WO2020117081A1 - Peptides for use in prevention and treatment of inflammation - Google Patents

Peptides for use in prevention and treatment of inflammation Download PDF

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Publication number
WO2020117081A1
WO2020117081A1 PCT/PL2019/050072 PL2019050072W WO2020117081A1 WO 2020117081 A1 WO2020117081 A1 WO 2020117081A1 PL 2019050072 W PL2019050072 W PL 2019050072W WO 2020117081 A1 WO2020117081 A1 WO 2020117081A1
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Prior art keywords
dal
peg
peptide
lys
leu
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PCT/PL2019/050072
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French (fr)
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Paulina KOSIKOWSKA-ADAMUS
Adam LESNER
Joanna KOZIEL
Anna GOLDA
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Uniwersytet Gdański
Uniwersytet Jagiellonski
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Publication of WO2020117081A1 publication Critical patent/WO2020117081A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the present invention relates to peptides for medical use in limiting excessive local inflammation caused by bacterial infections, as well as a systemic inflammatory response, such as sepsis, inflammation, accompanied by an increase in the inflammatory mediators, including cytokines, reactive nitrogen species.
  • the present invention relates to peptide- conjugates for use in the prevention and treatment of an inflammation.
  • the human system is permanently exposed to pathogenic microorganisms, as a result of which, in the course of the evolution it has developed a number of efficient mechanisms preventing and fighting infections.
  • the human system is unable to eradicate a pathogen and curb an infection, which may result in an excessive development of an inflammation into a systemic one. Therefore, one seeks efficient pharmaceuticals preventing uncontrolled development of inflammation.
  • the initial response of the immune system to an inflammation caused by e.g. an infection is activation of the immunocompetent cells (macrophages, monocytes, neutrophils, dendritic cells).
  • the factor activating an immune response are the so-called pathogen-associated molecular patterns - PAMP, including e.g. lipid A (a component of lipopolysaccharide (LPS)), peptidoglycan (PGN) or lipotechoic acid (LTA).
  • LPS lipopolysaccharide
  • PPN peptidoglycan
  • LTA lipotechoic acid
  • the PAMP structures are recognised by receptors on the surface of immune cells, the so-called pattern recognition receptors - PRR, including Toll-like receptors - TLR.
  • TLR Recognition of antigens by TLR triggers a cascade of biochemical reactions, as a result of which proinflammatory cytokines are released, such as interleukin 1-a and b (IL-loc, IL-Ib) interleukin 6, 8, 10 (IL-6, IL-8, IL-10), tumour necrosis factor a (TNF-a), which is described in Mogensen, T. H. (2009). "Pathogen recognition and inflammatory signalling in innate immune defenses.” Clin Microbiol Rev 22(2): 240-273. These factors are also released in the case of inflammations caused by factors other than bacteria.
  • IL-loc, IL-Ib interleukin 6, 8, 10
  • TNF-a tumour necrosis factor a
  • the said conjugates are composed of two amino acid sequences corresponding to the following peptides: dalargin (DAL) and DK5, covalently bound to each other by means of a PEG linker (the molecule (or moiety) of the 8-amino-3 6- dioxaoctanoic acid).
  • DAL dalargin
  • DK5 the molecule (or moiety) of the 8-amino-3 6- dioxaoctanoic acid.
  • Dalargin (Tyr-o/a-Gly-Phe-Leu-Arg-OH), hereinafter referred to as DAL, is a synthetic analogue of the endogenic opioid peptide of Leu-enkephalin.
  • the physiological activity of DAL in peripheral tissues results from the interaction with m and 5 - opioid receptors, which is described in an article by Penchev, Pospisek and co. "Activity profiles of Dalargin and Its Analogues in Mu-, delta- and Kappa-Opioid Receptor Selective Bioassays." Br J Pharmacol 128(3): 569-576 (1999).
  • Dalargin is known as an efficient medicine for the treatment of the ulcer disease or acute pancreatitis. Administered by means of a drip bag or intravenously, the medicine decreased pancreas swelling and relived pain.
  • the patent RU 2 341 282 discloses a mixture of octreotide (0.1 mg/kg) and dalargin (0.8 and 0.9 mg/kg) capable of impeding the enzymatic activity of the pancreas.
  • the peptide DK5 (lle-Lys-Lys-lle-Leu-Ser-/ys-lle-Lys-Lys-Leu-Leu-NH2) is a synthetic analogue of a naturally occurring temporin - ICEb, an agent obtained from the skin mucus of the Rana amurensis and Rana dybowskii frogs.
  • Chem Biol Drug Des (2012) 79(5): 653-662 revealed that the agent displays a high activity impeding an increase in the number of gram-negative and gram-positive strains, and a low haemolytic activity relative to human erythrocytes.
  • DAL-PEG-DK5 and DK5-PEG-DAL conjugates are capable of preventing a pathological inflammation and fighting an inflammation, including systemic one as sepsis induced by bacterial infection.
  • the efficiency of the agents was monitored by means of evaluating the level of the inflammatory mediators, including cytokines, reactive nitrogen species, and was confirmed through in vitro ( DAL-PEG-DK5 and DK5-PEG-DAL) and in vivo (DAL-PEG-DK5 exclusively) models.
  • the research was conducted on a model of an inflammation caused by a bacterial endotoxin - LPS. These results found confirmation in an in vitro model using peripheral blood infected by E. coli bacteria, which proves that the observations made using LPS are proper and reflects clinical conditions observedduring systemic infections.
  • the conducted experimental researches hence, confirm the efficiency of the agents in decreasing the level of inflammatory mediators, especially in the case of an inflammation caused by a Gram-negative bacteria infection.
  • a systemic inflammation is understood as an inflammation caused e.g. by an infection accompanied by activation of innate immunity Toll-like receptors - TLR and/or release of pro-inflammatory cytokines (incl. TNF-ot, IL-6, IL-8) and formation of reactive nitrogen species (incl. nitrogen oxide - NO) and other inflammatory mediators.
  • pro-inflammatory cytokines incl. TNF-ot, IL-6, IL-8
  • reactive nitrogen species incl. nitrogen oxide - NO
  • the present invention relates especially to medical use in prevention of inflammation caused by a bacterial infection.
  • the said medical use relates both to humans and animals.
  • the present invention also relates to a peptide - compound - having the following formula 1:
  • the present invention relates especially to a medical use in the treatment of an inflammation caused by a bacterial infection.
  • the said use relates both to humans and animals.
  • the present invention also relates to a peptide - compound - having the following formula 2:
  • the present invention relates especially to a medical use in an inflammation caused by a bacterial infection.
  • the said use relates both to humans and animals.
  • the present invention also relates to a peptide - compound having the following formula 2:
  • lle-Lys-Lys-lle-Leu-Ser-/ys-lle-Lys-Lys-Leu-Leu- 020c-Tyr-a/a-Gly-Phe-Leu-Arg- NH 2 for medical use in the treatment of inflammation, especially in the treatment of a systemic inflammatory response, including sepsis.
  • the said use relates especially to the treatment of an inflammation caused by a bacterial infection.
  • the anti-inflammatory activity of conjugates was examined using the cell line of murine macrophages (RAW 264.7) and primary human macrophages (monocyte derived macrophages, hMDMs), stimulated by a bacterial lipopolysaccharide (LPS) isolated from the cells of strain Escherichia coli bacteria. Based on the obtained results it was concluded, that the DAL-PEG-DK5 conjugate effectively decreases the level of expression of the inflammatory mediators TNF-a, IL-6, IL-8, and reduces the formation of reactive nitrogen (NO) species accompanying a nitrosative cell stress.
  • the anti-inflammatory activity of DK5-PEG-DAL was lower compared with DAL-PEG-DK5.
  • DAL-PEG-DK5 conjugate is capable to reduce significantly the level of the released pro-inflammatory cytokines in response to the presence of bacterial antigens - toxins (LPS, LTA), the cell wall components - PGN (peptidoglycan).
  • LPS bacterial antigens - toxins
  • PGN peptidoglycan
  • the formula 1 and formula 2 present the structure of the compounds - peptides - according to the invention, in configuration, position in compounds, from item 1 to 19.
  • the DAL-PEG-DK5 and DK5-PEG-DAL conjugates were obtained by means of solid phase method of synthesis applying Fmoc (fluorenyl-9-methoxycarbonyl) chemistry.
  • Fmoc fluorenyl-9-methoxycarbonyl
  • TentaGel S RAM Riv Polymere GmbH, Germany
  • the DAL-PEG-DK5 and DK5-PEG-DAL synthesis consist of the following stages:
  • Table 1 The list and order of incorporation of the amino acid residues in the course of the synthesis of the DAL-PEG-DK5 and DK5-PEG-DAL conjugates.
  • the raw peptide preparation was then purified by Reverse Phase High Performance Liquid Chromatography (RP-HPLC) in order to obtain homogeneous preparations of the DAL- PEG-DK5 and DK5-PEG-DAL conjugates.
  • RP-HPLC Reverse Phase High Performance Liquid Chromatography
  • the molecular weight of the conjugates was confirmed by means of Matrix-Assisted Laser Desorpotion Ionisation-Time of Flight Mass Spectrometry.
  • phase B The compounds were separated in a gradient of 1-80% of phase B in phase A for 30 min at 1 ml/min flow.
  • Phase A 0.1% TFA solution in water
  • phase B 80% acetonitrile solution in phase A.
  • Detection was performed at 226 nm.
  • the Shimadzu HPLC system and the Phenomex Jupiter 4pm Proteo 90A (250 x 4.6 mm) column were used for the analysis.
  • the chromatograms presented in the application (Fig. 3,4) represent the analyte fraction corresponding to the DAL-PEG-DK5 and DK5-PEG-DAL conjugates with specified retention times, respectively: 22.735 min. and 19.767 min.
  • the aim of the experiments was to determine the effect of DAL-PEG-DK5 and DK5-PEG- DAL on the reduction of the level of pro-inflammatory response of the mouse macrophages (RAW 264.7) in the presence of LPS ; LTA and PGN.
  • the activity of conjugates in this range was compared against the activity of their components (DK5, DAL).
  • the cells were stimulated accordingly: 10 ng/ml LPS, 10 pg/ml LTA or 2 mg/ml PGN in the presence of the tested compounds at 25 pg/ml or without peptides.
  • the expression of TNF-a cytokine and the concentration of nitric oxide (NO) released were measured by ELISA or Griess tests, respectively.
  • the DAL-PEG-DK5 conjugate is characterized by the best (in comparison to the results obtained for DAL and DK5) ability to inhibit the expression of inflammatory response markers (the lowest concentration of inflammatory TNF-a, NO mediators) caused by selected TLR receptor agonists (Fig. 5).
  • the activity of the DK5-PEG-DAL conjugate in the presence of LPS was comparable for the values obtained for DAL-PEG-DK5 (Fig. 5, LPS panel), however, it was significantly lower (at the control sample level, without the tested compounds) in the presence of LTA and PGN, comparable with the values obtained for DAL and DK5.
  • the level of macrophages activation was determined by measuring the concentration of TNF-a, IL-6, IL-8 and IL-10 cytokines released by cells stimulated with LPS (10 ng/ml). The analysis was performed 20 hours after stimulation with bacterial endotoxin. The experiments were conducted with the use of human monocyte-derived macrophages (hMDM) to which the tested compounds were added at a concentration of 10 pg/ml. The obtained results presented in Fig. 6 clearly show that DAL-PEG-DK5 and DK5-PEG-DAL peptides significantly inhibit the expression of proinflammatory cytokines (TNF-a, IL-6, IL- 8, IL-10) (Fig. 6A).
  • DAL-PEG-DK5 conjugate effectively reduces the level of expression of inflammatory markers in primary human macrophages in the presence of LPS and E. coli infection. Moreover, DAL-PEG-DK5 conjugate effectively neutralizes endotoxin in an environment resembling physiological conditions - an experiment conducted with the use of whole human blood. 3. Determination of the IC50 inhibition coefficient for the expression of inflammatory mediators (TNF-a and NO) for DAL-PEG-DK5 and DK5-PEG-DAL conjugates and their components
  • the DAL-PEG-DK5 peptide is 9 times more active in the human macrophage model and 5 times more active in the mouse model than the initial antimicrobial peptide DK5.
  • the DK5-PEG- DAL a positive effect on TNF-a cytokine level reduction was observed, but its effect on NO concentration was comparable to the values obtained for the native DK5 peptide (Fig. 7 and Table 2).
  • Table 2 The values of the inhibition coefficient (IC50) of macrophages stimulation induced by LPS.
  • phagocytes were stimulated with endotoxin in the presence of DAL-PEG-DK5 (concentrations as above).
  • the level of the released TNF-a and NO was measured 20 hours after stimulation.
  • the results showed, that the release of TNF-a and NO was significantly inhibited when the DAL-PEG-DK5 was administered simultaneously with LPS or when cells were pre-incubated with the conjugate before the addition of LPS.
  • No anti inflammatory effect was observed for DAL-PEG-DK5, when the peptide was added to the cells after their previous LPS pre-stimulation (Fig. 8 A,B).
  • the TLR4 receptor is a transmembrane protein belonging to the TLR receptor family and is present on the surface of many cells of the immune system (neutrophils, macrophages, dendritic cells, B-cells, monocytes, mast cells). TLR4 is a receptor that recognizes lipopolysaccharide. Activation of TLR4 receptor requires e.g. extracellular cooperation of the MD2 protein. Under the influence of LPS, the TLR4/MD2 complex is dimerized, which is the initial stage in the cascade activating the nuclear factor NF-kB.
  • NF-kB is a transcription factor, which after penetration into the cell nucleus induces expression of genes encoding proinflammatory cytokines.

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Abstract

The subject matter of invention relates to peptide having the formula (I): Tyr-ala-GIy-Phe-Leu-Arg-O2Oc-lle-Lys-Lys-lle-Leu-Ser-lys-lle-Lys-Lys-Leu-Leu-NH2 and peptide having the formula (II): lle-Lys-Lys-lle-Leu-Ser-lys-lle-Lys-Lys-Leu-Leu- 020c-Tyr-ala-Gly-Phe-Leu-Arg- NH2 for use in the prevention of inflammation.

Description

Peptides for use in prevention and treatment of inflammation
The present invention relates to peptides for medical use in limiting excessive local inflammation caused by bacterial infections, as well as a systemic inflammatory response, such as sepsis, inflammation, accompanied by an increase in the inflammatory mediators, including cytokines, reactive nitrogen species. The present invention relates to peptide- conjugates for use in the prevention and treatment of an inflammation.
Background Art
The human system is permanently exposed to pathogenic microorganisms, as a result of which, in the course of the evolution it has developed a number of efficient mechanisms preventing and fighting infections. However, in many cases the human system is unable to eradicate a pathogen and curb an infection, which may result in an excessive development of an inflammation into a systemic one. Therefore, one seeks efficient pharmaceuticals preventing uncontrolled development of inflammation.
The initial response of the immune system to an inflammation caused by e.g. an infection is activation of the immunocompetent cells (macrophages, monocytes, neutrophils, dendritic cells). The factor activating an immune response are the so-called pathogen-associated molecular patterns - PAMP, including e.g. lipid A (a component of lipopolysaccharide (LPS)), peptidoglycan (PGN) or lipotechoic acid (LTA). The PAMP structures are recognised by receptors on the surface of immune cells, the so-called pattern recognition receptors - PRR, including Toll-like receptors - TLR. Recognition of antigens by TLR triggers a cascade of biochemical reactions, as a result of which proinflammatory cytokines are released, such as interleukin 1-a and b (IL-loc, IL-Ib) interleukin 6, 8, 10 (IL-6, IL-8, IL-10), tumour necrosis factor a (TNF-a), which is described in Mogensen, T. H. (2009). "Pathogen recognition and inflammatory signalling in innate immune defenses." Clin Microbiol Rev 22(2): 240-273. These factors are also released in the case of inflammations caused by factors other than bacteria. Small concentration in the system of these inflammatory mediators, which are released as a response to microbe components (PAMP), activates a correct response of the immune system, neutralising the infection. However, the effect of an abrupt growth in the number of pathogens is an excessive systemic inflammatory response aimed at a quick suppression of the infection. This results in a release of a bigger number of pathogenic factors formed as a result of decomposition of bacteria and in further activation of an inflammation. This leads to a septic shock. In such case, excessive stimulation of the immune system may have a lethal effect or cause permanent damage to internal organs. Therefore, in the event of a sepsis it is important not only to immediately administer an antibiotic, but also medicines neutralising the activity of inflammatory mediators or blocking the cell receptors for such mediators.
Inventors of the Faculty of Chemistry of the University of Gdahsk has been working on designing and synthesising biologically active peptides for many years. The publication entitled "Synthesis and Evaluation of Biological Activity of Antimicrobial--Pro- Proliferative Peptide Conjugates," PLoS One 10(10): e0140377 presents the results of experiments with analysing the antimicrobial and pro-proliferative properties of developed peptide conjugates consisting of selected antimicrobial peptides - AMP and peptides having other properties, including those stimulating the wound healing processes, where, among other things, the activity of the DAL-PEG-DK5 and DK5-PEG-DK5 conjugates was analysed. The said conjugates are composed of two amino acid sequences corresponding to the following peptides: dalargin (DAL) and DK5, covalently bound to each other by means of a PEG linker (the molecule (or moiety) of the 8-amino-3 6- dioxaoctanoic acid). An in vitro model for these compounds has confirmed the activity impeding the development of gram-positive and gram-negative bacteria and Candida yeasts. The said publication describes only an analysis of the impact of compounds on an growth of microbes and the influence of conjugates on the proliferation and migration of human keratinocytes and fibroblasts. The publication does not analyse the anti inflammatory properties of conjugates, whose mechanism differs from the mechanism determining the antimicrobial properties of conjugates, and no anti-inflammatory properties arise from the described analysis.
Dalargin (Tyr-o/a-Gly-Phe-Leu-Arg-OH), hereinafter referred to as DAL, is a synthetic analogue of the endogenic opioid peptide of Leu-enkephalin. The physiological activity of DAL in peripheral tissues results from the interaction with m and 5 - opioid receptors, which is described in an article by Penchev, Pospisek and co. "Activity profiles of Dalargin and Its Analogues in Mu-, delta- and Kappa-Opioid Receptor Selective Bioassays." Br J Pharmacol 128(3): 569-576 (1999).
DAL in its native form (not conjugated with a nano-molecular carrier) does not cross the blood-brain barrier. In (Grigorevskii, Korotkina and co. 1989 "[Dalargin in the prevention of acute postoperative pancreatitis]." Klin Khir(ll): 9-12.; Georgadze, Fomin i wsp. 1990 [Treatment of peptic ulcer hemorrhage with leucine enkephalin analog dalargin]." Khirurgiia (Mpsk)(3): 39-43; Grekova, Reznikov i wsp. 1994 "[The effect of dalargin on the course of experimental cardiac arrhythmias]." Eksp Klin Farmakol 57(2): 24-26.; Dontsov 2015" [The Antioxidant Effect of Dalargin in Patients with Coronary Heart Disease and Metabolic Syndrome]." Eksp Klin Farmakol 78(7): 3-6) it was disclosed that this compound inhibits the release of digestive enzymes, relieves pain, reduces swelling after pancreatic surgery, regulates the heart beats and shows a local antioxidation activity in the case myocardial infarction. However, dalargin was not disclosed as being capable of reducing inflammation, including that caused by a bacterial infection.
Dalargin is known as an efficient medicine for the treatment of the ulcer disease or acute pancreatitis. Administered by means of a drip bag or intravenously, the medicine decreased pancreas swelling and relived pain. The patent RU 2 341 282 discloses a mixture of octreotide (0.1 mg/kg) and dalargin (0.8 and 0.9 mg/kg) capable of impeding the enzymatic activity of the pancreas.
The peptide DK5 (lle-Lys-Lys-lle-Leu-Ser-/ys-lle-Lys-Lys-Leu-Leu-NH2) is a synthetic analogue of a naturally occurring temporin - ICEb, an agent obtained from the skin mucus of the Rana amurensis and Rana dybowskii frogs. The publication by Shang, D., X. Li and co. entitled "Design of Potent, Non-Toxic Antimicrobial Agents Based Upon the Structure of the Frog Skin Peptide, Temporin-lCEb From Chinese Brown Frog, Rana Chensinensis." Chem Biol Drug Des (2012) 79(5): 653-662 revealed that the agent displays a high activity impeding an increase in the number of gram-negative and gram-positive strains, and a low haemolytic activity relative to human erythrocytes.
The above research does not indicate that the said agent has anti-inflammatory properties. Disclosure of Invention
In the course of an experimental research conducted by the research team of the Faculty of Chemistry of the University of Gdansk (dr inz. Paulina Kosikowska-Adamus, prof, dr hab. Adam Lesner) and the team of researchers from the Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University (dr hab. Joanna Koziet, prof. UJ, dr Anna Golda), it unexpectedly turned out that the DAL-PEG-DK5 and DK5-PEG-DAL conjugates are capable of preventing a pathological inflammation and fighting an inflammation, including systemic one as sepsis induced by bacterial infection. The efficiency of the agents was monitored by means of evaluating the level of the inflammatory mediators, including cytokines, reactive nitrogen species, and was confirmed through in vitro ( DAL-PEG-DK5 and DK5-PEG-DAL) and in vivo (DAL-PEG-DK5 exclusively) models. The research was conducted on a model of an inflammation caused by a bacterial endotoxin - LPS. These results found confirmation in an in vitro model using peripheral blood infected by E. coli bacteria, which proves that the observations made using LPS are proper and reflects clinical conditions observedduring systemic infections. The conducted experimental researches, hence, confirm the efficiency of the agents in decreasing the level of inflammatory mediators, especially in the case of an inflammation caused by a Gram-negative bacteria infection.
A systemic inflammation is understood as an inflammation caused e.g. by an infection accompanied by activation of innate immunity Toll-like receptors - TLR and/or release of pro-inflammatory cytokines (incl. TNF-ot, IL-6, IL-8) and formation of reactive nitrogen species (incl. nitrogen oxide - NO) and other inflammatory mediators.
The subject matter of the invention is a peptide - compound - having the following formula 1:
Tyr-o/o-Gly-Phe-Leu-Arg-020c-lle-Lys-Lys-lle-Leu-Ser-/ys-lle-Lys-Lys-Leu-Leu-NFl2 for medical use in prevention of inflammation, particularly for use in preventing a systemic inflammatory response, including prevention of sepsis. The present invention relates especially to medical use in prevention of inflammation caused by a bacterial infection. The said medical use relates both to humans and animals. The present invention also relates to a peptide - compound - having the following formula 1:
Tyr-a/a-Gly-Phe-Leu-Arg-020c-!le-Lys-Lys-lle-Leu-Ser-/ys-lle-Lys-Lys-Leu-Leu-IMH2 for medical use in the treatment of inflammation, especially in the treatment of a systemic inflammatory response, including sepsis. The present invention relates especially to a medical use in the treatment of an inflammation caused by a bacterial infection. The said use relates both to humans and animals.
The present invention also relates to a peptide - compound - having the following formula 2:
lle-Lys-Lys-lle-Leu-Ser-/ys-lle-Lys-Lys-Leu-Leu- 020c-Tyr-a/a-Gly-Phe-Leu-Arg- NH2 for medical use in prevention of inflammation, especially in the prevention of a systemic inflammatory response, including sepsis. The present invention relates especially to a medical use in an inflammation caused by a bacterial infection. The said use relates both to humans and animals.
The present invention also relates to a peptide - compound having the following formula 2:
lle-Lys-Lys-lle-Leu-Ser-/ys-lle-Lys-Lys-Leu-Leu- 020c-Tyr-a/a-Gly-Phe-Leu-Arg- NH2 for medical use in the treatment of inflammation, especially in the treatment of a systemic inflammatory response, including sepsis. The said use relates especially to the treatment of an inflammation caused by a bacterial infection.
The anti-inflammatory activity of conjugates was examined using the cell line of murine macrophages (RAW 264.7) and primary human macrophages (monocyte derived macrophages, hMDMs), stimulated by a bacterial lipopolysaccharide (LPS) isolated from the cells of strain Escherichia coli bacteria. Based on the obtained results it was concluded, that the DAL-PEG-DK5 conjugate effectively decreases the level of expression of the inflammatory mediators TNF-a, IL-6, IL-8, and reduces the formation of reactive nitrogen (NO) species accompanying a nitrosative cell stress. The anti-inflammatory activity of DK5-PEG-DAL was lower compared with DAL-PEG-DK5. Importantly, however, single DAL and DK5 peptides displayed no anti-inflammatory properties, as in the majority of the experiments they didn't impact the level of the cytokines released or the reactive nitrogen species. The in vivo experiments using the mouse model of LPS-induced septic shock were conducted using the most active conjugate derivative - DAL-PEG-DK5. These experiments proved the effectiveness of DAL-PEG-DK5 in neutralising the effects caused by the LPS toxin. An intraperitoneal injection - administration LPS from E. coli in the concentration of 0.1 mg/g resulted in a 90% death-rate of mice within 26 hours. While administration of LPS together with the studied DAL-PEG-DK5 conjugate (25 mg/ml) resulted in 100% survival of mice and a reduced level of pro-inflammatory mediators (IFN- g, MCP-1) measured in the blood serum. Detailed analysis of the results are presented in the description of the examples and embodiments.
The results of the researches have confirmed that DAL-PEG-DK5 conjugate is capable to reduce significantly the level of the released pro-inflammatory cytokines in response to the presence of bacterial antigens - toxins (LPS, LTA), the cell wall components - PGN (peptidoglycan). Moreover, it was concluded that the anti-inflammatory effect for DAL- PEG-DK5 agent results from the ability of direct neutralization of a bacterial endotoxin, which loses its ability to activate the host's cells this way.
The results of in vitro also confirm the anti-inflammatory efficiency of both conjugates - DAL-PEG-DK5 and DK5-PEG-DAL - in reduction of the level of releasing pro-inflammatory cytokines and other inflammatory mediators.
Terms and abbreviations used in the description have the following meaning:
AMP - antimicrobial peptides
DCM-dichlorometan
DIPEA- N,IM-Diisopropylethylamine
DMF- dimethylformamide
hMDM - human monocyte derived macrophages
HPLC - high-performance liquid chromatography
IC50- concentration inhibiting activity/biological process etc. in 50% relative to control
IFN-g - interferon g
IL - interleukin
LPS- lipopolysaccharide
LTA - Lipoteichoic acid MCP-1 - macrophage chemoattractant protein-1
MOI- multiplicity of infection
NF-kB - nucleus transcription factor kappa B
TFA - trifluoroacetic acid
TLR-toll- Toll-like receptors
TNF-a tumour necrosis factor a
PAMP - pathogen-associated molecular patterns
PEG- 8-amino-3,6-dioxa-octanoic acid
PGN-peptidoglycan.
The formula 1 and formula 2 present the structure of the compounds - peptides - according to the invention, in configuration, position in compounds, from item 1 to 19.
Tyr-a/a-Gly-Phe-Leu-Arg-020c-lle-Lys-Lys-lle-Leu-Ser-/ys-lle-Lys-Lys-Leu-Leu-l\IH2
Figure imgf000009_0001
Formula 1 - DAL-PEG-DK5
lle-Lys-Lys-lle-Leu-Ser-/ys-lle-Lys-Lys-Leu-Leu- 020c-Tyr-o/o-Gly-Phe-Leu-Arg- NH2
Figure imgf000009_0002
Formula 2 - DK5-PEG-DAL Brief Description of Drawings
The present invention is described in more detail in examples and the following figures fig.1-fig.10, where fig.l-fig.4 present a confirmation of manufacture of conjugates, while fig.5-fig.10 present the efficiency of their activity according to the medical use.
Examples
Example 1
Chemical synthesis of the DAL-PEG-DK5 and DK5-PEG-DAL conjugates
The DAL-PEG-DK5 and DK5-PEG-DAL conjugates were obtained by means of solid phase method of synthesis applying Fmoc (fluorenyl-9-methoxycarbonyl) chemistry. To this, TentaGel S RAM (Rapp Polymere GmbH, Germany) polymer resin was used as a solid support with a substitution degree of 0.24 mmol/g. Both compounds were synthesized manually.
The DAL-PEG-DK5 and DK5-PEG-DAL synthesis consist of the following stages:
1. Removal of the Fmoc protecting group from the the amino group of the TentaGel S RAM resin by shaking with a 20% solution of piperidine in DMF. Rinsing the resin three times using: DMF, isopropanol, DCM, respectively.
2. Incorporation of the first Fmoc-protected amino acid residue. In the case of DAL- PEG-DK5, a solution of Fmoc-Leu-OH in DMF was added to the reaction vessel containing the activated for coupling (after step 1) TentaGel S RAM resin, for DK5-PEG-DAL-Fmoc- Arg(Pbf)-OH in DMF. Furthermore, agents activating carboxylic groups of F-moc protected amino acid residues - HOBt/HBTU and DIPEA were added to the solution. The molar ratio of particular components of the reaction mixture was (1:1: 1:2), i.e. 1 mol of the protected amino acid: lmol HOBt:l mol HBTU: 2 DIPEA moles. 3-times the excess of reagents was used relative to the degree of the level of resin substitution. The acylation reaction was performed by means of shaking the resin with the reaction mixture for 1.5 hours in room temperature. Following 3-time rinsing of the resin (DMS/isopropanol, DMC), the reaction was repeated. The progress of the acylation reaction was controlled by means of the colorimetric chloranil test detecting free amino groups on the surface of the resin grain.
3. The deprotection of the amino acid residue covalently attached - bound to the resin was conducted as specified in item 1, using a 20% piperidine in DMF and rinsing the resin three times by means of DMF, isopropanol, DCM following deprotection.
4. The incorporation of the subsequent F-moc protected amino acid residue was performed according to the same procedure as that described in item 2. Subsequently, the removal of the Fmoc protective group of the incorporated residue was performed according to the procedure described in item 1 or 2
The order of incorporating the amino acid residues during the synthesis of DAL-PEG-DK5 and DK5-PEG-DAL is shown in Table 1
Table 1. The list and order of incorporation of the amino acid residues in the course of the synthesis of the DAL-PEG-DK5 and DK5-PEG-DAL conjugates.
Figure imgf000011_0001
Figure imgf000012_0001
5. Following incorporation of the last residue from the amino acid sequence (item 19, Table 2) and removing the Fmoc protection, the DAL-PEG-DK5 and DK5-PEG-DAL peptides were removed from the resin with simultaneous removal of side-chain protecting groups by means of the TFA/Fenol/Triisopropylosilane/H20 (88:5:2:5. v/v) mixture.
The raw peptide preparation was then purified by Reverse Phase High Performance Liquid Chromatography (RP-HPLC) in order to obtain homogeneous preparations of the DAL- PEG-DK5 and DK5-PEG-DAL conjugates.
The molecular weight of the conjugates was confirmed by means of Matrix-Assisted Laser Desorpotion Ionisation-Time of Flight Mass Spectrometry.
The mass spectra for DAL-PEG-DK5 and DK5-PEG-DK5 (Fig. 1,2) presented in the application indicate the presence of a dominant ion with a mass-to-charge ratio (m/z) of 2277.9 Da, which is consistent with the theoretical molecular mass calculated for the conjugates, respectively: 2277.9 Da for DAL-PEG-DK5 and 2276.9 Da for DK5-PEG-DAL, for which the extra charge in the mass spectrum results from the presence of a pseudo- molecular ion [M+H]+. The homogeneity of the final preparations was examined using the high-performance reversed-phase liquid chromatography (RP-FIPLC) technique. The compounds were separated in a gradient of 1-80% of phase B in phase A for 30 min at 1 ml/min flow. Phase A: 0.1% TFA solution in water, phase B: 80% acetonitrile solution in phase A. Detection was performed at 226 nm. The Shimadzu HPLC system and the Phenomex Jupiter 4pm Proteo 90A (250 x 4.6 mm) column were used for the analysis. The chromatograms presented in the application (Fig. 3,4) represent the analyte fraction corresponding to the DAL-PEG-DK5 and DK5-PEG-DAL conjugates with specified retention times, respectively: 22.735 min. and 19.767 min.
The confirmation of chemical identity and homogeneity of DAL-PEG-DK5 and DK5-PEG- DAL preparations is shown in fig.1-fig.4.
Example 2
Examination of the anti-inflammatory properties of DAL-PEG-DK5 and DK5-PEG-DAL conjugates - in vitro confirmation of preventive and therapeutic activity of a local and systemic inflammation
I. In vitro experiments:
1. Effects of DAL-PEG-DK5 and DK5-PEG-DAL conjugates on TNFa- cytokine release and reactive forms of nitrogen (NO) in the mouse macrophages model in the presence of LPS, LTA and PGN
The aim of the experiments was to determine the effect of DAL-PEG-DK5 and DK5-PEG- DAL on the reduction of the level of pro-inflammatory response of the mouse macrophages (RAW 264.7) in the presence of LPS; LTA and PGN. The activity of conjugates in this range was compared against the activity of their components (DK5, DAL). The cells were stimulated accordingly: 10 ng/ml LPS, 10 pg/ml LTA or 2 mg/ml PGN in the presence of the tested compounds at 25 pg/ml or without peptides. After 20 hours, the expression of TNF-a cytokine and the concentration of nitric oxide (NO) released were measured by ELISA or Griess tests, respectively. On the basis of the obtained results it was found that the DAL-PEG-DK5 conjugate is characterized by the best (in comparison to the results obtained for DAL and DK5) ability to inhibit the expression of inflammatory response markers (the lowest concentration of inflammatory TNF-a, NO mediators) caused by selected TLR receptor agonists (Fig. 5). The activity of the DK5-PEG-DAL conjugate in the presence of LPS was comparable for the values obtained for DAL-PEG-DK5 (Fig. 5, LPS panel), however, it was significantly lower (at the control sample level, without the tested compounds) in the presence of LTA and PGN, comparable with the values obtained for DAL and DK5.
2. Effects of DAL-PEG-DK5 and DK5-PEG-DAL on the level of proinflammatory cytokine release in human macrophage model in the presence of LPS and suspension of
Escherichia coli bacteria
The level of macrophages activation was determined by measuring the concentration of TNF-a, IL-6, IL-8 and IL-10 cytokines released by cells stimulated with LPS (10 ng/ml). The analysis was performed 20 hours after stimulation with bacterial endotoxin. The experiments were conducted with the use of human monocyte-derived macrophages (hMDM) to which the tested compounds were added at a concentration of 10 pg/ml. The obtained results presented in Fig. 6 clearly show that DAL-PEG-DK5 and DK5-PEG-DAL peptides significantly inhibit the expression of proinflammatory cytokines (TNF-a, IL-6, IL- 8, IL-10) (Fig. 6A). Additionally, the influence of peptides on the level of expression of the TNF -a cytokine in full peripheral blood stimulated with LPS (10 ng/ml) and in cell culture of E. co//-infected macrophages (MOI 1:50; MOI - multiplicity of infection) was investigated. In both cases, the DAL-PEG-DK5 conjugate most effectively inhibited the release of the TNF- a cytokine. The activity of DK5-PEG-DAL conjugate in these experiments was lower, but better than in the case of free DAL or DK5 (Fig. 6 B,C).
The obtained results clearly indicate, that DAL-PEG-DK5 conjugate effectively reduces the level of expression of inflammatory markers in primary human macrophages in the presence of LPS and E. coli infection. Moreover, DAL-PEG-DK5 conjugate effectively neutralizes endotoxin in an environment resembling physiological conditions - an experiment conducted with the use of whole human blood. 3. Determination of the IC50 inhibition coefficient for the expression of inflammatory mediators (TNF-a and NO) for DAL-PEG-DK5 and DK5-PEG-DAL conjugates and their components
In order to determine the anti-inflammatory effect of individual compounds, their concentration that cause 50% inhibition of LPS-induced macrophage stimulation were established in the meaning of inhibitory concentration of 50% (IC50) of macrophages stimulation caused by LPS was determined. Human (hMDM) and mouse (RAW 264.7) macrophages were incubated in the presence of LPS (10 ng/ml) or a mixture of LPS (10 ng/ml) with individual peptides (analysed concentration range from 2.5 to 50 pg/ml). After 20 hours the concentration of the TIMF-cr cytokine and NO level in cellular supernatants was analysed. The obtained results confirmed, that the DAL-PEG-DK5 peptide is 9 times more active in the human macrophage model and 5 times more active in the mouse model than the initial antimicrobial peptide DK5. in case of the DK5-PEG- DAL a positive effect on TNF-a cytokine level reduction was observed, but its effect on NO concentration was comparable to the values obtained for the native DK5 peptide (Fig. 7 and Table 2).
Table 2: The values of the inhibition coefficient (IC50) of macrophages stimulation induced by LPS.
ICSe [m§M] MUDMs RAW264
DK5 18,68 17.70
DAL no * no
DK5-PEG-DAL 15.58 19-83
DAL-PEG-DK5 2.015 3.Q9E
SCU ** no no
* no - not determined
** SCR - scrambled peptide, a peptide of mixed type designed on the basis of DAL- PEG-DK5 sequence. In experiments it serves as a control, confirming the relationship between the peptide sequence and its biological activity. The activity of the DK5-PEG-DAL compound was confirmed in in vitro tests - its influence on the decrease of LPS-induced release of proinflammatory cytokines TNF- a, IL-6, IL-10 was found in the concentration of 10 pg/ml.
4. Determination of potential mechanism of LPS neutralization by DAL-PEG-DK5 a. Blocking direct interaction of LPS with the surface of macrophage cells In order to determine whether the most active form of DAL-PEG-DK5 conjugate prevents macrophages from recognising LPS, an experiment was performed in which hMDM and RAW 264.7 cells were pre-incubated for 2 hours with LPS, followed by the addition of DAL-PEG-DK5 at 10 pg/ml for hMDM or 25 pg/ml for RAW 264.7. Alternatively, both macrophage lines were pre-incubated in the presence of the peptide added at the concentrations as above and then the cells were stimulated with LPS. In the third variant, phagocytes were stimulated with endotoxin in the presence of DAL-PEG-DK5 (concentrations as above). The level of the released TNF-a and NO was measured 20 hours after stimulation. The results showed, that the release of TNF-a and NO was significantly inhibited when the DAL-PEG-DK5 was administered simultaneously with LPS or when cells were pre-incubated with the conjugate before the addition of LPS. No anti inflammatory effect was observed for DAL-PEG-DK5, when the peptide was added to the cells after their previous LPS pre-stimulation (Fig. 8 A,B). These results suggest that DAL- PEG-DK5 prevents LPS interactions with the surface of macrophage cells. This hypothesis was further confirmed in flow cytometry experiments, in which fluorescent labelled LPS (AF488-LPS) was incubated with RAW 264.7 macrophages (5 x 105 cells/ml) without peptide or in the presence of DAL-PEG-DK5 at 25 pg/ml. The histogram shown in Fig. 8C shows that DAL-PEG-DK5 effectively blocks LPS interactions with macrophage surface, because only 14% of the examined cell pool was LPS-positive (Fig. 8C). In the case of control cells (pre-stimulation of LPS without peptide), a positive fluorescence signal was obtained for 74% of macrophages. b. Blocking of TLR4/MD2 complexes and NF-kB signal transduction pathway activation by DAL-PEG-DK5
The TLR4 receptor is a transmembrane protein belonging to the TLR receptor family and is present on the surface of many cells of the immune system (neutrophils, macrophages, dendritic cells, B-cells, monocytes, mast cells). TLR4 is a receptor that recognizes lipopolysaccharide. Activation of TLR4 receptor requires e.g. extracellular cooperation of the MD2 protein. Under the influence of LPS, the TLR4/MD2 complex is dimerized, which is the initial stage in the cascade activating the nuclear factor NF-kB.
To determine the effect of DAL-PEG-DK5 on the TLR4/MD2 complex dimerization process, specific anti-TLR4/MD2 antibodies binding exclusively to the monomeric form of the TLR4/MD2 complex were used. Cell selection performed using flow cytometry technique, showed that the presence of DAL-PEG-DK5 in macrophage suspension prevents LPS activation of TLR4 receptor at the stage of TRLR4/MD2 complex dimerization (Fig. 9A). The influence of DAL-PEG-DK5 conjugate on NF-kB activation was studied using RAW 263.7 macrophage reporter culture. NF-kB is a transcription factor, which after penetration into the cell nucleus induces expression of genes encoding proinflammatory cytokines. The results of the experiment showed that DAL-PEG-DK5 conjugate 6 h after stimulation of LPS cells significantly inhibits NF-kB activation. This effect does not occur in the case of mixed DAL-PEG-DK5 (SCR - scrambled peptide) (Fig. 9B).
II. In vivo experiments - examination of the properties in prevention and treatment of systemic sepsis
Effect of DAL-PEG-DK5 conjugate on survival of C57BL/6 mice sensitized with D- galactosamine (D-GaIN) in LPS-induced septic shock conditions. LPS at 0.1 pg/ml was administered intraperitoneally. The results of the experiment show that administration of LPS alone caused 90% lethal effect, whereas simultaneous administration of LPS and DAL- PEG-DK5 at 25 mg/ml caused survival of all mice from this group (Fig. 10A). The results of the in vivo experiment correspond to the results of in vitro studies, where it was proved that simultaneous administration of LPS and DAL-PEG-DK5 significantly reduces the level of the TNF-a cytokine release and reactive forms of nitric oxide (NO) (Fig. 8A,B). The analysis of cytokine levels in serum collected from mice administered with endotoxin simultaneously with DAL-PEG-DK5 showed a decrease in the level of IFN-y, MCP-1 (monocyte chemoattractant protein -1, chemotactic protein of monocytes) pro- inflammatory cytokines. In contrast, the level of IL-6 and IL-10 cytokines increased and the TNF-a concentration remained unchanged. The above data confirm the potential effectiveness of the DAL-PEG-DK5 peptide conjugate in neutralisation of LPS endotoxin and prevention of complications accompanying septic shock (Fig. 10B).

Claims

Claims
1. Peptide having the formula 1:
Tyr-a/a-Gly-Phe-Leu-Arg-020c-lle-Lys-Lys-lle-Leu-Ser-/ys-lle-Lys-Lys-Leu-Leu-NH2 for use in the prevention of inflammation.
2. Peptide having the formula 1 for use according to claim 1, wherein the peptide is used in the prevention of systemic inflammatory response.
3. Peptide having the formula 1 for use according to claim 2, wherein the peptide is used in the prevention of sepsis.
4. Peptide having the formula 1 for use according to claims 1-3, wherein the peptide is used in the prevention of an inflammation caused by a bacterial infection.
5. Peptide having the formula 1:
Tyr-a/a-Gly-Phe-Leu-Arg-020c-lle-Lys-Lys-lle-Leu-Ser-/ys-lle-Lys-Lys-Leu-Leu-NH2 for use application in the treatment of inflammation.
6. Peptide having the formula 1 for use according to claim 5, wherein the peptide is used in the treatment of a systemic inflammatory response.
7. Peptide having the formula 1 for use according to claim 6, wherein the peptide is used in the treatment of sepsis.
8. Peptide having the formula 1 for use according to claims 5-7, wherein the peptide is used in the treatment of inflammation caused by a bacterial infection.
9. Peptide having the formula 2:
lle-Lys-Lys-lle-Leu-Ser-/ys-lle-Lys-Lys-Leu-Leu- 020c-Tyr-o/o-Gly-Phe-Leu-Arg- NH2 for use in the prevention of inflammation.
10. Peptide having the formula 2 for use according to claim 9, wherein the peptide is used in the prevention of systemic inflammatory response.
11. Peptide having the formula 2 for use according to claim 8, wherein the peptide is used in the prevention of sepsis.
12. Peptide having the formula 2 for use according to claim 9-11, wherein the peptide is used in the prevention of an inflammation caused by a bacterial infection.
13. Peptide having the formula 2:
lle-Lys-Lys-lle-Leu-Ser-/ys-lle-Lys-Lys-Leu-Leu- 020c-Tyr-o/a-Gly-Phe-Leu-Arg- NH2 for use in the treatment of inflammation.
14. Peptide having the formula 2 for use according to claim 13, wherein the peptide is used in the treatment of systemic inflammatory response.
15. Peptide having the formula 2 for use according to claim 14, wherein the peptide is used in the treatment of sepsis.
16. Peptide having the formula 2 for use according to claim 13-15, wherein the peptide is used in the treatment of inflammation caused by a bacterial infection.
PCT/PL2019/050072 2018-12-06 2019-12-04 Peptides for use in prevention and treatment of inflammation WO2020117081A1 (en)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
A. GOLDA ET AL: "Conjugate of Enkephalin and Temporin Peptides as a Novel Therapeutic Agent for Sepsis", BIOCONJUGATE CHEMISTRY, vol. 29, no. 12, 10 December 2018 (2018-12-10), US, pages 4127 - 4139, XP055675177, ISSN: 1043-1802, DOI: 10.1021/acs.bioconjchem.8b00763 *
PAULINA KOSIKOWSKA ET AL: "Synthesis and Evaluation of Biological Activity of Antimicrobial - Pro-Proliferative Peptide Conjugates", PLOS ONE, vol. 10, no. 10, 16 October 2015 (2015-10-16), pages e0140377, XP055675172, DOI: 10.1371/journal.pone.0140377 *

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