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WO1999033957A1 - Enzymes modifiees comprenant un domaine polyanionique - Google Patents

Enzymes modifiees comprenant un domaine polyanionique Download PDF

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Publication number
WO1999033957A1
WO1999033957A1 PCT/DK1998/000569 DK9800569W WO9933957A1 WO 1999033957 A1 WO1999033957 A1 WO 1999033957A1 DK 9800569 W DK9800569 W DK 9800569W WO 9933957 A1 WO9933957 A1 WO 9933957A1
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Prior art keywords
enzyme
modified
modified enzyme
polyanionic domain
polyanionic
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PCT/DK1998/000569
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English (en)
Inventor
Heinz-Josef Deussen
Claus Crone Fuglsang
Arne Agerlin Olsen
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Novo Nordisk A/S
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Publication date
Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to EP98962294A priority Critical patent/EP1042454A1/fr
Priority to JP2000526615A priority patent/JP2002526029A/ja
Priority to AU17515/99A priority patent/AU1751599A/en
Publication of WO1999033957A1 publication Critical patent/WO1999033957A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/84—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds obtained by reactions otherwise than those involving only carbon-carbon unsaturated bonds
    • A61K8/88—Polyamides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • 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)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • 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)
    • C12N9/2405—Glucanases
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
    • C12N9/2454—Dextranase (3.2.1.11)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/57—Compounds covalently linked to a(n inert) carrier molecule, e.g. conjugates, pro-fragrances

Definitions

  • the present invention relates to modified enzymes comprising a polyanionic domain, to methods for producing such modified enzymes, to oral compositions comprising such modified enzymes, and to the use of such oral care compositions for the prevention and/or removal of dental plaque.
  • Dental plaque is a mixture of bacteria, epithelial cells, leukocytes, macrophages and other oral exudate that is formed on the surface of teeth.
  • the formation of dental plaque leads to dental caries, gingival inflammation, periodontal disease, and eventually tooth loss.
  • Said bacteria produce highly branched polysaccharides, which together with micro-organisms from the oral cavity form an adhesive matrix for the continued proliferation of plaque.
  • plaque As plaque continues to accumulate, rock hard white or yellowish deposits arise. These deposits are called calcified plaque, calculus or tartar, and are formed in the saliva from plaque and minerals, in particular calcium.
  • Oral polysaccharides are produced from sucrose introduced into the mouth, e.g. as a food or beverage constituent, by the action of cariogenic micro-organisms such as Streptococcus mutans or Streptococcus sanguis growing in the oral cavity.
  • Said oral polysaccharides comprise water-soluble dextran having large portions of ⁇ -l,6-glycosidic linkages, and a major component of water-insoluble extra-cellular polysaccharides called "mutan" comprised of a backbone with ⁇ -l,3-glycosidic linkages and branches with ⁇ -1, 6-glycosidic linkages.
  • Mutan binds to hydroxylapatite (constituting the hard outer porous layer of the teeth) and to acceptor proteins on the cell surface of said cariogenic bacteria adhering to the tooth surface.
  • various enzymes e.g. a dextranase and/or a mutanase
  • oral care compositions and products and a number of oral care products containing various enzymes, including glucanases, oxidoreductases such as oxidases and peroxidases, are known.
  • a problem with the known enzyme-containing oral care products is the fact that the enzymes generally do not bind to components of the teeth or plaque, which means that enzymes applied e.g. by means of a toothpaste are relatively quickly removed from the teeth and mouth. This in turn means that such enzymes are able to act only for a limited amount of time, and that their full potential for the maintenance of oral hygiene by e.g. combating plaque is not realised.
  • Chu and Orgel Bioconjugate Chem.
  • a modified enzyme comprising one or more polyanionic domains binds to hydroxylapatite in the teeth, thereby allowing the enzyme in an oral care composition to exert a prolonged enzymatic action.
  • the present invention thus relates to a modified enzyme comprising an enzyme and at least one poly- anionic domain, wherein the enzyme comprises or is covalently attached to each said polyanionic domain.
  • a second aspect the invention relates to an oral care composition comprising such modified enzymes.
  • a third aspect the invention relates to the use of a composition or oral care product comprising the modified enzymes of the invention for the prevention or treatment of a dental disease, in particular for preventing the formation of dental plaque or removing dental plaque.
  • modified enzyme refers to an enzyme comprising or covalently attached to at least one polyanionic domain.
  • the attachment may be effected by coupling a polyanionic domain to various groups in the enzyme by chemical or recombinant DNA techniques, or the polyanionic domain may be inserted into one or more sites of the enzyme by means of recombinant DNA technology.
  • At least one polyanionic domain is covalently attached to a carboxylate group and/or an amino group of the enzyme.
  • the enzyme moiety is chemically modified by coupling a polyanionic domain to the carboxyl group of Glutamic acid and/or Aspartic acid residues in the enzyme and/or to one or more C-terminal carboxyl groups in the enzyme.
  • the modified enzyme is produced by means of recombinant DNA technology, i.e. the polyanionic domain constitutes an extension of the enzyme in question by being bound to one or more C- and/or N-terminal groups in the enzyme, or the polyanionic domain is incorporated into one or more sites in the enzyme.
  • polyanionic domain is intended to mean a molecule or moiety having a net negative charge at pH 7 and being capable of being covalently bound to an enzyme.
  • the polyanionic domain may be incorporated into the amino acid sequence of the enzyme itself.
  • Suitable domains which may be used according to the invention are peptides comprising from 1 to 150 amino acid residues, such as from 1 to 100, e.g. from 1 to 50, preferably from 2 to 40, such as from 2 to 30, e.g. from 2 to 20, more preferably from 3 to 15, such as from 3 to 10. Any naturally-occurring amino acid may be incorporated in the domains' peptide structure.
  • the domain is a peptide
  • the peptide domain possesses a net negative charge at pH 7.
  • the peptide domain must include at least one Glutamic acid and/or Aspartic acid residue, e.g. from 1 to 150, such as from 1 to 100, e.g. from 1 to 50, preferably from 2 to 40, such as from 2 to 30, e.g. from 2 to 20, more preferably from 3 to 15, such as from 3 to 10.
  • polyanionic peptide domains are polyglutamic acid and polyaspartic acid comprising a total of from 2 to 100 Glutamic acid and/or Aspartic acid residues, such as from 3 to 75, e.g. from 3 to 50, preferably from 3 to 40, such as from 3 to 30, e.g. from 3 to 20, more preferably from 3 to 15, such as from 3 to 10, e.g. from 4 to 8.
  • polyanionic peptides containing polyglutamic acid and/or polyaspartic acid together with at least one amino acid with an uncharged side chain will also be efficient domains.
  • amino acids with an uncharged side chain may be incorporated in the polyanionic peptide in several ways.
  • Glu and/or Asp residues and amino acids with an uncharged side chain such as Alanine, Valine, Leucine, Isoleucine, Methionine, Phenylalanine, Tryptophan, Proline, Glycine, Serine, Threonine, Cysteine, Tyrosine, Aspargine, and/or Glutamine.
  • amino acids with an uncharged side chain may optionally be located together in one or more groups of 2 to 50 residues, preferably 3 to 25 residues, such as 3 to 10 residues, e.g. 4 to 8 residues.
  • Suitable polyglutamic acids and polyaspartic acids are Glu-Glu, (Glu) 3 , (Glu) , (Glu) 5 , (Glu) 6 , (Glu) 7 , (Glu) 8 , (Glu) 9 , (Glu) 10 , Asp-Asp, (Asp) 3 , (Asp) 4 , (Asp) 5 , (Asp) 6 , (Asp) 7 , (Asp) 8 , (Asp) 9 , (Asp) 10 , Glu-Asp, (Glu- As P) 2/ (Glu-Asp) 3 , (Glu-Asp) 4 , (Glu-Asp) 5 , Asp-Glu, (Asp-Glu) 2 , (Asp-Glu) 3 , (Asp-Glu) 4 , (Asp-Glu) 5 , Xaa-Glu, (Xaa-Glu) 2 , (Xaa-Glu) 2
  • n is an integer in the range of from 1 to 15, preferably in the range of from 1 to 8, such as from 1 to 5, e.g. from 1 to 3
  • m is an integer in the range of from 1 to 50, preferably from 2 to 40, such as from 2 to 30, e.g. from 2 to 20, more preferably from 3 to 15, such as from 3 to 10
  • each R is independently selected from the group consisting of hydrogen, Cx-g-alkyl, hydroxy, amino, and halogen such as fluoro, chloro, iodo and bromo.
  • R is hydrogen.
  • Ci-s-alkyl used alone or as part of another group designates a straight, branched or cyclic saturated hydrocarbon group having from one to six carbon atoms such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, cyclohehexyl, etc.
  • C 2 _ 6 -alkenyl designates a hydrocarbon group having from two to six carbon atoms, which may be straight, branched or cyclic and may contain one or more double bonds such as vinyl, allyl, 1-butenyl, 2-butenyl, iso- butenyl, 1-pentenyl, 2-pentenyl, 4-pentenyl, 3-methyl-l-butenyl, 2-hexenyl, 5-hexenyl, cyclohexenyl, 2,3-dimethy1-2-butenyl, etc. , and which may have the cis and/or trans configuration.
  • polyanionic domains which are envisaged to be suitable for the purpose of the invention are polyphosphates, polysulfonic acids, and polycarboxylic acids.
  • polyphosphate is intended to mean a molecule comprising at least two and preferably at least three phosphate groups. If a phosphate group of such a polyphosphate is used for coupling to an amine group in the polypeptide, the polyphosphate should then preferably contain at least 3 phosphate groups. Preferred polyphosphates are aminated polyphosphates.
  • polysulfonic acid is intended to mean a molecule comprising at least two and preferably at least three sulfonic acid groups. If a sulfonic acid group of such a polysulfonic acid is used for coupling to an amine group in the polypeptide, the polysulfonic acid should then preferably contain at least 3 sulfonic acid groups.
  • Preferred polysulfonic acids are aminated polysulfonic acids.
  • polycarboxylic acid is intended to mean a molecule comprising at least two and preferably at least three carboxyl groups. If a carboxyl group of such a polycarboxylic acid is used for coupling to an amine group in the polypeptide, the polycarboxylic acid should then contain at least 3 carboxyl groups.
  • An example of a suitable polycarboxylic acid is citric acid.
  • a preferred class of polycarboxylic acid is an aminated polycarboxylic acid. Examples of aminated polycarboxylic acids are aminated polycarboxylic alkanes and derivatives thereof, aminated polycarboxylic sugars, aminated polycarboxylic alcohols and aminated polycarboxylic polyalcohols.
  • aminated polycarboxylic acids are aminated poly(vinyl acetate-co-crotonic acid) , aminated polygalacturonic acid, and aminated poly(aerylamide-co-acylic acid) .
  • aminated polycarboxylic acids such as aminated polycarboxylic alkanes, aminated polycarboxylic sugars, aminated polycarboxylic alcohols and aminated polycarboxylic polyalcohols, should have at least one amino group per molecule, but they may suitably also have more than one amino group per molecule.
  • the polyanionic domain may be covalently coupled to the enzyme by various methods which, of course, will depend on the actual chosen attachment group or groups in the enzyme and the polyanionic domain, respectively. Thus, for the person skilled in the art, a broad class of chemical coupling techniques are available. However, preferred methods for chemically coupling the polyanionic domain to the enzyme are e.g. those described in G.T Hermanson "Bioconjugate Techniques", Academic Press,
  • the general strategy for coupling a polyanionic domain to an enzyme usually comprises reacting one or more functional groups in the enzyme with one or more functional groups in the polyanionic domain, optionally with the aid of suitable catalysts or other coupling promoting agents.
  • Another strategy commonly applied in coupling procedures involves the transformation of functional groups in the enzyme and/or the polyanionic domain into reactive groups and subsequently coupling the reactants, i.e. the enzyme and the polyanionic domain.
  • suitable coupling reaction techniques which can be employed for the production of the modified enzymes are e.g. reaction techniques using amine groups, thiol groups, carboxylate groups, hydroxyl groups, aldehyde/ketone groups, active hydrogen groups and photo-reactive groups.
  • amine groups are capable of reacting with e.g. isothiocyanates, isocyanates, acyl azides, NHS-esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, arylating agents, imidoesters, anhydrides, acid groups activated with carbodiimides, and photoreactive groups such as aryl azides, benzophenones, diazo compounds and diaziridine derivatives, the formation of such groups in the enzyme or the polyanionic domain may be used to covalently couple the polyanionic domain to the enzyme.
  • thiol-reactive groups such as e.g. haloacetyls, alkyl halide derivatives, maleimides, aziridines, acryloyl derivatives, arylating agents, thiol-disulfide exchange reagents such as pyridyl disulfides, TNB-thiol, and disulfide reductants may conveniently be used for the formation of covalent bonds between the polyanionic domain and the enzyme, through thiol groups in the enzyme or the polyanionic domain.
  • thiol-reactive groups such as e.g. haloacetyls, alkyl halide derivatives, maleimides, aziridines, acryloyl derivatives, arylating agents, thiol-disulfide exchange reagents such as pyridyl disulfides, TNB-thiol, and disulfide reductants may conveniently be used for the formation of covalent bonds between the polyanionic domain and the
  • Suitable coupling strategies include the use of carboxylate-reactive groups such as diazoalkanes, diazoacetyl compounds, CDI and carbodiimides; hydroxyl-reactive groups such as epoxides, oxiranes, CDI, N,N'-disuccinimidylcarbonate, N- hydroxysuccinimidyl chloroformate, alkyl halogens, isocyanates, and formation of reactive aldehyde groups from the hydroxyl groups by means of periodate oxidation or enzymatic oxidation; aldehyde/ketone reactive groups such as hydrazine and reactions such as Schiff-base formation, reductive amination, and Mannich condensation; active hydrogen-reactive groups such as diazonium derivatives and iodination reactions.
  • carboxylate-reactive groups such as diazoalkanes, diazoacetyl compounds, CDI and carbodiimides
  • hydroxyl-reactive groups such as epoxid
  • the polyanionic domain is covalently bound to the enzyme by means of a C-N bond, the carbon atom preferably originating from the enzyme and the nitrogen atom preferably originating the polyanionic domain.
  • the covalent bond is a peptide bond, wherein the carbon atom preferably originates from the enzyme and the nitrogen atom preferably originates the peptide.
  • a particularly preferred coupling agent for the coupling reaction is a carbodiimide, e.g. l-ethyl-3- (3-dimethyl- aminopropyl) carbodiimide (EDC) .
  • EDC electroactive polymer
  • Methods of conjugating proteins with domains using EDC can be implemented according to manufacturers' descriptions (e.g. Pierce Instructions 0475 C, 22980 X; 22981 X; EDC) using either the protocol for "Use of EDC for coupling of Haptens/small ligands to carrier Proteins" or "Protocol for Efficient Two- Step coupling of Proteins in Solution Using EDC and N- hydroxysuccinimide or sulfo-N-hydroxysucciminide” .
  • the polyanionic domain contains free amine groups
  • such groups may conveniently be protected by methods well-known in the art, see e.g. J. Jones “The Chemical Synthesis of Peptides”, Clarendon Press, Oxford, 1991, and M. Bodanszky and A. Bodanszky “The Practice of Peptide Synthesis", Springer-Verlag, Berlin, 1994.
  • amine groups of the polyanionic domain such as a peptide
  • BOC tert-butyloxycarbonyl
  • the protecting groups may be removed using standard techniques, such as removing the BOC protecting group with e.g. trifluoro acetic acid.
  • the enzyme may be dissolved, or transferred by dialysis or desalting by size exclusion chromatography in a coupling buffer, for example 50 mM MES pH 5.0 containing 200 mM sodium chloride.
  • the polyanionic domain e.g. a peptide and/or a polycarboxylic acid, may be dissolved in the coupling buffer as well.
  • the conjugation reaction may proceed by mixing enzyme and domain to a final concentration of e.g. 3 mg/ml for both enzyme and domain, followed by mixing with e.g. 5 mg of EDC per mg of enzyme.
  • the conjugation reaction then runs for e.g. about 2 hours at room temperature with continuous stirring.
  • the reaction is terminated by removal of surplus reagent either by desalting by size exclusion chromatography or by extensive dialysis, e.g. against 0.2 M ammonium acetate pH 6.9 at 5°C.
  • the resulting derivative may then be stored at 5°C.
  • the enzyme is first activated by EDC in the "Two-Step Coupling of Proteins" method, followed by removal of excess EDC by dialysis or desalting.
  • the conjugation reaction may proceed by mixing activated enzyme and the domain, e.g. peptide and/or polycarboxylic acid, and the derivative can be subsequently purified using standard procedures.
  • the degree of modification or incorporation of domains may, of course, be controlled by adjustments in the initial enzyme, domain and/or carbodiimide concentration. Variations in pH or temperature of the coupling buffer may also be used to optimise the conjugation reaction for a specific enzyme.
  • Active site protection by substrate, substrate analogues or reversible inhibitors may be used to control the modification reaction.
  • the enzyme may be modified through attachment of the above-mentioned domains to the carbohydrate part of glycosylated enzymes.
  • Periodate oxidation of carbohydrates is a well-established classical technology for generation of aldehyde groups which readily react with amino groups on the polyanionic domain, initially generating a Schiff base.
  • the reaction product can be stabilised by standard methods, e.g. by reduction using NaBH 4 or NaCNBH 3 (see e.g. G.T Hermanson, Bioconjugate Techniques, Academic Press, 1996) .
  • This process may be performed as a one- step or two-step procedure, and a number of parameters may be varied to optimise the reaction conditions for a specific enzyme/or a specific application.
  • the enzyme may be modified by substitution and/or addition of one or more amino acids by means of recombinant DNA-technology.
  • the invention therefore further relates to a modified enzyme comprising a modified enzyme.
  • the enzyme modification may e.g. be: i) insertion of at least one Glutamic acid and/or Aspartic acid residues in one or more sites of the enzyme, such as insertion of e.g. from 1 to 10 Glutamic acid and/or Aspartic acid residues, preferably from 1 to 7 Glutamic acid and/or Aspartic acid residues, e.g.
  • amino acid sequences which can constitute the extension may be such as described earlier, e.g. polyglutamic acid and polyaspartic acid comprising a total of from 2 to 100 Glutamic acid and/or Aspartic acid residues, such as from 3 to 75, e.g. from 3 to 50, preferably from 3 to 40, such as from 3 to 30, e.g. from 3 to 20, more preferably from 3 to 15, such as from 3 to 10, e.g from 4 to 8.
  • the above-mentioned insertions and N- and C-terminal extensions may conveniently be carried out by means of recombinant DNA-technology using general methods and principles known to the person skilled in the art. Oral Care Compositions
  • the oral care compositions or products of the invention have as a primary function the prevention and/or removal of dental plaque by the enzymatic action of modified enzymes bound to hydroxylapatite of the teeth, such compositions or products may also directly or indirectly have other oral care functions at the same time, e .g. the prevention of dental cavities, gingivitis and periodontal disease in general.
  • the enzyme moiety of the modified enzymes according to the invention may be any enzyme suitable for the desired purpose. It is in particular an enzyme selected from the group consisting of oxidoreductases such as oxidases and peroxidases, proteases, Upases, glucanases, esterases, deaminases, ureases and polysaccharide hydrolases, or a mixture thereof.
  • Preferred enzyme activities for oral care compositions are glucanases activities, such as an ⁇ -glucosidase activity, such as dextranase, mutanase, and/or pullulanase activity.
  • glucanases activities such as an ⁇ -glucosidase activity, such as dextranase, mutanase, and/or pullulanase activity.
  • glucanases include the enzymes in the enzyme class EC 3.2.1, in particular: glucan 1,4- ⁇ -glucosidase (3.2.1.3), cellulase (3.2.1.4), endo-
  • glucanases examples include ⁇ -1, 3-glucanases derived from Trichoderma harz ⁇ anum; ⁇ -l,6-glucanases derived from a strain of Paecilomyces; ⁇ -glucanases derived from Bacillus subtili ⁇ ; ⁇ -glucanases derived from Humicola insolens; ⁇ -glucanases derived from Aspergillus niger; ⁇ -glucanases derived from a strain of Trichoderma; ⁇ -glucanases derived from a strain of Oerskovia xanthineolytica ; exo-l,4- ⁇ -D- glucosidases (glucoamylases) derived from Aspergillus niger.
  • microbial amylases such as ⁇ -amylases derived from Bacillus ⁇ ubtilis ; ⁇ -amylases derived from Bacillus amyloliquefaciens ,' ⁇ -amylases derived from Bacillus stearothermophilus; ⁇ -amylases derived from Aspergillus oryzae ; ⁇ -amylases derived from non-pathogenic microorganisms.
  • contemplated suitable glucanases include ⁇ - galactosidases derived from Aspergillus niger; Pentosanases, xylanases, cellobiases, cellulases, hemi-cellulases derived from Humicola insolens; cellulases derived from Trichoderma reesei ; cellulases derived from non-pathogenic mold; pectinases, cellulases, arabinases, hemi-celluloses derived from Aspergillus niger; dextranases derived from Penicillium lilacinum; endo-glucanase derived from non-pathogenic mold; pullulanases derived from Bacillus acidopullyticus ; ⁇ - galactosidases derived from Kluyveromyces fragilis; xylanases derived from Trichoderma reesei .
  • glucanases include Alpha-Gal®, Bio-Feed® Alpha, Bio-Feed® Beta, Bio-Feed® Plus, Novozyme® 188, Carezyme®, Celluclast®, Cellusoft®, Ceremyl®, Citrozym®, Denimax®, Dezyme®, Dextrozyme®, Finizym®, Fungamyl®, Gamanase®, Glucanex®, Lactozy ®, Maltogenase®, Pentopan®, Pectinex®, Promozyme®, Pulpzyme®, Novamyl®, Termamyl®, AMG (Amyloglucosidase Novo) , Sweetzyme®, Aquazym® (all enzymes available from Novo Nordisk A/S) .
  • Other carbohydrases are available from other companies.
  • glucanase variants are contemplated as the enzyme moiety.
  • Another group of enzymes of interest are Oxidoreductases
  • IUBMB International Union of Biochemistry and Molecular Biology
  • Examples include oxidoreductases selected from those classified under the Enzyme Classification (E.C.) numbers: Glycerol-3-phosphate dehydrogenase _NAD+_ (1.1.1.8), Glycerol-3- phosphate dehydrogenase _NAD(P) + _ (1.1.1.94), Glycerol-3- phosphate 1-dehydrogenase _NADP_ (1.1.1.94), Glucose oxidase (1.1.3.4), Hexose oxidase (1.1.3.5), Catechol oxidase (1.1.3.14), Bilirubin oxidase (1.3.3.5), Alanine dehydrogenase (1.4.1.1), Gluta ate dehydrogenase (1.4.1.2) , Glutamate dehydrogenase _NAD (P) + _ (1.4.1.3), Glutamate dehydrogenase _NADP + _ (1.4.1.4), L-Amino acid dehydrogena
  • Said Glucose oxidases may be derived from Aspergillus niger.
  • Said Laccases may be derived from Polyporus pinsitus, Myceliophtora thermophila, Coprinus cinereus, Rhizoctonia ⁇ olani , Rhizoctonia praticola, Scytalidium thermophilum and Rhus o verni cifera .
  • Bilirubin oxidases may be derived from Myrothechecium verrucaria .
  • the Peroxidase may be derived from e .g. Soy bean, Horseradish or Coprinus cinereus. 5
  • the Protein Disulfide reductase may be any mentioned in any of WO 95/00636, WO 95/01425 and WO 95/01420 (Novo Nordisk A/S) including Protein Disulfide reductases of bovine origin, Protein Disulfide reductases derived from Aspergillus oryzae or Aspergillus niger, and DsbA or DsbC derived from Escherichia coli .
  • oxidoreductases include GluzymeTM (enzyme available from Novo Nordisk A/S) . However, other oxidoreductases are available from others.
  • lipases i.e. enzymes classified under the Enzyme Classification number E.C. 3.1.1 (Carboxylic Ester Hydrolases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)
  • E.C. 3.1.1 Carboxylic Ester Hydrolases
  • IUBMB International Union of Biochemistry and Molecular Biology
  • lipases selected from those classified under the Enzyme Classification (E.C.) numbers: 3.1.1 (i.e. so-called Carboxylic Ester Hydrolases), including (3.1.1.3) Triacylglycerol lipases, (3.1.1.4.) Phosphorlipase A 2 ⁇
  • lipases examples include lipases derived from the following microorganisms.
  • the indicated patent publications are incorporated herein by reference: Humicola, e.g. H. brevispora, H. lanuginosa, H. brevis var. thermoidea and H. insolens (US 4,810,414)
  • Pseudomonas e.g. Ps. fragi, Ps. stutzeri, Ps. cepacia and Ps. fluorescens (WO 89/04361) , or Ps. plantarii or Ps. gladioli
  • Fusarium e.g. F. oxysporum (EP 130,064) or F. solani pisi
  • Mucor also called Rhizomucor
  • M. iehei EP 238 023
  • Chromobacterium especially C. viscosum
  • Aspergillus especially A. niger
  • Candida e.g. C. cylindracea (also called C. rugosa) or C. antarctica (WO 88/02775) or C. antarctica lipase A or B (WO 94/01541 and WO 89/02916) .
  • Geotricum e.g. G. candidum (Schimada et al., (1989), J.
  • Penicillium e.g. P. camembertii (Yamaguchi et al., (1991), Gene 103, 61-67).
  • Rhizopus e.g. R. delemar (Hass et al., (1991), Gene 109, 107-113) or R. niveus (Kugimiya et al., (1992) Biosci. Biotech. Biochem 56, 716-719) or R. oryzae.
  • Bacillus e.g. B. subtilis (Dartois et al., (1993) Biochemica et Biophysica acta 1131, 253-260) or B. stearothermophilus (JP 64/7744992) or B. pumilus (WO 91/16422) .
  • Specific examples of readily available commercial lipases include Lipolase®, Lipolase® Ultra, Lipozyme®, Palatase®, Novozym® 435, Lecitase® (all available from Novo Nordisk A/S) .
  • Examples of other lipases are Lumafast®, Ps. mendocian lipase from Genencor Int. Inc.; Lipomax®, Ps.
  • Pseudoalcaligenes lipase from Gist Brocades/Genencor Int. Inc. ; Fusarium solani lipase (cutinase) from Unilever; Bacillus sp. lipase from Solvay enzymes. Other lipases are available from other companies.
  • lipase variants are contemplated as the suitable enzymes. Examples of such are described in e.g. WO 93/01285 and WO 95/22615.
  • the modified enzyme of the invention has an enzymatic activity that is at least 1% of the catalytic activity of the free enzyme, preferably at least 2%, such as at least 5%, e.g. at least 10%, more preferably at least 20%, such as at least 30%, e.g. at least 40%, still more preferably at least 50%, such as at least 60%, e.g. at least 70%, even more preferably at least 80%, such as at least 90%, e.g.
  • the modified enzyme is substantially identical to the catalytic activity of the free enzyme, as determined according to "Methods of Enzymatic Analysis", 3rd. Edition, vol. 1-10, 1984, Verlag Chemie, Weinheim. Methods for determining the activity of different types classes of enzymes are found e.g. in the following volumes of this book: Oxidoreduktaser: vol. 3
  • Lipaser vol. 6 It is also contemplated that other enzyme activities may be included in the oral care compositions of the invention, either in addition to or instead of e.g. a dextranase and/or mutanase, for example proteases, such as papain, endoglucosidases, lipases, amylase and mixtures thereof.
  • the dextranase may be derived from a strain of the filamentous fungal genus Paecilomyces , in particular a strain of Paecilomyces lilacinum. Paecilomyces lilacium dextranase (available from Novo Nordisk A/S) .
  • a mutanase suitable for use e.g. in combination with a dextranase in an oral care composition of the invention may be produced by filamentous fungi from the group including Trichoderma, in particular from a strain of Trichoderma harzian- um, such as Trichoderma harzianum CBS 243.71, or Penicillium, in particular a strain of Penicillium funiculosum, such as Penicillium funiculosum NRRL 1768, or a strain of Penicillium lilacinum, such as Penicillium lilacinum NRRL 896, or a strain of Penicillium purpurogenum , such as the strain of Penicillium purpurogenum CBS 238.95, or a strain of the genus Pseudomonas , or a strain of Flavobacterium sp., or a strain of Bacillus circulanse or a strain of Aspergillus sp., or a strain of Strepto
  • An oral care composition of the invention may suitably have incorporated an amount of enzyme moiety, e.g. dextranase and/or mutanase, equivalent to an enzyme activity, calculated as enzyme activity units in the final oral care product, in the range of from 0.001 KDU to 1000 KDU/ml, preferably from 0.01 KDU/ l to 500 KDU/ l, especially from 0.1 KDU/ml to 100 KDU/ml, and from 0.001 MU/ml to 1000 MU/ml, preferably from 0.01 MU/ml to 500 MU/ml, especially from 0.01 MU/ml to 100 MU/ml and from 0.01 MU/ml to 100 MU/ml, respectively.
  • enzyme moiety e.g. dextranase and/or mutanase
  • the modified enzymes should show sufficient enzymatic activity at temperatures between 20°C and 45°C, especially around 37°C, as the temperature prevailing in the human mouth lies within this interval.
  • Oral care products As explained above, the present invention also relates to oral care compositions and products comprising a modified enzyme as described herein.
  • the oral care product may have any suitable physical form (i.e. paste, gel, liquid, powder, ointment, tablet, chewing gum, etc.).
  • An "oral care product” can be defined as a product which can be used for maintaining or improving the oral hygiene in the mouth of humans and animals, by preventing formation of dental plaque, removing dental plaque, preventing and/or treating dental diseases, etc.
  • Oral care products according to the invention also encompass products for cleaning dentures, artificial teeth and the like.
  • Toothpastes and tooth gels typically include abrasive polishing materials, foaming agents, flavouring agents, humectants, binders, thickeners, sweetening agents, whitening/bleaching/stain removing agents, water, and optionally enzymes.
  • Tooth washes, including plaque removing liquids typically comprise a water/alcohol solution, flavouring agents, humectants, sweeteners, foaming agents, colorants, and optionally enzymes.
  • Abrasive polishing material can also be incorporated into a dentifrice product of the invention.
  • Suitable abrasive polishing material includes alumina and hydrates thereof, such as alpha alumina trihydrate, magnesium trisilicate, magnesium carbonate, kaolin, aluminosilicates, such as calcined aluminum silicate and aluminum silicate, calcium carbonate, zirconium silicate, and also powdered plastics, such as polyvinyl chloride, polya ides, polymethyl methacrylate, polystyrene, phenol-formaldehyde resins, melamine-for aldehyde resins, urea-formaldehyde resins, epoxy resins, powdered polyethylene, silica xerogels, hydrogels and aerogels and the like.
  • alumina and hydrates thereof such as alpha alumina trihydrate, magnesium trisilicate, magnesium carbonate, kaolin, aluminosilicates, such as calc
  • abrasive agents are calcium pyrophosphate, water-insoluble alkali metaphosphates, dicalcium phosphate and/or its dihydrate, dicalcium orthophosphate, tricalcium phosphate, particulate hydroxylapatite and the like. It is also possible to employ mixtures of these substances.
  • the abrasive material may be present in an amount of from 0 to 70% by weight, preferably from 1% to 70%.
  • the abrasive material content typically lies in the range of from 10% to 70% by weight of the final toothpaste product.
  • Humectants are employed to prevent loss of water from e .g. toothpastes.
  • Suitable humectants for use in oral care products according to the invention include the following compounds and mixtures thereof: glycerol, polyol, sorbitol, polyethylene glycols (PEG), propylene glycol, 1,3-propanediol, 1,4-butanediol, hydrogenated partially hydrolysed polysaccharides and the like.
  • Humectants are in general present in an amount of from 0% to 80%, preferably 5 to 70% by weight in toothpaste.
  • Silica, starch, tragacanth gum, xanthan gum, extracts of Irish moss, alginates, pectin, cellulose derivatives, such as hydroxyethyl cellulose, sodium carboxymethyl cellulose and hydroxypropyl cellulose, polyacrylic acid and its salts, and polyvinylpyrrolidone are examples of suitable thickeners and binders that may be used to stabilise the dentifrice product.
  • Thickeners may be present in toothpastes, creams and gels in an amount of from 0.1 to 20% by weight, and binders in an amount of from 0.01 to 10% by weight of the final product.
  • soaps as well as anionic, cationic, non- ionic, amphoteric and/or zwitterionic surfactants can be used. These may be present at levels of from 0% to 15%, preferably from 0.1 to 13%, more preferably from 0.25 to 10% by weight of the final product.
  • Surfactants are only suitable to the extent that they do not exert an inactivation effect on the modified enzymes.
  • Surfactants include fatty alcohol sulphates, salts of sulphonated mono- glycerides or fatty acids having 10 to 20 carbon atoms, fatty acid-albumen condensation products, salts of fatty acids amides and taurines and/or salts of fatty acid esters of isethionic acid.
  • Suitable sweeteners include saccharin.
  • Flavours such as spearmint
  • Whitening/bleaching agents include H 2 0 2 and may be added in amounts less that 5%, preferably from 0.25 to 4%, calculated on the basis of the weight of the final product. Water is usually added in an amount sufficient to give the product, e.g. a toothpaste, a flowable form.
  • water-soluble anti-bacterial agents such as chlorhexidine digluconate, hexetidine, alexidine, quaternary ammonium anti-bacterial compounds and water-soluble sources of certain metal ions such as zinc, copper, silver and tin (e .g. zinc, copper and stannous chloride, and silver nitrate) may also be included.
  • certain metal ions such as zinc, copper, silver and tin (e .g. zinc, copper and stannous chloride, and silver nitrate) may also be included.
  • Also contemplated according to the invention is the addition of compounds which can be used as a fluoride source, dyes/colorants, preservatives, vitamins, pH-adjusting agents, anti-caries agents, desensitizing agents etc.
  • Enzymes provide several benefits when used for cleansing of the oral cavity.
  • Proteases break down salivary proteins, which are adsorbed onto the tooth surface and form the pellicle, the first layer of resulting plaque.
  • Proteases along with lipases destroy bacteria by lysing proteins and lipids which form the structural components of bacterial cell walls and membranes.
  • Dextranase breaks down the organic skeletal structure produced by bacteria that forms a matrix for bacterial adhesion.
  • Proteases and amylases not only prevent plaque formation but also prevent the development of calculus by breaking up the carbohydrate- protein complex that binds calcium, preventing mineralization.
  • a toothpaste produced from an oral care composition of the invention may typically comprise the following ingredients: Abrasive material 10 to 70%
  • Modified enzyme(s) 0.0001% to 20%
  • a mouth wash produced from an oral care composition of the invention may typically comprise the following ingredients: 0-20% Humectant
  • the mouth wash may be in non-diluted form (i.e. to be diluted before use) or in ready-to-use form.
  • the invention relates to the use of the composition of the invention or an oral care product of the invention for preventing the formation of plaque or for removing dental plaque.
  • Using a product of the invention typically involves applying a safe and effective amount of said product to the oral cavity. These amounts (e .g. from 0.3 to about 2 grams), if it is a toothpaste or tooth gel, is kept in the mouth for a suitable period of time, e.g. from about 15 seconds to about 12 hours. It will be clear from the description above that even though a modified enzyme-containing oral care composition or product as such may only be kept in the mouth for a limited period of time, for example about 1-3 minutes for a toothpaste or mouthwash, the modified enzymes nevertheless become bound to tooth surfaces and therefore are able to exert an enzymatic action for an extended period of time.
  • a suitable period of time e.g. from about 15 seconds to about 12 hours.
  • the oral care composition and products of the present invention can be made using methods which are common in the oral product area.
  • Hydroxyapatite disks are prepared by compressing 250 mg of hydroxyapatite in a disk die at about 5,900 kg (13,000 lbs) of pressure for 5 minutes. The disks are then sintered at 600°C for 4 hours and finally hydrated with sterile de-ionised water.
  • HAP disks are sterilised at 180°C for two hours, hydrated with the sterilised de-ionised water and placed in a lid of Nunc tube (10 ml volume) .
  • KDU dextranase activity
  • One Kilo Novo Dextranase Unit (1 KDU) is the amount of enzyme which breaks down dextran forming reducing sugar equivalent to 1 g maltose per hour in Novo Nordisk' method for determination of dextranase based on the following standard conditions:
  • the dextranase in the reaction mixture was activated by addition of l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC; see Table 1) for one hour at ambient temperature.
  • EDC l-ethyl-3-(3-dimethylaminopropyl) carbodiimide
  • the activated dextranase was purified by size-exclusion chromatography on a PD 10 column (Pharmacia) . 50 mg of polyglutamic acid (M r 1000 D ) (Sigma # pl818) was then added, and the coupling was allowed to proceed for 20 hours at room temperature.
  • the reaction was terminated and excess reagent was removed by dialysis for 65 hours against a sodium acetate buffer (10 mM at pH 5.5) .
  • the sodium acetate buffer was changed several times during this period.
  • the degree of reaction was followed by isoelectric focusing.
  • An enzyme stock solution of Lipolase was diluted in 50 mM MES buffer containing 250 mM NaCl at pH 6.0. The final concentration of Lipolase in the reaction mixture was 10 mg enzyme per ml.
  • the Lipolase in the reaction mixture was activated by addition of l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC; see Table 1) for one hour at ambient temperature.
  • EDC l-ethyl-3-(3-dimethylaminopropyl) carbodiimide
  • the activated Lipolase was purified by size-exclusion chromatography on a PD 10 column (Pharmacia) . 50 mg of polyglutamic acid (M r 2000-15000 D ) (Sigma # P4636) was then added, and the coupling was allowed to proceed for 20 hours at room temperature.
  • reaction was terminated and excess reagent was removed by dialysis for 16 hours against a sodium phosphate buffer (10 mM at pH 7) .
  • 500 ⁇ l 10 mg/ml hydroxylapatit (HAP) in 50 mM Britton- Robinson buffer (at pH 4, 5, 6, 7, 8 and 9) was added 500 ⁇ l enzyme (Lipolase, or EDC-poly-Glu modified Lipolase (conjugate no. 3 or no. 4 in example 3) diluted in water to A 2 go 0.1. The resulting mixture was incubated for 30 minutes at room temperature while stirring. Then the samples were centrifuged at 14,000G for 4 minutes and 500 ⁇ l of the supernatant was diluted into 1.5 ml water.
  • enzyme Lipolase, or EDC-poly-Glu modified Lipolase (conjugate no. 3 or no. 4 in example 3)
  • the enzyme concentration was then measured by fluorescence spectroscopy using the LS50B spectrometer from Perkin Elmer (excitation: 280 nm, emission: 340 nm) . Controls were included without HAP addition. Binding was calculated relative to the control.
  • the present results show an improved binding of the EDC-poly- Glu modified Lipolase (Conjugate No. 3) to hydroxylapatit in the entire pH-range.
  • the Lipolase in the reaction mixture was activated by addition of l-ethyl-3- (3-d ethylaminopropyl) carbodiimide (EDC; see Table 1) for two hours at ambient temperature. After two hours the activated Lipolase was purified by size-exclusion chromatography on a PD 10 column (Pharmacia) .
  • the conjugate no. 6 was produced essentially through a similar route though by addition of 2.5 ml of the oligomer of DL-2-amino-3-phosphonobutyricic acid per ml of EDC-activated- Lipolase. The coupling was allowed to proceed for 16 hour at ambient temperature followed by dialysis as described above and and storage of conjugate no. 6 at 5°C.
  • the resulting mixture was incubated for 30 minutes at room temperature while stirring. Then the samples were centrifuged at 14,000G for 4 minutes and 500 ⁇ l of the supernatant was diluted into 1.5
  • the enzyme concentration was then measured by fluorescence spectroscopy using the LS50B spectrometer from Perkin Elmer (excitation: 280 nm, emission: 340 nm) . Controls were included without HAP addition. Binding was calculated relative to the control.

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Abstract

La présente invention concerne une enzyme modifiée comprenant une enzyme et au moins un domaine polyanionique, tel qu'un acide polyglutamique, un acide polyaspartique ou un acide polycarboxilique, l'enzyme comprenant ou étant fixée par liaison covalente à chaque domaine polyanionique. Cette invention concerne également des compostions de soins buccaux comprenant de telles enzymes modifiées et l'utilisation de ces compositions de soins dans la prévention ou le traitement des maladies dentaires, en particulier pour prévenir ou éliminer la plaque dentaire. Ces enzymes modifiées peuvent par ailleurs se fixer à l'hydroxylapatite sur les dents.
PCT/DK1998/000569 1997-12-29 1998-12-21 Enzymes modifiees comprenant un domaine polyanionique WO1999033957A1 (fr)

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EP98962294A EP1042454A1 (fr) 1997-12-29 1998-12-21 Enzymes modifiees comprenant un domaine polyanionique
JP2000526615A JP2002526029A (ja) 1997-12-29 1998-12-21 ポリアニオン性ドメインを含んで成る修飾された酵素
AU17515/99A AU1751599A (en) 1997-12-29 1998-12-21 Modified enzymes comprising a polyanionic domain

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6264925B1 (en) * 1996-10-11 2001-07-24 Novozymes A/S Cellulose binding domains (CBDs) for oral care products
WO2001049255A3 (fr) * 1999-12-30 2002-02-21 Univ Louisville Res Found Procedes et compositions permettant d'inhiber l'adherence des les micro-organismes
US20100158889A1 (en) * 2004-06-10 2010-06-24 Saint Louis University, A Non-Profit Organization Enhancing the effect of therapeutic proteins on the central nervous system
US7972593B2 (en) 2004-06-10 2011-07-05 Saint Louis University Delivery of therapeutic agents to the bone

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4138476A (en) * 1977-08-03 1979-02-06 The United States Of America As Represented By The Secretary Of The Navy Plaque dispersing enzymes as oral therapeutic agents by molecular alteration
WO1982003008A1 (fr) * 1981-03-04 1982-09-16 Reynolds Eric Charles Inhibition de la carie

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4138476A (en) * 1977-08-03 1979-02-06 The United States Of America As Represented By The Secretary Of The Navy Plaque dispersing enzymes as oral therapeutic agents by molecular alteration
WO1982003008A1 (fr) * 1981-03-04 1982-09-16 Reynolds Eric Charles Inhibition de la carie

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6264925B1 (en) * 1996-10-11 2001-07-24 Novozymes A/S Cellulose binding domains (CBDs) for oral care products
WO2001049255A3 (fr) * 1999-12-30 2002-02-21 Univ Louisville Res Found Procedes et compositions permettant d'inhiber l'adherence des les micro-organismes
AU782624B2 (en) * 1999-12-30 2005-08-18 Board Of Trustees Of Miami University Methods and compositions for inhibiting adhesion by microorganisms
US20100158889A1 (en) * 2004-06-10 2010-06-24 Saint Louis University, A Non-Profit Organization Enhancing the effect of therapeutic proteins on the central nervous system
US7972593B2 (en) 2004-06-10 2011-07-05 Saint Louis University Delivery of therapeutic agents to the bone
US8226940B2 (en) * 2004-06-10 2012-07-24 Saint Louis University Enhancing the effect of therapeutic proteins on the central nervous system

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