Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• • 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
  • 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
  • A61K8/86—Polyethers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
• • 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
• • 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
  • A61K2800/70—Biological properties of the composition as a whole
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
  • A61Q19/10—Washing or bathing preparations
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q5/00—Preparations for care of the hair
  • A61Q5/02—Preparations for cleaning the hair
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q5/00—Preparations for care of the hair
  • A61Q5/06—Preparations for styling the hair, e.g. by temporary shaping or colouring
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q5/00—Preparations for care of the hair
  • A61Q5/10—Preparations for permanently dyeing the hair
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q5/00—Preparations for care of the hair
  • A61Q5/12—Preparations containing hair conditioners
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q9/00—Preparations for removing hair or for aiding hair removal
  • A61Q9/02—Shaving preparations

Definitions

• the present invention relates to modified enzymes, a skin care composition comprising said modified enzyme and ingredients known to be used in skin care composition, a skin care product comprising a skin care composition of the invention and the use of said modified enzyme for improving the stability and/or for reducing the sensitization potential of enzyme.
• Enzymes for Skin Care The beneficial potential action of treating the skin with enzymes in the form of vegetables and fruits, such as cucumber, tomato, carrots, banana etc., have been known for a long period of time.
• enzymes were not introduced into commercial skin care products before the 1970 'ies, partly due to a limited knowledge about enzymes but also because enzymes were considered to have an unsatisfactory stability and also some disadvantageous properties in skin care products. For instance, cellulases were found to change the viscosity of lotions and creams containing carboxymethylcellulose; lipases resulted in changes in creams containing fatty acids esters; proteases were found to breakdown protein ingredients and to cause loss in viscosity.
• the Human Skin The human skin is composed of several layers.
• the top layer, the Epidermis contains the fibrous protein keratin and functions as a sort of protective cover from the environment.
• the outer layer of the Epidermis is formed from organised cell death from the granular layer which lies underneath. In the granular layer numerous enzymes are released which convert the dead cell material to keratin.
• the Coriu (dermis) is connected to the Epidermis by way of the basal membrane and links the skin to the rest of the body through the circulatory system.
• the Corium is equipped with blood vessels, nerve fibres and lymphatic vessels and comprises a fibrous network of mainly collagen fibres with a limited amounts of elastin and reticulin fibres.
• Modified enzymes for personal care products As mentioned above some enzymes have an unsatisfactory stability and may under certain circumstances - dependent on the way of contact - cause an immune response, typically an IgG and/or IgE response.
• Said patent concerns non-immunogenic polypeptides, such as enzymes and peptide hormones coupled to polyethylene glycol (PEG) or polypropylene glycol. At least 15% of polypeptides' physiological activity is maintained.
• PEG polyethylene glycol
• Ppropylene glycol At least 15% of polypeptides' physiological activity is maintained.
• GB patent no. 1,183,257 (Crook et al.) describes chemistry for conjugation of enzymes to polysaccharides via a triazine ring. Further, techniques for maintaining of the enzymatic activities of enzyme-polymer conjugates are also known in the art.
• WO 93/15189 (Veronese et al.) concerns a method for main- taining the activity in polyethylene glycol-modified proteolytic enzymes by linking the proteolytic enzyme to a macromolecularized inhibitor.
• the conjugates are intended for medical applications.
• EP 183 503 (Beecham Group PLC) discloses a development of the above concept by providing conjugates comprising pharmaceutically useful proteins linked to at least one water-soluble polymer by means of a reversible linking group.
• EP 471,125 discloses skin care products comprising a parent protease (the Bacillus protease Esperase®) coupled to polysaccharides through a triazine ring to improve the thermal and preservation stability.
• the coupling technique used is de- scribed in the above mentioned GB patent no. 1,183,257 (Crook et al.) .
• JP 3083908 describes a skin cosmetic material contains a transglutaminase from guinea pig liver modified with one or more water-soluble substance such as PEG, starch, cellulose etc. The modification is performed by activating the polymeric molecules and coupling them to the enzyme.
• the composition is claimed to be mild to the skin.
• modified enzyme conjugates suitable for use in skin care products .
• the present inventors have found that when using modified enzyme with an activity suitable for skin care certain claims must be imposed on the enzyme and polymeric molecule to obtain improved stability and a reduced sensitisation potential while still having a substantial residual enzymatic activity maintained.
• the invention relates to a modified enzyme having from 4 to 70 polymeric molecules, with a molecule weight from 1 to 35 kDa, coupled covalently to the surface of parent enzymes having a molecule weight from 15 to 100 kDa.
• the parent enzyme has a molecule weight from 15 to 35 kDa from 4 to 20 polymeric are coupled covalently should be coupled to the surface of the enzyme.
• the molecule weight of the parent enzyme lie in the range from 35 to 60 kDa from 7 to 40, preferably 10 to 30 polymeric molecules are coupled to the surface of said parent enzyme.
• the parent enzyme has a molecule weight from 60 to 80 kDa from 10 to 50, preferably 13 to 40 polymeric molecules are coupled to the surface of said parent enzyme.
• polymeric molecules are coupled to the surface of parent enzymes having a molecule weight from 80 to 100 kDa.
• polymeric molecules are coupled to the amino groups (-NH 2 ) on the enzyme's surface and a the N-terminal amino group.
• polymeric molecules may also be coupled to the carboxylic acid groups (-COOH) of amino acids in the enzyme chain positioned on the surface.
• Preferred attachment groups are Lysine residues and the amino groups at the N-terminal.
• Carboxylic acid attachment groups may be the carboxylic acid group of Aspartate or Glutamate and the C-terminal COOH-group.
• the number of "attachment groups” counts in the present application the number of the amino groups of Lysine residue in the polypeptide chain plus the N-terminal amino group.
• the parent enzyme of the invention may be a hydrolase, including proteases, in particular subtilisins, or lipase, or an Oxidoreductase, including laccases and Superoxide dismutase.
• the invention relates to skin care composition
• a modified enzyme of the invention further ingredients being used in skin care products.
• the invention relates to skin care product comprising a skin care composition of the invention.
• the skin care product of the invention has improved stability and reduced sensitisation potential in comparison to corresponding skin care products (with parent enzymes) .
• reduced sensitisation potential means in the context of the present invention "reduced allergenicity” which means that the amount of produced IgE (in humans, and molecules with comparable effects in specific animals) , which can lead to an allergic state, is decreased when inhaling a modified enzyme of the invention in comparison to the corresponding parent en- zymes.
• skin care products cover all personal care products used for cleansing, care and/or beautification of the skin of the body and further other products, such as hair care products, which during use might come in contact with the skin or respiratory system. Also corresponding products for animals are contemplated according to the present invention.
• skin care products contemplated according to the present invention are soap, cosmetics, skin creams, skin gels, skin milk, skin lotion, cleansing cream, cleansing lotion, cleansing milk, cold cream, cream soap, makeup base, milky lotion, pack, calamine lotion, T zone essence, hand cream, essence powder, whitening powder, powder soap, cake soap, transparent soap, lip cream, lipstick, nourishing essence, creamy foundation, face powder, powder eye-shadow, powder foundation, nail polish remover, hair tonic, hair liquid, hair cream, hair gel, hair treatment, hair setting preparations, hair dyes, hair colorants, scalp treatment, shampoo, balsam, hair rinse, hair spray sun oil, sun screen, shaving foam and gel, shaving cream, baby oil, acne care products, antiperspirants, insect repellents, deodorants etc.
• Assessment of allergenicity may be made by inhalation tests, comparing the effect of intratracheally (into the trachea) administrated parent enzymes with the corresponding modified enzymes according to the invention.
• a number of in vivo animal models exist for assessment of the allegenicity of enzymes. Some of these models give a suitable basis for hazard assessment in man. Suitable models include a guinea pig model and a mouse model. These models seek to identify respiratory allergens as a function of elicitation reactions in- prised in previously sensitised animals. According to these models the alleged allergens are introduced intratracheally into the animals.
• a rat strain suitable for intratracheal exposure to polypeptides and enzymes is the Brown Norway strain. Brown Norway rats produce IgE as the allergic response.
• the BALB/C mice strain is suitable for determining the IgE response caused by subsctaneous injection.
• mice such as rats, rabbits etc. may also be used for comparable studies.
• Figure 1 shows the kinetics of the specific anti-PD498 IgE response in BALB/C mice after immunization with modified PD498- SPEG, unmodified PD498 and Glycine-SPEG 15,000.
• Figure 2 shows the IgGi level of modified PD498-SPEG and unmodified PD498 of administrated intratrachaeally to Dunkin Hartley guinea pigs.
• Figure 3 shows the IgGi levels of 3 ⁇ g, 30 ⁇ g and 300 ⁇ g of modified PD498-SPEG 5,000 in the Dunkin Hartley guinea pigs IT dose response study (B 3.0 ⁇ g; ⁇ 30 ⁇ g; T 300 ⁇ g) .
• the 0.3 ⁇ g dose curve is ommitted due to nor response at all.
• Figure 4 shows the IgGi levels of 0.3 ⁇ g, 3.0 ⁇ g and 30 ⁇ g of unmodified parent PD498 in the Dunkin Hartley guinea pigs IT dose response study (• 0.3 ⁇ g; ⁇ 3.0 ⁇ g; T 30 ⁇ g) .
• EP 471,125 a Bacillus protease Esperase® (available from Novo Nordisk A/S) is conjugated through a triazine ring with a 40 kDa dextran (Example 1) and a 50 kDa pullulan (Example 2) .
• Said Bacillus protease i.e. Esperase®
• the attachment groups are present as two amino groups (i.e. two Lysine residues on the surface of the 3D structure) and one N-terminal amino group.
• Enzyme weight versus the number of polymeric molecules The present invention is based to the general principle that the larger the surface area is and/or the weight of the enzyme is the more polymeric molecules must be coupled to the surface of the enzyme to obtain improved stability, a substantial residual enzymatic activity and/or a reduced sensitisation potential.
• EP 471,125 describes coupling of few (i.e. up to 3) heavy (i.e. 40 and 50 kDa) polymeric molecules to the surface of the microbial protease Esperase® having a molecule weight of about 28 kDa.
• the invention related to a modified enzyme suitable for skine care having from 4 to 70 polymeric molecules, with a molecule weight from 1 to 35 kDa, coupled covalently to the surface of a parent enzyme with a molecule weight from 15 to 100 kDa.
• enzymes having a molecule weight of from 15 to 35 kDa which is typical for many microbial enzymes, such bacterial proteases of e . g.
• Bacillus origin are coupled covalently with from 4 to 20 polymeric molecules .
• the modified enzyme may have 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 polymeric molecules covalently coupled to the surface of 3D structure of the parent enzyme (including the N-terminal amino group) .
• the preferred ratio between the weight and/or surface area of the enzyme, the number of coupled polymeric molecules and the weight of the polymer is displayed below in Table 1.
• the molecule weight of the polymeric molecules may according to the invention be within the ranges between 1 and 35 kDa. However, if the polymeric molecules get to light and/or to few the epitope(s) in question of the enzyme's surface may not be shielded sufficiently resulting in an immune response.
• the preferred molecule weight of the polymeric molecule lies according to the present invention between 4 to 25 kDa, especially 6 to 25 kDa, such as 8 to 20 kDa.
• a “substantially” maintained residual activity means that more than 20%, 30% or 40%, better more than 50%, 60% or 70%, even better between 70% or 80%, up to between 80% and 90% and even up to 100%, of the activity of the enzyme is maintained.
• loss of enzymatic activity of modified enzymes might be a consequence of impeded access of the substrate to the active site in the form of spatial hindrance of the substrate by bulky/heavy polymeric molecules to the catalytic cleft of the enzyme. It might also, at least partly, be due to disadvantageous structural changes of the 3D structure of the enzyme. When coupling few bulky/heavy polymeric molecules to the enzyme surface it might cause uneven interactions on different parts of the enzyme molecule. This might lead to that the enzyme structure is pulled partly out of it normal configuration which in most cases will result in loss of enzymatic activity.
• the modified protease described in EP 471,125 has few (i.e. up to 3 polymeric molecules) heavy/bulky polymeric molecules (i.e. 40 and 50 kDa polysaccharides) coupled to amino groups on the enzyme's surface.
• the loss of enzymatic activity observed i.e. 45% to 67% residual enzymatic activity
• the bulky/heavy polymeric molecules coupled to the enzyme's surface might further impede the access of the substrate to the activity site of the enzyme resulting in the reduction of the maintained enzymatic activity.
• polymeric molecules i.e. more than 4
• a relatively low molecule weight i.e. 1-35 kDa
• the enzyme's surface in the case of enzymes with a molecule weight from 15 to 35 kDa
• the polymeric molecules are spread broadly over the surface of the enzyme, with the exception of the area close to the active site.
• “spread broadly” means positioned so that the polymeric molecules coupled to the attachment groups of the enzyme shield different parts of the enzyme surface, preferable the whole or close to the whole surface area away of the active site, to make sure that the relevant epitope(s) in question being recognisable are shielded and hereby not recognised by the immune system's antibodies. It is believed that the surface area of interaction between the enzyme and an antibody lies in the range about 500 A 2 (26 x 19A) (see Sheriff et al. (1987) , Proc. Natl. Acad. Sci. USA, Vol. 84, p. 8075). Two or more attachment groups on the enzyme should preferably not lie close to each other as it will probably result in that only one polymeric molecule will be coupled.
• polymeric molecules are coupled in a close distance of the active site.
• the distance depends on the bulkiness of the polymeric molecules, as impeded access by the bulky polymeric molecules to the activity site is undesired. Therefore, the more bulky the polymeric molecules are the longer distance from the active site should the polymeric molecules be coupled. Generally seen it is preferred that no polymeric molecules are attached within 5 A, preferred 10 A from the active site.
• enzymes having coupled polymeric molecules at (a) known epitope(s) recognisable by the immune system or close to known epitope(s) recognisable by the immune system or close to said epitope are also considered advantageous according to the invention. If the position of the epitope (s) is (are) unknown it is advantageous to coupled as many polymeric molecules to the attachment groups available on the surface of the enzyme. It is preferred that said attachment groups are spread broadly over the surface of the enzyme in a suitable distance from the active site. Modified enzymes fulfilling the above claims to the distribution of coupled polymeric molecules on the surface of the enzyme are preferred according to the invention. Especially such enzymes having no or only very few polymeric molecules (i.e. 0 to 2) coupled within a distance of 0 to 5 A, preferably 0 to 10 A from the active site are preferred.
• the polymeric molecules coupled to the enzyme may be any suitable polymeric molecule, including natural and synthetic homo-polymers, such as polyols (i.e. poly-OH) , polyamines (i.e. poly-NH 2 ) and polycarboxyl acids (i.e. poly-COOH) , and further hetero-polymers i.e. polymers comprising one or more different coupling groups e.g. a hydroxyl group and amine groups.
• polyols i.e. poly-OH
• polyamines i.e. poly-NH 2
• polycarboxyl acids i.e. poly-COOH
• hetero-polymers i.e. polymers comprising one or more different coupling groups e.g. a hydroxyl group and amine groups.
• polymeric molecules include polymeric molecules selected from the group comprising polyalkylene oxides (PAO) , such as polyalkylene glycols (PAG) , including polyethylene glycols (PEG) , methoxypolyethylene glycols (mPEG) and polypropy- len glycols, PEG-glycidyl ethers (Epox-PEG) , PEG-oxycarbonylimi- dazole (CDI-PEG) , Branced PEGs, poly-vinyl alcohol (PVA) , poly- carboxylates, poly-(vinylpyrolidone) , poly-D,L-amino acids, polyethylene-co-maleic acid anhydride, polystyrene-co-malic acid anhydrid, dextrans including carboxymethyl-dextrans , heparin, homologous albumin, celluloses, including methylcellulose, carbo- xymethylcellulose, ethylcellulose, hydroxyethoxy
• Preferred polymeric molecules are non-toxic polymeric molecules such as (m) polyethylene glycol ((m)PEG) which further requires a relatively simple chemistry for its covalently coupling to attachment groups on the enzyme's surface.
• polyalkylene oxides such as polyethylene oxides, such as PEG and especially mPEG
• PAO polyalkylene oxides
• PEG polyethylene oxides
• mPEG polyethylene oxides
• PEG and especially mPEG are the preferred polymeric molecules, as these polymeric molecules, in comparison to polysaccharides such as dextran, pullulan and the like, have few reactive groups capable of cross-linking.
• methoxypolyethylene glycols may advantageously be used. This arise from the fact that methoxyethylene glycols have only one reactive end capable of conjugating with the enzyme. Consequently, the risk of cross-linking is less pronounced. Further, it makes the product more homogeneous and the reaction of the polymeric molecules with the enzyme easier to control.
• polymeric molecules to be conjugated with the enzyme are not active it must be activated by the use of a suitable method.
• the polymeric molecules may be coupled to the enzyme through a linker. Suitable linkers are well known to the skilled person.
• Coupling polymeric molecules to the free acid groups of enzymes can be performed with the aid of diimide and for example amino-PEG or hydrazino-PEG (Pollak et al. , (1976), J. Amr. Chem..
• Coupling polymeric molecules to hydroxy groups are generally very difficult as it must be performed in water. Usually hydrolysis predominates over reaction with hydroxyl groups.
• Coupling polymeric molecules to free sulfhydryl groups can be reached with special groups like maleimido or the ortho- pyridyl disulfide.
• vinylsulfone US patent no. 5,414,135, (1995), Snow et al.
• Accessible Arginine residues in the polypeptide chain may be targeted by groups comprising two vicinal carbonyl groups. Techniques involving coupling electrophilically activated PEGs to the amino groups of Lysines are also be useful. Many of the usual leaving groups for alcohols give rise to an amine linkage. For instance, alkyl sulfonates, such as tresylates (Nilsson et al. , (1984), Methods in Enzymology vol. 104, Jacoby, W. B., Ed., Academic Press: Orlando, p. 56-66; Nilsson et al., (1987), Methods in Enzymology vol. 135; Mosbach, K. , Ed.; Academic Press: Orlando, pp.
• tresylates such as tresylates (Nilsson et al. , (1984), Methods in Enzymology vol. 104, Jacoby, W. B., Ed., Academic Press: Orlando, p. 56-66; Nils
• Organic sulfonyl chlorides e.g. Tresyl chloride
• Tresyl chloride effectively converts hydroxy groups in a number of polymers, e.g. PEG, into good leaving groups (sulfonates) that, when reacted with nucleophiles like amino groups in polypeptides allow stable linkages to be formed between polymer and polypeptide.
• the reaction conditions are in general mild (neutral or slightly alkaline pH, to avoid denaturation and little or no disruption of activity) , and satisfy the non-destructive requirements to the polypeptide.
• Tosylate is more reactive than the mesylate but also more unstable decomposing into PEG, dioxane, and sulfonic acid (Zalipsky, (1995), Bioconjugate Chem., 6, 150-165). Epoxides may also been used for creating amine bonds but are much less reactive than the above mentioned groups. Converting PEG into a chloroformate with phosgene gives rise to carbamate linkages to Lysines. This theme can be played in many variants substituting the chlorine with N-hydroxy succinimide (US patent no. 5,122,614, (1992); Zalipsky et al., (1992), Biotechnol. Appl. Biochem., 15, p.
• isocyanates and isothiocyanates may be employed yielding ureas and thioureas, respectively.
• Amides may be obtained from PEG acids using the same leaving groups as mentioned above and cyclic imid thrones (US patent no. 5,349,001, (1994), Greenwald et al.). The reactivity of these compounds are very high but may make the hydrolysis to fast.
• PEG succinate made from reaction with succinic anhydride can also be used.
• the hereby comprised ester group make the conjugate much more susceptible to hydrolysis (US patent no. 5,122,614, (1992), Zalipsky). This group may be activated with N- hydroxy succinimide.
• peptides do not comprise many Lysines it may be advantageous to attach more than one PEG to the same Lysine. This can be done e.g. by the use of 1, 3-diamino-2-propanol.
• PEGs may also be attached to the amino-groups of the enzyme with carbamate linkages (WO 95/11924, Greenwald et al.). Lysine residues may also be used as the backbone.
• conjugates of the invention described above may be prepared on the basis of selected parent enzymes using any suitable technique known in the art.
• the term "parent" enzyme is intended to indicate any uncoupled enzyme (i.e. an enzyme to be modified).
• the enzyme may preferably be of microbial origin, such as bacterial, filamentous fungus or yeast origin.
• the parent enzyme may be a naturally-occurring (or wild- type) enzyme or may be a variant thereof.
• the 3-dimensional structure of the enzyme is of interest in connection with assessing/selecting suitable parent enzymes to be modified.
• the 3-dimentional structure may be an X-ray structure, an NMR structure or a model-built structure.
• the Brookhaven Databank may be the source of X-ray and NMR- structures.
• a model-built structure may be produced by the person skilled in the art if one or more 3D-structure(s) exist (s) of homologous enzyme (s) sharing at least 30% sequence identity with the enzyme in question.
• 3D-structure(s) exist (s) of homologous enzyme (s) sharing at least 30% sequence identity with the enzyme in question.
• Several software packages such as the "Homology 95.0" package from Biosym, exist which may be employed to construct a model structure.
• Typical actions required for the construction of a model structure are: alignment of homologous sequences for which 3D- structures exist, definition of Structurally conserveed Regions (SCRs) , assignment of coordinates to SCRs, search for structural fragments/loops in structure databases to replace Variable Regions, assignment of coordinates to these regions, and structural refinement by energy minimization. Regions containing large inserts (>3 residues) relative to the known 3D- structures are known to be quite difficult to model, and structural predictions must be considered with care.
• SCRs Structurally conserveed Regions
• this structure serves as an essential prerequisite for the identifying suitable parent enzymes which when modified has a reduced allergenicity and a substantially maintained residual enzymatic activity.
• Preferred enzymes for skin care products are enzymes having a substantially enzymatic activity in the pH range used in the skin care product.
• the enzyme activity The parent enzyme may have any activity known to be used for skin care.
• Contemplated enzymes including Oxidoreductases including Oxidoreductases
• Transferases (E.C. 2), such as transglutaminases
• PDI Isomerases
• Contemplated proteolytic enzymes includes selected from the group of acidic aspartic proteases, cysteine proteases, serine proteases, such as subtilisins, or metallo proteases, with the above indicated properties (i.e. number of attachment groups, position of attachment groups etc.).
• the subtilisin PD498 has a molecule weight of 29 kDa and is shown in SEQ ID NO. 2.
• PD498 has 12 Lysine groups for attachment on the surface of the enzyme plus one N-terminal amino group.
• preferred enzyme has Lysine spread broadly over the enzyme's surface.
• PD498 has no Lysine residues in a distance of 0-10 A from the active site which makes it especially suitable in modified form. Further, the Lysine residues are spread broadly on the surface of the enzyme (i.e. away from the active site) .
• the enzyme Subtilisin DY has a molecule weight of 27 kDa and has 12 amino groups (i.e. Lysine residues) on the surface of the enzyme and one N-terminal amino group (see SEQ ID NO. 3) .
• the parent protease Lion Y has a molecule weight of 46 kDa and has 14 amino groups (i.e. Lysine residues) on the surface of the enzyme plus one N-terminal amino group (see SEQ ID NO. 4) .
• the neutral metallo protease Thermolysin has a molecule weight of 34 kDa and has 11 amino groups (i.e. Lysine residues) on the surface plus one N-terminal amino group. (See SEQ ID NO 5)
• Contemplated lipolytic enzymes include include Humicola lanugino ⁇ a lipases, e.g. the one described in EP 258 068 and EP 305 216, Humicola insolens, a Rhizomucor miehei lipase, e.g. as described in EP 238 023, Absidia sp. lipolytic enzymes (WO 96/13578), a Candida lipase, such as a C. antarctica lipase, e.g. the C. antarctica lipase A or B described in EP 214 761, a Pseudomonas lipase such as a P. alcaligenes and P.
• Humicola lanugino ⁇ a lipases e.g. the one described in EP 258 068 and EP 305 216
• Humicola insolens e.g. as described in EP 238 023
• Absidia sp. lipolytic enzymes WO 96/1
• pseudoalcaligenes lipase e.g. as described in EP 218 272
• a P. cepacia lipase e.g. as described in EP 331 376
• a Pseudomonas sp. lipase as disclosed in WO 95/14783
• a Bacillus lipase e.g. a
• WO 91/16422 B . pumilus lipase
• Other types of lipolytic include cutinases, e.g. derived from Pseudomonas mendocina as described in WO 88/09367, or a cutinase derived from Fusarium solani pi ⁇ i (e . g . described in WO 90/09446) .
• Laccases Contemplated laccases include the laccases disclosed in WO 96/00290 and WO 95/33836 from Novo Nordisk.
• Transglutaminases Suitable transferases include any trnsglutaminases disclosed in WO 96/06931 (Novo Nordisk A/S) and WO 96/22366 (Novo Nordisk A/S) .
• suitable protein disulfide isomerases include PDIs described in WO 95/01425 (Novo Nordisk A/S).
• the invention relates to skin care compositions comprising a modified enzyme of the invention and ingredients known to be used in skin care compositions
• a number of enzyme activities are known to be used skin care compositions.
• Proteases are effective ingredients in skin cleaning products. Proteases remove the upper layer of dead keratinous skin cells and thereby makes the skin look brighter and more fresh. Further, proteases also improves the smoothness of the skin.
• Proteases are used in toiletries, bath and shower products, including shampoos, conditioners, lotions, creams, soap bars, toilet soaps, and liquid soaps.
• Lipases can be applied for cosmetic use as active ingredients in skin cleaning products and anti-acne products for removal of excessive skin lipids, and in bath and shower products such as creams and lotions as active ingredients for skin care.
• Lipases can also be used in hair cleaning products (e.g. shampoos) for effective removal of sebum and other fatty material from the surface of hair.
• hair cleaning products e.g. shampoos
• oxidase usually glucose oxidase
• substrate e.g. glucose
• peroxidase usually lactoperoxidase
• oxidoreductases are oxidative hair dyeing using oxidases, peroxidases and laccases (See e.g. WO 96/00290 or WO 95/33836 from Novo Nordisk) .
• the free radicals activate chain reactions that leads to destruction of fatty membranes, collagen, and cells.
• Protein disulfide isomerase is also an oxidoreductase. It may be utilised for waving of hair (reduction and reoxidation of disulfide bonds in hair) and repair of spoiled hair (where the damage is mainly reduction of existing disulfide bonds) .
• Transglutaminase Skin care compositions for application to human skin, hair or nails comprise (a) an amino-functional active ingredient, (b) transglutaminase to catalyse crosslinking of the active ingredient to the skin, hair or nails, and (c) a carrier is known from US patent no. 5,490,980.
• a cosmetic composition suitable for application to mammalian skin, hair or nails comprising: (a) at least one corneocyte envelope protein in an amount sufficient to provide a protective layer on said skin, hair or nails; (b) a transglutaminase in an amount sufficient to form covalent bonds between the corneocyte envelope protein and externally exposed corneocyte proteins present in the stratum corneum of said skin, hair or nails; (c) calcium ions in an amount sufficient to activate the transglutaminase; and (d) a cosmetically ac- ceptable vehicle, wherein the composition comprises an emulsion having two phases and wherein the corneocyte envelope protein is contained in one of the phases and the transglutaminase is contained within the other phase (see US patent no. 5,525,336).
• JP 3083908 describes a skin cosmetic material contains a transglutaminase modified with a water-soluble substance.
• the modifying substance is, e.g., one or more of polyethylene glycol, ethylene glycol, propylene glycol, glycerine, polyvinyl alcohol, glucose, sucrose, alginil acid, carboxymethyl cellulose, starch, and hydroxypropyl cellulose.
• the modification is done, e.g., by introducing reactive groups and bonding to the enzyme. For providing a material mild to the skin, causing less time-lapse discolouring and odorising, and having good effects of curing rough skin, retaining moisture, and conditioning the skin beautifully.
• the invention relates to a skin care product comprising a skin care composition of the invention.
• skin care products are defined above.
• a skin care product of the invention may comprise from an effective amount of modified enzymes of the invention. Such effective amounts known to the skilled person may will often lie in the range from above 0 to 5% of the final skin care product.
• Contemplated skin care products of the invention include, without being limited thereto, the following products: soap, cosmetics, skin creams, skin milk, skin lotion, skin gel, cleansing cream, cleansing lotion, cleansing milk, cold cream, cream soap, makeup base, milky lotion, pack, calamine lotion, T zone essence, hand cream, essence powder, whitening powder, powder soap, cake soap, transparent soap, lip cream, lipstick, nourishing essence, creamy foundation, face powder, powder eyeshadow, powder foundation, nail polish remover, hair tonic, hair liquid, hair cream, hair gel, hair treatment, hair setting preparations, hair dyes, hair colorants, scalp treatment, shampoo, balsam, hair rinse, hair spray sun oil, sun screen, shaving foam, shaving cream, baby oil, acne care products, antiperspirants, insect repellents, deodorants etc.
• ingredients used in skin care products is meant to cover all ingredients which are known to be used in skin care product formulations. Examples of such ingredients ingredients can be found in "Cosmetics and Toiletries” edited by Wilfried
• Antioxidants 2, 6-bis(l, 1-Dimethylethyl) - 0.1-0.3
• Plasticizers Stearyl mono/diglycerides 0-10
• Foam bath and shower bath Ingredients Examples % %
• Foam stabilizers Fatty acid alkanol amides 0.2-2 0-4
• Active agents Vegetable extracts 0-1 0-1
• Body lotion oil-in-water type
• skin lotion for application on the wet skin
• Emulsifiers Cetyl/Stearyl alcohol polyglycol ethers 1 1 --33 — -
• Vegetable extracts 0. .5- -3 Enzymes Protease/Lipase 0- -5 Water Balance
• Foam boosters Fatty acid ethanol amides 0.5-2.5 Conditioners Quaternized hydroxyethyl 0.4-1 cellulose
• Anti-dandruff agents 0-1 Preservatives 5-Bromo-5-nitro-l , 3-dioxane 0.1-0.3
• Surfactants Fatty alcohol poly- glycol ethers 0.1-0.2 1.5-2.5 Cetyl trimethyl ammonium chloride 0.5-1 Dimethyl benzyl stearyl ammonium 0.5-1 chloride
• Consistency regulators Fatty alcohols 1-2.5 2.5-3.5 Thickeners Methyl hydroxypropyl cellulose 0.3-0.6 0.4-0.8
• Dyestuffs ⁇ 0.1 ⁇ 0.1 pH-Regulators Acids/Bases 0,1-1 0.1-1 Fragrances 0.2-0.5 0.2-0.5 Enzymes Protease/Lipase 0-5 0-5 Water Balance Balance
• Emulsifiers Etboxylated castor oil 0 . 1- -0 . . 5 Fragrances 0 . 1- -0 . , 2 Dyestuffs ⁇ 0 . 1 Enzymes Lipase 0 >-E Water Bal ance
• the invention relates to the use of a modified enzyme of the invention for reducing the sensitisation potential of skin care products by reducing the IgE response when the skin care product is used.
• PD498 Protease of subtilisin type shown in WO 93/24623. The sequence of PD498 is shown in SEQ ID NO. 1 and 2.
• Subtilisin DY Protease of the subtilisin type shown in SEQ ID NO. 4 isolated from Bacillus sp. variant (Detzel et al. (1993), Archives of Biophysics, Vol. 302, No. 2, p. 499-502).
• ELISA reagents Horse Radish Peroxidase labelled anti-rat-Ig (Dako, DK, P162, #
• Mouse anti-rat IgE (Serotec MCA193; dilution 1:200).
• Rat anti-mouse IgE (Serotec MCA419; dilution 1:100).
• Biotin-labelled mouse anti-rat IgGi monoclonal antibody (Zymed 03-9140; dilution 1:1000)
• Tresyl chloride (2, 2, 2-triflouroethansulfonyl chloride) (Fluka)
• Tween 20 Poly oxyethylene sorbitan mono laurate (Merck cat no.
• OPD o-phenylene-diamine, (Kementec cat no. 4260) Test Animals:
• the Brown Norway rats (BN) weighed at the starting time more than 5 250 grams and at termination approximately 450 grams.
• mice about 20 grams (purchased from Bomholdtgaard, Ry, DK))
• mice Female Balb/C mice (20 grams) are immunized by subcutaneous injection of 50 ⁇ l of a 0.9% (wt./vol.) NaCI solution containing 25 ⁇ l of PD498, PD498-SPEG 5,000 and Glycine-SPEG-15, 000 0 respectively. The amount of protein for each batch are measured by the NanoOrange Protein Quantification test (Molecular Probes Europe N-6666) . Immunizations were performed every second week over a period of three month. Blood samples (200 ⁇ l) were collected from the eye one week after the immunization. Serum is 5 obtained by blood clothing and centrifugation.
• Antigen is incubated with % dilutions of sera in Dilution Buffer. Make those solutions just before adding them to the wells. Keep some wells free for Dilution Buffer only (Blanks) . 0 Incubate at least 1 hour at room temperature. 50 ⁇ l/well. Shake gently. Wash the plates 3 times in Washing buffer.
• a three layer sandwich ELISA is used to determine relative concentrations of specific IgE serum antibodies.
• 30) Coat the ELISA-plate with 10 ⁇ g rat anti-mouse IgE or mouse anti-rat IgE/ l buffer 1.
• ELISA procedure for determination of IgG j _ positive guinea pigs ELISA microtiter plates are coated with rabbit anti-PD498 1:8000 in carbonate buffer (pH 9.6) and incubated over night at 4°C. The next day the plates is blocked with 2% BSA for 1 hour and washes 3 times with PBS Tween 20.
• A4187 is applied and incubated for 1 hour, washed 2 times in PBS Tween 20 and 1 time with diethanol amine buffer.
• the marked alkaline phosphatase is developed using p- nitrophenyl phosphate for 30 minutes at 37°C or until appropriate 5 colour has developed.
• the reaction is stopped using Stop medium (K 2 HP0 4 /HaH 3 buffer comprising EDTA (pH 10)) and read at OD 405/650 using a ELISA reader.
• Stop medium K 2 HP0 4 /HaH 3 buffer comprising EDTA (pH 10)
• Double blinds are included on all ELISA plates. l ⁇ Positive and negative sera values are calculated as the average blind values added 2 times the standard deviation. This gives an accuracy of 95%.
• test animals are Brown Norway rats (BN) in groups of 10. Weight at time of start is more than 250 grams and at termination approximately 450 grams.
• the animals are stimulated once a week for 10 consecutive weeks.
• a three layer sandwich ELISA is used to determine relative 35 concentrations of specific antibodies.
• the immunizing molecule is used as coating antigen with 10 ⁇ g per ml and 50 ⁇ l per well, in neutral phosphate buffer, incubated overnight at 4°C. All remaining binding spots on the well surface are blocked in 2 % skim milk, 200 ⁇ l per well in phosphate buffer for at least 30 minutes at room temperature (RT) . All seras to be tested with this antigen are added at 50 ⁇ l per well to this plate using a 8-channel pipette in dilution series from 10 x diluted followed by 3-fold dilutions. Dilutions are made in phosphate buffer with 0.5 % skim milk and 0.05% Tween20, incubated 2 hours on agitation platform at RT.
• the "tracer” molecule is biotinylated Mouse anti Rat IgE 50 ⁇ l per well and diluted 2000 x in phosphate buffer with 0.5 % skim milk and 0.05% Tween 20, incubated 2 hours on an agitation platform at RT. Control (blank) was identical sequence but without rat sera. 50 ⁇ l per well streptavidin horse raddish peroxidase, diluted 2000 x was incubated 1 hour on an agitation platform. Colouring substrate at 50 ⁇ l per well is OPD (6 mg) and H 2 0 2 (4 ⁇ l of a 30% solution) per 10 ml citrate buffer pH 5.2. The reaction is stopped using 100 ⁇ l per well 2 N H 2 S0 4 . All readings on SLT at 486 nm and 620 nm as reference. Data is calculated and presented in Lotus.
• Electrophoretic separation of proteins was performed by standard methods using 4-20% gradient SDS poly acrylamide gels (Novex) . Proteins were detected by silver staining. The molecule weight was measured relative to the mobility of Mark-12® wide range molecule weight standards from Novex.
• Proteases cleave the bond between the peptide and p- nitroaniline to give a visible yellow colour absorbing at 405 nm.
• Buffer e.g. Britton and Robinson buffer pH 8.3
• Substrate 100 mg suc-AAPF-pNa is dissolved into 1 ml dimethyl sulfoxide (DMSO) . 100 ⁇ l of this is diluted into 10 ml with Britton and Robinson buffer.
• DMSO dimethyl sulfoxide
• the substrate and protease solution is mixed and the absorbance is monitored at 405 nm as a function of time and ABS 405 rim/rain.
• the temperature should be controlled (20-50°C depending on protease) . This is a measure of the protease activity in the sample.
• Activation of mPEG 15,000 with N-succinimidyl carbonate mPEG 15,000 was suspended in toluene (4 ml/g of mPEG) 20% was distilled off at normal pressure to dry the reactants azeotropically.
• Dichloromethane dry 1 ml/g mPEG
• phosgene in toluene (1.93 M 5 mole/mole mPEG) was added and mixture stirred at room temperature over night. The mixture was evaporated to dryness and the desired product was obtained as waxy lumps.
• the molecule weight of the obtained derivative was approximately 100 kDa, corresponding to about 13 moles of mPEG attached per mole PD498. Compared to the parent enzyme, residual activity was close to 100% towards peptide substrate (succinyl-Ala-Ala-Pro-Phe-p- Nitroanilide) .
• Subtilisin DY was conjugated to mPEG 5,000 with N- succinimidyl carbonate using the same procedure as described in Example 3.
• mice were stimulated subcutaneously (SC) with modified PD498-SPEG 5,000, parent unmodified PD498 and Glycine- SPEG 15,000 prepared as described in the examples above.
• Sera from immunized mice were tested in a specific IgE ELISA (described above) to elucidate whether the molecules could activated the immune response system giving rise to a specific IgE response (See Figure 1) .
• Figure 2 shows the IgGi levels of Dunkin Hartley guinea pigs during the trail period of 10 weeks. As can be seen the level of IgGi of tne modified PD498 is not detectable before tapping no. #7 (Ta p-7) eqv. to 7 weeks. The IgGi level was not significantly increased upon successive stimulations with the modified PD498.
• modified PD498-SPEG 5,000 were tested in guinea pigs by IT trails.
• the guinea pigs were stimilated once a week for 10 consecutive weeks.
• Groups of 10 guinea pigs were stimulated intratrachaeally (IT) with 0.3 icrograms, 3 micrograms, 30 icrograms, 300 micrograms of:
• MOLECULE TYPE DNA (genomic)
• ORIGINAL SOURCE
• Leu Asp Ser lie Ala Ser Gly He Arg Tyr Ala Ala Asp Gin Gly Ala 115 120 125
• TCA TTC TCC AAT TAC GGA ACG TGG GTG GAT GTC ACT GCT CCA GGT GTG 624 Ser Phe Ser Asn Tyr Gly Thr Trp Val Asp Val Thr Ala Pro Gly Val 195 200 205

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  • 1998-01-12 EP EP98900274A patent/EP0954572A1/de not_active Withdrawn
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