Inflammation inhibiting compounds

Abstract

The present invention relates to the use of compounds selected from the group consisting of Lys-(D)Pro-Thr, N-acyl Lys-(D)Pro-Thr, C-amide Lys-(D)Pro-Thr, and C-esters of Lys-(D)Pro-Thr; or a pharmaceutically acceptable salt of said compound for the treatment of inflammatory disorders. The invention also relates to the use of ?MSH for inducing tolerance.

Claims

1. A method for treatment of inflammation resulting from a disorder selected from the group consisting of enterocolitis, allergic reactions, food allergies, rheumatoid arthritis, multiple sclerosis, asthma, contact allergies, rhinitis, atopic dermatitis, transplant rejection, scleroderma, and vasculitis, comprising the step of administering to a patient in need of such treatment, a pharmaceutical composition comprising a therapeutically effective amount of a compound selected from the group consisting of Lys-(D)Pro-Thr [K(D)PT] (KPT), N-acyl Lys-(D)Pro-Thr, C-amide Lys-(D)Pro-Thr, and C-esters of Lys-(D)Pro-Thr; or a pharmaceutically acceptable salt of said compound, wherein said therapeutically effective amount is in the range from 20 ?g/kg of body weight to 10 mg/kg of body weight.

2. The method according to claim 1, wherein said inflammation arises in the skin and said pharmaceutical composition is in the form of an ointment or cream.

3. The method according to claim 2, wherein said compound is present in the ointment or cream in a concentration of 1 ?M to 1 mM.

4. The method according to claim 1, wherein said pharmaceutical composition is formulated for intraperitoneal, intravenous or oral administration.

5. The method according to claim 1, wherein said pharmaceutical composition further comprises at least one additional compound selected from the group consisting of Lys-Pro, N-acyl Lys-Pro, C-amide Lys-Pro, C-esters of Lys-Pro, Lys-(D)Pro-Thr [K(D)PT] (KPT), N-acyl Lys-(D)Pro-Thr, C-amide Lys-(D)Pro-Thr, and C-esters of Lys-(D)Pro-Thr.

6. A method for the treatment of inflammation of the skin resulting from a disorder selected from the group consisting of allergic reactions, rheumatoid arthritis, contact allergies, rhinitis, atopic dermatitis and scleroderma and comprising the step of administering to a patient in need of such treatment, a medicament formulated for topical application, said medicament containing a pharmaceutical composition comprising a therapeutically effective amount of a compound selected from the group consisting of Lys-(D)Pro-Thr [K(D)PT](KPT), N-acyl Lys-(D)Pro-Thr, C-amide, Lys-(D)Pro-Thr, and C-esters of Lys-(D) Pro-Thr; or a pharmaceutically acceptable salt of said compound, wherein said compound is present in said preparation in a concentration of from 1 ?M to 1 mM.

7. The method according to claim 6, wherein said medicament is an ointment or cream.

8. The method according to claim 6, wherein said pharmaceutical composition further comprises at least one additional compound selected from the group consisting of Lys-Pro, N-acyl Lys-Pro, C-amide Lys-Pro, C-esters of Lys-Pro, Lys-(D)Pro-Thr [K(D)PT] (KPT), N-acyl Lys-(D)Pro-Thr, C-amide Lys-(D)Pro-Thr, and C-esters of Lys-(D)Pro-Thr.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows that intravenous injection of ?MSH, KPV or KPT suppresses the CHS sensitization phase.

(2) FIG. 2 shows that intravenous administration of ?MSH, KPV or KPT is able to induce tolerance.

(3) FIG. 3 illustrates IL-10 secretion by human PBL 24 hours after treatment with ?MSH, KPV or KPT.

(4) FIG. 4 illustrates IL-10 secretion by human PBL 48 hours after treatment with ?MSH, KPV or KPT.

(5) FIG. 5 shows that THP-1 cells express receptors for ?MSH.

(6) FIGS. 6A to 6D show that unlabeled ?MSH is able to displace biotin-labeled ?MSH from binding sites on THP-1 cells in a competitive assay.

(7) FIGS. 7A to 7D show that unlabeled KPV is able to displace biotin-labeled ?MSH from binding sites on THP-1 cells in a competitive assay.

(8) FIGS. 8A to 8D show that unlabeled KPT is able to displace biotin-labeled ?MSH from binding sites on THP-1 cells in a competitive assay.

(9) FIGS. 9A and 9B show the expression of cell adhesion molecules (CAMS) on the surface of HMEC-1 cells 24 hours after treatment by TNF?+?MSH (FIG. 9A) or TNF?+KPT (FIG. 9B).

(10) FIGS. 9C and 9D show the adhesion of lymphocytes to HDMEC (chromium release assay).

(11) FIG. 9C: molt4 T lymphocytes; FIG. 9D: JY B lymphocytes.

(12) FIG. 10 shows the effect of ?MSH, KP, KPV or KPT on NF-?B activation in LPS-treated HMEC-1 cells.

(13) FIG. 11A shows that the number of E selectin-expressing vessels in tissue sections is reduced by ?MSH treatment.

(14) FIG. 11B shows that the number of petechial lesions on the ears of LPS-treated mice is reduced by ?MSH treatment.

(15) FIG. 12 shows that in vitro treatment of BMDC with ?MSH or KP suppresses CHS and can induce tolerance.

(16) FIG. 13A shows that in an NF-?B band shift assay the intensity of the NF-?B p65/p50 heterodimer band is reduced by various ?MSH-derived peptides.

(17) FIG. 13B shows the effect of ?MSH, KP or K on the CHS reaction and the effect of ?MSH or KP on the induction of tolerance in Ba1bC mice.

(18) FIG. 14 shows the suppression of CHS by T cells which have been contacted in vitro with antigen-loaded and ?MSH or derivative-treated DC.

(19) FIG. 15 shows the induction of tolerance by T cells which have been contacted in vitro with antigen-loaded and ?MSH DC.

(20) FIGS. 16A and 16B show the up-regulation of CTLA-4 on T cells after contact with antigen-loaded and ?MSH or derivative-treated DC. FIG. 16A: CD4 positive T cells; FIG. 16B: CD8 positive T cells.

(21) FIG. 17 shows the effect of K(D)PT in the therapeutic treatment of DSS-Colitis.

(22) FIG. 18 shows significant reduction of histological signs of inflammation by K(D)PT.

(23) FIG. 19 shows significant therapeutic effects of rectally applied K(D)PT.

(24) FIG. 20 significant reduction of histological score and mucosal IL-1? expression in K(D)PT-treated animals.

(25) FIG. 21 shows that clinical signs of CD45RBhigh-transfer colitis are significantly reduced by K(D)PT-treatment.

(26) FIG. 22 shows the effect of KdPT on colitis in IL-10 deficient mice.

(27) FIG. 23 shows the effect of KdPT on Imiquimod-induced Psoriasis-like disease in mice.

(28) FIG. 24 shows the effect of KdPT on Psoriasis in a transplant model.

(29) FIGS. 25A and 25B further illustrate the effect of KdPT on Psoriasis in a transplant model.

(30) FIGS. 26A, 26B, and 26C show the effect of Effect of KdPT on the number of regulatory CD4+CD25+Foxp3+?T-cells (as measured by flow cytometry, A), as well as on the expression of Foxp3, IL-10, IFN? and TNF?(qRT-PCR, B).

(31) FIGS. 27A, 27B, 27C, and 27D show the effect of KdPT on CD4+CD25+Foxp3+?Treg in blood of psoriasis patients.

(32) FIGS. 28A and 28B show the suppressive effect of CD4+CD25+Foxp3+ T-cells after treatment with K(D)PT.

(33) FIGS. 29A, 29B, and 29C show the effect of KdPT on human Th17-cells and expression of Th17-typical parameters.

(34) FIGS. 30A and 30B show the effect of KdPT on the expression of murine Th17-typical parameters.

(35) FIGS. 31A, 31B, and 31C show the effect of KdPT on dendritic cells.

(36) FIGS. 32A and 32B show the effect of KdPT-treated DC on CD4+?T-cells.

(37) FIGS. 33A and 33B show that CD4+?T-cells display suppressive properties in vitro and in vivo after treatment with KdPT-treated DC.

(38) FIGS. 34A, 34B, and 34C show the effect of KdPT on CD8+?T-cells from DNFB-sensitised mice/contact allergy experiment.

(39) FIGS. 35A and 35B show the effect of KdPT on number and degranulation of human mast cells in the hair follicle.

DETAILED DESCRIPTION OF THE INVENTION

(40) The following examples are intended to explain the invention in more detail. As used herein, the term KdPT or KPT refers to the isomer (L)Lys-(D)Pro-(L)Thr.

(41) The experimental data below shows that the peptides KdPT and KP are effective in the treatment of multiple inflammatory disorders as they interfere with basic mechanisms of inflammation. In particular, Examples 7 and 8 make it plausible that KP and KdPT are effective against various autoimmune disorders. Examples 7 and 8 show that KdPT and KP lead to the induction of tolerant dendritic cells and subsequently to the generation of regulatory T cells. Antigen recognition by the dendritic cells and the resulting activation of T cells is of central importance for autoimmune disorders. KdPT and KP influence these steps such that the result is not an immune activation but a tolerance induction.

(42) Furthermore, KdPT has the effect that activation of transcription factor NF-?B (which is relevant for many inflammatory and immune reactions) is reduced, see Example 5. Also, this aspect supports the notion that KdPT is effective in the treatment of various different autoimmune disorders as well as allergies including enterocolitis, Crohn's disease, ulcerative colitis, psoriasis, rheumatoid arthritis, multiple sclerosis, vasculitis, allergic reactions, food allergies, asthma, contact allergies, rhinitis, atopic dermatitis, transplant rejection, scleroderma, fibroses and inflammatory disorder of the blood vessels. Autoimmune diseases particularly include but are not limited to multiple sclerosis, vasculitis, rheumatoid arthritis, psoriasis, Crohn's disease and ulcerative colitis.

Example 1

(43) Mice

(44) 7 to 10 week old female Balb/C mice were obtained from Charles River (Sulzfeld, Germany) and kept in compliance with government regulations.

(45) Administration of ?MSH or KPV or KPT or KP:

(46) ?MSH and the peptides were stored as aliquots at ?20? C. until used. Before the injection, the particular compound was dissolved in PBS, 0.1% mouse serum, and stored on ice until injected i.v. into the tail vein of the mice. 5 ?g of ?MSH or 1.5 ?g of peptide (KP: 50 ?g) per mouse were injected 2 hours before the sensitization.

(47) Determination of CHS and Tolerance:

(48) The mice were sensitized by spreading 75 ?l of 0.5% DNFB in acetone/olive oil (4:1) on the shaven abdomen of naive mice. CHS was induced by applying 10 ?l of 0.3% DNFB to the ears of the mice on both sides of one ear. CHS was determined by the degree of auricular swelling of the hapten-exposed ear compared with the other, control-treated ear and was measured using spring-loaded dividers 24 hours after hapten exposure. Mice whose ears were exposed to hapten without previous sensitization served as negative controls. In order to determine whether the injection of ?MSH or peptides before hapten administration leads to induction of tolerance, mice underwent i.v. injection (abdomen) or exposure (left ear) as described with ?MSH or peptides 2 hours before the sensitization. To confirm the ?MSH-induced suppression of CHS, mice were exposed to hapten on one ear 7 days after the sensitization, and the auricular swelling response was determined 24 to 36 hours later. 14 days later, the same mice were sensitized once again on the shaven back (now in the absence of exogenous ?MSH), and investigated for their ability to induce a CHS response by a second exposure to hapten on the right ear one week later.

(49) Topical preparations of ?MSH were used in some experiments. In these experiments, application to the mice took place at the sensitization site (abdomen) immediately before or 3 hours or 24 hours before the sensitization.

(50) Result:

(51) i.v. injection of ?MSH and of KPV or KPT or KP inhibits the ability of the mice to induce a CHS response to DNFB exposure taking place 7 days later. These mice thus developed no DNFB-specific sensitization. KPT suppressed the CHS response most effectively (see FIGS. 1 and 13B).

(52) In order to distinguish between temporary immunosuppression and specific immunological tolerance, mice were sensitized a second time and exposed to hapten. Mice injected with ?MSH or KPV or KPT before the first sensitization could not be sensitized even by administration of a second sensitizing dose of hapten, which indicates that these mice have developed tolerance to DNFB. KPV showed a weak effect, whereas ?MSH and KPT and KP inhibited the auricular swelling response very greatly (see FIGS. 2 and 13B).

Example 2

(53) Material and Methods:

(54) Mononuclear cells (PBMC) were separated from human buffy coats by Ficoll Hypaque density gradient centrifugation. Cells (1?106 per ml), cultivated in RPMI 1640 with antibiotics and 10% FCS, were either not treated or stimulated with ?MSH or the peptides KPV or KPT with or without IL-1? (10 U/ml). The supernatants of the PBMC cultures were collected after incubation for 24 or 48 hours and stored at ?20? C. until used further. A commercially available ELISA was employed to detect IL-10.

(55) Results:

(56) Human PBMC which were untreated or had been treated with various concentrations of ?MSH or peptides produced only low concentrations of IL-10 (5-10 pg/ml) after incubation for 24 hours. ?MSH (10.sup.?11 M), KPV (10.sup.?8 to 10.sup.?9M) and KPT (10.sup.?8 to 10.sup.?9M) evidently induced IL-10 production (see FIG. 3).

(57) The human PBMC produced significant amounts of IL-10 after incubation for 48 hours. ?MSH, KPV and KPT significantly increased the production of IL-10 by human PBLC. There was no essential difference between ?MSH and the peptides (see FIG. 4).

(58) The results which are shown prove that the peptide KPT is able, like ?MSH and KPV, to inhibit the sensitization of CHS after intravenous administration and to induce hapten-specific tolerance. KPT is also able to induce IL-10 in vivo and in vitro. The data also make it probable that the immunosuppressant effect of ?MSH in vivo depends not just on IL-10 induction.

Example 3

(59) Material and Methods:

(60) All the steps were carried out at 0 to 4? C. The monocytic cell line THP-1 was washed once in PBS, once one acidic glycine buffer (50 mM glycine, 100 mM sodium chloride, pH 3) and three times with RPMI. The cells 2.5?10.sup.6 per ml) were then resuspended in 100 ?l of RPMI/1% BSA and transferred into 96-well microtiter plates. After addition of biotin-labeled ?MSH (10.sup.?10 M), the cells were incubated at 4? C. for 1 hour, washed once with PBS, resuspended in 100 ?l of PBS/1% BSA and incubated with FITC-labeled streptavidin (40 ?g/ml) in the dark at 4? C. for 30 minutes. After a last washing step, the cells were resuspended in PBS. The amount of bound biotin-labeled ?MSH was analysed using a flow cytometer. In control experiments, the cells were incubated without biotin-labeled ?MSH but in the presence of FITC-streptavidin. Dead cells were excluded by adding propidium iodide shortly before the FACS analysis. The specificity of the binding of the biotin-labeled MSH was determined by adding unlabeled ?MSH (10.sup.?6 to 10.sup.?12M) or KPV or KPT (10.sup.?6 to 10.sup.?12M).

(61) Results:

(62) According to FACS analysis with biotin-labeled ?MSH, unstimulated THP-1 cells express significant amounts of binding sites which are specific for ?MSH compared with control mixtures incubated only with FITC-streptavidin. The concentration of ?MSH employed in this experiment was 10.sup.?10 M (see FIG. 5).

(63) In order to determine whether THF-1 cells express one of the known melanocortin receptors (MC), RT-PCR was carried out with MC-1-, MC-2-, MC-3- and MC-4-specific primers. Total RNA was obtained from THP-1 cells. A PCR product specific for MC-1 with an expected length of 416 bp was detected (Rajora et al., 1996, J. Leuk. Biol., 59, 248). PCR products specific for MC-2, MC-3 or MC-4 were not detected. The results show that THP-1 cells express MC-1 which, in contrast to other melanocortin receptors, is specific for ?MSH and ACTH.

(64) In order to investigate whether the binding sites expressed on THP-1 are specific for ?MSH, competition experiments were carried out with ?MSH or KPV or KPT.

(65) The specific binding was measured by incubating THP-1 cells with biotin-labeled ?MSH (10.sup.?10 M) and various concentrations of unlabeled ?MSH or peptides. Unlabeled ?MSH in a concentration of 10.sup.?6 significantly suppressed ?MSH binding. No significant suppression was observable when ?MSH was employed in concentrations of 10.sup.?6M, 10.sup.?10 M or 10.sup.?12M (FIGS. 6A to 6D).

(66) When unlabeled KPV was employed, a significant inhibition was observable only at a concentration of 10.sup.?6 M (see FIGS. 7A to 7D).

(67) In the case of the peptide KPT, a significant inhibition of ?MSH binding was observable at each of the tested concentrations. (10.sup.?6 to 10.sup.?12M, see FIGS. 8A to 8D).

(68) These results show that the KPT peptide binds to the melanocortin receptor on THP-1 cells which is specific for ?MSH, which indicates that ?MSH and KPT have a common binding site. However, since KPT shows competition for receptor even at very low concentrations, it is probable that this peptide in fact has a higher affinity for MC-1 receptor than MSH.

Example 4

(69) Material and Methods:

(70) Human dermal microvascular endothelial cells (HDMEC) and the cell line HMEC-1 (human microvascular endothelial cell line 1) were treated either with TNF? or LPS in the presence or absence of one of the peptides. The cells were harvested for RNA isolation after 3 and after 6 hours after treatment or harvested either for adhesion molecule ETA or FACS analysis 3, 6, 16 or 24 hours after treatment. RNA underwent reverse transcription, and samples were subjected to a PCR for E selectin, ICAM-1, VCAM or for ?-actin as housekeeping gene in order to carry out a semiquantitative determination. For the lymphocyte adhesion assay, the endothelial cells were seeded in dishes and incubated with .sup.51Cr-labeled lymphocytes. After a washing step, the amount of remaining lymphocytes bound to the EC layer was determined by measuring the radioactivity in the samples.

(71) Results:

(72) Treatment of the endothelial cells with ?MSH or KPT inhibited the LPS- or TNF?-induced expression of adhesion molecules. This effect was observed in a concentration range from 10.sup.?6 to 10.sup.?12 M ?MSH or peptide. The peptide KPT had the strongest effect on the expression of adhesion molecule mRNA.

(73) The LPS- or TNF?-induced surface expression of adhesion molecules was reduced to, a small extent by all the agonists. These data were obtained both by EIA, in which case whole cells were employed, and by FACS with specific antibodies (see. FIGS. 9A and 9B, which show EIA data).

(74) ?MSH significantly reduces the binding of T and B cells to LPS- or TNF-?MSH-treated EC layers (see FIGS. 9C and 9D). Taken together, these results show that ?MSH has an effect on the adhesion of lymphocytes to EC and thus also reduces the extravasation of lymphocytes in conditions of tissue inflammation. This is supported by the in vivo data on localized vasculitis.

Example 5

(75) Material and Methods:

(76) Epidermal cells (ECs) or normal human keratinocytes (HNK) were treated with IL-1, LPS or TNF? in the presence or absence of peptides. After 15 or 30 minutes, the nuclear proteins were obtained and subjected to an electrophoretic mobility shift assay (EMSA) with radiolabeled oligonucleotide with NF-?B-specific binding sequence. Unlabeled oligonucleotide was used as competitor. In some experiments, antibodies against the p65 or p50 chain of NF-?B were used in order to confirm the identity of the detected bands as either p50 homodimer or p50/p65 heterodimer.

(77) Result:

(78) Addition of the peptides in TNF?- or LPS-treated ECs and in IL-1-treated HNKs leads to a reduced activation of the transcription factor NF-?B (see FIGS. 10 and 13A). This in turn leads to a diminution in the transcription of the genes for numerous proinflammatory mediators (cytokines, chemokines, adhesion molecules, etc.). The identity of the observed bands in the EMSA as NP-?B heterodimer was confirmed by using anti-p65 antibody.

Example 6

(79) Material and Methods:

(80) Mice were treated with LPS by s.c. injection on one ear. This preparatory injection induces a long-lasting rise in E selectin expression at the site of the LPS injection. 24 hours later, a second LPS dose was injected i.p. (challenge). This second LPS injection leads to rapid vessel necrolysis and to the formation of petechial lesions which are easily measurable because of their size and number. ?MSH (25 ?g) was administered at the time of the preparatory LPS injection.

(81) Result:

(82) Injection of ?MSH at the time of the preparatory LPS administration inhibits the induction of local E selectin expression in the ear (see FIG. 11A) and significantly reduces the number and size of the petechial lesions formed after the challenge injection of LPS (see FIG. 11B).

Example 7

(83) Material and Methods:

(84) Bone marrow dendritic cells (BMDC) were isolated from the femoral bones of mice and treated with IL-4 and GMCSF for 6 or 9 days. On day 6 or 9, the cells were treated with ?MSH (2?10.sup.?11 M) or the peptide KP (2?10.sup.?6M) 3 hours and 2.5 hours before reinjection into naive mice with the same genetic background. 2 hours before reinjection, the cells were treated with hapten (1 mM DNBS, the water-soluble form of DNFB). Immediately before the reinjection, the cells were washed 2? with PBS. 5?10.sup.?5 cells were injected i.v. into each animal. Control cells were treated either with DNBS alone or with ?MSH alone or were left untreated. 5 days after injection, the animals were contacted with DNFB on the ear, and the ear thickness was measured the next day. 2 weeks later, the animals were resensitized with DNFB and again recontacted on the ear 5 days later. Finally, the auricular swelling was measured.

(85) Results:

(86) Recipient animals injected with untreated cells or ?MSH-treated cells showed no immune response after the first challenge, as expected. Recipient animals injected with DNBS-treated BMDC showed an appropriate CHS reaction at the time of the first challenge. This reaction was suppressed in animals injected with cells previously treated with TNBS and ?MSH or TNBS and KP in vitro. Thus, contact of DCs with ?MSH or peptide is sufficient to induce inhibition of CHS (see FIG. 12).

(87) At the time of the second challenge and of the corresponding resensitization, animals again injected with DNBS-treated cells showed no immune response, whereas animals injected with DNBS/?MSH-treated cells showed no immune response, which indicates that the ?MSH-induced tolerance is likewise mediated by DC (see FIG. 12).

Example 8

(88) Immunotherapy with ?MSH or ?MSH-Derivative-Treated Dendritic Cells or T Cells

(89) Dendritic cells (DC) were isolated (from blood, bone marrow or tissue). It is, however, also possible to use cell mixtures containing DC (e.g. epidermal cell mixtures) and cultivated in the presence of GM-CSF and IL-4 (preferably: 250-1000 ?/ml for each of the substances).

(90) After a maturation period (preferably 6-9 days), the cells are loaded with antigen (concentration depends on the particular antigen, likewise period) and treated with ?MSH or derivatives thereof. The derivatives correspond at least to amino acids 12 and 13 of ?MSH (Lys-Pro), with preference for Lys-Pro-Val-containing derivatives. D and L configurations of the AA are possible, likewise conservative AA exchanges. This leads inter alia to the possibility also of using Lys-Pro-Thr which is derived from IL-1?, and derivatives thereof with N-terminal extensions. Derivatives with C-terminal extensions can also be employed. Addition of the peptide can take place before addition of the antigen, at the same time, later, once or more than once (the preferred dose depends on the particular peptide, for ?MSH e.g. 10.sup.?8 M to 10.sup.?14 M).

(91) The cells treated in this way are then injected i.v. into the recipient organism (i.p. or s.c. would also be possible); mouse: 2?10.sup.5 cells approximate lower limit. Depending on the antigen, it is sufficient to undertake a single injection or necessary to undertake a plurality of injections in this case. It is also possible that the injections need to be repeated after lengthy periods (no data yet available on this).

(92) An alternative possibility is to bring DC into contact with T cells outside the body and then to inject the mixture or the T cells. In this case, the antigen loading of the DC can take place before the contact with the T cells or during it. The T cells may moreover originate from individuals which have already been sensitized to the particular antigen. The lower limit in the mouse is about 1 million T cells, with more cells being preferred (FIGS. 14 and 15).

(93) Advantages of such a mode of use are the prevention of every type of unwanted immune response, which are antigen-specific and in which antigen-specific lymphocytes (B or T cells) play a pathogenetic part.

(94) These include inter alia allergies, autoimmune diseases, chronic inflammations or implantations. A cure of preexistent disorders is also possible if sufficiently large numbers of cells are employed.

(95) The surprising results can, without wishing to be bound to one theory, be regarded as the fact that ?MSH is a potent immunomodulator and has numerous antiinflammatory properties. These include inter alia its property of reducing the expression of costimulatory molecules on DC. Similar properties are also shown by the ?MSH derivatives of the invention, including the C-terminal tripeptide and the dipeptide Lys-Pro. Derivatives with a different amino acid composition (conservative AA exchanges) also have comparable properties, and these include in particular the Lys-Pro-Thr derived from IL-1?(so that it is to be presumed that N-terminally (analogous to ?MSH) extended peptides with sequence colinear to IL-1? have the same effect).

(96) ?MSH, as well as the derivatives, are able to induce hapten-specific tolerance in vivo. DC are professionally antigen-presenting cells which are able to induce numerous types of immune responses and which also determine the course of such responses. These immune responses include in particular the T-cell-mediated immune responses.

(97) It has now been possible to show that in vitro treatment of DC or DC/T cell mixtures with an antigen in the presence of ?MSH or derivatives leads to the cells likewise inducing hapten-specific tolerance after injection into an organism.

(98) The mechanism in this case appears to be that the antigen presentation by the DC is modulated by ?MSH or derivatives in such a way that suppressor T cells are generated. It was thus possible to show that T cells in appropriate mixtures show high expression of CTLA-4 (FIGS. 16A and 16B). This is one of the surface molecules which characterize suppressor T cells.

(99) It is possible with DC or T cells generated in this way to preventively impede autoimmune diseases, chronic inflammations or allergies. A cure of a preexistent pathological condition is also conceivable if sufficiently large numbers of cells are used.

(100) The compounds of the invention can also be used for tumor treatment by means of an in situ activation of dendritic cells. It is also possible that this method can be used for tolerization in the presence of the peptides of the invention.

Example 9

(101) Therapeutic Treatment of Colitis Using K(D)PT

(102) Colitis in C57BL/6 mice was induced using dextrane sodium sulfate (2% DSS in drinking water for 5 days); 1st set-up: i.p. application of 10 ?g K(D)PT beginning on day 4 of DSS application; 2nd set-up: rectal application of 10 ?g K(D)PT beginning one day prior to DSS application; control animals were treated with PBS; and observed parameters were body weight, histology, IL-1? expression.

(103) In another study of chronic CD45RB high transfer-colitis using Rag2-deficient mice (n=5); transfer of 1?10.sup.6 colitogenic T-cells; Daily application of 10 ?g KDPT i.p from day 23 after cell transfer; Control animals were treated with PBS; parameters measured were body weight, histology, IL-1? expression.

(104) As shown in FIG. 17, K(D)PT was shown to be effective in therapeutic treatment of DSS-colitis. Colitis was induced by DSS application on day 0. On day 4 of DSS feeding all animals showed clinical signs of colitis including weight loss. In the following days weight loss was significantly more pronounced in PBS-treated mice, reaching a maximum on day 9, with 26.9%?1.1% vs. 11.6%?4.4% in K(D)PT treated mice. On day 12 after DSS-start K(D)PT-treated animals had regained 97.2%?0.26% of initial weight, PBS-treated animals only 79.3%?4.2%. Mucosal expression of IL-1?mRNA in K(D)PT-treated animals was 65% lower (4.14?1.2 fold induction) than in PBS-treated mice (11.2?1.7 fold induction (p<0.01)).

(105) As shown in FIG. 18, significant reduction of histological signs of inflammation by K(D)PT was observed. Histological analysis of colonic tissue revealed reduced inflammation in K(D)PT treated mice. K(D)PT-treated animals showed fewer ulcerations, less epithelial denudations, leukocyte infiltration and submucosal edema, as well as a significantly lower inflammatory score of 7.25?4.42, as compared to PBS treated (25.0?2.81 (p<0.001)).

(106) As shown in FIG. 19, significant therapeutic effects of rectally applied K(D)PT was observed. Colitis was induced by DSS application on day 0. Treatment with K(D)PT was started one day prior to DSS-treatment. Maximum weight loss in the K(D)PT-treated group was 14.8%?2.6 vs. 20.6%?3.6 in the PBS-treated group. K(D)PT-treated animals quickly regained weight and at the end of the observation period had a body weight of 98.7%?2.7 of the initial weight, while PBS-treated animals reached only 90.8%?2.8 (p<0.02) of initial weight.

(107) As shown in FIG. 20, significant reduction of histological score and mucosal IL-1? expression in K(D)PT-treated animals was observed. Histological evaluation of the distal colon third showed an inflammatory score of 12.4?1.83 for K(D)PT-treated animals and of 20.4?2.93 for PBS-treated animals (p<0.04). Mucosal expression of IL-1?mRNA in the distal third of the colon of K(D)PT-treated animals was reduced significantly (p<0.002).

(108) As shown in FIG. 21, clinical signs of CD45RBhigh-transfer colitis are significantly reduced by K(D)PT-treatment. In the CD45RBhigh-transfer colitis model K(D)PT-treated animals recovered significantly faster and gained weight almost immediately after start of treatment (on day 23 after cell transfer), while PBS-treated animals further lost weight. From day 31 after cell transfer until the end of the observation period the K(D)PT group showed significantly higher weight (p<0.05). At the end of the experiment K(D)PT-treated animals had regained 99.3%?1.9 of their initial body weight, PBS-treated animals only 90.7%?2.6. Histological analysis confirmed these result, showing significantly higher inflammatory activity in control group animals.

Example 10

(109) IL-10 Deficient Colitis

(110) Interleukin (IL)-10?/? mice spontaneously develop intestinal inflammation characterized by discontinuous transmural lesions affecting the small and large intestine and by dysregulated production of proinflammatory cytokines. (Rennick, Donna M., and Madeline M. Fort. Lessons From Genetically Engineered Animal Models. XII. IL-10-deficient (IL-102/2) mice and intestinal inflammation. Am J Physiol Gastrointest Liver Physiol 278: G829-G833, 2000. See also Rennick, Donna M., Madeline M. Fort and Natalie L\J. Davidson. Studies with IL-10?/? mice: an overview J of Leukocyte Biology, Vol. 61, 389-396: 1997; both references of which are herein incorporated by reference in their entireties.)

(111) The uncontrolled generation of IFN-?-producing CD41 T cells (Th1 type) has been shown to play a causal role in the development of enterocolitis affecting these mutants. These genetically engineered mutants exhibit distinct pathological changes and cytokine profiles that have been associated with either Crohn's disease (CD) or ulcerative colitis (UC) in humans. These experiments using animal models of animal models of inflammatory bowel disease (IBD), Crohn's disease (CD) or ulcerative colitis (UC) demonstrate the efficacy of the inflammatory compounds of the present invention in the treatment of Inflammatory bowel disease, Crohn's disease, and ulcerative colitis.

(112) Inflammatory bowel disease (IBD) describes two distinct idiopathic inflammatory disorders of the intestine, ulcerative colitis and Crohn's disease. Ulcerative colitis is characterized by periods of active and inactive disease, a pattern observed in 80-90% of patients with this disease. Crohn's disease is also typified by repetitive cycles of active and quiescent disease with clinical patterns that vary according to disease location and clinical manifestations (inflammatory, fibrostenotic, and fistulizing). The primary goal of treatment is to induce and maintain remission in a safe and efficacious fashion. Currently the armamentarium of agents used to treat IBD includes derivatives of 5-aminosalicylic acid (5-ASA), corticosteroids, immune modulators such as azathioprine (AZA) or 6-mercaptopurine (6-MP) and methotrexate, cyclosporine (CyA), antitumor necrosis factor (TNF) antibodies such as infliximab, adalimumab or certolizumab pegol, and monoclonal antibodies against the cellular adhesion molecule ?4-integrin like natalizumab. The instant specification demonstrates that upon administration of KdPT in the IL-10 deficient mice (FIG. 22), weight loss and inflammation were significantly reduced in KdPT treated animals.

(113) Animals

(114) Homologous IL-10 gene-deficient mice were purchased from Jackson Laboratories (strain name: C3Bir.129P2(B6)-Il10tm1Cgn/Lt; Bar Harbor, Me., USA). Experiments described here were performed with 8-9 weeks old female mice. All animals were kept under pathogen-free conditions at 24? C. with a controlled 12 h day-night cycle and had free access to standard diet and drinking water. The animal studies were approved by the local animal subjects committee, University of M?nster.

(115) Induction of Colitis in IL-10?/? Mice

(116) The targeted disruption of the IL-10 gene in mice leads to spontaneous development of a chronic enterocolitis. In order to aggravate the developing colitis piroxicam was given from day 1 until day 7 and from day 15 until 22 at a dose of 60 mg/250 g of food and 80 mg/250 g, respectively. Piroxicam was mixed with rodent-chow. PBS-diluted KdPT was given daily by oral administration beginning from day 0 before start of piroxicam administration until the end of the experiment. Control mice received PBS only. Disease activity was monitored daily by body weight measurement. At the end of experiment mice were sacrificed and the colons removed. Colons were opened, embedded in O.C.T. and kept frozen at ?80? C. until further use. Sections (5 ?m) were stained with H&E and analysed by two blinded investigators.

(117) KdPT Attenuates Colitis in NSAID-Treated IL-10-Deficient Mice

(118) IL-10-deficient mice are known to develop a spontaneous colitis that can be aggravated by oral administration of a non-steroidal drug such as piroxicam (see supra). Accordingly, IL-10-deficient mice received an effective oral dose of KdPT (100 ?g daily) beginning from day 0 after administration of piroxicam until the end of the experiment on day 31 while control animals received PBS. Immediately after starting the first piroxicam treatment all placebo-treated animals showed a progressive weight loss up to 19.9?3.8% on day 27. However, the KdPT-treated group exhibited a significantly less pronounced loss of body weight of 8.4?2.9% (p<0.05) on day 27 confirming the protective effect of KdPT regarding intestinal inflammation.

(119) Histologic examination on day 31 after starting KdPT application revealed less severe inflammation with reduced involvement of the submucosa and a lower number of crypt abscesses in KdPT-treated animals than in controls. Histological scoring according to Berg et al confirmed a significantly higher score in control mice (13.6?3.2) vs. KdPT-treated animals (4.8?1.5; p<0.01).

(120) FIG. 22 shows the effect of KdPT on colitis in IL-10 deficient mice. 8-9 week old IL-10 deficient mice were fed with chow containing Piroxicam (day 1 to 7, 60 mg/250 g chow and day 15 to 22, 80 mg/250 g chow), in order to amplify the spontaneous enterocolitis observed in these animals. PBS or KdPT in PBS (100 ?g/animal/day, p.o.), respectively, were given from day 0 until the end of the experiment. Disease activity was observed by daily weighing; at the end of the experiment the colon was explanted and subjected to histological evaluation (scale according to Berg et al.). Weight loss and inflammation were significantly reduced in KdPT treated animals.

Example 11

(121) Effect of KdPT on Imiquimod-Induced Psoriasis-Like Disease in Mice

(122) The experiment was conducted as indicated in FIG. 23 and clearly reduced skin reddening and -scaling are visible as well as a histologically clearly reduced epidermal thickness. KdPT is roughly equipotent to an anti-TNF?-antibody.

Example 12

(123) Effect of KdPT on Psoriasis in a Transplant Model

(124) Immunodeficient mice (BNX mice) were transplanted with skin from psoriatic human donors. Animals with viable transplant were injected with PBMCs from the same donor, resulting in appearance of a psoriatic phenotype within the transplant. Animals were treated as follows:

(125) Group 1: Betamethasone, topically, 2? daily, Group 2: Vehicle, i.p. 100 ?L/d, Group 3: KdPT i.p. 1 ?g/d, Group 4: KdPT i.p. 10 ?g/d, all treatments were kept up for three weeks. As shown in FIG. 24:

(126) A) Clear reduction of epidermal thickness by KdPT.

(127) B) Significant reduction of Ki67-expression by KdPT.

(128) As shown in FIGS. 25A and 25B, immunodeficient mice (BNX mice) were transplanted with skin from psoriatic donors. Animals with viable transplant were injected with PBMCs from the same donor, resulting in appearance of a psoriatic phenotype within the transplant. Animals were treated as follows:

(129) Group 1: Vehicle, i.p. 100 ?L/d, Group 2: Betamethasone, topically, 2? daily, Group 3: KdPT i.p. 10 ?g/d, all treatments were kept up for three weeks.

(130) FIG. 25A depicts the clear reduction of epidermal thickness and Ki67-expression by KdPT.

(131) FIG. 25B shows that treatment with KdPT does not result in weight loss as compared to treatment with betamethasone.

Example 13

(132) Effect of KdPT on the Number of Regulatory CD4+CD25+Foxp3+?T-Cells (as Measured by Flow Cytometry, A), as Well as on the Expression of IL-10, IFN? and TNF?(qRT-PCR, B)

(133) FIGS. 26A and 26B show increased numbers of regulatory T cells in mice treated with ?-MSH or K(D)PT after Imiquimod-induced psoriasis-like skin inflammation. FIG. 26C shows increased expression of regulatory T cells markers in mice treated with ?-MSH or K(D)PT after Imiquimod-induced psoriasis-like skin inflammation.

Example 14

(134) Effect of KdPT on CD4+CD25+Foxp3+?Treg in Blood of Psoriasis Patients

(135) Clearly elevated numbers of CD4+CD25+Foxp3+ T-cells (A) and clearly higher expression of IL-10 as well as concomitant reduction of IFN? expression (B). FIGS. 27A and 27B show the effects of ?-MSH and K(D)PT on the number of regulatory T cells from psoriasis patients. FIGS. 27C and 27D show the effects of ?-MSH and K(D)PT on regulatory T cells from psoriasis patients. While the expression of Foxp3 and IL-10 are upregulated the expression of IFN-? is downregulated substantially.

Example 15

(136) Suppressive Effect of CD4+CD25+Foxp3+ T-Cells after Treatment with K(D)PT.

(137) In FIGS. 28A and 28B, PBMC were isolated from peripheral blood of 3 healthy donors and 3 psoriasis patients and stimulated for 48 h with ?-MSH or K(D)PT (10.sup.?9 molar) Subsequently, CD4+CD127? regulatory T cells were purified by magnetic cell separation and co-cultured at a 1:1 ratio with freshly isolated CD4+CD25 effector T cells from the same donor/patient.

Example 16

(138) Effect of KdPT on Th17-Zellen and Expression of Th17-Typical Parameters

(139) In FIGS. 29A to 29C, PMBC were isolated from peripheral blood of 8 healthy donors and 8 psoriasis patients and stimulated for 48 h with ?-MSH or K(D)PT (10-9 molar). Subsequently, CD4+ T cells were purified by magnetic cell separation and subjected to RNA-isolation and reverse transcription. The expression of IL-17, IL-21, IL-22, IL-23R, as well as RORc was quantified by realtime-PCR. FIG. 29A shows the effects of ?-MSH and K(D)PT on Th-17 cells from psoriasis patients. FIGS. 29B and 29C show the expression of Th-17 associated genes in CD4+ T cells from psoriasis patients and healthy donors after stimulation with ?-MSH or K(D)PT.

Example 17

(140) Effect of KdPT on the Expression of Th17-Typical Parameters

(141) FIG. 30A presents the results from quantitative RT-PCR for several Th17 markers; murine ROR?t is equivalent to human RORc. Reduced Expression of Th17 markers in mice treated with ?-MSH or K(D)PT after Imiquimod-induced psoriasis-like skin inflammation. FIG. 30B depicts cytokine by post-experiment harvested and purified CD4+?T-cells points to suppression of Th17 phenotype while showing increased level for anti-inflammatory IL.-10.

Example 18

(142) Effect of KdPT on Dendritic Cells

(143) In FIGS. 31A to 31C, KdPT treated DC displayed elevated levels of parameters, which stand for a tolerizing dendritic cell phenotype. Among these are the cell surface protein CD205 as well as the cytokines IL-6 and IL-10. Expression of surface molecules involved in antigen presentation, namely CD80, CD86 and MHC class II, on the other hand is downregulated.

Example 19

(144) Effect of KdPT-Treated DC on CD4+?T-Cells

(145) In FIGS. 32A and 32B, after treatment with KdPT-treated dendritic cells CD4+?T-cells show elevated levels of regulatory markers.

Example 20

(146) CD4+?T-Cells Display Suppressive Properties after Treatment with KdPT-Treated DC

(147) FIGS. 33A and 33B show that CD4+?T-cells after co-culture with ?-MSH, KPV or KdPT become anergic and exert a suppressive effect on the proliferation of CD4+CD25? responder T-cells. Also in FIGS. 33A and 33B, it is shown that CD4+?T-cells (1?10.sup.6 cells), contacted in vitro with ?-MSH, KPV or KdPT-treated DC, upon reinjection into sensitised recipient mice (Hapten: DNFB) show a suppressive effect on contact allergy.

Example 21

(148) Effect of KdPT on CD8+?T-Cells from DNFB-Sensitised Mice/Contact Allergy Experiment

(149) In FIGS. 34A to 34C, C57BL/6 mice were sensitised with DNFB in vivo and treated either with KdPT or Dexamethasone. At the end of the experiments CD8+?T-cells were isolated and characterised. CD8+?T-cells from KdPT treated animals proliferated more slowly and secrete lower amounts of Th1-type cytokines, without a shift to the Th2-type, as can be deduced from the although suppressed secretion of IL-4. The clearly enhanced expression of IL-10 additionally indicates that the obtained T-cells mediate an altogether dampening effect in this allergic reaction.

Example 22

(150) Effect of KdPT on Number and Degranulation of Human Mast Cells in the Hair Follicle

(151) In FIGS. 35A and 35B, (m) Each Leder esterase-stained hair follicle was quantitatively analyzed regarding its number of mast cells (nondegranulated/degranulated) and mena was calculated. Representative experiment of protection assay, n=10-18 hair follicles per group, data are mean?SEM*p<0.05. (n-p) Representative photodocument of a Leder esterase stained hair follicle (cytoplasmic mast cell granules stained red, green counterstain); (o) nondegranulated mast cell, (p) degranulated mast cell (original magnification?400) (q-s) Representative photodocument of c-KIT staining (green signal/flourescein isothiocyanate) (original magnification?200)

(152) The results of these experiments clearly demonstrate the efficacy of KdPT in treating various autoimmune disorders.

(153) (1) KdPT is effective in at least two model systems for different autoimmune disorders.

(154) Administration of KdPT in the IL-10 deficient mice (FIG. 22) shows that weight loss and inflammation were significantly reduced in KdPT treated animals (FIG. 1). This is an animal model of Crohn's disease.

(155) In two models for psoriasis comparable results could be obtained. In FIG. 23, a psoriasis-like condition was induced in mice by administration of Imiquimod/Aldara. FIG. 24 and FIGS. 25A and 25B show a transplant model of psoriasis. In both cases significant effects of KdPT on skin condition and molecular parameters are evident.

(156) (2) KdPT increases the number of regulatory CD4+CD25+Foxp3+ T cells.

(157) In the Imiquimod model for psoriasis it was shown that the treatment of the animals with KdPT also results in significant increase in the number of regulatory CD4+CD25+Foxp3+ T cells, as well as in a significant upregulation of IL-10. At the same time, IFN? and TNF? expression is suppressed (FIG. 26C). These changes strongly suggest that there is an effect of KdPT on the number of regulatory T cells and on the expression of cytokines which are involved in many autoimmune disorders.

(158) In case of humans, a similar situation can be found. In PBMC from the blood of psoriasis patients administration of KdPT significantly increased the number of CD4+CD25+Foxp3+ T cells (FIG. 27A-27B). These cells express less IL-10 and less IFN?(FIG. 27C-27D). In addition, the regulatory T cells from the blood of these patients exhibit substantial suppressive properties after treatment with KdPT (FIG. 28A-28B).

(159) The importance of regulatory T cells of this type, and in particular of Foxp3+ for human autoimmune disorders and the maintenance of self-tolerance has been discussed for several years in scientific journals (e.g. Sakaguchi et al. 2006 Immunol Rev 212:8-27; Valencia et al. 2007 Nat Clin Pract Rheum 3:619-626; Buckner J H 2010 Nat Rev 10:849-859). For various autoimmune disorders either reduced numbers of Treg cells or a reduced Treg activity is observed. Both things are obviously corrected by KdPT. The number of CD4+CD25+Foxp3+ T cells is increased and due to the increase in IL-10 production an increase in activity can be assumed. In addition, KdPT-induced CD4+CD25+Foxp3+ T cells show a substantial suppressive phenotype.

(160) (3) KdPT reduces the number of Th17 cells.

(161) Apart from the regulatory T cells, Th17 cells which have only recently been identified as a distinct cell population, have been discussed as playing a role in autoimmune disorders and also in allergic disorders in transplant rejection (e.g. Wang et al. 2011 Rheumatol Int January 11 epub; Tokura et al. 2010 J UOEH 32:317-328, Oh et al. 2011 Eur J Immunol 41:392-402, Shilling et al. 2011 Semin Immunpathol February 1 epub, Jadidi-Niaragh et al. 2011 Scand J Immunol doi: 10.1111/j.1365-3083.2011.02536.x. epub, Korn et al. 2009 Annu Rev Immunol 27:485-517, Lee et al. 2010 J Invest Dermatol 130:2540-2, Di Cesare et al. 2009 J Invest Dermatol 1339-50, Mesquita et al. Braz J Med Biol Res 42:476-486.).

(162) With a view to Th17 cells it could be shown in samples from psoriasis patients that in the blood of these patients an increased number of Th17 cells could be reduced to the normal level of healthy individuals by KdPT (FIG. 29). Also on a molecular level this effect could be observed. Central cytokines such as IL-17 but also IL-21, IL-22 and IL-23 as well as transcription factor RORc were produced at reduced levels due to the influence of KdPT on CD4+ T cells (FIGS. 29B and 29C). Comparable data could be generated in the murine Imiquimod psoriasis model (FIGS. 30A and 30B).

(163) The observed reduction on IL-17 expression alone suggests that KdPT, similar to the anti-IL-17 antibody AIN457 currently under development (Hueber et al. 2010 Science Translat Med 2(52):52-72) would have a broad effect in the treatment of autoimmune disorders. In addition it could be found that KdPT mediates its effect via a broader mechanism than only the isolated inhibition of IL-17 expression, as demonstrated by the attached data.

(164) Of particular interest is also the relatedness of Th17 cells and regulatory T cells which allows an interplay between both cells types (Korn et al. 2009 Annu Rev Immunol 27:485-517 Mai et al. 2010 Front Biosci 15:986-1006). This can be derived also from the data shown here, see in particular direct comparison of FIG. 27 (A-D) and FIG. 29 (A-C) for the human case; as well as comparison of FIG. 26 (A-C) and FIG. 30 (A-B) for the murine model.

(165) (4) KdPT induces tolerance.

(166) In addition to the data on regulatory T cells and Th17 cells in animals and in the blood of psoriasis patients it should be mentioned that the effect of KdPT is mediated via an interaction with dendritic cells (DC) which represent the central type of antigen presenting cells. Treatment of DC with KdPT results in formation of a tolerating phenotype which elicits the above-described phenomena in interaction with T cells.

(167) Dendritic cells generated in vitro by KdPT treatment express less co-stimulatory molecules (CD80, CD86), less molecules of the MEW II complex and more CD205 and IL-10 (FIGS. 31A-31C).

(168) CD4+ T cells which are contacted with these dendritic cells show a higher expression of regulatory molecules, such as Foxp3, Neuropilin-1(Nrp-1), IL-10 and CTLA-4 (FIG. 32) and act suppressive in proliferation assays (FIGS. 32A-32B). Also in vivo these T cells are capable of acting suppressively (FIGS. 33A-33B).

(169) (5) Further Effects

(170) With a view to allergic disorders it could be shown that KdPT has a direct effect on CD8+ T cells. CD8+ T cells which were isolated from in vivo sensitized and KdPT treated mice do proliferate less strongly than T cells from na?ve mice, from only sensitized animals or sensitized and Dexamethason treated animals (FIGS. 34A-34C).

(171) Besides that, KdPT further appears to have an effect on mast cells, as shown on the basis of explanted hair follicles. In IFN? stimulated hair follicles more and in particular more degranulated mast cells can be found than in hair follicles which have been stimulated with IFN? and KdPT (Meyer et al. 2009 Br J Dermatol 161:1399-1424) (FIGS. 35A and 35B). These data represent a further link to the anti-allergic effect of KdPT already shown in the parent application.

(172) In summary, ample experimental evidence strongly indicate that the peptides KP and KdPT are effective in the treatment of multiple autoimmune disorders and allergic reactions. This was shown by way of model systems for two specific autoimmune disorders. It is further supported by data regarding regulating T cells and Th17 cells. This supports the view that this effect can be observed in many inflammatory autoimmune disorders and allergic reaction.