US20150175940A1 - Compositions and methods for reducing cat allergens in the environment - Google Patents

Compositions and methods for reducing cat allergens in the environment Download PDF

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US20150175940A1
US20150175940A1 US14/570,310 US201414570310A US2015175940A1 US 20150175940 A1 US20150175940 A1 US 20150175940A1 US 201414570310 A US201414570310 A US 201414570310A US 2015175940 A1 US2015175940 A1 US 2015175940A1
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fel
protease
formulation
proteases
subtilisin
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George Wells
Delphine Tissot-Favre
Ebenezer Satyaraj
Juergen Eck
Daniel Meyer
Torsten Ertongur-Fauth
Alexander Pelzer
Scott Sherrill
Peichuan Sun
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Nestec SA
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Nestec SA
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Assigned to NESTEC SA reassignment NESTEC SA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: TISSOT-FAVRE, DELPHINE, SATYARAJ, EBENEZER, SUN, Peichuan, SHERRILL, SCOTT, WELLS, GEORGE, ERTONGUR-FAUTH, Torsten, ECK, JUERGEN, MEYER, DANIEL
Priority to US15/945,131 priority patent/US10889785B2/en
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K1/00—Housing animals; Equipment therefor
    • A01K1/015—Floor coverings, e.g. bedding-down sheets ; Stable floors
    • A01K1/0152—Litter
    • A01K1/0155—Litter comprising organic material
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/40—Feeding-stuffs specially adapted for particular animals for carnivorous animals, e.g. cats or dogs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/40—Feeding-stuffs specially adapted for particular animals for carnivorous animals, e.g. cats or dogs
    • A23K50/42—Dry feed
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/40—Feeding-stuffs specially adapted for particular animals for carnivorous animals, e.g. cats or dogs
    • A23K50/48—Moist feed
    • 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)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L9/00—Disinfection, sterilisation or deodorisation of air
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38609—Protease or amylase in solid compositions only
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38618—Protease or amylase in liquid compositions only
    • 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
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D2111/00—Cleaning compositions characterised by the objects to be cleaned; Cleaning compositions characterised by non-standard cleaning or washing processes
    • C11D2111/10—Objects to be cleaned
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21062—Subtilisin (3.4.21.62)

Definitions

  • the invention relates generally to reducing or preventing cat allergies or their symptoms, and specifically to compositions and methods for reducing the major cat allergen, Fel 1, from the environment.
  • Fel d 1 elicits IgE responses in 90-95% of patients with cat allergy (van Ree et al., 1999, J. Allergy Clin. Immunol. 104: 1223-1230) and accounts for 60-90% of the total allergenic activity in cat dander (Kleine-Tebbe, et al., 1993 Int. Arch. Allergy Immunol. 100: 256-262).
  • Posttranslational modification may also affect allergenicity, such as by introducing new epitopes or altering solubility, stability, size, susceptibility toward proteases, and/or uptake and processing by antigen-presenting cells (Aalberse, 2000, supra). Although glycosylation affects many of these processes, it is not determinative of allergenicity in and of itself. Many allergens are not glycosylated, whereas some are heavily glycosylated (Aalberse, 2000, supra). As a result, determination of the basis for allergenicity may require a detailed structural study of the allergen, and even that may not yield definitive results.
  • Fel d 1 is a 35-kDa tetrameric glycoprotein formed by two heterodimers. Each 18-kDa dimer is composed of two covalently linked chains derived from two independent genes, chain 1, comprising 70 residues, and chain 2 (of which there are two isoforms), comprising 90 or 92 residues. The three-dimensional structure of Fel d 1 has been determined (Kaiser et al., 2003, J. Biol. Chem. 278: 37730-37735, Kristensen et al., 1997, Biol. Chem. 378: 899-908).
  • the fold of the protein was found to bear a striking resemblance to that of uteroglobin, a steroid-inducible, cytokine-like molecule with anti-inflammatory and immunomodulatory properties.
  • the relative localization of three IgE epitopes was determined on the molecular surface of Fel d 1, at residues 15-28 (chain 2), 117-130, and 138-151 (chain 1), respectively (Kaiser et al., 2003, supra).
  • Fel d 1 is produced by sebaceous glands and squamous epithelial cells and is transferred to the fur by licking and grooming (Bartholome et al., 1985, J. Allergy Clin. Immunol. 76: 503-506; Charpin et al., 1991, J. Allergy Clin. Immunol. 88: 77-82; Dabrowski et al., 1990, J. Allergy Clin. Immunol. 86: 462-465). Fel d 1 is also present in the salivary, perianal, and lachrymal glands (Anderson et al., 1985, J. Allergy Clin. Immunol.
  • the allergen is present within and on the animal, and is also carried by small airborne particles to surfaces within the environment occupied by the cat.
  • Tolerance strategies entail establishment or reestablishment of a less harmful or more productive responses to exogenous allergens.
  • Tolerance-inducing strategies traditionally have involved allergen immunotherapy, wherein the sensitized individual is intentionally exposed to the allergen in a controlled manner, for example through a series of injections, or through oral or nasal absorption. Immunotherapy has been in use for over 100 years, and has been successful, although it may take years to establish an acceptable level of tolerance. While potentially effective in the specific individual receiving the treatment, tolerance strategies are expensive, invasive, time-consuming, and require experts such as doctors, immunologists, and the like, for administration. Treatments for tolerance also involve a certain level of risk associated with adverse reactions and negative outcomes.
  • U.S. Pat. No. 7,704,532 discloses methods purported to mitigate allergic reactions in humans and other susceptible animals by directly contacting the allergens, including cat allergens, with a composition containing an acidic salt solution, including salts of aluminum, calcium and/or magnesium, to clothing, surfaces, interiors, furniture, plant bedding, plants and the like.
  • U.S. Pat. No. 5,826,546 discloses a waterless method for shampooing a pet comprising using a foamable shampoo composition in combination with a dispenser capable of dispensing the composition as a foam.
  • the composition may contain one or more of: anionic detergents, nonionic detergents, amphoteric detergents, preservatives, antimicrobial agents, antioxidants, mild soaps, surfactants, skin conditioners such as aloe extracts, fragrances, agents for treating flea infestation such as melaleuca oil, a pH adjuster such as citric acid, depending on the particular need of the pet.
  • anionic detergents such as cat Fel d 1
  • U.S. 2011/0135750 discloses methods and compositions purported to denature allergens, such as cat Fel d 1, the composition comprising a combination of calcium salts and lanthanum salts.
  • 2004/0007251 discloses wet and dry wipe cleaners comprising an additive, such as a lectin, a protease and/or an enzyme inhibitor purportedly capable of binding to or cleaving an allergen, such as cat Fel d 1, and removing it from a surface.
  • U.S. 2006/0142394 discloses methods for inhibiting dust mite feces and denaturing animal hair keratin and/or plant pollen or spores using composition comprising an enzyme capable of breaking down polypeptides such that they cannot elicit an allergenic effect on humans.
  • 8,454,953 discloses methods for reducing or preventing allergies or symptoms of allergic reactions to allergens comprising contacting the source of the allergen with a composition comprising a molecule capable of inhibiting the ability of the allergen to bind to mast cells in an animal predisposed to having an allergic response.
  • the molecule can be an antibody specific for the allergen, such as an antibody specific for the cat Fel d 1 allergen.
  • One such method is performed by first cleaning surfaces in the environment and then applying a water-based solution that contains an ingredient derived from fruit and vegetable seed extracts, using a spray applicator to surfaces such as mattresses, carpeting, upholstered furniture, rugs and window treatments (see MASTERBLEND® Special Report on RESPONSIBLECARE SYSTEMTM ALLERGY RELIEF TREATMENTTM, url masterblend.net).
  • APDC, Inc is performed by first cleaning surfaces in the environment and then applying a water-based solution that contains an ingredient derived from fruit and vegetable seed extracts, using a spray applicator to surfaces such as mattresses, carpeting, upholstered furniture, rugs and window treatments (see MASTERBLEND® Special Report on RESPONSIBLECARE SYSTEMTM ALLERGY RELIEF TREATMENTTM, url masterblend.net).
  • AUER-RX® Anti-Allergen Spray which is a liquid composition said to be derived from plants and naturally occurring organic compounds, containing chlorine dioxide.
  • Another method involves directly applying a composition comprising shampoo and skin conditioning ingredients directly to an animal to remove allergens, including cat Fel d 1, on the animal's fur and skin (see ALLERPET®, produced by Allerpet, Inc., New York, N.Y.).
  • compositions that allow sufficient control of the level and/or potency of Fel d 1 to reduce, minimize, or prevent an allergic response in individuals predisposed to having such a response.
  • One or more of these other objects can be achieved using formulations, products, kits and methods comprising protease enzymes that interact with Fel d 1 and substantially degrade allergenic epitopes on the Fel d 1, thereby reducing or eliminating its allergenicity.
  • FIG. 1 SDS-PAGE showing that Fel d 1 is completely degraded by a Bacillus licheniformis subtilisin.
  • Natural Fel d 1 (1.9 ⁇ g) was incubated overnight with 0.1, 1 or 10 rfu s ⁇ 1 of Bacillus licheniformis subtilisin (Alcalase®, Novozymes) activity at 37° C. either in Tris/HCl buffer (200 mM, pH 7.8) or ammonium acetate buffer (100 mM, pH 4.0.
  • the subtilisin (10 rfu s ⁇ 1 ) without Fel d 1 and Fel d 1 without the subtilisin served as controls.
  • FIG. 2 Histogram showing residual Fel d 1 concentration after protease-catalyzed degradation as determined by ELISA.
  • Natural Fel d 1 (2.5 ⁇ g) was incubated overnight with all tested commercial proteases (except for BLAP) in 20 ⁇ l of the optimal buffer at minimally required activities to achieve complete degradation of Fel d 1 (c.f. Table 2).
  • the samples were diluted and analyzed by ELISA with time intervals of 20 and 30 minutes. As a result of the dilution, an initial concentration of 12 ng ml ⁇ 1 of Fel d 1 was expected (i.e. in the absence of degradation).
  • the negative control contained no Fel d 1
  • the positive controls contained Fel d 1 incubated with the different buffers: Pos Ctrl 1: Tris/HCl buffer (200 mM, pH 7.8), Pos Ctrl 2: sodium phosphate buffer (100 mM, pH 7.8), Pos Ctrl 3: sodium phosphate buffer (100 mM pH 6.0), Pos Ctrl 4: ammonium acetate buffer (100 mM, pH 4.0). Protease-catalyzed degradations were performed in duplicates.
  • FIG. 3 Histogram showing keratin-hydrolyzing activity of the proteases as determined by TNBS assay. Keratin powder was incubated overnight with the hit proteases at concentrations and under reaction conditions that had been found to be optimal for Fel d 1 degradation (c.f. Table 2). Keratin-degradation products were determined spectrophoto-metrically at 405 nm in 10-fold dilutions of the samples after coupling reaction with picrylsulfonic acid (TNBS). The data represent the difference between absorbance determined in the samples and in controls with the same buffer but without protease. Average and standard deviation were determined from three independent repetitions of the experiment.
  • FIG. 4 Histogram showing celltoxic effect of proteases on human keratinocytes as determined by Sulforhodamin B and WST-1 assays.
  • Confluently grown primary human keratinocytes were incubated overnight with hit proteases (in cell cultivation medium) at concentrations that were identified to be minimally required to degrade 125 ⁇ g m ⁇ 1 of Fel d 1 (c.f. Table 2).
  • hit proteases in cell cultivation medium
  • remaining cell vitality was determined for all cells per well (adherent and detached cells) by staining (of metabolically active cells) with WST-1.
  • the remaining cell vitality of the adherent cells alone was then determined by staining (of the proteins of living cells) with Sulfrohodamin B.
  • Cells that were not incubated with proteases served as control (corresponds to 100% remaining cell vitality). Average and standard deviation were determined from three independent repetitions of the experiment.
  • FIG. 5 Histogram showing effect of isopropanol on the protease-catalyzed degradation of Fel d 1.
  • Hit proteases were incubated overnight with 0, 2.5%, 5.0%, 7.5%, 10.0% and 12.5% of isopropanol at concentrations and under reaction conditions that were found to be optimal for Fel d 1 degradation (c.f. Table 2).
  • the samples were diluted and analyzed by ELISA with a time interval of 20 minutes.
  • the indicated residual Fel d 1 concentration represents the percentage of Fel d 1 determined for the samples as compared to the Fel d 1 for the respective buffer controls with maximal isorpropanol concentration but without protease. Average and standard deviation were determined from three independent repetitions of the experiment.
  • FIG. 6 Graph showing effect of Tween® 20 on the protease-catalyzed degradation of Fel d 1.
  • Hit proteases were incubated overnight with 0, 10%, 20%, and 30% of Tween® 20 at concentrations and under reaction conditions that were found to be optimal for Fel d 1 degradation (c.f. Table 2).
  • the samples were diluted and analyzed by ELISA with a time interval of 20 minutes.
  • the indicated residual Fel d 1 concentration represents the percentage of Fel d 1 determined for the samples as compared to the Fel d 1 for the buffer controls with the respective Tween® 20 concentrations but without protease. Average and standard deviation were determined from three independent repetitions of the experiment.
  • FIG. 7 Histogram showing effect of cysteine and different concentrations of skim milk on the papain-catalyzed degradation of different concentrations of Fel d 1 in artificial cat saliva during overnight incubation at 37° C. 1.25, 12.5 and 125 ⁇ g ml ⁇ 1 natural Fel d 1 was incubated overnight with 4.65 ⁇ g ml ⁇ 1 of papain (corresponds to 60 rfu s ⁇ 1 in 20 ⁇ l reaction mix, c.f. Table 2) at 37° C. in artificial cat saliva with 0, 71, 714 and 7140 ⁇ g ml ⁇ 1 skim milk with and without 40 mM of cysteine.
  • FIG. 8 Histogram showing determination of Fel d 1 by ELISA after incubation for 1 hour and overnight at 37° C. with different concentrations of Bacillus licheniformis subtilisin (Protex® 6L) in artificial cat saliva (extreme conditions). 125 ⁇ g ml ⁇ 1 natural Fel d 1 was incubated for 0 min, 1 hour or 20 hours (overnight) with 0, 9.5, 95, 950 and 9500 ⁇ g ml ⁇ 1 of the subtilisin at 37° C. in artificial cat saliva (extreme conditions). Reaction was stopped by addition of 1 mM PMSF. Samples were diluted and analyzed by ELISA with a time interval of 30 minutes. As a result of the dilution, a maximal concentration of 12 ng ml ⁇ 1 of Fel d 1 was expected (i.e., in the absence of degradation). Average and standard deviation were determined from two independent repetitions of the experiment.
  • FIG. 9 Histogram showing results of testing papain and Bacillus licheniformis subtilisin (Protex 6L) at 1000-fold increased concentrations for the degradation of Fel d 1 in artificial cat saliva after incubation for 5 to 60 min. 4.5 mg ml ⁇ 1 of papain and 9.5 mg ml ⁇ 1 of the subtilisin were incubated for 0, 5, 10, 15 and 60 min with Fel d 1 in artificial cat saliva at 37° C. under normal and extreme conditions. Reaction was stopped by addition of 100 ⁇ M E64 (for papain) and 1 mM PMSF (for subtilisin). Samples were diluted and analyzed by ELISA with a time interval of 30 minutes.
  • the indicated residual Fel d 1 concentration represents the percentage of Fel d 1 determined for the samples as compared to the Fel d 1 for the respective buffer controls without protease. Average and standard deviation were determined from three independent repetitions of the experiment. Abbr.: n.d., not determined
  • FIG. 10 Histogram showing results of testing different combinations of papain and Bacillus licheniformis subtilisin (Protex® 6L) concentrations (at constant ratio) for the degradation of Fel d 1 in artificial cat saliva after incubation for 0 to 60 min.
  • the different combinations of papain and subtilisin concentrations (4.5 and 9.5 ⁇ g ml ⁇ 1 ; 45 and 95 ⁇ g ml ⁇ 1 ; 450 and 950 ⁇ g ml ⁇ 1 ; 4500 and 9500 ⁇ g ml ⁇ 1 of papain and subtilisin, respectively) were incubated for 0, 5, 10, 15 and 60 min with Fel d 1 in artificial cat saliva at 37° C. under normal and extreme conditions.
  • Reaction was stopped by addition of 100 ⁇ M E64 and 1 mM PMSF. Samples were diluted and analyzed by ELISA with a time interval of 30 minutes. The indicated residual Fel d 1 concentration represents the percentage of Fel d 1 determined for the samples as compared to the Fel d 1 for the respective buffer controls without protease. Average and standard deviation were determined from two independent repetitions of the experiment.
  • FIG. 11 Histogram showing results of testing different combinations of papain and Bacillus licheniformis subtilisin (Protex® 6L) concentrations (with constant papain concentration) for the degradation of Fel d 1 in artificial cat saliva after incubation for 0 to 10 min.
  • the different combinations of papain and subtilisin concentrations (4500 and 1188 ⁇ g ml ⁇ 1 ; 4500 and 2375 ⁇ g ml-1; 4500 and 4750 ⁇ g ml-1; 4500 and 9500 ⁇ g ml-1 of papain and subtilisin, respectively) were incubated for 0, 5 and 10 min with Fel d 1 in artificial cat saliva at 37° C. under normal and extreme conditions.
  • Reaction was stopped by addition of 100 ⁇ M E64 and 1 mM PMSF. Samples were diluted and analyzed by ELISA with a time interval of 30 minutes. The indicated residual Fel d 1 concentration represents the percentage of Fel d 1 determined for the samples as compared to the Fel d 1 for the respective buffer controls without protease. Average and standard deviation were determined from two independent repetitions of the experiment.
  • FIG. 12 Histogram showing results of testing different combinations of papain and Bacillus licheniformis subtilisin (Protex® 6L) concentrations (with constant subtilisin concentration) for the degradation of Fel d 1 in artificial cat saliva after incubation for 0 to 10 min.
  • the different combinations of papain and subtilisin concentrations (565 and 1188 ⁇ g ml ⁇ 1 ; 1125 and 1188 ⁇ g ml ⁇ 1 ; 2250 and 1188 ⁇ g ml ⁇ 1 ; 4500 and 1188 ⁇ g ml ⁇ 1 of papain and subtilisin, respectively) were incubated for 0, 5 and 10 mM with Fel d 1 in artificial cat saliva at 37° C. under normal and extreme conditions.
  • Reaction was stopped by addition of 100 ⁇ M E64 and 1 mM PMSF. Samples were diluted and analyzed by ELISA with a time interval of 30 minutes. The indicated residual Fel d 1 concentration represents the percentage of Fel d 1 determined for the samples as compared to the Fel d 1 for the respective buffer controls without protease. Average and standard deviation were determined from two independent repetitions of the experiment.
  • allergy is synonymous with “allergic response” or “allergic reaction”.
  • Each of the terms refers to a state of immune responsiveness in an animal specific to an exogenous antigen (or “allergen”) that is not otherwise harmful to the animal.
  • a “symptom” of an allergic response refers to any measure of the aforesaid immune responsiveness, e.g. on the molecular level (including measurement of a activity or expression of a protein, or transcript or gene), the cellular level, organ level, systemic level, or organism level. Such symptoms can comprise one or more such levels.
  • Symptoms may include generalized phenomena such as inflammation, respiratory complaints, swelling, or distress typically associated with allergy, rhinitis, edema, and allergic skin disorders including but not limited to atopic dermatitis (e.g. eczema), urticaria (e.g. hives) and angioedema, and allergic contact dermatitis.
  • atopic dermatitis e.g. eczema
  • urticaria e.g. hives
  • angioedema e.g. hives
  • More specific phenomena that are “symptoms” of an allergic response include any measurable or observable change, for example at the cellular level, including but not limited to local or systemic changes in cell populations, eosinophilia, recruitment and/or activation of immune cells, including, for example, mast cells and/or basophils, changes in antigen-presenting cells (including but not limited to FccRI-bearing dendritic cells), intracellular or molecular changes, including measurement or observations of one or more steps in an immunological cascade, release of intracellular compounds that mediate an allergic response (e.g. mediators), and changes in one or more cytokines (e.g.
  • IL-3, IL-5, IL-9, IL-4, or IL-13 or related compounds or antagonists thereof.
  • IL-3, IL-5, IL-9, IL-4, or IL-13 or related compounds or antagonists thereof.
  • the term “environment” as used herein has three components as they relate to the cat allergen Fel d 1. These are sometimes referred to as “around the cat,” “on the cat” and “in the cat.”
  • the environment “around the cat” refers to a local environment of an individual who may suffer from cat allergies, and/or a local environment inhabited by a cat. For example a house, room, car, office, hotel, yard, garage, and the like, could each be “environments” as used herein. Any inanimate surface on which an allergen may be disposed is considered part of the environment. Airborne particles containing the allergen are also considered part of the environment.
  • the environment can also comprise a part or all of an animal that is the source of an allergen, e.g., the cat's skin, fur or saliva on the cat's skin or fur (“on the cat”) or the cat's oral cavity or saliva therein (“in the cat”).
  • an allergen e.g., the cat's skin, fur or saliva on the cat's skin or fur (“on the cat”) or the cat's oral cavity or saliva therein (“in the cat”).
  • an “individual” means an individual animal of any species or kind, including a human.
  • single package means that the components of a kit are physically associated in or with one or more containers and considered a unit for manufacture, distribution, sale, or use.
  • Containers include, but are not limited to, bags, boxes or cartons, bottles, packages of any type, design, or material, over-wrap, shrink-wrap, affixed components (e.g. stapled, adhered, or the like), or combinations of any of the foregoing.
  • a single package kit may provide containers of individual compositions and/or food compositions physically associated such that they are considered a unit for manufacture, distribution, sale, or use
  • kits means that the components of a kit are associated by directions on one or more physical or virtual kit components instructing the user how to obtain the other components, e.g., in a bag or other container containing one component and directions instructing the user to go to a website or personal device application (“app”), contact a recorded message or a fax-back service, view a visual message, or contact a caregiver or instructor to obtain, for example, instructions on how to use the kit, or safety or technical information about one or more components of a kit.
  • apps personal device application
  • Examples of information that can be provided as part of a virtual kit include instructions for use; safety information such as material safety data sheets; poison control information; information on potential adverse reactions; clinical study results; dietary information such as food composition or caloric composition; general information on physical activity, exercise, metabolism, endurance and the like.
  • safety information such as material safety data sheets; poison control information; information on potential adverse reactions; clinical study results; dietary information such as food composition or caloric composition; general information on physical activity, exercise, metabolism, endurance and the like.
  • dry matter basis means that an ingredient's concentration or percentage in a composition is measured or determined after any free moisture in the composition has been removed.
  • Ranges are used herein in shorthand, so as to avoid having to set out at length and describe each and every value within the range. Any appropriate value within the range can be selected, where appropriate, as the upper value, lower value, or the terminus of the range.
  • compositions, methods, articles of manufacture, kits and packages are provided for reducing or eliminating the cat allergen Fel d 1 from the environment.
  • the invention springs in part from the inventors' discovery that certain enzymes, particularly proteolytic enzymes (proteases), are able to degrade the antigenic epitopes from Fel d 1, thereby reducing or eliminating its allergenic effect.
  • One aspect of the invention features a formulation for reducing or eliminating allergenicity of Fel d 1, comprising at least one protease that interacts with the Fel d 1 and substantially degrades allergenic epitopes on the Fel d 1.
  • the effective proteases are selected from serine proteases, thiol proteases, aspartyl proteases, zinc metalloproteases, or any combination thereof.
  • the serine proteases include subtilisin, proteinase K, trypsin, alpha-chymotrypsin, endoproteinase Glu-C and endoproteinase Lys-C. If subtilisin is used, it may be sourced from a species of Bacillus selected from B. licheniformis, B. clausii, B. haloudurans, B. lentus, B. amyloliquefaciens, B. subtilis or any combination thereof.
  • the thiol proteases can include bromelain, papain and ficin, which are obtained from plant sources, e.g., pineapple, papaya and fig plants.
  • the aspartyl proteases can include bovine chymosin, endothiapepsin (e.g., from Chryphonectria parasitica ), mucorpepsin/rennin (e.g., from Mucor miehei ) pepsin (e.g., of porcine origin) and aspergillopepsin (e.g., from Trichoderma reesei, Aspergillus niger or Aspergillus oryzae ).
  • bovine chymosin e.g., from Chryphonectria parasitica
  • mucorpepsin/rennin e.g., from Mucor miehei
  • pepsin e.g., of porcine origin
  • aspergillopepsin e.g., from Trichoderma reesei, Aspergillus niger or Aspergillus oryzae .
  • the zinc metalloproteases can include thermolysin (e.g., from Bacillus thermoproteolyticus rokko or Geobacillus sp.) and endoproteinase Asp-N, (e.g., from Flavobacterium meningosepticum ).
  • thermolysin e.g., from Bacillus thermoproteolyticus rokko or Geobacillus sp.
  • endoproteinase Asp-N e.g., from Flavobacterium meningosepticum
  • proteases suitable for use in the present invention.
  • the proteases may be purified from a biological source or they can be obtained from commercial sources. Certain exemplary commercial sources are shown in the table; many others are available.
  • the protease, or combination of proteases, in the formulation reduces Fel d 1 binding to anti-Fel d 1 antibodies by at least 50% under conditions applicable to the protease or combination thereof, as described in the Examples, as measured by one or more of ELISA, SDS-PAGE, or any other known method for detecting the presence of the antigen and/or antigenic epitopes or the binding of the antigen to Fel d 1-specific antibodies.
  • the proteases can include one or more of subtilisin, proteinase K, alpha-chymotrypsin, trypsin, endoproteinase Lys-C, bromelain, papain, ficin, chymosin, endothiapepsin, mucorpepsin/rennin, pepsin, aspergillopepsin, thermolysin and endoproteinase Asp-N.
  • the protease, or combination of proteases, in the formulation reduces Fel d 1 binding to anti-Fel d 1 antibodies by at least 90% under conditions applicable to the protease or combination thereof, as described in the Examples, as measured by one or more of ELISA, SDS-PAGE, or any other known method for detecting the presence of the antigen and/or antigenic epitopes or the binding of the antigen to Fel d 1-specific antibodies.
  • the proteases can include one or more of subtilisin (e.g., from Bacillus licheniformis, B. clausii, B. lentus B. amyloliquefaciens and/or B.
  • subtilis subtilis ), trypsin (e.g., porcine), alpha-chymotrypsin (e.g., bovine), bromelain, papain, ficin, chymosin (e.g., bovine), endothiapepsin, mucorpepsin/rennin, pepsin, aspergillopepsin (e.g., from Trichoderma reesei, Aspergillus niger or Aspergillus oryzae ), thermolysin and endoproteinase Asp-N.
  • trypsin e.g., porcine
  • alpha-chymotrypsin e.g., bovine
  • bromelain papain
  • ficin ficin
  • chymosin e.g., bovine
  • endothiapepsin mucorpepsin/rennin
  • pepsin e.g., aspergillopepsin (e.
  • the proteases include one or more of papain, subtilisin from B. licheniformis , aspergillopepsin from A. oryzae , endoproteinase Asp-N, bromelain, ficin, alpha-chymotrypsin, endothiapepsin from Cryphonectria parasitica, pepsin and thermolysin from Geobacillus sp.
  • the proteases include one or more of papain, subtilisin from B. licheniformis, aspergillopepsin from A. oryzae and endoproteinase Asp-N.
  • proteases may be particularly suitable for use in compositions containing alcohol or certain detergents.
  • the protease, or combination of proteases reduces Fel d 1 binding to anti-Fel d 1 antibodies by at least 50% in the presence of up to 7.5% isopropanol under reaction conditions as set forth for the respective classes of enzymes in Table 2.
  • These proteases can include one or more of ficin, bromelain, papain, aspergillopepsin from Aspergillus oryzae , or endoproteinase Asp-N.
  • the protease, or combination of proteases reduces Fel d 1 binding to anti-Fel d 1 antibodies by at least 50% in the presence of non-ionic detergent, e.g., polysorbate 20 (Tween® 20) at up to 10%, under reaction conditions as set forth in Table 2.
  • non-ionic detergent e.g., polysorbate 20 (Tween® 20)
  • These proteases can include one or more of aspergillopepsin from A. oryzae , endoproteinase Asp-N, subtilisin from B. licheniformis, papain, bromelain and ficin. I was thinking the range should be broader?
  • the formulation can further comprise an additive that enhances the efficiency of the enzyme in degrading the Fel d 1.
  • the additive is cysteine or calcium salts/ions (Ca 2+ ), or a combination of cysteine and calcium salts/ions, or compounds that form cysteine or calcium salts/ions in situ.
  • the formulation is disposed within a composition selected from, for example: liquid, solid or powder cleaning agent, spray, moist cloth, wipe, sponge, water-dissolvable tablet, filter, food, oil or water supplement, vacuum cleaner filter or additive, granule, detergent, carpet and room deodorizer, litter, litter additive, mitt, additive for non-woven products, washing machine pod (tablet), multi chamber liquid tablets.
  • the formulations can be disposed of in an oral preparation.
  • the formulation can be disposed within a toy, e.g. cat toy, including edible and non-edible toys.
  • the formulation can comprise a granule, powder or tablet that is reconstituted with a liquid (e.g., water, buffer or other liquid) prior to use.
  • the formulation can comprise a liquid or spray that can be applied to a surface or on the animal.
  • the spray does not aerosolize. Suitable sprays that do not aerosolize can be made in accordance with methods known in the art.
  • the formulations contain enzymes that are generally recognized as safe for use in foods and cosmetics.
  • Such formulations are particularly suitable for use in soaps, shampoos, foams/mousses, powders, sprays, conditioners, rinses, gels, lotions, collars, dispersants, moist mitts, wipes, dentifrices and/or mouthwashes, or any other compositions suitable for applying to the skin, hair, fur or oral cavity, or disposed within an edible composition, or formulated for adding to any such compositions.
  • the protease has substantially no interaction with substances that include keratin, collagen, elastin, fibronectin, other proteins and fibers or fabric (in clothing, carpet, upholstery, curtains and bedding).
  • Suitable proteases that are inactive on such proteins include, but are not limited to papain and pepsin.
  • the protease may be active against keratin and may be useful in reducing keratin buildup that can occur when the pet is shampooed.
  • keratin a group consisting of keratin, keratin, keratin, keratin, aspergillopepsin, endothiapepsin, certain subtilisins, thermolysin, endoproteinase Asp-N and alpha-chymotrypsin were found to degrade keratin (Example 2).
  • Another aspect of the invention features a method of making a formulation for reducing or eliminating allergenicity of Fel d 1.
  • the method comprises combining at least one protease that interacts with the Fel d 1 and substantially degrades allergenic epitopes on the Fel d 1 with a medium in which the protease is active or can be made active prior to use.
  • the medium can be any medium that satisfies the aforementioned requirement, including but not limited to liquids, solids, granules, powders, moist cloths, wipes, mitts, sponges, water-dissolvable tablets, filters, foods, dietary supplements, beverages, concentrates to add to foods and beverages, vacuum cleaner filters or additives, detergents, carpet, upholstery and room deodorizers, litters, litter additives, mitts, washing machine pods (tablet), to name just a few.
  • the proteases can include any of the proteases discussed hereinabove, or any combination thereof, in an amount suitable to degrade the Fel d 1 sufficiently to inhibit it from binding to anti-Fel d 1 antibodies during the time the formulation is exposed to the Fel d 1 in the environment (around the cat, on the cat or in the cat).
  • formulations intended to be applied to an environment and removed shortly thereafter e.g., within minutes or hours
  • a cleaning agent, pet shampoo or oral product should contain a concentration of protease(s) sufficient to degrade Fel d 1 within that time period.
  • the kinetic analyses discussed in the Examples provide concentration ranges of the proteases to achieve such a result. For example, kinetic analysis of papain and subtilisin from B.
  • licheniformis in artificial cat saliva revealed that, under normal cat saliva conditions, more than 80% of Fel d 1 was degraded within 5 minutes by papain at 4.5 mg ml ⁇ 1 and by the subtilisin at 9.5 mg ml ⁇ 1 .
  • Formulations intended to remain in place for an extended time such as an upholstery or fabric spray or a leave-in mousse, gel, shampoo or spray, would require a lesser concentration of protease(s).
  • Fel d 1 degradation by any method known in the art.
  • degradation of Fel d 1 can be measured by ELISA and/or SDS-PAGE, or combinations thereof.
  • ELISA assays are used to determine the amount of binding of Fel d 1 to Fel d 1—specific antibodies following exposure to the protease(s).
  • protease activity is measured and standardized using a standard protease assay. Using such an assay, protease activities can be standardized across proteases and assay conditions.
  • a typical protease activity assay system includes a protease substrate and a suitable buffer to support protease activity, and may also include diluents or solvents and other agents useful for the activity of proteases (e.g., cysteine).
  • the substrate is designed such that cleavage of the substrate by the protease generates a detectable product.
  • one type of protease assay utilizes a protein, e.g., casein, derivatized to contain fluorophore that is quenched until the protease cleaves the protein. Upon cleavage by the protease, the fluorophore is separated from the quencher and yields a quantitatively detectable fluorescence signal.
  • a protein e.g., casein
  • an assay may utilize a succinylated protein, such as casein, as a substrate. Hydrolysis of this substrate in the presence of protease results in the release of peptide fragments with free terminal amino groups. These peptides are reacted with trinitrobenzene sulfonic acid (TNBS), followed by measurement of the absorbance increase that results from the formation of yellow colored TNB-peptide adducts.
  • TNBS trinitrobenzene sulfonic acid
  • the enzyme activities determined by a protease assay can be expressed generally as relative product units per second and normalized to the amount of enzyme preparation (rpu s ⁇ 1 g ⁇ 1 ).
  • the enzyme activities can be expressed as relative fluorescence units per second and normalized to the amount of enzyme preparation (rfu).
  • the method comprises combining the requisite amount of the protease with the medium.
  • the amount of protease to include in the formulation will depend on whether it is prepared in “ready to use” form, or as a concentrate for later dilution.
  • Another aspect of the invention features a method of reducing or eliminating allergenic Fel d 1 from the environment.
  • the method comprises contacting an element of the environment where Fel d 1 is present with a formulation comprising at least one protease that interacts with the Fel d 1 and substantially degrades allergenic epitopes on the Fel d 1, thereby reducing or eliminating allergenic Fel dl from the environment.
  • the environment is “around the cat.”
  • the Fel d 1 is present on an inanimate surface and the formulation is applied to the surface. Typical surfaces can include counters, floors, walls, furniture, upholstery and clothing, to name a few.
  • the Fel d 1 is airborne and the formulation is contacted with the air.
  • the formulation can be disposed within a filter through which air passes, such as an air filter for a fan, heater or air conditioner, or a vacuum cleaner filter.
  • the environment is “on the cat.” and the formulation is applied to the portion of the animal on which the Fel d 1 is present.
  • the Fel d 1 may be present on the hair, fur or external skin of the animal, or on saliva deposited on the hair, fur or skin of the animal.
  • the environment is “in the cat,” typically in the mouth of the animal where Fel d 1—containing saliva is produced, and the formulation is applied as a dentifrice, rinse, food, treat, film or strip foam or spray or beverage.
  • an article of manufacture comprising a formulation that includes at least one protease that interacts with the Fel d 1 and substantially degrades allergenic epitopes on the Fel d 1, and instructions for its use in reducing or eliminating allergenic Fel dl from the environment.
  • the product can be formulated as a liquid, solid or powder cleaning agent, spray, moist cloth, wipe, sponge, water-dissolvable tablet, detergent, carpet or fabric deodorizer, litter, litter additive, mitt, additive for non-woven or woven products, washing machine pod (tablet), multicompartment liquid tablet, for application to an inanimate surface.
  • the product can be formulated as an air filter additive for contacting airborne Fel d 1.
  • the product is formulated for application or administration to an animal that produces the Fel d 1.
  • the product can be formulated as a shampoo, conditioner, rinse, mousse, gel, spray, lotion or powder for application to hair, fur or external skin of the animal.
  • the product can be formulated as a dentifrice, food, treat, or additive to the animal's food or water, for application to the mouth of the animal.
  • Such products and articles of manufacture as disclosed herein can be effective for inactivating Fel d 1 in an environment.
  • the products and/or articles can reduce or inactivate Fel d 1 by at least 10% in their respective environments.
  • Fel d 1 can be inactivated by the products and/or articles by at least 50%.
  • Fel d 1 can be inactivated by at least 1%, 5%, 15%, 20%, 30%, 40%, 60%, 70%, 80%, 90%, or even by at least 95%.
  • the product comprises a food or other edible composition for reducing or eliminating the allergenicity of the cat allergen Fel d 1.
  • the food product is a dry pet food or pet treat comprising a protease-containing formulation.
  • the formulation can be applied by dusting or coating the formulation onto the dry food composition prior to packaging or shipping. Because the food product is dry, activity of the proteases can be preserved during shipping and storage.
  • the formulation can also be provided as a concentrate that is dissolved prior to feeding, or a sachet or pouch containing a powdered or granular protease preparation that can be sprinkled onto a food composition or mixed into water or other liquid beverages.
  • the formulation can be provided as a liquid formulation that can be applied, for example, directly to a food composition (dry, moist or intermediate), or to water or other liquid beverages.
  • kits for reducing or eliminating the allergenicity of the cat allergen Fel d 1 comprise one of the aforementioned protease-containing formulations and instructions for their use in removing allergenic Fel d 1 from the environment.
  • the kit comprises a composition for cleaning or otherwise removing Fel d 1 from the environment around the cat, such as a surface of an inanimate object.
  • a composition for cleaning or otherwise removing Fel d 1 from the environment around the cat such as a surface of an inanimate object.
  • these include, for instance, a liquid, solid or powder cleaning agent, spray, moist cloth, wipe, sponge, carpet and room deodorizer, granule or detergent, said composition comprising the Fel d 1-degrading formulation, along with instructions for use.
  • the protease formulations can be provided in concentrated form and the instructions will contain directions for dilution.
  • a multi-component kit is provided.
  • a cleaning kit can comprise a cleaning agent in one container and a protease formulation in another, and the instructions can direct the user on how to combine the components prior to use.
  • Such embodiments can be particularly beneficial if the proteases in the formulation are sensitive to ingredients in the cleaning agent, such that they would be inactivated by the cleaning agent with prolonged
  • the kit may comprise a composition for washing fabric, such as a liquid, solid or powder, water-dissolvable tablet or washing machine pod (tablet), said composition comprising the Fel d 1-degrading formulation, along with instructions for use.
  • the kit comprises an air filter, such as a vacuum cleaner filter or additive, comprising the Fel d 1-degrading formulation, along with instructions for use.
  • the protease formulations again can be provided in concentrated form and the instructions can contain directions for dilution.
  • a multi-component kit is provided.
  • a laundry kit can comprise a laundry detergent in one container and a protease formulation in another, and the instructions can direct the user on how to combine the components prior to use.
  • An air filter kit can comprise the air filter and the protease formulation, and instructions for combining the two to reduce or eliminate airborne Fel d 1.
  • the kit comprises treatments for “on the cat,” e.g., fur, hair or skin.
  • Such kits can include a soap, shampoo, powder, spray, conditioner, rinse, mousse, gel, lotion, collar, dispersant or moist mitt or wipe suitable for applying to the skin, hair or fur or formulated for adding to any such compositions, along with instructions for use.
  • the protease-containing formulation can be included within the fur/hair treatment composition, or it can be provided separately, as a concentrate or otherwise, and mixed with the fur/hair treatment prior to use.
  • the kits can also contain a combination of fur/hair treatments.
  • a kit can comprise a mitt or cloth for wiping the fur and a liquid protease formulation.
  • kits can comprise a shampoo and a rinse, spray, gel or mousse wherein the shampoo is a standard pet shampoo that does not contain the protease formulation and the rinse, spray, gel or mousse contains the protease formulation.
  • the kit comprises an oral composition, such as a liquid, solid or powder, moist cloth, wipe, dentifrice or mouthwash, said composition comprising the Fel d 1-degrading formulation, suitable for applying to the oral cavity, or formulated for adding to any such compositions, along with instructions for use.
  • the kit comprises a dental kit comprising a water-dissolvable tablet that can be administered to the animal, such as by dissolving in drinking water.
  • the kit comprises an edible form of a composition described herein in a sachet or pouch attached to or recommended to be used alongside to a food composition, such as a pet food package, along with instructions for mixing the edible composition into the food, adding the composition to the food, or dissolving, mixing, or adding the composition to a fluid that is to be administered to the animal receiving the food, such as drinking water.
  • a food composition such as a pet food package
  • the kit comprises at least a food composition described herein comprising the Fel d 1-degrading formulation, along with instructions for use.
  • a concentrated form of the composition is provided, and also provided is a tool or device for conveniently measuring a suitable amount of the concentrate for mixing with, adding to, diluting, or dissolving with a food or fluid to be provided to the animal.
  • the composition in edible form is provided in a convenient dosage in a series of identical packages, such that one package of the composition is added to one package (e.g. can) of pet food without a requirement for measuring.
  • Such kits can be provided such that for each package of pet food in a point-of-sale package, there is one package of Fel d 1-degrading formulation provided. For example, twelve cans of food and twelve packages of composition are packaged together in a single kit.
  • the kit can comprise a composition, in concentrated or other form, instructions for use, including, if required, instructions for preparation of a suitable dilution, and optionally one or more of a diluent or extender, a tool or measuring device for preparing a suitable dilution, and an applicator such as a sprayer, duster, wipe, or the like.
  • a suitable dilution including, if required, instructions for preparation of a suitable dilution, and optionally one or more of a diluent or extender, a tool or measuring device for preparing a suitable dilution, and an applicator such as a sprayer, duster, wipe, or the like.
  • an applicator such as a sprayer, duster, wipe, or the like.
  • kits may include devices, applicators, dilutors, and the like that are automatic or the automate the dosing, dilution, mixing, addition, or application of the composition for an appropriate use.
  • the kits described herein they can be provided as sachets or bundled with other products to maximize convenience, compliance, and efficiency of use and purchase.
  • the kits can include, or be bundled with any or all of food for the pet, bedding for pet, shampoo or cosmetic items for the pet, medicine for the pet.
  • kits can also be provided as virtual kits.
  • the kit provides instructions in a virtual environment in combination with one or more physical kit components, such as those described above.
  • the kit contains at least one composition described herein, and other components, including optional components.
  • the kits may contain the kit components in any of various combinations and/or mixtures.
  • the kit contains a packet containing one or more compositions and a container of food for consumption by an animal.
  • the kit may contain additional items such as a device for mixing the compositions and ingredients or a device for containing the admixture, e.g., a food bowl.
  • the compositions are mixed with additional nutritional supplements such as vitamins and minerals that promote good health in an animal. Further information and instructions are provided in the virtual environment that is provided to the purchaser—i.e. directions to a website, faxback server, or an included computer readable device such as a CD-ROM and/or an application (“app”) for a device.
  • the invention provides a communication means, or a means for communicating information about or instructions for one or more of the formulations, methods, compositions, articles of manufacture, products and/or kits described herein for reducing the allergenicity of, or the amount of allergenic Fel d 1 from the environment.
  • the information pertains to formulations, compositions, articles of manufacture or products of the present invention.
  • the information pertains to methods or kits useful for practicing the invention described herein.
  • the information relates to combinations of any of the foregoing.
  • the communication means comprises one or more of text information, audio information, still or moving images, including animations, or video.
  • the communication means comprises one or more of a printed document, a static or dynamic electronic document, for example a hypertext document, a computer readable or digital storage medium, including but not limited to electronic, optical, or magnetic media of any type, audio information, an audio, audiovisual or visual display or presentation, or video information however encoded, wherein the communication means displays or contains information or instructions comprising any of the aforesaid.
  • the communication means comprises a web site, an FAQ (Frequently Asked Questions) page or file, an electronic file or collection of two or more electronic files of the same or different types, an email or email file, a visual display, kiosk, brochure, advertisement, package or product label, package or product insert, handout, public announcement, audiotape or electronic audio file embodied in any machine-readable or computer-readable medium, a videotape, videodisk, or electronic video file embodied in any machine readable or computer-readable medium, DVD, CD-ROM, app, or the like, or any combination of the foregoing containing such information or instructions.
  • FAQ Frequently Asked Questions
  • Useful information includes one or more of (1) methods and techniques for combining and administering the allergen-specific molecules and/or other components, (2) contact information for allergic animals or their guardians or caregivers to use if they have a question about the kit, the composition, or its use; (3) nutritional information about food compositions, and other components provided in any kit, (4) safety information including for example emergency information, and further contacts in the event of adverse reaction; poison control, material data safety sheets, (5) information useful for reordering, for example through automatic fulfillment systems; (6) general information about allergies, environmental allergens, and methods for minimizing or eliminating specific environmental allergens.
  • Useful instructions can include amounts for mixing and administration amounts and frequency.
  • the communication means is useful for instructing on the benefits of using the present invention and communicating the approved methods for administering the invention to an animal.
  • Another aspect of the invention provides a package comprising any one or more of the formulations, compositions, products and/or kits described herein.
  • the package has affixed thereto a label containing a word or words, picture, symbol, design, acronym, slogan, phrase, or other device, or combination thereof (the label “device”), that indicates that the contents of the package contains a protease formulation for reducing or eliminating allergenic Fel d 1 from the environment.
  • Candidate proteases were selected from several different families of proteases, including serine proteases, thiol proteases, aspartic acid proteases and zinc metalloproteases.
  • the ENZCHEK® Protease Assay Kit green fluorescence from Invitrogen, Inc. (Carlsbad Calif.) was used, which is based on casein as substrate, derivatized to contain fluorophore that is quenched until cleaving by the protease.
  • the enzyme activities determined by the protease assay were expressed as relative fluorescence units per second and normalized to the amount of enzyme preparation (rfu s ⁇ 1 g ⁇ 1 ).
  • different buffers and pH conditions were tested at 37° C.
  • Table 2 provides the pH of the buffer with the best performance, and the activity (increase of fluorescence per second, rfu s ⁇ 1 ) per amount of protease preparation (g ⁇ 1 ) as determined by the ENZCHEK® Protease Assay.
  • proteases tested showed optimal activity in reaction buffers at pH 7.8.
  • the aspartic acid proteases were optimally active at pH 4.0 or pH 6.0.
  • Some serine and aspartic acid proteases were activated by the addition of calcium salt/ions (Ca 2 ).
  • the thiol proteases were significantly more active when cysteine was added to the reaction buffer.
  • Endoproteinase Lys-C was the only protease that was not active in the ENZCHEK® Protease Assay under the tested conditions.
  • Fel d 1 degradation To characterize Fel d 1 degradation, different concentrations of candidate proteases were incubated with Fel d 1 at 37° C. overnight (-18 hours), followed by separation of proteins by gel electrophoresis (SDS-PAGE under non-reducing conditions) and COOMASSIE® staining.
  • a typical reaction mix to test Fel d 1 degradation contained 2.5 ⁇ g of natural Fel d 1 (nFel d 1, LOTOX® Natural Feld d 1 was obtained from Indoor Biotechnologies (LTN-FD1-1)) and varying concentrations of candidate proteases in a total reaction volume of 20 ⁇ l (final Fel d 1 concentration of 125 ⁇ g ml ⁇ 4 ).
  • Fel d 1 has been reported to be a 35 kDa tetrameric glycoprotein formed by two heterodimers of 18 kDa. Therefore, Fel d 1 degradation was assessed by the extent of disappearance of the Fel d 1 protein band at approximately 18 kDa. For most proteases, conditions were optimized to promote complete degradation of Fel d 1 with minimal protease activity. For all tested proteases, the minimal activity (rfu s ⁇ 1 ) as well as the corresponding amount of enzyme ( ⁇ g) that was required to completely degrade 2.5 ⁇ g Fel d 1 in 20 ⁇ l of buffer (with optimal pH for the respective protease) was estimated based on the absence of the Fel d 1 band on the SDS-PAGE gel. The results are summarized in Table 2.
  • subtilis 3525 U g ⁇ 1 4.97 * 10 8 pH 7.8 ⁇ 100 ⁇ 0.20 Protex ® 89L
  • Subtilisin B . subtilis 1930 U g ⁇ 1 1.53 * 10 7 pH 7.8 10-100 0.65-6.5 BLAP (Henkel)
  • Various serine proteases candidates were tested for their ability to degrade Fel d 1.
  • the Fel d 1 proteolytic activity of a subtilisin from B. licheniformis (bl) was determined.
  • Natural Fel d 1 (1.9 ⁇ g) was incubated with 0.1, 1 or 10 rfu s ⁇ 1 of the bl subtilisin overnight at 37° C. in 20 ⁇ l of either Tris/HCl buffer (200 mM, pH 7.8) or ammonium acetate buffer (100 mM, pH 4.0).
  • the subtilisin (10 rfu s ⁇ 1 ) without Fel d 1 and Fel d 1 without the subtilisin served as controls.
  • Fel d 1 was completely degraded by 10 rfu s ⁇ 1 of the bl subtilisin in Tris/HCl buffer, but not in ammonium acetate buffer conditions as determined by SDS-PAGE and COOMASSIE® staining ( FIG. 1 ).
  • natural Fel d 1 (2.5 ⁇ g) was incubated overnight at 37° C. with different amounts of papain, bromelain or ficin in sodium phosphate buffer (100 mM, pH 7.8) containing 40 mM cysteine. Buffer with cysteine alone, enzyme without Fel d 1 in cysteine-free buffer and Fel d 1 without enzyme (with and without cysteine) served as controls.
  • the thiol proteases bromelain, papain and ficin were all able to completely degrade Fel d 1 as determined by SDS-PAGE and COOMASSIE® staining.
  • the optimal conditions for bromelain, papain and ficin determined by the ENZCHEK® Protease Assay include a pH of 7.8 in the presence of cysteine.
  • the addition of 40 mM cysteine increased the activities of each of bromelain, papain and ficin by factors of 270, 60 and 460, respectively, as determined by the ENZCHEK® Protease Assay.
  • Table 2 shows the minimum amounts of these proteases required for degradation of 2.5 ⁇ g Fel d 1 in 20 ⁇ l cysteine-containing buffer.
  • Some of the candidate proteases were tested for their abilities to degrade 2.5 ⁇ g of natural Fel d 1 in 20 ⁇ l optimal reaction buffer at 37° C. within short incubation time points by adding a protease inhibitor after one or two hours to stop the reaction. Reactions were visualized by SDS-PAGE followed by COOMASSIE® staining. It was found that 50 rfu s ⁇ 1 (2.1 ⁇ g) of a bl subtilisin (ALCALASE® 2.4L) was sufficient to completely degrade Fel d 1 within one hour of incubation when using 1 mM phenylmethylsulfonyl fluoride (PMSF) as inhibitor.
  • PMSF phenylmethylsulfonyl fluoride
  • Fel d 1 specific antibodies were obtained from Indoor Biotechnologies Ltd (Indoor Biotechnologies Ltd (Cardiff, Wales). ELISA conditions were determined such that a reliable quantitation of natural Fel d 1 in a range of concentrations from 0.3 to 12 ng ml ⁇ 1 was possible.
  • Various candidate proteases as indicated in FIG. 2 , were incubated with Fel d 1 (2.5 ⁇ g) overnight in 20 ⁇ l of the optimal buffer at minimally required activities to achieve complete degradation of Fel d 1 (See optimized conditions reported in Table 2).
  • Fel d 1 concentration of 12 ng ml ⁇ 1 (initial substrate concentration) and analyzed by ELISA at two different assay reading time points (20 and 30 minutes). After incubating Fel d 1 with a candidate protease, plates coated with anti-Fel d 1 antibody were used to capture Fel d 1. Then, an enzyme-linked detecting antibody that binds Fel d 1 was added, followed by the addition of substrate, which was enzymatically converted to a detectable signal.
  • the proteases degrade Fel d 1, such that Fel d 1 epitopes required for binding to anti-Fel d 1 antibody are no longer present and/or accessible, the anti-Fel d 1 antibodies would not bind Fel d 1 and no signal would be detected by ELISA.
  • the ELISA was repeated but with the addition of a defined amount of Fel d 1. SDS-PAGE experiments as described above were conducted on all tested proteases to confirm Fel d 1 degradation and ELISA results.
  • Results are shown in FIG. 2 .
  • subtilisin serine proteases as well as trypsin and alpha-chymotrypsin; the thiol proteases papain, bromelain and ficin; the aspartic acid proteases mucorpepsin/rennin, pepsin, aspergillopepsin, endothiapepsin and chymosin; and the zinc-metalloproteases thermolysin (from Geobacillus ) and endoproteinase Asp-N. Degradation of Fel d 1 to completion could be achieved by increasing the concentration of several other of the proteases.
  • Fel d 1 degradation by proteases can suppress IgE mediated allergic reactions. Most of the tested proteases were able to completely degrade Fel d 1 and suppress the binding of the resultant degradation products to epitope specific antibodies.
  • Representative proteases from these experiments capable of completely degrading Fel d 1 include papain, subtilisin from B. licheniformis (bl subtilisin, PROTEX® 6L), aspergillopepsin from A.
  • oryzae (ao aspergillopepsin, PROTEX® 50FP), endoproteinase Asp-N, bromelain, ficin, alpha-chymotrypsin, endothiapepsin from Cryphonectria parasitica (cp endothiapepsin, THERMOLASE®), pepsin and thermolysin from Geobacillus sp. (PROTEX® 14L).
  • Table 3 summarizes the properties of these “hit” proteases, with details provided in the following Examples.
  • Keratin azure (Sigma-Aldrich, K8500) is composed of azure dye-impregnated sheep's wool keratin and is used as a keratin substrate to quantify protease activity. Keratin azure was incubated overnight with hit proteases under optimal conditions (as determined in the ENZCHEK® protease assay) and at protease concentrations that were found to completely degrade 125 ⁇ g ml ⁇ 1 Fel d 1 (Table 2).
  • oryzae aspergillopepsin, PROTEX® 50FP
  • endothiapepsin from Cryphonectria parasitica cp endothiapepsin, THERMOLASE®
  • pepsin were not active, as they degraded less than 1% of keratin azure in average (Table 3).
  • keratin degrading activity of hit proteases To further characterize the keratin degrading activity of hit proteases, an assay was performed that colorimetrically detects keratin degradation products by using a coupling reaction with trinitrobenzene sulfonic acid (TNBS) reagent. This assay enabled the determination of hydrolyzing activity of all tested enzymes using keratin powder from sheep's wool as substrate. Papain and pepsin showed no keratin degrading activity. All other hit proteases were active against keratin substrate. The keratin degrading activity of Geobacillus thermolysin was at least two-fold higher compared to all other tested proteases ( FIG. 3 ).
  • TNBS trinitrobenzene sulfonic acid
  • the results from the azure keratin assay described above correlated with the results from the TNBS-based keratin assay, with the exception of the two aspartic acid proteases ao aspergillopepsin and cp endothiapepsin (Table 3).
  • the buffer conditions may have affected the keratin degrading activity measurements as determined by the TNBS assay.
  • the ammonium acetate buffer 100 mM, pH 4.0
  • the Endoproteinase Asp-N buffer which was supplemented by the manufacturer
  • the TNBS-based assay determines proteolysis directly on keratin
  • the keratin azure assay uses a derivatized keratin substrate, which might further explain inconsistencies between the results of the two assays for some of the tested hit proteases.
  • the cytotoxic effects of the hit proteases were tested on keratinocytes.
  • a primary cell line of human keratinocytes was cultured to confluence in microplates and tested for cell viability after overnight incubation with the hit proteases at minimal concentrations found to completely degrade 125 ⁇ g ml ⁇ 1 Fel d 1 (Table 2).
  • the proteases were directly diluted in the cell culture medium. After overnight incubation with the proteases, water-soluble tetrazolium WST-1 was added to the cell culture (confluent layer and medium) (Cell proliferation reagent WST-1, Roche Diagnostics).
  • the colorless WST-1 is reduced into a yellow dye (formazan) due to electron transfer catalyzed by mitochondrial dehydrogenases of vital cells.
  • yellow dye indicates cell vitality of both adherent and detached cells.
  • Cell vitality of adherent cells alone was determined by staining with sulfrohodamin B after overnight incubation with proteases.
  • the red color formation is a measure of protein concentration, which correlates with the adherent cell concentration.
  • Cells that were not incubated with proteases served as a control. For all tested proteases, comparable results were obtained for both assays ( FIG.
  • the results of the cytotoxicity assay were less graduated compared to the results of the TNBS keratin degradation assay ( FIG. 3 ). Proteases either had a significant cytotoxic effect or failed to elicit cytotoxicity. Nevertheless, correlations between the results of the two assay were present. For example, papain and pepsin showed no effect in either assay. On the other hand, the cytotoxic effects of bromelain, ficin, Geobacillus thermolysin and endoproteinase Asp-N on keratinocytes were confirmed by the TNBS assay.
  • keratinocytes were incubated in cell culture medium overnight with papain in concentrations 1000-fold greater compared to the minimum required protease concentration shown in Table 2. This resulted in significant cytotoxic effects, with 31% and 8% cell vitality as determined by the WST-1 and sulfurhodamin B assays, respectively. Most cosmetic applications do not require overnight incubation. Microscopy revealed that no significant cell detachment occurred after the first hour of incubation, even in the presence of 1000-fold increased concentration of papain, indicating the absence of cytotoxic effects at this earlier time point.
  • protease preparations were tested with cellulose azure (Sigma-Aldrich, St. Louis, Mo.), which is a substrate analogous to keratin azure. This experiment was performed to estimate the effect of the hit protease preparations on cotton textiles. Proteolytic hydrolysis of cellulose was determined using cellulose azure as surrogate substrate. The assay was performed in duplicate and as proposed by the manufacturer. The cellulose hydrolysis activity corresponds to the release of the azure dye, which was determined spectrophotometrically at 575 nm.
  • Typical cleansing agents contain solvents, such as denatured ethanol or isopropanol, as disinfectants and to dissolve oil and grease.
  • the isopropanol concentration of a typical bath tub cleaner is 10 to 15%.
  • solvents such as isopropanol can inactivate enzymes such as proteases. Therefore, hit proteases were tested for activity in isopropanol-containing media. Because cleaning agents are typically diluted by water, the Fel d 1 degrading activity of the hit proteases was tested in the presence of isopropanol at concentrations between 0 and 12.5%.
  • Ao aspergillopepsin (PROTEX® 50FP), pepsin and cp endothiapepsin were inactivated by more than 90% in the presence of 12.5% isopropanol.
  • bromelain or ficin appear to be particularly suitable proteases to degrade Fel d 1 present in dust or on surfaces, using an isopropanol-containing cleaning agent. These proteases actively degraded Fel d 1, even in isopropanol concentrations that exceed those in typical cleaning applications.
  • bromelain and ficin are generally regarded as safe when used in cosmetics (see above) or in nutrition processing (e.g., as meat tenderizer).
  • Fel d 1 concentration on the fur of the cat include protease-containing shampoos or dry powders, foams/mousses among other compositions.
  • a total of 67 mg of Fel d 1 per cat has been estimated.
  • the large amount of Fel d 1 present on the cat indicates the significant benefit afforded by reducing the amount of Fel d 1 in or on the cat's fur and/or skin.
  • the use of conventional pet shampoos on cats has been reported to reduce Fel d 1 concentration in the fur as well as the airborne Fel d 1 concentration in the cat's home.
  • the efficacy of conventional shampoos can be increased by the addition of proteases as described herein.
  • Non-ionic surfactants are the second largest surfactant class, and have either polyether or polyhydroxyl as a polar group to increase water solubility.
  • Non-ionic surfactants are widely used in topical applications because, they have a reduced ability to cause irritation when compared to anionic surfactants. This property makes non-ionic surfactants attractive for protease-containing pet shampoos.
  • TWEEN® 20 polysorbate 20
  • E-432 approved food additive
  • 1NCI index International Nomenclature of Cosmetic Ingredients
  • TWEEN® 20 The effect of TWEEN® 20 on Fel d 1 degrading activity of the hit proteases was tested after overnight incubation with 0, 10%, 20%, and 30% TWEEN® 20 and at protease concentrations and under reaction conditions optimal for Fel d 1 degradation as shown in Table 2.
  • the samples were diluted and analyzed by ELISA with an assay reading time point of 20 minutes.
  • the indicated residual Fel d 1 concentration represents the percentage of Fel d 1 determined for the samples compared to the Fel d 1 for the buffer controls with the respective TWEEN® 20 concentrations but without protease. Averages and standard deviations were determined from three independent repetitions. All tested hit proteases displayed tolerance to TWEEN® 20.
  • a pH neutral skin product has a pH of approximately 5.5, which corresponds to the pH on the skin.
  • the Fel d 1 degrading activity of the aspartic acid protease ao aspergillopepsin (PROTEX® 50FP) was not affected by TWEEN® 20 even at the highest tested concentration of 30% ( FIG. 6 ). Although, this protease was found to have the highest activity at pH 4.0 (See Table 2), it still retained 46% activity at pH 6.0 in the ENZCHEK® protease assay.
  • ao aspergillopepsin (PROTEX® 50FP), which did not show degradation activity when keratin azure was used as substrate (Table 3), showed only minimal activity against keratin ( FIG.
  • Papain may also be a suitable alternative to ao aspergillopepsin (PROTEX® 50FP) in shampoo applications. Although papain is less tolerant to TWEEN® 20 than ao aspergillopepsin (PROTEX® 50FP), it remains active at expected TWEEN® 20 concentrations of 2% during application ( FIG. 6 ). Papain showed comparable Fel d 1 degradation activity at pH 7.4 and pH 6.0, rendering it likely to be suitable for use in a pH neutral shampoo. Papain was not active against keratin in the TNBS assay ( FIG. 3 ) and did not induce significant cytotoxicity against keratinocytes ( FIG. 4 ).
  • Papain was not active on cellulose azure substrate, and is therefore likely not to exhibit degradation activity against cotton textiles (Table 3). Papain is also used at high concentrations in nutrients (meat tenderizer, BBQ sauces) and cosmetics (enzyme peeling) without requiring heat inactivation before use. Papain is likely applicable as a shampoo ingredient, even if the cat accidentally ingests the shampoo during the washing procedure.
  • protease-containing product for use in a cat's mouth, including dentifrices, rinses, beverages, foods or treats, strips or films. Such products can reduce Fel d 1 concentration in the mouth of the cat, which is a major source of the allergen.
  • a model for cat saliva was established. The preparation of artificial cat saliva was based on published artificial human saliva preparations (McKnight-Hanes & Whitford, 1992, Caries Res. 26: 345-350) (Table 4).
  • the pH was adjusted to 7.5, which is the pH of cat saliva.
  • the concentration of Fel d 1 in cat saliva was determined to be between 0.3 to 45 ⁇ g/ml (whole cat population) and 2.2 to 12.4 ⁇ g/ml for an intermediate group, which is represented by one third of the whole population.
  • the maximum Fel d 1 concentration in the artificial cat saliva was defined as 125 ⁇ g ml ⁇ 1 , as this concentration was used in all previous experiments, although it exceeded the predicted maximum Fel d 1 concentration in cat saliva by a factor of 2 to 3.
  • Skim milk which contains approximately 35% protein, was used to simulate the protein content in the saliva. Skim milk also contains other components such as carbohydrates, which are related to the complex composition of real saliva and are therefore predicted to render the model more realistic.
  • Human saliva from different individuals has been reported to contain between 0.67 and 2.37 mg ml ⁇ 1 of protein (as determined by different techniques with bovine serum albumin as standard). This estimate was used as an approximation for the protein content in cat saliva. A maximum protein content of 2.5 mg ml ⁇ 1 was defined for /the artificial cat saliva, which was represented by 7.14 mg ml ⁇ 1 skim milk.
  • cysteine has been found to activate certain proteases for Fel d 1 degradation. The effect was most prominent for thiol proteases, such as papain, bromelain and ficin (see above).
  • the concentration of papain would need to be increased to completely degrade Fel d 1 at the highest skim milk concentration (and in the presence of cysteine). Moreover, an increased papain concentration would likely result in a faster Fel d 1 degradation, which would be preferred in order to degrade Fel d 1 in the saliva within the time frame in which the cat consumes the protease-containing pet food or beverage, or is otherwise orally exposed to a protease-containing formulation.
  • the first type represented “extreme conditions,” and contained 125 ⁇ g ml ⁇ 1 of Fel d 1 (2 to 3-fold higher concentration than the maximum concentration determined in cat saliva) and 7140 ⁇ g ml ⁇ 1 of skim milk (corresponding to the maximally determined protein content in human saliva). These allergen and protein concentrations are above expected levels and are provided to estimate the surplus activity of proteases in a pet food application.
  • the second type represented “normal conditions,” and contained 12.5 ⁇ g m ⁇ 1 of Fel d 1 (representing the upper border of Fel d 1 concentrations as determined in the intermediate cat group) and 4.5 ⁇ g ml ⁇ 1 of skim milk (corresponding to the average protein concentration in human saliva of 1.6 ⁇ g mr ⁇ 1 ). These allergen and protein concentrations represent an average level expected in the saliva of most cats.
  • bl subtilisin amounts that were 10-fold, 100-fold, and 1000-fold greater than 9.5 ⁇ g bl subtilisin, which was the minimum concentration of protease required to degrade 125 ⁇ g ml ⁇ 1 Fel d 1 under optimal conditions during an overnight incubation (Table 2) were tested in artificial cat saliva under extreme conditions for Fel d 1 degradation after one and 20 hours of incubation at 37° C. ( FIG. 8 ).
  • 125 ⁇ g ml ⁇ 1 natural Fel d 1 was incubated for 0 min, 1 hour or 20 hours (overnight) with 0, 9.5, 95, 950 and 9500 ⁇ g ml ⁇ 1 of bl subtilisin at 37° C.
  • Fel d 1 degradation by papain and bl subtilisin was kinetically analyzed in artificial cat saliva under normal and extreme conditions at shorter incubation times ( FIG. 9 ).
  • Papain and 9.5 mg ml ⁇ 1 of bl subtilisin were incubated for 0, 5, 10, 15 and 60 min with Fel d 1 in artificial cat saliva at 37° C. under normal and extreme conditions (Table 4). Reactions were stopped by addition of 100 ⁇ M E64 (for papain) and 1 mM PMSF (for bl subtilisin). Samples were diluted and analyzed by ELISA with an assay reading time point of 30 minutes.
  • the residual Fel d 1 concentration represents the percentage of Fel d 1 determined for the samples as compared to the Fel d 1 for the respective buffer controls without protease. Averages and standard deviations were determined from three independent repetitions. Most of the Fel d 1 was degraded already after 5 min with bl subtilisin (9.5 mg ml ⁇ 1 ) in artificial cat saliva under both normal and extreme conditions. The experiments revealed that bl subtilisin was somewhat more effective than papain. However, under normal conditions, which correspond to the average composition of cat saliva, more than 80% of Fel d 1 was degraded by papain (4.5 mg ml ⁇ 1 ) within 5 minutes ( FIG. 9 ).
  • both papain and bl subtilisin present in the concentrations tested, would be predicted to degrade the allergen by more than 80% within 5 minutes.
  • this excess enzyme promotes the accelerated degradation of Fel d 1, such that most of it is degraded within a few minutes.
  • the two proteases were tested in combination for their ability to synergistically degrade Fel d 1 in artificial cat saliva at the concentrations previously tested for each protease alone (4500 ⁇ g ml ⁇ 1 of papain and 9500 ⁇ g ml ⁇ 1 of bl subtilisin, FIG. 9 ).
  • Fel d 1 degradation was tested at protease concentrations reduced by a factor of 10, 100 and 1000. 450 ⁇ g ml ⁇ 1 papain and 950 ⁇ g ml ⁇ 1 bl subtilisin were minimally required to completely degrade Fel d 1 within one hour ( FIG. 10 ).
  • the minimum protease combination of 565 ⁇ g ml ⁇ 1 papain and 1188 ⁇ g ml ⁇ 1 bl subtilisin would be sufficient to reduce Fel d 1 content in the saliva of an average cat by approximately 80% within 10 minutes of incubation, e.g. while the cat is eating the pet food or treat, or drinking a beverage containing the proteases, or otherwise being orally exposed to the protease-containing formulation.
  • a wipe prototype containing functional proteases was developed and tested for inactivation of Fel d 1.
  • a batch of cotton wipes (20 cm ⁇ 20 cm) containing active PROTEX® 6L was manufactured and tested for Fel d 1 inactivation. The wipe was successful in degradation of Fel d 1. The specifics are discussed in detail below.
  • the concentration of PROTEX® 6L required for the wipe was determined.
  • the required enzyme concentration is dependent on the amount of Fel d 1 that has to be inactivated by the prototype.
  • protease PROTEX® 6L combines all characteristics required for development of application prototypes, including those of Examples 6-10: It shows only weak effects on keratin hydrolysis, no effects on viability of keratinocytes, no cellulose activity but high tolerability towards non-ionic detergents. Therefore, PROTEX® 6L was used for the development of the present prototypes (wipes, cat litter, shampoo, cleansing liquid).
  • Molton fabric consists of 100% cotton fibers. Its roughened surface and its high water absorption make it to a versatile cleaning tissue. The size of one final wipe is approx. 20 cm ⁇ 20 cm.
  • immobilization of enzymes on solid material can be achieved by covalent coupling of the enzyme via a chemical reaction.
  • a first screening of conditions for the immobilization of PROTEX® 6L used not only the enzyme and carrier (cotton wipe) but also two cross-linkers since the carrier material (cotton) does not possess groups that readily form covalent bonds.
  • the cross-linker used is a 2-component system: a diamine (pentaethylenehexamine, PEHA) and a dialdehyde (glutaraldehyde, Ga.). They are combined in a fixed ratio and reference is mostly made to the glutaraldehyde concentration to indicate the amount used in proportion to the enzyme.
  • the 2-component cross-linking system has to match the amount of protein. It was found that the amount used in proportion to PROTEX® 6L is critical as too low amounts will not cross-link all the enzyme or will results in unstable formulations. Too high amounts will give overcross-linking reducing conformational freedom and subsequently reducing the activity of the enzyme.
  • Cross-linker amounts and conditions as well as enzyme loadings amounts and conditions were experimentally determined. After which, big-scale reactions were performed to cross-link PROTEX® 6L on complete wipes (20 cm ⁇ 20 cm).
  • liquid PROTEX® 6L (9.24 ml of 4600 ELU/ml PROTEX® 6L) was mixed with 83.2 ml phosphate buffer (100 mM, pH 6.5) and glutaraldehyde was added (133 mM final concentration).
  • Cross-linking of the enzyme occurred by incubating the wipe in the reaction mixture over-night at room-temperature. The wipe was washed with water and the liquid is squeezed out and washed again with water and 3% PEG. Finally, the wipe is dried with a stream of air (21° C.) for 5 h.
  • PROTEX® 6L was immobilized on 10 cotton wipes, another 10 control wipes were produced the same way, with the exception that no enzyme was added to the reaction mixture. The wipes were then tested for application. The capability of Fel d 1 inactivation was determined and shown in Example 10 (Table 6).
  • Cat litter prototypes containing functional proteases were developed and tested for inactivation of Fel d 1. Specifically, 260 g cat litter (perlite) containing active PROTEX® 6L was manufactured and tested for Fel d 1 inactivation. The cat litter was successful in degradation of Fel d 1. The specifics are discussed in detail below.
  • the concentration of PROTEX® 6L required for the prototype was determined.
  • the required enzyme concentration is dependent on the amount of Fel d 1 that has to be inactivated by the prototype.
  • the amount of Fel d 1 that should be inactivated by the cat litter is approx. 250 mg.
  • PROTEX® 6L “Purina Tidy Cats non clumping cat litter Instant Action” was tested for the immobilization of PROTEX® 6L.
  • the cat litter is based on the clay mineral bentonite.
  • PROTEX® 6L was diluted with water and added to bentonite to check possible immobilization conditions and absorption of the enzyme. However, adding water to PROTEX® 6L dilutes down the 50% glycerol present in this enzyme mixture and makes the bentonite fall apart.
  • Sand with a particle size of 1.2 mm was tested as another carrier which could be principally be mixed with bentonite. Again two cross-linkers were used besides the enzyme. Sand was used to demonstrate the viability of the approach where the enzyme is immobilized on a second carrier (sand), which could then be mixed with other cat litter particles. A maximum loading of 200 ⁇ l PROTEX® 6L was used per gram of sand. However, the activity and recovery was relatively low, most likely because sand lacks pores and has a small surface area. Leaching of the enzyme is low and thus sand is in principle a suitable material for covalent linkage.
  • Perlite was chosen as a third carrier. Due to its particle size of 2.8-6 mm, it could also principally be mixed with bentonite. Again two cross-linkers (GA and PEHA) were used besides the enzyme to induce covalent bonding. First immobilization experiments showed that the recovery as well as the leaching of the enzyme PROTEX® 6L is relatively good for enzyme loadings in the lower range. Enzyme loadings above 500 ⁇ l /g give significant coloration of the perlite particles but with loadings in the lower range it maintains a bright white color.
  • perlite For cross-linking of PROTEX® 6L to cat litter material, perlite turned out to be superior to sand and bentonite. Perlite permits high enzyme loading, while keeping enzyme leaching low. Moreover, its particle size allows homogenous mixing with other cat litter material. Therefore, perlite was found to be an excellent material for cross-linking of PROTEX® 6L and finally 260 g perlite containing PROTEX® 6L were produced.
  • liquid PROTEX® 6L 60 ml of 4600 ELU/ml PROTEX® 6L was mixed with 472.8 ml cold water, PEHA (adjusted to pH 7.0, 20 mM final concentration, 4° C.) and glutaraldehyde (133 mM final concentration)was added, mixed well and added to 300 g perlite.
  • Cross-linking of the enzyme occurred by incubating perlite particles in the reaction mixture overnight at room-temperature.
  • Perlite was washed with water, 5 mM phosphate buffer, and water with 3% PEG. Finally, perlite particles were dried over-night at room temperature.
  • PROTEX® 6L was immobilized on 260 g perlite, another small control sample (1 g perlite) was produced the same way, with the exception that no enzyme was added to the reaction mixture. Perlite was then tested for application. The capability of Fel d 1 inactivation was determined and shown in Example 10 (Table 6).
  • Cleansing liquid prototypes containing functional proteases were developed and tested for inactivation of Fel d 1. Specifically, 1 Liter of cleansing liquid and 50 ml of enzyme concentrate containing active PROTEX® 6L were manufactured and tested for Fel d 1 inactivation. The prototypes were successful in degradation of Fel d 1. The specifics are discussed in detail below.
  • the concentration of PROTEX® 6L required for every prototype was determined.
  • the required enzyme concentration is dependent on the amount of Fel d 1 that has to be inactivated by the respective prototype.
  • PROTEX® 6L concentration that is required for total Fel d 1 digestion
  • the activity of a PROTEX® 6L dilution series was analyzed by ELISA.
  • PROTEX® 6L Units are based on ethyl-L-lactate assay (ELU), determined by manufacturer Genencore) are capable to completely digest 2.5 ⁇ g Fel d 1 under optimal reaction conditions (Tris-HCl buffer, pH 7.8). Based on these calculations, including the initial calculations for the shampoo in Example 9, the concentration of PROTEX® 6L was adjusted accordingly for the cleansing liquids.
  • PROTEX® 6L Based on the Fel d 1 concentration on hard surfaces, the required amount of PROTEX® 6L was determined and incorporated into the basic cleansing liquid formulation (KAR-001).
  • water 960 g
  • 10 g phenoxyethanol preservative
  • 30 g Zusolat 1008/85 fatty alcohol available from Zschimmer & Schwarz GmbH & Co KG
  • 10 g EUXYL® PE 9010 were added and stirred. If necessary pH had to be adjusted to 8.0 by using NaOH.
  • PROTEX® 6L 9 ml of 4600 ELU/ml PROTEX® 6L was added (KAR-001+E).
  • the advantage is that the enzyme could be mixed with any other surface cleanser, which could either be provided by the customer.
  • storing the enzyme in an optimized liquid formulation and not in surface cleanser formulation could stabilize the enzyme and lead to increased enzymatic stability. Therefore, an enzyme concentrate consisting of 20 x higher concentrated PROTEX® 6L (in comparison to the ready-to-use surface cleanser KAR-001+E) and enzyme stabilizer propylene glycol was developed.
  • the capability of Fel d 1 inactivation (after dilution of the 20 ⁇ KAK-001+E in 1 ⁇ surface cleanser) was determined and is shown in Example 10 (Table 6).
  • the concentration of PROTEX® 6L required for the prototype was determined.
  • the required enzyme concentration is dependent on the amount of Fel d 1 that has to be inactivated by the prototype.
  • the Fel d 1 amount on the cat is approx. 2 ⁇ g/mg cat hair (data provided by Nestl—Purina). Since the weight of hair per cat is in average 121 g (Avner DB et al., J Allergy Clin Immunol, 1997) the total amount of Fel d 1 on the cat that should be inactivated by the shampoo is approx. 250 mg
  • PROTEX® 6L concentration that is required for total Fel d 1 digestion
  • the activity of a PROTEX® 6L dilution series was analyzed by ELISA.
  • 3.8 U/ml PROTEX® 6L (Units are based on ethyl-L-lactate assay (ELU), determined by manufacturer Genencore) are capable to completely digest 2.5 ⁇ g Fel d 1 under optimal reaction conditions (Tris-HCl buffer, pH 7.8).
  • ELU ethyl-L-lactate assay
  • water (980 g) was added to a beaker glass, then 10 g phenoxyethanol (preservative), followed by 10 g betaine (KFS-002) or 10 g SLS (KFS-004a) and 10 g PERLASTAN® L30 were added and stirred. If necessary, pH had to be adjusted to 5.5 (KFS-002) or 7.5 (KFS-004a) by using NaOH.
  • KFS-002/004a 9 ml PROTEX® 6L (of 4600 ELU/ml PROTEX® 6L) was added (KFS-002/004a+E).

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EP3082849B1 (fr) 2019-11-06
CN105873603A (zh) 2016-08-17
WO2015092663A1 (fr) 2015-06-25
BR112016013800A2 (pt) 2017-08-08
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