WO2002040512A2 - Human beta-defensin-3 - Google Patents

Human beta-defensin-3 Download PDF

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Publication number
WO2002040512A2
WO2002040512A2 PCT/EP2001/013174 EP0113174W WO0240512A2 WO 2002040512 A2 WO2002040512 A2 WO 2002040512A2 EP 0113174 W EP0113174 W EP 0113174W WO 0240512 A2 WO0240512 A2 WO 0240512A2
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WIPO (PCT)
Prior art keywords
arg
cys
lys
hbd
gly
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PCT/EP2001/013174
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German (de)
French (fr)
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WO2002040512A3 (en
Inventor
Wolf-Georg Forssmann
Enno KLÜVER
Jose-Ramon Conejo-Garcia
Knut Adermann
Robert Bals
Hans-Jürgen MÄGERT
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Ipf Pharmaceuticals Gmbh
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Priority to AU2002227925A priority Critical patent/AU2002227925A1/en
Publication of WO2002040512A2 publication Critical patent/WO2002040512A2/en
Publication of WO2002040512A3 publication Critical patent/WO2002040512A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to a peptide, the hBD-3 (human beta-defensin-3) and its derivatives and fragments, and to processes for their preparation.
  • the peptides according to the invention can be provided as medicaments and can be used to ward off and combat pathogenic germs.
  • the object on which the invention is based is to provide peptides with special physiological effects which are highly therapeutically active and which are accessible in a relatively simple manner.
  • the peptides are said to be particularly effective against pathogenic germs which have developed resistance to known antibiotics.
  • the antibiotic peptides according to the invention should also be well tolerated and allow easy application.
  • the object of the invention is achieved by the antibiotic protein human beta-defensin-3 (hBD-3, also referred to as hBD-5) with the amino acid sequence Seq. No. 1 : Zx-Cys-Arg-Val-Arg-Gly-Gly-Arg-Cys-Ala-Val-Leu-Ser-Cys-Leu-Pro-Lys-Glu-Glu-Gln-Ile-Gly-Lys-Cys-Ser- Thr-Arg-Gly-Arg-Lys-C / sC / s-Xi, where Zi and Xi represent amino acids or polypeptides from 1 to 50 amino acids, as well as their natural, synthetic and pharmacologically acceptable derivatives, in particular amidated, acylated, phosphorylated, glycosylated and cyclic derivatives, as well as their derivatives and fragments with biological activity resulting from amino acid substitution, deletion or insertion, which are derived from the amino acid sequence.
  • Preferred according to the invention are hBD-3 with the Seq. No. 2:
  • the invention also relates to fragments of hBD-3 of the amino acid sequence Seq. No. 5:
  • Z 2 , X x and X 2 represent amino acids or polypeptides from 1 to 50 amino acids, and their synthetic and pharmacologically acceptable derivatives, in particular cyclic, amidated, acylated, phosphorylated and glycosylated derivatives, and their by amino acid substitution, deletion or - insertion derived derivatives and fragments with biological effect, which are derived from the amino acid sequence.
  • hBD-3 fragments of the invention are preferably those of the sequence Seq. No. 7:
  • the invention also relates to derivatives of hBD-3 or fragments of hBD-3 in which individual amino acids have been exchanged such that the peptide has a higher positive charge overall.
  • Derivatives in which one of the four glycines from sequence no. 1 and / or the alanine, which in sequence No. 1 follows cysteine 2 and / or the leucine or proline, which in sequence no. 1 followed by cysteine 3 are substituted by basic amino acids.
  • the designated seven amino acids can also be replaced by aromatic amino acids.
  • the invention relates to derivatives of the peptides with the sequences 2 to 9, in which the glycine, alanine, leucine and / or proline which is homologous to sequence 1 has been replaced.
  • cysteines 1 and 2 are bridged, cysteines 3 and 4 being linked to cysteines 5 and 6 via a disulfide bond in each case.
  • the hBD-3 or hBD-3 fragments or derivatives according to the invention are capable of bridging cysteine bridges between the six cysteines in sequence no. 1 to train.
  • the cysteine bridges are formed between cysteine 1 and cysteine 2, cysteine 3 and cysteine 6 and / or cysteine 4 and cysteine 5.
  • Numbers 1 to 6 correspond to the six cysteines that are shown in sequence no. 1 from the N to the C terminus are included.
  • the numbers 1 to 6 remain unchanged and denote the cysteines which are homologous to those in sequence 1.
  • the fragment of sequence No. 9 cysteines 3 to 6 because the section containing cysteines 1 and 2 is missing.
  • the cysteines are shown in italics for easier identification.
  • Preferred peptides of the invention are, in particular, fragments of sequence 6 in which
  • Cysteine 3 and cysteine 6 do not form cysteine bridges and cysteine 4 and cysteine 5 form a cysteine bridge, or
  • Cysteine 3 and cysteine 6 form a cysteine bridge and cysteine 4 and cysteine 5 do not form a cysteine bridge, or
  • Cysteine 3 and cysteine 6 form a cysteine bridge and cysteine 4 and cysteine 5 form a cysteine bridge.
  • Preferred peptides of the invention are also hBD-3 or derivatives of hBD-3, in which the cysteines 3 to 6 are linked / not linked in one of the four ways listed above.
  • cysteine 1 and cysteine 2 can be connected via a cysteine bridge or form no cysteine bridge.
  • the proteins according to the invention can be obtained by chemical synthesis, by genetic engineering production or by isolation from natural sources.
  • the invention relates in particular to a process for the preparation of hBD-3 or derivatives or fragments of hBD-3, the hBD-3 or hBD-3 fragments or derivatives being obtained by solid-phase and / or liquid-phase synthesis from the protected amino acids contained in it, manufactured, deblocked, and purified by chromatography.
  • cysteine bridges can be introduced by oxidation.
  • the introduction can be targeted at specific positions by preventing the reaction of non-linked cysteines by means of suitable protective groups.
  • hBD-3 or derivatives or fragments of hBD-3 are produced by expression in prokaryotic or eukaryotic cells and subsequent chromatographic purification using known methods.
  • Various expression vectors are available for secretory or direct cytoplasmic expression.
  • the hBD-3 according to the invention or derivatives or fragments can be isolated from body fluids, preferably from human blood, hemofiltrate or hemodialysate, using a chromatography method. Hemofiltrate or hemodialysate can be filtered from human blood. This is particularly advantageous since hemofiltrate has so far been regarded as worthless and has been discarded. In this way, a source for economic exploitation is easily developed.
  • the invention also relates to nucleic acids coding for hBD-3 or its derivatives or fragments, in particular DNA and RNA.
  • the invention also relates to vectors or plasmids which contain such nucleic acids and fragments which are suitable as antisense nucleotides, in particular to coding nucleic acids complementary fragments with a length of 12-26 nucleotides.
  • the invention also relates to a medicament containing hBD-3 or derivatives or fragments of hBD-3, or nucleic acids which code for hBD-3 and vectors or plasmids which contain such nucleic acids.
  • the invention also relates to the use of hBD-3 or derivatives or fragments of hBD-3 for the manufacture of a medicament for antimicrobial treatment, for defense and combating pathogenic germs, for the treatment of respiratory diseases, in particular those caused by infections with the bacteria Burkholderia cepacia and Pseudomonas aeroginosa are triggered or intensified, for the treatment of cystic fibrosis, for the treatment of inflammatory diseases of the gastrointestinal tract, the urogenital tract, for sepsis, for gastrointestinal infections, in particular triggered by Heliobacter pylori, for the treatment and defense against gram-positive and gram-negative bacteria, yeast, Heliobacter pylori, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa or Burkholderia cepacia.
  • nucleic acids that code for hBD-3, derivatives or fragments, and vectors or plasmids that contain such nucleic acids can also be used, for example in connection with gene therapy.
  • the nucleic acids can preferably also be used as antisense oligonucleotides.
  • the proteins according to the invention are in particular able to restrict or prevent the bacterial invasion in inflammatory diseases of the respiratory tract.
  • the peptides according to the invention are analogues of the body's own substances, they have a high tolerance for the organism.
  • the peptide hBD-3 according to the invention and its derivatives and fragments are also particularly suitable for long-term therapy for infectious diseases of the respiratory tract caused by pathogenic germs, in particular Burkholderia cepacia and Pseudomonas aeroginosa. Suitable because they have an excellent biological effectiveness and, on the other hand, do not trigger an immune reaction even with long-term treatment.
  • hBD-3 its derivatives and fragments are also suitable for inducing apoptosis and therefore for treating malignant melanomas and other tumors, such as tumors in the gastrointestinal area.
  • apoptosis as programmed cell death, plays an important role in a wide variety of biological processes that require the targeted elimination of unwanted or unnecessary cells. Examples include the removal of unnecessary cells during development and differentiation processes, the killing of cells in the context of immune reactions and the destruction of damaged cells. The targeted induction of apoptosis with the help of certain chemotherapeutic agents is used to treat tumors.
  • the protein p53 plays a key role in the initiation of apoptotic processes. It is activated in a not yet fully understood manner by damaged deoxyribonucleic acid (DNA) and other signals mediated in connection with cellular stress. Activated p53 probably stimulates the release of cytochrome C from mitochondria with the participation of the factor Bax. Cytochrome C triggers the oligomerization of the factor Apaf-1, which then binds to and activates procaspase-9, which inevitably leads to cell death (Jones, P.A., Nature 409: 141, 2001).
  • mutations in the gene for p53 occur in many aggressive and chemoresistant tumors and are usually associated with a poor prognosis for the patient and ineffective chemotherapy.
  • mutations in the p53 gene are only rarely observed in the likewise aggressive and chemoresistant melanomas.
  • p53 can be upregulated normally by chemotherapy drugs, but this does not result in apoptosis. It has been shown that this defect in the apoptotic signaling cascade is based on the apoptosis effector Apaf-1 being switched off, which is due to the methylation of an enhancer region of the gene and thus to a repression of the transcription (Soengas et al., Nature 409 : 207, 2001).
  • the possibility of up-regulation of Apaf-1 is therefore an important step in the treatment of malignant melanoma and other tumors.
  • the disulfide bridging Cysl-Cys2; Cys3-Cys6; Cys4-Cys5 made it possible to synthesize the biologically active fragments of the peptide described above without having to make any further changes to the molecule. These are shorter N-terminal fragments, which contain the disulfide bridge Cysl-Cys2, and longer C-terminal fragments, which contain the other two disulfide bridges.
  • hBD-3 or derivatives and fragments is preferably carried out in suitable pharmaceutical application forms, in particular lyophilized and taken up in mannitol in sterile ampoules for dissolution in saline and / or infusion solutions and inhalation solutions, and / or in combination with other antibiotic active substances. Mixtures of different active ingredients according to the invention can of course also be used.
  • the peptides can be used as a highly pure substance or - if sufficient for the particular use - within a partially purified peptide mixture.
  • the pharmaceutical preparation contains the peptide according to the invention or a physiologically acceptable salt.
  • the form and composition of the drug containing the peptide depends on the mode of administration.
  • the peptide can be administered parenterally, intranasally, orally and by inhalation.
  • HBD-3 or its derivatives or fragments are preferably packaged either as a solution or as a lyophilisate for dissolution immediately before use.
  • Drug preparation can also contain excipients that are due to filling technology, contribute to the solubility, stability or sterility of the drug or increase the efficiency of absorption into the body.
  • the peptide was cleaved from the carrier resin by adding a mixture of trifluoroacetic acid / ethanedithiol / water 94: 3: 3 (v / v / v) and precipitated with tert-butyl methyl ether.
  • the peptide prepurified by HPLC was oxidized with the help of dimethyl sulfoxide (DMSO, 20%) at pH 6.
  • DMSO dimethyl sulfoxide
  • the introduction of the disulfide bridges can be carried out selectively by using two Fmoc-Cys (Acm) derivatives during the synthesis and linking the corresponding cysteines using iodine after the introduction of the other two disulfide bridges.
  • Acm Fmoc-Cys
  • the characterization of the synthesized peptide includes the detection of the intramolecular disulfide linkage according to the following procedure.
  • hBD-3 was proteolytically broken down by simultaneous exposure to trypsin and chymotrypsin.
  • the disulfide bridges remain intact, and an analysis of the interlinked cysteine-containing fragments allows the derivation of the original linking pattern.
  • the peptide fragments obtained were isolated by HPLC and identified both by mass spectrometry and with the aid of amino acid sequence analysis (FIG. 4, Tab. 1).
  • the sequence analysis also made it possible to clearly assign the disulfide bridging to the two neighboring cysteines, a constellation that generally causes particular analytical difficulties.
  • the disulfide bridging Cysl-Cys2; Cys3-Cys6; Cys4-Cys5 made it possible to synthesize two sections of the peptide separately without having to make any further changes to the molecule. It is a shorter N-terminal fragment that contains the Cysl-Cys2 disulfide bridge and a longer C-terminal fragment that contains the other two disulfide bridges. The synthesis and subsequent investigation of these two fragments serves to assign biological effects of the peptide hBD-3 to specific molecular regions.
  • the peptide was cleaved from the carrier resin by adding a mixture of trifluoroacetic acid / ethanedithiol / water 94: 3: 3 (v / v / v) and precipitated with tert-butyl methyl ether.
  • the peptide prepurified by HPLC was oxidized with the aid of dimethyl sulfoxide (DMSO, 20%) at pH 6.
  • DMSO dimethyl sulfoxide
  • the reaction product was purified by HPLC and characterized using analytical HPLC, mass spectrometric analysis, capillary zone electrophoresis and amino acid sequence analysis (Fig. 5-7).
  • the disulfide bridge can alternatively be introduced by oxidation with atmospheric oxygen in the basic pH range, but dimers are formed as by-products.
  • the peptide sequence mentioned was carried out on a carrier resin preloaded with lysine using an automatic peptide synthesis apparatus (ABI 433A) using the in situ method by adding HBTU [2- (1H-benzotriazol-l-yl) -l, 1,3,3 - Tetramethyluronium hexafluorophosphate] formed hydroxy-benzotriazole ester synthesized.
  • HBTU 2- (1H-benzotriazol-l-yl) -l, 1,3,3 - Tetramethyluronium hexafluorophosphate
  • the peptide was cleaved from the carrier resin by adding a mixture of trifluoroacetic acid / ethanedithiol / water 94: 3: 3 (v / v / v) and precipitated with tert-butyl methyl ether.
  • the peptide requires selective introduction of the two disulfide bridges. Simultaneous oxidation with atmospheric oxygen or DMSO provides a mixture of two folding isomers in equal proportions. Therefore, two of the four cysteines (Cysl ⁇ and Cys36) were used as Fmoc-Cys (Acm) derivatives in the peptide synthesis, the other two as Fmoc-Cys (Trt) derivatives.
  • the peptide prepurified by HPLC was oxidized at pH 6 using dimethyl sulfoxide (DMSO, 20%).
  • the reaction product was purified by HPLC and subsequently oxidized with iodine in the acidic pH range.
  • the fully oxidized end product was purified by HPLC and characterized using analytical HPLC, mass spectrometric analysis, capillary zone electrophoresis and amino acid sequence analysis (Fig. 8-10).
  • the synthetic peptide hBD-3 (14-40) [Cysl8-36 / Cys28-35] with known cysteine linkage, which is predetermined due to the synthesis, provided another possibility for verifying the disulfide bridging of hBD-3 determined.
  • Fragment was obtained by analogous proteolysis of hBD-3 (14-40) [Cysl8-36 / Cys28-35]. The identity of the two fragments, and thus identical disulfide bridging, could be shown by comigration in capillary zone electrophoresis (FIG. 11).
  • Table 1 shows the results of the mass spectrometric identification of the peptide fragments after proteolysis of hBD-3 with trypsin / chymotrypsin.
  • the antimicrobial activity of the peptides according to the invention was examined using the determination of the minimum inhibitory concentration (MIC) against human pathogenic Gram-positive and Gram-negative bacteria.
  • the minimum inhibitor concentration is the minimum peptide concentration required to completely inhibit the growth of microorganisms after an incubation period of 18 ⁇ 2 h.
  • the minimum inhibitory concentration was performed based on the recommendations (M7-A3) issued by the NCCLS (National Committee for Clinical Laboratory Standards), using chemically synthesized peptides of the invention.
  • the minimum inhibitory concentrations are given in the table below in [ ⁇ g / ml]. The following peptides were tested:
  • Test medium 1 / concentrated Mueller Hinton Broth
  • the peptides according to the invention effectively inhibit the growth of both Gram-positive and Gram-negative bacteria.
  • Human pathogens such as Pseudomonas aeruginosa, Staphylococcus aureus and Klebsiella pneumoniae are effectively killed.
  • the growth of antibiotic-resistant germs such as. B. Streptococcus pneumoniae (penicillin resistance), Enterococcus faecalis (vancomycin resistance) and Burkholderia cepacia (multi-resistant) is effectively inhibited by the peptides according to the invention.
  • the ability of antimicrobial peptides to attach to and permeabilize plasma membranes is often considered to be the mechanism of action of these substances. It is desirable that bacterial Membranes are selectively damaged, whereas the host's cells should remain unaffected.
  • the hemolysis test is used as a simple model for examining the cytotoxic effect of a peptide on eukaryotic cells.
  • Hemolysis [%] [A 450 nm (peptide) - A 450 nm (negative control)] / [A 450 nm (1% Tween 20) - A 450 nm (negative control)] * 100
  • TSB medium with 287 mM glucose was used as the test medium.
  • the isotonic glucose concentration is intended to prevent unspecific hemolysis in the hypotonic environment (osmoprotection).
  • DNA-microarray hybridization allows the detection by a certain factor of up- or down-regulated genes, whether hBD-3 plays a role in triggering apoptosis.
  • a cell line or primary cells are stimulated with the factor to be examined and a control sample is left unstimulated.
  • the messenger ribonucleic acid mRNA is isolated from both samples and, in the presence of two fluorescence-labeled nucleotides (Cy-3 for the stimulated sample, Cy-5 for the control), is transcribed into cDNA first strand ("copy deoxyribonucleic acid"). If genes are up-regulated by the factor to be examined, their transcripts are overrepresented in the stimulated sample compared to the control sample.
  • the transcripts are underrepresented in downregulation. These relationships are reflected accordingly in the labeled cDNAs.
  • the labeled cDNAs from stimulated and unstimulated cells are "hybridized” in one approach with DNA samples ("spots") of a multiplicity (several thousands) of different genes arranged in a punctiform manner on a suitable carrier, the cDNAs each corresponding to the spots of theirs corresponding genes "get stuck".
  • the signal strengths which can subsequently be detected with a special "analyzer” are proportional to the amounts of cDNAs hybridized with the respective spots.
  • SKMC 6723 Human skeletal muscle cells (SKMC 6723, obtained from Bio Whittaker, Walkerswill, MD, USA) were stimulated with 50 ⁇ g hBD-3 per ml medium for 90 minutes. A control sample remained unstimulated. The mRNA was then isolated from stimulated sample and control using standard methods and in the presence of Cy-3 (stimulated sample) or Cy-5-labeled (control) nucleotides rewritten into cDNA first strand. Both cDNAs were combined and hybridized together with the DNA spots on the array of NEN according to the manufacturer's instructions. The array was then sent to NEN for evaluation.
  • hBD-3 thus represents an activator of the gene for Apaf-1.
  • the human melanoma cell line SKMEL-28 was used to investigate the influence of hBD-3 on the induction of apoptosis.
  • the apoptosis was detected using a specific ELISA from Röche (Mannheim) ("Cell Death Detection ELISA", order no. 1 544 675).
  • the principle of this ELISA is based on the detection of fragmented DNA typical of apoptosis (Zhang JH, Xu M (2000). DNA fragmentation in apoptosis. Cell Res. 10: 205-211) and bound histones.
  • special microtiter modules are coated with monoclonal mouse antibodies directed against histones.
  • cell lysate is added to the test and fragmented, histone-associated DNA - if available - binds to the antibodies.
  • a second mouse monoclonal antibody directed against DNA is added, which is conjugated with a peroxidase.
  • the amount of binding antibodies is proportional to the amount of fragmented DNA present in the original cell lysate within a certain range. After repeated washing steps, this can be detected indirectly by the peroxidase-catalyzed formation of a substance absorbing in the range of 405 nm from the substrate ABTS (2,2'-azino-di- [3-ethylbenzthiazoline sulfonate]) (measurement on a spectrophotometer).
  • SKMEL-28 cells were cultivated specifically in "six-well culture plates" (2.2 ml of medium per well) and in the exponential growth phase, 10 5 cells per ml and at different concentrations were also used hBD-3 adjusted (0, 1, 3, 10, 30, 100 ⁇ g / ml). After five hours of incubation, the cells were treated further in accordance with the manufacturer's protocol and apoptoses which had finally occurred were determined indirectly by the peroxidase-catalyzed color reaction on the photometer.
  • SKMEL-28 cells were cultivated in 75 cm 2 culture bottles with a volume of 20 ml of medium and treated with 10 ⁇ g / ml of hBD-3 in accordance with the above-mentioned conditions during the exponential growth phase.
  • the DNA of the cells was then isolated according to standard protocols, completely electrophoretically separated on an agarose gel and, after ethidium bromide staining, made visible under UV transmitted light.
  • the pattern of a ladder of genomic DNA fragments typical of cells passed into apoptosis was shown, the sizes of which represented multiples of approximately 180 bp (for an overview, see Zhang and Xu, 2000).
  • the DNA of the untreated cells was, as expected for undegraded DNA, in the high-molecular range.
  • hBD-3 can be used as a therapeutic agent for the treatment of melanoma and other types of cancer. Characters :
  • Capillary fused silica capillary, 30 cm ⁇ 50 ⁇ m, uncoated buffer system: 0.1 M phosphate buffer with polymer modifier, pH 2.5 Measurement parameters: constant voltage 120 kV; UV detection at 200 nm
  • Capillary fused silica capillary, 30 cm ⁇ 50 ⁇ m, uncoated buffer system: 0.1 M phosphate buffer with polymer modifier, pH 2.5 Measurement parameters: constant voltage 120 kV; UV detection at 200 nm Figure 7:
  • Capillary fused silica capillary, 30 cm ⁇ 50 ⁇ m, uncoated buffer system: 0.1 M phosphate buffer with polymer modifier, pH 2.5 Measurement parameters: constant voltage 120 kV; UV detection at 200 nm
  • Capillary fused silica capillary, 30 cm ⁇ 50 ⁇ m, uncoated buffer system: 0.1 M phosphate buffer with polymer modifier, pH 2.5 Measurement parameters: constant voltage 120 kV; UV detection at 200 nm.
  • Figure 12 Hemolytic activity of hBD-3 rCys6-13 / Cys! 8-36 / Cvs28-35].
  • the hemolytic activity of hBD-3 [Cys6-13 / Cysl8-36 / Cys28-35] was determined via the release of hemoglobin from erythrocytes, which can be quantified spectrophotometrically.
  • the antimicrobial, hemolytically active peptide MBI-28 (Piers et al., 1994, Antimicrob. Agents Chemother. 38: 2311-2316) served as a positive control.

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Abstract

The invention relates to a peptide, hBD-3 (human beta-defensin-3) and to derivatives and fragments thereof, in addition to a method for producing the same. The inventive peptides can be made available as medicaments and can be used to resist and combat pathogenic germs. They are particularly useful for treating bacterial diseases of the respiratory system, in particular for infections caused by Burkholderia cepacia and Pseudomonas aeroginosa and for treating cystic fibrosis. The inventive peptides can also be used to treat inflammatory diseases of the gastro-intestinal tract, urogenital tract and to treat septicaemia.

Description

Humanes beta-Defensin-3 Human beta-defensin-3
Die Erfindung betrifft ein Peptid, das hBD-3 (humanes beta-Defensin-3) und seine Derivate und Fragmente sowie Verfahren zu ihrer Herstellung. Die erfindungsgemäße Peptide können als Arzneimittel bereitgestellt werden und zur Abwehr und Bekämpfung von pathogenen Keimen verwendet werden.The invention relates to a peptide, the hBD-3 (human beta-defensin-3) and its derivatives and fragments, and to processes for their preparation. The peptides according to the invention can be provided as medicaments and can be used to ward off and combat pathogenic germs.
Die Zahl der bekannten Wirkstoffe zur Behandlung von pathogenen Keimen ist begrenzt. Dies ist in hohem Maße problematisch, da auf der einen Seite pathogene Keime zunehmend Resistenzen gegen Antibiotika ausbilden, während andererseits nur noch selten neue antibiotische Wirkstoffe entwickelt werden. Daher sind bereits die ersten Fälle von Erregern bekannt, die sich mit keinem der bekannten antibiotischen Wirkstoffe behandeln lassen.The number of known active substances for the treatment of pathogenic germs is limited. This is highly problematic since pathogenic germs are increasingly becoming resistant to antibiotics, while on the other hand, new antibiotic agents are rarely developed. Therefore, the first cases of pathogens are known that cannot be treated with any of the known antibiotic agents.
Die der Erfindung zugrunde liegende Aufgabe ist es, Peptide mit besonderen physiologischen Wirkungen bereitzustellen, die in hohem Maße therapeutisch wirksam sind und die auf relativ einfache Art zugänglich sind. Die Peptide sollen insbesondere wirksam sein gegen pathogene Keime, die gegen bekannte Antibiotika Resistenzen ausgebildet haben. Die erfindungsgemäßen antibiotischen Peptide sollten außerdem gut verträglich sein und eine einfache Applikation ermöglichen.The object on which the invention is based is to provide peptides with special physiological effects which are highly therapeutically active and which are accessible in a relatively simple manner. The peptides are said to be particularly effective against pathogenic germs which have developed resistance to known antibiotics. The antibiotic peptides according to the invention should also be well tolerated and allow easy application.
Überraschenderweise wird die erfindungsgemäße Aufgabe gelöst durch den antibiotisch wirkenden Eiweißstoff humanes beta-Defensin-3 (hBD-3, auch bezeichnet als hBD-5) mit der Aminosäurensequenz Seq. No. 1 : Zx-Cys-Arg-Val-Arg-Gly-Gly-Arg-Cys-Ala-Val-Leu-Ser-Cys-Leu-Pro-Lys-Glu- Glu-Gln-Ile-Gly-Lys-Cys-Ser-Thr-Arg-Gly-Arg-Lys-C/s-C/s-Xi, wobei Zi und Xi Aminosäuren oder Polypeptide aus 1 bis 50 Aminosäuren darstellen, sowie deren natürliche, synthetische und pharmakologisch verträgliche Derivate, insbesondere amidierte, acylierte, phosphorylierte, glycosylierte und zyklische Derivate, sowie deren durch Aminosäuresubstitution, -deletion oder -insertion entstandene Derivate und Fragmente mit biologischer Wirkung, die aus der Aminosäurensequenz abgeleitet werden.Surprisingly, the object of the invention is achieved by the antibiotic protein human beta-defensin-3 (hBD-3, also referred to as hBD-5) with the amino acid sequence Seq. No. 1 : Zx-Cys-Arg-Val-Arg-Gly-Gly-Arg-Cys-Ala-Val-Leu-Ser-Cys-Leu-Pro-Lys-Glu-Glu-Gln-Ile-Gly-Lys-Cys-Ser- Thr-Arg-Gly-Arg-Lys-C / sC / s-Xi, where Zi and Xi represent amino acids or polypeptides from 1 to 50 amino acids, as well as their natural, synthetic and pharmacologically acceptable derivatives, in particular amidated, acylated, phosphorylated, glycosylated and cyclic derivatives, as well as their derivatives and fragments with biological activity resulting from amino acid substitution, deletion or insertion, which are derived from the amino acid sequence.
Erfindungsgemäß bevorzugt sind hBD-3 mit der Seq. No. 2:Preferred according to the invention are hBD-3 with the Seq. No. 2:
Leu-Gln-Lys-Tyr-Tyr-Cys-Arg-Val-Arg-Gly-Gly-Arg-C/s-Ala-Val-Leu-Ser-Cys- Leu-Pro-Lys-Glu-Glu-Gln-Ile-Gly-Lys-Cys-Ser-Thr-Arg-Gly-Arg-Lys-Cys-Cys- Arg-Arg-Lys-Lys,Leu-Gln-Lys-Tyr-Tyr-Cys-Arg-Val-Arg-Gly-Gly-Arg-C / s-Ala-Val-Leu-Ser-Cys- Leu-Pro-Lys-Glu-Glu-Gln- Ile-Gly-Lys-Cys-Ser-Thr-Arg-Gly-Arg-Lys-Cys-Cys-Arg-Arg-Lys-Lys,
Seq. No. 3:Seq. No. 3:
Gly-Ile-Ile-Asn-Thr-Leu-Gln-Lys-Tyr-Tyr-Cys-Arg-Val-Arg-Gly-Gly-Arg-Cys-Gly-Ile-Ile-Asn-Thr-Leu-Gln-Lys-Tyr-Tyr-Cys-Arg-Val-Arg-Gly-Gly-Arg-Cys
Ala-Val-Leu-Ser-Cys-Leu-Pro-Lys-Glu-Glu-Gln-Ile-Gly-Lys-Cys-Ser-Thr-Arg-Ala-Val-Leu-Ser-Cys-Leu-Pro-Lys-Glu-Glu-Gln-Ile-Gly-Lys-Cys-Ser-Thr-Arg-
Gly-Arg-Lys-Cys-Cys-Arg-Arg-Lys-Lys oder Seq. No. 4:Gly-Arg-Lys-Cys-Cys-Arg-Arg-Lys-Lys or Seq. No. 4:
Met-Arg-Ile-His-Tyr-Leu-Leu-Phe-Ala-Leu-Leu-Phe-Leu-Phe-Leu-Val-Pro-Val- Pro-Gly-His-Gly-Gly-Ile-Ile-Asn-Thr-Leu-Gln-Lys-Tyr-Tyr-Cys-Arg-Val-Arg-Gly- Gly-Arg-Cys-Ala-Val-Leu-Ser-Cys-Leu-Pro-Lys-Glu-Glu-Gln-Ile-Gly-Lys-Cys- Ser-Thr-Arg-Gly-Arg-Lys-Cys-Cys-Arg-Arg-Lys-Lys.Met-Arg-Ile-His-Tyr-Leu-Leu-Phe-Ala-Leu-Leu-Phe-Leu-Phe-Leu-Val-Pro-Val- Pro-Gly-His-Gly-Gly-Ile-Ile- Asn-Thr-Leu-Gln-Lys-Tyr-Tyr-Cys-Arg-Val-Arg-Gly-Gly-Arg-Cys-Ala-Val-Leu-Ser-Cys-Leu-Pro-Lys-Glu-Glu- Gln-Ile-Gly-Lys-Cys-Ser-Thr-Arg-Gly-Arg-Lys-Cys-Cys-Arg-Arg-Lys-Lys.
Gegenstand der Erfindung sind auch Fragmente des hBD-3 der Aminosäurensequenz Seq. No. 5:The invention also relates to fragments of hBD-3 of the amino acid sequence Seq. No. 5:
Z!-Cys-Arg-\/al-Arg-Gly-Gly-Arg-Cys-X2 oder Seq. No. 6:Z ! -Cys-Arg - \ / al-Arg-Gly-Gly-Arg-Cys-X 2 or Seq. No. 6:
Z2-Cys-Leu-Pro-Lys-Glu-Glu-Gln-Ile-Gly-Lys-Cys-Ser-Thr-Arg-Gly-Arg-Lys-
Figure imgf000004_0001
wobei Zi, Z2, Xx und X2 Aminosäuren oder Polypeptide aus 1 bis 50 Aminosäuren darstellen, sowie deren synthetische und pharmakologisch verträgliche Derivate, insbesondere zyklische, amidierte, acylierte, phosphorylierte und glycosylierte Derivate, sowie deren durch Aminosäuresubstitution, -deletion oder -insertion entstandene Derivate und Fragmente mit biologischer Wirkung, die aus der Aminosäurensequenz abgeleitet werden. hBD-3-Fragmente der Erfindung sind bevorzugt solche der Sequenz Seq. No. 7:Z 2 -Cys-Leu-Pro-Lys-Glu-Glu-Gln-Ile-Gly-Lys-Cys-Ser-Thr-Arg-Gly-Arg-Lys-
Figure imgf000004_0001
where Zi, Z 2 , X x and X 2 represent amino acids or polypeptides from 1 to 50 amino acids, and their synthetic and pharmacologically acceptable derivatives, in particular cyclic, amidated, acylated, phosphorylated and glycosylated derivatives, and their by amino acid substitution, deletion or - insertion derived derivatives and fragments with biological effect, which are derived from the amino acid sequence. hBD-3 fragments of the invention are preferably those of the sequence Seq. No. 7:
Leu-Gln-Lys-Tyr-Tyr-Cys-Arg-Val-Arg-Gly-Gly-Arg-Cys-Ala-Val-Leu-Ser,Leu-Gln-Lys-Tyr-Tyr-Cys-Arg-Val-Arg-Gly-Gly-Arg-Cys-Ala-Val-Leu-Ser,
Seq. No. 8:Seq. No. 8th:
Gly-Ile-Ile-Asn-Thr-Leu-Gln-Lys-Tyr-Tyr-Cys-Arg-Val-Arg-Gly-Gly-Arg-Cys- Ala-Val-Leu-Ser oder Seq. No. 9:Gly-Ile-Ile-Asn-Thr-Leu-Gln-Lys-Tyr-Tyr-Cys-Arg-Val-Arg-Gly-Gly-Arg-Cys-Ala-Val-Leu-Ser or Seq. No. 9:
Ala-Val-Leu-Ser-Cys-Leu-Pro-Lys-Glu-Glu-Gln-Ile-Gly-Lys-Cys-Ser-Thr-Arg- Gly-Arg-Lys-Cys-Cys-Arg-Arg-Lys-Lys.Ala-Val-Leu-Ser-Cys-Leu-Pro-Lys-Glu-Glu-Gln-Ile-Gly-Lys-Cys-Ser-Thr-Arg- Gly-Arg-Lys-Cys-Cys-Arg-Arg- Lys-Lys.
Gegenstand der Erfindung sind auch Derivate von hBD-3 oder Fragmenten von hBD-3, bei denen einzelne Aminosäuren so ausgetauscht wurden, dass das Peptid insgesamt eine höhere positive Ladung aufweist. Bevorzugt sind insbesondere Derivate, bei denen eines der vier Glycine aus Sequenz No. 1 und/oder das Alanin, das in Sequenz No. 1 auf Cystein 2 folgt und/oder das Leucin oder Prolin, die in der Sequenz No. 1 auf Cystein 3 folgen gegen basische Aminosäuren substituiert sind. Die bezeichneten sieben Aminosäuren können erfindungsgemäß auch gegen aromatische Aminosäuren ausgetauscht sein. Gegenstand der Erfindung sind analog Derivate der Peptide mit den Sequenzen 2 bis 9, bei denen das zur Sequenz 1 homologe Glycin, Alanin, Leucin und/oder Prolin ausgetauscht wurde(n).The invention also relates to derivatives of hBD-3 or fragments of hBD-3 in which individual amino acids have been exchanged such that the peptide has a higher positive charge overall. Derivatives in which one of the four glycines from sequence no. 1 and / or the alanine, which in sequence No. 1 follows cysteine 2 and / or the leucine or proline, which in sequence no. 1 followed by cysteine 3 are substituted by basic amino acids. According to the invention, the designated seven amino acids can also be replaced by aromatic amino acids. Analogously, the invention relates to derivatives of the peptides with the sequences 2 to 9, in which the glycine, alanine, leucine and / or proline which is homologous to sequence 1 has been replaced.
Überraschenderweise konnte festgestellt werden, dass biologische Aktivität auch bei einer Verbrückung der Cysteine 1 und 2 vorliegt, wobei Cysteine 3 und 4 mit Cysteinen 5 und 6 über je eine Disulfidbindung verbunden sind. Dies steht im Gegensatz zu früheren Darstellungen bisher bekannter Defensine [Selsted & Harwig, Journal of Biological Chemistry 264 (1989) 4003-4007; Tang & Selsted, Journal of Biological Chemistry 268 (1993) 6649-6653]. Die erfindungsgemäßen hBD-3 oder hBD-3-Fragmente oder Derivate sind in der Lage, Cysteinbrücken zwischen den sechs Cysteinen in Sequenz No. 1 auszubilden. In einer bevorzugten Ausführungsform erfolgt die Bildung der Cysteinbrücken zwischen Cystein 1 und Cystein 2, Cystein 3 und Cystein 6 und/oder Cystein 4 und Cystein 5. Die Nummern 1 bis 6 entsprechen dabei den sechs Cysteinen, die in der Sequenz No. 1 vom N- zum C- Terminus hin enthalten sind. Bei Fragmenten oder bei h.BD-3, die zum Beispiel N-terminal zusätzliches Cystein beinhalten, bleiben die Nummern 1 bis 6 unverändert und bezeichnen die Cysteine, die zu denen in Sequenz 1 homolog sind. So enthält zum Beispiel das Fragment der Sequenz No. 9 die Cysteine 3 bis 6, da der Abschnitt fehlt, der die Cysteine 1 und 2 enthält. Zur einfacheren Identifizierung sind die Cysteine in den angegebenen Sequenzen kursiv aufgeführt.Surprisingly, it was found that biological activity is also present when cysteines 1 and 2 are bridged, cysteines 3 and 4 being linked to cysteines 5 and 6 via a disulfide bond in each case. This stands in contrast to earlier representations of previously known defensins [Selsted & Harwig, Journal of Biological Chemistry 264 (1989) 4003-4007; Tang & Selsted, Journal of Biological Chemistry 268 (1993) 6649-6653]. The hBD-3 or hBD-3 fragments or derivatives according to the invention are capable of bridging cysteine bridges between the six cysteines in sequence no. 1 to train. In a preferred embodiment, the cysteine bridges are formed between cysteine 1 and cysteine 2, cysteine 3 and cysteine 6 and / or cysteine 4 and cysteine 5. Numbers 1 to 6 correspond to the six cysteines that are shown in sequence no. 1 from the N to the C terminus are included. For fragments or for h.BD-3, which contain, for example, additional N-terminal cysteine, the numbers 1 to 6 remain unchanged and denote the cysteines which are homologous to those in sequence 1. For example, the fragment of sequence No. 9 cysteines 3 to 6 because the section containing cysteines 1 and 2 is missing. The cysteines are shown in italics for easier identification.
Bevorzugte Peptide der Erfindung sind insbesondere Fragmente der Sequenz 6, bei denenPreferred peptides of the invention are, in particular, fragments of sequence 6 in which
■ Cystein 3 und Cystein 6 keine Cysteinbrücken ausbilden und Cystein 4 und Cystein 5 eine Cysteinbrücke ausbilden, oder■ Cysteine 3 and cysteine 6 do not form cysteine bridges and cysteine 4 and cysteine 5 form a cysteine bridge, or
■ Cystein 3 und Cystein 6 eine Cysteinbrücke ausbilden und Cystein 4 und Cystein 5 keine Cysteinbrücke ausbilden, oder■ Cysteine 3 and cysteine 6 form a cysteine bridge and cysteine 4 and cysteine 5 do not form a cysteine bridge, or
■ keine Cysteinbrücke ausgebildet ist, oder■ no cysteine bridge is formed, or
■ Cystein 3 und Cystein 6 eine Cysteinbrücke ausbilden und Cystein 4 und Cystein 5 eine Cysteinbrücke ausbilden.■ Cysteine 3 and cysteine 6 form a cysteine bridge and cysteine 4 and cysteine 5 form a cysteine bridge.
Bevorzugte Peptide der Erfindung sind auch hBD-3 oder Derivate von hBD-3, bei denen die Cysteine 3 bis 6 nach einer der vier oben aufgeführten Möglichkeiten verknüpft/nicht verknüpft sind. Dabei können Cystein 1 und Cystein 2 über eine Cysteinbrücke verbunden sein oder keine Cysteinbrücke ausbilden. Die erfindungsgemäßen Eiweißstoffe können durch chemische Synthese, durch gentechnologische Produktion oder durch Isolierung aus natürliche Quellen gewonnen werden.Preferred peptides of the invention are also hBD-3 or derivatives of hBD-3, in which the cysteines 3 to 6 are linked / not linked in one of the four ways listed above. Here, cysteine 1 and cysteine 2 can be connected via a cysteine bridge or form no cysteine bridge. The proteins according to the invention can be obtained by chemical synthesis, by genetic engineering production or by isolation from natural sources.
Gegenstand der Erfindung ist insbesondere ein Verfahren zur Herstellung von hBD-3 oder Derivaten oder Fragmenten von hBD-3, wobei das hBD-3 oder hBD-3-Fragmente oder Derivate durch Festphasen- und/oder Flüssigphasen- Synthese aus den geschützten Aminosäuren, die in ihr enthalten sind, herstellt werden, deblockiert werden, und durch Chromatographie aufgereinigt werden.The invention relates in particular to a process for the preparation of hBD-3 or derivatives or fragments of hBD-3, the hBD-3 or hBD-3 fragments or derivatives being obtained by solid-phase and / or liquid-phase synthesis from the protected amino acids contained in it, manufactured, deblocked, and purified by chromatography.
Erfindungsgemäß können Cysteinbrücken durch Oxidation eingeführt werden Die Einführung kann gezielte an bestimmten Positionen erfolgen, indem die Reaktion von nicht zu verknüpfenden Cysteinen durch geeignete Schutzgruppen verhindert wird.According to the invention, cysteine bridges can be introduced by oxidation. The introduction can be targeted at specific positions by preventing the reaction of non-linked cysteines by means of suitable protective groups.
In einem weiterem erfindungsgemäßen Verfahren erfolgt die Herstellung von hBD-3 oder Derivaten oder Fragmenten von hBD-3 durch Expression in prokaryontischen oder eukaryontischen Zellen und anschließende chromatographische Reinigung unter Verwendung bekannter Methoden. Hierfür stehen verschiedene Expressionsvektoren zur sekretorischen oder direkten cytoplasmatischen Expression zur Verfügung.In a further method according to the invention, hBD-3 or derivatives or fragments of hBD-3 are produced by expression in prokaryotic or eukaryotic cells and subsequent chromatographic purification using known methods. Various expression vectors are available for secretory or direct cytoplasmic expression.
Das erfindungsgemäße hBD-3 oder Derivate oder Fragmente können aus Körperflüssigkeiten, vorzugsweise aus menschlichem Blut, Hemofiltrat oder Hemodialysat über Chromatographie-Verfahren isoliert werden. Hemofiltrat oder Hemodialysat kann aus menschlichem Blut abfiltriert werden. Dies ist besonders vorteilhaft, da Hemofiltrat bislang als wertlos angesehen und verworfen wurde. So erschließt sich auf einfache Art eine Quelle für die wirtschaftliche Verwertung.The hBD-3 according to the invention or derivatives or fragments can be isolated from body fluids, preferably from human blood, hemofiltrate or hemodialysate, using a chromatography method. Hemofiltrate or hemodialysate can be filtered from human blood. This is particularly advantageous since hemofiltrate has so far been regarded as worthless and has been discarded. In this way, a source for economic exploitation is easily developed.
Gegenstand der Erfindung sind auch Nucleinsäuren, kodierend für hBD-3 oder seine Derivate oder Fragmente, insbesondere DNS und RNS. Die Erfindung betrifft auch Vektoren oder Plasmide, die solche Nucleinsäuren enthalten und Fragmente, die sich als Antisense-Nucleotide eignen, insbesondere zu kodierenden Nucleinsäuren komplementäre Fragmente mit einer Länge von 12-26 Nucleotiden.The invention also relates to nucleic acids coding for hBD-3 or its derivatives or fragments, in particular DNA and RNA. The invention also relates to vectors or plasmids which contain such nucleic acids and fragments which are suitable as antisense nucleotides, in particular to coding nucleic acids complementary fragments with a length of 12-26 nucleotides.
Gegenstand der Erfindung ist auch ein Arzneimittel, enthaltend hBD-3 oder Derivate oder Fragmente von hBD-3, oder Nucleinsäuren, die für hBD-3 kodieren und Vektoren oder Plasmide, die solche Nucleinsäuren enthalten.The invention also relates to a medicament containing hBD-3 or derivatives or fragments of hBD-3, or nucleic acids which code for hBD-3 and vectors or plasmids which contain such nucleic acids.
Die Erfindung betrifft auch die Verwendung von hBD-3 oder Derivaten oder Fragmenten von hBD-3 zur Herstellung eines Arzneimittels zur antimikrobiellen Behandlung, zur Abwehr und Bekämpfung von pathogenen Keimen, zur Behandlung von Erkrankungen der Atemwege, insbesondere die durch Infektionen mit den Bakterien Burkholderia cepacia und Pseudomonas aeroginosa ausgelöst oder verstärkt werden, zur Behandlung der cystischen Fibröse, zur Behandlung entzündlicher Krankheiten des Magen-Darm-Traktes, des Urogenitaltraktes, bei Sepsis, bei gastrointestinalen Infektionen, insbesondere durch Heliobacter pylori ausgelöst, zur Behandlung und Abwehr von gram-positiven und gram-negativen Bakterien, Hefen, Heliobacter pylori, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa oder Burkholderia cepacia. Dabei können in weiteren Ausführungsformen auch Nucleinsäuren, die für hBD-3, Derivate oder Fragmente kodieren, sowie Vektoren oder Plasmide, die solche Nucleinsäuren enthalten, eingesetzt werden, zum Beispiel im Zusammenhang mit einer Gentherapie. Die Nucleinsäuren können vorzugsweise auch als Antisense-Oligonucleotide eingesetzt werden.The invention also relates to the use of hBD-3 or derivatives or fragments of hBD-3 for the manufacture of a medicament for antimicrobial treatment, for defense and combating pathogenic germs, for the treatment of respiratory diseases, in particular those caused by infections with the bacteria Burkholderia cepacia and Pseudomonas aeroginosa are triggered or intensified, for the treatment of cystic fibrosis, for the treatment of inflammatory diseases of the gastrointestinal tract, the urogenital tract, for sepsis, for gastrointestinal infections, in particular triggered by Heliobacter pylori, for the treatment and defense against gram-positive and gram-negative bacteria, yeast, Heliobacter pylori, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa or Burkholderia cepacia. In further embodiments, nucleic acids that code for hBD-3, derivatives or fragments, and vectors or plasmids that contain such nucleic acids can also be used, for example in connection with gene therapy. The nucleic acids can preferably also be used as antisense oligonucleotides.
Die erfindungsgemäßen Eiweißstoffe sind insbesondere in der Lage, die bakterielle Invasion bei Entzündungserkrankungen der Atemwege einzuschränken oder zu verhindern. Insbesondere weil die erfindungsgemäßen Peptide Analoga körpereigener Substanzen sind, weisen sie eine hohe Verträglichkeit für den Organismus auf. Das erfindungsgemäße Peptid hBD-3 und seine Derivate und Fragmente sind besonders auch für die Langzeittherapie bei Infektionserkrankungen der Atemwege durch pathogene Keime, insbesondere Burkholderia cepacia und Pseudomonas aeroginosa, geeignet, da sie über eine ausgezeichnete biologische Wirksamkeit verfügen und andererseits auch bei Dauerbehandlung keine Immunreaktion auslösen.The proteins according to the invention are in particular able to restrict or prevent the bacterial invasion in inflammatory diseases of the respiratory tract. In particular, because the peptides according to the invention are analogues of the body's own substances, they have a high tolerance for the organism. The peptide hBD-3 according to the invention and its derivatives and fragments are also particularly suitable for long-term therapy for infectious diseases of the respiratory tract caused by pathogenic germs, in particular Burkholderia cepacia and Pseudomonas aeroginosa. Suitable because they have an excellent biological effectiveness and, on the other hand, do not trigger an immune reaction even with long-term treatment.
Überraschenderweise eignen sich hBD-3, seine Derivate und Fragmente auch zur Einleitung der Apoptose und daher zur Behandlung maligner Melanome und anderer Tumore, wie Tumore des gastrointestinalen Bereichs. Im Gegensatz zur Nekrose erfüllt die Apoptose als programmierter Zelltod eine wichtige Funktion bei unterschiedlichsten biologischen Vorgängen, welche die gezielte Eliminierung unerwünschter oder überflüssiger Zellen erfordern. Als Beispiele seien die Beseitigung nicht benötigter Zellen bei Entwicklungs- und Differenzierungsvorgängen, die Abtötung von Zellen im Rahmen von Immunreaktionen und die Vernichtung geschädigter Zellen genannt. Für die Therapie von Tumoren macht man sich die gezielte Herbeiführung der Apoptose mit Hilfe bestimmter Chemotherapeutika zunutze.Surprisingly, hBD-3, its derivatives and fragments are also suitable for inducing apoptosis and therefore for treating malignant melanomas and other tumors, such as tumors in the gastrointestinal area. In contrast to necrosis, apoptosis, as programmed cell death, plays an important role in a wide variety of biological processes that require the targeted elimination of unwanted or unnecessary cells. Examples include the removal of unnecessary cells during development and differentiation processes, the killing of cells in the context of immune reactions and the destruction of damaged cells. The targeted induction of apoptosis with the help of certain chemotherapeutic agents is used to treat tumors.
Das Protein p53 nimmt bei der Einleitung apoptotischer Prozesse eine Schlüsselfunktion ein. Es wird in noch nicht vollständig geklärter Weise durch geschädigte Desoxyribonucleinsäure (DNA) und andere in Zusammenhang mit zellulärem Stress vermittelte Signale aktiviert. Wahrscheinlich unter Beteiligung des Faktors Bax stimuliert aktiviertes p53 die Freisetzung von Cytochrom C aus Mitochondrien. Cytochrom C triggert die Oligomerisierung des Faktors Apaf-1, der daraufhin an Procaspase-9 bindet und diese aktiviert, was unweigerlich zum Zelltod führt (Jones, P.A., Nature 409: 141, 2001).The protein p53 plays a key role in the initiation of apoptotic processes. It is activated in a not yet fully understood manner by damaged deoxyribonucleic acid (DNA) and other signals mediated in connection with cellular stress. Activated p53 probably stimulates the release of cytochrome C from mitochondria with the participation of the factor Bax. Cytochrome C triggers the oligomerization of the factor Apaf-1, which then binds to and activates procaspase-9, which inevitably leads to cell death (Jones, P.A., Nature 409: 141, 2001).
Mutationen im Gen für p53 kommen in der Tat bei vielen aggressiven und chemoresistenten Tumoren vor und gehen meist mit einer schlechten Prognose für den Patienten und unwirksamer Chemotherapie einher. In den ebenfalls aggressiven und chemoresistenten Melanomen werden Mutationen im p53-Gen hingegen nur selten beobachtet. Hier kann p53 durch Chemotherapeutika normal hochreguliert werden, was jedoch keine Apoptose nach sich zieht. Es wurde gezeigt, dass dieser Defekt in der apoptotischen Signalkaskade auf einer Ausschaltung des Apoptose-Effektors Apaf-1 beruht, die auf die Methylierung einer Enhancer-Region des Gens und damit auf eine Repression der Transkription zurückzuführen ist (Soengas et al., Nature 409: 207, 2001). Die Möglichkeit einer Hochregulation von Apaf-1 ist daher ein wichtiger Schritt für die Behandlung maligner Melanome und anderer Tumore.Indeed, mutations in the gene for p53 occur in many aggressive and chemoresistant tumors and are usually associated with a poor prognosis for the patient and ineffective chemotherapy. However, mutations in the p53 gene are only rarely observed in the likewise aggressive and chemoresistant melanomas. Here p53 can be upregulated normally by chemotherapy drugs, but this does not result in apoptosis. It has been shown that this defect in the apoptotic signaling cascade is based on the apoptosis effector Apaf-1 being switched off, which is due to the methylation of an enhancer region of the gene and thus to a repression of the transcription (Soengas et al., Nature 409 : 207, 2001). The possibility of up-regulation of Apaf-1 is therefore an important step in the treatment of malignant melanoma and other tumors.
Die bei der Substanzklasse der Defensine bislang nicht beobachtete Disulfidverbrückung Cysl-Cys2; Cys3-Cys6; Cys4-Cys5 eröffnete die Möglichkeit, die oben bezeichneten biologisch aktiven Fragmente des Peptids zu synthetisieren, ohne dabei weitere Veränderungen am Molekül vornehmen zu müssen. Es handelt sich dabei um kürzere N-terminale Fragmente, die die Disulfidbrücke Cysl-Cys2 enthalten, sowie um längere C-terminale Fragmente, die die beiden übrigen Disulfidbrücken beinhalten.The disulfide bridging Cysl-Cys2; Cys3-Cys6; Cys4-Cys5 made it possible to synthesize the biologically active fragments of the peptide described above without having to make any further changes to the molecule. These are shorter N-terminal fragments, which contain the disulfide bridge Cysl-Cys2, and longer C-terminal fragments, which contain the other two disulfide bridges.
Die Verwendung von hBD-3 oder Derivaten und Fragmenten erfolgt dabei vorzugsweise in geeigneten galenischen Applikationsformen, insbesondere lyophilisiert und in Mannit aufgenommen in sterilen Ampullen zur Auflösung in Kochsalzlösung und/oder Infusionslösungen und Inhalationslösungen, und/oder in Kombination mit anderen antibiotischen Wirkstoffen. Natürlich können auch Gemische verschiedener erfindungsgemäßer Wirkstoffe eingesetzt werden. Die Peptide können als hochreiner Stoff oder - wenn für die bestimmte Verwendung ausreichend - innerhalb eines teilweise aufgereinigten Peptidgemisches verwendet werden.The use of hBD-3 or derivatives and fragments is preferably carried out in suitable pharmaceutical application forms, in particular lyophilized and taken up in mannitol in sterile ampoules for dissolution in saline and / or infusion solutions and inhalation solutions, and / or in combination with other antibiotic active substances. Mixtures of different active ingredients according to the invention can of course also be used. The peptides can be used as a highly pure substance or - if sufficient for the particular use - within a partially purified peptide mixture.
Die Arzneimittelzubereitung enthält das erfindungsgemäße Peptid oder ein physiologisch verträgliches Salz. Die Form und Zusammensetzung des Arzneimittels, welches das Peptid enthält, richtet sich nach der Art der Verabreichung. Das Peptid kann parenteral, intranasal, oral und mittels Inhalation verabreicht werden. Vorzugsweise werden hBD-3 oder seine Derivate oder Fragmente entweder als Lösung oder als Lyophilisat zur Auflösung unmittelbar vor Gebrauch, konfektioniert. DieThe pharmaceutical preparation contains the peptide according to the invention or a physiologically acceptable salt. The form and composition of the drug containing the peptide depends on the mode of administration. The peptide can be administered parenterally, intranasally, orally and by inhalation. HBD-3 or its derivatives or fragments are preferably packaged either as a solution or as a lyophilisate for dissolution immediately before use. The
Arzneimittelzubereitung kann außerdem Hilfsstoffe enthalten, die abfülltechnisch bedingt sind, einen Beitrag zur Löslichkeit, Stabilität oder Sterilität des Arzneimittels leisten oder den Wirkungsgrad der Aufnahme in den Körper erhöhen. Ausführunqsbeispiele:Drug preparation can also contain excipients that are due to filling technology, contribute to the solubility, stability or sterility of the drug or increase the efficiency of absorption into the body. EXEMPLARY EMBODIMENTS:
Synthese des humanen Defensins hBD-3:Synthesis of the human defensin hBD-3:
Für die Synthese des Peptids mit der FormelFor the synthesis of the peptide with the formula
Leu-Gln-Lys-Tyr-Tyr-Cys-Arg-Val-Arg-Gly-Gly-Arg-Cys-Ala-Val-Leu-Ser-Cys- Leu-Pro-Lys-Glu-Glu-Gln-Ile-Gly-Lys-Cys-Ser-Thr-Arg-Gly-Arg-Lys-Cys-Cys- Arg-Arg-Lys-Lys wurde die Festphasenmethode (Chan and White, Fmoc Solid Phase Peptide Synthesis. Oxford University Press, 2000) angewendet. Die genannte Peptidsequenz wurde an einem mit Lysin vorbeladenen Trägerharz mit Hilfe einer automatischen Peptidsynthese-Apparatur (ABI 433A) unter Verwendung der in situ durch Zugabe von HBTU [2-(lH-benzotriazol-l-yl)-l, 1,3,3- tetramethyluronium-Hexafluorophosphat] gebildeten Hydroxy-Benzotriazol- Ester synthetisiert. Verwendet wurden folgende Derivate der L-Aminosäuren im Überschuss (10 Äquivalente):Leu-Gln-Lys-Tyr-Tyr-Cys-Arg-Val-Arg-Gly-Gly-Arg-Cys-Ala-Val-Leu-Ser-Cys- Leu-Pro-Lys-Glu-Glu-Gln-Ile- Gly-Lys-Cys-Ser-Thr-Arg-Gly-Arg-Lys-Cys-Cys-Arg-Arg-Lys-Lys, the solid phase method (Chan and White, Fmoc Solid Phase Peptide Synthesis. Oxford University Press, 2000) was used , The peptide sequence mentioned was carried out on a carrier resin preloaded with lysine using an automatic peptide synthesis apparatus (ABI 433A) using the in situ method by adding HBTU [2- (1H-benzotriazol-l-yl) -l, 1,3,3 - Tetramethyluronium hexafluorophosphate] formed hydroxy-benzotriazole ester synthesized. The following derivatives of the L-amino acids in excess (10 equivalents) were used:
Fmoc-Ala Fmoc-LeuFmoc-Ala Fmoc-Leu
Fmoc-Arg(Pbf) Fmoc-Lys(Boc)Fmoc-Arg (Pbf) Fmoc-Lys (Boc)
Fmoc-Cys(Trt) Fmoc-ProFmoc-Cys (Trt) Fmoc-Pro
Fmoc-Gln(Trt) Fmoc-Ser(tBu)Fmoc-Gln (Trt) Fmoc-Ser (tBu)
Fmoc-Glu(OtBu) Fmoc-Thr(tBu)Fmoc-Glu (OtBu) Fmoc-Thr (tBu)
Fmoc-Gly Fmoc-Tyr(tBu)Fmoc-Gly Fmoc-Tyr (tBu)
Fmoc-Ile Fmoc-ValFmoc-Ile Fmoc-Val
Das Peptid wurde vom Trägerharz durch Zugabe einer Mischung von Trifluoressigsäure-Ethandithiol-Wasser 94 : 3 : 3 (v/v/v) abgespalten und mit tert-Butylmethylether gefällt. Zur Einführung der drei intramolekularen Disulfidbrücken wurde das per HPLC vorgereinigte Peptid mit Hilfe von Dimethylsulfoxid (DMSO, 20 %) bei pH 6 oxidiert. Aus der inhomogenen Produktmischung wurde das Hauptprodukt mittels HPLC isoliert und mit Hilfe von analytischer HPLC, massenspektrometrischer Analyse, Kapillarzonenelektrophorese und Aminosäuresequenzanalyse charakterisiert (Fig. 1-3).The peptide was cleaved from the carrier resin by adding a mixture of trifluoroacetic acid / ethanedithiol / water 94: 3: 3 (v / v / v) and precipitated with tert-butyl methyl ether. To introduce the three intramolecular disulfide bridges, the peptide prepurified by HPLC was oxidized with the help of dimethyl sulfoxide (DMSO, 20%) at pH 6. The main product was isolated from the inhomogeneous product mixture by means of HPLC and with the aid of analytical HPLC, mass spectrometric analysis, Capillary zone electrophoresis and amino acid sequence analysis characterized (Fig. 1-3).
Alternativ kann die Einführung der Disulfidbrücken selektiv vorgenommen werden durch Verwendung zweier Fmoc-Cys(Acm)-Derivate während der Synthese und Verknüpfung der entsprechenden Cysteine mittels Iod nach Einführung der anderen beiden Disulfidbrücken.Alternatively, the introduction of the disulfide bridges can be carried out selectively by using two Fmoc-Cys (Acm) derivatives during the synthesis and linking the corresponding cysteines using iodine after the introduction of the other two disulfide bridges.
Die Charakterisierung des synthetisierten Peptids beinhaltet den Nachweis der intramolekularen Disulfidverknüpfung nach folgender Vorgehensweise. hBD-3 wurde durch gleichzeitige Einwirkung von Trypsin und Chymotrypsin proteolytisch zerlegt. Dabei bleiben die Disulfidbrücken intakt, und eine Analyse der miteinander verknüpften cysteinhaltigen Fragmente erlaubt die Ableitung des ursprünglichen Verknüpfungsmusters. Die erhaltenen Peptidfragmente wurden per HPLC isoliert und sowohl massenspektrometrisch als auch mit Hilfe von Aminosäuresequenzanalyse identifizert (Fig.4, Tab.l). Die Sequenzanalyse ermöglichte zudem eine eindeutige Zuordnung der Disulfidverbrückung an den beiden benachbarten Cysteinen, einer Konstellation, die im Allgemeinen besondere analytische Schwierigkeiten bereitet.The characterization of the synthesized peptide includes the detection of the intramolecular disulfide linkage according to the following procedure. hBD-3 was proteolytically broken down by simultaneous exposure to trypsin and chymotrypsin. The disulfide bridges remain intact, and an analysis of the interlinked cysteine-containing fragments allows the derivation of the original linking pattern. The peptide fragments obtained were isolated by HPLC and identified both by mass spectrometry and with the aid of amino acid sequence analysis (FIG. 4, Tab. 1). The sequence analysis also made it possible to clearly assign the disulfide bridging to the two neighboring cysteines, a constellation that generally causes particular analytical difficulties.
Die bei der Substanzklasse der Defensine bislang nicht beobachtete Disulfidverbrückung Cysl-Cys2; Cys3-Cys6; Cys4-Cys5 eröffnete die Möglichkeit, zwei Teilstücke des Peptids getrennt zu synthetisieren, ohne dabei weitere Veränderungen am Molekül vornehmen zu müssen. Es handelt sich dabei um ein kürzeres N-terminales Fragment, das die Disulfidbrücke Cysl- Cys2 enthält, sowie um ein längeres C-terminales Fragment, das die beiden übrigen Disulfidbrücken beinhaltet. Die Synthese und folgende Untersuchung dieser beiden Fragmente dient der Zuordnung biologischer Wirkungen des Peptids hBD-3 zu bestimmten Molekülregionen.The disulfide bridging Cysl-Cys2; Cys3-Cys6; Cys4-Cys5 made it possible to synthesize two sections of the peptide separately without having to make any further changes to the molecule. It is a shorter N-terminal fragment that contains the Cysl-Cys2 disulfide bridge and a longer C-terminal fragment that contains the other two disulfide bridges. The synthesis and subsequent investigation of these two fragments serves to assign biological effects of the peptide hBD-3 to specific molecular regions.
Synthese des Peptids hBD-3 . 1-171) :Synthesis of the peptide hBD-3. 1-17 1 ):
Für die Synthese des Peptids mit der FormelFor the synthesis of the peptide with the formula
Leu-Gln-Lys-Tyr-Tyr-Cys-Arg-Val-Arg-Gly-Gly-Arg-Cys-Ala-Val-Leu-Ser wurde die Festphasenmethode (Chan and White, Fmoc Solid Phase Peptide Synthesis. Oxford University Press, 2000) angewendet. Die genannte Peptidsequenz wurde an einem mit Serin vorbeladenen Trägerharz mit Hilfe einer automatischen Peptidsynthese-Apparatur (ABI 433A) unter Verwendung der in situ durch Zugabe von HBTU [2-(lH-benzotriazol-l-yl)-l, 1,3,3- tetramethyluronium-Hexafluorophosphat] gebildeten Hydroxy-Benzotriazol- Ester synthetisiert. Verwendet wurden folgende Derivate der L-Aminosäuren im Überschuss (10 Äquivalente) :Leu-Gln-Lys-Tyr-Tyr-Cys-Arg-Val-Arg-Gly-Gly-Arg-Cys-Ala-Val-Leu-Ser the solid phase method (Chan and White, Fmoc Solid Phase Peptide Synthesis. Oxford University Press, 2000) was used. The peptide sequence mentioned was carried out on a carrier resin preloaded with serine using an automatic peptide synthesis apparatus (ABI 433A) using the in situ method by adding HBTU [2- (1H-benzotriazol-l-yl) -l, 1,3,3 - Tetramethyluronium hexafluorophosphate] formed hydroxy-benzotriazole ester synthesized. The following derivatives of the L-amino acids in excess (10 equivalents) were used:
Fmoc-Ala Fmoc-LeuFmoc-Ala Fmoc-Leu
Fmoc-Arg(Pbf) Fmoc-Lys(Boc)Fmoc-Arg (Pbf) Fmoc-Lys (Boc)
Fmoc-Cys(Trt) Fmoc-Tyr(tBu)Fmoc-Cys (Trt) Fmoc-Tyr (tBu)
Fmoc-Gin(Trt) Fmoc-Val Fmoc-GlyFmoc-Gin (Trt) Fmoc-Val Fmoc-Gly
Das Peptid wurde vom Trägerharz durch Zugabe einer Mischung von Trifluoressigsäure-Ethandithiol-Wasser 94 : 3 : 3 (v/v/v) abgespalten und mit tert-Butylmethylether gefällt.The peptide was cleaved from the carrier resin by adding a mixture of trifluoroacetic acid / ethanedithiol / water 94: 3: 3 (v / v / v) and precipitated with tert-butyl methyl ether.
Zur Einführung der Disulfidbrücke wurde das per HPLC vorgereinigte Peptid mit Hilfe von Dimethylsulfoxid (DMSO, 20 %) bei pH 6 oxidiert. Das Reaktionsprodukt wurde mittels HPLC gereinigt und mit Hilfe von analytischer HPLC, massenspektrometrischer Analyse, Kapillarzonenelektrophorese und Aminosäuresequenzanalyse charakterisiert (Fig. 5-7).To introduce the disulfide bridge, the peptide prepurified by HPLC was oxidized with the aid of dimethyl sulfoxide (DMSO, 20%) at pH 6. The reaction product was purified by HPLC and characterized using analytical HPLC, mass spectrometric analysis, capillary zone electrophoresis and amino acid sequence analysis (Fig. 5-7).
Die Disulfidbrücke kann alternativ durch Oxidation mit Luftsauerstoff im basischen pH-Bereich eingeführt werden, wobei als Nebenprodukte jedoch Dimere entstehen.The disulfide bridge can alternatively be introduced by oxidation with atmospheric oxygen in the basic pH range, but dimers are formed as by-products.
Synthese des Peptids hBD-3 (14-401 rCysl8-36/Cvs28-351 :Synthesis of the peptide hBD-3 (14-401 rCysl8-36 / Cvs28-351:
Für die Synthese des Peptids mit der FormelFor the synthesis of the peptide with the formula
Ala-Val-Leu-Ser-Cys-Leu-Pro-Lys-Glu-Glu-Gln-Ile-Gly-Lys-Cys-Ser-Thr-Arg- Gly-Arg-Lys-Cys-Cys-Arg-Arg-Lys-Lys wurde die Festphasenmethode (Chan and White, Fmoc Solid Phase Peptide Synthesis. Oxford University Press, 2000) angewendet. Die genannte Peptidsequenz wurde an einem mit Lysin vorbeladenen Trägerharz mit Hilfe einer automatischen Peptidsynthese-Apparatur (ABI 433A) unter Verwendung der in situ durch Zugabe von HBTU [2-(lH-benzotriazol-l-yl)-l, 1,3,3- tetramethyluronium-Hexafluorophosphat] gebildeten Hydroxy-Benzotriazol- Ester synthetisiert. Verwendet wurden folgende Derivate der L-Aminosäuren im Überschuss (10 Äquivalente):Ala-Val-Leu-Ser-Cys-Leu-Pro-Lys-Glu-Glu-Gln-Ile-Gly-Lys-Cys-Ser-Thr-Arg- Gly-Arg-Lys-Cys-Cys-Arg-Arg- Lys-Lys the solid phase method (Chan and White, Fmoc Solid Phase Peptide Synthesis. Oxford University Press, 2000) was used. The peptide sequence mentioned was carried out on a carrier resin preloaded with lysine using an automatic peptide synthesis apparatus (ABI 433A) using the in situ method by adding HBTU [2- (1H-benzotriazol-l-yl) -l, 1,3,3 - Tetramethyluronium hexafluorophosphate] formed hydroxy-benzotriazole ester synthesized. The following derivatives of the L-amino acids in excess (10 equivalents) were used:
Fmoc-Ala Fmoc-IleFmoc-Ala Fmoc-Ile
Fmoc-Arg(Pbf) Fmoc-LeuFmoc-Arg (Pbf) Fmoc-Leu
Fmoc-Cys(Trt) Fmoc-Lys(Boc)Fmoc-Cys (Trt) Fmoc-Lys (Boc)
Fmoc-Cys(Acm) Fmoc-ProFmoc-Cys (Acm) Fmoc-Pro
Fmoc-Gln(Trt) Fmoc-Ser(tBu)Fmoc-Gln (Trt) Fmoc-Ser (tBu)
Fmoc-Glu(OtBu) Fmoc-Thr(tBu)Fmoc-Glu (OtBu) Fmoc-Thr (tBu)
Fmoc-Gly Fmoc-ValFmoc-Gly Fmoc-Val
Das Peptid wurde vom Trägerharz durch Zugabe einer Mischung von Trifluoressigsäure-Ethandithiol-Wasser 94 : 3 : 3 (v/v/v) abgespalten und mit tert-Butyimethylether gefällt.The peptide was cleaved from the carrier resin by adding a mixture of trifluoroacetic acid / ethanedithiol / water 94: 3: 3 (v / v / v) and precipitated with tert-butyl methyl ether.
Das Peptid verlangt eine selektive Einführung der zwei Disulfidbrücken. Eine simultane Oxidation mit Luftsauerstoff oder DMSO liefert ein Gemisch von zwei Faltungsisomeren zu gleichen Anteilen. Daher wurden bei der Peptidsynthese zwei der vier Cysteine (Cyslδ und Cys36) als Fmoc-Cys(Acm)-Derivate eingesetzt, die beiden übrigen als Fmoc-Cys(Trt)-Derivate.The peptide requires selective introduction of the two disulfide bridges. Simultaneous oxidation with atmospheric oxygen or DMSO provides a mixture of two folding isomers in equal proportions. Therefore, two of the four cysteines (Cyslδ and Cys36) were used as Fmoc-Cys (Acm) derivatives in the peptide synthesis, the other two as Fmoc-Cys (Trt) derivatives.
Zur Bildung der ersten Disulfidbrücke wurde das per HPLC vorgereinigte Peptid mit Hilfe von Dimethylsulfoxid (DMSO, 20 %) bei pH 6 oxidiert. Das Reaktionsprodukt wurde über HPLC gereinigt und nachfolgend mit Iod im sauren pH-Bereich oxidiert. Das vollständig oxidierte Endprodukt wurde mittels HPLC gereinigt und mit Hilfe von analytischer HPLC, massenspektrometrischer Analyse, Kapillarzonenelektrophorese und Aminosäuresequenzanalyse charakterisiert (Fig. 8-10). Das synthetische Peptid hBD-3 (14-40) [Cysl8-36/Cys28-35] mit bekannter, weil synthesebedingt vorgegebener Cysteinverknüpfung lieferte eine weitere Möglichkeit zur Verifizierung der ermittelten Disulfidverbrückung von hBD-3. Die proteolytische Zerlegung von hBD-3 mit Chymotrypsin (pH 8) führte zu dem C-terminalen Peptidfragment Ser-Cys-Leu-Pro-Lys-Glu-Glu-Gln-Ile-Gly- Lys-Cys-Ser-Thr-Arg-Gly-Arg-Lys-Cys-Cys-Arg-Arg-Lys-Lys. DasselbeTo form the first disulfide bridge, the peptide prepurified by HPLC was oxidized at pH 6 using dimethyl sulfoxide (DMSO, 20%). The reaction product was purified by HPLC and subsequently oxidized with iodine in the acidic pH range. The fully oxidized end product was purified by HPLC and characterized using analytical HPLC, mass spectrometric analysis, capillary zone electrophoresis and amino acid sequence analysis (Fig. 8-10). The synthetic peptide hBD-3 (14-40) [Cysl8-36 / Cys28-35] with known cysteine linkage, which is predetermined due to the synthesis, provided another possibility for verifying the disulfide bridging of hBD-3 determined. The proteolytic decomposition of hBD-3 with chymotrypsin (pH 8) led to the C-terminal peptide fragment Ser-Cys-Leu-Pro-Lys-Glu-Glu-Gln-Ile-Gly-Lys-Cys-Ser-Thr-Arg- Gly-Arg-Lys-Cys-Cys-Arg-Arg-Lys-Lys. The same thing
Fragment wurde erhalten durch analoge Proteolyse von hBD-3 (14-40) [Cysl8-36/Cys28-35]. Die Identität der beiden Fragmente, und damit identische Disulfidverbrückung, konnte durch Komigration bei der Kapillarzonenelektrophorese gezeigt werden (Fig. 11). Fragment was obtained by analogous proteolysis of hBD-3 (14-40) [Cysl8-36 / Cys28-35]. The identity of the two fragments, and thus identical disulfide bridging, could be shown by comigration in capillary zone electrophoresis (FIG. 11).
Tabelle 1Table 1
Sequenzsequence
Fragment MWca|C MWexp bestätigt (Da) (Da)Fragment MW ca | C MWexp confirmed (Da) (Da)
GIu-Giu-Gin-rie-GIy-Lys 702.8 702.5GIu-Giu-Gin-rie-GIy-Lys 702.8 702.5
Cys-Leu-Pro-Lys Cys-Ser-Thr-Arg Cys-Cys-Arg 1301.5 1300.8 jaCys-Leu-Pro-Lys Cys-Ser-Thr-Arg Cys-Cys-Arg 1301.5 1300.8 yes
I r I i Ser-Cys-Leu-Pro-Lys Cys-Ser-Thr-Arg Cys-Cys-Arg 1388.6 1387.8 jaI r I i Ser-Cys-Leu-Pro-Lys Cys-Ser-Thr-Arg Cys-Cys-Arg 1388.6 1387.8 yes
Tyr-Tyr-Cys-Arg Cys-Ala-Val-Leu-Ser 1093.3 1092.8 aTyr-Tyr-Cys-Arg Cys-Ala-Val-Leu-Ser 1093.3 1092.8 a
Tyr-Tyr-Cys-Arg Cys-Ala-Val-Leu 1006.2 1005.8Tyr-Tyr-Cys-Arg Cys-Ala-Val-Leu 1006.2 1005.8
Tabelle 1 zeigt die Ergebnisse der massenspektrometrische Identifizierung der Peptidfragmente nach Proteolyse von hBD-3 mit Trypsin/Chymotrypsin.Table 1 shows the results of the mass spectrometric identification of the peptide fragments after proteolysis of hBD-3 with trypsin / chymotrypsin.
Antimikrobielle Wirkung der Peptide gegen Bakterien und HefenAntimicrobial effect of the peptides against bacteria and yeast
Die antimikrobielle Aktivität der erfindungsgemäßen Peptide wurde unter Anwendung der Bestimmung der minimalen Inhibitionskonzentration (MIC) gegen human pathogene Gram-positive und Gram-negative Bakterien untersucht. Die minimale Hemmstoffkonzentration bezeichnet die Peptidkonzentration, die minimal erforderlich ist, um das Wachstum von Mikroorganismen nach einer Inkubationszeit von 18 ± 2 h vollständig zu hemmen. Die minimale Inhibitionskonzentration wurde in Anlehnung an die von dem NCCLS (National Committee for Clinical Laboratory Standards) herausgegebenen Empfehlungen (M7-A3) durchgeführt, wobei chemisch synthetisierte Peptide der Erfindung verwendet wurden. Die minimalen Hemmkonzentrationen sind in der nachfolgenden Tabelle in [μg/ml] angegeben. Folgende Peptide wurden getestet:The antimicrobial activity of the peptides according to the invention was examined using the determination of the minimum inhibitory concentration (MIC) against human pathogenic Gram-positive and Gram-negative bacteria. The minimum inhibitor concentration is the minimum peptide concentration required to completely inhibit the growth of microorganisms after an incubation period of 18 ± 2 h. The minimum inhibitory concentration was performed based on the recommendations (M7-A3) issued by the NCCLS (National Committee for Clinical Laboratory Standards), using chemically synthesized peptides of the invention. The minimum inhibitory concentrations are given in the table below in [μg / ml]. The following peptides were tested:
Peptide: 1 : hBD-3 [Cys6-13/Cysl8-36/Cys28-35]Peptides: 1: hBD-3 [Cys6-13 / Cysl8-36 / Cys28-35]
LQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKKLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK
2: hBD-3, linear:2: hBD-3, linear:
LQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKKLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK
3 : hBD-3 (1-17) [Cys6-13] :3: hBD-3 (1-17) [Cys6-13]:
LQKYYCRVRGGRCAVLSLQKYYCRVRGGRCAVLS
4: [Cys28,35(Acm)] hBD-3 (14-40), linear: AVLSCLPKEEQIG KCSTRG RKCCRRKK4: [Cys28.35 (Acm)] hBD-3 (14-40), linear: AVLSCLPKEEQIG KCSTRG RKCCRRKK
5: [Cysl8,36(Acm)] hBD-3 (14-40) [Cys28-35] :5: [Cysl8.36 (Acm)] hBD-3 (14-40) [Cys28-35]:
AVLSCLPKE EQIG KCSTRG RKCCRRKKAVLSCLPKE EQIG KCSTRG RKCCRRKK
6: hBD-3 (14-40) [Cysl8-36/28-35] :6: hBD-3 (14-40) [Cysl8-36 / 28-35]:
AVLSCLPKEEQIGKCSTRGRKCCRRKKAVLSCLPKEEQIGKCSTRGRKCCRRKK
Acm: AcetamidomethylAcm: acetamidomethyl
Testmedium : 1/ konzentrierte Mueller Hinton BrothTest medium: 1 / concentrated Mueller Hinton Broth
Minimale Inhibitionskonzentrationen [μg/ml] :
Figure imgf000018_0001
nb: nicht bestimmtMinimum inhibition concentrations [μg / ml]:
Figure imgf000018_0001
nb: not determined
Die erfindungsgemässen Peptide inhibieren effektiv sowohl das Wachstum von Gram-positiven als auch von Gram-negativen Bakterien. Human pathogene Erreger, wie Pseudomonas aeruginosa, Staphylococcus aureus und Klebsiella pneumoniae werden effektiv abgetötet. Auch das Wachstum von antibiotikaresistenten Keimen wie z. B. Streptococcus pneumoniae (Penicillin Resistenz), Enterococcus faecalis (Vancomycin Resistenz) und Burkholderia cepacia (multiresistent) wird von den erfindungsgemäßen Peptiden effektiv inhibiert.The peptides according to the invention effectively inhibit the growth of both Gram-positive and Gram-negative bacteria. Human pathogens such as Pseudomonas aeruginosa, Staphylococcus aureus and Klebsiella pneumoniae are effectively killed. The growth of antibiotic-resistant germs such as. B. Streptococcus pneumoniae (penicillin resistance), Enterococcus faecalis (vancomycin resistance) and Burkholderia cepacia (multi-resistant) is effectively inhibited by the peptides according to the invention.
Test der erfindunqsgemäßen Peptide auf hämolvtische Aktivität:Test of the Peptides According to the Invention for Hemolytic Activity:
Die Fähigkeit antimikrobieller Peptide, sich an Plasmamembranen anzulagern und diese zu permeabilisieren, wird in vielen Fällen als Wirkmechanismus dieser Substanzen angesehen. Es ist wünschenswert, dass bakterielle Membranen selektiv geschädigt werden, wohingegen die Zellen des Wirtes unbeeinflusst bleiben sollten. Als einfaches Modell zur Untersuchung der zytotoxischen Wirkung eines Peptids auf eukaryotische Zellen wird der Hämolysetest genutzt.The ability of antimicrobial peptides to attach to and permeabilize plasma membranes is often considered to be the mechanism of action of these substances. It is desirable that bacterial Membranes are selectively damaged, whereas the host's cells should remain unaffected. The hemolysis test is used as a simple model for examining the cytotoxic effect of a peptide on eukaryotic cells.
Die Untersuchung der hämolytischen Aktivität der erfindungsgemäßen Peptide wurde in Anlehnung an Heimerhorst et al. (Heimerhorst et al., 1999, FEBS Lett. 449: 105-110) durchgeführt. Erythrozyten wurden aus dem citrathaltigen Vollblut eines gesunden Probanden durch Zentrifugation (1500 x g, 20 °C, 10 min) isoliert und mit Testmedium 200-fach verdünnt. Verschiedene Konzentrationen hBD-3 [Cys6-13/Cysl8-36/Cys28-35] wurden in einer 96-Well-Mikrotiterplatte mit V-förmig zulaufendem Boden vorgelegt und für 1 h bei 37 °C mit der verdünnten Erythrozytensuspension inkubiert. Durch anschließende Zentrifugation (1000 x g für 5 min) wurden die Erythrozyten abgetrennt. Die durch freigesetztes Hämoglobin gefärbten Überstände wurden in eine 96-Well-Mikrotiterplatte mit flachem Boden überführt und ihre Absorption bei einer Wellenlänge von λ=450 nm im Mikrotiterplatten-Leser bestimmt. Als Referenzwert für 100 %ige Hämolyse diente die Inkubation mit einer 1 %igen Lösung des Detergenz Tween-20. Nur mit Testmedium inkubierte Erythrozyten dienten als Negativkontrolle. Die Angabe der hämolytischen Aktivität von hBD-3 [Cys6-13/Cysl8-36/Cys28-35] erfolgte in Bezug auf die totale Hämolyse mit Tween-20 und wurde nach folgender Formel berechnet:The investigation of the hemolytic activity of the peptides according to the invention was based on Heimerhorst et al. (Heimerhorst et al., 1999, FEBS Lett. 449: 105-110). Erythrocytes were isolated from the citrate-containing whole blood of a healthy subject by centrifugation (1500 × g, 20 ° C., 10 min) and diluted 200-fold with test medium. Different concentrations of hBD-3 [Cys6-13 / Cysl8-36 / Cys28-35] were placed in a 96-well microtiter plate with a V-shaped bottom and incubated for 1 h at 37 ° C. with the diluted erythrocyte suspension. The erythrocytes were separated by subsequent centrifugation (1000 x g for 5 min). The supernatants stained by the released hemoglobin were transferred to a 96-well microtiter plate with a flat bottom and their absorption at a wavelength of λ = 450 nm was determined in the microtiter plate reader. Incubation with a 1% solution of the detergent Tween-20 served as the reference value for 100% hemolysis. Erythrocytes incubated only with test medium served as a negative control. The hemolytic activity of hBD-3 [Cys6-13 / Cysl8-36 / Cys28-35] was stated in relation to total hemolysis with Tween-20 and was calculated using the following formula:
Hämolyse [%] = [A450 nm (Peptid) - A 450 nm (Negativkontrolle)] / [A450 nm (1% Tween 20) - A450 nm (Negativkontrolle)] * 100Hemolysis [%] = [A 450 nm (peptide) - A 450 nm (negative control)] / [A 450 nm (1% Tween 20) - A 450 nm (negative control)] * 100
Als Testmedium diente TSB-Medium mit 287 mM Glukose. Die isotonische Glukosekonzentration soll einer unspezifischen Hämolyse im hypotonen Milieu vorbeugen (Osmoprotektion).TSB medium with 287 mM glucose was used as the test medium. The isotonic glucose concentration is intended to prevent unspecific hemolysis in the hypotonic environment (osmoprotection).
Die Ergebnisse sind in Figur 12 aufgeführt. Das Peptid hBD-3 [Cys6-13/Cysl8- 36/Cys28-35] weist in antimikrobiell wirksamen Konzentrationen keine nennenswerte hämolytische Aktivität auf. Effekt von hBD-3 auf Effektoren der Apoptose:The results are shown in Figure 12. The peptide hBD-3 [Cys6-13 / Cysl8- 36 / Cys28-35] has no significant hemolytic activity in antimicrobial concentrations. Effect of hBD-3 on effectors of apoptosis:
Es wurde mit der Methode der "DNA-Microarray-Hybridisierung", welche die Detektion durch einen bestimmten Faktor hoch- oder herunterregulierter Gene erlaubt, untersucht, ob hBD-3 eine Rolle bei der Auslösung der Apoptose zukommt. Dabei stimuliert man eine Zellinie bzw. Primärzellen mit dem zu untersuchenden Faktor und lässt eine Kontrollprobe unstimuliert. Aus beiden Proben wird die Botenribonucleinsäure mRNA isoliert und in Gegenwart zweier fluoreszenzmarkierter Nucleotide (Cy-3 für die stimulierte Probe, Cy-5 für die Kontrolle) in cDNA-Erststrang ("Copy-Desoxyribonucleinsäure") umgeschrieben. Werden nun durch den zu untersuchenden Faktor Gene hochreguliert, so sind deren Transkripte in der stimulierten Probe verglichen mit der Kontrollprobe überrepräsentiert. Umgekehrt sind die Transkripte bei einer Herunterregulation unterrepräsentiert. Diese Verhältnisse spiegeln sich entsprechend in den markierten cDNAs wieder. Nun werden die markierten cDNAs aus stimulierten und unstimulierten Zellen in einem Ansatz mit punktförmig auf einem geeigneten Träger geordnet aufgebrachten DNA- Proben ("Spots") einer Vielzahl (mehrere Tausend) unterschiedlicher Gene "hybridisiert", wobei die cDNAs jeweils ah den Spots der ihnen entsprechenden Gene "hängenbleiben". Die anschließend mit einem speziellen "Analyzer" detektierbaren Signalstärken sind proportional zu den Mengen mit den jeweiligen Spots hybridisierter cDNAs. Daher können durch den Vergleich des Hybridisierungsergebnisses aus der stimulierten mit dem der unstimulierten Probe (durch die unterschiedlichen Markierungen Cy-3 und Cy-5 unterscheidbar) alle die Gene detektiert werden, die durch den untersuchten Faktor in ihrer Aktivität beeinflusst wurden. Verwendet wurde ein von der Firma "NEN" (Boston, MA, USA) bezogener Array, der DNA-Proben von insgesamt 2400 Genen enthielt.It was examined with the method of "DNA-microarray hybridization", which allows the detection by a certain factor of up- or down-regulated genes, whether hBD-3 plays a role in triggering apoptosis. A cell line or primary cells are stimulated with the factor to be examined and a control sample is left unstimulated. The messenger ribonucleic acid mRNA is isolated from both samples and, in the presence of two fluorescence-labeled nucleotides (Cy-3 for the stimulated sample, Cy-5 for the control), is transcribed into cDNA first strand ("copy deoxyribonucleic acid"). If genes are up-regulated by the factor to be examined, their transcripts are overrepresented in the stimulated sample compared to the control sample. Conversely, the transcripts are underrepresented in downregulation. These relationships are reflected accordingly in the labeled cDNAs. Now the labeled cDNAs from stimulated and unstimulated cells are "hybridized" in one approach with DNA samples ("spots") of a multiplicity (several thousands) of different genes arranged in a punctiform manner on a suitable carrier, the cDNAs each corresponding to the spots of theirs corresponding genes "get stuck". The signal strengths which can subsequently be detected with a special "analyzer" are proportional to the amounts of cDNAs hybridized with the respective spots. Therefore, by comparing the hybridization result from the stimulated with that from the unstimulated sample (distinguishable by the different labels Cy-3 and Cy-5), all the genes can be detected which were influenced in their activity by the investigated factor. An array obtained from the company "NEN" (Boston, MA, USA) was used, which contained DNA samples from a total of 2400 genes.
Humane Skelettmuskelzellen (SKMC 6723, bezogen von Bio Whittaker, Walkerswill, MD, USA) wurden mit 50 μg hBD-3 pro ml Medium für 90 Minuten stimuliert. Eine Kontrollprobe blieb unstimuliert. Anschließend wurde nach Standardverfahren die mRNA aus stimulierter Probe und Kontrolle isoliert und in Gegenwart von Cy-3- (stimulierte Probe) bzw. Cy-5-markierten (Kontrolle) Nucleotiden in cDNA-Erststrang umgeschrieben. Beide cDNAs wurden vereint und gemeinsam mit den DNA-Spots auf dem Array von NEN entsprechend den Herstellerangaben hybridisiert. Der Array wurde anschließend zu Auswertung an NEN gesendet.Human skeletal muscle cells (SKMC 6723, obtained from Bio Whittaker, Walkerswill, MD, USA) were stimulated with 50 μg hBD-3 per ml medium for 90 minutes. A control sample remained unstimulated. The mRNA was then isolated from stimulated sample and control using standard methods and in the presence of Cy-3 (stimulated sample) or Cy-5-labeled (control) nucleotides rewritten into cDNA first strand. Both cDNAs were combined and hybridized together with the DNA spots on the array of NEN according to the manufacturer's instructions. The array was then sent to NEN for evaluation.
Es zeigte sich, dass das Gen für den Apoptose-Effektor Apaf-1 in den hBD-3- stimulierten Zellen um den Faktor 3,4 höher exprimiert wurde als in der Kontrolle. hBD-3 stellt damit einen Aktivator des Gens für Apaf-1 dar.It was shown that the gene for the apoptosis effector Apaf-1 was expressed in the hBD-3-stimulated cells by a factor of 3.4 higher than in the control. hBD-3 thus represents an activator of the gene for Apaf-1.
Einleitung der Apoptose bei Melanomzellen durch hBD-3:Initiation of apoptosis in melanoma cells by hBD-3:
Um den Einfluss von hBD-3 auf die Induktion der Apoptose zu untersuchen, wurde die humane Melanomzelllinie SKMEL-28 verwendet. Der Apoptose- Nachweis erfolgte mit Hilfe eines spezifischen ELISA der Firma Röche (Mannheim) ("Cell Death Detection ELISA", Best. Nr. 1 544 675). Das Prinzip dieses ELISAs beruht auf dem Nachweis Apoptose-typischer fragmentierter DNA (Zhang JH, Xu M (2000). DNA fragmentation in apoptosis. Cell Res. 10: 205-211) und daran gebundener Histone. Hierzu werden spezielle Mikrotiter- Module mit monoklonalen, gegen Histone gerichteten Maus-Antikörpern beschichtet. Anschließend wird zu testendes Zelllysat zugegeben und fragmentierte, Histon-assoziierte DNA - soweit vorhanden - bindet an die Antikörper. Nach entsprechenden Waschschritten wird ein zweiter, gegen DNA gerichteter monoklonaler Maus-Antikörper zugegeben, der mit einer Peroxidase konjugiert ist. Die Menge an bindenden Antikörpern ist dabei innerhalb eines bestimmten Bereiches proportional zur Menge im ursprünglichen Zellysat vorhandener fragmentierter DNA. Diese kann nach erneuten Waschschritten indirekt durch die Peroxidase-katalysierte Bildung einer im Bereich von 405 nm absorbierenden Substanz aus dem Substrat ABTS (2,2'-Azino-di-[3-ethylbenzthiazolin-sulfonat]) erfasst werden (Messung am Spektralphotometer). Zum Nachweis der hBD-3-induzierten Apoptose wurden konkret in "Sechs-Well-Kulturplatten" (2,2 ml Medium je Vertiefung) SKMEL-28 Zellen kultiviert und noch in der exponentiellen Wachstumsphase auf 105 Zellen pro ml sowie auf unterschiedliche Konzentrationen an hBD-3 eingestellt (0, 1, 3, 10, 30, 100 μg/ml). Nach fünf Stunden Inkubation wurden die Zellen entsprechend des Herstellerprotokolls weiterbehandelt und schließlich eingetretene Apoptosen indirekt durch die Peroxidase katalysierte Farbreaktion am Photometer bestimmt.The human melanoma cell line SKMEL-28 was used to investigate the influence of hBD-3 on the induction of apoptosis. The apoptosis was detected using a specific ELISA from Röche (Mannheim) ("Cell Death Detection ELISA", order no. 1 544 675). The principle of this ELISA is based on the detection of fragmented DNA typical of apoptosis (Zhang JH, Xu M (2000). DNA fragmentation in apoptosis. Cell Res. 10: 205-211) and bound histones. For this purpose, special microtiter modules are coated with monoclonal mouse antibodies directed against histones. Then cell lysate is added to the test and fragmented, histone-associated DNA - if available - binds to the antibodies. After corresponding washing steps, a second mouse monoclonal antibody directed against DNA is added, which is conjugated with a peroxidase. The amount of binding antibodies is proportional to the amount of fragmented DNA present in the original cell lysate within a certain range. After repeated washing steps, this can be detected indirectly by the peroxidase-catalyzed formation of a substance absorbing in the range of 405 nm from the substrate ABTS (2,2'-azino-di- [3-ethylbenzthiazoline sulfonate]) (measurement on a spectrophotometer). To detect hBD-3-induced apoptosis, SKMEL-28 cells were cultivated specifically in "six-well culture plates" (2.2 ml of medium per well) and in the exponential growth phase, 10 5 cells per ml and at different concentrations were also used hBD-3 adjusted (0, 1, 3, 10, 30, 100 μg / ml). After five hours of incubation, the cells were treated further in accordance with the manufacturer's protocol and apoptoses which had finally occurred were determined indirectly by the peroxidase-catalyzed color reaction on the photometer.
Die Auswertung von drei unabhängigen Versuchen ergab, dass bereits bei hBD-3-Konzentrationen ab 10 μg/ml eine signifikante Erhöhung der Absorption gegenüber der Nullkontrolie zu verzeichnen war. Sie betrug rund 2,0 A405E (Absorptionseinheiten bei 405 nm), während die Absorption bei der Nullkontrolie niedriger als 0,2 A405E war. Erhöhte hBD-3 Konzentrationen von 30 und 100 μg/ml führten zu keiner zusätzlichen Absorptionserhöhung. Damit wird eine Einleitung der Apoptose in der humanen Melanomzelllinie SKMEL-28 durch hBD-3 in Konzentrationen ab 10 μg/ml gezeigt.The evaluation of three independent experiments showed that a significant increase in the absorption compared to the zero control was already recorded at hBD-3 concentrations from 10 μg / ml. It was around 2.0 A 405 E (absorption units at 405 nm), while the absorption at the zero control was lower than 0.2 A 405 E. Increased hBD-3 concentrations of 30 and 100 μg / ml did not lead to an additional increase in absorption. An initiation of apoptosis in the human melanoma cell line SKMEL-28 by hBD-3 in concentrations from 10 μg / ml is shown.
Um die Versuchsergebnisse in einem zweiten unabhängigen Experiment zu verifizieren, wurden SKMEL-28 Zellen in 75 cm2 Kulturflaschen mit einem Volumen von 20 ml Medium kultiviert und noch innerhalb der exponentiellen Wachstumsphase entsprechend den oben genannten Bedingungen mit 10 μg/ml hBD-3 behandelt. Die DNA der Zellen wurde anschließend nach Standardprotokollen isoliert, komplett auf einem Agarosegel elektrophoretisch aufgetrennt und nach Ethidiumbromid-Färbung unter UV-Durchlicht sichtbar gemacht. Auch hier zeigte sich das für in die Apoptose übergegangene Zellen typische Muster einer Leiter genomischer DNA-Fragmente, deren Größen Vielfache von ca. 180 Bp darstellten (zur Übersicht siehe Zhang und Xu, 2000). Die DNA der unbehandelten Zellen befand sich wie für undegradierte DNA erwartet hingegen im hochmolekularen Bereich.In order to verify the test results in a second independent experiment, SKMEL-28 cells were cultivated in 75 cm 2 culture bottles with a volume of 20 ml of medium and treated with 10 μg / ml of hBD-3 in accordance with the above-mentioned conditions during the exponential growth phase. The DNA of the cells was then isolated according to standard protocols, completely electrophoretically separated on an agarose gel and, after ethidium bromide staining, made visible under UV transmitted light. Here, too, the pattern of a ladder of genomic DNA fragments typical of cells passed into apoptosis was shown, the sizes of which represented multiples of approximately 180 bp (for an overview, see Zhang and Xu, 2000). The DNA of the untreated cells was, as expected for undegraded DNA, in the high-molecular range.
Die ermittelten Daten belegen eine Apoptose-auslösende Wirkung von hBD-3 in Melanomzelllinien bereits ab Konzentrationen von 10 μg pro ml Kulturmedium. Daher ist hBD-3 als Therapeutikum für die Behandlung von Melanomen und andere Krebsarten einsetzbar. Figuren :The data obtained demonstrate an apoptosis-inducing effect of hBD-3 in melanoma cell lines from concentrations of 10 μg per ml culture medium. Therefore hBD-3 can be used as a therapeutic agent for the treatment of melanoma and other types of cancer. Characters :
Figur 1 :Figure 1:
Analytische HPLC von hBD-3.Analytical HPLC of hBD-3.
Säule: Vydac C18, 4.6x250 mm, 5 μm, 300 ÄColumn: Vydac C18, 4.6x250 mm, 5 μm, 300 Ä
Flussrate: 0.8 mL/minFlow rate: 0.8 mL / min
Eluent A: 0.07% TrifluoressigsäureEluent A: 0.07% trifluoroacetic acid
Eluent B: 0.07% Trifluoressigsäure in Acetonitril/Wasser 80:20Eluent B: 0.07% trifluoroacetic acid in acetonitrile / water 80:20
Gradient: 0 - 5 min: 10 % B, 5 - 35 min: 2 % B/minGradient: 0 - 5 min: 10% B, 5 - 35 min: 2% B / min
Figur 2:Figure 2:
Kapillarzonenelektrophorese von hBD-3.Capillary zone electrophoresis of hBD-3.
System: Biofocus 3000 (Bio-Rad)System: Biofocus 3000 (Bio-Rad)
Kapillare: fused silica capillary, 30 cmχ50 μm, unbeschichted Puffersystem: 0.1 M Phosphatpuffer mit Polymer modifier, pH 2.5 Messparameter: konstante Spannung 120 kV; UV-Detektion bei 200 nmCapillary: fused silica capillary, 30 cmχ50 μm, uncoated buffer system: 0.1 M phosphate buffer with polymer modifier, pH 2.5 Measurement parameters: constant voltage 120 kV; UV detection at 200 nm
Figur 3 :Figure 3:
Massenspektrometrische Analyse von hBD-3.Mass spectrometric analysis of hBD-3.
(Elektrospray-Massenspektrometrie, Perkin-Elmer Sciex API III)(Electrospray mass spectrometry, Perkin-Elmer Sciex API III)
Figur 4:Figure 4:
HPLC-Analyse der proteolytischen Zersetzung von hBD-3 mit Trypsin/Chymdtrypsin mit Zuordnung der identifizierten Fragmente.HPLC analysis of the proteolytic decomposition of hBD-3 with trypsin / chymdtrypsin with assignment of the identified fragments.
Säule: Vydac C18, 4.6x250 mm, 5 μm, 300 ÄColumn: Vydac C18, 4.6x250 mm, 5 μm, 300 Ä
Flussrate: 0.8 mL/minFlow rate: 0.8 mL / min
Eluent A: 0.07% TrifluoressigsäureEluent A: 0.07% trifluoroacetic acid
Eluent B: 0.07% Trifluoressigsäure in Acetonitril/Wasser 80 :20Eluent B: 0.07% trifluoroacetic acid in acetonitrile / water 80:20
Gradient: 0 - 5 min : 10 % B, 5 - 35 min : 2 % B/minGradient: 0-5 min: 10% B, 5-35 min: 2% B / min
Figur 5:Figure 5:
Analytische HPLC von hBD-3 (1-17).Analytical HPLC of hBD-3 (1-17).
Säule: Vydac C18, 4.6x250 mm, 5 μm, 300 ÄColumn: Vydac C18, 4.6x250 mm, 5 μm, 300 Ä
Flussrate: 0.8 mL/minFlow rate: 0.8 mL / min
Eluent A: 0.07% TrifluoressigsäureEluent A: 0.07% trifluoroacetic acid
Eluent B: 0.07% Trifluoressigsäure in Acetonitril/Wasser 80: 20Eluent B: 0.07% trifluoroacetic acid in acetonitrile / water 80:20
Gradient: 0 - 5 min : 10 % B, 5 - 35 min : 2 % B/minGradient: 0 - 5 min: 10% B, 5 - 35 min: 2% B / min
Figur 6 :Figure 6:
Kapillarzonenelektrophorese von hBD-3 (1-17).Capillary zone electrophoresis of hBD-3 (1-17).
System: Biofocus 3000 (Bio-Rad)System: Biofocus 3000 (Bio-Rad)
Kapillare: fused silica capillary, 30 cmχ50 μm, unbeschichted Puffersystem: 0.1 M Phosphatpuffer mit Polymer modifier, pH 2.5 Messparameter: konstante Spannung 120 kV; UV-Detektion bei 200 nm Figur 7:Capillary: fused silica capillary, 30 cmχ50 μm, uncoated buffer system: 0.1 M phosphate buffer with polymer modifier, pH 2.5 Measurement parameters: constant voltage 120 kV; UV detection at 200 nm Figure 7:
Massenspektrometrische Analyse von hBD-3 (1-17).Mass spectrometric analysis of hBD-3 (1-17).
(Elektrospray-Massenspektrometrie, Perkin-Elmer Sciex API III)(Electrospray mass spectrometry, Perkin-Elmer Sciex API III)
Figur 8:Figure 8:
Analytische HPLC von hBD-3 (14-40) [Cysl8-36/Cys28-35].Analytical HPLC of hBD-3 (14-40) [Cysl8-36 / Cys28-35].
Säule: Vydac C18, 4.6x250 mm, 5 μm, 300 ÄColumn: Vydac C18, 4.6x250 mm, 5 μm, 300 Ä
Flussrate: 0.8 mL/minFlow rate: 0.8 mL / min
Eluent A: 0.07% TrifluoressigsäureEluent A: 0.07% trifluoroacetic acid
Eluent B: 0.07% Trifluoressigsäure in Acetonitril/Wasser 80:20Eluent B: 0.07% trifluoroacetic acid in acetonitrile / water 80:20
Gradient: 0 - 5 min : 10 % B, 5 - 35 min: 2 % B/minGradient: 0-5 min: 10% B, 5-35 min: 2% B / min
Figur 9:Figure 9:
Kapillarzonenelektrophorese von hBD-3 (14-40) [Cysl8-36/Cys28-35].Capillary zone electrophoresis of hBD-3 (14-40) [Cysl8-36 / Cys28-35].
System: Biofocus 3000 (Bio-Rad)System: Biofocus 3000 (Bio-Rad)
Kapillare: fused silica capillary, 30 cmχ50 μm, unbeschichted Puffersystem: 0.1 M Phosphatpuffer mit Polymer modifier, pH 2.5 Messparameter: konstante Spannung 120 kV; UV-Detektion bei 200 nmCapillary: fused silica capillary, 30 cmχ50 μm, uncoated buffer system: 0.1 M phosphate buffer with polymer modifier, pH 2.5 Measurement parameters: constant voltage 120 kV; UV detection at 200 nm
Figur 10:Figure 10:
Massenspektrometrische Analyse von hBD-3 (14-40) [Cysl8-36/Cys28-35].Mass spectrometric analysis of hBD-3 (14-40) [Cysl8-36 / Cys28-35].
(Elektrospray-Massenspektrometrie, Perkin-Elmer Sciex API III)(Electrospray mass spectrometry, Perkin-Elmer Sciex API III)
Figur 11 :Figure 11:
Vergleich der Peptidfragmente Ser-Cys-Leu-Pro-Lys-Glu-Glu-Gln-Ile-Gly-Lys- Cys-Ser-Thr-Arg-Gly-Arg-Lys-Cys-Cys-Arg-Arg-Lys-Lys nach proteolytischer Zersetzung von hBD-3 bzw. hBD-3 (14-40) [Cysl8-36/Cys28-35] mit Chymotrypsin (pH -8, 4 h) durch Kapillarzonenelektrophorese; a - Peptidfragment aus hBD-3; b - Peptidfragment aus hBD-3 (14-40) [Cysl8- 36/Cys28-35]; c - Komigration beider Fragmente.Comparison of the peptide fragments Ser-Cys-Leu-Pro-Lys-Glu-Glu-Gln-Ile-Gly-Lys-Cys-Ser-Thr-Arg-Gly-Arg-Lys-Cys-Cys-Arg-Arg-Lys-Lys after proteolytic decomposition of hBD-3 or hBD-3 (14-40) [Cysl8-36 / Cys28-35] with chymotrypsin (pH -8, 4 h) by capillary zone electrophoresis; a - peptide fragment from hBD-3; b - peptide fragment from hBD-3 (14-40) [Cysl8- 36 / Cys28-35]; c - Comigration of both fragments.
System: Biofocus 3000 (Bio-Rad)System: Biofocus 3000 (Bio-Rad)
Kapillare: fused silica capillary, 30 cmχ50 μm, unbeschichted Puffersystem: 0.1 M Phosphatpuffer mit Polymer modifier, pH 2.5 Messparameter: konstante Spannung 120 kV; UV-Detektion bei 200 nm.Capillary: fused silica capillary, 30 cmχ50 μm, uncoated buffer system: 0.1 M phosphate buffer with polymer modifier, pH 2.5 Measurement parameters: constant voltage 120 kV; UV detection at 200 nm.
Figur 12: Hämolytische Aktivität von hBD-3 rCys6-13/Cys!8-36/Cvs28-35].Figure 12: Hemolytic activity of hBD-3 rCys6-13 / Cys! 8-36 / Cvs28-35].
Die hämolytische Aktivität von hBD-3 [Cys6-13/Cysl8-36/Cys28-35] wurde über die Freisetzung von Hämoglobin aus Erythrozyten bestimmt, die spektralphotometrisch quantifizierbar ist. Als Positivkontrolle diente das antimikrobiell wirksame, hämolytisch aktive Peptid MBI-28 (Piers et al., 1994, Antimicrob. Agents Chemother. 38: 2311-2316). The hemolytic activity of hBD-3 [Cys6-13 / Cysl8-36 / Cys28-35] was determined via the release of hemoglobin from erythrocytes, which can be quantified spectrophotometrically. The antimicrobial, hemolytically active peptide MBI-28 (Piers et al., 1994, Antimicrob. Agents Chemother. 38: 2311-2316) served as a positive control.

Claims

Patentansprüche claims
1. Humanes beta-Defensin-3 (hBD-3) mit der Aminosäurensequenz Seq. No. 1 :1. Human beta-defensin-3 (hBD-3) with the amino acid sequence Seq. No. 1 :
Zi-Cys-Arg-Val-Arg-Gly-Gly-Arg-Cys-Ala-Val-Leu-Ser-Cys-Leu-Pro-Lys- Glu-Glu-Gln-Ile-Gly-Lys-Cys-Ser-Thr-Arg-Gly-Arg-Lys-Cys-Cys-X1( wobei Zi und Xi Aminosäuren oder Polypeptide aus 1 bis 50 Aminosäuren darstellen, sowie deren natürliche, synthetische und pharmakologisch verträgliche Derivate, insbesondere amidierte, acylierte, phosphorylierte, glycosylierte und zyklische Derivate, sowie deren durch Aminosäuresubstitution, -deletion oder -insertion entstandene Derivate und Fragmente mit biologischer Wirkung, die aus der Aminosäurensequenz abgeleitet werden.Zi-Cys-Arg-Val-Arg-Gly-Gly-Arg-Cys-Ala-Val-Leu-Ser-Cys-Leu-Pro-Lys- Glu-Glu-Gln-Ile-Gly-Lys-Cys-Ser- Thr-Arg-Gly-Arg-Lys-Cys-Cys-X 1 ( where Zi and Xi represent amino acids or polypeptides from 1 to 50 amino acids, as well as their natural, synthetic and pharmacologically acceptable derivatives, in particular amidated, acylated, phosphorylated, glycosylated and cyclic derivatives, as well as their derivatives and fragments with biological activity resulting from amino acid substitution, deletion or insertion, which are derived from the amino acid sequence.
2. hBD-3 nach Anspruch 1, das eine der Sequenzen Seq. No. 2:2. hBD-3 according to claim 1, which one of the sequences Seq. No. 2:
Leu-Gln-Lys-Tyr-Tyr-Cys-Arg-Val-Arg-Gly-Gly-Arg-Cys-Ala-Val-Leu-Ser- Cys-Leu-Pro-Lys-Glu-Glu-Gln-Ile-Gly-Lys-Cys-Ser-Thr-Arg-Gly-Arg-Lys- Cys- Cys- Arg -Arg -Lys-LysLeu-Gln-Lys-Tyr-Tyr-Cys-Arg-Val-Arg-Gly-Gly-Arg-Cys-Ala-Val-Leu-Ser- Cys-Leu-Pro-Lys-Glu-Glu-Gln-Ile- Gly-Lys-Cys-Ser-Thr-Arg-Gly-Arg-Lys-Cys-Cys-Arg -Arg -Lys-Lys
Seq. No. 3 :Seq. No. 3:
Gly-Ile-Ile-Asn-Thr-Leu-Gln-Lys-Tyr-Tyr-Cys-Arg-Val-Arg-Gly-Gly-Arg-Gly-Ile-Ile-Asn-Thr-Leu-Gln-Lys-Tyr-Tyr-Cys-Arg-Val-Arg-Gly-Gly-Arg-
Cys-Ala-Val-Leu-Ser-Cys-Leu-Pro-Lys-Glu-Glu-Gln-Ile-Gly-Lys-Cys-Ser-Cys-Ala-Val-Leu-Ser-Cys-Leu-Pro-Lys-Glu-Glu-Gln-Ile-Gly-Lys-Cys-Ser-
Thr-Arg-Gly-Arg-Lys-Cys-Cys-Arg-Arg-Lys-Lys oder Seq No. 4:Thr-Arg-Gly-Arg-Lys-Cys-Cys-Arg-Arg-Lys-Lys or Seq No. 4:
Met-Arg-Ile-His-Tyr-Leu-Leu-Phe-Ala-Leu-Leu-Phe-Leu-Phe-Leu-Val- Pro-Val-Pro-GIy-His-Gly-Gly-Ile-Ile-Asn-Thr-Leu-Gln-Lys-Tyr-Tyr-Cys- Arg-Val-Arg-Gly-Gly-Arg-Cys-Ala-Val-Leu-Ser-Cys-Leu-Pro-Lys-Glu-Glu- Gln-Ile-Gly-Lys-Cys-Ser-Thr-Arg-Gly-Arg-Lys-Cys-Cys-Arg-Arg-Lys-Lys aufweist.Met-Arg-Ile-His-Tyr-Leu-Leu-Phe-Ala-Leu-Leu-Phe-Leu-Phe-Leu-Val- Pro-Val-Pro-GIy-His-Gly-Gly-Ile-Ile- Asn-Thr-Leu-Gln-Lys-Tyr-Tyr-Cys- Arg-Val-Arg-Gly-Gly-Arg-Cys-Ala-Val-Leu-Ser-Cys-Leu-Pro-Lys-Glu-Glu- Gln-Ile-Gly-Lys-Cys-Ser-Thr-Arg-Gly-Arg-Lys-Cys-Cys-Arg-Arg-Lys-Lys.
3. hBD-3-Fragmente mit einer der Aminosäurensequenzen Seq. No. 5: Z!-Cys-Arg-Val-Arg-Gly-Gly-Arg-Cys-X2 oder Seq. No. 6:3. hBD-3 fragments with one of the amino acid sequences Seq. No. 5: Z ! -Cys-Arg-Val-Arg-Gly-Gly-Arg-Cys-X 2 or Seq. No. 6:
Z2-Cys-Leu-Pro-Lys-Glu-Glu-Gln-Ile-Gly-Lys-Cys-Ser-Thr-Arg-Gly-Arg- Lys- ys-Cys-Xx wobei Zi, Z2, Xi und X2 Aminosäuren oder Polypeptide aus 1 bis 50 Aminosäuren darstellen, sowie deren synthetische und pharmakologisch verträgliche Derivate, insbesondere zyklische, amidierte, acylierte, phosphorylierte und glycosylierte Derivate, sowie deren durch Aminosäuresubstitution, -deletion oder -insertion entstandene Derivate und Fragmente mit biologischer Wirkung, die aus der Aminosäurensequenz abgeleitet werden.Z 2 -Cys-Leu-Pro-Lys-Glu-Glu-Gln-Ile-Gly-Lys-Cys-Ser-Thr-Arg-Gly-Arg-Lys-ys-Cys-Xx where Zi, Z 2 , Xi and X 2 represent amino acids or polypeptides from 1 to 50 amino acids, as well as their synthetic and pharmacologically acceptable derivatives, in particular cyclic, amidated, acylated, phosphorylated and glycosylated derivatives, and their derivatives and fragments with biological activity resulting from amino acid substitution, deletion or insertion, which are derived from the amino acid sequence.
4. hBD-3-Fragmente nach Anspruch 3, die eine der Sequenzen Seq. No. 7:4. hBD-3 fragments according to claim 3, which one of the sequences Seq. No. 7:
Leu-Gln-Lys-Tyr-Tyr-Cys-Arg-Val-Arg-Gly-Gly-Arg-Cys-Ala-Val-Leu-Ser oder Seq. No. 8:Leu-Gln-Lys-Tyr-Tyr-Cys-Arg-Val-Arg-Gly-Gly-Arg-Cys-Ala-Val-Leu-Ser or Seq. No. 8th:
Gly-Ile-Ile-Asn-Thr-Leu-Gln-Lys-Tyr-Tyr-Cys-Arg-Val-Arg-Gly-Gly-Arg- Cys-Ala-Val-Leu-Ser oder Seq.°No. 9:Gly-Ile-Ile-Asn-Thr-Leu-Gln-Lys-Tyr-Tyr-Cys-Arg-Val-Arg-Gly-Gly-Arg-Cys-Ala-Val-Leu-Ser or Seq. ° No. 9:
Ala-Val-Leu-Ser-Cys-Leu-Pro-Lys-Glu-Glu-Gln-Ile-Gly-Lys-Cys-Ser-Thr- Arg-Gly-Arg-Lys-Cys-Cys-Arg-Arg-Lys-Lys aufweisen.Ala-Val-Leu-Ser-Cys-Leu-Pro-Lys-Glu-Glu-Gln-Ile-Gly-Lys-Cys-Ser-Thr- Arg-Gly-Arg-Lys-Cys-Cys-Arg-Arg- Have Lys-Lys.
5. Derivate oder Fragmente des hBD-3 nach Anspruch 1 oder 2, dadurch gekennzeichnet, dass einzelne Aminosäuren so ausgetauscht wurden, dass das Peptid insgesamt eine höhere positive Ladung aufweist.5. derivatives or fragments of hBD-3 according to claim 1 or 2, characterized in that individual amino acids have been exchanged so that the peptide has a higher overall positive charge.
6. Derivate oder Fragmente des hBD-3 nach Anspruch 5, dadurch gekennzeichnet, das eines der vier Glycine aus Sequenz No. 1 und/oder das Alanin, das in Sequenz No. 1 auf Cystein 2 folgt und/oder das Leucin oder Prolin, die in der Sequenz No. 1 auf Cystein 3 folgen gegen basische oder aromatische Aminosäuren substituiert sind, wobei bei Fragmenten des hBD-3 die entsprechenden zur Sequenz No. 1 homologen Aminosäuren substituiert sind.6. derivatives or fragments of hBD-3 according to claim 5, characterized in that one of the four glycins from sequence No. 1 and / or the alanine, which in sequence No. 1 follows cysteine 2 and / or the leucine or proline, which in sequence no. 1 followed by cysteine 3 are substituted for basic or aromatic amino acids, with fragments of the hBD-3 corresponding to sequence no. 1 homologous amino acids are substituted.
7. hBD-3 oder hBD-3-Fragmente oder Derivate nach einem der Ansprüche 1 bis 6, dadurch gekennzeichnet, dass diese eine Cysteinbrücke zwischen Cystein 1 und Cystein 2, eine Cysteinbrücke zwischen Cystein 3 und Cystein 6 und/oder eine Cysteinbrücke zwischen Cystein 4 und Cystein 5 enthalten, wobei die Nummern 1 bis 6 den sechs Cysteinen oder den zu ihnen homologen Cysteinen entsprechen, die in der Sequenz in Anspruch 1 vom N- zum C- Terminus hin enthalten sind.7. hBD-3 or hBD-3 fragments or derivatives according to one of claims 1 to 6, characterized in that they have a cysteine bridge between cysteine 1 and cysteine 2, a cysteine bridge between cysteine 3 and cysteine 6 and / or a cysteine bridge between cysteine 4 and cysteine 5, the numbers 1 to 6 corresponding to the six cysteines or the homologous cysteines which are contained in the sequence in claim 1 from the N- to the C-terminus.
8. hBD-3 oder Derivate von hBD-3 nach einem der Ansprüche 1, 2, 5 oder 6 oder Fragmente der Sequenz 6 nach Anspruch 3, dadurch gekennzeichnet, dass8. hBD-3 or derivatives of hBD-3 according to one of claims 1, 2, 5 or 6 or fragments of sequence 6 according to claim 3, characterized in that
■ Cystein 3 und Cystein 6 keine Cysteinbrücken ausbilden und Cystein 4 und Cystein 5 eine Cysteinbrücke ausbilden, oder■ Cysteine 3 and cysteine 6 do not form cysteine bridges and cysteine 4 and cysteine 5 form a cysteine bridge, or
■ Cystein 3 und Cystein 6 eine Cysteinbrücke ausbilden und Cystein 4 und Cystein 5 keine Cysteinbrücke ausbilden, oder■ Cysteine 3 and cysteine 6 form a cysteine bridge and cysteine 4 and cysteine 5 do not form a cysteine bridge, or
■ keine Cysteinbrücke ausgebildet ist, oder■ no cysteine bridge is formed, or
■ Cystein 3 und Cystein 6 eine Cysteinbrücke ausbilden und Cystein 4 und Cystein 5 eine Cysteinbrücke ausbilden.■ Cysteine 3 and cysteine 6 form a cysteine bridge and cysteine 4 and cysteine 5 form a cysteine bridge.
9. Verfahren zur Herstellung von hBD-3 oder Derivaten oder Fragmenten von hBD-3 nach einem der Ansprüche 1 bis 8, dadurch gekennzeichnet, dass das hBD-3 oder hBD-3-Fragmente oder Derivate durch Festphasen- und/oder Flüssigphasen-Synthese aus den geschützten Aminosäuren, die in der enthalten sind, herstellt werden, deblockiert werden, und durch Chromatographie aufgereinigt werden.9. The method for producing hBD-3 or derivatives or fragments of hBD-3 according to one of claims 1 to 8, characterized in that the hBD-3 or hBD-3 fragments or derivatives by solid phase and / or liquid phase synthesis are prepared, deblocked, and purified by chromatography from the protected amino acids contained in the.
10. Verfahren nach Anspruch 9, dadurch gekennzeichnet, dass Cysteinbrücken durch Oxidation eingeführt werden, wobei gegebenenfalls eine gezielte Einführung der Cysteinbrücken durch Verwendung von Schutzgruppen an nicht zu verknüpfenden Cysteinen erfolgt. 10. The method according to claim 9, characterized in that cysteine bridges are introduced by oxidation, where appropriate a targeted introduction of the cysteine bridges is carried out by using protective groups on non-linked cysteines.
11. Verfahren zur Herstellung von hBD-3 oder Derivaten oder Fragmenten von hBD-3 nach einem der Ansprüche 1 bis 8, dadurch gekennzeichnet, dass dieses in prokaryontischen oder eukaryontischen Zellen exprimiert und chromatographisch gereinigt wird.11. A process for the preparation of hBD-3 or derivatives or fragments of hBD-3 according to one of claims 1 to 8, characterized in that it is expressed in prokaryotic or eukaryotic cells and purified by chromatography.
12. Verfahren zur Herstellung des hBD-3 oder seiner Derivate oder Fragmente nach einem der Ansprüche 1 bis 8, dadurch gekennzeichnet, dass es aus Körperflüssigkeiten, insbesondere Blut, Hemodialysat oder Hemofiltrat über Chromatographie-Verfahren isoliert wird.12. A process for the preparation of the hBD-3 or its derivatives or fragments according to one of claims 1 to 8, characterized in that it is isolated from body fluids, in particular blood, hemodialysate or hemofiltrate by means of a chromatography process.
13. Nucleinsäuren, kodierend für hBD-3 oder seine Derivate oder Fragmente nach einem der Ansprüche 1 bis 8.13. Nucleic acids coding for hBD-3 or its derivatives or fragments according to one of claims 1 to 8.
14. Vektoren oder Plasmide, enthaltend Nucleinsäuren nach Anspruch 13.14. Vectors or plasmids containing nucleic acids according to claim 13.
15. Arzneimittel, enthaltend hBD-3 oder Derivate oder Fragmente von hBD- 3 nach einem der Ansprüche 1 bis 8, Nucleinsäuren nach Anspruch 13 oder Vektoren oder Plasmide nach Anspruch 14.15. Medicament containing hBD-3 or derivatives or fragments of hBD-3 according to one of claims 1 to 8, nucleic acids according to claim 13 or vectors or plasmids according to claim 14.
16. Diagnostikmittel, enthaltend hBD-3 oder Derivate oder Fragmente von hBD-3 nach einem der Ansprüche 1 bis 6, Nucleinsäuren nach Anspruch 13 oder Vektoren oder Plasmide nach Anspruch 14.16. Diagnostic agent containing hBD-3 or derivatives or fragments of hBD-3 according to one of claims 1 to 6, nucleic acids according to claim 13 or vectors or plasmids according to claim 14.
17. Verwendung von hBD-3 oder Derivaten oder Fragmenten von hBD-3 nach einem der Ansprüche 1 bis 8, von Nucleinsäuren nach Anspruch 13 oder Vektoren oder Plasmiden nach Anspruch 14 zur Herstellung eines Arzneimittels zur antimikrobiellen Behandlung, zur Abwehr und Bekämpfung von pathogenen Keimen, zur Behandlung von Erkrankungen der Atemwege, insbesondere die durch Infektionen mit den Bakterien Burkholderia cepacia und Pseudomonas aeroginosa ausgelöst oder verstärkt werden, zur Behandlung der cystischen Fibröse, zur Behandlung entzündlicher Krankheiten des Magen-Darm-Traktes, des Urogenitaltraktes, bei Sepsis, bei gastrointestinalen Infektionen, insbesondere durch Heliobacter pylori ausgelöst, zur Behandlung und Abwehr von gram-positiven und gram-negativen Bakterien, Hefen, Heliobacter pylori, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa oder Burkholderia cepacia, zur Auslösung der Apoptose, zur Behandlung maligner Melanome und von Tumoren.17. Use of hBD-3 or derivatives or fragments of hBD-3 according to one of claims 1 to 8, of nucleic acids according to claim 13 or vectors or plasmids according to claim 14 for the manufacture of a medicament for antimicrobial treatment, for defense and control of pathogenic germs , for the treatment of diseases of the respiratory tract, especially those triggered or intensified by infections with the bacteria Burkholderia cepacia and Pseudomonas aeroginosa, for the treatment of cystic fibrosis, for the treatment of inflammatory diseases of the gastrointestinal tract, the urogenital tract, for sepsis, for gastrointestinal tract Infections, especially triggered by Heliobacter pylori, for the treatment and defense against gram-positive and gram-negative bacteria, yeasts, Heliobacter pylori, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa or Burkholderia cepacia, for inducing apoptosis, for treating malignant melanomas and tumors.
18. Verwendung von hBD-3 oder Derivaten und Fragmenten von hBD-3 nach Anspruch 17 in galenischen Applikationsformen, insbesondere lyophilisiert und in Mannit aufgenommen in sterilen Ampullen zur Auflösung in Kochsalzlösung und/oder Infusionslösungen und Inhalationslösungen, und/oder in Kombination mit anderen antibiotischen Wirkstoffen. 18. Use of hBD-3 or derivatives and fragments of hBD-3 according to claim 17 in galenic application forms, in particular lyophilized and taken up in mannitol in sterile ampoules for dissolution in saline and / or infusion solutions and inhalation solutions, and / or in combination with other antibiotic agents.
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EP3111951A4 (en) * 2015-03-26 2017-09-27 Seoul National University R&DB Foundation Anticancer functional peptide for inhibiting proliferation of cancer stem cell, and use thereof
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KR20200026122A (en) * 2018-08-31 2020-03-10 주식회사 나이벡 Use of Peptides having Ability to inhibit multiple disease biomarkers' expression
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