HK1139078A - Lys-pro and lys-pro-thr for the manufacture of a cosmetic composition - Google Patents

Description

The tridecapeptide α-melanocyte-stimulating hormone (αMSH) is derived from the precursor hormone pro-opiomelanocortin (POMC). Several biologically active peptide hormones such as β-lipotropin, adrenocorticotropin (ACTH), β-endorphin and the melanootropins (α, β and γMSH) are derived from the POMC gene product. Proteolytic enzymes of various specificities are necessary to process these peptides. In addition, posttranslational modifications such as acetylation can occur.

The effects of αMSH and other POMC peptides on the various tissues are mediated by a family of specific receptors, the melanocortin (MC) receptors, which belong to the group of G-protein coupled receptors. Five different melanocortin receptors (MC-1 to MC-5) have been cloned.

For example, the proliferation, differentiation and cytokine production of melanocytes are to be influenced by αMSH.

For example, αMSH is thought to down-regulate several pro-inflammatory cytokines, while the production of the anti-inflammatory cytokine IL-10 is stimulated by αMSH, thus αMSH has an important role in suppressing immune and inflammatory responses. Several studies suggest that the immunomodulatory and anti-inflammatory effects of αMSH are mediated by the C-terminal region of MSH (amino acids 11-13: Lys-Pro-Val) since administration of the C-terminal tripeptide is sufficient to induce these effects (Catania Lippe and Bhraj, 1993, Rev. et al. 15, Rev. 564-5176; J. 254, 1996; J. 254, Rev. 564, et al. 257, J. 254, and Immunol.

WO 88/00833 reveals the use of the tripeptide Lys-Pro-Val to produce a drug for the treatment of inflammation.

One of the functions of the present invention is to provide additional anti-inflammatory compounds.

Surprisingly, the tripeptide Lys-Pro-Thr was found to have anti-inflammatory properties, and even smaller compounds such as Lys-Pro and Lys, unlike expected, showed beneficial properties.

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The compound used according to the invention may be lysine or the lysine-proline dipeptide, but the lysine-proline-threonine tripeptide (= KPT) is preferred.

The amino acids of the compounds used in accordance with the invention can have either the (L) or the (D) configuration. (L) Lys-D) Pro-L) Thr, (L) Lys-L) Pro-D) Thr, (L) Lys-D) Pro-D) Thr, (L) Lys-L) Pro-L) Thr, (D) Lys-D) Pro-L (Thr) (D) Lys-D) Pro-D) Thr, (D) Lys-L-Pro-L-Th, (D) Lys-L-Pro-D) Thr, The compounds used according to the invention may also have amino acid exchange, where one of the amino acids has been conservatively replaced.

The compound of the formula (I) of the invention may be chemically modified at the N-terminus and/or C-terminus, for example by an acyl group, preferably an acetyl group at the N-terminus and/or an amidification or esterification at the C-terminus. Other protective groups known to themselves are also possible. The modifications may also affect the amino group in the side chain of lysine or the hydroxyl group of threonine. Other modifications are also possible on the side of the NH2 group, e.g. extension to a glycine, and further amino acids up to the length of α-MSH.

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The above compounds can be used to treat all types of acute or chronic inflammation, including acute and chronic inflammation, e.g. of the skin, psoriasis, atopic dermatitis, allergic reactions of all kinds, from rhinitis to contact allergies to asthma and food allergies, autoimmune diseases, fibrosis and scleroderma and transplant rejection, as well as vascular diseases. Preferably, the compounds are used to treat inflammatory conditions of the skin. In this case, it is preferable to administer the compound in the form of an ointment or cream as a topical stimulation.

In a preferred embodiment, the peptides of the invention can also be used for inflammatory bowel diseases, including inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, as well as short-term irritation of the bowel caused by minor food poisoning.

In another preferred embodiment, the compounds of the invention may be used to treat inflammatory diseases of inflammation occurring in areas of the body in contact with the outside world, in particular the mucous membranes of the oral and gastrointestinal tract and the lungs.

The compounds used in accordance with the invention are, however, also systemically effective in treating or preventing inflammation. The compound is then preferably administered intraperitoneally, intravenously or orally. The dose of an application is usually 20 μg to 10 mg/kg body weight, preferably 100 μg to 1 mg/kg body weight.

Finally, the compounds mentioned may also be used in sprays, for example for inhalation for the treatment of respiratory infections.

It is possible that several different formula (I) compounds may be used for treatment, in which case at least two different formula (I) compounds are used for the treatment of inflammation.

The compounds of formula (I) can also be used to manufacture a drug to treat and/or prevent inflammation. All of the above embodiments are included in this use in an analogue manner. The compound is usually mixed with a pharmaceutically compatible carrier or diluent.

The compounds of formula (I) can also be added to food to reduce the allergic potential of certain food ingredients. The invention therefore also concerns the use of a compound of formula (I) as a food additive. The concentration in food may then be 1 μM to 1 mM.

According to the invention, it is also possible to use a compound of formula (I) as a non-pharmaceutical additive in cosmetics, for example, creams for irritated skin or after sunbathing containing a compound of formula (I) may be used.

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There are several types of antigen-presenting cells. The preferred cells according to the present invention are dendritic cells or Langerhans cells. It is not necessary that the antigen-presenting cells be present in a preparation free of other components or cells. The antigen-presenting cells may also be provided in a mixture with other cells. For example, epidermal cells containing Langerhans cells as antigen-presenting cells are preferred. Dendritic cells from bone marrow may also be isolated or dendritic cells produced by in vitro-culture precursor cells known in themselves, e.g. PBMC. Methods for the provision of antigen-presenting cells, e.g. in J. Laurent et al. (1991:75-1689) of J. Immunol.

The cells are then exposed in vitro to αMSH or a biologically active derivative or fragment thereof. Biologically active derivatives or fragments of αMSH are, for example, chemical modifications of αMSH, fragments of αMSH containing Lys, Lys-Pro, Lys-Pro-Val or Lys-Pro-Thr, or compounds containing any of these substances. Various modifications are conceivable as long as the biological activity of αMSH - the ability to induce tolerance - is substantially maintained.

After the cells have been exposed to αMSH or a biologically active derivative or fragment thereof, or before or at the same time, the cells are in vitro exposed to the antigen against which tolerance is to be induced. The antigen may be a protein to which there is a risk of an allergic reaction. For example, if the antigen's bite against which the immune response is directed is known, the cells can only be exposed to the specific bite. For example, the length of the peptides may be 7 to 20 amino acids, preferably 7 to 15 amino acids.

The antigen-presenting cells can be washed and mixed with a pharmaceutically compatible carrier or diluent following the above steps, and then the cells can be introduced into a patient or a mammal, resulting in tolerance to the hapten or antigen used.

Another aspect of the invention is the use of αMSH or a biologically active derivative or fragment thereof to produce a drug to induce tolerance to an antigen. Preferably, the resulting drug contains cells that are available by the in vitro production of cells that can confer tolerance described above.

The inventors were also able to show that, for example, the peptide KPT prevents contact hypersensitivity reactions (CHS reactions) and induces an allergen-specific, long-lasting tolerance. In the case of CHS reactions, two stages are distinguished: an initial (induction phase) with an antigen is the basis for a subsequent CHS reaction, which leads to a further induction reaction (CHS reaction) which can be used to prevent the onset of a reaction (inhibition, inhibition, etc.) and to prevent further exposure to CHS (inhibition, inhibition, etc.).

Lys-Pro-Thr has also been found to reduce the expression of costimulatory molecules on dendritic cells, most likely part of the mechanism for CHS suppression and tolerance induction, while increasing the secretion of the anti-inflammatory IL-10 by monocytes, which is also part of the mechanism for allergic contact eczema.

Without being bound in any way to a theory, the compounds of the invention could bind to β-adrenergic receptors. Furthermore, it can be assumed that the peptides of the invention are capable of binding to the IL-1 receptor type 1. It can also not be excluded that the peptides of the invention bind to other receptors, such as the κ-opioid receptor. On the basis of this assumption, it is assumed that the compounds of the invention could bind to several receptors which, if activated by their original ligands, would all pro-inflammatory enter the receptor by way of the original receptor. The invention of B-L-α-peptides activates these receptors.

Figure 1 shows that intravenous injection of αMSH, KPV or KPT suppresses the sensitization phase of CHS.

Figure 2 shows that intravenous administration of αMSH, KPV or KPT may induce tolerance.

Figure 3 illustrates IL-10 secretion by human PBLs 24 hours after treatment with αMSH, KPV or KPT.

Figure 4 shows IL-10 secretion by human PBL 48 hours after treatment with αMSH, KPV or KPT.

Figure 5 shows that THP-1 cells express receptors for αMSH.

Figures 6a to d, 7a to d and 8a to d show that in a competitive assay unlabeled αMSH, KPV or KPT can displace biotin-labeled αMSH from binding sites on THP-1 cells.

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Figure 9b shows the adhesion of lymphocytes to HDMEC (Chromium Release Assay).

Figure 10 shows the effect of αMSH, KP, KPV or KPT on NF-κB activation in LPS-treated HMEC-1 cells.

Figure 11a shows that the number of E-selectin-expressing vessels in tissue sections is reduced by αMSH treatment.

Figure 11b shows that the number of petechial lesions in the ears of LPS-treated mice is reduced by αMSH treatment.

Figure 12 shows that in vitro treatment of BMDC with αMSH or KP can suppress and induce tolerance of CHS.

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Figure 13b shows the effect of αMSH, KP or K on the CHS response and the effect of αMSH or KP on tolerance induction in BalbC mice.

Figure 14 shows CHS suppression by T cells exposed in vitro to antigen-laden and αMSH or derivative-treated DC.

Figure 15 shows the induction of tolerance by T cells in vitro exposed to antigen-laden and αMSH DC.

Figure 16 shows the upregulation of CTLA-4 on T cells after contact with antigen-laden and αMSH or derivative-treated DC. A: CD4 positive T cells; B: CD8 positive T cells.

The following examples are intended to explain the invention in more detail.

Example 1 The mice:

Female Balb/C mice aged 7 to 10 weeks were obtained from Charles River (Sulzfeld, Germany) and kept in accordance with federal regulations.

Administration of αMSH or KPV or KPT or KP:

5 μg αMSH or 1.5 μg peptide (KP: 50 μg) per mouse were injected 2 hours prior to sensitisation.

Determination of CHS and tolerance:

To induce CHS, the ears of the mice were coated with 10 μl 0.3% DNFB on both sides of one ear. CHS was determined by the degree of swelling of the ear exposed to hapten compared to that of the other treated ear and measured with a spring-stretched grip 24 hours after hapten exposure. Mice whose ears were not previously exposed to hapten were described as negative controls. To determine whether the injection of αHMS or the application of the hapten leads to the injection, females were tested for their ability to tolerate the use of αHMS or the application of the hapten 2 days before or 2 days after exposure to the other treated ear and their ability to respond to the same response (response to a BHS) was determined by a CHS test conducted by the WHO and a CHS test conducted 24 hours after exposure to the right side of the abdomen (response to the right side of the abdomen) and a BHS test conducted by the WHO.

In some experiments, topical preparations of αMSH were used, in which the mice were coated at the sensitisation site (abdomen) immediately before or 3 hours or 24 hours before sensitisation.

The result:

IV injection of αMSH as well as KPV or KPT or KP inhibited the ability of the mice to induce a CHS response to DNFB exposure 7 days later, and these mice did not develop DNFB-specific sensitization.

In order to distinguish between temporary immunosuppression and specific immunological tolerance, mice were sensitized and exposed to hapten a second time; mice injected with αMSH or KPV or KPT before the first sensitization could not be sensitized even by the application of a second sensitizing hapten dose, suggesting that these mice developed tolerance to DNFB. KPV showed a weak effect, whereas αMSH and KPT and the earlobe very much inhibited KP response (see Figures 2 and 13b).

Example 2 Materials and methods:

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Results:

Human PBMCs that were not treated or treated with varying concentrations of αMSH or peptides produced only low levels of IL-10 (5-10 pg/ ml) after 24 hours of incubation. αMSH (10-11 M), KPV (10-8 to 10-9 M) and KPT (10-8 to 10-9 M) obviously induced IL-10 production (see Figure 3).

After 48 hours of incubation, the human PBMCs produced significant amounts of IL-10. αMSH, KPV and KPT significantly increased the production of IL-10 by human PBLC.

The results demonstrated show that the peptide KPT, like αMSH and KPV, after intravenous administration can inhibit CHS sensitization and induce hapten-specific tolerance. KPT is also capable of inducing IL-10 in vivo and in vitro. The data also suggest that the immunosuppressive effect of αMSH in vivo is not dependent on IL-10 induction alone.

Example 3 Materials and methods:

After addition of biotin-labelled αMSH (10-10 M), the cells were incubated for 1 hour at 4°C, washed once with PBS, resuspended in 100 μl of PBS/1% BSA and resuspended with FITC-labelled Streptomycin (40 μl/ ml) for up to 30 minutes at 4°C. The cells were incubated in a short time before the last incubation but were incubated in a controlled sample of α-H10-6MMS (10-10 MMS) or in a sample of 10-12 μl of BMS (10-6 MMS) The cells were incubated by a biotin-controlled test with a dose of 10-12 μl of α-H10-6MMS (10-6 MMS) or a biotin-controlled test with a dose of 10-12 μl of α-H10-6MMS (10 MMS).

Results:

Following FACS analysis with biotin-labeled αMSH, unstimulated THP-1 cells express significant amounts of binding cells specific to αMSH compared to control approaches incubated with FITC streptavidin alone.

To determine whether THP-1 cells express any of the known melanocortin receptors (MCs), RT-PCR was performed with MC-1, MC-2, MC-3 and MC-4 specific primers. Total RNA was obtained from THP-1 cells. A MC-1 specific PCR product with an expected length of 416 bp was detected (Rajora et al., 1996, J. Leuk. Biol., 59, 248). PCR products specific to MC-2, MC-3 or MC-4 were not detected. The results show that THP-1 cells express MC-1, which is specific to αMSH and ACTH, unlike other melanocortin receptors.

To investigate whether the binding cells expressed on THP-1 are specific for αMSH, competitive experiments were performed with αMSH or KPV or KPT. Specific binding was determined by incubating THP-1 cells with biotin-labelled αMSH (10-10 M) and varying concentrations of unlabelled αMSH or peptides. Unlabelled αMSH at a concentration of 10-8 M significantly suppressed αMSH binding. When αMSH was used at concentrations of 10-6 M, 10-10 M or 10-12 M, no significant suppression was observed (Figures 6a to 6d).

When unlabeled KPV was used, significant inhibition was observed only at a concentration of 10-6 M (see Figures 7a to 7d).

In the case of the peptide KPT, a significant inhibition of αMSH binding was observed at each of the test concentrations (10-6 to 10-12 M, see Figures 8a to 8d).

These results show that the peptide KPT binds to the melanocortin receptor on THP-1 cells, which is specific for αMSH, suggesting that αMSH and KPT share a common binding site.

Example 4 Materials and methods:

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Results:

Treatment of endothelial cells with αMSH or KPT inhibited LPS or TNFα-induced expression of adhesion molecules, which was observed in a concentration range of 10-6 to 10-12 M αMSH or peptide.

LPS or TNFα-induced surface expression of adhesion molecules was slightly reduced by all agonists, both by EIA using whole cells and by FACS with specific antibodies (see Figure 9a showing EIA data).

In combination, these results show that αMSH has an effect on the adhesion of lymphocytes to EC and thus also reduces the extravasation of lymphocytes in states of tissue inflammation, supported by in vivo data on localized vasculitis.

Example 5 Materials and methods:

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The result:

In TNFα or LPS-treated ECs and IL-1-treated HNKs, the addition of the peptides results in reduced activation of the transcription factor NF-κB (see Figures 10 and 13a), which in turn leads to a decrease in gene transcription for numerous proinflammatory mediators (cytokines, chemokines, adhesion molecules, etc.).

Example 6 Materials and methods:

Mice were treated with LPS by s.c. injection into an ear. This preparatory injection induced a long-lasting increase in E-selectin expression at the LPS injection site. 24 hours later, a second dose of LPS was injected intravenously (Challenge). This second LPS injection led to rapid vascular necrosis and the formation of petechial lesions, which can be easily detected due to their size and number. αMSH (25 μg) was administered at the time of the preparatory LPS injection.

The result:

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Example 7 Materials and methods:

Dendritic cells from bone marrow (BMDC) were isolated from the femur bones of mice and treated with IL-4 and GM-CSF for 6 or 9 days. On day 6 and 9 respectively, the cells were treated with αMSH (2 x 10-11 M) or the peptide KP (2 x 10-6 M) for 3 hours and 2.5 hours prior to reinjection in naïve mice with the same genetic background. 2 hours prior to reinjection, the cells were treated with haptosis (1 mM DNBS, the water-soluble form of DNFB). Immediately prior to reinjection, the cells were re-washed with PBS 2 times. 5 x 10-5 cells of ONF were injected i.v. Finally, the cells were either injected with DNBS alone or αMSH or untreated. 5 days after the injection, the control animals were treated with OBSD. 5 days later, the animals were washed and re-activated with OBSD.

Results:

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Example 8 Err1:Expecting ',' delimiter: line 1 column 43 (char 42)

Dendritic cells (DC) have been isolated (from blood, bone marrow or tissue) but cell mixtures containing DC can also be used (e.g. epidermal cell mixtures) and cultured in the presence of GM-CSF and IL-4 (preferably 250-1000 μ/ml for each substance).

After a period of maturation (preferably 6-9 days), the cells are loaded with antigens (concentration depends on the respective antigen, dito period) and treated with αMSH or derivatives thereof. The derivatives correspond to at least the amino acids 12 and 13 of αMSH (Lys-Pro), preferably are derivatives containing Lys-Pro-Val. D- and L-configuration of the AS are possible, dito conservative AS exchange. This leads, among other things, to the fact that the Lys-Pro-Thr derived from IL-1β can also be used, as well as the N-terminally prolonged derivatives of this.

The treated cells are then injected into the recipient body intravenously (i.p. or s.c. would also be possible); mouse: 2 x 105 cells approximately at the lower limit. Depending on the antigen, a single injection or several injections may be sufficient.

Alternatively, it is possible to bring DC into contact with T cells outside the body and then inject the mixture or T cells. In this case, the antigen loading of DC can be done before or during contact with the T cells. The T cells can also come from individuals already sensitized to the respective antigen. In the mouse, the lower limit is about 1 million T cells, preferably more cells (Figures 14 and 15).

The advantages of this method are the prevention of any type of antigen-specific adverse immune reaction in which antigen-specific lymphocytes (B or T cells) play a pathogenetic role, including allergies, autoimmune diseases, chronic inflammation or implantation.

The surprising results can be seen without being bound to a theory, in that αMSH is a potent immune modulator and has numerous anti-inflammatory properties, including its ability to reduce the expression of co-stimulating molecules at DC. Similar properties are also shown by the derivatives of the invention of αMSH up to the C-terminal tripeptide and also the dipeptide Lys-Pro. Derivatives with other amino acid composition (conservative AS-exchange) also have comparable properties, including in particular the lys-pro-thr derived from IL-1β (so that it is assumed that the N-terminal (αH analogue) peptide is also co-expressed with the IL-1β sequence).

In vivo, αMSH, as well as its derivatives, are capable of inducing hapten-specific tolerance. DCs are professional antigen-presenting cells capable of inducing numerous types of immune responses and also determining the course of such responses.

It has now been shown that in vitro treatment of DC or DC-T cell mixtures with an antigen in the presence of αMSH or derivatives causes these cells to induce hapten-specific tolerance after injection into an organism.

The mechanism here seems to be that the antigen presentation of DC is modulated by αMSH or derivatives to generate suppressive T cells, and T cells in appropriate mixtures have been shown to have high expression of CTLA-4 (Fig. 16).

The use of such DC or T cells can prevent autoimmune diseases, chronic inflammation or allergies, and also prevent rejection of immunity or transplanted cells.

The compounds of the invention may also be used for the treatment of tumours by in situ activation of dendritic cells, which may also be used for toleration in the presence of the peptides of the invention.

The present invention relates to the following articles (1) to (18): (1) Use of a compound of formula (I) Other X is a hydroxyl group, an amino group, an alkoxy, pro or pro-thr, or a pharmaceutically compatible salt thereof, for the treatment and/or prevention of inflammatory diseases.(2) Use after (1), characterised by the compound of formula (I) acylating at the N-terminus and/or amidating or esterifying at the C-terminus.(3) Use after (1) or (2), characterised by the inflammation being selected from the group consisting of inflammation of the skin or vessels, allergic reactions, autoimmune diseases, fibrosis, scleroderma and graft rejection.(4) Use after (1), or (3), characterised by the inflammation being selected from the group consisting of psoriasis,Dermatitis, rhinitis, contact allergies, asthma and food allergies.(5) Use after (1), (2), (3) or (4) characterised by inflammation of the skin.(6) Use after (1), (2), (3), (4) or (5) characterised by the compound of formula (I) being administered in the form of an ointment or cream.(7) use after (6) characterised by the compound of formula (I) being contained in the ointment or cream at a concentration of 1 μM to 1 mM.(8) use after (1) to (5) characterised by the compound of formula (I) being administered by the peritoneal, intravenous or oral route.(9) use after (8) characterised by the compound being administered at a concentration of 20 μm/kg bodyweight to 10 mg/kg bodyweight (I) of the formula.(10) Use in accordance with (1), (2), (3), (4), (5), (6), (7), (8) or (9) characterised by the use of at least 2 different compounds of formula (I).(11) Use of a compound of formula (I) as a food additive.(12) Use of a compound of formula (I) to produce a cosmetic composition.(13) In vitro production process of cells that can confer tolerance to an antigen, including the following steps: (a) Provision of antigen-presenting cells; (b) Contact of the cells with αMSH or a biologically active derivative or fragment thereof; and (c) Contact of the cells with the antigen; whereby steps (b) and (c) may be performed in any order or simultaneously.(iii) The test method is a test method characterised by the presence of antigen presenting cells, which are essentially dendritic cells. (iii) The test method is characterised by the presence of antigen presenting cells, which are essentially epidermal cells. (iv) The test method is characterised by the presence of cells in step (b) in contact with a compound containing αMSH, Lys-Pro-Val, Lys-Pro-Thr, Lys-Pro or Lys. (v) The test method is characterised by the presence of cells in step (b) containing αMSH, Lys-Pro-Val, Lys-Pro-Thr, Lys-Pro or Lys. (v) The test method is characterised by the presence of cells in step (b) containing αMSH, Lys-Pro-Val, Lys-Pro-Thr, Lys-Pro or Lys. (v) The test method is characterised by the presence of cells in step (b) containing cells obtained by a procedure (13), or by the presence of cells obtained by a procedure (14), or by the presence of cells obtained by a procedure (16).

Claims (7)

1. Use of a compound selected from the group consisting of the dipeptide lysine-proline, the tripeptide lysine-proline-threonine and their salts to produce a cosmetic composition.
2. Use according to claim 1, characterised by the compound being acylated at the N-terminus and/or amidized or esterified at the C-terminus.
3. Use according to claim 1 or 2, characterised by the compound being the tripeptide lysine-proline-threonine.
4. Use according to claim 3, characterised by the compound being the tripeptide (L) Lys- ((D) Pro- ((L) Thr).
5. Use according to one of claims 1 to 4 characterised by the cosmetic composition being a cream.
6. Use according to claim 5, characterised by the presence of the compound in the cream at a concentration of 1 μm to 1 mM.
7. Use according to one of the previous claims, characterised by the use of at least two different compounds of the compounds defined in claim 1.