DE102008062136A1 - Pharmaceutical composition based on peptide of camel milk - Google Patents

Abstract

The invention relates to peptides which are e.g. can be isolated from camel's milk, and can be used as an active ingredient in pharmaceutical compositions, in particular for use in medical indications which can be treated by inhibiting human DPP4, CD26, zAAP and / or DPP8 and / or DPP9, in particular type 2 diabetes mellitus, Obesity and / or cardiovascular diseases, as well as diseases caused by T cells, or which are treatable by influencing T cells, and in particular by inhibiting DPP4 or its cell-bound form CD26 and / or APN and / or zAAP are treatable, eg Autoimmune diseases. Furthermore, the invention relates to pharmaceutical compositions for use in neuronal diseases that are treatable by inhibiting DPP4 or CD26 and / or inhibiting APN and / or zAAP and / or in neuronal diseases, by inhibiting DPP8 and / or DPP9.

Description

The Invention relates to peptides, the z. B. isolatable from camel milk are, and used as an active ingredient in pharmaceutical compositions may be, especially for use in medical Indications caused by inhibition of human DPP4, CD26, zAAP and / or DPP8 and / or DPP9 can be treated, in particular Type 2 diabetes mellitus, obesity and / or cardiovascular disease, as well as Diseases caused by T cells or by Influence of T cells are treatable, and in particular by Inhibition of DPP4 or its cell-bound form CD26 and / or APN and / or zAAP are treatable, e.g. B. autoimmune diseases. Farther For example, the invention relates to pharmaceutical compositions for use in neuronal diseases caused by inhibition of DPP4 or of CD26 and / or inhibition of APN and / or zAAP and / or neuronal Diseases that are treatable by inhibiting DPP8 and / or DPP9.

Next the active compounds, the invention also relates to the production of pharmaceutical Compositions containing at least one of the peptides as an active ingredient in pharmaceutically acceptable formulations, the use of the peptides for the preparation of pharmaceutical compositions, as well Process for the preparation of the peptides for the pharmaceutical Use.

Furthermore, the invention relates to the use of the compounds with inhibitory activity against DPP4 for the preparation of pharmaceutical compositions against medical indications, which can be influenced by inhibition and / or inactivation of the DPP4 and / or the membrane-bound form of DPP4, CD26. This applies in addition to diabetes and obesity in particular immunological indications, eg. For example, those which are influenced by chemokines, in particular the RANTES group (regulated activation, normal T-cell expressed and secreted), in particular from the group of RANTES, the SDF-1α (stromal cell-derived factor 1) ( Proost et al., 1998 ; Shioda et al., 1998 ), Eotaxin ( Struyf et al., 1999 ), LD78β ( Proost et al., 2000 ), MIP-1β (macrophage 11 inflammatory protein-1β) ( Guan et al., 2004 ), MCP-2 (monocyte chemotactic protein-2), IP-10 (interferon-inducible protein 10) and MDC (macrophage-derived chemokine) ( De Meester et al., 1999 ; Lambeir et al., 2001 ). Because the activity of RANTES is influenced by proteolytic processing, and the inhibition of this proteolytic processing by inhibition of DPP4 or of CD26 allows the use of the compounds with inhibitory activity on the proteolytic activity of DPP4 and / or inhibitory effect on the proteolytic activity of DPP4 -like regulatory peptidases as active ingredient in pharmaceutical compositions.

State of the art

Drucker et al. (Nature Reviews 109-110 (2007)) describe the effect of sitagliptin phosphate, which is used as a drug in type 2 diabetes mellitus. The mechanism of action of sitagliptin is based on the inhibition of human dipeptidyl peptidase 4 (DPP4), which inactivates the insulin secretion of beta islet cells promoting glucagon-like peptide 1 (GLP 1) and the glucose-dependent insulinotropic polypeptide (GIP) by proteolysis. The inhibition of the proteolytic activity of DPP4 therefore leads to a prolonged activity of the endogenous glucoregulatory incretins GIP and GLP 1.

traditional Healing procedures in the Arab world and Africa put camel milk as Food for combating cardiovascular diseases and diabetes.

Kappeler et al. (J. Dairy Science 2084-2093 (1999)) describe the cloning and amino acid sequencing of lactophorin A and its variant lactophorin B from camel milk. For lactophorin, good emulsifying properties and inhibition of spontaneous lipolysis by lipoprotein lipase were found. The lactophorin was not glycosylated and showed sequence similarities to glycosylation-dependent cell adhesion molecule-1 proteins (GlyCAM-1). The amino acid sequences of lactophorin A and B are deposited under accession number AJ131714 at EMBL and GenBank, respectively.

Deacon ( Diabetes, 2181-2189 (2004) ) describes that inhibition of DPP4 may have antidiabetic activity due to decreased proteolysis of GLP1. Inhibitors of DPP4 are valine pyrrolidide and isoleucine thiazolidide. Since the DPP4 inhibitor valine pyrrolidide also improved glucose tolerance in mouse mutants lacking the GLP1 receptor, it is believed that inhibition of DPP4 also reduces proteolysis of exogenous GIPS. Administration of the DPP4 inhibitor isoleucine thiazolidide to Vancouver sugar diabetic fat rats also shows a decrease in total body weight in addition to the improvement in glucose tolerance and the improved response of β cells.

The EP 0995440 describes pharmaceutical compositions containing an inhibitor of DPP4 for increasing blood sugar levels. Inhibitors of DPP4 are generally called inhibitors, substrates, pseudo substrates, expression inhibitors, binding proteins and antibodies, in particular alanylpyrrolidide and isoleucyl-thiazolidide, N-valyl-prolyl and O-benzoylhydroxylamine, in particular as fumarate.

The WO 99/67278 describes precursors of isoleucyl thiazolidide for use as inhibitor of DPP4.

The WO 99/38501 describes the use of dipeptides, namely Pro-Pro, Ala-Pro and D-Ala-L-Ala, as inhibitors of DPP4 or of GLP1.

The WO 01/68603 describes inhibitors of DPP4 having a pyrroliding group with a bound cyclopropyl group.

WO 01/40180 describes compounds having a pyrrolidine moiety substituted with a nitrile group for use as an inhibitor of DPP4.

Object of the invention

Across from the aforementioned prior art, it is an object of the invention to provide an alternative inhibitor of human DPP4, and pharmaceutical compositions containing the inhibitor against DPP4 or CD26 and / or DPP4-like or CD26-like Contain enzymes as an active ingredient. A preferred task is in the provision of an inhibitor of human DPP4 for use in the manufacture of pharmaceutical compositions for the medical use in diabetes, especially diabetes mellitus (Type 2 diabetes), and for use in other indications, which are more human by inhibiting the proteolytic activity DPP4 and DPP4-free enzymes and / or inhibition of human APN are treatable.

A particularly preferred object is to provide an active ingredient for use in the manufacture of pharmaceutical compositions, which inhibits both human DPP4 and human aminopeptidase N (APN, CD13 aminopeptidase) and / or the human zAAP and / or the human DPP8 and / or DPP9 inhibited.

General description of invention

The Invention solves the above problem by pharmaceutical Compositions with peptides isolatable from camel's milk and N-terminal amino acid sequence consisting of of the group comprising VPQ, KPQ, QPT and EPK or consists of a peptide having one of the sequences selected from the Group comprising VPQ, KPQ, QPT and EPK, or peptides which, after proteolytic cleavage, have an N-terminal Have amino acid sequence, the VPQ, KPQ, QPT or EPK or a peptide having one of the sequences VPQ, KPQ, QPT or EPK, for medical use for inhibiting the DPP4 and DPP4-analogue peptidases, preferably in combination with the inhibition of APN, zAAP and / or DPP8 and DPP9, in particular in the medical indication diabetes mellitus, obesity and / or cardiovascular diseases, as well as medical indications, which are treatable by influencing T cells, in particular Indications due to inhibition of CD26 and / or zAAP of T cells are treatable. Furthermore, the invention relates to the use of peptides having an N-terminal amino acid sequence, which is selected from the group, the VPQ, KPQ, QPT and EPK comprises or consists of one of the sequences selected from the group which includes VPQ, KPQ, QPT and EPK, or Peptides which, after proteolytic cleavage, have an N-terminal amino acid sequence which is or consists of VPQ, KPQ, QPT or EPK Production of pharmaceutical compositions, in particular for use in the aforementioned medical indications.

As DPP4-like enzymes and peptidases or, equivalently, DPP4-analogous enzymes and peptidases, in the present case in particular peptidases are designated with regulatory function, which preferably have a similar to the DPP4 amino acid sequence, for. B. comprise or consist of at least 80 to 90% identical sequence. DPP4-analogous peptidases preferentially act in vivo on the same substrates as DPP4, and are particularly those peptidases which cleave in vivo GLP-1, GLP-2 and / or GIP and / or NPY and / or PYY. DPP4-analogous peptidases are particularly preferably those in which the inhibition of their proteolytic activity is one of the medical indications according to the invention positively influenced by cations.

The Analysis and identification of individual substances from camel milk has surprisingly demonstrated that isolated lactophorin A and / or lactophorin B, and derived peptides with a specific amino acid sequence inhibitory activity against the human DPP4 and DPP4 analog peptidases, and preferably also against the human aminopeptidase N (APN) and / or against the human cytosolic alanylaminopeptidase (zAAP) and / or against human dipeptide peptidase 8 (DPP8) and / or dipeptide peptidase 9 (DPP9). This inhibitory effect the abovementioned peptidases, in particular with respect to DPP4, was detected in in vitro tests and in vitro in cell culture, as well as in the experimental animal. The pharmaceutical efficacy of lactophorin A and lactophorin B from camel milk and derived peptides, especially those which have an amino acid sequence at the N-terminus from the group comprising VPQ, KPQ, QPT and EPK in animal experiments for the indications diabetes mellitus type 2, as well as for the indications obesity and cardiovascular diseases, especially those that are common with diabetes mellitus and / or obesity occur as well Indications caused by T cells. Preferably have the peptides of the invention in addition to the effect against DPP4 also a significant inhibitory activity against a or more from the group, human APN, human zAAP, human DPP8 and / or DPP9 so that amplification of the pharmaceutical effect of the peptides by their inhibition of at least two peptidases from the aforementioned group is achieved.

Especially could be shown in animal experiments that the invention Peptides even when administered orally against accumulation of storage fat tissue, z. B. are effective against obesity.

It it is assumed that the effectiveness of inventive Peptides against obesity in addition to the reduction of proteolysis Insulin is also based on the fact that neuropeptide Y (NPY) and / or Peptide YY (PYY) in inhibiting the activity of DPP4 and whose membrane-bound forms are less degraded or longer available. As a result, a by NPY and / or PYY increased satiety feeling stronger or stay longer.

The Effectiveness of inventive peptides against neuronal Diseases is based on the current state of knowledge that inhibits the inhibition of DPP8 and / or DPP9 diseases by causes or enhances the activity of DPP8 and / or DPP9 become. These neuronal diseases include in particular Anxiety, depression and Alzheimer's.

The Peptides according to the invention can by oral administration or by administration by injection, e.g. B. i. m. or I. v., of fusion peptides, especially in pharmaceutical acceptable formulation, with fusion peptides at least one amino acid sequence or a tripeptide VPQ, KPQ, QPT and EPK at the N-terminus, or consist of, or the fusion peptides have an amino acid sequence at least one peptide is generated by proteolytic cleavage, the N-terminal one amino acid sequence or one of the tripeptides VPQ, KPQ, QPT and EPK. The proteolytic Cleavage of the fusion peptides after their oral administration can z. B. done by natural digestive enzymes. A preferred one Fusion peptide whose proteolytic cleavage by digestive enzymes Peptides according to the invention having an inhibitory effect produced at least against DPP4, is lactophorin A or lactophorin B, the z. B. by heterologous expression in microorganisms or eukaryotic cells is produced, or isolated from camel milk is.

Also the use of the compounds of the invention for the preparation of pharmaceutical compositions for treatment HIV infections are due to the inhibition of cell-bound DPP4, CD26 forms of T lymphocytes. Because CD26 or DPP4 interacts with the HIV1 transactivator protein Tat and the HIV1 envelope protein GP120. Accordingly, the inventive Compounds also for the preparation of pharmaceutical compositions, or used as an active ingredient for the treatment of diseases, influenced by influencing the T-cell-mediated immune response be, for. Against autoimmune diseases, especially psoriasis, as well as asthma. The effect of the invention Peptides in medical indications caused by reduction of T-cells are treatable, based on the current state of knowledge on the influence of CD26 and / or zAAP of T cells. These Effect on T cells is as well as the inhibition of proteolytic Activity of DPP4, DPP4-like peptidases, APN, zAAP and / or DPP8 or DPP9 limited in vivo, e.g. B. with a half-life in vivo of up to about 4 h, so that negative Side effects of the peptides of the invention also are temporary and therefore do not occur or do not significantly in particular the possible increase in metastasis are limited, or not or not significantly occurs.

The peptides according to the invention, their fusion peptides may be compared to known ones Inhibitors of DPP4 are therefore beneficial because their action in the organism after administration of limited duration, e.g. B. on a main duration of action from about 1 to 4 hours. This limited or compared to z. B. sitagliptin phosphate significantly shorter main duration of action or half-life causes the unwanted reduction of natural cell-cell adhesion significantly lower or diminished. Therefore, the time-limited main duration of effect becomes the peptides of the invention as inhibitors The DPP4 currently considered as a reason for the side effect the peptides according to the invention for the production of Neoplasms and in particular the side effect of reinforcement the metastasis of existing tumors is significantly lower, than with known DPP4 inhibitors.

The Use of the peptides according to the invention for the production of pharmaceutical compositions or as an active ingredient in pharmaceutical Compositions for diabetes mellitus, obesity and so on Connected cardiovascular diseases, neuronal Diseases and / or diseases caused by HIV therefore have the advantage low side effects, especially a lower risk of neoplastic New diseases and / or in particular a lower risk of Metastasis of existing neoplasms, e.g. In endometrial adenocarcinoma, Melanoma, prostate carcinoma, haematological tumors, e.g. B. acute lymphoblastic leukemia and Hogdekin syndrome, gastrointestinal Carcinoma, human hepatocellular carcinoma, lung adenocarcinoma, Thyroid carcinoma, papillary and follicular thyroid carcinoma and ovarian cancer. As a cause of the low impact the peptides according to the invention for amplification or metastasis of at least these neoplasms is currently believed that decreased expression of DPP4 / CD26 on neoplastic Cells favored by the presence of an inhibitor of DPP4 being affected. So z. B. the presence of the invention Peptides for reducing migration and invasion activity, or the malignancy of neoplastic cells in comparison to the effect of z. As sitagliptin phosphate, since the reduction of Cell-cell adhesion by inhibition of DPP4 by the invention Peptides reduced or occurs for a limited time.

The temporary effect of the invention Peptides are currently attributed to that the active ingredients have no synthetic derivatizations and can be degraded by the organism. Therefore are unwanted Side effects when administering the compounds for medical purposes essentially not observable in humans or in comparison Significantly reduced with known inhibitors of DPP4.

The peptides and fusion peptides according to the invention, their proteolytic cleavage products according to the invention Peptides can be obtained by conventional synthetic methods or from natural sources, in particular from camel milk be isolated. Peptides that are not the complete sequence of lactophorin A or lactophorin B, e.g. By proteolytic cleavage of lactophorin A or lactophorin B, optionally prepared with subsequent purification become. Synthetic methods can be chemical peptide synthesis or the heterologous expression of the peptides of a DNA sequence encoding this, the z. B. from the amino acid sequence is derived and functional between a promoter and a Terminator is arranged as an expression cassette and in a host organism is expressed.

Preferably are pharmaceutical compositions which are an inventive Containing peptide as an active ingredient, formulated for oral administration, in particular as an enteric-coated formulation.

amino acid sequences of peptides in this application are from the N-terminus to the C-terminus specified.

Detailed description of the invention

basis The present invention is that the human GPP4 in vitro and inhibited in vivo by peptides that are one of the tripeptides VPQ (SEQ ID NO: 3), KPQ (SEQ ID NO: 4), QPT (SEQ ID NO: 5) or EPK (SEQ ID NO: 6) at the N-terminus or consist thereof.

Preferred fusion peptides which contain one of the tripeptides and are converted by proteolytic cleavage to peptides containing one of the tripeptides VPQ (SEQ ID NO: 3) KPQ (SEQ ID NO: 4), QPT (SEQ ID NO: 3). comprise or 5) or EPC (SEQ ID NO. 6) at the N-terminus consist of, the amino acid sequence of lactophorin A MKFFAVLLLASLAATSLASLNEPKDEIYMESQPTDTSAQVIMSNHQVSSEDLSMEPSISREDLVSKDDVVIKSARRHQNQNPKLLHPVPQESSFRNTATQSEETKELTPGAATTLEGKLVELTHKIIKNLENTMRETMDFLKSLFPHASEVVKPQ have No. (SEQ ID. 1) or the amino acid sequence of lactophorin B MKFFAVLL LASLAATSLASLNAAQVIMSNHQVSSEDLSMEPSISREDLVSKDDVVIKSARRHQNQNPKLLHPVPQESSFRNTATQSEETKELTPGAATTLEGKLVELTHKIIKNLENTMRETMDFLKSLFPHASEVVKPQ (SEQ ID NO: 2). Further fusion peptides according to the invention, which are converted by proteolytic cleavage into peptides containing one of the tripeptides VPQ (SEQ ID NO: 3), KPQ (SEQ ID NO: 4), QPT (SEQ ID NO: 5) or EPC (SEQ ID NO. 6) at the N-terminus or consist thereof, z. B. N-terminal and / or C-terminal to at least one of the tripeptides VPQ (SEQ ID NO: 3) KPQ (SEQ ID NO: 4), QPT (SEQ ID NO: 5) or EPK ( SEQ ID NO: 6) have peptide-linked amino acid sequences, particularly to form an amino acid sequence recognized by peptidases, e.g. B. digestive enzymes, in particular trypsin, chymotrypsin or pepsin.

invention Peptides therefore include Lactophorin A and / or Lactophorin B also fusion proteins natural or artificial Amino acid sequence, without reaction or after proteolytic cleavage at least one of the tripeptides VPQ (SEQ ID NO: 3) KPQ (Seq. ID No. 4), QPT (SEQ ID NO: 5) or EPK (SEQ ID NO: 6) at its N-terminus to assume such a three-dimensional structure that they significantly inhibit the proteolytic activity of DPP4.

For the proteolytic cleavage product of lactophorin A also became when administered orally, an inhibitory effect on DPP4 was found. This cleavage product containing the amino acid sequence EPKDEIYMESQPTDTS (SEQ ID NO: 7) and a portion of the amino acid sequence of lactophorin A with the N-terminal tripeptide EPK (SEQ. ID. NO. 6) shows an inhibitory effect in vitro and in vivo APN. Correspond have compositions according to the invention preferably a peptide with N-terminal tripeptide EPK (Seq. ID No. 6), e.g. See SEQ ID NO: 4, in combination with a peptide containing the tripeptide VPQ (SEQ ID NO: 3) N-terminal or consists of.

The Peptides according to the invention also include such Variants and modifications of their amino acid sequences, the the inhibitory activity against DPP4 or CD26 was not significant affect. In particular, variants have one or several exchanges, deletions or insertions of amino acids, especially conservative exchanges. The invention will now be described of examples with reference to the figures explained in more detail, at them

1 a graphic representation of the in vitro inhibition of DPP4 by peptide according to the invention,

2 a graphic representation of the in vitro inhibition of DPP4 by peptide according to the invention,

3 shows the relative inhibition of APN1 by peptide according to the invention,

4 FIG. 2 shows a graphic representation of the in vitro inhibition of DPP4 by peptide according to the invention and of actinonin as comparison substance,

5 FIG. 2 shows a graphic representation of the in vitro inhibition of DPP8 and DPP9 by the peptide according to the invention and the DPP4 inhibitor actinonin as comparison substance,

6 shows a graphic representation of the in vitro inhibition of zAAP by peptide according to the invention,

7 a graphic representation of the effect of the peptide according to the invention on the mitochondrial activity,

8th shows the inhibition of DPP4 by inventive peptide in vivo, and

9 shows the effect of peptides of the invention on the reduction of the increase in the total weight in mice as experimental animals.

Example 1: Inhibition of DPP4 by fusion peptide

When fractionating camel milk peptides, lactophorin A and B were found to have an inhibitory effect on human DPP4. A First In Vitro Trial of Purified Human Recombinant DPP4 Derived from Korom ( Transplantation 63, 1495-1500 (1997) ) by expression in Cell culture was prepared and isolated, showing that lactophorin A at 100 ng / mL (conc1), 200 ng / mL (conc2), 500 ng / mL (conc3), 1 ug / mL (conc4) and 10 ug / mL (conc ) inhibits DPP4. The result is in 1 and shows that lactophorin in the concentrations used significantly inhibits the proteolytic activity of DPP4. For comparison, the activity of DPP4 is shown without addition of a peptide in the otherwise identical experiment. The proteolytic activity was determined as the conversion of one of the substrates GlyPro-AMC (Gly-Pro-Aminomethylcourmarin) or Ala-Pro-Rhodamine 110 (R110).

As a general rule were inventive peptides in purified Form stored in HEPES buffer (100 mM HEPES, pH 7.5), preferably at -20 ° C, which also affects the storage stability the peptides in solution at temperatures below 0 ° C, preferably at -5 to -70 ° C shows. Tests were performed at 37 ° C. The measurement Hydrolysis of added substrate was done after incubation added substrate for 2 min at 37 ° C with enzyme and inhibitor.

For lactophorin A, the following parameters were determined for the substrate GlyPro-AMC in vitro: value SA V _max 6.59349 × 10 ³ 1.566 × 10 ³ k _m 1.70005 × 10 ^-4 4,829 × 10 ^-5 k _i 1.23528 × 10 ^-3 7.461 × 10 ^-5

The K _i value was calculated to be 1.235 × 10 ^-3 mg / mL. Similar values could be determined for Lactophorin B.

A comparison of the inhibitory effect of Lactophorin A on isolated recombinant human DPP4 in the in vitro experiment is in 2 compared to the known DPP4 inhibitor Lys-Z (nitro) pyrrolidide. To measure the activity, the hydrolysis of subsequently added Ala-Pro-Rhodamin110 (R110) was determined by fluorimetry after incubation with Lactophorin A or Lys-Z (nitro) pyrrolidide for 2 hours. The results are in

2 demonstrated that lactophorin A inhibits human DPP4. The Lactophorin A IC50 was determined to be 1.77 μM for GlyPro-AMC and 0.143 μM for Lys-Z (nitro) pyrrolidide.

Upon measuring the inhibitory effect of lactophorin A on the hydrolysis substrate gly-pro-aminomethylcoumarin (GlyPro-AMC), an IC50 = 6.9 μM was found for lactophorin A, and of 0.39 μM for the known DPP4 inhibitor actinonine. The data is in 4 shown.

By proteolytic fragmentation of lactophorin A and lactophorin B obtained peptides were also on the inhibition of DPP4 analyzed. It was found that the inhibitory effect of lactophorin exerted essentially by peptides which is N-terminal one of the amino acid sequences VPQ (Seq. ID No. 3) KPQ (SEQ ID NO: 4), QPT (SEQ ID NO: 5) or EPK (SEQ. ID No. 6). These peptides may be considered proteolytic Cleavage products of fusion peptides arise which are one of the amino acid sequences VPQ (SEQ ID NO: 3) KPQ (SEQ ID NO: 4), QPT (SEQ ID NO: 5) or EPK (SEQ ID NO: 6).

From the following isolated peptides it was possible to show that peptides having one of the amino acid sequences VPQ (SEQ ID NO: 3), KPQ (SEQ ID NO: 4), QPT (SEQ ID NO: 5) or EPK (SEQ ID NO: 6) N-terminal, have the inhibitory effect according to the invention on DPP4 and / or aminopeptidase N:
VPQ (SEQ ID NO: 3) and the natural proteolytic cleavage product of lactophorin A with N-terminal SEQ ID NO: 6, namely SEQ ID NO: 7.

Example 2: Inhibition of APN by Lactophorin A

The Activity of lactophorin A and B, as well as the tripeptide SEQ ID NO: 3 on APN was detected in the in vitro test.

Human recombinant APN following Lendecke et al. (BioChem 384 (2003)) was prepared and isolated in the buffer of Example 1 with Lactophorin A at 10 μg / mL (2 in 3 ), 5 μg / mL (3 in 3 ) or 100 ng / mL (4 in 3 ). For comparison, the activity of APN without additive is shown (1 in 3 ). The activity of APN was determined spectrophotometrically as the conversion of the substrate AMC to 0 min and 90 min, the results are summarized in the following table: Inhibition of APN by Lactophorin A Conc. 0 min 90 min Activity over 90 min 10 μg / mL 0.0437 0.0594 0,017 5 μg / mL 0.0506 0.0688 0.0132 0.1 μg / mL 0.0456 .6066 0.556

The Values show that lactophorin A affects the activity of APN increasingly inhibited with increasing concentration. As a positive control Actinonin was used for the inhibition.

Example 3: Inhibition of DPP8 / DPP9 by lactophore A

DPP8 / DPP9 was derived from livers of CD26-KO mice (Black6, after Marguet et al., PNAS (2000) ), partially solubilized in PBS with 1% Triton X100. The mixture of DPP8 / DPP9 was incubated in HEPES buffer with lactophorin A at 37 ° C for 2 h. Subsequently, the substrate Gly-Pro-AMC was added and as control the DPP4-specific inhibitor 3 and the DPP8 / DPP9 specific inhibitor 4, as of Lankas et al. (Diabetes 54 (10), 2988-2994 (2005)) described.

The results are in 5 and demonstrate that Lactophorin A inhibits DPP8 and / or DPP9. For DPP8 / DPP9, an IC50 of 21 μM was determined for lactophorin A, an IC50 of 0.009 μM for the DPP8 / DPP9 inhibitor and an IC50> 31.25 μM for the DPP4 inhibitor.

The Inhibition of DPP8 / DPP9 was also possible for the peptides of SEQ ID Nos. 3 to 6 and inventive fusion peptides whose amino acid sequence is N-terminal one Sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID No. 6 had.

Example 4: Inhibition of zAAP by Lactophorin A

zAAP was purified from cultured Jurkat cells. zAAP was in HEPES buffer with 0.25 mM DTT with lactophorin A at 37 ° C incubated for 2 h. Subsequently, the substrate became Ala-rhodamine 110 (R110) was added and the hydrolysis after 60 min spectrometrically certainly.

The results are in 6 and demonstrate that Lactophorin A inhibits zAAP. For lactophorin A, an IC50 of> 62.5 μM was determined under these experimental conditions with respect to zAAP.

The Inhibition of zAAP was also possible for the peptides of SEQ ID Nos. 3 to 6 and fusion peptides according to the invention whose amino acid sequence is N-terminal one Sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID No. 6 had.

Example 5: Cytotoxicity of peptides

To determine the cytotoxicity of peptides of the invention, representative Lactophorin A was used in the live staining with trypan blue (Celltiter-Blue test) with cultivated U937 cells. The results are in 7A for cells without additive, for cells supplemented with lactophorin A or for control with sodium azide (Na-azide) in 7B , each in log 2 dilution steps. The result shows, using the example of lactophorin A, that peptides according to the invention have no significant cytotoxic effect.

Example 6: Inhibitory effect of Peptides on DPP4 in vivo

The inhibitory effect of peptides according to the invention against DPP4 was measured in blood plasma of a rat to which a peptide according to the invention had previously been administered by hydrolysis of a substrate of DPP4. As an example of peptides of the invention, lactophorin A was used in saline (0.154 M, 0.1% BSA (bovine serum albumin)). Blood plasma was injected from a catheterized rat after fasting overnight (time 0). To the in 8th At time points, blood samples were taken and heparinized. For the analysis of the activity of DPP4, 100 μL heparinized blood was spiked with 10 μL 1 M citrate buffer, pH 3.0, centrifuged and cooled, or stored at -20 ° C until analyzed.

25 mg lactophorin A were dissolved in 5 mL 0.1% BSA in saline and aliquots of 500 μL frozen at -20 ° C. From this solution of Lactophorin A, 10 μL (0.05 mg) was added to the plasma samples (approximately 50 μL). For the test, 20 μL of the Lactophorin A-added plasma was mixed with 80 μL HEPES buffer and the substrate Gly-Pro-pNA and spectrophotometrically measured after incubation for 2 min at 30 ° C for 1 min at 30 ° C. The result of this test is in 8th shown. It can be seen that, starting from the original activity of 12.28 mU / mL (-10 min and 0 min) DPP4 in rat plasma, the activity of DPP4 was reduced to 7.46 mU / mL by injecting Lactophorin A into the plasma and decreased 10 minutes after the injection, followed by a slight increase to about 9 mU / mL. The original activity of DPP4 was not recovered in the 120 mm of observation. Overall, therefore, this experiment shows that lactophorin A inhibited DPP4 in serum by about 40% during the first 10 min after the administration of the injection, followed by a subsequent inhibition of about 25% for the next 110 min. The results are in 8th shown. The long-term study revealed that about 4 hours after administration of the peptide, the original activity of DPP4 was restored. Therefore, it can be seen from this experiment that peptides according to the invention, at least in these concentrations, have the proteolytic activity of DPP4 in vivo within a short time after administration, e.g. B. inhibit within about 10 mm, this inhibitory effect is effective for at least about 110 mm, and after about 4 hours the inhibitory effect on DPP4 is completed. This result demonstrates the advantage of the peptides according to the invention that the inhibitory effect on the proteolytic activity of at least DPP4 is limited in time or reversible in the mammalian organism.

The inhibitory effect of peptides of the invention was able to use the example of lactophorin A also in plasma a healthy Rat are detected when stabilized with citrate buffer Plasma Lactophorin A and substrate added for the DPP4 has been. Specifically, blood samples were catheterized from a healthy one Rat taken who still no inventive Peptide was administered. From the samples was as described above Plasma prepared and stabilized with citrate buffer. As a substrate for the DPP4 activity, Gly-Pro-pNA was in HEPES buffer used. Individual serum samples were 0.05 mg lactophorin A added. It could be shown that lactophorin A in the serum the activity of DPP4 from about 11.4 mU / mL to 3.51 mU / mL inhibited.

Comparative tests in which only buffer was added, or lactophorin A without citrate buffer showed that the inhibitory effect of Lactophorin A substantially was independent of the buffer substances.

In the following table the experimental approaches are shown in detail: activity Plasma +10 μL citrate buffer Plasma +10 μL citrate buffer + 10 μL HEPES buffer Plasma +10 μL citrate buffer + 0.05 mg lactophorin A in HEPES buffer Plasma + 0.05 mg Lactophorin A without citrate buffer DPP4 (mU / mL) 11.40 10.53 3.51 4.39 AE / min 0,013 0.12 0,004 0.005

This Experiment shows that peptides of the invention the Activity of DPP4 in mammalian plasma containing no DPP4 inhibitor was administered.

Example 7: Effect of peptides against Obesity in mammals

The Effect of peptides according to the invention against obesity of mammals was shown using the example of lactophorin A, the 6 out of 12 mice (Balb6) with an initial one Weight of 20 g was administered orally with the feed. The mice were fed standard food and water ad libitum. A weight control of the mice before and after the experiment 3 weeks showed that the oral administration of inventive Peptide the body weight and especially the part of body fat reduced. For the experiment, 6 animals were given 0.5 mg / kg lactophorin A mixed into the feed per day, 6 control animals received no further additives to the food.

The result is in 9 demonstrated and demonstrated that the administration of peptide according to the invention under otherwise identical feeding and movement conditions leads to a significantly reduced weight gain, here with the example of Lactophorin A. In each paired bars in 9 the leftmost bar represents the final weight of the mice given Lactophorin A and the right one represents the untreated mice.

The Reduction of the total weight increase with inventive Peptide treated mice were compared to untreated ones Mice under otherwise identical conditions was approx. 5 g each. Anatomical analyzes showed that with the invention Peptide treated mice significantly reduced Share of storage grease, while the rest no significant anatomical or histological changes were observed in untreated mice. A detailed histological analysis of the experimental animals revealed in that the administration of the peptide according to the invention did not result in tissue abnormalities, especially neoplasms.

It follows Sequence listing according to WIPO St. 25. This can of the official publication platform of the DPMA become.

QUOTES INCLUDE IN THE DESCRIPTION

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Cited patent literature

• EP 0995440 [0008]
• WO 99/67278 [0009]
• WO 99/38501 [0010]
• WO 01/68603 [0011]
• WO 01/40180 [0012]

Cited non-patent literature

• Proost et al., 1998 [0003]
• - Shioda et al., 1998 [0003]
• - Struyf et al., 1999 [0003]
• - Proost et al., 2000 [0003]
• Guan et al., 2004 [0003]
• De Meester et al., 1999 [0003]
• Lambeir et al., 2001 [0003]
• - Drucker et al. (Nature Reviews 109-110 (2007)) [0004]
• - Kappeler et al. (J. Dairy Science 2084-2093 (1999)) [0006]
• - Diabetes, 2181-2189 (2004) [0007]
• Transplantation 63, 1495-1500 (1997) [0043]
• Lendecke et al. (BioChem 384 (2003)) [0053]
• Marguet et al., PNAS (2000) [0055]
• - Lankas et al. (Diabetes 54 (10), 2988-2994 (2005)) [0055]

Claims (21)

A pharmaceutical composition having an active ingredient for inhibiting the proteolytic activity of human DPP4, characterized in that the active ingredient is a peptide having N-terminal amino acid sequence selected from the group comprising VPQ (SEQ ID NO: 3) KPQ (SEQ ID NO: 4), QPT (SEQ ID NO: 5) and EPK (SEQ ID NO: 6). Pharmaceutical composition according to claim 1, characterized in that the active ingredient is a peptide consisting of an amino acid sequence selected from the group VPQ (SEQ ID NO: 3) KPQ (SEQ ID NO: 4), QPT (SEQ ID NO: 4); 5) and EPK (SEQ ID NO: 6). Pharmaceutical composition according to claim 1, characterized in that the active ingredient is a fusion peptide, by proteolysis by a natural digestive enzyme is converted to a peptide having an N-terminal amino acid sequence which is selected from the group comprising VPQ (SEQ. ID. 3) KPQ (SEQ ID NO: 4), QPT (SEQ ID NO: 5) and EPK (SEQ ID NO: 4). 6). A pharmaceutical composition according to claim 1 or 3, characterized in that the active ingredient Seq.-ID No. 7 or consists of. Pharmaceutical composition according to claim 3, characterized in that the active ingredient is the amino acid sequence of lactophorin A (SEQ ID NO: 1) or the amino acid sequence of lactophorin B (SEQ ID NO: 2). Pharmaceutical composition according to one of the preceding Claims, characterized in that they also have the activity human APN, human zAAP and / or human DPP8 and / or DPP9 inhibits. Pharmaceutical composition according to one of the preceding Claims, characterized in that they are for oral Administration or for injection. Pharmaceutical composition according to one of the preceding Claims, characterized in that they are for use formulated against a medical indication selected is among diabetes mellitus, obesity, cardiovascular disease and T-cell mediated diseases including autoimmune diseases. A pharmaceutical composition according to any one of the claims 1 to 7, characterized in that they are for use against Neuronal diseases are formulated by inhibition of human DPP8 and / or DPP9 are treatable. Pharmaceutical composition according to claim 9, characterized in that the neuronal disease is selected is from anxiety, depression and Alzheimer's. Use of a peptide for the preparation of a pharmaceutical Composition according to one of the preceding claims, characterized in that the peptide N-terminal an amino acid sequence which is selected from the group comprising the VPQ (Seq. ID No. 3) KPQ (SEQ ID NO: 4), QPT (SEQ ID NO: 5) and EPK (SEQ. ID No. 6). Use according to claim 11, characterized that the peptide the activity of the human APN, the human zAAP and / or DPP8 and / or DPP9. Use according to one of claims 11 to 12, characterized in that the peptide consists of an amino acid sequence that is selected from the group that contains VPQ (Seq. ID No. 3) KPQ (SEQ ID NO: 4), QPT (SEQ ID NO: 5) and EPK (SEQ. ID No. 6). Use according to one of claims 11 to 12, characterized in that the peptide from Seq. ID No. 1, SEQ ID NO: 2, or SEQ ID NO: 3. Use according to any one of claims 11 to 13, characterized in that the peptide is a fusion peptide which is converted by proteolysis by a natural digestive enzyme to a peptide having N-terminal amino acid sequence selected from the group consisting of VPQ ( Seq. ID No. 3) KPQ (SEQ ID NO: 4), QPT (SEQ ID NO: 5) and EPK (SEQ ID NO: 6). Use according to claim 13 or 15, characterized the peptide has the amino acid sequence SEQ ID NO: 7 or consists of. Use according to claim 16, characterized that the peptide is the amino acid sequence of lactophorin A (SEQ ID NO: 1) or the amino acid sequence of lactophorin B (SEQ ID NO: 2). Use according to one of claims 11 to 17, characterized in that the composition for oral Administration or for injection. Use according to one of claims 11 to 18, characterized in that they are for use against A medical indication is formulated that is selected is diabetes mellitus, obesity, cardiovascular disease, including neuronal diseases and T-cell mediated diseases Autoimmune diseases. Process for the preparation of a pharmaceutical A composition according to any one of claims 1 to 10 or for use according to any one of claims 11 to 19, characterized by providing an isolated peptide, having an amino acid sequence whose N-terminus the group selected is the VPQ (Seq. ID No. 3) KPQ (SEQ ID NO: 4), QPT (SEQ ID NO: 5) and EPK (SEQ ID NO: 6), Mixing the peptide into a pharmaceutically acceptable formulation. Method according to claim 20, characterized in that that the isolated peptide by expression in a host organism and isolation from the host organism and / or from its culture medium becomes.