Classifications

• • 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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• 2001
  • 2001-11-14 WO PCT/EP2001/013174 patent/WO2002040512A2/de not_active Application Discontinuation
  • 2001-11-14 AU AU2002227925A patent/AU2002227925A1/en not_active Abandoned

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