AU736806B2 - A modified enzyme for skin care - Google Patents

Description

WO 98/30682 WO 9830682PCT/DK98/00015 Title:A modified enzyme for skin care FIELD OF THE INVENTION 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.

BACKGROUND OF THE INVENTION Since ancient time man has enjoyed taking baths and showers. This has not changed. For most people today bathing and showering are part of the daily rituals performed to maintain a good body hygiene and to obtain a pleasant scent. Certain people also regard a refreshing shower or bath in the morning as an important and necessary psychological experience without which they just cannot wake up.

A vast number of products for body care and maintenance of a good body hygiene, e.g. for cleansing and moisturising all parts of the body, are found on the consumer market. A few of these products comprise modified enzymes as an active ingredient.

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.

However, 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.

Furthermore, also the high costs of enzymes at that time wO98/30682 PCTK9/0001n 2 inhibited the application of enzymes in such personal care products.

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 Corium (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.

It is today generally recognised that the stability of polypeptides are improved and the immune response are reduced when polypeptides, such as enzymes, are coupled to polymeric molecules.

Techniques for conjugating polymeric molecules to polypeptides are well known in the art.

One of the first suitable commercially techniques was described back in the early 1970'ies (US patent no. 4,179,337).

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.

GB patent no. 1,183,257 (Crook et al.) describes chemistry for conjugation of enzymes to polysaccharides via a triazine ring.

wOr 8/3068) P 9 v outQWuuI3 1 1IJkJJOIUUU1 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 maintaining the activity in polyethylene glycol-modified proteolytic enzymes by linking the proteolytic enzyme to a macromolecularized inhibitor. The conjugates are intended for medical applications.

It has been found that the attachment of polypeptides to polymeric molecules in general has the effect of reducing the activity of the polypeptide or interfering with the interaction between the polypeptide and its substrate. 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 (Kanebo) 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 described 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.

Short Summary of the general knowledge based on prior art Techniques for coupling one or more polymeric molecules to a polypeptide molecule are known in the art. Further, it is known that such modified enzyme-polymer conjugates have a reduced immune response and have an improved stability.

SUMMARY OF THE INVENTION It is the object of the present invention to provide improved 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.

s The inventors found that the number and weight of the polymeric molecules coupled to the surface of the enzyme must be balanced with the weight and/or surface area of the enzyme.

Further, the position of coupling the polymeric molecules are also of importance.

Herein disclosed is 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 a parent enzyme having a to molecule weight from 15 to 100 kDa.

In the case of a parent enzyme having a molecule weight from 15 to 35 kDa, from 4 to polymeric molecules are coupled covalently to the surface of the enzyme.

If the molecule weight of the parent enzyme lies in the range from 35 to 60 kDa from 7 to preferably 10 to 30 polymeric molecules are coupled to the surface of said parent enzyme.

Likewise, is the parent enzyme has a molecule weight from 60 to 80 kDa from 10 to preferably 13 to 40 polymeric molecules are coupled to the surface of said parent enzyme.

From 15 to 70, preferably 18 to 60 polymeric molecules are coupled to the surface of parent enzymes having a molecule weight from 80 to 100 kDa.

Normally polymeric molecules are coupled to the amino groups (-NH 2 on the enzyme's 20 surface and a the N-terminal amino group. However, 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.

0.:0 Carboxylic acid attachment groups may be the carboxylic acid group of Aspartate or *00: Glutamate and the C-terminal COOH-group.

o0 25 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.

paThe parent enzyme may be a hydrolase, including proteases, in particular subtilisins, or lipase, or an Oxidoreductase, including laccases and Superoxide dismutase.

According to a first embodiment of the invention, there is provided a modified enzyme 30 characterised by having from 4 to 70 polymeric molecules with a molecular weight from 1 to covalently coupled to the surface of the protease, wherein the protease has an amino acid 0 sequence of SEQ ID NO: 2.

In a second aspect the invention relates to skin care compositions, comprising a modified enzyme of the invention further ingredients being used in skin care products.

Thus, according to a second embodiment of the invention, there is provided a skin care composition comprising a modified enzyme according to the invention, and further ingredients known to be used in skin care products.

SRA In a third aspect the invention relates to skin care product comprising a skin care Scomposition of the invention.

[I:\DayLib\LIBA]03973.dac:mrr Thus, according to a third embodiment of the invention, there is provided a skin care product comprising a skin care composition according to the invention, wherein the product is selected from the group of soap, cosmetics, skin creams, skin milk, skin lotion, skin gel, cleansing cream, cleansing lotion, cleansing milk, cold cream, cream soap, make-up 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, shaving cream baby oil, 1o acne care products, antiperspirants, insect repellents, deodorants etc; and the use of a modified enzyme according to the invention for reducing the sensitisation potential of skin care products.

The skin care product of the invention has improved stability and reduced sensitisation potential in comparison to corresponding skin care products (with parent enzymes).

According to a fourth embodiment of the invention, there is provided a synthetic detergent composition, comprising a modified enzyme according to the invention, and further ingredients known to be used in synthetic detergents; and the use of a modified enzyme according to the invention for reducing the sensitisation potential of synthetic detergents.

The term "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 S 20 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 enzymes.

In the context of the present invention "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.

sa Specific examples of 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 30 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 [I:\DayLib\L1BA]03973.doc:mrr W3 o/tnKR 1PT/r rno nn 6 lL 70 UUUJD spray sun oil, sun screen, shaving foam and gel, shaving cream, baby oil, acne care products, antiperspirants, insect repellents, deodorants etc.

Assessment of allergenicity 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 induced in previously sensitised animals. According to these models the alleged allergens are introduced intratracheally into the animals.

A suitable strain of guinea pigs, the Dunkin Hartley strain, do not as humans, produce IgE antibodies in connection with the allergic response. However, they produce another type of antibody the IgGlA and IgGlB (see e.g. Prento, ATLA, 19, p. 8-14, 1991), which are responsible for their allergenic response to inhaled polypeptides including enzymes. Therefore, when using the Dunkin Hartley animal model, the relative amount of IgGlA and IgGIB is a measure of the allergenicity level.

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.

More details on assessing respiratory allergens in guinea pigs and mice is described by Kimber et al.,(1996), Fundamental and Applied Toxicology, 33, p. 1-10.

Other animals such as rats, rabbits etc. may also be used for comparable studies.

BRIEF DESCRIPTION OF THE DRAWING Figure 1 shows the kinetics of the specific anti-PD498 IgE WO 98/30l682 PCTID98/l"onnm 7 •IUJntl OIUUU A 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 jig, 30 4g and 300 ig of modified PD498-SPEG 5,000 in the Dunkin Hartley guinea pigs IT dose response study (M 3.0 jg; A 30 4g; V 300 4g). The 0.3 jg dose curve is ommitted due to nor response at all.

Figure 4 shows the IgG 1 levels of 0.3 4g, 3.0 jig and 30 jg of unmodified parent PD498 in the Dunkin Hartley guinea pigs IT dose response study (M 0.3 jg; A 3.0 jg; V 30 ig).

DETAILED DESCRIPTION OF THE INVENTION It is the object of the present invention to provide modified enzymes suitable for skin care.

As mentioned above it is known to couple polymeric molecules to enzymes to improve the stability and to reduced the sensitisation potential of polypeptides, including enzymes. One of the problems arising when coupling polymeric molecules to enzymes are the loss of enzymatic activity.

According to the above mentioned EP 471,125 (Kanebo) 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 Esperase®) has 3 accessible amino (-NH 2 attachment group to which polymeric molecules (in this case polysaccharides) may be coupled. The attachment groups are present as two amino groups two Lysine residues on the surface of the 3D structure) and one N-terminal amino group. When coupling up to 3 polymeric molecules to said protease (a modification rate in the range of 68% to 71%, determined by the TNBS method (Haynes et al., (1967), Biochemistry 6, p. 641)) the residual enzymatic activity maintained is asserted to lie in the range from 45% (see Example 4) to 67% (see Example 3).

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(s) to obtain WO98/30682 PCr'TID/00015o/hint Wfl 021-40682 1P(TlfltO~nnn.1 C 8 improved stability and a reduced sensitisation potential while still having a substantial residual enzymatic activity maintained. The inventors found that the number and/or weight of the polymeric molecules coupled to the surface of the enzyme must be balanced with the weight and/or surface area of the enzyme.

Further, the position (on the surface) of coupling the polymeric molecules are also of importance.

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.

If only few polymeric molecules are coupled to a heavy enzyme with a large surface area said few polymeric molecules are not capable of shielding hiding/covering) the epitope(s) on the enzyme's surface responsible for the immune response resulting in the antibody formation, especially IgE antibodies.

The above mentioned EP 471,125 (Kanebo) describes coupling of few up to 3) heavy 40 and 50 kDa) polymeric molecules to the surface of the microbial protease Esperase® having a molecule weight of about 28 kDa.

In the first aspect 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.

According to the present invention 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.

In other words, 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).

WO 98/30682 lPC' T[ I/D98/0015 WO 98/30682.9 aPVTssWGoQvvt, According to the invention 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.

Table 1 Molecule weight of Number of polymeric Average molecule enzyme (Mw) molecules coupled to weight of the kDa the enzyme polymeric molecules kDa to 35 4-20 1-35 to 60 7-40 1-35 to 80 10-50 1-35 to 100 15-70 1-35 more than 100 more than 20 1-35 The molecule weight of the polymeric molecules may according to the invention be within the ranges between 1 and 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.

All polymer molecule weights mentioned are average molecule weights.

Position of the coupled polymeric molecules Virtually all ionized groups, such as the amino group of Lysine residues, are on the surface of the polypeptide molecule (see for instance Thomas E. Creighton, (1993), "Proteins", W.H.

Freeman and Company, New York). Therefore, the number of readily accessible attachment groups amino groups) on the enzyme's surface typically equals the number of Lysine residues in the primary structure of the enzyme plus the N-terminus amino group.

When choosing a parent enzyme for skin care compositions and products to be conjugated it is advantageous to use an enzyme with the number of attachment groups referred to above in Table WO 98/30682 PDfFK98/00n1 10 u L, .1.

Sensitisation potential vs. maintained residual enzymatic activity Especially for enzymes, in comparison with other proteins and polypeptides, there is a conflict between reducing the immune system's response toward enzymes and maintaining a substantial residual enzymatic activity as the activity of enzymes are connected with interaction between a substrate and the active site in a cleft in the enzyme structure.

According to the invention 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.

Without being limited to any theory 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 (Kanebo) has few up to 3 polymeric molecules) heavy/bulky polymeric molecules 40 and 50 kDa polysaccharides) coupled to amino groups on the enzyme's surface. The loss of enzymatic activity observed 45% to 67% residual enzymatic activity) might be a due to uneven interaction on different part of the enzyme's surface, causing the enzyme to be pulled out of it normal parent state configuration. Further, 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.

WO/30682l gA~ PD K9II~'Onn WO 98/306817 DrTrinivnO In 11 When coupling a larger number of less bulky/heavy polymeric molecules to the enzyme surface the disadvantageous impact of the polymeric molecules is believed to be less pronounced, as the forces having affect on the enzyme structure are more evenly/uniformly distributed over a larger area on the surface of the enzyme. The impact of the polymeric molecules on the loss of activity are hereby less pronounced.

Consequently, it is preferred to couple more polymeric molecules more than 4) with a relatively low molecule weight 1-35 kDa) to the enzyme's surface (in the case of enzymes with a molecule weight from 15 to 35 kDa).

In a preferred embodiment of the invention the polymeric molecules are spread broadly over the surface of the enzyme, with the exception of the area close to the active site. In the present context "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.

To ensure a minimal loss of enzymatic activity it is preferred not to couple polymeric molecules 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.

Further, enzymes having coupled polymeric molecules at (a) known epitope(s) recognisable by the immune system or close to WO 98/30682 PCT/DK98/001n 12 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 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 molecule The polymeric molecules coupled to the enzyme may be any suitable polymeric molecule, including natural and synthetic homo-polymers, such as polyols poly-OH), polyamines (i.e.

poly-NH 2 and polycarboxyl acids poly-COOH), and further hetero-polymers i.e. polymers comprising one or more different coupling groups e.g. a hydroxyl group and amine groups.

Examples of suitable 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 polypropylen glycols, PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), Branced PEGs, poly-vinyl alcohol (PVA), polycarboxylates, 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, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose carboxyethylcellulose and hydroxypropylcellulose, hydrolysates of chitosan, starches such as hydroxyethyl-straches and hydroxy propylstarches, glycogen, agaroses and derivates thereof, guar gum, pullulan, inulin, xanthan gum, carrageenin, pectin, alginic acid hydrolysates and bio-polymers.

Preferred polymeric molecules are non-toxic polymeric WO 98/30682 PC^TIDK98o /nnn c 1 3 O I V n i .i e .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.

Generally seen polyalkylene oxides (PAO), such as polyethylene oxides, such as 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.

Even though all of the above mentioned polymeric molecules may be used according to the invention the methoxypolyethylene glycols (mPEG) 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.

Activation of polymers If the 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.

Methods and chemistry for activation of polymeric molecules as well as for conjugation of proteins are intensively described in the literature. Commonly used methods for activation of insoluble polymers include activation of functional groups with cyanogen bromide, periodate, glutaraldehyde, biepoxides, epichlorohydrin, divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine etc. (see R.F. Taylor, (1991), "Protein immobilisation. Fundamental and applications", Marcel Dekker, S.S. Wong, (1992), "Chemistry of Protein Conjugation and Crosslinking", CRC Press, Boca Raton; G.T. Hermanson et al., (1993), "Immobilized Affinity Ligand Techniques", Academic Press, Some of the methods concern activation of insoluble polymers but are also applicable to activation of soluble polymers e.g. periodate, trichlorotriazine, sulfonylhalides, divinylsulfone, carbodiimide etc. The functional groups being Wn 98/062 PC'T/TK9/0015 14 amino, hydroxyl, thiol, carboxyl, aldehyde or sulfydryl on the polymer and the chosen attachment group on the protein must be considered in choosing the activation and conjugation chemistry which normally consist of i) activation of polymer, ii) conjugation, and iii) blocking of residual active groups.

In the following a number of suitable polymer activation methods will be described shortly. However, it is to be understood that also other methods may be used.

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..

Soc., 98, 289-291) or diazoacetate/amide (Wong et al., (1992), "Chemistry of Protein Conjugation and Crosslinking", CRC Press).

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 orthopyridyl disulfide. Also vinylsulfone (US patent no. 5,414,135, (1995), Snow et al.) has a preference for sulfhydryl groups but is not as selective as the other mentioned.

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. Ed., Academic Press: Orlando, p. 56-66; Nilsson et al., (1987), Methods in Enzymology vol. 135; Mosbach, Ed.; Academic Press: Orlando, pp. 65-79; Scouten et al., (1987), Methods in Enzymology vol. 135, Mosbach, Ed., Academic Press: Orlando, 1987; pp 79-84; Crossland et al., (1971), J. Amr. Chem.

Soc. 1971, 93, pp. 4217-4219), mesylates (Harris, (1985), supra; Harris et al., (1984), J. Polym. Sci. Polym. Chem. Ed. 22, pp 341-352), aryl sulfonates like tosylates, and para-nitrobenzene sulfonates can be used.

Wn O8/30682 PC''TIDK'ofnn I u OIUUL1y Organic sulfonyl chlorides, e.g. 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. In addition to high conjugation yields, 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. 100-114; Monfardini et al., (1995), Bioconjugate Chem., 6, 62-69, with imidazole (Allen et al., (1991), Carbohydr. Res., 213, pp 309-319), with paranitrophenol, DMAP (EP 632 082 Al, (1993), Looze, etc. The derivatives are usually made by reacting the chloroformate with the desired leaving group. All these groups give rise to carbamate linkages to the peptide.

Furthermore, 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 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 Nhydroxy succinimide.

Furthermore, a special linker can be introduced. The oldest Wn 98/3068i2 DCIKII98/ In e o16 7ou being cyanuric chloride (Abuchowski et al., (1977), J. Biol.

Chem., 252, 3578-3581; US patent no. 4,179,337, (1979), Davis et al.; Shafer et al., (1986), J. Polym. Sci. Polym. Chem. Ed., 24, 375-378.

Coupling of PEG to an aromatic amine followed by diazotation yields a very reactive diazonium salt which in situ can be reacted with a peptide. An amide linkage may also be obtained by reacting an azlactone derivative of PEG (US patent no. 5,321,095, (1994), Greenwald, R. thus introducing an additional amide linkage.

As some 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 Lysine residues may also be used as the backbone.

The parent enzyme The 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 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 wildtype) enzyme or may be a variant thereof.

Assessing/selecting suitable parent enzyme 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 NMRstructures.

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 sharing at least 30% sequence identity WO 98/30682 DCrfT/DK/tnn e 1 7 x l U M O IU U Il WO 98/30l682 1Df-ftn01 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 3Dstructures exist, definition of Structurally Conserved 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 3Dstructures are known to be quite difficult to model, and structural predictions must be considered with care.

Having obtained the 3D- structure of the enzyme in question, or a model of the structure based on homology to known structures, 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 1, "Enzyme Nomenclature, (1992), Academic Press, Inc.), such as laccase and Superoxide dismutase (SOD); Hydrolases E.C.

3, including proteases, especially subtilisins, and lipolytic enzymes; Transferases, such as transglutaminases (TGases); Isomerases such as Protein disulfide Isomerases (PDI).

Hydrolases Proteolytic enzymes Contemplated proteolytic enzymes includes selected from the group of acidic aspartic proteases, cysteine proteases, serine proteases, such as subtilisins, or metallo proteases, with the WO9 83062 Dr IKno ,nnn T. w 018 '-AZ OIUUUAI above indicated properties number of attachment groups, position of attachment groups etc.).

Specific examples of suitable parent proteases having a suitable number of attachment groups are indicated in Table 2 below: Table 2 Enzyme Number of Molecule Reference attachment weight groups kDa PD498 13 29 Seq. ID No. 2 WO 93/24623 Savinase® 6 27 von der Osten et al., (1993), Journal of Biotechnology, 28, p. Proteinase K 9 29 Gunkel et al., (1989), Eur. J. Biochem, 179, p. 185-194 Proteinase R 5 29 Samal et al, (1990), Mol. Microbiol, 4, p. 1789-1792 Proteinase T 14 29 Samal et al., (1989), Gene, 85, p. 329-333 Subtilisin DY 13 27 Betzel et al. (1993), Arch. Biophys, 302, no.

2, p. 499-502 Lion Y 15 46 SEQ ID NO. 4 JP 04197182-A Rennilase® 39 Available from Novo Nordisk A/S Jal6 5 28 WO 92/17576 Thermolysin 12 34 Titani et al., (1972) Nature New Biol. 238, p. 35-37, and SEQ ID NO Alcalase® 10 27 von der Osten et al., WO98/30682 PCTIDK98/00015 19 (a natural (1993), Journal of subtilisin Biotechnology, 28, Carlberg variant) p. 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. As mentioned above 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 away from the active site).

The enzyme Subtilisin DY has a molecule weight of 27 kDa and has 12 amino groups 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 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 Lysine residues) on the surface plus one N-terminal amino group. (See SEQ ID NO Lipolytic enzymes Contemplated lipolytic enzymes include include Humicola lanuginosa 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.

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 B. subtilis lipase (Dartois et al., (1993) Biochemica et Biophysica acta 1131, 253-260), a B. stearothermophilus lipase (JP 64/744992) and a B. pumilus lipase (WO 91/16422). Other types of lipolytic include cutinases, e.g. derived from Pseudomonas mendocina as described in WO 88/09367, or a cutihase derived from WO 98/30682 PCTID/00015nni Fusarium solani pisi described in WO 90/09446).

Oxidoreductases Laccases Contemplated laccases include the laccases disclosed in WO 96/00290 and WO 95/33836 from Novo Nordisk.

Transferases Transglutaminases Suitable transferases include any trnsglutaminases disclosed in WO 96/06931 (Novo Nordisk A/S) and WO 96/22366 (Novo Nordisk A/S).

Isomerases Protein Disulfide Isomerase Without being limited thereto suitable protein disulfide isomerases include PDIs described in WO 95/01425 (Novo Nordisk

A/S).

Enzyme activities suitable for Skin Care In the second aspect 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 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 Lipases can be applied for cosmetic use as active WO 98/30682 H/flr./lMltnO ,nln. i 2% 1 ZI J/IJR /OIUUU1 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.

Oxidoreductases The most common oxidoreductase for personal care purposes is an oxidase (usually glucose oxidase) with substrate (e.g.

glucose) that ensures production of H 2 0 2 which then will initiate the oxidation of for instance SCN~ or I" into antimicrobial reagents (SCNO~ or I2) by a peroxidase (usually lactoperoxidase). This enzymatic complex is known in nature from e.g. milk and saliva.

It is being utilised commercially as anti-microbial system in oral care products (mouth rinse, dentifrice, chewing gum) where it also can be combined with an amyloglucosidase to produce the glucose. These systems are also known in cosmetic products for preservation.

Another application of oxidoreductases are oxidative hair dyeing using oxidases, peroxidases and laccases (See e.g. WO 96/00290 or WO 95/33836 from Novo Nordisk).

Free radicals formed on the surface of the skin (and hair) known to be associated with the ageing process of the skin (spoilage of the hair).

The free radicals activate chain reactions that leads to destruction of fatty membranes, collagen, and cells.

The application of free radical scavengers such as Superoxide dismutase into cosmetics is well-known L.

Goldemberg, DCI, Nov. 93, p. 48-52).

Protein disulfide isomerase (PDI) 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).

Transqlutaminase WO 98/30682 PrT/DK98l/000 22 Skin care compositions for application to human skin, hair or nails comprise an amino-functional active ingredient, transglutaminase to catalyse crosslinking of the active ingredient to the skin, hair or nails, and a carrier is known from US patent no. 5,490,980.

A cosmetic composition suitable for application to mammalian skin, hair or nails comprising: at least one corneocyte envelope protein in an amount sufficient to provide a protective layer on said skin, hair or nails; 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; calcium ions in an amount sufficient to activate the transglutaminase; and a cosmetically acceptable 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, 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, 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 Skin Care Products of the invention In the third aspect the invention relates to a skin care product comprising a skin care composition of the invention. The term "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.

W I/302n on PTD/f V faitt^*a uM Jo uui W 2 3 A14 L.LUYOI1UI 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.

General skin care product formulations The term "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 Umbach and published by Ellis Horwood, Limited, England, (1991), and "Surfactants in Consumer Products", edited by J. Falbe and published by Spring-Verlag, (1987).

In the following a non exhausting list of guide formulations are listed. These provide an overwiev of formulations of important skin care products contemplated according to the invention.

Toilet soap Ingredients Examples Surfactants Soap (sodium salt) 83 -87 Sequestering agents Ethylenediamine tetraacetate 0.1-0.3 Consistency regulators Sodium chloride approx.

Dyestuffs 0.1 Optical brighteners 0.1 Antioxidants 2,6-bis(l,1-Dimethylethyl)- 0.1-0.3 4-methyl phenol(BHT) WO 98/30682 Whitening agents Fragrances Enzymes Water 24 Titanium dioxide Protease/Lipase PCT/DK98/00015 0.1-0.3 1.0-2.0 Balance Syndet (Synthetic Detergents) Ingredients Examples Surfactants Lauryl sulfate Lauryl sulfo succinate Refatting agents Fatty alcohols Plasticizers Stearyl mono/diglycerides Fillers Starches Active agents Salicylic acid 30-50 1-12 10-20 0-10 0-10 0-1 0.2 0-2 Balance Dyestuffs Fragrances Enzymes Water Protease/Lipase Foam bath and shower bath Ingredients Examples Surfactants Refatting agents Enzymes Lauryl ether sulfate Coco amidopropyl dimethyl betaine Ethoxylated fatty acids Fatty alcohols Ethoxylated fatty alcohols Protease/Lipase Foam bath 10-20 2-4 0.5-2 0.5-3 0.5-5 0-5 Shower bath 10-12 2-4 0-4 Ingredients Foam stabilizers Examples Fatty acid alkanol amides Quaternized hydroxypro- Foam bath 0.2-2 Shower bath 0-4 Conditioners WO 98/30682 WO 9830682PCT/DK98/00015 pyl cellulose Thickeners Sodium chloride Pearlescent agents Ethyleneglycol stearate Active agents Vegetable extracts s Preservatives 5-Bromo-5-nitro-1, 3dioxane 0-3 0-2 0-1 0-0.5 0-3 0-1 Dyestuffs Fragrances Enzymes Water 0.1 0.1-0.2 0. 3-3 0-5 Balance 0.1 0.1 0.3-2 Balance Protease/Lipase Skin cream (water-in-oil type and oil-in-water type) Ingredients Examples% Emulsifiers Fatty derivatives Moisturizers Stabilizers Preservatives Sorbitane sesquioleate Aluminum stearate Triethanolamine stearate Cetyl/Stearyl alcohol polyglycol ethers Isopropyl palmitate Cetyl/Stearyl alcohol 2-Octyl dodecanol Stearic/Palmitic acid Caprylic/Capric acid triglycerides Glycerine stearate Glycerine Sorbitol Poly (hydroxy carboxylic acids) Propyleneglycol Magnesium sulfate p-Hydroxy benzoic acid ester Protease/ Lipase Water-in-oil/ Oil-in-water type type 1-2 1-2 1-5 2-10 5-10 1-5 1-5 1-3 0-3 0-2 3-7 0-3 0.5-2 0-0.8 0.2- 0.4 0-5 0-3 0.2-0.4 Enzymes WO 98/30682 WO 9830682PCTIDK98/00015 Water Balance Balance Body lotion (oil-in-water type) and skin lotion for on the wet skin Ingredients Examples Body lotion application Skin lotion Emulsifiers Fatty derivatives Moisturizers Thickeners Preservatives Enzymes Water Cetyl/Stearyl alcohol polyglycol ethers Sorbitane monolaurate Sodium stearate Sodium lauryl ether sulfate 2-Octyl dodecanol Paraflin oils Bees wax Isooctyl stearate Isopropyl palmitate Glycerine Sorbitol Polyacrylates Methyl hydroxypropyl cellulose p-Hydroxy benzoic acid ester Protease/Lipase 1 -3 0.5-1 1-3 0.5-1 3-7 3-5 0-0.3 0-0.3 1-2 0.5-2 20-25 5-10 0-1 0-0.5 0.2-0.4 0.2-0.4 0-5 Balance Balance Face lotion Ingredients Surfactants Refatting agents Solubilizers Cleaning and refreshing Examples Magnesium lauryl ether sulfate Di-n-butyl adipate Castor oil polyglycol ethers Ethanol 0.2-0.5 1-2 0.1-1 0-15 WO 98/30682 PCT/DK98/00015 components Moisturizers Preservatives Adstringents Antiirritants Enzymes Water 27 Glycerine Sorbitol p-Hydroxy benzoic acid ester Vegetable extracts Panthenol Allantoine Vegetable extracts Protease/Lipase 0.2-0.4 0-1 0-0.2 0.5-3 Balance Hair shampoo Ingredients Examples Surfactants Lauryl ether sulfate Coco fatty acid amidopropyl dimethyl betaine Fatty acid polyglycol esters Foam boosters Fatty acid ethanol amides Conditioners Quaternized hydroxyethyl cellulose Protein hydrolysates Refatting agents Ethoxylated lanolin alcohols Additives Anti-dandruff agents Preservatives 5-Bromo-5-nitro-l,3-dioxane Pearlescent agents Ethyleneglycol stearate Dyestuffs pH-Regulators Acids/Bases Fragrances Enzymes Protease/Lipase Water 12-16 0-2 0.5-2.5 0.4-1 0.2-1 0.2-1 0-1 0.1-0.3 0-2 0.1 0.1-1 0.3-0.5 Balance Hair Hair rinse and hair conditioner Ingredients Examples Hair rinse conditioner Fatty alcohol poly- Surfactants WO 98/30682 PCT/DK98/00015 Refatting agents Consistency regulators Thickeners Conditioners Preservatives Dyestuffs pH-Regulators Fragrances Enzymes Water 28 glycol ethers Cetyl trimethyl ammonium chloride Dimethyl benzyl stearyl ammonium chloride Cetyl/Stearyl mono/ diglyceride Fatty alcohols Methyl hydroxypropyl cellulose Quaternized hydroxyethyl cellulose p-Hydroxy benzoic acid ester Acids/Bases Protease/Lipase 0.1-0.2 1.5-2.5 0.5-1 0.5-1 0.5-1.5 1.5-2.5 1-2.5 2.5-3.5 0.3-0.6 0.4-0.8 0.1-0.3 0.3-0.4 0.1-0.3 1 0,1-1 0.2-0.5 0-5 Balance 0.1-0.3 <0.1 0.1-1 0.2-0.5 Balance Hair dyes Ingredients Component 1: Surfactants Consistency Examples Alkaline dyeing cream Lauryl ether sulfate Ethoxylated castor oil Fatty alcohols 1-4 1-2 8-10 regulators Reductants Sodium sulfite Buffers Ammonium chloride Sequestrants 1-Hydroxyethane-1,1diphosphonic acid Alkaline agents Ammonia Oxidation dyestuffsDeveloping agents Coupling agents 0.8-1.2 0.5-1 0.1-0.2 1.2-2 1 1 Balance Enzyme Water Laccase WO 98/30682 PCT/DK98/00015 Component II: Surfactants Oxidants Stabilizers Thickeners Enzyme Water 29 Hydrogen peroxide dispersion Lauryl ether sulfate Hydrogen peroxide 1-Hydroxyethane-1,1diphos phonic acid Polyacrylates Laccase 0.5-1 6-9 1-1.5 Balance Shaving cream Ingredients Soaps Fatty components Stabilizers Examples Palmitic/Stearic acid Potassium hydroxide Sodium hydroxide Coconut oil Polyethyleneglycol Sodium tetraborate Sodium silicate Sorbitol Protease 30-40 5-7 1-2 5-10 0-2 0-0.5 0-0.5 0-3 Balance Enzyme Water Shaving lotion Ingredients Disinfecting and phonic acid Refatting agents Solubilizers Adstringents Antiirritants Stabilizers Examples Ethanol 40-80 Di-n-butyl adipate Ethoxylated castor oil Vegetable extracts Panthenol Vegetable extracts Glycerine Sorbitol Propyleneglycol Protease 1-2 0.5-1 1-10 0-0.5 0-2 0-3 Balance Enzymes Water WO 98/30682 30 Examples Fatty alcohols WO 9830682PCT/DK98/00015 Hair pomade Ingredients Consistency regulators Mineral fats Antioxidants Fragrances Dyestuffs Enzymes Emollients Ethoxylated lanolin alcohols Vaseline Branched chain paraffins 2,6-bis(1,1-Dimethylethyl)- 4-methyl phenol (BHT) 3-6 45-52 10-18 0.5- 1 0.2-0.4 0.1 Balance Lipase Glycerine Setting lotion Ingredients Solvents Film forming components Softening agents Conditioners Antistatics Emulsifiers Fragrances Dyestuffs Enzymes Water Examples Isopropanol Vinyl pyrrolidone/vinyl acetate copolymers Vinyl pyrrolidone/dimethyl amino ethyl methacrylate Protein hydrolysates Cetyl trimethyl ammonium chloride Etboxylated castor oil Lipase 12-20 2-3.5 0.2-1 0.2-0.5 0.1-0.5 0.1-0.5 0.1-0.2 0.1 Balance In a final aspect 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.

MATERIAL AND METHODS WO 98/30682 PCTID/00/015i 31 Materials Enzymes: 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, 031; dilution 1:1000).

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) Biotin-labelled rat anti-mouse IgG1 monoclonal antibody (Serotec MCA336B; dilution 1:1000) Streptavidin-horse radish peroxidase (Kirkegard Perry 14-30-00; dilution 1:1000).

Solutions: Stop-solution (DMG-buffer) Sodium Borate, borax (Sigma) 3,3-Dimethyl glutaric acid (Sigma) CaCl2 (Sigma) Tresyl chloride (2,2,2-triflouroethansulfonyl chloride) (Fluka) Tween 20: Poly oxyethylene sorbitan mono laurate (Merck cat no.

822184) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (Fluka) N-Hydroxy succinimide (Fluka art. 56480)) Phosgene (Fluka art. 79380) Lactose (Merck 7656) PMSF (phenyl methyl sulfonyl flouride) from Sigma Succinyl-Alanine-Alanine-Proline-Phenylalanine-para-nitroanilide (Suc-AAPF-pNP) Sigma no. S-7388, Mw 624.6 g/mole.

Colouring substrate: OPD: o-phenylene-diamine, (Kementec cat no. 4260) Wn 9R0682 PCT/DK9'8/00i«1 32 Test Animals: Brown Norway rats (from Charles River, DE) The Brown Norway rats (BN) weighed at the starting time more than 250 grams and at termination approximately 450 grams.

Dunkin Hartley guinea pigs (from Charles River, Wiga Gmbh Sulzfeld 1, Sandhofer Weg, DE).

Male Dunkin Hartley, which are sero negative for Parainfluenza 3, E. cuniculi, K pneumonia and P multocida. The animal weighed at the starting time 350-450 grams Female BALB/C mice (about 20 grams) (purchased from Bomholdtgaard, Ry, DK)) Equipment: XCEL II (Novex) ELISA reader (UVmax, Molecular Devices) HPLC (Waters) PFLC (Pharmacia) column, Mono-Q, Mono S from Pharmacia, SW.

SLT: Fotometer from SLT LabInstruments Size-exclusion chromatograph (Spherogel TSK-G2000 SW).

Size-exclusion chromatograph (Superdex 200, Pharmacia, SW) Amicon Cell Methods: Immunization of BALB/C mice Female Balb/C mice (20 grams) are immunized by subcutaneous injection of 50 gl of a 0.9% (wt./vol.) NaCl solution containing p1 of PD498, PD498-SPEG 5,000 and Glycine-SPEG-15,000 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 jl) were collected from the eye one week after the immunization. Serum is obtained by blood clothing and centrifugation.

ELISA procedure to determine relative concentrations of IqG1 antibodies in BALB/C mice W o/nn' PCTKM9n U /wfl OQI1A RUO33 OUUU1; 1) Coat the ELIAS-plates with 1 Hg protein/ml in coating buffer Incubate over night at 4 0 C, or at least 3 hours at room temperature. 50 i/well. Shake gently.

2) Empty the plates and block with blocking buffer at least hour at room temperature. 200 p./well. Shake gently. Wash the plates 3 times with Washing Buffer.

3) 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).

Incubate at least 1 hour at room temperature. 50 p./well. Shake gently. Wash the plates 3 times in Washing buffer.

4) Dilute biotin-labelled rat anti-mouse IgGI monoclonal antibody or biotin-labelled mouse anti-rat IgG1 monoclonal antibody in Dilution Buffer. Incubate at room temperature at least 1 hour. p./well. Shake gently. Wash the plates 3 times in Washing Buffer.

Dilute Streptavidin-horse radish peroxidase in Dilution Buffer. Incubate at room temperature at least 1 hour. 50 p/well.

Shake gently. Wash the plates 3 times in Washing Buffer.

6) Mix 0.6 mg ODP/ml 0.4 p. H 2 0 2 /ml in substrate Buffer. Make the solution just before use. Incubate for 10 minutes. pl/well.

7)To stop the reaction: add Stop Solution. 50 p/well.

8) Read the plates at 492 nm with 620 nm as reference.

Data is calculated and presented in Lotus software.

ELISA procedure to determine relative concentrations of IqE antibodies in BALB/C mice A three layer sandwich ELISA is used to determine relative concentrations of specific IgE serum antibodies.

1) Coat the ELISA-plate with 10 pg rat anti-mouse IgE or mouse anti-rat IgE/ml buffer 1.

pl/well. Incubate over night at 4°C.

2) Empty the plates and block with Blocking buffer at least hour at room temperature.

200 p./well. Shake gently. Wash the plates 3 times with Washing Buffer.

3) Incubate with mouse/rat sera, starting from undiluted and UO O/10c682 lnrP/Tl'o IA e VV .F 7OuIUU 3 4 J lMYOIUUVJU continue with 2-fold dilutions. Keep some wells free for buffer 4 only (blanks). 50 p /well.

Incubate for 30 minutes at room temperature. Shake gently. Wash the plates 3 times in Washing Buffer.

4) Dilute the enzyme in Dilution buffer to the appropriate protein concentration. Incubate for 30 minutes at room temperature. Shake gently. Wash the plates 3 times in Washing Buffer.

Dilute specific polyclonal anti-enzyme antiserum serum (pig) for detecting bound antibody in Dilution buffer.

50p1/well. Incubate for 30 minutes at room temperature. Shake gently. Wash the plates 3 times in Washing Buffer.

6) Dilute Horseradish Peroxidase-conjugated anti-pig-antibody in Dilution buffer. 50 p /well.

Incubate at room temperature for 30 minutes. Shake gently.

Wash the plates 3 times in Washing Buffer.

7) Mix 0.6 mg ODP/ml 0.4 pl H 2 0 2 /ml in substrate Buffer. Make the solution just before use. Incubate for 10 minutes. pl/well.

8)To stop the reaction: add Stop Solution. 50 p /well.

9 Read the plates at 492 nm with 620 nm as reference.

Data is calculated and presented in Lotus.

ELISA procedure for determination of IqG 1 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 1 pg/ml PD498 is added to the plates and incubated for 1 hour, then washed 3 times with PBS All guinea pig sera samples and controls are applied to the ELISA plates with 2 pl sera and 98 pl PBS, incubated for 1 hour and washed 3 times with PBS Tween Then goat anti-guinea pig IgGi (1:4000 in PBS buffer (Nordic Immunology 44-682)) is applied to the plates, incubated for 1 hour and washed with PBS tween Alkaline phosphatase marked rabbit anti-goat 1:8000 (Sigma WO 98/30682f PCT/DK98/00015 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 pnitrophenyl phosphate for 30 minutes at 370°C or until appropriate colour has developed.

The reaction is stopped using Stop medium (K 2

HPO

4 /HaH 3 buffer comprising EDTA (pH 10)) and read at OD 405/650 using a ELISA reader.

Double blinds are included on all ELISA plates.

Positive and negative sera values are calculated as the average blind values added 2 times the standard deviation. This gives an accuracy of Intratracheal (IT) stimulation of rats For IT administration of molecules disposable syringes with a 2 long metal probe are used. This probe is instilled in the trachea of the rats approximately 1 cm below the epiglottis, and 0.1 ml of a solution of the molecules is deposited. The animals are stimulated 4 times, with 5 days between the last stimulation and exsanguination.

The test animals are Brown Norway rats (BN) in groups of Weight at time of start is more than 250 grams and at termination approximately 450 grams.

Intratracheal (IT) stimulation of quinea pigs For IT administration of molecules disposable syringes with a 2 long metal probe are used. This probe is instilled in the trachea of the guinea pigs approximately 1 cm below the epiglottis, and 0.1 ml of a solution of the molecules is deposited. The animals are stimulated once a week for consecutive weeks.

ELISA IqE test system (for Brown Norway rats) A three layer sandwich ELISA is used to determine relative concentrations of specific antibodies.

The immunizing molecule is used as coating antigen with gg per ml and 50 1l per well, in neutral phosphate buffer, incubated overnight at 4°C. All remaining binding spots on the WA 9f/3n682 P rI/DKoi/nnu t 36 IJLmZ.7OIUUJ well surface are blocked in 2 skim milk, 200 Il per well in phosphate buffer for at least 30 minutes at room temperature 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% incubated 2 hours on agitation platform at RT. The "tracer" molecule is biotinylated Mouse anti Rat IgE 50 jil 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.

.l per well streptavidin horse raddish peroxidase, diluted 2000 x was incubated 1 hour on an agitation platform. Colouring substrate at 50 pl1 per well is OPD (6 mg) and H 2 0 2 (4 pl of a solution) per 10 ml citrate buffer pH 5.2. The reaction is stopped using 100 p1l per well 2 N H 2

SO

4 All readings on SLT at 486 nm and 620 nm as reference. Data is calculated and presented in Lotus.

Determination of the molecule weight 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.

Protease activity Analysis with Suc-Ala-Ala-Pro-Phe-pNa: Proteases cleave the bond between the peptide and pnitroaniline 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 pl of this is diluted into 10 ml with Britton and Robinson buffer.

Analysis The substrate and protease solution is mixed and the absorbance is monitored at 405 nm as a function of time and Wn OS/30682 PCT/rKo/ 001 W O i lf lll- 37 J1/3 Af68 IP 7C UIIIJ.3&

ABS

4 0 5 n/min. The temperature should be controlled (20-50°C depending on protease). This is a measure of the protease activity in the sample.

EXAMPLES

Example 1 Activation of mPEG 15,000 with N-succinimidyl carbonate mPEG 15,000 was suspended in toluene (4 ml/g of mPEG) was distilled off at normal pressure to dry the reactants azeotropically. Dichloromethane (dry 1 ml/g mPEG) was added when the solution was cooled to 30 0 C and phosgene in toluene (1.93 M 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.

After evaporation dichloromethane and toluene dry 3 ml/g mPEG) was added to re-dissolve the white solid. N-Hydroxy succinimide (2 mole/mole mPEG.) was added as a solid and then triethylamine (1.1 mole/mole mPEG). The mixture was stirred for 3 hours. initially unclear, then clear and ending with a small precipitate. The mixture was evaporated to dryness and recrystallised from ethyl acetate (10 ml) with warm filtration to remove salts and insoluble traces. The blank liquid was left for slow cooling at ambient temperature for 16 hours and then in the refrigerator over night. The white precipitate was filtered and washed with a little cold ethyl acetate and dried to yield 98 NMR Indicating 80 90% activation and 5 o/oo (w/w) HNEt 3 Cl. 1 H-NMR for mPEG 15,000 (CDC13) 6 1.42 t 4.8 CH 3 i HNEt 3 Cl), 2.84 s 3.7 succinimide), 3.10 dq 3.4 CH 2 i HNEt 3 Cl), 3.38 s 2.7 CH 3 i OMe), 3.40* dd (I 4.5 o/oo, 13C satellite), 3.64 bs (I 1364 main peak), 3.89* dd (I 4.8 o/oo 13C satellite), 4.47 dd (I 1.8, CH 2 in PEG). No change was seen after storage in a desiccator at 22 0 C for 4 months.

Example 2 Activation of mPEG 5,000 with N-succinimidyl carbonate Activation of mPEG 5,000 with N-succinimidyl carbonate was WO 98/3n62 TVE1 1 no fD A jtljLI Ufl OQI.flOO1 &3 8 E. IJYOiuij performed as described in Example 1.

Example 3 Conjugation of PD498 protease with activated mPEG 5,000 200 mg of PD498 was incubated in 50 mM NaBorate, pH 10, with 1.8 g of activated mPEG 5,000 with N-succinimidyl carbonate (prepared according to Example in a final volume of 20 ml.

The reaction was carried out at ambient temperature using magnetic stirring. Reaction time was 1 hour. The reaction was stopped by adding DMG buffer to a final concentration of 5 mM dimethyl glutarate, 1 mM CaC1 2 and 50 mM borate, pH 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).

Example 4 Conjugation of Subtilisin DY protease with activated mPEG 5,000 Subtilisin DY was conjugated to mPEG 5,000 with Nsuccinimidyl carbonate using the same procedure as described in Example 3.

Example BALB/C mice subcutaneous (SC) trails BALB/C 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).

Four 2-weekly immunizations were sufficient to elicit an IgE response to PD498.

The 2-weekly immunization scheme was continued for 3 month.

At the end of the study, seven immunizations were performed. As shown in Figure 1, the anti-PD498 IgE levels in BALB/C mice with Win 98/306o2 PwTIDl/no0nni e Af, OQi lIuuQ 3 9Q O s I JQ t..739 UUU parent unmodified PD498 increased up to immunization and stayed then rather constant. In contrast thereto, no specific IgE response was detected in mice immunized with modified PD498-SPEG 5,000.

Example 6 Allergenicity IT-trails of PD498-SPEG 5,000 in guinea pigs Dunkin Hartley guinea pigs were stimulated with 1.0 pg purified PD498 and 1.0 u.g modified PD498-SPEG 5,000 by intratracheal installation.

Sera from immunized Dunkin Hartley guinea pigs were tested during the trail period in a specific IgG 1 ELISA (described above) to elucidate whether the molecules could activated the immune response system giving rise to a specific IgG 1 response indicating an allergic response (See figure The assay level was 1:50 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 IgG 1 of the modified PD498 is not detectable before tapping no.

#7 (Ta p-7) eqv. to 7 weeks. The IgG 1 level was not significantly increased upon successive stimulations with the modified PD498.

Example 7 Dose-response intratrachaeal trails (IT) in quinea pigs The potential allergic response of 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.

Before the first intratrachaeal stimulation a blood test was collected from each Dunkin Hartley guinea pig using the ELIAS for guinea pigs described above. This was done to make sure that there were no unspecific binding of sera in ELISA.

Groups of 10 guinea pigs were stimulated intratrachaeally (IT) with 0.3 micrograms, 3 micrograms, 30 micrograms, 300 micrograms of: parent PD498, and modified PD498-SPEG 5,000.

WA ORnnR2 De'l/'fOiAn 4..0 z hI L, YOIJU13 The following solutions were used for blind tests 0.9% NaCl (Blind test for the parent PD498), and 300 micrograms PEG 5,000 in 0.9% NaCl corresponding to the amount of PEG in PD498-SPEG 5,000 (blind test for the modified PD498-SPEG).

Sera from all tested guinea pigs were tested in the IgGi ELISA (described above). The result of the IT trails for the modified PD498-SPEG 5,000 are shown in Figures 3. The result of the trails for the unmodified parent PD498 is shown in Figure 4.

As can be seen by comparing Figures 3 and 4 the response of the guinea pigs stimulated intratracheally with the modified enzyme is reduced in comparison to guinea pigs having been exposed intratracheally with the parent enzyme.

As will be apparent to those skilled in the art, in the light of the foregoing disclosure, many alterations and modifications are possible in the practice of this invention without departing from the spirit or scope thereof. Accordingly, the scope of the invention is to be construed in accordance with the substance defined by the following claims.

WO 98/30682 PCT/DK98/00015 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Novo Nordisk A/S STREET: Novo Alle CITY: Bagsveard COUNTRY: Denmark POSTAL CODE (ZIP): DK-2880 TELEPHONE: +45 4444 8888 TELEFAX: +45 4449 3256 (ii) TITLE OF INVENTION: A modified enzyme for skin care (iii) NUMBER OF SEQUENCES: 4 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (EPO) INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 840 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: STRAIN: Bacillus sp. PD498, NCIMB No. 40484 (ix) FEATURE: NAME/KEY: CDS LOCATION:1..840 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

TGG

Trp 1 TCA CCG AAT Ser Pro Asn CCT TAC TAT TCT Pro Tyr Tyr Ser GCT TAC Ala Tyr 10 CAG TAT GGA CCA CAA Gln Tyr Gly Pro Gln AAC ACC TCA Asn Thr Ser CAA ACG GTG Gln Thr Val CCT GCT GCC TGG Pro Ala Ala Trp GTA ACC CGT Val Thr Arg GCG GTC CTT GAT Ala Val Leu Asp

TCC

Ser 40 GGA GTG GAT TAT Gly Val Asp Tyr GGA AGC AGC ACT Gly Ser Ser Thr AAC CAC CCT GAT Asn His Pro Asp GAC AGG GAC AAT Asp Arg Asp Asn CTT GCA Leu Ala AGA AAA GTA ATA Arg Lys Val Ile

AAA

Lys GGG TAC GAC TTT Gly Tyr Asp Phe

AAC

Asn CCA ATG GAT CTT Pro Met Asp Leu GGA CAT GGT Gly His Gly ACC CAT Thr His GTA GCC Val Ala GTT GCC GGT ACT Val Ala Gly Thr GCT GCT GAT ACG Ala Ala Asp Thr AAT GGA ATT GGC Asn Gly Ile Gly GGT ATG GCA Gly Met Ala CCA GAT Pro Asp ACG AAG ATC Thr Lys Ile GCC GTA CGG GTC Ala Val Arg Val GAT GCC AAT GGA Asp Ala Asn Gly AGT GGC TCA Ser Gly Ser 110 CAA GGG GCA Gln Gly Ala 288 336 384 432 CTT GAC AGC ATT GCC TCA GGT Leu Asp Ser Ile Ala Ser Gly

ATC

Ile 120 CGC TAT GCT GCT Arg Tyr Ala Ala AAG GTA Lys Val 130 CTC AAC CTC TCC Leu Asn Leu Ser

CTT

Leu 135 GGT TGC GAA TGC Gly Cys Glu Cys TCC ACA ACT CTT Ser Thr Thr Leu AAG AGT GCC GTC GAC TAT GCA TGG AAC AAA GGA GCT GTA GTC GTT GCT WO 98/36f82 PCT/DK98/0nnni 42 Lys Ser Ala Val Asp Tyr Ala Trp Asn Lys Gly Ala Val Val Val Ala 145 150 155 160 GCT GCA GGG AAT GAC AAT GTA TCC CGT ACA TTC CAA CCA GCT TCT TAC 528 Ala Ala Gly Asn Asp Asn Val Ser Arg Thr Phe Gln Pro Ala Ser Tyr 165 170 175 CCT AAT GCC ATT GCA GTA GGT GCC ATT GAC TCC AAT GAT CGA AAA GCA 576 Pro Asn Ala Ile Ala Val Gly Ala Ile Asp Ser Asn Asp Arg Lys Ala 180 185 190 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 AAC ATA GCA TCA ACC GTT CCG AAT AAT GGC TAC TCC TAC ATG TCT GGT 672 Asn Ile Ala Ser Thr Val Pro Asn Asn Gly Tyr Ser Tyr Met Ser Gly 210 215 220 ACG TCC ATG GCA TCC CCT CAC GTG GCC GGT TTG GCT GCT TTG TTG GCA 720 Thr Ser Met Ala Ser Pro His Val Ala Gly Leu Ala Ala Leu Leu Ala 225 230 235 240 AGT CAA GGT AAG AAT AAC GTA CAA ATC CGC CAG GCC ATT GAG CAA ACC 768 Ser Gln Gly Lys Asn Asn Val Gln Ile Arg Gln Ala Ile Glu Gln Thr 245 250 255 GCC GAT AAG ATC TCT GGC ACT GGA ACA AAC TTC AAG TAT GGT AAA ATC 816 Ala Asp Lys Ile Ser Gly Thr Gly Thr Asn Phe Lys Tyr Gly Lys Ile 260 265 270 AAC TCA AAC AAA GCT GTA AGA TAC 840 Asn Ser Asn Lys Ala Val Arg Tyr 275 280 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 280 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Trp Ser Pro Asn Asp Pro Tyr Tyr Ser Ala Tyr Gin Tyr Gly Pro Gin 1 5 10 Asn Thr Ser Thr Pro Ala Ala Trp Asp Val Thr Arg Gly Ser Ser Thr 25 Gin Thr Val Ala Val Leu Asp Ser Gly Val Asp Tyr Asn His Pro Asp 40 Leu Ala Arg Lys Val Ile Lys Gly Tyr Asp Phe lie Asp Arg Asp Asn 50 55 Asn Pro Met Asp Leu Asn Gly His Gly Thr His Val Ala Gly Thr Val 70 75 Ala Ala Asp Thr Asn Asn Gly Ile Gly Val Ala Gly Met Ala Pro Asp 90 Thr Lys Ile Leu Ala Val Arg Val Leu Asp Ala Asn Gly Ser Gly Ser 100 105 110 Leu Asp Ser'Ile Ala Ser Gly Ile Arg Tyr Ala Ala Asp Gin Gly Ala 115 120 125 Lys Val Leu Asn Leu Ser Leu Gly Cys Glu Cys Asn Ser Thr Thr Leu 130 135 140 A 2 WO 98/30682 PCT/DK98/00015 Lys 145 Ala Pro Ser Asn Thr 225 Ser Ala Asn Ser Ala Ala Gly Asn Ala Phe Ser 195 Ile Ala 210 Ser Met Gin Gly Asp Lys Ser Asn 275 Val Asn Ile 180 Asn Ser Ala Lys Ile 260 Lys Gly 155 Phe Ser Val Tyr Leu 235 Gin Phe

INFOR

(i) RMATION FOR SEQ ID NO: 3: (ii) (vi) (xi) Ala 1 Gln Thr Ser Thr 65 Leu Ser Ala Ser Gly 145 SEQUENCE CHARACTERISTICS: LENGTH: 274 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein ORIGINAL SOURCE: STRAIN: Bacillus sp. variant SEQUENCE DESCRIPTION: SEQ ID NO: 1: Gin Thr Val Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val 5 10 Ala Gln Gly Tyr Lys Gly Ala Asn Val Lys Val Gly Ile Ile Asp 25 Gly Ile Ala (Ala/Ser) Ser His Thr Asp Leu Lys Val Val Gly Gly Ala 40 Phe Val Ser Gly Glu Ser Tyr Asn Thr Asp Gly Asn Gly His Gly 55 His Val Ala Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val 70 75 Gly Val Ala Pro Asn Val Ser Leu Tyr Ala Ile Lys Val Leu Asn 90 Ser Gly Ser Gly Thr Tyr Ser Ala Ile Val Ser Gly Ile Glu Trp 100 105 110 Thr Gin Asn Gly Leu Asp Val Ile Asn Met Ser Leu Gly Gly Pro 115 120 125 Gly Ser Thr Ala Leu Lys Gln Ala Val Asp Lys Ala Tyr Ala Ser 130 135 140 Ile Val Val Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Ser 150 155 160 W 9ln 0 R P'T/DKi9/0in1n 44 Gin Asn Thr Ile Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val 165 170 175 Gly Ala Val Asp Ser Asn Lys Asn Arg Ala Ser Phe Ser Ser Val Gly 180 185 190 (Ala/Ser) Glu Leu Glu Val Met Ala Pro Gly Val Ser Val Tyr Ser Thr Tyr 195 200 205 ,7 Pro Ser Asn Thr Tyr Thr Ser Leu Asn Gly 210 215 His Val Ala Gly Ala Ala Ala Leu Ile Leu 225 230 Ser Ala Ser Gln Val Arg Asn Arg Leu Ser 245 250 Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu 260 265 Ala Gin INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 433 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: STRAIN: Bacillus sp. Y (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Thr Ser Met Ala Ser Pro 220 Ser Lys Tyr Pro Thr Leu 235 240 Ser Thr Ala Thr Asn Leu 255 Ile Asn Val Glu Ala Ala 270 Asn 1 Tyr Leu Lys Pro 65 Leu Met Leu Trp Asp 145 Asn Asn Asp Val Ala Arg Gly Ile Val Lys Ala Asp Val Ala Gln Asn Asn 5 10 Tyr. Gly Gly Arg Ala Leu His Gly Gly Met Ser Gly 100 Gin Ala Pro Val Val Arg Pro Asn 165 Thr Val Gin Asn Tyr Thr 70 Ala Gly Trp Asn Asn 150 Ser Gly Gly Asp Ala His Pro Leu Asn Gly 135 Asn Gly Ala Leu Val 25 Ser Met Gly Arg Ala Gly Ala Asn 90 Gly Leu 105 Gly Ala Tyr Thr Met Thr Ile Ser 170 Glu Asn Ala Val His Glu Thr Asn Ser Val 75 Leu Val Pro Ser Arg Ile Ala Asn 140 Val Leu 155 Ala Pro Tyr Arg Ala Ala Asn Leu Phe Asn His 125 Ser Phe Gly Pro Asp Phe Ala Gly Gin Leu 110 Thr Arg Ala Thr Ser Thr Arg Ser Asn Ser Asn Asn Gin Ala Ala 175 Phe Gly Gly Asp Ala Ile Thr Ser Val Gly 160 Lys Gly wO 9R/0AR. Dfvrebno f 180 185 190 Ser Ile Ala Asp Asn Pro Asn His Ile Ala Gln Phe Ser Ser Arg Gly 195 200 205 Ala Thr Arg Asp Gly Arg Ile Lys Pro Asp Val Thr Ala Pro Gly Thr 210 215 220 Phe Ile Leu Ser Ala Arg Ser Ser Leu Ala Pro Asp Ser Ser Phe Trp 225 230 235 240 Ala Asn Tyr Asn Ser Lys Tyr Ala Tyr Met Gly Gly Thr Ser Met Ala 245 250 255 Thr Pro Ile Val Ala Gly Asn Val Ala Gln Leu Arg Glu His Phe Ile 260 265 270 Lys Asn Arg Gly Ile Thr Pro Lys Pro Ser Leu Ile Lys Ala Ala Leu 275 280 285 Ile Ala Gly Ala Thr Asp Val Gly Leu Gly Tyr Pro Ser Gly Asp Gin 290 295 300 Gly Trp Gly Arg Val Thr Leu Asp Lys Ser Leu Asn Val Ala Tyr Val 305 310 315 320 Asn Glu Ala Thr Ala Leu Ala Thr Gly Gln Lys Ala Thr Tyr Ser Phe 325 330 335 Gln Ala Gln Ala Gly Lys Pro Leu Lys Ile Ser Leu Val Trp Thr Asp 340 345 350 Ala Pro Gly Ser Thr Thr Ala Ser Tyr Thr Leu Val Asn Asp Leu Asp 355 360 365 Leu Val Ile Thr Ala Pro Asn Gly Gln Lys Tyr Val Gly Asn Asp Phe 370 375 380 Ser Tyr Pro Tyr Asp Asn Asn Trp Asp Gly Arg Asn Asn Val Glu Asn 385 390 395 400 Val Phe Ile Asn Ala Pro Gln Ser Gly Thr Tyr Ile Ile Glu Val Gin 405 410 415 Ala Tyr Asn Val Pro Ser Gly Pro Gln Arg Phe Ser Leu Ala Ile Val 420 425 430 His INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 316 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: STRAIN: Bacillus Thermoproteolyticus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Ile Thr Gly Thr Ser Thr Val Gly Val Gly Arg Gly Val Leu Gly Asp 1 5 10 Gln Lys Asn Ile Asn Thr Thr Tyr Ser Thr Tyr Tyr Tyr Leu Gln Asp 25 Asn Thr Arg Gly Asp Gly Ile Phe Thr Tyr Asp Ala Lys Tyr Arg Thr 40 7O0IU1JL ufl ftli2Ael 46 w WO 46 Thr Leu Pro Gly Ser Leu Trp Ala Asp Ala Asp Asn Gin Phe Phe Ala 55 Ser Tyr Asp Ala Pro Ala Val Asp Ala His Tyr Tyr Ala Gly Val Thr 65 70 75 Tyr Asp Tyr Tyr Lys Asn Val His Asn Arg Leu Ser Tyr Asp Gly Asn 90 Asn Ala Ala Ile Arg Ser Ser Val His Tyr Ser Gin Gly Tyr Asn Asn 100 105 110 Ala Phe Trp Asn Gly Ser Giu Met Val Tyr Gly Asp Gly Asp Gly Gin 115 120 125 Thr Phe Ile Pro Leu Ser Gly Gly Ile Asp Vai Val Ala His Glu Leu 130 135 140 Thr His Ala Vai Thr Asp Tyr Thr Ala Gly Leu Ile Tyr Gln Asn Giu 145 150 155 160 Ser Giy Ala Ile Asn Giu Ala Ile Ser Asp Ile Phe Giy Thr Leu Val 165 170 175 Giu Phe Tyr Ala Asn Lys Asn Pro Asp Trp, Giu Ile Giy Glu Asp Vai 180 185 190 Tyr Thr Pro Gly Ile Ser Gly Asp Ser Leu Arg Ser Met Ser Asp Pro 195 200 205 Ala Lys Tyr Gly Asp Pro Asp His Tyr Ser Lys Arg Tyr Thr Gly Thr 210 215 220 Gin Asp Asn Gly Gly Val His Ile Asn Ser Gly Ile Ile Asn Lys Ala 225 230 235 240 Ala Tyr Leu Ile Ser Gin Gly Gly Thr His Tyr Gly Val Ser Vai Val 245 250 255 Gly Ile Gly Arg Asp Lys Leu Gly Lys Ilie Phe Tyr Arg Ala Leu Thr 260 265 270 Gin Tyr Leu Thr Pro Thr Ser Asn Phe Ser Gin Leu Arg Ala Ala Ala 275 280 285 Val Gin Ser Ala Thr Asp Leu Tyr Gly Ser Thr Ser Gin Giu Val Ala 290 295 300 ~8/00015 3053135 SerValLysGinAlaPhe Asp Ala Val Gly Val Lys

Claims (30)

1. A modified enzyme characterised by having from 4 to 70 polymeric molecules with a molecular weight from 1 to 35kDa covalently coupled to the surface of the protease, wherein the protease has an amino acid sequence of SEQ ID NO: 2.

2. The modified enzyme according to claim 1, characterised in that from 4 to 20 polymeric molecules are covalently coupled to the surface of said protease.

3. The modified enzyme according to claim 2, wherein from 4 to 20 polymeric molecules are covalently coupled to the surface of the 3-D structure of the protease.

4. The modified enzyme according to claim 3, wherein the number of polymeric molecules is 13 to 18. The modified enzyme according to any one of claims 1 to 4, wherein the polymeric molecules have a molecular weight between 4 and

6. The modified enzyme according to claim 5, wherein the polymeric molecules have a molecular weight between 6 and

7. The modified enzyme according to claim 5, wherein the polymeric molecules have a molecular weight between 8 and

8. The modified enzyme according to any one of claims 1 to 7, wherein the polymeric molecule is selected from the group comprising natural or synthetic homo- and heteropolymers.

9. The modified enzyme according to claim 8, wherein the polymeric molecule is selected 20 from the group comprising synthetic polymeric molecules including Branched PEGs, poly-vinyl alcohol (PVA), poly-carboxyl acids, poly-(vinylpyrolidone) and poly-D, L-amino acids.

10. The modified enzyme according to claim 8, wherein the polymeric molecule is selected from the group comprising natural occurring polymeric molecules including dextrans, including carboxymethyl-dextrans, and celluloses such as methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, and hydrolysates of chitosan, starches, such as hydroxyethyl-starches, hydroxypropyl-starches, glycogen, agarose, guar gum, inulin, pullulans, xanthan gums, carrageenan, pectin and alginic acid.

11. The modified enzyme according to any one of claims 1 to 10, wherein the enzyme is coupled to one or more of the following groups on the activated polymer: amino, hydroxyl, thiol, carboxyl, aldehyde or sulfhydryl.

12. The modified enzyme according to any one of claims 1 to 11, wherein the polymeric molecules are coupled to the enzyme via a linker.

13. The modified enzyme according to claim 12, wherein the linker is a triazine ring.

14. The modified enzyme according to any one of claims 1 to 13, wherein the enzyme is of 37 4 robial origin, filamentous fungus or yeast origin. S S 55 5 SS S 55 C06940 48 The modified enzyme according to claim 14, wherein the enzyme is of bacterial origin.

16. The modified enzyme according to any one of claims 1 to 15, wherein the polymeric molecules are coupled to the enzyme through an amino group (-NH 2 positioned on the surface of the enzyme. s 17. The modified enzyme according to claim 16, wherein the polymeric molecules are coupled to the enzyme at the N-terminal amino group or Lysine residues positioned on the surface of the enzyme.

18. The modified enzyme according to any one of claims 1 to 17, wherein the polymeric molecule(s) is(are) coupled to the enzyme more than 5A, from the active site of the enzyme. o 19. The modified enzyme according to claim 18, wherein the polymeric molecule(s) is(are) coupled to the enzyme more than 10 A, from the active site of the enzyme. A modified enzyme as defined in claim 1, substantially as hereinbefore described with reference to any one of the examples.

21. A process for preparing a modified enzyme characterised by having from 4 to polymeric molecules with a molecular weight from 1 to 35kDa covalently coupled to the surface of the protease, wherein the protease has an amino acid sequence of SEQ ID NO: 2, substantially as hereinbefore described with reference to any one of the examples.

22. A modified enzyme characterised by having from 4 to 70 polymeric molecules with a S molecular weight from 1 to 35kDa covalently coupled to the surface of the protease, wherein the 20 protease has an amino acid sequence of SEQ ID NO: 2, prepared by a process according to claim 21.

23. A synthetic detergent composition, comprising a modified enzyme according to any one S: of claims 1 to 20 or 21 and further ingredients known to be used in synthetic detergents.

24. A synthetic detergent composition comprising a modified enzyme as defined in claim 1, substantially as hereinbefore described with reference to any one of the examples.

25. A skin care composition comprising a modified enzyme according to any one of claims 1 to 20 or 21 and further ingredients known to be used in skin care products. o. 26. A skin care composition comprising a modified enzyme as defined in claim 1, substantially as hereinbefore described with reference to any one of the examples.

27. A skin care product comprising a skin care composition according to claim 25 or claim 26, wherein the product is selected from the group of soap, cosmetics, skin creams, skin milk, skin lotion, skin gel, cleansing cream, cleansing lotion, cleansing milk, cold cream, cream soap, make-up 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 iquid, hair cream, hair gel, hair treatment, hair setting preparations, hair dyes, hair colorants, scalp CO6940 49 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.

28. A skin care product comprising a skin care composition according to claim 25 or claim 26, wherein the product is selected from the group including shampoos, conditioners, lotions, creams, soap bars, toilet soaps, and liquid soaps.

29. A skin care product comprising a modified enzyme as defined in claim 1, substantially as hereinbefore described with reference to any one of the examples. The use of a modified enzyme according to any one of claims 1 to 20 or 21 for reducing the sensitisation potential of skin care products.

31. The use of a modified enzyme according to any one of claims 1 to 20 or 21 for the manufacture of a skin care product with reduced sensitisation potential.

32. A modified enzyme according to any one of claims 1 to 20 or 21, when used for preparing a skin care product with reduced sensitisation potential.

33. A method of preparing a skin care product including a protease and with reduced sensitisation potential, comprising combining a modified enzyme according to any one of claims 1 to or 21, as the protease, with further ingredients known to be used in said skin care product.

34. The use of a modified enzyme according to any one of claims 1 to 20 or 21 for reducing S: the sensitisation potential of a synthetic detergent.

35. The use of a modified enzyme according to any one of claims 1 to 20 or 21 for the 20 manufacture of a synthetic detergent with reduced sensitisation potential.

36. A modified enzyme according to any one of claims 1 to 20 or 21, when used for preparing a synthetic detergent with reduced sensitisation potential.

37. A method of preparing a synthetic detergent including a protease and with reduced sensitisation potential, comprising combining a modified enzyme according to any one of claims 1 to 25 20 or 21, as the protease, with further ingredients known to be used in said synthetic detergent. Dated 16 May, 2001 Novozymes ANS 9 •9 Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON C06940