WO2023089544A1 - Composition pharmaceutique comprenant une endolysine ou une artilysine et un agent anticancéreux - Google Patents

Composition pharmaceutique comprenant une endolysine ou une artilysine et un agent anticancéreux Download PDF

Info

Publication number
WO2023089544A1
WO2023089544A1 PCT/IB2022/061122 IB2022061122W WO2023089544A1 WO 2023089544 A1 WO2023089544 A1 WO 2023089544A1 IB 2022061122 W IB2022061122 W IB 2022061122W WO 2023089544 A1 WO2023089544 A1 WO 2023089544A1
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
cancer
pharmaceutical composition
amino acid
endolysin
Prior art date
Application number
PCT/IB2022/061122
Other languages
English (en)
Inventor
Markus Graf Matuschka Von Greiffenclau
Original Assignee
Sasinapas Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sasinapas Co., Ltd. filed Critical Sasinapas Co., Ltd.
Publication of WO2023089544A1 publication Critical patent/WO2023089544A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01017—Lysozyme (3.2.1.17)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00033—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2462—Lysozyme (3.2.1.17)

Definitions

  • the present invention relates to a pharmaceutical composition comprising an endolysin or an artilysin and an anti-cancer agent.
  • the present invention relates to a pharmaceutical composition according to this invention for use as a medicament and for use in the treatment of a proliferative disease or disorder.
  • Cancer is a leading cause of death worldwide, accounting for nearly 10 million deaths in 2020. According to the World Health Organization the most common in 2020 (in terms of new cases of cancer) were: breast cancer (2.26 million cases), lung cancer (2.21 million cases), colon and rectum cancer (1.93 million cases), prostate cancer (1.41 million cases), skin cancer (non-melanoma) (1.20 million cases) and stomach cancer (1.09 million cases). Therefor, investigation of new strategies of cancer diagnosis and therapeutics is needed.
  • protein refers synonymously to the term "polypeptide”.
  • protein refers to a linear polymer of amino acid residues linked by peptide bonds in a specific sequence.
  • the amino-acid residues of a protein may be modified by e.g. covalent attachments of various groups such as carbohydrates and phosphate.
  • Other substances may be more loosely associated with the polypeptide chains, such as heme or lipid, giving rise to the conjugated proteins which are also comprised by the term “protein” as used herein.
  • proteins There are various ways in which the polypeptide chains fold have been elucidated, in particular with regard to the presence of alpha helices and beta-pleated sheets.
  • protein refers to all four classes of proteins being all-alpha, all-beta, alpha/beta and alpha plus beta.
  • fusion protein refers to an expression product resulting from the fusion of two nucleic acid sequences. Such a protein may be produced, e.g., in recombinant DNA expression systems.
  • fusion protein refers to a fusion of a first amino acid sequence, in particular an endolysin, autolysin and/or other peptidoglycan hydrolase, with a second or further amino acid sequence.
  • the second or further amino acid sequence is preferably a peptide stretch, in particular a cationic and/or polycationic peptide.
  • said second and/or further amino acid sequence is foreign to and not substantially homologous with any domain of the first amino acid sequence.
  • amysin refers to a fusion protein comprising an endolysin and a peptide stretch, in particular a cationic and/or polycationic peptide.
  • peptide refers to short peptides consisting of from about 2 to about 100 amino acid residues, more preferably from about 4 to about 50 amino acid residues, more preferably to about 5 to 30 amino acid residues, wherein the amino group of one amino acid residue is linked to the carboxyl group of another amino acid residue by a peptide bond.
  • a peptide may have a specific function.
  • a peptide can be a naturally occurring peptide or a synthetically designed and produced peptide.
  • the peptide can be, for example, derived or removed from a native protein by enzymatic or chemical cleavage, or can be prepared using conventional peptide synthesis techniques (e.g., solid phase synthesis) or molecular biology techniques (see Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)).
  • Preferred synthetically produced peptides are e.g. cationic, polycationic, amphipathic or hydrophobic peptides.
  • Preferred naturally occurring peptides are e.g. antimicrobial peptides.
  • cationic peptide refers to a peptide having positively charged amino acid residues.
  • a cationic peptide has a pKa-value of 9.0 or greater.
  • at least four of the amino acid residues of the cationic peptide can be positively charged, for example, lysine or arginine.
  • “Positively charged” refers to the side chains of the amino acid residues which have a net positive charge at about physiological conditions.
  • cationic peptide refers also to polycationic peptides.
  • polycationic peptide refers to a synthetically designed and produced peptide composed of mostly positively charged amino acid residues, in particular lysine, arginine and/or histidine residues, more preferably lysine and/or arginine residues.
  • a peptide is composed of mostly positively charged amino acid residues if at least about 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95 or about 100 % of the amino acid residues are positively charged amino acid residues, in particular lysine and/or arginine residues.
  • the amino acid residues being not positively charged amino acid residues can be neutrally charged amino acid residues and/or negatively charged amino acid residues and/or hydrophobic amino acid residues.
  • the amino acid residues being not positively charged amino acid residues are neutrally charged amino acid residues, in particular serine and/or glycine.
  • AMP antimicrobial peptide
  • anti-bacterial peptide refers in particular to any peptide having anti-bacterial, anti-fungal, anti-mycotic, anti- parasitic, anti-protozoal, anti-viral, anti-infectious, anti-infective and/or germicidal, algicidal, amoebicidal, microbicidal, bactericidal, fungicidal, parasiticidal, protozoacidal, protozoicidal properties, in particular sushi peptides and defensin.
  • the antimicrobial peptide may be a member of the RNAse A super family, a defensin, cathelicidin, granulysin, histatin, psoriasin, dermicidine or hepcidin.
  • the antimicrobial peptide may be naturally occurring in insects, fish, plants, arachnids, vertebrates or mammals.
  • sushi peptide refers to complement control proteins (CCP) having short consensus repeats.
  • CCP complement control proteins
  • the sushi module of sushi peptides functions as a protein -protein interaction domain in many different proteins. Peptides containing a Sushi domain have been shown to have antimicrobial activities.
  • sushi peptides are naturally occurring antimicrobial peptides.
  • amhiphatic peptide refers to synthetic peptides having both hydrophilic and hydrophobic functional groups.
  • amphiphatic peptides may be e.g. alpha helical, having predominantly non polar side chains along one side of the helix and polar residues along the remainder of its surface.
  • hydrophobic group refers to chemical groups such as amino acid side chains which are substantially water insoluble, but soluble in an oil phase, with the solubility in the oil phase being higher than that in water or in an aqueous phase.
  • amino acid residues having a hydrophobic side chain interact with one another to generate a nonaqueous environment.
  • Examples of amino acid residues with hydrophobic side chains are valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, cysteine, alanine, tyrosine, histidine, threonin, serine, proline and glycine residues.
  • endolysin refers to an enzyme which is suitable to hydrolyse bacterial cell walls.
  • endolysin as used herein comprise naturally occurring endolysins, e.g. encoded by bacteriophages or bacterial viruses as well as recombinant endolysins.
  • the recombinant endolysins may be shuffled, i.e. composed of heterologous domains like CBDs or EADs derived from different endolysins.
  • Endolysins comprise at least one “enzymatically active domain” (EAD) having at least one of the following activities: endopeptidase, N-acetyl-muramoyl-L-alanine-amidase (amidase), N-acetyl-muramidase, N- acetyl-glucosaminidase (lysozyme) or transglycosylases.
  • EAD enzymatically active domain
  • the endolysins may contain also regions which are enzymatically inactive, and bind to the cell wall of the host bacteria, the so-called CBDs (cell wall binding domains).
  • CBDs cell wall binding domains.
  • the endolysin may contain one, two or more CBDs.
  • the term “endolysin” as used herein refers also to enzymes having at least one EAD but no CBDs.
  • the cell wall binding domain is able to bind different components on the surface of bacteria.
  • the cell wall binding domain is a peptidoglycan binding domain and binds to the bacteria’s peptidoglycan.
  • EAD refers to the enzymatically active domain of an endolysin.
  • the EAD is responsible for hydrolysing bacterial peptidoglycans. It exhibits at least one enzymatic activity of an endolysin.
  • the EAD can also be composed of more than one enzymatically active module.
  • EAD is used herein synonymously with the term “catalytic domain”.
  • cancer cells may have a slightly elevated surface content of negatively- charged immobilized molecules (e.g., sialic acid), which is 30%-50% more than normal cells, but this is hardly comparable to the elevated levels of glycolysis and lactate secretion, that is 30 times higher than normal cell levels (Dubyak 2004; Gadsby et al. 2009). Therefore, the negative charge generated on the cancer cells is due to different sugar metabolism compared to normal cells.
  • negatively- charged immobilized molecules e.g., sialic acid
  • Gram-positive and Gram-negative bacteria cells exhibit a negative charge of the cell surface.
  • Bacterial membranes are negatively charged with lipids such as phosphatidylglycerol, cardiolipin, or phosphatidyl serine.
  • the outer membrane of a Gramnegative bacteria is also negatively charged as it contains anionic lipopolysaccharides.
  • mammalian cell membranes consist largely of zwitterionic phospholipids (neutral in net charge) such as phosphatidylethanolamine, phosphatidylcholine or sphingomyelin.
  • Antimicrobials against Gram-positive and Gram-negative bacteria cells like endolysins and artilysins are positively charged and thus, the electrostatic interaction between these components plays a great role in the lysis process of bacteria.
  • Such electrostatic interaction may be used between negatively charged cancer cells and positively charged antimicrobials, like endolysins and artilysins.
  • Endolysins are peptidoglycan hydrolases encoded by bacteriophages (or bacterial viruses), however there are also recombinant endolysins which are engineered dependent on the desired functions. They are synthesized during late gene expression in the lytic cycle of phage multiplication and mediate the release of progeny virions from infected cells through degradation of the bacterial peptidoglycan. They are either B(l,4)-glycosylases (lysozymes), transglycosylases, amidases or endopeptidases. Antimicrobial application of endolysins was already suggested in 1991 by Gasson (GB2243611).
  • endolysins are built up from of at least one “enzymatically active domain” (EAD) having at least one of the following activities: endopeptidase, N-acetyl-muramoyl-L-alanine-amidase (amidase), N-acetyl- muramidase, N-acetyl-glucosaminidase (lysozyme) or transglycosylases.
  • EAD enzymatically active domain
  • the endolysins may contain also regions which are enzymatically inactive, and bind to the cell wall of the host bacteria, the so-called CBDs (cell wall binding domains).
  • LPS lipopolysachharide
  • Artilysins comprise an endolysin fused to a peptide with lipopolysachharide (LPS) or in general membrane disrupting activity.
  • LPS is a major component of the outer membrane of Gram-negative bacteria. It increases the negative charge of the cell membrane and protects the membrane from certain kinds of chemical attack. To a certain degree said LPS protects the membrane of Gram-negative bacteria also from endolysins added from outside of the bacteria.
  • the LPS can be disrupted by peptide stretches having a LPS disrupting activity as e.g. positively charged peptides.
  • said peptide stretches may be involved in the outer membrane protein transport mechanism, a destabilisation of structural outer membrane proteins and/or in lipid-dependent destabilisation.
  • a peptide stretch having LPS disrupting activity or in general membrane disrupting activity promotes the passage of an endolysin fused to said peptide stretch through the outer membrane of Gram-negative bacteria. After the promoted pass of the endolysin through the outer membrane of Gramnegative bacteria, the cell wall of the Gram-negative bacterium can be more easily be disrupted or desintegrated by the endolysin due to degradation of the peptidoglycan layer followed by osmotic lysis when the internal cell pressure of the bacterium cannot longer be resisted.
  • Gram-positive bacteria In contrast to Gram-negative bacteria, Gram-positive bacteria do not possess an outer membrane.
  • the cytoplasmic membrane is surrounded by an up to 25 nm thick layer of peptidoglycan (which is only up to 5 nm for Gram-negative bacteria) which forms the cell wall.
  • Main purpose of the cell wall of Gram-positives is to maintain bacterial shape and to counteract the internal bacterial cell pressure.
  • Peptidoglycan, or murein is a polymer consisting of sugars and amino acids.
  • the sugar component consists of alternating residues of P-(l,4) linked N-acetylglucosamine and N-acetylmuramic acid residues compose the sugar components.
  • a peptide chain of three to five amino acids is attached to the N-acetylmuramic acid.
  • the peptide chain can be cross-linked to the peptide chain of another strand forming a 3D mesh-like layer.
  • the peptide chain may contain D- and L- amino acid residues and the composition may vary for different bacteria.
  • the cell wall of Gram-positive bacteria composed of peptidoglycan may be disrupted by enzymes like endolysins and the EAD of artilysins representing peptidoglycan hydrolases.
  • an endolysin or artilysin is engineered, i.e. the specific EAD and CBD as well as a peptide with LPS disrupting or membrane disrupting activity with the desired activity is chosen and combined.
  • This specificity of endolysins and artilysins for bacteria may be transferred to the different cancer cell types and thus, enables an engineering of endolysins or artilysins with specific targeting activity for specific cancer cell types.
  • the selectivity of the endolysins and artilysins for cancer cells is based on the opposite charge, i.e. a negativ charge of cancer cells compared to healthy mammalian cells consisting largely of zwitterionic phospholipids (neutral in net charge).
  • Endolysins and artilysins are thus selected or engineered to bind and lysate Gram-negative or Gram-positive bacteria cells.
  • the artilysins according to the present invention may be engineered to target different, specific cancer cells and thus, function as vehicle for anti-cancer agents which may be delivered to the specific target site
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an endolysin or an artilysin and an anti-cancer agent.
  • the endolysin or the endolysin part of the artilysin according to the present invention is preferably encoded by bacteriophages specific for Gram-negative bacteria such as Gramnegative bacteria of bacterial groups, families, genera or species comprising strains pathogenic for humans or animals like Enterobacteriaceae (Escherichia, especially E. coli, Salmonella, Shigella, Citrobacter, Edwardsiella, Enterobacter, Elafnia, Klebsiella, especially K. pneumoniae, Morganella, Proteus, Providencia, Serratia, Yersinia), P seudomonadaceae Pseudomonas, especially P.
  • Enterobacteriaceae Esscherichia, especially E. coli, Salmonella, Shigella, Citrobacter, Edwardsiella, Enterobacter, Elafnia, Klebsiella, especially K. pneumoniae, Morganella, Proteus, Providencia, Serratia, Yersinia
  • aeruginosa Burkholderia, Stenotrophomonas, Shewanella, Sphingomonas, Comamonas), Neisseria, Moraxella, Vibrio, Aeromonas, Brucella, Francisella, Bordetella, Legionella, Bartonella, Coxiella, Haemophilus, Pasteurella, Mannheimia, Actinobacillus, Gardnerella, Spirochaetaceae (Treponema and Borrelia), Leptospiraceae, Campylobacter, Helicobacter, Spirillum, Streptobacillus, Bacteroidaceae (Bacteroides, Fusobacterium, Prevotella, Porphyromonas), Acinetobacter, especially A. baumanii.
  • the endolysin or the endolysin part of the artilysin is encoded by bacteriophages specific for Gram-positive bacteria such as Gram-positive bacteria of bacterial groups, families, genera or species comprising strains pathogenic for humans or animals, in particular of the phylum Actinobacteria, in particular of the class Actinobacteridae, in particular of the order Actinomycetales, in particular of the families Actinomycineae: Actinomycetaceae (Actinomyces, Mobiluncus), Corynebacterineae : Mycobacteriaceae (Mycobacterium), Nocardiaceae, Corynebacteriaceae, Frankineae: Frankiaceae, Micrococcineae: Brevibacteriaceae and Propionibacteriaceae
  • bacteriophages specific for Gram-positive bacteria such as Gram-positive bacteria of bacterial groups, families, genera or species comprising strains pathogenic for humans or animals, in particular of the
  • Bacilli Bacilli
  • Bacillales Bacillaceae (Bacillus), Listeriaceae (Listeria), Staphylococcaceae (Staphylococcus, Gemella, Jeotgalicoccus) and of the order Lactobacillales, in particular of the families: Enterococcaceae (Enterococcus), Lactobacillaceae (Lactobacillus, Pediococcus), Leuconostocaceae (Leuconostoc), Streptococcaceae (Lacto
  • the endolysins or the artilysin according to the present invention comprise modifications and/or alterations of the amino acid sequences.
  • Such alterations and/or modifications may comprise mutations such as deletions, insertions and additions, substitutions or combinations thereof and/or chemical changes of the amino acid residues, e.g. biotinylation, acetylation, PEGylation, chemical changes of the amino-, SH- or carboxyl-groups.
  • the peptide of the artilysin according to the present invention may be linked to the endolysin part by additional amino acid residues e.g. due to cloning reasons.
  • said additional amino acid residues may be not recognized and/or cleaved by proteases.
  • said peptide may be linked to the enzyme by at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid residues.
  • the peptide fused on the N-terminus of the endolysin part of the artilysin according to the invention further comprises additional amino acids on its N- terminus.
  • the peptide comprises the amino acid methionine (Met), or methionine, glycine and serine (Met-Gly-Ser) or alanine, methionine and glycine (Ala-Met-Gly).
  • the peptide is linked to the N-terminus of the endolysin part by the additional amino acid residues, in particular glycine and serine (Gly-Ser).
  • the peptide is linked to the C-terminus of the endolysin part by the additional amino acid residues, in particular glycine and serine (Gly-Ser).
  • the peptide with membrane and/or LPS disrupting activity comprises a positively charged peptide, which comprises one or more of the positively charged amino acids being lysine, arginine and/or histidine.
  • a positively charged peptide which comprises one or more of the positively charged amino acids being lysine, arginine and/or histidine.
  • more than 80%, preferably more than 90%, preferably 100% of the amino acids in said peptide are positively charged amino acids.
  • the cationic peptide is fused at the N-terminal and/or the C-terminal end of the artilysin, thus enhancing the cationicity of the latter proteins.
  • the artilysin comprises an endolysin and a peptide fused thereto said peptide comprising about 3 to about 50, more preferably about 5 to about 20, for instance about 5 to about 15 amino acid residues and at least 20, 30, 40, 50, 60 or 70%, more preferably at least 80%, for instance at least 90% of the said amino acid residues are either arginine or lysine residues.
  • the peptide of the artilysin is fused to the N-terminus and/or to the C-terminus of the endolysin.
  • said peptide is only fused to the N- terminus of the endolysin.
  • artilysins having a peptide both on the N-terminus and on the C-terminus. Said peptides on the N-terminus and on the C-terminus can be the same or distinct peptides.
  • the peptide of the artilysin according to the present invention is preferably covalently bound to the enzyme.
  • said peptide consists of at least 5, more preferably at least of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or at least 100 amino acid residues.
  • a peptide comprising about 5 to about 100 amino acid residues, about 5 to about 50 or about 5 to about 30 amino acid residues.
  • the peptide is selected from the group of cationic peptides, polycationic peptides, hydrophobic peptides, antimicrobial peptides and amphiphatic peptides.
  • the fused peptide is a cationic and/or polycationic peptide, which comprises one or more of the positively charged amino acid residues of lysine, arginine and/or histidine, in particular of lysine and/or arginine.
  • lysine arginine
  • histidine in particular of lysine and/or arginine.
  • more than about 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95 or 99 % of the amino acid residues in said peptide stretch are positively charged amino acid residues, in particular lysine and/or arginine residues.
  • peptides consisting of about 100 % positively charged amino acid residues, in particular arginine and/or lysine residues, wherein preferably about 60 % to about 70 % of said positively charged amino acid residues are lysine residues and about 30% to about 40 % of said positively charged amino acid residues are arginine residues.
  • Peptides consisting of either only arginine or only lysine are also preferred.
  • the cationic peptide comprises beside the positively charged amino acid residues, in particular lysine and/or arginine residues, neutrally charged amino acid residues, in particular glycine and/or serine residues.
  • the cationic peptides comprise beside the positively charged amino acid residues, in particular lysine and/or arginine residues, hydrophobic amino acid residues, in particular valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, cysteine, alanine, tyrosine, histidine, threonin, serine, proline and glycine residues, more preferably alanine, valine, leucine, isoleucine, phenylalanine, and/or tryptophan residues.
  • the peptide is an antimicrobial peptide comprising a positive net charge and around 50% hydrophobic amino acids.
  • the antimicrobial peptides are amphiphatic, with a length of about 12 to about 50 amino acid residues.
  • the antimicrobial peptides are naturally occurring in insects, fish, plants, arachnids, vertebrates or mammals.
  • the antimicrobial peptide may be naturally occurring in radish, silk moth, wolf spider, frog, preferably in Xenopus laevis, Rana frogs, more preferably in Rana catesbeiana, toad, preferably Asian toad Bufo bufo gargarizans, fly, preferably in Drosophila, more preferably in Drosophila melanogaster, in Aedes aegypti, in honey bee, bumblebee, preferably in Bombus pascuorum, flesh fly, preferably in Sarcophaga peregrine, scorpion, horseshoe crab, catfish, preferably in Parasilurus asotus, cow, pig, sheep, porcine, bovine, monkey and human.
  • the peptide is a sushi peptide which is described by Ding JL, Li P, Ho B Cell Mol Life Sci. 2008 Apr;65(7-8): 1202-19.
  • the Sushi peptides structural characterization and mode of action against Gram- negative bacteria.
  • Preferred sushi peptides of the fusion protein are sushi peptides SI and S3 and multiples thereof; FASEB J. 2000 Sep; 14(12): 1801- 13.
  • the peptide is a hydrophobic peptide, which comprises at least 90 % of the hydrophobic amino acid residues of valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, cysteine, alanine, tyrosine, histidine, threonin, serine, proline and/or glycine.
  • the hydrophobic peptide of the fusion protein consist of about 90 % to about 95 %, or of about 90 to about 100%, or of about 95 % to about 100 % of the hydrophobic amino acid residues of valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, cysteine, alanine, tyrosine, histidine, threonin, serine, proline and/or glycine.
  • the peptide is an amphiphatic peptide, which comprises one or more of the positively charged amino acid residues of lysine, arginine and/or histidine, combined to one or more of the hydrophobic amino acid residues of valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, cysteine, alanine, tyrosine, histidine, threonin, serine, proline and/or glycine.
  • Side chains of the amino acid residues are oriented in order that cationic and hydrophobic surfaces are clustered at opposite sides of the peptide.
  • amino acids in said peptide are positively charged amino acids.
  • amino acid residues in said peptide are hydrophobic amino acid residues.
  • the amphiphatic peptide is fused at the N-terminal and/or the C-terminal end of the enzyme having cell wall degrading activity, thus enhancing the amphiphaticity of the latter proteins.
  • the peptide is an amphiphatic peptide consisting of at least 5, more preferably at least of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acid residues.
  • At least about 30, 40, 50, 60 or 70% of the said amino acid residues of the amphiphatic peptide are either arginine or lysine residues and/or at least about 30, 40, 50, 60 or 70% of the said amino acid residues of the amphiphatic peptide are of the hydrophobic amino acids valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, cysteine, alanine, tyrosine, histidine, threonin, serine, proline and/or glycine.
  • the peptide is an amphiphatic peptide comprising beside the positively charged amino acid residues, in particular lysine and/or arginine residues, hydrophobic amino acid residues, in particular valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, cysteine, alanine, tyrosine, histidine, threonin, serine, proline and glycine residues, more preferably alanine, valine, leucine, isoleucine, phenylalanine, and/or tryptophan residues.
  • amphiphatic peptides consisting of about 10 % to about 50 %, or about 20 % to about 50 %, or about 30 % to about 45 % or about 5 % to about 30 % positively charged amino acid residues, in particular lysine and/or arginine residues and of about 50 % to about 85 %, or about 50 % to about 90 %, or about 55 % to about 90 %, or about 60 % to about 90 %, or about 65 % to about 90 % hydrophobic amino acid residues, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, cysteine, alanine, tyrosine, histidine, threonin, serine, proline and glycine residues, more preferably alanine, valine, leucine, isoleucine, phenylalanine, and/or tryptophan residues
  • the peptide is no tag such as a His-tag, Strep-tag, Avi-tag, Myc-tag, Gst-tag, JS- tag, cystein-tag, FLAG-tag or other tags known in the art and no thioredoxin or maltose binding proteins (MBP).
  • tag such as a His-tag, Strep-tag, Avi-tag, Myc-tag, Gst-tag, JS- tag, cystein-tag, FLAG-tag or other tags known in the art and no thioredoxin or maltose binding proteins (MBP).
  • MBP thioredoxin or maltose binding proteins
  • the artilysin or endolysin according to the present invention may comprise in addition such tag or tags.
  • the artilysins according to the present inventions are fusion proteins. Fusion proteins are constructed by linking at least two nucleic acid sequences using standard cloning techniques as described e.g. by Sambrook et al. 2001, Molecular Cloning: A Laboratory Manual. Such a protein may be produced, e.g., in recombinant DNA expression systems. Such fusion proteins according to the present invention can be obtained by fusing the nucleic acids for endolysin and the respective peptide stretch.
  • the peptides of the artilysin according to the present invention comprise modifications and/or alterations of the amino acid sequences.
  • Such alterations and/or modifications may comprise mutations such as deletions, insertions and additions, substitutions or combinations thereof and/or chemical changes of the amino acid residues, e.g. biotinylation, acetylation, PEGylation, chemical changes of the amino-, SH- or carboxyl- groups.
  • the anti-cancer agent according to the present invention refers to any agent suitable in the treatment of any cancer.
  • the anti-cancer agent according to the present invention refers to a chemotherapeutic agent, a radiotherapy agent, an anti-cancer vaccine, an antibody, an immunotherapy agent and/or a cell therapy agent.
  • the anti-cancer agent according to the present invention refers to alkylating agents, antimetabolites, natural products, and hormones.
  • the anti-cancer agent according to the present invention refers to RNA, DNA, protein, peptide, immune cell, viral vector and antibodies. More preferably the RNA refers to mRNA, self-amlifying mRNA, siRNA, nonreplicating unmodified and modified mRNA and modified mRNA.
  • the anti-cancer agent is coupled to the artilysin as vehicle via covalent binding, electrostatic interactions, hydrogen bonding, hydrophobic interactions and base stacking in a manner similar to protein-DNA interactions.
  • the artilysin and/or anti-cancer agent may be provided in the pharmaceutical composition according to the present invention included in lipid nanoparticles, solid lipid nanoparticles, polyplexes or cationic nanoemulsions.
  • a further aspect of the present invention is related to a pharmaceutical composition according to the present invention comprising an endolysin or an artilysin and an anti-cancer agent for use in the treatment of a proliferative disease.
  • proliferative disease characterized by an abnormal proliferation of cells is used herein in a broad sense to include any disorder that requires control of the cell cycle.
  • the proliferative disease refers to cancers, leukaemias, cardiovascular disorders such as restenosis and cardiomyopathy, auto-immune disorders such as glomerulonephritis and rheumatoid arthritis, dermatological disorders such as psoriasis, anti-inflammatory, anti-fungal, antiparasitic disorders such as malaria, emphysema and alopecia.
  • the pharmaceutical composition of the present invention may be administered directly to a tumor or to a wide variety of locations including, for example, into sites such as the cerebral spinal fluid, bone marrow, joints, arterial endothelial cells, rectum, buccal/sublingual, vagina, the lymph system, to an organ selected from the group consisting of lung, liver, pancreas, spleen, skin, blood and brain, or to a site selected from the group consisting of tumors and interstitial spaces.
  • the composition may be administered intraocularly, intranasally, sublingually, orally, topically, intravesically, intrathecal, intravenously, intraarterially e.g.
  • intrahepatic artery intraperitoneally, intracranially, intramuscularly, intraarticularily or subcutaneously.
  • Other representative routes of administration include gastroscopy, ECRP and colonoscopy, which do not require full operating procedures and hospitalization, but may require the presence of medical personnel.
  • the present invention relates to a method of treating a disorder, disease or condition in a subject in need of treatment and/or prevention, which method comprises administering to said subject an effective amount of a pharmaceutical composition according to the present invention.
  • the subject may be a human or an animal.
  • the dosage and route of administration used in a method of treatment (or prophylaxis) according to the present invention depends on the specific disease or target site to be treated.
  • the route of administration may be for example oral, topical, nasopharyngeal, parenteral, inhalational, intravenous, intramuscular, intrathecal, intraspinal, endobronchial, intrapulmonal, intraosseous, intracardial, intraarticular, rectal, vaginal or any other route of administration.
  • a formulation may be used that protects the active compounds from environmental influences such as proteases, oxidation, immune response etc., until it reaches the target site. Therefore, the formulation may be capsule, dragee, pill, powder, suppository, emulsion, suspension, gel, lotion, cream, salve, injectable solution, syrup, spray, inhalant or any other medical reasonable galenic formulation.
  • the galenic formulation may comprise suitable carriers, stabilizers, flavourings, buffers or other suitable reagents.
  • the formulation may be a lotion, cream, gel, salve or plaster
  • the formulation may be saline solution to be applied via a spray to the nose.
  • a pharmaceutical pack comprising one or more compartments, wherein at least one compartment comprises one or more artilysins according to the present invention and one or more anti-cancer agents or a composition according to the present invention.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Oncology (AREA)
  • Zoology (AREA)
  • Communicable Diseases (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne une composition pharmaceutique comprenant une endolysine ou une artichaulysine et un agent anticancéreux. De plus, la présente invention concerne une composition pharmaceutique selon l'invention destinée à être utilisée en tant que médicament et destinée à être utilisée dans le traitement d'une maladie ou d'un trouble prolifératifs.
PCT/IB2022/061122 2021-11-18 2022-11-18 Composition pharmaceutique comprenant une endolysine ou une artilysine et un agent anticancéreux WO2023089544A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP21208926.2 2021-11-18
EP21208926 2021-11-18

Publications (1)

Publication Number Publication Date
WO2023089544A1 true WO2023089544A1 (fr) 2023-05-25

Family

ID=78821011

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2022/061122 WO2023089544A1 (fr) 2021-11-18 2022-11-18 Composition pharmaceutique comprenant une endolysine ou une artilysine et un agent anticancéreux

Country Status (1)

Country Link
WO (1) WO2023089544A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2243611A (en) 1988-07-13 1991-11-06 Agricultural & Food Res Use of viral enzymes
WO2011134998A1 (fr) * 2010-04-27 2011-11-03 Lysando Holding Ag Procédé de réduction de biofilms
WO2012142180A1 (fr) * 2011-04-12 2012-10-18 Tianxin Wang Procédés de détection et méthodes de traitement de maladies
WO2015145463A1 (fr) * 2014-03-27 2015-10-01 Gangagen, Inc. Compositions dérivée de phages pour un meilleur traitement contre les mycobactéries
WO2019068875A1 (fr) * 2017-10-06 2019-04-11 Micreos Human Health B.V. Traitement d'un état de santé associé à une infection par une bactérie oncogène

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2243611A (en) 1988-07-13 1991-11-06 Agricultural & Food Res Use of viral enzymes
WO2011134998A1 (fr) * 2010-04-27 2011-11-03 Lysando Holding Ag Procédé de réduction de biofilms
WO2012142180A1 (fr) * 2011-04-12 2012-10-18 Tianxin Wang Procédés de détection et méthodes de traitement de maladies
WO2015145463A1 (fr) * 2014-03-27 2015-10-01 Gangagen, Inc. Compositions dérivée de phages pour un meilleur traitement contre les mycobactéries
WO2019068875A1 (fr) * 2017-10-06 2019-04-11 Micreos Human Health B.V. Traitement d'un état de santé associé à une infection par une bactérie oncogène

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
COOPER BETHANY M. ET AL: "Peptides as a platform for targeted therapeutics for cancer: peptide-drug conjugates (PDCs)", CHEMICAL SOCIETY REVIEWS, vol. 50, no. 3, 21 December 2020 (2020-12-21), UK, pages 1480 - 1494, XP093025721, ISSN: 0306-0012, DOI: 10.1039/D0CS00556H *
DING JLLI PHO B, CELL MOL LIFE SCI, vol. 65, no. 7-8, April 2008 (2008-04-01), pages 1202 - 19
FASEB J, vol. 14, no. 12, September 2000 (2000-09-01), pages 1801 - 13
HOPPENZ PAUL ET AL: "Peptide-Drug Conjugates and Their Targets in Advanced Cancer Therapies", FRONTIERS IN CHEMISTRY, vol. 8, 7 July 2020 (2020-07-07), XP093025718, DOI: 10.3389/fchem.2020.00571 *
LE, WENJUN ET AL., BIOPHYSICS REPORT, vol. 5, February 2019 (2019-02-01)
SAMBROOK ET AL., MOLECULAR CLONING: A LABORATORY MANUAL, 2001
SAMBROOK, J. ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR PRESS

Similar Documents

Publication Publication Date Title
EP2655610B1 (fr) Agents antimicrobiens
EP2445515B1 (fr) Agents antimicrobiens
EP2702070B1 (fr) Nouveaux agents antimicrobiens
EP2470648B1 (fr) Nouvel obpglys d'endolysine
CA2765810C (fr) Proteines de fusion d'endolysine antimicrobiennes
EP2563916B1 (fr) Procédé de réduction de biofilms
EP2331687B1 (fr) Agents antimicrobiens
EP2635676B1 (fr) Nouvelle endolysine
US11208643B2 (en) Engineered gram-negative endolysins
EP2679677A1 (fr) Procédé et appareil d'amélioration d'images
CN112166190A (zh) 新抗微生物蛋白
CN112204054A (zh) 新抗微生物融合蛋白
WO2023089544A1 (fr) Composition pharmaceutique comprenant une endolysine ou une artilysine et un agent anticancéreux

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22823148

Country of ref document: EP

Kind code of ref document: A1