JP2002526434A - Personal care compositions containing a subtilisin enzyme conjugated to a water-insoluble substrate - Google Patents

Abstract

(57) Abstract: The present invention includes a water-insoluble substrate, a plurality of protease G enzymes and a method of bonding each of the enzymes to the substrate in a permanent manner, wherein the enzyme is bonded to the substrate in an amount of about 0%. .01 g / cm ² ~ about 1000 g / cm ² personal care composition comprising about.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[Industrial applications]

The present invention relates to personal care compositions containing a specifically substituted subtilisin enzyme associated with a wipe substrate. Embodiments of the present personal care composition include:
Includes personal care wipes and personal care skin masks. The composition provides improved cleansing and skin conditioning by the activity of the active protein, while minimizing the risk of the user having an allergic reaction with the active protein.

[0002]

[Problems to be solved by the invention]

There is an increasing number of commercially available active protein-containing products. Most of such products utilize enzymes as active proteins. An enzyme is a protein that reacts with a compound or substrate to degrade the compound. Enzymes can be divided into numerous types based on the type of substrate with which the enzyme reacts. Each type of enzyme usually catalyzes the cleavage of a different chemical bond as a result of specific selection of activity. Enzymes of the lipase type are known, but not limited to, to hydrolyze ester bonds formed between hydrocarbon and polyalcohol backbone substrates. Examples of such substrates are mono-, di- and triglyceride polyglycerol esters. Enzymes of the protease type are known to be capable of hydrolyzing proteins. Naturally occurring and bioengineered protease enzymes are household cleaners for hydrolyzing protein stains and stains, personal care products for removing skin stains and grime,
It is mixed with mouthwash products that help remove plaque in the mouth and pharmaceuticals that act on unwanted proteins in the body.

[0003] Current commercial cleaning products are known to be more effective by mixing protease enzymes. U.S. Pat. No. 4,261,8 to Hora et al.
No. 68, Tai, U.S. Pat. No. 4,404,115, Letton
Et al., U.S. Pat. No. 4,318,818; European Patent Application 130,756 (1985).
Issued on January 9, 1998) and U.S. Pat. No. 5,030,378 to Venegas.
All references disclose the use of protease enzymes in cleansing or cleaning products.

However, it has also been found that many active proteins, including enzymes, can be antigens and can cause allergic reactions in humans under certain conditions. The human immune system can produce specific antibodies by exposure to active proteins. This process of producing specific antibodies is described as "immunization" when a clinically useful response is obtained. However, when the response induces hypersensitivity, it is described as "sensitization." Allergic sensitization to active proteins is seen in environments where humans are regularly exposed to proteins. Such environments include manufacturing facilities where employees can be exposed to dust or aerosols containing the active protein without control, or markets where consumers repeatedly use products containing the active protein that occasionally cause allergic reactions. No.

[0005] Currently, allergic reactions to active proteins can be minimized by limiting the choice of such proteins used in products to human sources. While this approach minimizes the problem of allergenicity, it is not a complete solution because it makes it impossible to find active proteins that also have the desired active properties. Another way to reduce allergic reactions is to reduce the size of the protein molecule (see Japanese Patent Publication No. 4,112,753). However, reducing the size can also cause an apparent decrease in enzyme activity. A third alternative to reducing allergenicity is by mapping epitopes to deliver proteins with reduced allergenicity, and by altering the protein amino acid sequence. This usually requires a lot of development time and money.

[0006] In the medical field, proposals have been made to reduce the immunogenicity of proteins by yet another method. This method involves the attachment of a non-reactive polymer to the protein. U.S. Pat. No. 4,179,337 to Davis et al. Discloses a substantially linear polyethylene glycol (PEG) or polypropylene glycol (PPG).
It describes the binding of the polymer moiety to the enzyme. Conjugation with PEG / PPG has been shown to reduce the allergenicity of the enzyme, retaining only 15% of the physiological activity. Olsen et al., PCT Patent Application WO 96/17929 (199)
Issued June 13, 6) describes modification by conjugation with a suitable polymer. Olsen's application describes a modified enzyme which shows that the allergenicity is reduced by about 25% to 66% compared to the parent enzyme, while the activity of the parent enzyme is maintained between 39% and 100%.

[0007] US Patent Application No. 08 / 903,298 discloses the use of enzymes that have been modified by adding a pair of polyethylene glycol polymer moieties to reduce allergenicity while exhibiting high enzyme activity. The modified enzyme is then used in a wipe application in combination with a fibrous substrate. The modified enzyme does not adhere to the substrate. Reduction of allergenicity is achieved through modification of the enzyme.

[0008] US patent application Ser. No. 09 / 088,912 describes polymerization chemical modification of a subtilisin enzyme at one or more of three specific epitope regions known to mask the immunogenic determinants of the enzyme. Disclosed.

[0009] Another attempt to reduce the allergenicity of the active protein has been to granulate, coat, or dissolve it to prevent the active protein from scattering into the air. U.S. Pat. No. 4,556,554 to Calvo discloses a cosmetic composition comprising an enzyme immobilized by attachment to polymer-based particles. The particles attached to the enzyme are dispersed in a cosmetic vehicle. When the vehicle is used on the skin, the enzyme is released from the substrate and thereby reactivated. While such methods address the exposure of consumers to proteins scattered in the air, such methods involve releasing enzymes deposited on the skin and prolonging contact with tissue. The associated substantial risks still remain.

[0010] Canadian Patent No. 1,229,808 (issued December 1, 1987) teaches that enzymes, specifically β-galactosidase and β-glucosidase, are immobilized on cellulosic substrates. At that time, the enzyme is immobilized by being adsorbed on an agarose gel coating the substrate. UK Patent Application GB 2,240,040, issued July 24, 1991, teaches the immobilization of enzymes on a substrate. The enzyme is covalently bonded to the substrate therein to provide a dressing. The activity of enzymes used in biological devices such as biosensors, bioseparators and bioreactors was enhanced by using site-specific attachment of the enzyme to the device surface. Huang et al., "Improving the Activity of Immobilized Subtilisi
n by Site-specific Attachment to Surface ")", Analytical Chemistry No. 6
See Vol. 9, No. 22, November 15, 1997. Juan, Serine 24
It teaches the mutation of 9 or serine 145 to cysteine and immobilization of the subtilisin enzyme via coupling to silica beads functionalized with amino groups. It would be highly desirable to develop compositions that provide improved levels of protein activity and maintenance of a hypoallergic response to exposure to the active protein. If this could be achieved, it would provide consumers with a safer way to take advantage of the benefits of protein engineering. It is an object of the present invention to provide a wiping composition that delivers this improved activity and maintains stimulation of the immune system and consequent reduction in activation.

[0011]

[Means for Solving the Problems]

The present invention is directed to a personal care composition comprising a plurality of specifically substituted subtilisin BPN 'enzymes, a mutant form of the enzyme, and a water-insoluble substrate wherein each enzyme is permanently attached to the substrate as a binding method. respect, the personal care composition, said enzyme comprises from about 0.01 g / cm ² ~ about 1000 g / cm ² to the substrate.

[0012]

DESCRIPTION OF THE INVENTION

The personal care compositions of the present invention include a subtilisin BPN 'enzyme and a derivative modified by replacing one cysteine amino acid group that is permanently attached to a water-insoluble substrate. The compositions provide a convenient way to take advantage of the specialized activity of the subtilisin BPN 'enzyme and its derivatives, wherein the enzyme is bound to a substrate,
Thereby minimizing the risk of any allergic reaction. Preferred embodiments of the present composition are very effective for cleaning sweat, sebum, dead skin cells, fats and oils from the skin, and moisturizing the skin.

[0013] Without being limited by theory, the permanent binding of the enzyme to the substrate may cause them to come into contact with the skin during use and act on its surface. Is possible. Thereafter, as the wipe or mask is removed from the skin, all enzymes are lifted off the skin surface and removed and treated with the wipe used, thereby aerosolizing and extending the risk of skin exposure. Decrease. The active protein reacts with the compound, which has specific reactivity during contact with the skin, leaving nothing on the skin that stimulates the immune system after use and then forms antibodies responsible for allergic reactions. Absent. As used herein, the term "amino acid sequence" refers to the specific configuration of the amino acids that make up a protein. The following is a list of abbreviations for the amino acids used herein:

[0014]

[Table 1]

As used herein, the term “mutation” refers to a genetic change in an organism,
The amino acid sequence of the enzyme produced by the organism changes as well. Mutations in organisms often alter the properties of the enzyme. As used herein, the term "wild-type" refers to an enzyme produced by a non-mutated host.

As used herein, the term “variant” refers to an enzyme that has a sequence that differs from the amino acid sequence of a wild-type enzyme due to a genetic mutation in the host producing the enzyme.

[0017] All percentages and ratios used herein are by weight unless otherwise indicated, and all measurements are made at 25 ° C unless otherwise specified. The present invention may include, consist of, or consist essentially of optional materials and components as well as the essential materials and components described herein. A list of the essential components of the personal care composition of the present invention, as well as suitable, but not exclusive, and optional components is described in detail below.

Water-insoluble substrate The product of the present invention comprises a water-insoluble substrate. The term "water-insoluble" means that the substrate does not dissolve in water or does not decompose when immersed in water. A water-insoluble substrate is a means or vehicle for delivering the active protein of the present invention to the skin for cleaning and moisturizing, and removing substantially all protein from the skin.

A wide variety of materials can be used as a substrate. The following features are desirable, but not limited to: (i) sufficient wet strength for use, (ii) sufficient attrition,
(Iii) sufficient bulk and porosity, (iv) sufficient thickness and (V) adequate size. Examples of suitable insoluble substrates that meet the above criteria include nonwoven substrates, woven substrates, hydraulically entangled substrates, air entangled substrates, natural sponges, synthetic sponges, and mesh meshes made of polymers. And the like, but are not limited thereto. A preferred embodiment uses a nonwoven substrate because it is economical and readily available in a variety of materials. By nonwoven is meant that the layer is not woven into the fabric, but rather is made up of fibers, such as those formed in sheets, mats and pad layers.
The fibers can be random (i.e., arranged randomly) or they can be combed (i.e., combed primarily in one direction). Further, the nonwoven substrate can be constructed by combining layers of randomly combed fibers.

The nonwoven substrate may be composed of various natural and synthetic materials. The term natural means that the material is derived from plants, animals, insects, or by-products of plants, animals, and insects. The term synthetic means that the material is obtained primarily from a variety of artificial materials or further modified natural materials. Conventional basic starting materials are usually
A fibrous fabric comprising any common synthetic or natural fabric length fibers or mixtures thereof.

Examples of natural materials useful in the present invention include, but are not limited to, silk fibers, keratin fibers, and cellulosic fibers. Examples of keratin fibers include, but are not limited to, those selected from the group consisting of wool fibers, camel hair fibers, and the like. Examples of cellulosic fibers include, but are not limited to, those selected from the group consisting of wood pulp fibers, cotton fibers, hemp fibers, jute fibers, flax fibers, and mixtures thereof.

Examples of synthetic materials useful in the present invention include acetate fibers, acrylic fibers, cellulose ester fibers, acrylic fibers, polyamide fibers, polyester fibers, polyolefin fibers, polyvinyl alcohol fibers, rayon fibers, polyurethane foams, and the like. But are not limited to those selected from the group consisting of: Some examples of such synthetic materials include fibers derived from Acrylan, Creslan and Acrylonitrile, acrylics such as Orlon; cellulose ester fibers such as cellulose acetate, Arnel and Asere; polyamides such as nylon (eg, nylon 6, nylon 66, nylon 610, etc.); fortrel, codel and polyethylene terephthalate fibers, polyesters such as Dacron; polyolefins such as polypropylene, polyethylene; polyvinyl acetate fibers; polyurethane foams and mixtures thereof. Such and other suitable fibers and nonwoven materials made therefrom are commonly described by Riedel, "Nonwoven Bonding Methods and Materials.
) Nonwoven World (1987), "Americana Encyclopedia (Th
e Encyclopedia Americana) "(Vol. 11, pp. 147-153 and 26, 566).
581 (1984); U.S. Patent No. 4,891,2 to Thaman et al.
No. 27, issued January 2, 1990, and U.S. Pat. No. 4,891,228, all of which are incorporated herein by reference.

Nonwoven substrates made from natural materials consist of fabrics or sheets most commonly formed on a fine wire screen made of a liquid suspension of fibers. C.
A. "The Encyclopedia of Chemi," by CA Hampel and others
stry) Third Edition, 1973, pp. 793-795 (1973), "The Encyclopedia Americana, Vol. 21, pp. 376-383 (1984).
Year), and G. A. GA Smook's Handbook of Pulp and Paper Technologies, Pulp and Paper Industry Association (Tec)
hnical Association for the Pulp and Paper Industry (1986), all of which are incorporated herein by reference.

Substrates made of natural materials useful in the present invention can be obtained from a wide variety of commercial products. Examples of suitable commercial paper layers useful herein include James River (
James River, an embossed, dry-formed cellulosic compound layer having a basis weight of about 71 gsy, commercially available from Green Bay, Wisconsin, Airtex ™, and Walkysoft US. A. (Walkis
oft USA) (Mount Holly, NC), including, but not limited to, embossed, dry-formed, cellulosic compound Walkisoft ™ having a basis weight of about 75 gsy. Not done.

[0025] Methods for making nonwoven substrates are well known in the art. Typically, such nonwoven substrates are air-twisted by first cutting the fibers or filaments to the desired length, passing them through a stream of water or air, and depositing them on a screen containing air or water containing fibers. It can be made by a process of water twisting, melt blow molding, co-forming, spunbonding, or carding. The resulting layer, regardless of production or composition, undergoes at least one treatment of several types of bonding to anchor the individual fibers together to form a supporting fabric by itself. . In the present invention, the various methods, for example, the nonwoven layer, can be made by hydro-winding, thermal bonding, and combinations thereof. Furthermore, the substrate of the present invention can be composed of a single layer or a plurality of layers. Further, multi-layer substrates can include films and other non-fibrous materials.

[0026] Nonwoven substrates made of synthetic materials useful in the present invention can also be obtained from a wide variety of commercial products. Examples of suitable nonwoven layer materials useful herein include HEF
40-047 (a perforated material containing about 50% rayon and 50% polyester, entrapped with water, having a basis weight of about 43 g per square yard (gsy), Veratech. Inc., Walpole, Mass.) (Walpole)), HEF140-102 (porous material containing about 50% rayon and 50% polyester, water entrapped, with a basis weight of about 56 gsy, sold by Bellatech, Inc. Novonet (trademark) 149-61
6 (It is a grid material that contains about 100% polypropylene and is bonded by heat.
gsy basis weight, sold by Bellatech, Inc.), Novonet (
149-801 (about 69% rayon, about 25% polypropylene, and about 6%).
% Cotton, thermally bonded latticed material, having a basis weight of about 75 gsy, sold by Bellatech, Inc., Novonet ™ 149-191 (about 69% rayon, about 25% A thermally bonded checkered material comprising polypropylene and about 6% cotton, having a basis weight of about 100 gsy and sold by Bellatech, Inc.), HEF Nubtex® 149-801. (About 10
It is a perforated material containing 0% polyester and smashed with water and has a basis weight of about 70 gsy, sold by Bellatech, Inc.), key back (Keybak
) ™ V 951V (about 75% rayon, about 25% acrylic fiber, a dry, perforated material having a basis weight of about 43 gsy, from Chicopee, New Brunswick, NJ) Sold), Keyback ™ 1368 (perforated material containing about 75% rayon, about 25% polyester, perforated and having a basis weight of about 39 gsy, sold from Chicopee above), Dura Duralace (TM) 1236 (porous material containing about 100% rayon, entrapped with water and having a basis weight of about 40 gsy to about 115 gsy and sold from Chicopee above), Duralace (Trademark) 5904 (
It contains about 100% polyester and is a material with holes entangled with water.
Sontaro 8868 (having a basis weight of about 115 gsy and sold by Chicopee above), containing about 50% cellulose and about 50% polyester, and being entrapped with water and having a basis weight of about 60 gsy. Dupont Chemical Company (D
commercially available from upon Chemical Corp.), and the like, but are not limited thereto.

[0027] Alternatively, the water-insoluble substrate is disclosed in European Patent No. E published March 27, 1996.
Polymer mesh sponges described in P702550A1 may also be used, all of which are incorporated herein by reference. Polymer sponges include multiple layers of extruded tubular mesh networks made from strong, flexible polymers, such as polymer addition of olefin monomers and polyamide addition of polycarboxylic acids.
Although such polymer sponges are designed to be used in combination with liquid detergents, these types of sponges can also be used as the water-insoluble substrates of the present invention.

The substrate can be in a wide variety of forms, including flat pads, thick pads, thin sheets, spheres, and irregular shapes, with surface areas ranging from a few square inches to hundreds of square inches. it can. The exact size will depend on the desired use and product characteristics. Particularly convenient are square, round, rectangular or oval pads,
The surface portion is about 1 square inch to about 144 square inches, preferably about 10 square inches to about 120 square inches, more preferably about 30 square inches to about 80 square inches and a thickness of about 1 mil to about 500 mil, preferably About 5mil to about 250mil,
More preferably from about 10 mil to about 100 mil. The water-insoluble substrate of the present invention may include two or more layers, each having a different tissue structure and abrasion. Different weaves can be made by different combinations of materials, or by different manufacturing processes or combinations thereof. Substrates having a dual texture can be made to provide a polishing surface for exfoliation and a soft, absorbent surface for gentle cleaning. Further, substrates with separate layers can be processed to different colors, which allows the user to better distinguish the surfaces.

Specific Substituted Subtilisin BPN 'Enzyme-"Protease G" An essential component of the present invention comprises a plurality of specifically substituted subtilisin BPN' enzymes (
Hereinafter referred to as protease G). Protease G, the surface to about 0.01 g / cm ² ~ about 1000 g / cm ² of water-insoluble substrate, preferably about 0.
05 g / cm ² ~ about 100 g / cm ² and most preferably from about 0.1 g / cm ² ~ about 1,
Present at levels in the range of 0 g / cm ² .

Usually, protease enzymes are available from the International Union of Biochemistry and Molecular Biology (International Un
Recommendation of ion of Biochemistry and Molecular Biology: IUBMB (1992)
Year) according to Enzyme Classification number. C. 3.4
(Carboxylate hydrolase). Related proteases are also described in PCT Publication: The Procter & Gamble Company.
Gamble Company) WO95 / 30010 (published November 9, 1995), Procter & Gamble Company WO95 / 30011 (published November 9, 1995), Procter & Gamble Company WO95 / 29979 (published November 9, 1995)
(Published November 9, 1995).

The subtilisin enzyme is derived from the microorganism Bacillus alcalophilus (Bacillus alcal
ophilus), Bacillus amyloliquefacien
s), Bacillus amylosaccharicus, Bacillus licheniformis, Bacillus lentus (Baci)
llus lentus) and a protease enzyme naturally produced from Bacillus subtilis. One known subtilisin enzyme is BPN '. Wild-type BPN 'from Bacillus amyloliquefaciens is characterized by the following amino acid sequence:

[0032]

[Table 2]

Some variants of BPN ′ are also known. Several related variants, hereinafter referred to all as "protease A", are disclosed in Venegas U.S. Pat. No. 5,030,378 (issued Jul. 9, 1991) The following a. ) Gly at position Gly166 is Asn, Ser, Lys, Arg, His
, Gln, Ala or Glu, Gly at position Gly169 is substituted with Ser, Met at position Met222 is Gln, Phe, Cys, His, Asn,
Substituted with Glu, Ala, or Thr; or b. )) Gly at position Gly166 is substituted with Lys, and Met at position Met222 is substituted with Cys; or c.) ) BPN 'amino acid sequence with mutations in which Gly at position Gly160 is replaced with Ala and Met at position Met222 is replaced with Ala.

An additional variant of BPN ′, previously designated “Protease B”, is the European Patent EP-B-251,446 of Genencor International, Inc., San Francisco, Calif. (1994 December
(Published on Jan. 7, 1988), the following amino acid Tyr2
1, Thr22, Ser24, Asp36, Ala45, Ala48, Ser4
9, Met50, His67, Ser87, Lys94, Val95, Gly9
7, Ser101, Gly102, Gly103, Ile107, Gly110
, Met124, Gly127, Gly128, Pro129, Leu135,
Lys170, Tyr171, Pro172, Asp197, Met199, S
one or more of er204, Lys213, Tyr214, Gly215, and Ser221, or an amino acid as described above and Asp32, Ser33, Tyr
104, Ala152, Asn155, Glu156, Gly166, Gly1
69, Phe189, Tyr217, Met222 are characterized by a mutated wild-type BPN 'amino acid in which two or more of the amino acids are mutated, wherein both mutations are Asp32, Ser33, Tyr104, Ala152, Asn
155, Glu156, Gly166, Gly169, Phe189, Tyr2
17, and cannot occur on the amino acids of Met222.

Another BPN ′ mutant protease, hereinafter referred to as “Protease D”, is Genencor International's WO 95/10615 (1995).
(Published April 20, 2008), and a mutation at the position of Asn76 of the wild-type BPN 'amino acid and Asp99, Ser101, Gln103, Tyr104,
Ser105, Ile107, Asn109, Asn123, Leu126, G
ly127, Gly128, Leu135, Glu156, Gly166, Gl
u195, Asp197, Ser204, Gln206, Pro210, Ala
216, Tyr217, Asn218, Met222, Ser260, Lys2
65 and / or Ala274 in combination with mutations at one or more other amino acid positions selected from the group consisting of:

Another BPN ′ mutant protease, hereinafter referred to as “Protease F”,
No. 4,760,025 (issued Jul. 26, 1988) to Estel et al .; Asp32, Ser33, His64, Tyr.
104, Asn155, Glu156, Gly166, Gly169, Phe1
89, Tyr217, and Met222 characterized as a wild-type BPN ′ amino acid mutated at one or more amino acid positions selected from the group consisting of:

The enzyme protease G used in the personal care composition of the present invention is BPN ′
Includes both enzymes and variants of the BPN 'enzyme which have been specifically substituted or have cysteine amino acids inserted in the amino acid sequence. Cysteine is the most preferred replacement amino acid because it does not occur in the wild-type subtilisin BPN 'or derivative thereof for substitutions at the desired epitope region. Substituted or inserted amino acids provide a suitable site for attachment to a substrate of the invention at a specific site within the enzyme.

[0038] Preferably, the substitution or insertion should be made at a location in the epitope region that is at a point on the protein remote from the active site of the protein. Subtilisin BP
In N 'and its derivatives, the active site is Asn32, His64, and S64.
It is spatially specified by a triple of er221. In the protease G enzyme, cysteine is replaced at a point away from the above triad. Preferred epitope regions to be replaced are Asp140-Val150 and Ala230-Leu2.
50 areas. Examples of cysteine substitution include Ser145, Asn24
It can be, but is not limited to, either 0 or Ser249. Cysteine is most preferred as a substituted amino acid since it does not occur with wild-type subtilisin BPN 'or its derivatives for substitutions at the desired epitope region.

Binding Method The active protein is bound to the water-insoluble substrate by any suitable binding method. Binding methods include any physical or chemical method that permanently binds the active protein to the substrate. Many such methods are known in the art.

Physical Capture One method of binding the active proteins of the present invention to a substrate is to physically capture the protein within the body of the substrate. A preferred method of capture is to coat the substrate surface and seal the protein. Any adhesive or polymerizing agent known in the art may be used to seal the protein to the substrate. A preferred coating is poly-2-hydroxyethyl acrylate formed by separately applying the initiator of 2-hydroxyethyl acrylate monomer and iron (II) sulfate heptahydrate. A solution of either a) active protein and adhesive or b) active protein and monomer / initiator combination is sprayed uniformly onto the substrate surface. A second adhesive or monomer / initiator or separate initiator may be needed to achieve sufficient bonding. The substrate is then dried such that a polymer is formed. The substrate is then rinsed sufficiently to remove any free protein.

Proteins Bound to the Polymerizable Gel Coating on the Substrate Proteins may be bound to the substrate by a chemical bond that binds to the polymerizable coating on the substrate. The protein associates with a polymerizable bond that is covalently associated with the polymer coating. One embodiment of the polymerizable linkage is the Shearwater Polymer Company (
Polyethylene glycol (P) sold by Shearwater Polymers, Inc.
EG) -maleimide. PEG-maleimide must be used with cysteine amino acids, so the active protein may be used when protease G. PEG-maleimide may also be used for conjugation with a poly-2-hydroxyethyl acrylate coating. Preferred acrylate-PEG-
The general structure of the maleimide linkage is represented by the following formula:

[0042]

Embedded image

Yet another embodiment of a cysteine-containing protein that binds to a polymerizable coating on a wipe substrate comprises PEG-maleimide covalently linked to a polyethyleneimine coating by N-hydroxysuccinimide and has the following formula: expressed:

[0044]

Embedded image

The binding method involving the binding protein is such that the protein to be bound is more mobile,
Physical capture is preferred because there is less coverage with a polymer coating, both of which provide additional protein activity

Proteins that Covalently Bind to Activated Device Surfaces Another method of coupling proteins of the invention is to covalently bind to activation sites on the surface. For this bonding method, the substrate is preferably a cellulosic material. The chemical bond can be with any bifunctional compound that reacts with the substrate and the protein. A preferred chemical bond is ethylenediamine / N-γ-maleimidobutyryloxysuccinimide ester (GMBS). The general structure of a preferred ethylenediamine / GMBS bond is represented by the following formula:

[0047]

Embedded image

Proteins Covalently Bonded to Device Surfaces via Polymerizable Bonds Yet another method of binding active proteins to the substrate of the personal care wipes of the present invention is to covalently bind to activated sites on the substrate surface. It is a polymerizable bond.
A preferred linkage is ethylene diamine / polyethylene glycol-maleimide. The general structure to which the ethylenediamine / PEG-maleimide linkage is directly attached is represented by the following formula:

[0049]

Embedded image

Optional Ingredients The wipe composition of the present invention can include a wide range of optional ingredients. CTFA
International Cosmetic Ingredient Dictionary (CTFA)
The sixth edition (1995) is hereby incorporated by reference in its entirety, which describes a wide variety of cosmetic and medicinal ingredients commonly used in the skin care industry and is used in the compositions of the present invention. However, the present invention is not limited to this. Examples of functional classification of components are described on page 537 of this reference, but are not limited thereto. Examples of such functional classifications include: abrasives, anti-acne agents,
Anti-coating agents, antimicrobial agents, antioxidants, binders, biological additives, extenders, chelating agents, chemical additives, coloring agents, cosmetic astringents, cosmetic biocides, denaturants , Chemical astringents, emulsifiers, topical analgesics, film forming agents, fragrance components, humectants, softening agents (cationic and nonionic polymers, co-surfactants, lipid humectants, hydrocarbon oils, silicone oils, waxes ), Opacifiers, plasticizers, preservatives, propellants, reducing agents, skin bleaching agents, skin conditioning agents (emollients, moisturizers, other and occlusion),
Skin protective agent, solvent, foam enhancer, hydrotrope, solubilizer, stabilizer, suspending agent, sunscreen, surfactant (anionic, cationic, amphoteric, bipolar), ultraviolet light absorber And thickeners (aqueous and non-aqueous). Examples of other functional classes of materials useful in the present invention that are known to those skilled in the art include solubilizers, sequestrants, and keratolytics.

Method of Use The personal care composition of the present invention can be used for personal cleansing, cosmetic skin treatment and / or
Or it is useful for skin conditioning. The present invention may take the form of a personal care wipe or personal care skin mask. Typically, wipes are used to expose the area to be cleaned to the active enzyme for a relatively short period of time. In use, the wipe is brought into contact with or wipes the skin in need of treatment and then removed. Typical amounts of the present wipes useful for cleaning are about 1 to about 4 wipes per use, preferably about 1 to about 2 wipes. Skin masks are used to expose the area to be treated for a relatively long time. Typical amounts of the present skin masks useful for cleaning are from about 1 to about 2 masks per use, preferably one mask per use.

[0052]

Examples and manufacturing method

The following examples further describe and illustrate embodiments within the scope of the present invention. In the following examples, all ingredients are listed at the activity level. These examples are provided for illustrative purposes only, and many modifications of the present invention are possible without departing from the spirit and scope of the present invention and should be construed as limiting the invention. is not. Components are identified by chemical name, ie, CTFA name. Examples of wipes associated with the active proteins of the present invention are set forth below, but are not limited thereto.

Example 1 Protease G Captured in Rayon / PET-Based Acrylate Gel Protease G was treated with 10 mM monopotassium phosphate (KH ₂ PO ₄ ) buffer (pH 5.
Purify and concentrate to a concentration of 3 mg / ml in 5). Add 2-hydroxyethyl acrylate (HEA) to a final molarity of 1.3M. 50 mM hydrogen peroxide (H ₂ O ₂ ) is added to a final molar concentration of 2.5 mM H ₂ O ₂ . Spray the protease F / HEA / H ₂ O ₂ solution evenly onto the rayon / PET sheet until the sheet is saturated. Uniformly sprayed with iron (II) sulfate heptahydrate (FeSO ₄ · 7H ₂ O) solution onto the sheet. Let the sheet sit for 5 minutes. Continuous 0.01M
Rinse completely in a KH ₂ PO ₄ solution bath until all free protein is removed.
Dry the sheet.

Example 2 Polymerizable Coating Bound Protease G Approximately 2.5 mg of Protease G in 10 mM KH ₂ PO ₄ buffer (pH 5.5)
/ Ml and concentrate. Acrylate-PEG ₃₄₀₀ -maleimide (Shearwater Polymer) is added in a molar ratio of 15: 1 or more. The pH of the solution is raised to 7 with dilute sodium hydroxide (NaOH). Protease G and acrylate-PEG-maleimide are reacted at room temperature for 1-2 hours. Lowering the pH of the solution to 5.5 with dilute phosphoric acid (H ₃ PO _4). 2-hydroxyethyl acrylate (HEA) was added to the protease G-PEG-maleimide solution at a final molarity of 1.
Add to 3M. A 50 mM H ₂ O ₂ solution was added to protease G-PEG / HE.
Solution A is added so that a final molarity of 2.5 mM H ₂ O ₂ is present. The solution
Spray onto rayon / PET sheet until sheet is saturated. Separately, 50
mM iron (II) sulfate heptahydrate and (FeSO ₄ · 7H ₂ O) solution entire surface sprayed to the sheet until uniformly sprayed. Let the sheet sit for 5 minutes. The sheet is then thoroughly rinsed through a continuous bath of 0.01 M KH ₂ PO ₄ until all free Protease G has been removed. Dry the sheet.

[0055]Example 3 Protease G directly covalently binds to activated device surface Soak the rayon / PET sheet in a 10% (w / v) hydrated NaOH solution
1cm^TwoShake with an automatic shaker for 10 to 15 minutes using 1 ml of the solution per time. Shi
3 times with deionized water under suction, 1cm sheet at a time^TwoWash with about 1ml per
U. The sheet is washed 5 times with acetone under suction. Aceto sheet between washes.
Soak for 1 minute. 10% (w / v) p-toluenesulfonyl chloride / acetate
Reaction with shaking for 25-30 minutes with a ton solution (~ 1 ml / cm^Two). Extra
To remove the p-toluenesulfonyl chloride, the sheet is treated with acetone under suction.
Rinse three times. The sheet was placed in a 2.2 M ethylenediamine / acetone solution (pH 13 to 1).
React while shaking for 2 hours in 4). Suction to remove excess EDA
Rinse the sheet three times with acetone underneath. Dry the sheet under suction, N, N-dimethyl
Rinse with formamide (DMF) 5 times. Sheets are placed in DMF between washes
Soak for 1 minute. The sheet was treated with 10% (w / v) N-γ-maleimidobutyryloxys
Reaction with shaking imide (GMBS) overnight (~ 1 ml / cm ^Two ). The sheet is rinsed three times with dry DMF under suction. 0.0
1M KH_TwoPO_FourRinse 5 times with buffer (pH 7). Buffer between rinses
Soak the sheet for 1 minute. The sheet was treated with 3 mg / ml protease G / 10 mM KH._TwoP
O_FourBuffer solution (pH 7) (~ 1 ml / cm^Two) For about 1-2 hours. Sheet
0.01M KH_TwoPO_FourRinse three times with buffer (pH 5.5). Dry the sheet
You.

[0056]Example 4 Protease G covalently bonded to activated device surface via polymer bond Soak the rayon / PET sheet in a 10% (w / v) hydrated NaOH solution
1cm^TwoShake with an automatic shaker for 10 to 15 minutes using 1 ml of the solution per time. Shi
Wash the plate 3 times with deionized water under suction (~ 1 ml / cm per wash)^Two). Sucking
The sheet is washed five times with acetone underneath. Put the sheet in acetone between washings.
Soak for a minute. The sheet was dissolved in 10% (w / v) p-toluenesulfonyl chloride / acetone.
Reaction while shaking with liquid for 25-30 minutes (~ 1 ml / cm^Two). Extra p-salt
Three times with acetone under suction to remove toluenesulfonyl bromide
soon. The sheet is prepared as a 2.2 M ethylenediamine / acetone solution (pH 13-14)
With shaking for 2 hours. Under vacuum to remove excess EDA
And rinse the sheet three times with acetone. 0.2M sodium borate under suction
Rinse 5 times with buffer buffer (pH 8.5). Buffer the sheet between rinses
Soak for 1 minute. The sheet is treated with 3-5% (w / v) maleimide-PEG₃₄₀₀-NHS (
Shearwater Polymer) /0.2 M sodium borate buffer (pH 8.5)
) For 1-2 hours while shaking (~ 1 ml / cm)^Two). Extra P sheet
10 mM KH under suction to remove EG_TwoPO_Four5 times with buffer solution (pH 7)
Rinse The sheet was treated with 3 mg / ml protease G / 10 mM KH._TwoPO_FourBuffer solution (pH
7) (~ 1ml / cm^Two) For about 1-2 hours. Seat 0.01M KH _Two PO_FourRinse three times with buffer (pH 5.5). Dry the sheet.

[0057]Example 5 Protea covalently bonded to the coated device surface via polymer bonds Gose G Shake the rayon / PET sheet in a polyethylene imine 500ppm solution tank
1 hour at room temperature (sheet 1cm^TwoPer ~ 1 to 2 ml). Rayon / PET
The sheet is placed in two successive 0.2 M sodium borate buffer (pH 12) solution baths.
Rinse (~ 5-10ml / cm^Two). Sheet into two consecutive 0.2M sodium borate
Rinse with a solution buffer (pH 8.5) solution tank. The sheet was treated with 5 mg / ml NHS-PEG- ₃₄₀₀ Shake with maleimide / 0.2 M sodium borate buffer (pH 8.5).
Then, react at room temperature. Rinse the sheet with four deionized water baths (~ 5-10
ml / cm^Two). Seat two 0.01M KH_TwoPO_FourBuffer solution (pH 7-7.5)
Rinse with solution bath. Separately, protease G was added to 0.01 M KH_TwoPO_FourBuffer solution (pH
Purify and concentrate to 1-2 mg / ml in 7-7.5). "PEGylated
While shaking the sheet with Protease G solution (pH 7-7.5) for 1 hour at room temperature.
And react. To remove unreacted, unbound protease G,
Five consecutive 0.01M KH_TwoPO_Four(PH 5.5) Completely soak in solution bath
Go. Dry the sheet.

Examples 6-9 Personal care wipes composition products are prepared as follows.

[0059]

[Table 3]

The water-insoluble substrate had a basis weight of about 60 gsy and contained 50% rayon and 50% polyester, was approximately 6 inches by 7.6 inches, had a thickness of about 20 mil, and was demonstrated by Examples 1-7. A nonwoven substrate with pores through which water has a binding active protein. In a suitable vessel, mix the ingredients of Phase A at room temperature to form a dispersion and heat to 65 ° C. with stirring. Mix the ingredients of Phase B in another suitable container and heat to 65 ° C. As soon as the temperature is the same, the ingredients of phase B are mixed in a container containing the ingredients of phase A and then cooled to 45 ° C. The ingredients of Phase C are mixed in a separate container at room temperature. Next, the mixture of phase C is added to the vessel containing the mixture of phases A and B at room temperature. 1.5 g of the resulting solution is sprayed on each substrate. Alternatively, the substrate can be lightly immersed in the resulting solution. The treated substrate is then dried in an oven to a constant weight. In another method, the treated substrate is brought to a certain weight
Dry at 45 ° C. in a convection oven. In other embodiments, other substrates such as woven substrates, wet interlaced substrates, natural sponges, synthetic sponges, or plastic meshes. Another embodiment may be in the form of a personal care skin mask.

Examples 10 to 13 Personal care wipes are manufactured as follows.

[0062]

[Table 4]

The water-insoluble substrate had a basis weight of about 60 gsy and contained 50% rayon and 50% polyester, was approximately 6 inches by 7.6 inches, had a thickness of about 20 mils, and was demonstrated by Examples 1-7. A nonwoven substrate with pores through which water has a binding active protein. In a suitable vessel, mix the ingredients of Phase A at room temperature to form a dispersion and heat to 65 ° C. with stirring. Mix the ingredients of Phase B in another suitable container and heat to 65 ° C. As soon as the temperature is the same, the ingredients of phase B are mixed in a container containing the ingredients of phase A and then cooled to 45 ° C. Next, the mixture of phase C is added to the vessel containing the mixture of phases A and B at room temperature. Spray 1.5 g of the resulting solution onto each substrate. In another method, the substrate is lightly immersed in the resulting solution. The treated substrate is then dried in an oven to a constant weight. In another method, the treated substrate is dried in a convection oven at 45 ° C. to constant weight. In other embodiments, other substrates such as woven substrates, wet interlaced substrates, natural sponges, synthetic sponges, or plastic meshes. Another embodiment may be in the form of a personal care skin mask. Examples 14 to 17

[0064]

[Table 5]

The water-insoluble substrate has a basis weight of about 60 gsy, contains 50% rayon and 50% polyester, is approximately 6 inches by 7.6 inches, has a thickness of about 20 mils, and has the binding activity shown by Examples 1-7. A nonwoven substrate with pores through which water passes with proteins. In a suitable vessel, mix the ingredients of Phase A at room temperature to form a dispersion and heat to 65 ° C. with stirring. Mix the ingredients of Phase B in another suitable container and heat to 65 ° C. As soon as the temperature is the same, the ingredients of phase B are mixed in a container containing the ingredients of phase A and then cooled to 45 ° C. The ingredients of Phase C are mixed in a separate container at room temperature. Next, the mixture of phase C is added to the vessel containing the mixture of phases A and B at room temperature. Spray 1.5 g of the resulting solution onto each substrate. In another method, the substrate is lightly immersed in the resulting solution. The treated substrate is then dried in an oven to a constant weight. In another method, the treated substrate is dried in a convection oven at 45 ° C. to constant weight. In other embodiments, other substrates such as woven substrates, wet interlaced substrates, natural sponges, synthetic sponges, or plastic meshes. Another embodiment may be in the form of a personal care skin mask. Examples 18 to 21

[0066]

[Table 6]

A water-insoluble substrate having a basis weight of about 60 gsy, containing 50% rayon and 50% polyester, was approximately 6 inches by 7.6 inches, had a thickness of about 20 mil, and was demonstrated by Examples 1-7. A nonwoven substrate with pores through which water has a binding active protein. In a suitable vessel, mix the ingredients of Phase A at room temperature to form a dispersion and heat to 65 ° C. with stirring. Mix the ingredients of Phase B in another suitable container and heat to 65 ° C. As soon as the temperature is the same, the ingredients of phase B are mixed in a container containing the ingredients of phase A and then cooled to 45 ° C. Next, the mixture of phase C is added to the vessel containing the mixture of phases A and B at room temperature. Spray 1.5 g of the resulting solution onto each substrate. In another method, the substrate is lightly immersed in the resulting solution. The treated substrate is then dried in an oven to a constant weight. In another method, the treated substrate is dried in a convection oven at 45 ° C. to constant weight. In other embodiments, other substrates such as woven substrates, wet interlaced substrates, natural sponges, synthetic sponges, or plastic meshes. Another embodiment may be in the form of a personal care skin mask.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // C08L 101: 00 C08L 101: 00 (71) Applicant ONE PROCTER & GANBLE PLAZA, CINCINNATI, OHIO, UNITED STATES OF AMERICA (72) Inventor Allcock, Andrew Campbell Cincinnati, Ohio, USA Cincinnati, Drake, Avenue 3837 F-term (Reference) 4C083 AC012 AC122 AC342 AC532 AC642 AC662 AC682 AC692 AC782 AC852 AD021 AD042 AD071 AD072 AD091 AD092 AD111 AD111 AD441 AD471 AD472 CC02 DD12 EE09 4F006 AA02 AA12 AA19 AA37 AA38 AB01 AB24 AB35 4L031 AA02 AA12 AA18 AB34 BA33 BA34 BA39 DA12 DA13 4L033 AA02 AA07 AB07 AC10 AC15 CA18 CA23

Claims (16)

[Claims] 1. A method comprising: (a) a water-insoluble substrate; (b) a plurality of protease G enzymes; and (c) a bonding method in which each enzyme and the substrate are permanently attached. about 0.01 g / cm ² ~ about 1000 g / cm ² personal care wipes composition comprising (wipe composition). 2. A method comprising: (a) a water-insoluble substrate; (b) a plurality of protease G enzymes; and (c) a bonding method in which each enzyme and the substrate are permanently attached. about 0.01 g / cm ² ~ about 1000 g / cm ² personal Care skin mask composition comprising. 3. The method according to claim 2, wherein the water-insoluble substrate is silk, keratin, cellulose, acetate, acrylic, cellulose ester, acrylic fiber, polyamide, polyester, polyolefin, polyvinyl alcohol, wood pulp, cotton, hemp, jute, flax,
The personal care wipe according to claim 1 or 2, comprising one or more materials selected from the group consisting of acrylics, nylon, polyester, polypropylene, polyethylene, polyvinyl acetate, polyurethanes, rayon and mixtures thereof. Composition. 4. The personal care wipe according to claim 1, wherein said water-insoluble substrate comprises a nonwoven sheet selected from the group consisting of rayon fibers, cellulose fibers, polyester fibers and mixtures thereof. Composition. 5. The method according to claim 1, wherein the water-insoluble substrate has two or more different tissue structures.
5. A personal care wiping composition according to any of the preceding claims, comprising sheets each having a respective texture). 6. The method according to claim 1, wherein the bonding method is a method of physically capturing, linking with a polymerizable gel.
6.) A personal care wipe according to claim 1, wherein the wipe is selected from the group consisting of: a covalent bond to the activated surface, a covalently bonded tether to the activated surface, and a tether covalently bonded to the coating surface. Composition. 7. The personal care wiping composition according to claim 1, wherein the bonding method is physical entrapment in a polymerizable coating. 8. The personal care wiping composition according to claim 1, wherein the polymerizable coating is poly-2-hydroxyethyl acrylate. 9. The personal care wiping composition according to claim 1, wherein the bonding method is a tether to a polymerizable coating. 10. The personal care wiping composition according to claim 1, wherein the tether is polyethylene glycol-maleimide, and the polymerizable coating comprises poly-2-hydroxyethyl acrylate. 11. The bond is polyethylene glycol-maleimide,
A personal care wiping composition according to any preceding claim, wherein the polymerizable coating comprises polyethyleneimine. 12. The personal care wiping composition according to claim 1, wherein the bonding method is a covalent bond with an activation site on a substrate. 13. The personal care wiping composition according to claim 1, wherein the covalent bond is N-γ-maleimidobutyryloxysuccinimide ester. 14. The personal care wiping composition according to claim 1, wherein the bonding method is a polymerizable tether covalently bonded to an activation site on a substrate surface. 15. The personal care wiping composition according to claim 1, wherein the polymerizable tether comprises polyethylene glycol-maleimide. 16. A method for moisturizing the skin comprising contacting the personal care wiping composition of claim 1 with the skin in need of treatment.