Patent Application
20050214358
The present invention relates to a composition for promoting the development of a nervous system, a method for promoting the development of a nervous system, and a use of a specific compound for producing a composition for promoting the development of a nervous system.
A protein involved in intracellular transport of fatty acids is, for example, a fatty acid-binding protein (FABP). FABP is a cytoplasmic protein binding to a long-chain fatty acid and having a molecular weight between 14 kDa and 16 kDa. The protein includes several types having sequence homology, such as a liver type (L-FABP), an intestine type (I-FABP), and a heart-type (H-FABP) according to locations from which the proteins were initially isolated.
In particular, the heart-type fatty acid-binding protein (H-FABP) is a protein with a molecular weight of approximately 15 kDa, which is distributed over a broad range of tissues such as heart, skeletal muscle, stomach, testes, kidney, and mammary glands. The protein is known to be involved in intracellular transport of polyunsaturated fatty acids (linoleic acid, arachidonic acid, and linolenic acid) (non-patent documents 1 to 4). In addition, because the H-FABP content of myocardial cells is high, H-FABP is utilized as a marker for cardiomyopathy such as myocardial infarction (patent documents 1 to 3).
In the meantime, examples of therapeutic agents for affective disorders such as ADHD include atomoxetine (non-patent document 5) as a drug for central stimulation and methylphenidate (non-patent document 6) and amphetamine (non-patent document 7) as psychotropic drugs.
[Non-Patent Document 1]
An object of the present invention is to provide a composition for promoting the development of a nervous system, a method for promoting the development of a nervous system, a use of a specific compound for producing a composition for promoting the development of a nervous system, and the like. Another object of the present invention is to provide a composition for preventing or treating a disease in which the expression of a pheromone receptor is involved, a method for preventing or treating a disease in which the expression of a pheromone receptor is involved, a use of a specific compound for producing a composition for preventing or treating a disease in which the expression of a pheromone receptor is involved, and the like.
In one aspect, the present invention provides a composition comprising a fatty acid-binding protein (FABP) fraction as an active ingredient for promoting mammalian nervous system development. Preferably, the above FABP fraction is a heart-type fatty acid-binding protein (H-FABP) fraction. More preferably, the above FABP fraction is H-FABP. Preferably, the above composition is in a form appropriate for nasal administration, oral administration, or administration by spraying. Furthermore, preferably, the above composition is in the form of spray, powder, liquid, gel, cream, foam, jelly, crystal, liposome, or suspension. Preferably, the above composition is to be administered to an infant and more preferably to a newborn. Furthermore, preferably, the above composition is to be added to a food. In another aspect, the present invention provides a food comprising the above composition, particularly synthetic milk.
In another aspect, the present invention relates to a method for promoting the development of a mammalian nervous system, comprising administering an FABP fraction to a mammal. Preferably, the above FABP fraction is a heart-type fatty acid-binding protein (H-FABP) fraction. More preferably the above FABP fraction is H-FABP. Preferably, the above administration is nasal administration, oral administration, or administration by spraying. Furthermore, preferably, the above FABP fraction is administered in the form of spray, powder, liquid, gel, cream, foam, jelly, crystal, liposome, or suspension. Preferably, administration is performed for an infant, and more preferably for a newborn. Furthermore, preferably, the above FABP fraction is added to a food, and then the fraction contained in the food is administered.
In another aspect, the present invention relates to a use of an FABP fraction for producing a composition for promoting the development of the nervous system. Preferably, the above FABP fraction is a heart-type fatty acid-binding protein (H-FABP) fraction. More preferably the above FABP fraction is H-FABP. Preferably, the above composition for promoting the development of a nervous system is in a form appropriate for nasal administration, oral administration, or administration by spraying. Furthermore, preferably, the above composition for promoting the development of a nervous system is in the form of spray, powder, liquid, gel, cream, foam, jelly, crystal, liposome, or suspension. Preferably the above composition for promoting the development of a nervous system is to be administered to an infant, and more preferably a newborn. Furthermore, preferably, the above composition for promoting the development of a nervous system is added to a food, and then the composition contained in the food is administered.
In another aspect, the present invention provides a composition for promoting mammalian pheromone receptor expression, comprising an FABP fraction as an active ingredient. Preferably, the above pheromone receptor is a V2R type pheromone receptor and more preferably VR1, VR4, or VR1 and VR4 of the V2R type pheromone receptor. Preferably, the above FABP fraction is a heart-type fatty acid-binding protein (H-FABP) fraction and more preferably the above FABP fraction is H-FABP. Preferably, the above composition is in a form appropriate for nasal administration, oral administration, or administration by spraying. Furthermore, preferably the above composition is in the form of spray, powder, liquid, gel, cream, foam, jelly, crystal, liposome, or suspension. Preferably, the above composition is to be administered to an infant and more preferably to a newborn. Furthermore, preferably, the above composition is to be added to a food. In another aspect, the present invention provides a food containing the above composition, particularly synthetic milk.
In another aspect, the present invention provides a method for promoting mammalian pheromone receptor expression, comprising administering an FABP fraction to a mammal. Preferably, the above pheromone receptor is a V2R type pheromone receptor, and more preferably VR1, VR4, or VR1 and VR4 of the V2R type pheromone receptor. Preferably, the above FABP fraction is a heart-type fatty acid-binding protein (H-FABP) fraction, and more preferably the above FABP fraction is H-FABP. Preferably, the above administration is nasal administration, oral administration, or administration by spraying. Furthermore, preferably, the above FABP fraction is administered in the form of spray, powder, liquid, gel, cream, foam, jelly, crystal, liposome, or suspension. Preferably, administration is performed for an infant, and more preferably for a newborn. Furthermore, preferably, the above FABP fraction is added to a food, and then the fraction contained in the food is administered.
In another aspect, the present invention relates to a use of an FABP fraction for producing a composition for promoting the expression of a pheromone receptor. Preferably, the above pheromone receptor is a V2R type pheromone receptor, and more preferably VR1, VR4, or VR1 and VR4 of the V2R type pheromone receptor. Preferably, the above FABP fraction is a heart-type fatty acid-binding protein (H-FABP) fraction, and more preferably, the above FABP fraction is H-FABP. Furthermore, preferably, the above composition is in the form of spray, powder, liquid, gel, cream, foam, jelly, crystal, liposome, or suspension. Preferably, the above composition is in a form appropriate for nasal administration, oral administration, or administration by spraying. Preferably, the above composition is to be administered to an infant, and more preferably to a newborn. Furthermore, preferably, the above composition is added to a food and then the composition contained in the food is administered.
In another aspect, the present invention relates to a composition for preventing or treating a disease in which the expression of a pheromone receptor is involved, comprising an FABP fraction in an effective amount for preventing or treating a disease in which the expression of a pheromone receptor is involved. Preferably, the above pheromone receptor is a V2R type pheromone receptor, and more preferably VR1, VR4, or VR1 and VR4 of the V2R type pheromone receptor. Preferably, the above FABP fraction is a heart-type fatty acid-binding protein (H-FABP) fraction, and more preferably, the above FABP fraction is H-FABP. Furthermore, preferably, the above composition is in the form of spray, powder, liquid, gel, cream, foam, jelly, crystal, liposome, or suspension. Preferably, the above composition is in a form appropriate for nasal administration, oral administration, or administration by spraying. Furthermore, preferably, the above composition is to be added to a food. In another aspect, the present invention provides a food containing the above composition, particularly synthetic milk.
In another aspect, the present invention provides a method for preventing or treating a disease in which the expression of a pheromone receptor is involved, comprising administering an FABP fraction in an effective amount for preventing or treating a disease in which the expression of a pheromone receptor is involved to a subject who requires such prevention or treatment. Preferably, in the above method, the fraction is administered in the form of aerosol by spraying. Preferably, the above pheromone receptor is a V2R type pheromone receptor, and more preferably VR1, VR4, or VR1 and VR4 of the V2R type pheromone receptor. Preferably, the above FABP fraction is a heart-type fatty acid-binding protein (H-FABP) fraction, and more preferably, the above FABP fraction is H-FABP. Furthermore, preferably, the above FABP fraction is administered in the form of spray, powder, liquid, gel, cream, foam, jelly, crystal, liposome, or suspension. Furthermore, preferably, the above FABP fraction is added to a food and then the fraction contained in the food is administered.
In another aspect, the present invention provides a use of an FABP fraction for producing a composition for preventing or treating a disease in which the expression of a pheromone receptor is involved. Preferably, the above pheromone receptor is a V2R type pheromone receptor, and more preferably VR1, VR4, or VR1 and VR4 of the V2R type pheromone receptor. Preferably, the above FABP fraction is a heart-type fatty acid-binding protein (H-FABP) fraction, and more preferably, the above FABP fraction is H-FABP. Furthermore, preferably the above composition is in the form of spray, powder, liquid, gel, cream, foam, jelly, crystal, liposome, or suspension. Preferably, the above composition is in a form appropriate for nasal administration, oral administration, or administration by spraying. Furthermore, preferably, the above composition is added to a food and then the composition contained in the food is administered.
In still another aspect, the present invention provides a composition comprising a breast milk extract for promoting the development of a mammalian nervous system, for promoting mammalian pheromone receptor expression, or for preventing or treating a disease in which the expression of a pheromone receptor is involved. Preferably, the above pheromone receptor is a V2R type pheromone receptor, and more preferably VR1, VR4, or VR1 and VR4 of the V2R type pheromone receptor. Preferably, the above breast milk extract is a fraction containing a protein of less than 100 kDa and more preferably a fraction containing a protein between 10 kDa and 100 kDa. Preferably, the above composition is in a form appropriate for nasal administration, oral administration, or administration by spraying. Furthermore, preferably the above composition is in the form of spray, powder, liquid, gel, cream, foam, jelly, crystal, liposome, or suspension. Preferably the above composition is to be administered to an infant and more preferably a newborn. Furthermore, preferably the above composition is to be added to a food. In another aspect, the present invention provides a food containing the above composition, particularly synthetic milk. Furthermore, the present invention also provides: a use of a breast milk extract for producing the above composition; or a method for promoting the development of a mammalian nervous system, for promoting mammalian pheromone receptor expression, or for preventing or treating a disease in which the expression of a pheromone receptor is involved, which comprises administering a breast milk extract.
1. Definition
In this specification, the term “FABP fraction” means a fatty acid-binding protein (FABP) or a fragment thereof, a variant, a derivative, or an analogue, or a substance that binds to or is associated with FABP. The term “FABP fraction” is intended to also include one of these substances or a mixture of a plurality of these substances. For example, “FABP fraction” includes a functional derivative of FABP, a substance-bound FABP, and a complex of FABP and a substance.
The term “H-FABP fraction” means a heart-type fatty acid-binding protein (H-FABP) or a fragment thereof, a variant, a derivative, or an analogue, or a substance binding to or associated with H-FABP. Furthermore, the term “H-FABP fraction” is intended to also include one of these substances or a mixture of a plurality of these substances. For example, “H-FABP fraction” includes a functional derivative of H-FABP, a substance-bound H-FABP, and a complex of H-FABP and a substance.
The term “FABP” means a protein that is a fatty acid-binding protein (FABP), where such proteins are of a family of intracellular fat-binding proteins with a low molecular weight (14 to 15 kDa). Examples of FABP include a heart-type fatty acid-binding protein (H-FABP), a liver-type fatty acid-binding protein (L-FABP), an adipocyte-type fatty acid-binding protein (A-FABP), a myelin-type fatty acid-binding protein (M-FABP), an intestine-type fatty acid-binding protein (I-FABP), an epithelial fatty acid-binding protein (E-FABP), and a brain-type fatty acid-binding protein (B-FABP) (Veerkamp J H., Proc Nutr Soc. 1995 Mar., 54(1): 23-37; Banaszak L, Winter N, Xu Z, Bernlohr D A, Cowan S, Jones T A., Adv Protein Chem. 1994, 45, 89-151 (all contents are incorporated in this specification by reference in their entirety)). These proteins may be natural, synthetic, or semi-synthetic. Furthermore, when used in this specification, “FABP” indicates not only FABP, which is morphologically and constitutively completely identical to that existing in nature, but also FABP variants, derivatives, and analogues such as an FABP fragment; that is, a molecule of a portion of any FABP amino acid sequence, a protein having an amino acid sequence derived from the amino acid sequence of any FABP by deletion, substitution, and/or addition of one or a plurality of (preferably 1 to 5) amino acid residues, a protein consisting of a sequence that is at least 90% or more identical to any FABP amino acid sequence, and a protein obtained by modification of any FABP with compounds.
The term “H-FABP” means a heart-type fatty acid-binding protein (H-FABP) or its equivalent such as FABP3, a cardiac muscle-type fatty acid-binding protein, a skeletal muscle-type fatty acid-binding protein, or a mammary gland-derived growth inhibitor (MDGI) (Bohmer F D, Kraft R, Otto A, Wernstedt C, Hellman U, Kurtz A, Muller T, Rohde K, Etzold G, Lehmann W, et al., J Biol. Chem. 1987 Nov. 5, 262(31): 15137-43 (all contents are incorporated in this specification by reference in their entirety)). They may be natural, synthetic, or semi-synthetic.
The term “infant” means children of an age between immediately after birth and complete weaning. In the case of humans, generally the term means children of two and under. Furthermore, the term “newborn” means children under 1 month old.
The term “synthetic milk” means pseudo-breast milk other than breast milk itself. Examples of “synthetic milk” include milk-processing products for oral ingestion and milk preparations.
The term “mammal” means any animal classified as a mammal. Examples of “mammals” include humans, domestic animals, farm animals, and pet animals. Preferably a “mammal” is a human.
The term “subject” includes all persons who require treatment or prevention. Specifically, “subject” includes, in addition to persons who are currently having diseases, persons who may develop diseases in the future, persons who are diagnosed as being likely to develop diseases, and healthy persons.
“Effective amount” or “effective dose” for prevention or treatment means an amount necessary for obtaining a preventive or therapeutic effect, such as an amount necessary for promoting the development of a nervous system, particularly an amount necessary for promoting the expression of a pheromone receptor or an amount necessary for preventing or treating a disease in which the expression of a pheromone receptor is involved.
The term “disease in which the expression of a pheromone receptor is involved” includes all diseases that can be developed due to changes in the expression of pheromone receptors, such as disorders relating to emotion. The term “disorder relating to emotion” is interpreted in a wide sense and includes so-called behavioral disorders such as attention-deficit hyperactivity disorder (ADHD), attention deficit disorder (ADD), anxiety, panic disorder, depression, autism, dysgryphia, Tourette's syndrome, and the like.
2. Fatty Acid-Binding Protein (FABP)
Fatty acid-binding proteins are substances functioning as mediators for the transport of cytoplasmic hydrophobic compounds. The proteins are particularly involved in transport and metabolism of fat and are richly present in the cytoplasm. The proteins are known to bind to free fatty acids or CoA derivatives thereof. Among the members of this protein family, a liver type fatty acid-binding protein (L-FABP), an intestine-type fatty acid-binding protein (I-FABP), a heart-type fatty acid-binding protein (H-FABP), an adipocyte-type FABP (A-FABP), an epithelial fatty acid-binding protein (E-FABP), and the like are known.
H-FABP will be explained as follows, but the present invention is not limited thereto. For example, other FABPs can also be produced by a method similar to that employed for H-FABP.
3. Heart-Type Fatty Acid-Binding Protein (H-FABP)
H-FABP is a protein with a molecular weight of approximately 15 kDa distributed in a broad range of tissues such as heart, skeletal muscle, stomach, testis, kidney, and mammary gland. The protein is known to be involved in intracellular transport of polyunsaturated fatty acids (linoleic acid, arachidonic acid, and linolenic acid).
H-FABP has been purified from heart tissue (van Nieuwenhoven F A, Vork M M, Surtel D A, Kleine A H, van der Vusse G J, Glatz J F., J Chromatogr., 1991 Sep. 18, 570(1), 173-9), and can be purified from breast milk by a method known by persons skilled in the art (Grosse R, Boehmer F D, Langen P, Kurtz A, Lehmann W, Mieth M, Wallukat G., Methods Enzymol., 1991, 198, 425-40; Vitaly L. Spitsberg and R. C. Gorewit, Pakistan Journal of Nutrition 1(1): 43-48, 2002 (all contents are incorporated in this specification by reference in their entirety)). For example, H-FABP can be obtained by fractionation by centrifugation or column chromatography based on molecular size, or by affinity purification using antibodies.
Furthermore, H-FABP can be produced in large quantities by causing Escherichia coli, yeast, or the like to express H-FABP by gene recombination techniques, and then purifying the product (Peeters R A, Veerkamp J H, Geurts van Kessel A, Kanda T, Ono T., Biochem J. 1991 May 15, 276 (Pt 1), 203-7; Peeters R A, Ena J M, Veerkamp J H., Biochem J. 1991 Sep. 1, 278 (Pt 2), 361-4; Schaap F G, Specht B, van der Vusse G J, Borchers T, Glatz J F., J Chromatogr B Biomed Appl. 1996 Apr. 26, 679(1-2), 61-7 (all contents are incorporated in this specification by reference in their entirety)). These methods are already known and can be easily implemented by persons skilled in the art. The nucleotide sequence and the amino acid sequence encoding H-FABP can be easily obtained from, for example, gene sequence databases such as GENBANK. For example, in the DBGET database, bovine H-FABP has been registered under registration number [P10790], human H-FABP under registration number [P05413], mouse H-FABP under registration number [P11404], swine H-FABP under registration number [002772], and rat H-FABP under registration number [P07483]. Furthermore, a nucleotide sequence encoding H-FABP of an animal species other than those of the above animals can be obtained by a known method such as cloning and sequencing from genomic DNA or cDNA samples of a target animal species using the above known sequences as probes.
4. Composition
A composition containing the thus obtained FABP fraction can be used as a composition for promoting the development of a mammalian nervous system, for promoting mammalian pheromone receptor expression, or for preventing or treating a disease in which the expression of a pheromone receptor is involved.
5. Use as Food Additive
The composition containing the FABP fraction according to the present invention can be added as a food additive to a food, such as synthetic milk.
It is particularly preferred that the composition or the like according to the present invention be added to synthetic milk, particularly powdered milk, that the resultant be prepared in a manner similar to general preparation of synthetic milk or powdered milk, and that the preparation then be provided. Since H-FABP is a soluble protein, H-FABP is advantageous in that it can be easily mixed into synthetic milk.
Dosage varies depending on target animal species, body weight, and intake. Preferably, the composition in an amount approximately equivalent to that contained in breast milk is mixed. In general, dosage is approximately between 0.1% and 10% and preferably approximately between 0.5% and 2% (W/W) in a food, where such food is preferably synthetic milk, and particularly powdered milk.
The composition according to the present invention is in a form that can be added to a food, such as freeze-dried powder or liquid. They can be added to foods. Moreover, a food to which the composition is added, particularly synthetic milk, is also an object of the present invention. For example, this can be provided in the form of H-FABP-containing powdered milk wherein the composition is in the form of freeze-dried powder has been previously mixed with powdered milk.
Particularly when used in synthetic milk, H-FABP is known to bind to essential fatty acids including linoleic acid, arachidonic acid, and α-linolenic acid. In particular, α-linolenic acid is a raw material for biosynthesis of docosahexaenoic acid (DHA) and is a substance recognized to have usefulness. Since the solubility of fatty acid is low in an aqueous solution, the amount of fatty acid that can be ingested in a processed milk product for oral ingestion is limited. There is another advantage in that by mixing H-FABP, the water-soluble protein, with an essential fatty acid, H-FABP functions as a transport carrier for the essential fatty acid, so as to increase the solubility of the essential fatty acid into an aqueous solution.
6. Use as Pharmaceutical Composition
Furthermore, the thus obtained H-FABP or a fraction or a composition containing H-FABP can also be provided as a pharmaceutical composition. Preferably, the composition contains a pharmaceutically or physiologically acceptable carrier. Moreover, the composition according to the present invention can be used in combination with one or a plurality of other active ingredients, such as a combination with other substances acting on the nervous system or the like.
“Carrier” includes a pharmaceutically acceptable carrier, excipient, or stabilizer and is non-toxic to a subject or a mammal within the range of the amount or concentration thereof to be used. Examples of such carrier include: an organic acid buffer such as phosphate or citrate; an antioxidant such as ascorbic acid; a polypeptide; a protein such as serum albumin, gelatine, or immunoglobulin; a hydrophilic polymer such as polyvinylpyrrolidone; an amino acid such as glycine, glutamine, asparagine, arginine, or lysine; a carbohydrate such as a monosaccharide, e.g., glucose, arabinose, or mannose, a disaccharide, e.g., lactose, saccharose, or maltose, or a polysaccharide, e.g., dextran; a chelating agent such as EDTA; sugar alcohol such as xylitol, mannitol, or sorbitol; salts such as sodium chloride or calcium carbonate; or a nonionic surfactant such as polyethylene glycol, TWEEN (registered trademark), or PLURONIC (registered trademark). The aforementioned may also be used in combination.
The route of administration of the composition according to the present invention may be any method as long as a desired effect can be obtained therewith. The composition can be administered by oral administration, nasal administration, transmucosal administration, or administered by inhalation, such as by spraying. In particular, oral administration, nasal administration, and administration by inhalation are preferable.
The composition according to the present invention can be in a dosage form appropriate for the above routes of administration, such as spray, powder, liquid, gel, cream, foam, jelly, crystal, liposome, or another micelle, a solution, or a suspension. The composition can be administered via liquid agents, dispersing agents, emulsions, suspending agents, bio-adhesive gel or non-bio-adhesive gel, powders, microspheres, lozenge, tablets, chewing gum agents, tablets, capsules, dragee, sustained release preparations, or the like. Administration can take place in combination.
In particular, in the case of nasal administration or administration by inhalation, the composition can be administered via liquid formulations, semi-solid formulations, or dry formulations such as powder formulations using spray, aerosol, an aeration apparatus, an inhalator, or the like. Moreover, the composition can be administered in the form of liquid or a suspending agent sprayed from a spray vessel or in the form of aerosol spray sprayed from a pressurization vessel or the like using an appropriate nebula or the like.
When the composition is administered using aerosol, it is preferable that the formulation be in the form of small particles, such as small particles of 5 microns or less. Such particles can be obtained by a method known in the art. Active ingredients can be administered using an appropriate propellant (for example, a chlorofluorocarbon such as dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, or a gas such as carbon dioxide). A surfactant such as lectin may be contained in an aerosol agent. In the case of dry powder, for example, the composition can be administered as a powdered mixture of the composition and a dry base (for example, a starch derivative such as lactose, starch, hydroxypropyl methylcellulose, or polyvinylpyrrolidone (PVP)). These dosage forms can be prepared using techniques known in the art.
The dose of the composition according to the present invention may be any dose, as long as a desired effect can be obtained therewith, such as in the case of an effective amount for treatment or prevention.
Furthermore, H-FABP is a soluble protein and can be conveniently stored by frozen storage, freeze-dry storage, or the like. Hence, the composition is produced in the form of powder, the composition in the form of powder is suspended or dissolved in a water-soluble carrier or in water before administration, and then the resultant can be administered. In addition, the composition may also be in a form that enables preparation and storage in the form of small separated portions for single doses.
7. Method and Use According to the Present Invention
The above and following definitions, explanations, and the like relate mainly to the composition according to the present invention. In particular, they relate to a composition for promoting the development of a mammalian nervous system, a composition for promoting mammalian pheromone receptor expression, and a composition for preventing or treating a disease in which the expression of a pheromone receptor is involved. The definitions, explanations, and the like are also similarly applied directly to: the method according to the present invention, particularly the method for promoting the development of a mammalian nervous system, the method for promoting mammalian pheromone receptor expression, and the method for preventing or treating a disease in which the expression of a pheromone receptor is involved; and to a use according to the present invention, particularly a use for producing a composition for promoting the development of a nervous system, a use for producing a composition for promoting the expression of a pheromone receptor, a use for producing a composition for preventing or treating a disease in which the expression of a pheromone receptor is involved, and the like.
Hereinafter, examples of H-FABP, examples of VR1 and VR4 pheromone receptors existing in mouse vomeronasal organs (VNOs), and examples of administration to infants or newborns will be described below. However, the present invention is not limited thereto. In addition, other documents are cited at need as follows, but all contents thereof are incorporated in this specification by reference in their entirety.
Mammalian Vomeronasal Organ
The mammalian vomeronasal organ (VNO) is located at the base of the nasal septum and is connected to nasal cavity via a narrow duct. Sensory neurons in the VNO project their axons into the accessory olfactory bulb (AOB). It has been reported in mice that the presence of lesions or genetic inactivation factors in this VNO to AOB pathway may result in abnormal sex discrimination and mating behaviors (E. B. Keverne, Trends in Neurosciences 6, 381 (1983); L. Stowers, T. E. Holy, M. Meister, C. Dulac, G. Koentges, Science 295, 1493 (2002); B. G. Leypold et al., Proc Natl Acad Sci USA 99, 6376 (2002); K. Del Punta et al., Nature 419, 70 (2002)). Consequently, the VNO and the AOB are thought to be essential regions for the detection of pheromones that affect social communication, reproductive behaviors, and endocrine responses. Previous studies also indicated that a dam and her pups communicated with each other through pheromonal signals. It is now generally believed that there is a positive association between dam-pup bonding and breast-feeding (R. H. Porter, J. Winberg, Neurosci Biobehav Rev 23, 439 (1999); B. Schaal et al., Nature 424, 68 (2003)).
Recent studies have reported putative pheromone receptors cloned from the VNO(C. Dulac, R. Axel, Cell 83, 195 (1995); G. Herrada, C. Dulac, Cell 90, 763 (1997); H. Matsunami, L. B. Buck, Cell 90, 775 (1997); N. J. Ryba, R. Tirindelli, Neuron 19, 371 (1997)). These pheromone receptors are members of the G-protein-coupled family of receptors and have been classified into two large groups (R. Tirindelli, C. Mucignat-Caretta, N. J. Ryba, Trends Neurosci 21, 482 (1998); E. B. Keverne, Science 286, 716 (1999); C. Dulac, A. T. Torello, Nat Rev Neurosci 4, 551 (2003)). One type, the VIR type receptors, have short N-terminal extracellular domains. The other type, the V2R type receptors, have characteristically very long N-terminal extracellular domains. While VIRs respond to volatile pheromone molecules, V2Rs respond to nonvolatile pheromone molecules such as lipocalins (R. Tirindelli, C. Mucignat-Caretta, N. J. Ryba, Trends Neurosci 21, 482 (1998); E. B. Keverne, Science 286, 716 (1999); C. Dulac, A. T. Torello, Nat Rev Neurosci 4, 551 (2003)). These pheromone receptors are thought to be involved in pheromonal communication between a dam and her pups.
Mammal and Breast Milk
Mammals need breast feeding by their dams for a certain period after birth. However, in modern society, many non-human mammals are born in an artificial raising environment such as a zoo or a facility for the livestock industry. These mammals are often unable to be breast-fed by their dams due to their environment or the conditions of their dams.
Furthermore, even in the case of humans, many infants are completely unable to be breast-fed, or are insufficiently breast-fed by their mothers due to a variety of reasons. It is assumed that recently the number of such infants is on the increase. Such infants are often raised by hand-feeding with nutrients; that is, synthetic milk. Such synthetic milk contains nutrients that are thought to be necessary for the healthy growth of infants, such as micronutrients, e.g., various minerals, lactose as an energy source, and proteins.
However, recent studies have revealed that breast milk contains various substances to promote infants' biophylaxis, such as an immunoglobulin (IgA) for preventing bacteria or viruses from invading and infecting bodies, bifidus factors for growing intestinal bacteria, lactoferrin, lysozyme, and leucocytes having bactericidal action, and polyamine thought to suppress the onset of allergies. As shown by such findings, breast milk contains not only general nutrients, but also various functional substances. However, there may be many other unidentified breast milk ingredients with unknown functions. Addition of breast-milk-specific functional ingredients to synthetic milk and administration of such synthetic milk to infants are thought to allow the promotion of the growth of infants who are hand-fed with synthetic milk, without impact from insufficient breast feeding or from lack of ability to breast feed.
Composition and the Like According to the Present Invention
Through the administration of the composition according to the present invention, even in the case of infants who are not breast-fed, induction of the expression of pheromone receptors (particularly, the V2R type receptor) in vomeronasal organs is observed with distribution and levels similar to those observed in breast-fed newborns. Administration of the composition enables even infants who are not breast-fed to carry out appropriate pheromonal reception, so as to be able to promote normal growth.
Reception of pheromonal stimuli is thought to promote induction of the development of a nervous system. Hence, the composition and the like of the present invention can be used as an inducer for pheromone receptor expression, a promoter for the development of a nervous system, or a promoter for the formation of a bond between a mother and her child. Here, “formation of a bond between a mother and her child” means the formation of a psychological bond between a mother and her child, which is induced by neural stimuli. Insufficient formation of this bond may cause symptoms such as a child becoming unable to discriminate his or her mother from other individuals or a child becoming unable to feel love for his or her mother. Furthermore, when such an infant who has performed insufficient bond formation grows, abnormalities may be found in his or her sex-discrimination ability, reproductive behavior, or the like. Furthermore, when such an infant grows and has her child, she may be unable to form a sufficient mother-child bond with her child. Accordingly, if pheromone receptor expression is too late in terms of timing or is insufficient in terms of amount, such a disorder will be passed onto the next generation.
Such pheromonal stimuli are thought to also be involved in emotional development. According to the composition and the like of the present invention, a disease in which a pheromone receptor is involved, particularly, a disease relating to emotion, can be treated or prevented by promoting the development of a pheromone receptor and promoting the following emotional development.
Furthermore, non-human mammals are often kept in artificial environments as pet animals, animals in a zoo, or the like. In such cases, mammals may be unable to be breast-fed by dams. In this case, it is preferable to administer the composition of the present invention in addition to provision of synthetic milk.
Through the administration of H-FABP, pheromone receptor expression is induced similarly to the case of breast feeding, the following development of a nervous system is caused similarly to that in breast-fed animals, and emotional development and mother-child bond formation are promoted, so that the above disorders can be prevented or reduced.
Effect of the Invention
According to the composition, the method, or the use of the present invention, the development of a mammalian nervous system can be promoted. Furthermore, according to the composition, the method, or the use of the present invention, a disease in which the expression of a pheromone receptor is involved can be prevented or treated.
Materials and Methods
Preparation of Antibodies Against VR1, VR4, and H-FABP
Peptides of the COOH-termini of VR1 (NH2-CPDSNFIKNHKGKLLY-COOH: SEQ ID NO: 1) and VR4 (NH2-CPERNSTQKIREKSYF-COOH: SEQ ID NO: 2) of V2R type pheromone receptors of a mouse vomeronasal organ (VNO) and H-FABP (NH2-CGSVVSTRTYEKEA-COOH: SEQ ID NO: 3) were synthesized. The peptides were then conjugated to keyhole limpet hemocyanin (KLH) according to standard methods. Rabbits were immunized with them as antigens. Specific antibodies against VR1, VR4, and H-FABP were affinity-purified from the anti-sera using affinity columns (Sulfolink column: PIERCE).
Construction of Adenovirus Vectors
All of the entire coding sequences of the mouse pheromone receptors, VR1 and VR4, were amplified by the PCR method from the VR1 and VR4 cloned vectors. As VR1 primers, a forward primer: 5′-CCGGATCCTCGCCACCATGAAGCAGCTCTGCGC-3′ (SEQ ID NO: 4) and a reverse primer: 5′-GCCTCGAGTCAATAAAGCAATTTACCTTTGTGG-3′ (SEQ ID NO: 5) were used. As VR4 primers, a forward primer: 5′-CCCTCGAGCGCCACCATGTTCATTTTCATGGGAGTC-3′ (SEQ ID NO: 6) and a reverse primer: 5′-GCTCTAGATCAAAAATATGATTTTTCTCTGATCTTTTGGG-3′ (SEQ ID NO: 7) were used. The amplified products were cloned into pBluescript II(KS−)(Stratagene). The sequences were confirmed by sequencing. The BamH I-Xho I fragment of VR1 cDNA and the Xho I-Xba I fragment of VR4 cDNA were then inserted into adenovirus expression vectors. The vectors were then introduced into HeLaS3 cells using Lipofectamine 2000 (Invitrogen).
Nursing Newborn Pups by Hand-Feeding
ICR (Slc: ICR; NipponSLC, Japan) and H-FABP-deficient mice (B. Binas, H. Danneberg, J. McWhir, L. Mullins, A. J. Clark, FASEB J 13, 805 (1999)) were housed in a controlled environment under a regulated 12-hour light:dark cycle. The day upon which a vaginal plug was detected was considered to be day E0.5 (0.5-day old embryo). Day E19.5 was designated as postnatal day 0 (P0). Mouse breast milk was collected from ICR and H-FABP-deficient dams breast-feeding pups at P14 (P14 breast milk). The milk was subjected to molecular weight fractionation by ultracentrifugation using Ultrafree 0.5 [100 kDa nominal molecular weight limits (NMWL): Millipore], thereby obtaining 100 kDa or more breast milk fractions and 100 kDa or less breast milk fractions (fraction 1). Fraction 1 was separated by Ultrafree 0.5 (10 kDa NMWL: Millipore), thereby obtaining fraction 2 and fraction 3. Each fraction was stored at −80° C.
ICR mouse newborn pups were delivered by Caesasrian section on day E18.5, immediately isolated from dams, and placed on pure cotton. Newborn pups were maintained at 37° C. and swabbed until they began breathing. Immediately after confirmation of their ability to breathe through their noses, the newborn pups were hand-fed with P14 breast milk obtained from ICR dams, 20% (w/v) skim milk (SM: Wako), 20% (w/v) SM supplemented with breast milk fraction 1 at a 1:10 ratio, or P14 breast milk obtained from H-FABP-deficient dams. Specifically, drops of each nutrient were added to the pups' mouths, and it was visually confirmed that each pup had approximately 50 to 100 μl of the nutrient introduced into its stomach. The pups were kept under control until the next day (P0). The hand-feeding with each nutrient was performed on a litter of pups.
SDS-PAGE and Western Blotting
Tissues of adult ICR mice and HeLaS3 cells transfected with VR1 or VR4 expression adenovirus vectors were homogenized in 20 mM Tris-HCl buffer (pH 7.5) containing 250 mM sucrose, 10 mM EDTA (pH 8.0), 1 mM β-mercaptoethanol, and 0.2 mM phenylmethylsulphonyl fluoride on ice. The protein concentration of each homogenate sample was measured using a protein assay kit (Bio-Rad). Each homogenate (10 μg protein/lane) was subjected to 10% SDS-PAGE. The filtrate of P14 breast milk (breast milk of dams having P14 pups) or the biotinylated filtrate of P14 breast milk was subjected to 16.5% tricine-SDS-PAGE. Detection was carried out by Coomassie staining.
Gel was transferred to PVDF membranes (Immobilon: Millipore). Membranes were blocked overnight with PBS(−) containing 1% skim milk (Becton Dickinson) and 0.1% Tween 20 (Nacalai Tesque, Inc.) at 4° C. The membranes were caused to react with antibodies against VR1 (1 μg/ml) or VR4 (0.5 μg/ml) for 2 hours. Following several washes, immunoreactivity was detected using secondary antibodies (ECL kit: Amersham).
Immunostaining
Two days after the transfection with the VR1 or VR4 adenovirus expression vector, HeLaS3 cells were fixed in 4% (w/v) paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4), and subsequently washed with PBS(−). Day E18.5 embryos and P0 newborn pups were anesthetized and fixed by intracardiac perfusion with 4% PFA. The VNOs of 5-week-old ICR mice were also excised and then the VNOs were fixed overnight at 4° C. Samples were isolated as described in literature (M. Hashimoto et al., Hum Gene Ther 7, 149 (1996)), and then transverse-sectioned (10 μm thickness at 100 μm intervals) using a cryostat.
HeLaS3 cells and sections were washed with PBS(−), subjected to blocking with PBS(−) containing 1% skim milk and 0.1% Triton X-100 (Sigma) for 1 hour, and then caused to react with VR1 (2 μg/ml) or VR4 (1 μg/ml) antibodies. The neighboring sections were caused to react with anti-Gα0 IgG (1:200; MBL). Positive immunoreactivity was detected with a rhodamine-labeled anti-rabbit IgG (1:200; CAPPEL).
To examine the development of pheromone receptors, immunostaining was carried out using antibodies that specifically recognize VR1 and VR4 of V2R type pheromone receptors, respectively. Specific antibodies against the C-terminal intracellular domains of VR1 and VR4 were affinity-purified from the immunized rabbit antisera and analyzed by immunological analyses (data not shown), Western blotting, and immunohistochemical analysis.
Homogenate samples of HeLaS3 cells that had been transfected with vectors expressing VR1 and VR4 were subjected to Western blotting (
To determine the in vivo distribution of V2R type pheromone receptors, Western analysis was carried out on various tissue homogenates derived from adult ICR mice (
The bands corresponding to a putative receptor dimer were the most strongly detected. These results indicate that V2R type pheromone receptors (VR1 and VR4) exist in multimeric conformations. Furthermore, sections of adult mouse VNO were immunostained with anti-VR1 (
Induction of Expression of VR1 and VR4 after Birth
Using these specific antibodies, the distribution of VR1 and VR4 pheromone receptors within the VNOs of embryos and newborn pups was examined. VR1 and VR4 were undetectable in embryonic VNOs at embryonic day (E) 15.5, E17.5 (data not shown), and E18.5 (
Induction of VR1 and VR4 Expression by Breast Milk
The identity of the factor that rapidly induces upregulation of VR1 and VR4 expression remains unknown. However, any of the following 6 factors may serve as stimuli inducing the upregulation of VR1 and VR4 expression: parturition, first breath, dam's licking stimulation, dam's saliva, dam's odors, and breast-feeding. To examine this question, ICR mouse pups were delivered by Caesarean section on day E18.5 and then put on pure cotton, kept at 37° C., and stimulated to breathe by swabbing in a manner similar to a dam's licking. After first breath, the newborn pups were nursed with either skim milk or mouse breast milk (P14 breast milk) obtained from a dam nursing P14 pups, while being isolated from dams.
On the next day (postnatal day 0 (P0)), each newborn pup was fixed by intracardiac perfusion with 4% paraformaldehyde and sectioned transversely on a cryostat. Sections were stained with anti-VR1 and anti-VR4 antibodies. In newborn pups nursed with skim milk, anti-VR1 and anti-VR4 immunoreactivity (
Inducing Factor in Breast Milk: H-FABP
To identify the factor in breast milk that induces VR1 and VR4 expression, P14 breast milk was fractionated by ultracentrifugation using a molecular weight fractionation method into two fractions, a fraction (fraction 1) consisting of proteins of less than 100 kDa and a fraction consisting of proteins of 100 kDa or more. Fraction. 1 was liquid containing lactose, water, ions, water-soluble molecules, and whey proteins, and the other fraction was in the form of cream. Next, newborn pups derived from litters delivered by Caesarean section were divided into 4 groups: a group of pups that was breast-fed by dams, a group of pups that was hand-fed with P14 breast milk, a group of pups that was hand-fed with skim milk, and a group of pups that was hand-fed with skim milk supplemented with fraction 1 (SM+Fr.1). While VR1 and VR4 expression was induced in the group nursed with skim milk supplemented with fraction 1, VR1 and VR4 expression was not induced in the group nursed with only skim milk (“SM” in
To examine components of the proteins fractionated in fraction 1, fraction 1 was separated by SDS-PAGE (lane 1 in
When pups were hand-fed with P14 breast milk derived from H-FABP-deficient dams, the number of VR1- and VR4-positive VNO neurons was significantly reduced (VR1, P<0.01; VR4, P<0.005) (“breast milk (H-FABP−/−)” in
The present invention can be implemented in other various forms without departing from the spirit or principles of the present invention. Hence, the above examples are given merely for illustrative purposes in every respect, and should not be construed in a limited way. The scope of the present invention is defined by the scope of the claims, and is never restrained by description given in the specification. Furthermore, all modifications and variations within the scope of equivalents of the scope of the claims are within the scope of the present invention.
| Number | Date | Country | |
|---|---|---|---|
| 60555336 | Mar 2004 | US |
