.

One area was treated with placebo in a double-blind fashion, 10 minutes-labeled C.

Color photographs were taken 10, 20, 30 minutes after zero (T0).

The subject responded to the pruritus questionnaire 5 and 15 minutes after treatment.

In one study, subjects were tested with the treatments shown in tables 8 and 9.

Table 8: prick test sample set

	Left forearm	Right forearm
			Subject 1	A5	C2
Subject 1	A4	C1
			Subject 2	A2	C3
Subject 2	A5	C2
			Subject 3	A4	C1
Subject 3	A1	C3

Table 9: prick test treatment

Treatment of	Group of
		MPDY-11μg/ml	A1
MPDY-110μg/ml	A2
		Cream 10ppm	A4
Gel 10ppm	A5
		Cream W/O active material	C1
Gel W/O active material	C2
		DPBS	C3

Subjects with forms of pruritus sensation are provided and queried at different time intervals to indicate the level of pruritus for sensations from 0 (no response) to 4 (uncontrollable pruritus). The results are shown in Table 10 below.

Table 10: results

Furthermore, as is evident in fig. 39-42, the application of MPDY-1 over time significantly attenuated redness, inflammation, and pruritus compared to controls.

While the subject matter of the present disclosure has been described with reference to the above embodiments, it is to be understood that modifications and variations are included within the spirit and scope of the present disclosure. Accordingly, the disclosure is to be limited only by the following claims.

Example 9

Combining insulin and a PKC α inhibitor avoids adverse side effects resulting from treatment with insulin alone.

Wounds were made by incision on the back of 8-10 week old C57BL mice and treated with vehicle (PBS) control or with 1 μ M insulin (human monocomponent insulin, Eli Lilly, USA) or 1 μ M insulin with 1 μ M of SEQ ID NO: the mixture of PKC α inhibitor combination of 6 was treated daily for 7 days. After 7 days of trauma, all mice were sacrificed and the treated wounds were histologically analyzed for epidermal (proliferating cell nuclear antigen PCNA), angiogenesis, inflammation, proliferative capacity of epidermal cells and remodeling process at the wound opening.

As shown in table 11, treatment with insulin alone resulted in a large increase in the incidence of abnormal angiogenesis in the wound area compared to controls with PBS (60% and 25%, respectively). Since the wound healing process involves rapid proliferation of epidermal cells, these increased angiogenesis may also increase the risk of impaired wound closure by delaying the formation of normal granulation tissue. On the other hand, when insulin is identical to SEQ ID NO: 6, no abnormal angiogenesis was observed in the treated wound area.

Table 11: effect of insulin alone and insulin in combination with PKC α inhibitors on the severity of angiogenesis in a wound area.

Furthermore, treatment with insulin alone results in increased inflammation, hypernormal proliferation of epidermal cells, delayed differentiation of the spinous layers of epidermal cells, and increased scaling. When PKC α inhibitors are combined with insulin, no one of the adverse side effects of insulin therapy alone is observed.

Example 10PKC alpha inhibitors reduce wound inflammation

In wounds, a late and severe inflammatory response may inhibit the healing process, and thus preventing such inflammatory development may promote the wound healing process. Thus, the effect of PKC α inhibitors HO/02 and insulin on wound inflammation was tested in the following experiments.

Wounds were made on the backs of C57BL mice by incision and were treated daily for 7 days with the following: (i) PBS, control; (ii)1 μ M of SEQ ID NO: 6, a PKC α inhibitor; (iii)1 μ M insulin (human mono-component insulin, Eli Lilly, USA); or (iv) a mixture of 1 μ MPKC α inhibitor and 1 μ M insulin. Seven days after the trauma, all mice were sacrificed and the inflammation of the treated trauma was observed under a microscope. The resulting incidence of severe inflammation observed in the wound area is summarized in table 12.

As shown in table 12, administration of PKC α inhibitors to wounds resulted in a substantial (33.3%) reduction in the incidence of severe wound inflammation when compared to controls. Insulin alone has no anti-inflammatory effect under the experimental conditions.

TABLE 12

Treatment of	Incidence of Severe inflammation in wounds (%)
		PBS control	60
PKC alpha inhibitors	40
		Insulin	56
PKC alpha inhibitor + insulin	50

These results indicate that PKC α inhibitors may be used in the treatment of severe inflammation to control trauma. Confirmed SEQ ID NO: the ability of PKC α inhibitors of 6 to reduce inflammation, along with their ability to promote epidermal closure, dermal closure, and spatial differentiation of epidermal cells, makes them potentially the most effective therapeutic agents for wound healing.

Example 11 Effect of insulin in combination with PKC α inhibitor HO/02 in wound healing in STZ-induced diabetic mice

Full skin incisions (20mm) were made in the upper back of anesthetized Streptozotocin (STZ) (175mg/kg body weight) injected diabetic, citrate buffer injected (6 mice/group) non-diabetic and non-injected (6 mice/group) C57BL/6J mice. Periodic blood glucose monitoring was performed after STZ injection by measuring glucose levels from the tail vein. Only mice exhibiting blood glucose levels above 450mg/dl were included in the study. After incision, the wound was closed by applying PBS (7/6 mice/group), insulin (0.1 unit/ml) (6 mice/group), SEQ ID NO: PKC α inhibitor of 6 (1 μ g/m1) (7/5 mice/group) or a PKC α inhibitor comprising insulin and N-myristoylation of SEQ ID NO: 6 formulations of PKC α inhibitors (referred to herein as HO/03/03) (7/6 mice/group) were treated daily for STZ-induced diabetic wounds. Biopsies were collected 9 days after trauma. Wounds were excised from euthanized animals to assess wound healing parameters by histology and immunohistochemistry.

Epidermal closure of the wound was assessed by keratin 14 staining. Wound closure was considered when intact epidermal staining across the wound gap was observed. The dermis is considered to contract when both dermal edges are visible in a fixed domain at x100 magnification using H & E staining. Epidermal differentiation was assessed by keratin 1 staining. Wounds that showed positive staining across the entire wound gap were considered differentiated (K1 positive).

As shown in table 13, STZ-induced diabetic animals were impaired in wound healing. Comparison of important wound healing parameters in treated diabetic, untreated diabetic and non-diabetic mice shows that the group treated with formulation HO/03/03 shows a synergistic healing effect. The synergy was evident in all critical stages of healing including epidermal closure (71% vs. 17% p < 0.05 in the diabetic control), epidermal differentiation (28% vs. 0%) and dermal contraction (33% vs. 0%).

The organization of the subcutaneous tissue is assessed by the presence (or absence) of subcutaneous tissue at the two lateral edges of the wound. Granulation tissue formation was assessed by the presence of fibroblasts and collagen fibers in the wound bed. Wounds were considered positive for granulation tissue formation when a continuous layer of granulation tissue was present in the wound gap. Furthermore, we have defined 3 specific histological parameters characterizing severe inflammation in diabetic wounds: (i) abscess formation in the wound area, (ii) excessive leukocytosis (> 100 cells in fixed domain (x 200)) and (iii) high White Blood Cell (WBC)/Red Blood Cell (RBC) ratio in the vessels shown in fixed domain (x 200). When at least 2 of these parameters are present at the wound gap, the wound is considered severely inflamed.

Table 13: histological analysis of wound healing parameters 9 days after wound

Quantitative analysis of healing parameters was performed as described above.

Results are expressed as percentage of wounds in each group.

Table 14: histological analysis of wound healing parameters 9 days after wound

Quantitative analysis of healing parameters was performed as described above.

Results are expressed as percentage of wounds in each group.

As shown in table 14, diabetic animals showed damage on other wound healing parameters (e.g., organization of subcutaneous tissue at the wound edge, inflammation, and granulation tissue formation). Impaired healing parameters associated with diabetes, as evidenced by organization of subcutaneous tissue at the edge of the wound gap (43% versus 25%) and granulation tissue formation (86% versus 42%), were corrected by treatment with formulation HO/03/03. Treatment with HO/03/03 reduced the inflammatory response in the wound gap (28% vs. 67%). The healing effect of the formulation alone (insulin or the peptide of SEQ ID NO: 6) showed only partial healing effect.

The results outlined above demonstrate that formulation HO/03/03 exhibits synergy in overcoming diabetes-related healing lesions over multiple healing parameters.

Example 12 Effect of insulin in combination with PKC α inhibitor HO/02 in porcine skin model in wound healing in vivo

Several wound healing studies were performed in a porcine skin model system to further understand the wound healing process and the healing role of formulation HO/03/03.

A full 35-40mm skin incision was made in the back of anesthetized female swine (5 months of age, 60-70 kg). Ten symmetrical incisions were made on both sides of the dorsal region at the same distance from the spinal column (20 wounds total). The wound area was identified by applying 1m1PBS directly to the wound area (10 groups per group) or SEQ ID NO: a formulation of 1 μ g/ml of PKC α inhibitor (referred to herein as HO/03/03) (10 groups per group) was used to treat wounds twice a day per day. Wounds were excised from animals sacrificed on days 7 and 22 post-wound and morphologically, histologically and immunohistochemically evaluated.

As shown in table 15, treatment with formulation HO/03/03 accelerated wound healing by affecting epidermal migration and granulation tissue formation early in healing. Moreover, these wounds are large and susceptible to infection by environmental pathogens (animals are not kept in sterile conditions), however HO/03/03 appears to attenuate the inflammatory response at the wound gap thereby promoting the healing process.

Table 15: histological analysis of the utility of formulation HO/03/03 in pigs 7 days post-trauma.

Results are expressed as percentage of wounds in each group.

Table 16: histological analysis of the efficacy of formulation HO/03/03 in pigs 22 days post-trauma

Results are expressed as percentage of wounds in each group.

HO/03/03-treated wounds also exhibited accelerated healing when tested for the advanced healing stage. As shown in table 16, no difference was noted on epithelial closure, but dermal contraction and epidermal differentiation were significantly higher in the treated wounds. It is important to emphasize that these wounds are performed on the same animal, thereby contributing to the healing effect of the therapeutic marker.

It is readily understood from the results outlined above that formulation HO/03/03 promotes accelerated healing in the porcine skin model, affecting the healing parameters in the early as well as late stages of wound healing.

Claims (84)

1. A kit for treating an inflammatory disease or disorder in a subject, the kit comprising:

a) an inhibitor of PKC α, PKC epsilon, or PKC η; and

b) instructions for administering the PKC inhibitor to the subject.

2. The kit of claim 1, wherein the inhibitor is a polypeptide.

3. The kit of claim 2, wherein the polypeptide is between 5 and 20 amino acids in length.

4. The kit of claim 3, wherein the instructions specify that the polypeptide is administered parenterally, subcutaneously, intravenously, topically, orally, transmucosally, rectally, pulmonarily, nasally, or otically.

5. The kit of claim 3, wherein the instructions specify that the polypeptide is administered daily, weekly, biweekly, or monthly.

6. The kit of claim 1, wherein the inflammatory disease or disorder is selected from the group consisting of psoriasis, multiple sclerosis, rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, hashimoto's thyroiditis, myasthenia gravis, diabetes type I or II, asthma, inflammatory lung injury, inflammatory liver injury, inflammatory glomerular injury, atopic dermatitis, allergic contact dermatitis, irritant contact dermatitis, seborrheic dermatitis, sjogren's syndrome, keratoconjunctivitis, uveitis, inflammatory bowel disease, crohn's disease, ulcerative colitis, inflammatory diseases of joints, skin or muscles, acute or chronic idiopathic inflammatory arthritis, myositis, demyelinating diseases, chronic obstructive pulmonary disease, interstitial lung disease, interstitial nephritis and chronic active hepatitis.

Use of an inhibitor of PKC α, PKC epsilon, or PKC η in the manufacture of a medicament for treating an inflammatory disease or disorder.

8. The use of claim 7, wherein the inhibitor is a polypeptide.

9. The use of claim 8, wherein the polypeptide is between 5 and 20 amino acids in length.

10. The use of claim 9, wherein the polypeptide is administered parenterally, subcutaneously, intravenously, topically, orally, mucosally, rectally, pulmonarily, nasally, or otically.

11. The use according to claim 9, wherein the polypeptide is administered at a dose of about 0.1 to about 10000 micrograms per kilogram, preferably about 0.1 to about 1000 micrograms per kilogram and more preferably about 1.0 to about 50 micrograms per kilogram.

12. The use of claim 9, wherein the polypeptide is administered daily, weekly, biweekly, or monthly.

13. The use according to claim 7, wherein the inflammatory disease or disorder is selected from the group consisting of psoriasis, multiple sclerosis, rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, hashimoto's thyroiditis, myasthenia gravis, diabetes type I or II, asthma, inflammatory lung injury, inflammatory liver injury, inflammatory glomerular injury, atopic dermatitis, allergic contact dermatitis, irritant contact dermatitis, seborrheic dermatitis, sjogren's syndrome, keratoconjunctivitis, uveitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, inflammatory diseases of the joints, skin or muscles, acute or chronic idiopathic inflammatory arthritis, myositis, demyelinating diseases, chronic obstructive pulmonary disease, interstitial lung disease, interstitial nephritis and chronic active hepatitis.

14. A kit for treating an inflammatory disease or disorder in a subject, the kit comprising:

a) activators of PKC δ; and

b) instructions for administering to the subject an activator of the PKC δ.

15. The kit of claim 14, wherein the instructions indicate that the polypeptide is administered parenterally, subcutaneously, intravenously, topically, orally, transmucosally, rectally, pulmonarily, nasally, or otically.

Use of an activator of PKC δ in the manufacture of a medicament for treating an inflammatory disease or disorder.

17. The use of claim 16, wherein the polypeptide is administered parenterally, subcutaneously, intravenously, topically, orally, mucosally, rectally, pulmonarily, nasally, or otically.

18. The use of claim 16, wherein the polypeptide is administered at a dose of about 0.1 to about 10000 micrograms per kilogram, preferably about 0.1 to about 1000 micrograms per kilogram and more preferably about 1.0 to about 50 micrograms per kilogram.

19. The use according to claim 16, wherein the inflammatory disease or disorder is selected from the group consisting of psoriasis, multiple sclerosis, rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, hashimoto's thyroiditis, myasthenia gravis, diabetes type I or II, asthma, inflammatory lung injury, inflammatory liver injury, inflammatory glomerular injury, atopic dermatitis, allergic contact dermatitis, irritant contact dermatitis, seborrheic dermatitis, sjogren's syndrome, keratoconjunctivitis, uveitis, inflammatory bowel disease, crohn's disease, ulcerative colitis, inflammatory diseases of the joints, skin or muscles, acute or chronic idiopathic inflammatory arthritis, myositis, demyelinating diseases, chronic obstructive pulmonary disease, interstitial lung disease, interstitial nephritis and chronic active hepatitis.

20. A kit for treating itch in a subject, said kit comprising:

a) an inhibitor of PKC; and

b) instructions for administering the PKC inhibitor to the subject.

21. The kit of claim 20, wherein the inhibitor is an inhibitor of PKC α, PKC epsilon, or PKC η.

22. The kit of claim 21, wherein the inhibitor is a polypeptide.

23. The kit of claim 3 or 22, wherein the polypeptide comprises a sequence selected from SEQ ID NOs: 1-5, 14-19, 26, 27 and physiologically acceptable salts thereof, or a polypeptide selected from the group consisting of SEQ ID NOs: 6-13, 20, 21, 28 and 29.

24. The kit of claim 23, wherein the polypeptide comprises an N-terminal modification, a C-terminal modification, or a combination thereof, preferably the polypeptide is N-acylated. And more preferably, the polypeptide is N-myristoylated or N-palmitoylated.

25. The kit of claim 20, wherein the instructions specify that the polypeptide is to be administered orally.

26. The kit of claim 3 or 20, wherein the instructions specify that the polypeptide is administered at a dose of about 0.1 to about 10000 micrograms per kilogram.

Use of a PKC inhibitor in the manufacture of a medicament for the treatment of pruritus.

28. The use of claim 27, wherein the inhibitor is an inhibitor of PKC α, PKC epsilon, or PKC η.

29. The use of claim 28, wherein the inhibitor is a polypeptide.

30. The use of claim 9 or 29, wherein the polypeptide comprises a sequence selected from SEQ ID NO: 1-5, 14-19, 26, 27 and physiologically acceptable salts thereof, or a polypeptide selected from the group consisting of SEQ ID NOs: 6-13, 20, 21, 28 and 29.

31. The use of claim 30, wherein the polypeptide comprises an N-terminal modification, a C-terminal modification, or a combination thereof, preferably the polypeptide is N-acylated. And more preferably, the polypeptide is N-myristoylated or N-palmitoylated.

32. The use of claim 29, wherein the instructions specify that the polypeptide is to be administered orally.

33. The kit of claim 29, wherein the instructions specify that the polypeptide is administered at a dose of about 0.1 to about 10000 micrograms per kilogram.

34. A kit for treating itch in a subject, said kit comprising:

a) activators of PKC δ; and

b) instructions for administering to the subject an activator of the PKC δ.

35. The kit of claim 14 or 34, wherein the inhibitor is a polypeptide.

36. The kit of claim 35, wherein the polypeptide comprises a sequence selected from SEQ ID NOs: 33-30 and physiologically acceptable salts thereof, or a polypeptide selected from the group consisting of SEQ ID NO: 34-37.

37. The kit of claim 36, wherein the polypeptide comprises an N-terminal modification, a C-terminal modification, or a combination thereof, preferably the polypeptide is N-acylated. And more preferably, the polypeptide is N-myristoylated or N-palmitoylated.

38. The kit of claim 35, wherein the instructions specify that the polypeptide is to be administered orally.

39. The kit of claim 35, wherein the instructions specify that the polypeptide is administered at a dose of about 0.1 to about 10000 micrograms per kilogram.

40. The kit of claim 35, wherein the instructions specify that the polypeptide is administered daily, weekly, biweekly, or monthly.

Use of an activator of PKC δ in the manufacture of a medicament for the treatment of pruritus.

42. The use of claim 16 or 41, wherein the inhibitor is a polypeptide.

43. The use of claim 42, wherein the polypeptide comprises an amino acid sequence selected from SEQ ID NO: 33-30 and physiologically acceptable salts thereof, or a polypeptide selected from the group consisting of SEQ ID NO: 34-37.

44. The use of claim 43, wherein the polypeptide comprises an N-terminal modification, a C-terminal modification or a combination thereof, preferably the polypeptide is N-acylated. And more preferably, the polypeptide is N-myristoylated or N-palmitoylated.

45. The use of claim 42, wherein the polypeptide is administered orally.

46. The use of claim 42, wherein the polypeptide is administered at a dose of about 0.1 to about 10000 micrograms per kilogram.

47. The use of claim 42, wherein the polypeptide is administered daily, weekly, biweekly, or monthly.

48. A polypeptide consisting of SEQ ID NO: 3 or a physiologically acceptable salt thereof, wherein the polypeptide is N-myristoylated.

49. The polypeptide of claim 48, wherein the polypeptide is SEQ ID NO: 12.

50. a pharmaceutical composition comprising:

a) consisting of SEQ ID NO: 3 or a physiologically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein the polypeptide is N-myristoylated; and

b) a pharmaceutically acceptable carrier.

51. The pharmaceutical composition of claim 50, wherein the polypeptide is SEQ ID NO: 12.

52. consisting of SEQ ID NO: 3 or a physiologically acceptable salt thereof, in the manufacture of a medicament for the treatment of an inflammatory disease or disorder, wherein the polypeptide is N-myristoylated.

53. The use of claim 52, wherein the polypeptide is SEQ ID NO: 12.

54. a polypeptide comprising SEQ ID NO: 4 or a physiologically acceptable salt thereof.

55. The polypeptide of claim 54, wherein the polypeptide is N-myristoylated.

56. The polypeptide of claim 54, wherein the polypeptide is SEQ ID NO: 10 or 13.

57. A pharmaceutical composition comprising:

a) comprises the amino acid sequence of SEQ ID NO: 4 or a physiologically acceptable salt thereof; and

b) a pharmaceutically acceptable carrier.

58. The pharmaceutical composition of claim 57, wherein the polypeptide is N-myristoylated.

59. The pharmaceutical composition of claim 57, wherein the polypeptide is SEQ ID NO: 10 or 13.

60. Comprises the amino acid sequence of SEQ ID NO: 4 or a physiologically acceptable salt thereof in the manufacture of a medicament for the treatment of an inflammatory disease or disorder.

61. The use of claim 60, wherein the polypeptide is N-myristoylated.

62. The use of claim 60, wherein the polypeptide is SEQ ID NO: 10 or 13.

63. A polypeptide consisting of an amino acid sequence selected from SEQ ID NO: 30-33 or a physiologically acceptable salt thereof.

64. The polypeptide of claim 63, wherein the polypeptide is SEQ ID NO: 34-37.

65. A pharmaceutical composition comprising:

a) consisting of a sequence selected from SEQ ID NO: 30-33 or a physiologically acceptable salt thereof; and

b) a pharmaceutically acceptable carrier.

66. The pharmaceutical composition of claim 65, wherein the polypeptide is SEQ ID NO: 34-37.

67. Consisting of a sequence selected from SEQ ID NO: 30-33 or a physiologically acceptable salt thereof in the manufacture of a medicament for the treatment of an inflammatory disease or disorder.

68. The use of claim 67, wherein the polypeptide is SEQ ID NO: 34-37.

69. A kit for treating multiple sclerosis in a subject, the kit comprising:

a) an inhibitor of PKC α, PKC epsilon, or PKC η; and

b) instructions for administering the inhibitor to the subject.

70. The kit of claim 69, wherein the inhibitor is a polypeptide.

71. The kit of claim 70, wherein the polypeptide consists of a sequence selected from SEQ ID NO: 2. SEQ ID NO: 26 and physiologically acceptable salts thereof, or a polypeptide selected from the group consisting of seq id NO: 6 or SEQ ID NO: 28, wherein the polypeptide is N-myristoylated.

72. The kit of claim 70, wherein the instructions indicate that the polypeptide is administered intravenously, subcutaneously, or intraperitoneally.

73. The kit of claim 70, wherein the instructions specify that the polypeptide is administered at a dose of about 0.001 to about 50 micrograms per kilogram.

74. The kit of claim 70, wherein the instructions specify that the polypeptide is administered daily, weekly, biweekly, or monthly.

75. The kit of claim 70, wherein treatment of the subject results in a reduced number of contrasted lesions.

76. The kit of claim 70, wherein the subject has relapsing-remitting multiple sclerosis or secondary progressive multiple sclerosis.

77. use of an inhibitor of PKC α, PKC ε or PKC η in the manufacture of a medicament for the treatment of multiple sclerosis.

78. The use of claim 77, wherein said inhibitor is a polypeptide.

79. The use of claim 78, wherein the polypeptide consists of a sequence selected from SEQ ID NO: 2. SEQ ID NO: 26 and physiologically acceptable salts thereof, or a polypeptide selected from the group consisting of seq id NO: 6 or SEQ ID NO: 28, wherein the polypeptide is N-myristoylated.

80. The use of claim 78, wherein the polypeptide is administered intravenously, subcutaneously, or intraperitoneally.

81. The use of claim 78, wherein the polypeptide is administered at a dose of about 0.001 to about 50 micrograms per kilogram.

82. The use of claim 78, wherein the polypeptide is administered daily, weekly, biweekly, or monthly.

83. The use of claim 77, wherein treatment of the subject results in a reduced number of contrasted lesions.

84. The use of claim 77, wherein the subject has relapsing-remitting multiple sclerosis or secondary progressive multiple sclerosis.