EP1129201A1 - Traitement d'infections au moyen de proteines de fusion lysozyme - Google Patents

Traitement d'infections au moyen de proteines de fusion lysozyme

Info

Publication number
EP1129201A1
EP1129201A1 EP99967112A EP99967112A EP1129201A1 EP 1129201 A1 EP1129201 A1 EP 1129201A1 EP 99967112 A EP99967112 A EP 99967112A EP 99967112 A EP99967112 A EP 99967112A EP 1129201 A1 EP1129201 A1 EP 1129201A1
Authority
EP
European Patent Office
Prior art keywords
lysozyme
seq
composition
mice
bacterial
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP99967112A
Other languages
German (de)
English (en)
Inventor
Timothy Edward Weaver
Henry Toyin Akinbi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cincinnati Childrens Hospital Medical Center
Original Assignee
Cincinnati Childrens Hospital Medical Center
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
Priority claimed from US09/193,877 external-priority patent/US5993809A/en
Application filed by Cincinnati Childrens Hospital Medical Center filed Critical Cincinnati Childrens Hospital Medical Center
Publication of EP1129201A1 publication Critical patent/EP1129201A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to prophylactic and therapeutic uses of
  • the respiratory tract such as cystic fibrosis or the gastrointestinal tract such as
  • bacterial infections may have severe
  • colonization of the lungs indicates that their systemic immunity is essentially
  • the respiratory and gastrointestinal tracts are frequent sites of
  • the normal respiratory tract has
  • immunoglobulins IgA and IgM secretory immunoglobulins IgA and IgM, the proteins lactoferrin, betalysin and
  • fibronectin fibronectin, complement components and the enzyme lysozyme.
  • lysozyme is the best established antimicrobial substance
  • Human lysozyme is a naturally occurring enzyme that is known
  • Lysozyme is a small ( 1 5
  • Lysozyme can
  • Lysozyme produced by
  • polymorphonuclear leukocytes such as neutrophils, inhibits chemotaxis of
  • Lysozyme is also probably
  • Pulmonary surfactant a complex mixture of phospholipids and
  • Surfactant protein-B is one of the protein
  • This invention is directed to a composition for the prophylaxis or
  • composition for treating a bacterial infection in a mammal.
  • SP-B lysozyme/surfactant protein-B
  • composition prevents or treats a respiratory infection such as occurs frequently in individuals with cystic fibrosis, or may
  • Pseudomonas aeruginosa which is the major airway pathogen in patients with
  • cystic fibrosis Furthermore, the elevated lysozyme activity in bronchoalveolar
  • lavage fluid resulting from administration of lysozyme is not associated with
  • the invention is also directed to a method of preventing or
  • composition in a dosing regimen sufficient to prevent or treat the infection.
  • the route of administration may be parenteral, for example by inhalation, or
  • the invention is still further directed to a fusion protein SEQ ID NO: 1
  • the invention is still further directed to a fusion protein SEQ ID NO: 1
  • the invention is additionally directed to a method of treating a
  • the fusion protein may be administered by aerosol installation
  • the invention is also directed to a method of preventing or
  • the invention is additionally directed to a composition
  • a composition comprising
  • FIG. 1 is a histogram showing bacterial clearance from lungs of
  • transgenic lysozyme/surfactant protein-B fusion protein
  • FIG. 2A is a photograph showing expression of recombinant
  • FIG 2B is a photograph showing
  • mouse line probed with rat cDNA mouse line probed with rat cDNA.
  • FIG. 3 is a photograph showing analysis of lysozyme protein
  • FIG. 4 is a histogram of lysozyme activity in bronchoalveolar
  • FIG. 5A is a photograph showing lysozyme cellular localization in
  • FIG. 5B is a photograph showing lysozyme cellular
  • FIG. 6A is a photograph showing lung structure in transgenic mice
  • FIG. 6B is a photograph showing lung
  • FIG. 7 is a histogram illustrating the cellular composition of BAL
  • FIG 8 is a histogram showing clearance of Group B Streptococcus
  • FIG 9 is a histogram showing clearance of Pseudomonas
  • aeruginosa from the lungs at twenty-four hours post infection.
  • Rat lysozyme is a hydrophobic peptide of 1 48 amino acids SEQ ID NO:
  • Human lysozyme SEQ ID NO:5 also has 1 48 amino acids and has
  • Surfactant protein-B is a hydrophobic peptide of 79 amino
  • Human SP-B is synthesized by alveolar type II epithelial cells as a
  • propeptide of 1 77 amino acids propeptide of 1 77 amino acids, and a carboxy terminal (C-terminal)
  • propeptide of 102 amino acids The C-terminal propeptide has been shown to function in maintenance of the size of lamellar bodies which store SP-B and in
  • oligonucleotide primers based on the published sequence of the rat enzyme by
  • SP-B cDNA has previously been described (Glasser, S.W., et al. cDNA and deduced amino acid sequence of human pulmonary surfactant-associated
  • proteolipid SPL (Phe). Proc. Natl. Acad. Sci. USA 84:4007-401 1 , 1 987).
  • the synthesized cDNAs were either generated into a chimeric
  • SP-B human surfactant protein B
  • this chimeric molecule was a fusion protein of residues 1 -1 48 of rat lysozyme
  • transgenic mice expressing a transgene construct encoding the fusion protein
  • SP-C protein C
  • mice was restricted to the distal respiratory epithelium. Expression of the chimeric protein SEQ ID NO:3 was confirmed.
  • transgene product was detected in both lung homogenates and in
  • BAL bronchoalveolar lavage
  • chimeric protein SEQ ID NO:3 was not associated with altered lung structure
  • transgenic mouse lines were generated in which rat lysozyme SEQ ID NO: 1
  • SP-C human surfactant protein C
  • PBS sterile phosphate buffered saline
  • tuberculin syringes fitted with 27 gauge needles in preparation for the
  • mice Five to six week old mice maintained in clean rooms were used
  • mice were anesthetized with a mixture of
  • the two halves of the thyroid muscles were apposed at
  • mice were harvested, weighed, homogenized in PBS and plated on BAP
  • CFU/g gram of lung tissue
  • transgenic mice was even less than the number of bacteria that had been
  • transgenic mice had significantly enhanced
  • mice had 1 .99 ⁇ 1 .4 x 10 4 CFU/g of tissue, while BAP inoculated with
  • lung tissue from wild type (control) mice had 25.49 ⁇ 1 2.43 x 1 0 4 CFU/g
  • mice had 9.9 ⁇ 6.43 x 10 4 CFU/g tissue, while BAP
  • transgenic mice facilitated bacterial clearance from the airway.
  • mice carrying the lysozyme/surfactant protein-B fusion protein show that in mice carrying the lysozyme/surfactant protein-B fusion protein
  • mice which expressed a lysozyme/surfactant protein B fusion protein SEQ ID NO: 1
  • lysozyme transgene was assessed by Northern blot analysis of 2 ⁇ g of total
  • FIG. 2A complementary DNA (cDNA) (FIG. 2A) and rat cDNA (FIG. 2B) .
  • cDNA complementary DNA
  • FIG. 2B rat cDNA
  • mouse cDNA probe because of cross hybridization between rat lysozyme and mouse
  • FIG. 2A both the larger endogenous mouse lysozyme mRNA and rat
  • mice lysozyme mRNA were detected.
  • the mouse lysozyme mRNA was used as an
  • FIG. 3 shows
  • mice from transgenic line 3.5 had
  • lysozyme was used to generate a standard curve.
  • mice from transgenic line 3.5 had a 1 7.5-fold
  • transgenic line 3.5 versus transgenic line 2.6.
  • transgenic line 2.6 As predicted from Western
  • FIG. 5B wild type control littermates
  • Lysozyme was detected in Type II cells in wild type and
  • transgenic mice with more intense staining in Type II cells from transgenic
  • mice Transgenic mice, in addition, have expression in bronchiolar epithelial cells.
  • transgenic mice and wild type littermate controls were compared following an
  • mice intratracheal injection of 1 0 6 colony forming units (CFU) of GBS. All mice
  • transgenic line 2.6 (4.2 ⁇ 0.8 x 10 6 CFU/g lung tissue versus 7.1 ⁇ 0.6
  • mice received intratracheal injections with 1 0 7 CFU of Pseudomonas
  • FIG. 9 shows CFU/g lung tissue ⁇ SEM. Bacterial clearance was
  • the lysozyme/SP-B fusion protein SEQ ID NO:3 or SEQ ID NO:6
  • lysozyme SEQ ID NO: 1 of the invention may be used to treat
  • Cystic fibrosis is a systemic disease in which mucus secretion is
  • lysozyme or the lysozyme/surfactant protein-B fusion protein in vivo offers a
  • the method and composition of the invention may range from total prevention,
  • the lysozyme/SP-B fusion protein SEQ ID NO:3 or
  • SEQ ID NO:6 or recombinant lysozyme SEQ ID NO: 1 may be used to protect
  • Staphylococcus aureus Streptococcus species
  • Streptococcus Streptococcus
  • the invention may be used to combat gastrointestinal
  • the lysozyme/surfactant protein-B fusion protein SEQ ID NO:3 or
  • SEQ ID NO:6 may be administered by an enteral route to target the
  • enterocolitica Campy/obacter fetus, ssp. jej ' uni, and Helicobacter pylori.
  • lysozyme/SP-B may be formulated for oral administered as a solid or liquid in
  • saposin A structurally related saposin protein family
  • saposin B structurally related saposin protein family
  • saposin C saposin C
  • saposin D NK lysin
  • pore forming peptide of amoebapore A etc. could be generated.
  • Various modes of administration besides inhalation could be generated.
  • fusion protein SEQ ID NO:3 or SEQ ID NO:

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Communicable Diseases (AREA)
  • Pulmonology (AREA)
  • Oncology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicinal Preparation (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne une méthode et une composition destinées au traitement prophylactique et curatif d'infections bactériennes, en particulier au niveau des voies respiratoires. Une protéine de fusion de lysozyme et du propeptide C-terminal de la protéine tensioactive B (SP-B) avec les dix acides aminés précédents du peptide SP-B mature, ou le lysozyme recombinant seul, est administré à un patient via un support acceptable au plan pharmaceutique. La protéine de fusion seule ou le lysoszyme recombinant peut être sélectionné de manière à pouvoir être administré sur un site d'infection cible, tel que les poumons ou l'appareil gastro-intestinal. Ce procédé et cette composition permettent d'éviter certains problèmes associés à des traitements antibiotiques classiques, tels que la perte d'efficacité ou le développement de souches bactériennes résistant aux antibiotiques.
EP99967112A 1998-11-18 1999-11-18 Traitement d'infections au moyen de proteines de fusion lysozyme Withdrawn EP1129201A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US440742 1995-05-10
US09/193,877 US5993809A (en) 1998-11-18 1998-11-18 Lysozyme fusion proteins in infections
US193877 1998-11-18
US44074299A 1999-11-16 1999-11-16
PCT/US1999/027403 WO2000029588A1 (fr) 1998-11-18 1999-11-18 Traitement d'infections au moyen de proteines de fusion lysozyme

Publications (1)

Publication Number Publication Date
EP1129201A1 true EP1129201A1 (fr) 2001-09-05

Family

ID=26889453

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99967112A Withdrawn EP1129201A1 (fr) 1998-11-18 1999-11-18 Traitement d'infections au moyen de proteines de fusion lysozyme

Country Status (6)

Country Link
EP (1) EP1129201A1 (fr)
JP (1) JP2002530083A (fr)
AU (1) AU759743B2 (fr)
BR (1) BR9915218A (fr)
CA (1) CA2349837A1 (fr)
WO (1) WO2000029588A1 (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1194570A1 (fr) 1999-06-23 2002-04-10 PPL Therapeutics (Scotland) Limited Proteines de fusion incorporant un lysozyme
CA2588965C (fr) * 2003-12-18 2013-01-22 Werner Seeger Nouveaux activateurs chimeres du plasminogene et leur utilisation a des fins pharmaceutiques
WO2005108563A2 (fr) * 2004-04-19 2005-11-17 University Of Chicago Proteine hydrolysant le peptidoglycane encodée par bacteriophage n4
AU2006207998A1 (en) 2005-01-27 2006-08-03 Novartis Vaccines And Diagnostics Inc. A method for characterizing the efficacy of an agent targeting a primary cystic fibrosis defect
WO2012140676A2 (fr) * 2011-04-12 2012-10-18 Appaiah C B Polypeptides chimères antibactériens
CN104817616A (zh) * 2014-01-30 2015-08-05 陈光健 寡肽cd02及其制备方法和应用
CN104817618B (zh) * 2014-01-30 2018-07-03 陈光健 寡肽cd01及其制备方法和应用
CN114149986B (zh) * 2022-02-08 2022-05-06 中国科学院天津工业生物技术研究所 一种地衣芽孢杆菌溶菌酶突变体及其在虹鳟鱼保鲜中的应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3540075A1 (de) * 1985-11-12 1987-05-14 Boehringer Ingelheim Int Human-lysozym
DE3818094C1 (fr) * 1988-05-27 1989-07-20 Medichemie Ag, Ettingen, Ch
US5006343A (en) * 1988-12-29 1991-04-09 Benson Bradley J Pulmonary administration of pharmaceutically active substances

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0029588A1 *

Also Published As

Publication number Publication date
BR9915218A (pt) 2001-07-31
AU759743B2 (en) 2003-05-01
AU2345900A (en) 2000-06-05
WO2000029588A1 (fr) 2000-05-25
JP2002530083A (ja) 2002-09-17
CA2349837A1 (fr) 2000-05-25

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