WO2002061066A1 - Procede de production d'une enzyme a partir d'une source nouvelle - Google Patents

Procede de production d'une enzyme a partir d'une source nouvelle Download PDF

Info

Publication number
WO2002061066A1
WO2002061066A1 PCT/FI2002/000081 FI0200081W WO02061066A1 WO 2002061066 A1 WO2002061066 A1 WO 2002061066A1 FI 0200081 W FI0200081 W FI 0200081W WO 02061066 A1 WO02061066 A1 WO 02061066A1
Authority
WO
WIPO (PCT)
Prior art keywords
milk
enzyme preparation
composition
human
enzyme
Prior art date
Application number
PCT/FI2002/000081
Other languages
English (en)
Other versions
WO2002061066A8 (fr
Inventor
Pepe Karhumaa
Kari Kaunisto
Jukka Leinonen
Seppo Parkkila
Hannu Rajaniemi
Original Assignee
Oulun Yliopisto
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from FI20010193A external-priority patent/FI20010193A0/fi
Application filed by Oulun Yliopisto filed Critical Oulun Yliopisto
Priority to AU2002231817A priority Critical patent/AU2002231817A1/en
Publication of WO2002061066A1 publication Critical patent/WO2002061066A1/fr
Publication of WO2002061066A8 publication Critical patent/WO2002061066A8/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to a method for preparing an enzyme preparation comprising carbonic anhydrase NI (CA NI) and to the enzyme preparation comprising CA NI. Furthermore, this invention relates to pharmaceutical compositions and food compositions comprising the enzyme preparation. Still further the present invention relates to the use of the enzyme preparation of this invention as medicine and to the preparation of medicaments for the treatment of gastrointestinal disorders and compositions for prevention of caries. This invention concerns also microbial hosts and transgenic animals transferred to produce the enzyme preparation of this invention.
  • CA NI carbonic anhydrase NI
  • CAs carbonic anhydrases
  • Carbonic anhydrase VI (CA VI) is the only known secreted isoenzyme of this family (Fernley et al., 1979, Feldstein and Silverman, 1984 and Murakami and Sly 1987), which has been detected to date only in the saliva secreted by the serous acinar cells of mammalian parotid and submandibular glands (Parkkila et al., 1990).
  • CA VI salivary CA VI
  • the secretion of salivary CA VI is characterized by a circadian periodicity, the con- centration in saliva being very low during sleep and rapidly rising to the daytime levels after awakening (Parkkila et al., 1995; Kivela et al., 1997b).
  • CA VI is suggested to participate in protecting the teeth from caries (Kivela et al. 1999) and in neutralizing of excess acid in the mucous layer covering the esophageal and gastric epithelium (Parkkila et al., 1997). Thatcher et al (1998) identified gustin, a salivary factor implicated in taste bud growth as a developmental factor, as CA VI.
  • CA VI carbonic anhydrase
  • cDNA maybe isolated from a human salivary gland cDNA, mammary gland cDNA or genomic DNA libraries and expressed in a suitable expression host system.
  • the prior art does not describe any natural source, which would contain high amount of CA VI and from which CA VI could be isolated by an economical procedure in industrial scale.
  • CA VI is secreted to milk and that its concentration is approximately eight times higher in the colostrum than in the mature milk, the latter levels corresponding to those reported earlier for adult human saliva (Kivela et al. 1997).
  • CA VI is transferred to the infantile alimentary tract in the milk in concentrations which exceed several fold the levels found in adult human saliva.
  • Finnish Patent No. 103089 suggests the production of a nutritional product based on colostrum, from which fats and casein has been removed and creatin and L-carnitin added.
  • the patent publication does not mention CA VI.
  • CA VI can be isolated and purified from the milk of an efficiently milk-producing (non-human) animal, such as cow, goat or sheep or from human milk.
  • non-human animal such as cow, goat or sheep or from human milk.
  • the enzyme may be purified until homogeneity or until a chosen purity level is achieved depending on the use of the enzyme.
  • the method is mainly characterized by what is stated in the characterizing part of claim 1.
  • CA VI may be purified from mature milk, but more preferably it is purified from colostrum.
  • the mature milk or colostrum from which the enzyme is purified should not be treated with methods, for example standard sterilization methods, such as heating or pasteurization, which potentially inactivate the enzyme.
  • Another object of this invention is an enzyme preparation, which comprises CA NI isolated from milk. More specifically, the enzyme preparation is mainly characterized by what is stated in the characterizing part of claim 6.
  • the isolated CA NI from human milk is mainly characterized by what is stated in claim 13 and the isolated CA NI from cow's milk is mainly characterized by what is stated in claim 15.
  • One further object of this invention is to provide pharmaceutical compositions.
  • a pharmaceutical composition is mainly characterized by what is stated in claim 18.
  • composition for treating gastrointestinal disorders according to the present invention is mainly characterized by what is stated in the characterizing part of claim 24.
  • the composition may be in the form of a powder, tablet, granule, capsule, ball, a solution, or the like.
  • a single portion of the composition may comprise 10 - 500 ⁇ g of CA NI.
  • the composition may comprise an effective component selected from the group comprising bicarbonate, sucralfate, histamine 2 -receptor antagonist, such as cimetidine, ranitidine, famotidine, nizatidine; and proton pump inhibitor, such as omeprazole, lanzoprazole, pantoprazole, esomeprazole, rabeprazole.
  • the composition for preventing caries according to the present invention is mainly characterized by what is stated in the characterizing part of claim 28.
  • the composition may be a product selected from the group consisting of mouthrinses, tablets, toothpowders, toothpastes, prophylaxis pastes, emulsions, gels, chewing gums, lozenges, artificial saliva, and candies.
  • a single portion of the product may comprise 10 - 100 ⁇ g of CA NI.
  • the composition may comprise also xylitol and/or fluoride.
  • the enzyme preparation of this invention can be used for preparing food compositions or food additives intended to be added to foodstuffs.
  • the food composition of this invention is mainly characterized by what is stated in in the characterizing part of claim 33.
  • the food additive of this invention is mainly characterized by what is stated in the characterizing part of claim 35.
  • the enzyme preparation of this invention can be used for preparing infant milk formula compositions.
  • Infant milk formula compositions of this invention are mainly characterized by what is stated in the characterizing part of claim 37.
  • An infant milk formula composition according to this invention comprises CA NI in an amount typical for normal breast milk at a certain point of time up to six months, preferably up to three months from childbirth.
  • the composition may comprise CA NI 10 mg - 150 mg/1, preferably 30 mg - 100 mg/1, more preferably 60 mg-100 mg/1 ready for use infant milk formula composition.
  • CA NI may be added to ready for use infant milk formula composition before the use, or it may be added during production process of the infant milk formula composition.
  • the infant milk formula composition may be in liquid form or in powder form. If it is in powder form, ready for use product is made by adding a suitable amount of liquid, typically-waterj-to the powder.
  • One object of this invention is also a transgenic milk-producing (non-human) animal.
  • the animal may be an efficiently milk-producing animal, such as cow, sheep or goat, or related, or preferably closely related animals. Most preferably the animal is cow.
  • the animal is transferred to overproduce endogenous CA NI, CA NI from another milk-producing animal, or preferably human CA NI.
  • Transgenic animals according to this invention are mainly characterized by what is stated in claim 44.
  • Microbial hosts according to this invention are mainly characterized by what is stated in claim 46.
  • This invention provides also a method for producing a food composition, which comprises that the enzyme preparation of this invention is added to and/ or mixed with a foodstuff.
  • the food composition is a nutrient composition or an infant milk formula composition.
  • This invention provides also a method for producing a pharmaceutical composition, which comprises that the enzyme preparation of this invention is added to and/ or mixed with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is a composition for treating nutritional disorders or for the prevention of caries.
  • CA NI can be purified from milk by using a simple and efficient procedure.
  • the enzyme may be purified until homogeneity, but the enzyme preparation may contain small amount of natural ingredients of milk, because they do not have any harmful effect in various applications.
  • cow's colostrum the most important source for the enzyme, cow's colostrum, is available in high amounts, but there are no significant or larger uses for that product.
  • CA NI CA VI originating from milk in the prevention of caries.
  • No side effects are expected for CA VI from milk. It is not necessary to purify the enzyme until homogeneity, since small amount of natural ingredients of milk may not have any harmful effect in oral hygiene products.
  • CA NI has been proven to be a structurally and functionally stable enzyme.
  • the high stability is beneficial when the enzyme is added into solid matrices and has to be stored for long periods.
  • CA NI can be added to any carriers or matrices of the prior art suitable for oral use, such as toothpastes, tablets, mouthrinses etc., but it does not need to be combined with any potentially toxic antimicrobial agents or alcohols, which are commonly used in the oral hygiene products of the prior art. Already small concentrations of the enzyme in the matrices are effective.
  • CA NI is capable of neutralizing the acid in the upper alimentary canal by catalyzing the formation of carbon dioxide and water.
  • compositions or food additives which comprise CA NI from milk.
  • Food compositions of this invention can be used for treating gastrointestinal disorders.
  • the compositions can be in the form of a normal foodstuff (for example functional foodstuff) or in the form of a nutrient solution.
  • infant milk formula compositions of this invention comprise CA NI in concentrations typical for normal breast milk after childbirth.
  • the composition is useful for reducing the symptoms caused by the immaturity of the gastrointestinal tract of an infant.
  • the enzyme from milk is used, because no side effects are expected. There is no need to purify the enzyme preparation until homogeneity, since small ingredients from milk do not have any harmful effect in infant milk formula compositions.
  • FIG. 1 A: Western blot of the saliva of the human newborn infant (N saliva), human colostral milk (milk), human adult saliva (A saliva) and purified human salivary CA VI (CA VI) using anti-human CA VI antibody (anti-CA VI) and normal rabbit serum (NRS).
  • the anti-human CA VI antibody recognized the 42-kDa polypeptides of glycosylated CA VI in all the samples.
  • the 36-kDa polypeptide of the deglycosylated form of CA VI was visible in the purified human salivary CA VI sample. Control stainings using NRS were negative.
  • B Western blot of rat milk (milk), rat mammary glands and purified rat salivary CA VI (S CA VI) using antibody raised against rat CA VI.
  • the antibody recognized in all the samples similar CA VI 42- and 36-kDa polypeptides. From three different mammary gland specimens, the positive signal was strongest in the lactating gland (lac ), moderate in the gland from pregnant animal (preg.), and faintest in the resting gland (rest.).
  • FIG. 1 SDS-PAGE and Colloidal Coomassie Blue staining of total colostral milk (milk), CA purified from colostral milk (M CA VI; 0.2 ⁇ g left lane, 0.6 ⁇ g right lane) and CA purified from saliva (S CA VI).
  • M CA VI colostral milk
  • S CA VI saliva
  • the polypeptides of about 42 kDa mass are seen in CAs purified from both milk and saliva.
  • a polypeptide of similar size is also visible in the total milk sample.
  • FIG. 3 PNGase F treatment of human salivary and milk CA VI followed by SDS-PAGE and Colloidal Coomassie Blue staining. Without PNGase F treatment (-), the 42 kDa polypeptides for both salivary (S) and milk (M) CA VI are seen, corresponding to the glycosylated form of CA VI, but after digestion (+) the 36 kDa polypeptides for both samples are seen, indicating that the two glycopolypeptides have polypeptide cores of similar sizes.
  • Figure 4 Mean concentrations of CA NI in human colostrum and mature milk.
  • FIG. 1 Immunohistochemical staining of rat mammary glands using the anti-rat CA NI antibody (A,C,D) and ⁇ RS (B). Milk inside the alveoli (arrows) stained strongly for CA NI in the lactating gland (A) and moderately in the gland of the pregnant animal (C). No staining was seen inside the alveoli of the resting gland (D). The cytoplasm of the alveolar epithelium showed a faint staining intensity (D, arrowheads) in all the glands. Control staining of a lactating mammary gland with NRS was negative (B).
  • the enzyme preparation comprising CA VI can be purified from milk of a (non-human) animal producing efficiently milk, such as cow, sheep or goat or related, or preferably closely related animals. Isolation of the enzyme from human milk is naturally possible, but not practical due to its limited availability. Because cow's milk is usually available in high amounts, the milk is preferably cow's milk. The milk may be mature non-manufactured milk, but preferably it is colostrum because the amount of CA VI is clearly higher in colostrum. The milk should not be treated with methods which potentially inactivate the enzyme, such as heating or pasteurization.
  • the source for the enzyme is preferably colostrum a few days after calving.
  • colostrum is here meant milk, which is produced by human or a milk producing animal, such as cow, a few days after parturition. It is known that the amount of proteins in cow colostrum decreases rapidly within the first two days after parturition, leveling out by ten days. The amount of immunoglobulins is decreased within two days (Powell et al. 1984).
  • Colostrum means in this invention milk from human or milk from a milk producing animal, preferably cow, 0-10 days, preferably 0-5 days, most preferably 0-2 days after parturition.
  • a milk-producing animal such as cow is transferred to overexpress and secrete endogenous CA VI or express and secrete human or other CA VI into its milk.
  • the normal mature milk of the cow would be a suitable source for the purification of CA VI.
  • the transgenic animal can be transferred to express human CA NI, which may be more suitable than the natural CA NI of the transgenic animal for humans, in particular infants, or at least for some individuals.
  • CA NI may be purified from milk to homogeneity or partly so that the enzyme preparation may contain milk ingredients 0.1 - 10 w-%. Some normal milk components, such as proteins and fats, may be present in the preparation, if they do not have harmful effects in their application, as is the case in oral hygiene products or in infant milk formulas.
  • the amount of milk ingredients may be 0.1 - 10 w-%, preferably it is 0.1 - 5 w-% of the enzyme preparation.
  • Milk preferably colostrum
  • Milk is centrifuged to remove epithelial cells, particles, bacteria or cell debris and fat, preferably by 35 000 x g at 4°C for 30 min.
  • the clear fat-free supernatant is collected and mixed with buffer, preferably ice-cold 0.1 M Tris-SO 4 buffer, pH 8.7, containing a protease inhibitor, such as 1 mM benzamidine and the solution is subjected to affinity purification.
  • the inhibitor affinity chromatography is performed using CM Bio-Gel A or a corresponding matrix coupled to enzyme inhibitor, preferably p- aminomethyl benzenesulfonamide.
  • the displacing agent is removed by a suitable method such as dialysis. Further purification is possible by gel filtration or HPLC, but further purification is normally not needed.
  • the purification of the enzyme can be carried out by various protein chemistry methods well known to a person skilled in the art. However, the method described here and in the examples is useful in large scale and fast, the yield is high and there are only negligible amount of impurities left in the enzyme preparation.
  • the purified enzyme preparation may be used as such or it may comprise also stabilizers, such as glycerol, casein, sucrose or lactose.
  • enzyme preparation comprising CA NI is here meant an enzyme preparation comprising as the major activity CA NI.
  • the preparation may comprise in addition small amounts of non-harmful components still left after the purification process of the enzyme.
  • the amount of milk ingredients may be 0.1 - 10 w-%, preferably it is 0.1 - 5 w-% of the enzyme preparation.
  • the enzyme preparation of this invention may comprise natural CA NI from a milk-producing animal or recombinant CA NI if the animal is transferred to overexpress and produce its natural CA NI, express and produce CA NI of another milk- producing animal or human CA NI. Methods of transferring animals, such as cow, to express or overexpress desired nucleotide sequences are available and well known to a person skilled in the art (for example EP 471832).
  • This invention covers also enzyme preparations prepared in a microbial host transferred to produce milk CA NI, in particular cow's milk CA NI.
  • Many microbial host expression systems are available and well-known to a person skilled in the art.
  • Nucleotide sequence encoding milk CA NI can be integrated into a vector that allows the large-scale production of CA NI in a suitable microbial host, such as in fungal (e.g. yeast), bacterial or mammalian cells according to conventional methods such as described in Maniatis et al 1989 and Ausubel et al. (1989)
  • enzyme preparation may comprise additives, such as buffers and stabilizers.
  • additives such as buffers and stabilizers.
  • Carbonic anhydrase isoenzyme NI (CA NI) is secreted into human saliva by the serous acinar cells of the parotid and submandibular glands.
  • CA NI is the only known secreted isoenzyme of the CA gene family, and has several properties that distinguish it from the well-characterized cytoplasmic isoenzymes.
  • the enzyme molecule has two ⁇ -linked oligosaccharide chains, which can be cleaved by endo- ⁇ - ⁇ -acetylglucosaminidase F but not by endo- ⁇ - ⁇ -acetylglucosaminidase H, indicating that the oligosaccharides are of a complex type (Murakami & Sly 1987).
  • Neuraminidase has no effect on the endo- ⁇ -N-acetylglucos-aminidase F-digested protein, suggesting that CA VI has no O-linked oligosaccharide which contains neuraminidase- sensitive sialic acid residues (Murakami & Sly 1987).
  • the complete amino acid sequence of ovine CA VI was determined by Fernley et al. (1988), and the complete nucleotide sequence for human CA6 cDNA by Aldred et al. (1991).
  • the human CA6 gene is located on chromosome 1 (Aldred et al. 1991).
  • the human CA VI protein has a sequence identity of 35 % to human CA II, while residues involved at the active site of the enzyme are conserved.
  • Radioimmunoassay methods have shown the mean ⁇ SD concentration of CA VI in human parotid saliva to be 47.0 ⁇ 39.2 mg/1, in which case it represented about 3 % of total protein in the parotid saliva (Fernley et al. 1995).
  • secretion of CA VI into the saliva was observed to follow a circadian periodicity, its concentrations being very low during sleep and rising rapidly to the daytime level after awakening and breakfast (Parkkila S et al. 1995).
  • Carbonic anhydrase VI is the only secretory isoform in the CA gene family, having been identified previously in the saliva of several mammalian species. It is demonstrated here for the first time that CA VI is also present in mammalian milk.
  • the specific antibodies to salivary CA VI recognized polypeptides of similar molecular mass (42 kDa) in human and rat saliva and milk, the polypeptide was effectively purified from human milk on CA inhibitor affinity chromatography, and the entire amino acid sequence of the 42-kDa polypeptide obtained from MALDI-MS (40% coverage) was identical to salivary CA VI.
  • Immunohistochemical staining of a rat mammary gland showed that the enzyme is present in both the alveolar milk and the glandular epithelia. The fairly faint epithelial staining is likely to be due to the rapid secretion rate of the epithelia during late pregnancy and lactation. The immunoblots revealing the major 42-kDa polypeptide in rat mammary gland samples confirmed that the immunohistochemical results are specific to CA VI.
  • CA VI was also isolated from mature cow's milk. SDS-PAGE analysis of the purified CA VI fraction showed three polypeptide bands: 29 kDa, 32 kDa and 35 kDa. The 32 kDa band was strongest of these, suggesting that the polypeptide from cow's milk is smaller than in human milk.
  • Recent findings reported in the literature are in accordance with the conceptual CA VI functions as a growth factor. Henkin et al. (1999a) described a clinical disorder in which patients-had-decreased salivary CA V-I-concentrations associated with a loss and distortion of taste and smell after an influenza-like illness. The morphological chances in the taste buds presented apoptotic-like features (Henkin et al. 1999a).
  • CA VI nerve growth factor
  • CA VI glycoprotein and an acid-base modulating enzyme may also possess multifunctional properties such as antimicrobial (Hooper et al. 1995), anti-inflammatory, immunomodulating and mucosa- protecting effects (Parkkila et al. 1997).
  • compositions of this invention comprise CA VI in association with a pharmaceutically acceptable carrier.
  • CA VI may also be combined with known pharmaceuticals which are used to treat gastrointestinal disorders, in particular acid-peptic diseases (esophagitis, gastritis, gastric ulcer or duodenal ulcer) or to prevent caries.
  • compositions which comprise CA VI isolated from milk and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition according to this invention may comprise binders such as gum arabic, syrup, sorbitol, gelatin, tragacanth, and polyvinylpyrrolidone; excipients such as maize starch, calcium phosphate, lactose, sorbitol, and glycine; lubricants such as talc, polyethylene glycol, magnesium stearate and silica; disintegrators such as potato starch; and acceptable wetting agents such as sodium lauryl sulfate.
  • binders such as gum arabic, syrup, sorbitol, gelatin, tragacanth, and polyvinylpyrrolidone
  • excipients such as maize starch, calcium phosphate, lactose, sorbitol, and glycine
  • lubricants such as talc, polyethylene glycol, magnesium stearate and silica
  • disintegrators such as
  • the composition maybe a ⁇ t ⁇ inistered perorally in the form of a powder, tablets, granules, capsules, balls, a solution, or the like. Tablets, granules etc. may be coated by a method known in the art.
  • composition may also contain other effective components together with CAVI.
  • Such components include e.g. bicarbonate, sucralfate, histamine 2 -receptor antagonists (e.g. cimetidine, ranitidine, famotidine, nizatidine), and proton pump inhibitors (e.g. omeprazole, lanzoprazole, pantoprazole, esomeprazole, rabeprazole).
  • the dosage of CA VI may be 10 - 500 ⁇ g/ single portion, preferably it is 50 - 350 ⁇ g/ single portion, more preferably 100 - 200 ⁇ g/ single portion, which is administered 2 to 5 times a day.
  • the enzyme may be in the form of a powder capable of being re-dissolved in an adequate vehicle such as sterilized water containing no pyrogens.
  • CA VI concentration in saliva appears to protect from dental caries and CA VI is associated with the enamel pellicle, suggesting that CA VI prevents caries by accelerating the acid removal in the local microenvironment of the tooth surface as was shown by Leinonen et al. (1999) and Kivela et al. (1999b).
  • This invention describes a method for preventing or reducing the incidence of caries, which comprises that the surface of human teeth is contacted with an effective anti-caries amount of carbonic anhydrase isoenzyme VI, which advantageously is isolated from milk.
  • an effective anti-caries amount is meant here that the surface of teeth is contacted with 10 - 100 ⁇ g, preferably 50 -100 ⁇ g most preferably 50 - 75 ⁇ g of CA VI at a time.
  • the treatment can be carried out 1 to 10 times per day. Preferably the treatment is carried out 2 to 5 times per day. For example, if the treatment was carried out using single dosages of 50 to 100 ⁇ g three times a day, the total daily dosage would be 150 to 300 ⁇ g.
  • the aim of the CA VI treatment is to reach a concentration of 10-100 ⁇ g, preferably 50 -100 ⁇ g, most preferably 50 - 75 ⁇ g of CA VI of CA VI per ml of saliva.
  • This invention furthermore describes a composition for prevention of caries, which comprises-an-effective anti-caries amount of-CA VI-
  • CA VI enzyme may be added to any suitable carrier or matrix, which is used in the prior art in oral care products.
  • the carrier or matrix may be selected from the group of mouthrinses, tablets, toothpowders, toothpastes, prophylaxis pastes, emulsions, gels, chewing gums, lozenges, and artificial saliva.
  • a single dosage is chosen depending on the used matrix.
  • a chewing tablet, toothpaste or mouthrinse may be used twice a day in connection with cleaning the teeth.
  • a single dosage may thus comprise 10 to 100 ⁇ g of CA VI, preferably 50 to 100 ⁇ g, most preferably 50 to 75 ⁇ g .
  • a single dosage may safely be up to 1 mg. However, it is not economical to use dosages higher than 100 ⁇ g.
  • lower dosages than 50 ⁇ g can be used, but the effective amount is at least about 10 ⁇ g, preferably 50 ⁇ g, at a time.
  • Chewing gum maybe used 1 to 10 times a day and a single dosage may thus comprise 10 to 100 ⁇ g, preferably 20 to 30 ⁇ g.
  • CA VI may be added to artificial saliva used by patients with grave salivary hypofunction after receiving radiation therapy to the area of the head or neck, since these patients often suffer of rampant caries.
  • concentration of CA VI in artificial saliva should be 5-50 mg/1, preferably 10-20 mg/1.
  • CA VI as a natural component of the enamel pellicle attaches to the hydroxyapatite of the enamel.
  • CA VI treatment may be carried out before eating sweet food, candies etc. in order to accelerate the acid removal from the dental surface and to reduce demineralization of the dental hard tissues.
  • CA VI may be added also to candies.
  • the amount of CA VI in a single portion depends again on the amount of candies to be used in a day.
  • a single candy may comprise 10 to 50 ⁇ g, preferably 20 to 30 ⁇ g of CA VI.
  • a single dosage may in the case of candies or chewing gum be lower, because candies or chewing gums may be consumed especially by children in higher amounts than oral care products.
  • the weight of a tablet may vary from 0.05 g to 1 g, and it may comprise 0.2 to 0.01 w-% CA VI of the total composition, respectively.
  • a single dosage of paste or gel or powder may weigh 0.1 to 1 g, and it may thus contain 0.1 to 0.01 w-% CA VI of the total composition, respectively.
  • a single dosage of mouthrinse may weight 1 to 3 g, and it may comprise 0.01 to 0.005 w-% CA VI of the total composition, respectively.
  • the carrier for preventing or reducing the incidence of caries and comprising CA VI may preferably comprise also xylitol and/or fluoride.
  • the oral compositions can be formulated in any suitable application form, such as mouthrinses, tablets, toothpowders, toothpastes, prophylaxis pastes, emulsions, gels, chewing gums, lozenges, and artificial saliva.
  • Additives typically included in toothpastes and gels may be used in toothpaste and gel compositions in accordance with the invention.
  • Suitable additives include abrasive polishing materials, sudsing agents, flavoring agents, humectants, binders, sweetening agents, and water as described in US Patent No. 5,981,475, US Patent No. 5,801,226, US Patent No. 5,849,271 and US Patent No. 5,824,242, hereby incorporated by reference.
  • the composition may be relatively simple and natural comprising a matrix, which functions merely as a carrier and should not comprise any potentially toxic antimicrobial agents or alcohol, which would denature or inactivate the enzyme.
  • the oral compositions may comprise particulate abrasive materials including silicas, aluminas, calcium and magnesium carbonates, aluminosilicate, zirconium silicate, polymethylmethacrylate, powdered polyethylene, silica xerogels, hydrogels and aerogels and the like, dicalciumphosphates, calcium pyrophosphates, hydroxyapatites, trimeta- phosphates, insoluble hexametaphosphates and so on, in amounts between 0 and 70% by weight, preferably 1 to 70, usually 5 to 60. Higher levels, as high as 95%, may be used if the composition is a toothpowder.
  • particulate abrasive materials including silicas, aluminas, calcium and magnesium carbonates, aluminosilicate, zirconium silicate, polymethylmethacrylate, powdered polyethylene, silica xerogels, hydrogels and aerogels and the like, dicalciumphosphates, calcium
  • the toothpaste/powder compositions may comprise humectants such as glycerol, polyol, sorbitol, propyleneglycol, polyethyleneglycol, xylitol, lactitol and so on.
  • humectants such as glycerol, polyol, sorbitol, propyleneglycol, polyethyleneglycol, xylitol, lactitol and so on.
  • the humectants may be generally present in amounts of from 0 to 80%, preferably 15 to 70% by weight.
  • Binders and thickeners such as sodium carboxymethyl-cellulose, xanthan gum, gum arabic etc. may also be included, as well as synthetic polymers such as polyacrylates and carboxy- vinyl polymers.
  • Thickeners suitable for use in the invention include silica. Thickeners may be present in toothpastes and gels at 0.1 to 20% by weight.
  • Flavours such as peppermint and spearmint oils may also be included, as well as pH- adjusting agents, colouring agents, opacifying agents, preservatives, sweetening agents, such as xylitol or saccharin and so on. Flavors are usually included in toothpastes in low amounts, such as from 0.01 to about 5% by weight, especially from 0.1 % to 5% of the total composition.
  • Fluoride sources used in toothpastes are such as sodium fluoride, aminefluorides, sodium monofluorophosphate, stannous fluoride, zinc ammonium fluoride, tin ammonium fluoride, calcium fluoride and cobalt ammonium fluoride.
  • Preferred compositions of the invention include the fluoride source.
  • Fluoride ions are typically provided at a level of from 0 to 1500 ppm, preferably 50 to 1500 ppm, although higher levels up to about 3000 ppm may be used.
  • Anticaries agents include casein, plaque buffers such as urea, calcium lactate, calcium glycerophosphate and strontium polyacrylates.
  • Desensitising agents such as, potassium citrate, potassium tartrate, potassium chloride, potassium bicarbonate, potassium oxalate, potassium nitrate as well as strontium salts may also be included.
  • Buffers and salts to buffer the pH and ionic strength of the compositions may also be included. Liposomes and other encapsulates may also be used to improve delivery or stability of active ingredients.
  • the oral compositions may comprise anti-calculus agents such as alkalimetal pyrophosphates, hypophosphite-containing polymers, organic phosphonates, phospho- citrates etc.
  • anti-calculus agents such as alkalimetal pyrophosphates, hypophosphite-containing polymers, organic phosphonates, phospho- citrates etc.
  • compositions may further comprise other enzymes, such as dextranase and/or mutanase, amyloglucosidase, glucose oxidase with lactoperoxidase, neurarninidases, and hydrogen peroxide generating compounds such as potassium peroxydiphosphate.
  • enzymes such as dextranase and/or mutanase, amyloglucosidase, glucose oxidase with lactoperoxidase, neurarninidases, and hydrogen peroxide generating compounds such as potassium peroxydiphosphate.
  • additives that may be included are e.g. organic peroxyacids, effervescing systems, such as sodium bicarbonate/citric acid systems, colour change systems, and so on.
  • Water is also present in the compositions of this invention.
  • Water employed in the preparation of commercially suitable toothpastes should preferably be deionized and free of organic impurities.
  • Water generally comprises from about 10% to 50%, preferably from about 20% to 40%, by weight of the toothpaste compositions herein. These amounts of water include the free water which is added plus that which is introduced with other materials such as with sorbitol.
  • a toothpaste may be prepared by kneading the above-mentioned components with an appropriate amount of water.
  • moumtrinses comprise a water solution, flavor, humectant, sweetener, sudsing agent, and colorant. Alcohol should be avoided to maintain the enzyme activity.
  • the mouthwashes of the invention may comprise 0% to 20% by weight of a humectant, 0% to 2% emulsifying agent, 0% to 0.5% sweetening agent, such as xylitol or saccharin, 0% to 0.3% by weight flavoring agent, and the balance water.
  • the pH of the oral composition of the invention is generally in the range of from about 4.5 to about 10 and typically from about 5.5 to 9.
  • the pH of the compositions range from about 4.0 to about 9.5, with the preferred pH being from about 5.0 to about 9.0 and the most preferred pH being 6.5 to about 9.0.
  • the pH can be controlled with acid (e.g. citric acid or benzoic acid) or base (e.g. sodium hydroxide) or be buffered (e.g. with sodium citrate, benzoate, carbonate, or bicarbonate, disodium hydrogen phosphate, or sodium dihydrogen phosphate).
  • Food compositions according to this invention comprise CA VI mixed to any foodstuff, including drinking water, intended for human consumption.
  • Food additives mean any liquid or solid material intended to be added to a foodstuff.
  • the foodstuff is useful as a functional foodstuff or composition, such as a yoghurt-type product or nectar or soft drink.
  • it may be a nutrient solution, which may be used for patients nutrition in hospitals.
  • the amount of CA VI in the product is calculated on the basis of the description for pharmaceutical compositions above.
  • the amount used a day should not exceed the dosage of pharmaceutical compositions dosaged a day.
  • overdosing CA VI it is recommendable not to use the food compositions at the same time as the pharmaceutical compositions.
  • the infant milk formula composition of this invention comprises CA VI advantageously isolated from milk of a milk-producing animal, preferably cow "in an amount typical for normal breast milk up to six months, preferably up to three months from childbirth".
  • breast milk comprises after child birth the highest level of CA VI, as can be seen in Figure 4.
  • the amount of CA VI decreases gradually and is after 6 months post partum on the level of saliva, but is on lower level already after 3 months post partum.
  • the infant milk formula should contain CA VI in an amount which corresponds to the amount of CA VI in breast milk of a mother having a child of the same age.
  • the infant milk formula composition should comprise CA VI preferably 10 - 150 mg/1, preferably 30 - 100 mg/1, more preferably 60 - 100 mg/1 ready for use infant milk formula composition.
  • Ready for use infant milk formula composition means here a liquid composition or a powder, into which liquid has been added before use.
  • infant milk formula composition we mean here any nutritional formula or substitute composition, which is aimed for the feeding of an infant instead of breast milk.
  • the infant milk-formula composition may be any commercially available infant milk formula composition based for example on cow milk or soy milk. It may be based also on another mother's milk. Usually another mother's milk comprises lower levels of CA VI, because it is not available immidiately or shortly after the birth of a child.
  • CA VI is added to the infant milk formula composition in the amount which corresponds to the amount of CA VI in the breast milk of a mother having a child of the same age.
  • Different infant milk products can be prepared comprising of gradually decreasing amounts of CA VI.
  • the proportioning of CA VI is adjusted to the age of the child. This is preferably carried out by adding CA VI by the parents or nurses of a child to a ready for use infant milk product in an amount recommended for a child of that age.
  • nutritional supplement products products, which are aimed to be added to infant milk formula compositions and which are in liquid or in solid form.
  • Such products may be for example products which contain purified CA VI and optionally buffers and/or factors, which stabilize the product.
  • the product is preferably sterilized by filtering because heating could inactivate the enzyme.
  • the product may be provided in dropping bottles such as vitamins (Vitamin A for example) and may be stored in refrigerator before use.
  • the infant milk formula composition may be in liquid or powder form.
  • liquid normally water
  • the infant milk formula composition of this invention is recommended for children up to 6 months, preferably up to 3 months. However, it may be used also for elder children, if they have gastrointestinal disorders.
  • the formula provides energy about 67-70 kcal/100 ml.
  • Typical protein levels in commercially available infant formulas are 1.2 - 1.8 g/100 ml.
  • Conventional infant formula is formed by mixing 60 % sweet whey and 40 % casein, the ratio of the amounts of tryptophan to the total amount of large neutral amino acids (Phe, Tyr, Val, He, Leu) is about 4.4 - 4.7/100 ml
  • infant formula compositions are described in United States Patent No. 5,902, 617.
  • Commercially available infant milk formulas may comprise in 1000 g of the product: energy 676 - 670 cal, protein 15.0 - 19.0 g depending on the source, which may be cow's milk, reduced mineral whey and milk, soy protein isolate or casein hydrolysate containing Cys, Tyr, and Trp.
  • the infant milk formula may comprise fat 36.3 - 38 g, the source of which may be soy and coconut oils or medium chain triglycerides.
  • the infant formula composition may furthermore comprise polyunsaturated fatty acids 11 - 14 g, saturated fatty acids 16 - 18.2 g, linoleic acids 7400 - 10816 mg, carbohydrates 68.9 - 72.3 g.
  • the source of carbohydrates may be lactose or corn syrap and sucrose or sucrose and starch.
  • commercial infant formulas may comprise minerals, vitamins, other nutrients and 900 g water.
  • formulas based on a variety of protein and carbohydrate sources are also available.
  • the formulas may be in the form of liquid in a ready to feed concentration or a concentrate or dehydrated or lyophilized.
  • a method for preparing an infant milk formula composition is also available.
  • An-infant-milk formula composition is prepared, for example, by a process described, for example, in International Patent Publication No. WO 97/35488:
  • a) pasteurised milk is standardised by the addition of whey protein concentrate, minerals, water-soluble vitamins, trace elements and carbohydrates at high temperatures, for example at 60 °C; b) vegetable oil, oil-soluble emulsifiers, oil-soluble vitamins and anti-oxidants are mixed at high temperatures, for example at 60 °C; c) the oil mixture obtained from b) (an oil phase) is added to the standardised milk obtained from a) (a water phase) with sufficient agitation to allow mixing; d) the mixture obtained above is homogenised in two stages at high temperature and pressure, for example at 60 °C at 150 and 30 bar; e) the emulsion obtained above is cooled to a low temperature, for example to 5 °C f) if desired, water-soluble vitamins, minerals and trace emulsions are added to the cooled emulsion; g) the emulsion from e) is sterilised on-line at ultra high temperature (UHT) and/
  • Purified CA VI is added to the infant milk formula preparation at a suitable stage of the preparation.
  • CA VI is added at a relatively late stage in the preparation. This is because high temperatures are used at several points in the preparation, which would result in inactivation of CA VI.
  • the formula preparation should be filtered in order to prepare a sterile product instead of heating or pasteurizing.
  • CA VI is preferably added after sterilization in step g) or pasteurization in step h).
  • CA VI enzyme preparation is added to a ready for use infant milk formula composition before use.
  • - infant formula composition is prepared according to conventional methods; - CA NI is added to infant milk formula composition in an amount normal breast milk comprises CA NI at a certain point of time after cMldbirth; and
  • the infant milk formula composition is recovered from the production process.
  • CA NI as a nutritional supplement product or concentrate.
  • the enzyme may be packaged in a buffered solution, filtered with, for example, 0.2 micron filter to sterilize the concentrate, and the concentrate may be refrigerated when the container is opened.
  • the formulation may be added as drops per volume. The addition can be made at the time of feeding.
  • Colostral milk samples were obtained from 9 mothers on the 2-4th days post partum, and saliva samples were obtained at the same time from two 3-day-old infants prior to nursing. Mature milk samples were obtained from four mothers on the 90th day post partum. The procedures were carried out according to the provisions of the Declaration of Helsinki and informed consent was obtained from each mother. Rat milk samples were collected from the rats of Spraque-Dawley strain anesthetized with fentanyl-fluanisone (3ml/kg, Janssen Pharmaceutica, Beerse, Belgium) on the second day post partum and pooled. All the milk samples were stored frozen prior to use in the experiments.
  • Mammary gland specimens were taken from a sexually mature non-pregnant female rat, and from another two days before and two days after parturition.
  • the tissue samples were homogenized in ice-cold 0.1 M Tris-SO 4 buffer, pH 8.7, containing 1 mM PMSF, 1 mM benzamidine and 1 mM o- phenanthroline as protease inhibitors and used for western blotting, or fixed in Carnoy's fluid and embedded in paraffin as described (Karhumaa et al. 2000).
  • Human colostrum (15 ml) was centrifuged (35 000 x g) at 4°C for 30 min and the clear supernatant (10 ml) was collected and mixed with 30 ml of ice-cold 0.1 M Tris-SO 4 buffer, pH 8.7, containing 1 mM benzamidine as a protease inhibitor and subjected to affinity purification.
  • the inhibitor affinity chromatography was performed using CM Bio-Gel A coupled to p-aminomethyl benzenesulfonamide as described (Parkkila et al. 1990).
  • the protein sequencing of trypsin-digested polypeptides was carried out by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) followed by analysis with the ProFound and PeptideSearch programs.
  • the sequencing was performed in the HHMI Biopolymer/W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University. Deglycosylation studies
  • Purified milk and salivary CA NI (1 ⁇ g) were digested with PNGase F as described earlier (Petaja-Repo et al— 1991-) andthe-deglycosylated-andnon-deglycosylated proteins were subjected to SDS-PAGE followed by Colloidal Coomassie staining (Novex, San Diego, CA).
  • the mean intra-assay coefficient of variation (CN) of the present series was 9.4-% and the interassay CN determined in three assays was 9.7- %.
  • Immunocytochemistry Mammary gland samples fixed in Carnoy's fluid were sectioned at 5 ⁇ m and placed on gelatin-coated microscope slides. Immunohistochemical staining was performed using the biotin-streptavidin complex method as described (Karhumaa et al. 2000). The stained sections were examined and photographed with a Nikon Eclipse E600 microscope (Tokyo, Japan).
  • the CA VI levels in the saliva of two infants were 1.9 and 3.6 mg/1, about half of the concentration in adults (Parkkila et al 1993).
  • the mammary glands of a sexually mature nonpregnant female rat showed a faint positive reaction for CA VI in the alveolar epithelia, and the same was observed prior to and after parturition (Fig. 5A,C,D).
  • a strong reaction was seen in the alveolar milk after parturition, the reaction being slightly weaker prior to parturition (Compare figures 5A and C).
  • No reaction was seen in the alveolar lumen of the resting gland (Fig. 5D).
  • Western blotting of mammary gland homogenates showed the most intense band (reaction to CA VI) to be in the lactating gland, a moderate band in glandular tissue prior to parturition, and a faint band in the resting gland (Fig. IB).
  • CA VI was purified from fresh non-manufactured mature cow's milk. The purification was carried out essentially as described in example 1. Samples of CAs purified from cow's milk were subjected to SDS-PAGE under reducing conditions as described in example 1. The SDS-PAGE analysis gave three polypeptides bands: 29 kDa, 32 kDa, and 35 kDa. The 32 kDa band was strongest, which indicated that the polypeptide is smaller than CA VI from human milk.
  • CA VI is purified from the non-manufactured unsterilized cow's colostrum. The purification is carried out essentially as described in example 1.
  • a toothpaste is prepared by conventional methods as described here earlier or for example in-US Patent No. 5,98- 7-5 T US Patent No. -5 ⁇ 8 ⁇ l,226 r US-Patent-No. 5,849,271 and US Patent No. 5,824,242, hereby incorporated by reference.
  • To the product is added CA VI purified to homogenity 0.1 - 0.01 w-% of the total composition.
  • CA VI is purified from non-manufactured unsterilized cow's colostrum after 2 days from calving. The purification is carried out as described in example 1.
  • An infant milk formula composition is prepared by conventional methods as described, for example, in International Patent Publication WO 97/35488. To the ready for use infant milk formula is added purified CA VI to give a concentration between 10 - 150 mg/1.
  • Fernley RT Carbonic anhydrases secreted in the saliva; in Dodgson SJ, Tashian RE, Gros G, Carter ND (eds): The Carbonic Anhydrases: Cellular Physiology and Molecular Genetics. New York, Plenum Press, 1991, pp 365-373. Fernley RT, Farthing J, Cooper EJ: Radioimmunoassay for salivary carbonic anhydrase in human parotid saliva. Arch Oral Biol 1995;40:567-569.
  • Fernley RT, Wright RD, Coghlan JP Complete aminoacid sequence of ovine salivary carbonic anhydrase. Biochemistry 1988;27:2815-2820. Feldstein, J.B., and Silverman, D.N. (1984) J. Biol. Chem. 259, 5447-5453.
  • Salivary carbonic anhydrase isoenzyme VI is located in the human enamel pellicle. Caries Research 33:185- 190.
  • CA II carbonic anhydrase isoenzyme

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Dairy Products (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne un nouveau procédé de préparation d'une enzyme comprenant de l'anhydrase carbonique VI (CA VI). Le procédé comporte une étape d'isolement de l'anhydrase carbonique VI à partir du lait maternel ou à partir du lait d'un animal produisant du lait, tel une vache, chèvre ou brebis. On peut purifier l'anhydrase carbonique VI jusqu'à l'obtention d'une homogénéité ou d'un niveau de pureté choisi selon l'utilisation de la formulation enzymatique. La formulation enzymatique peut être utilisée pour la préparation de compositions pharmaceutiques, notamment, pour la prévention des caries ou pour la préparation des compositions alimentaires, notamment, une formule de composition de lait pour nourrissons.
PCT/FI2002/000081 2001-02-01 2002-02-01 Procede de production d'une enzyme a partir d'une source nouvelle WO2002061066A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002231817A AU2002231817A1 (en) 2001-02-01 2002-02-01 Method for producing an enzyme from a novel source

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
FI20010193A FI20010193A0 (fi) 2001-02-01 2001-02-01 Menetelmä entsyymin tuottamiseksi uudesta lähteestä
FI20010193 2001-02-01
FI20010845A FI20010845A (fi) 2001-02-01 2001-04-24 Menetelmä entsyymin tuottamiseksi uudesta lähteestä
FI20010845 2001-04-24

Publications (2)

Publication Number Publication Date
WO2002061066A1 true WO2002061066A1 (fr) 2002-08-08
WO2002061066A8 WO2002061066A8 (fr) 2003-11-06

Family

ID=26161118

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/FI2002/000081 WO2002061066A1 (fr) 2001-02-01 2002-02-01 Procede de production d'une enzyme a partir d'une source nouvelle

Country Status (3)

Country Link
AU (1) AU2002231817A1 (fr)
FI (1) FI20010845A (fr)
WO (1) WO2002061066A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1568356A1 (fr) * 2004-02-24 2005-08-31 Sara Lee/DE N.V. Prévention de l'érosion des dents par l'utilisation de protéine du colostrum
JP2007167022A (ja) * 2005-12-26 2007-07-05 Kaneka Corp 乳由来のタンパク質

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988004688A1 (fr) * 1986-12-23 1988-06-30 Howard Florey Institute Of Experimental Physiology Isoenzyme d'anhydrase carbonique

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988004688A1 (fr) * 1986-12-23 1988-06-30 Howard Florey Institute Of Experimental Physiology Isoenzyme d'anhydrase carbonique

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
DATABASE FSTA [online] KITCHEN B.J. ET AL.: "Milk enzymes their distribution and activity", XP002950423, accession no. STN Database accession no. 1970(08):P1072 *
DATABASE MEDLINE [online] NATIONAL LIBRARY OF MEDICINE; PARKKILA S. ET AL.: "Slivary carbonic anhydrase protects gastroesophageal mucosa from acid injury", XP002954984, Database accession no. 9149056 *
DIGESTIVE DISEASES AND SCIENCES, vol. 42, no. 5, May 1997 (1997-05-01), pages 1013 - 1019 *
JOURNAL OF DAIRY RESEARCH, vol. 37, no. 2, 1970, pages 279 - 288 *
JUKKA LEINONEN ET AL.: "Salivary carbonic anhydrase isoenzyme VI is located in the human enamel pellicle", CARIES RES., vol. 33, 1999, pages 185 - 190, XP002950453 *
JYRKI KIVELA ET AL.: "A low concentration of carbonic anhydrase isoenzyme VI in whole saliva is associated with caries prevalence", CARIES RES., vol. 33, 1999, pages 178 - 184, XP002950454 *
KATARINA CVEK ET AL.: "Localization of carbonic anhydrase in the goat mammary gland during involution and lactogenesis", JOURNAL OF DAIRY RESEARCH, vol. 65, 1998, pages 43 - 54, XP002950424 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1568356A1 (fr) * 2004-02-24 2005-08-31 Sara Lee/DE N.V. Prévention de l'érosion des dents par l'utilisation de protéine du colostrum
JP2007167022A (ja) * 2005-12-26 2007-07-05 Kaneka Corp 乳由来のタンパク質

Also Published As

Publication number Publication date
WO2002061066A8 (fr) 2003-11-06
FI20010845A0 (fi) 2001-04-24
FI20010845A (fi) 2002-08-02
AU2002231817A1 (en) 2002-08-12

Similar Documents

Publication Publication Date Title
US6740334B2 (en) Bone resorption suppressing agent
JP2003519167A (ja) 微生物による接着を阻害するための方法及び組成物
CA2349980C (fr) Methode de production de fractions contenant une forte concentration de cystatine, une proteine de base du lait, et produits de decomposition connexes
EP1010430A1 (fr) Substances a action preventive et agents d'amelioration de la parodontose
JP2001346519A (ja) 乳塩基性シスタチン高含有画分及びその分解物の製造方法
CA2523098C (fr) Promoteur de la production du collagene de la peau
ES2370122T3 (es) Alimento para estimular la diferenciación de osteoblastos e inhibir la diferenciación de osteoclastos.
JP2001510803A (ja) 母体の免疫分泌液、および人体の症状の治療および／または予防におけるその使用
US9063147B2 (en) Methods for isolation, use and analysis of ferritin
JP2004331566A (ja) 皮膚コラーゲン産生促進剤
RO114793B1 (ro) Peptida derivata de lipaza umana stimulata de sarea biliara si secventa de adn ce o codifica
WO2002061066A1 (fr) Procede de production d'une enzyme a partir d'une source nouvelle
AU2007259959B2 (en) Agent for maintaining the hardness of tooth structure
RU2379975C2 (ru) Добавка в состав для детского питания
JP4707401B2 (ja) ローヤルゼリー由来の抗酸化性ペプチド
AU2017206200A1 (en) Sensation-improving agent
AU5185301A (en) Method of producing fractions containing a high concentration of milk basic cystatin and decomposition products thereof
EP2668956A1 (fr) Agent améliorant les sens
JPH11269078A (ja) カルシウム吸収促進剤及びカルシウム補給飲食品
WO2002060269A1 (fr) Nouvelle formule de composition de lait pour nourrissons
JP2007055938A (ja) カルシウム可溶化剤
Leinonen Carbonic anhydrase isoenzyme VI: distribution, catalytic properties and biological significance
AU2023217563A1 (en) Combination, therapeutic uses and prophylactic uses
US20150250852A1 (en) Methods for isolation, use and analysis of ferritin
Adkins et al. University of California, Davis, Davis, California, USA

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
CFP Corrected version of a pamphlet front page
CR1 Correction of entry in section i

Free format text: IN PCT GAZETTE 32/2002 DUE TO A TECHNICAL PROBLEM AT THE TIME OF INTERNATIONAL PUBLICATION, SOME INFORMATION WAS MISSING (81). THE MISSING INFORMATION NOW APPEARS IN THE CORRECTED VERSION.

Free format text: IN PCT GAZETTE 32/2002 DUE TO A TECHNICAL PROBLEM AT THE TIME OF INTERNATIONAL PUBLICATION, SOME INFORMATION WAS MISSING (81). THE MISSING INFORMATION NOW APPEARS IN THE CORRECTED VERSION.

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP