JP2005119986A - Method for administering physiologically active substance, physiologically active substance-containing emulsion, and method for producing physiologically active substance-containing emulsion - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an excellent oral drug delivery technique utilizing an oil particle-absorbing action in a constant animal digestive canal. <P>SOLUTION: This method for administering the physiologically active substance is characterized by preparing an oil-in-water type emulsion whose oil drops are fine oil particles containing the physiologically active substance and having an average particle diameters targeted for an oil sphere-absorbing action in the digestive canals of animals, and then orally administering the emulsion to a non-human animal whose oil particle-absorbing action has been recognized. In the fine oil particles in the emulsion, the physiologically active substance is buried in the oil phase by the action of an oil-soluble emulsifier as such or while forming an extremely fine dispersion phase together with a small amount of water. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a technique for oral drug delivery to animals utilizing the oil-ball absorbing action which has been known relatively recently by the report of the present inventor. More specifically, the present invention relates to a method for administering a physiologically active substance that effectively utilizes the action of oil ball absorption in the digestive tract of certain animals, and a newly developed physiological activity that is particularly well suited to such a method of administration. The present invention relates to a substance-containing emulsion and a method for producing this physiologically active substance-containing emulsion.

  For a long time, various methods for administering vaccines, immunostimulatory substances, and the like have been proposed to prevent specific diseases of mammals such as humans, rabbits, mice, and domestic animals. In recent years, many attempts have been made to administer vaccines, substances exhibiting a certain pharmacological action, and various other physiologically active substances to cultured aquatic animals and the like to prevent or treat the disease.

  Methods such as vascular injection (intravenous injection) traditionally performed on humans and other mammals are drug delivery methods that can theoretically be expected to have excellent effects. There is a drawback of applying surgically invasive stress, and furthermore, this method is not easily applied to fish, shellfish, shellfish, and the like.

  Therefore, oral administration has become the mainstream as a method for administering physiologically active substances to fish, shellfish, shellfish and the like. And in designing the oral administration agent, one or two or more types of physiologically active substances to be administered and various auxiliary agents used in combination with each have different affinity (hydrophilicity or lipophilicity). Considering the points, etc., various forms of emulsion preparations are often devised in consideration of the stability of orally administered agents.

In JP-A-11-255664, peptidoglycan having a molecular weight of 10,000 or less is used as an active ingredient of an immunopotentiator for oral administration, and it is added as it is to a feed, to mammals and fish. A technique for administration to crustaceans (for example, black tiger, a kind of shrimp) is disclosed.

In JP-A-8-504811, the above-mentioned patent document 2 describes a particle that is made into a physiologically acceptable emulsion for drug delivery and emulsified in water of fluorochemical (for example, perfluorocarbon such as perfluorodecalin) and oil. , An emulsion comprising a surfactant and a drug solubilized in the emulsion is disclosed.

  The invention according to this Patent Document 2 consists of ambiguous and esoteric text expressions, but in short, a liquid fatty oil such as triglyceride 30% by weight or less as a matrix, and 75% by weight or less of a fluorochemical liquid, An emulsion system in which 30% by weight or less of a drug solution is dispersed by the action of a surfactant is described.

JP-A-7-53404 discloses the invention of an oil-type and water-type adjuvant mixture in which an antigenic substance and one adjuvant are oil-type and the other adjuvant is water-type. More specifically, the emulsion is a water-in-oil emulsion, and the dispersoid (eg, 30% by weight of water droplets) is made antigenic by adding an emulsifier to the matrix dispersion medium (eg, 70% by weight of oil). Substances and / or adjuvants are included.

  However, when a physiologically active substance is orally administered, a general problem that the effect of the administration is not sufficiently expressed has been recognized for some time. For example, it is pointed out that a part of the above-mentioned Patent Document 1 is difficult to produce a sufficient effect by oral administration of a physiologically active substance to fish. The reason for this is not always fully understood, but perhaps unlike vascular injection, oral administration results in absorption through the gastrointestinal tract, so that bioactive substances are digested before absorption or sufficient in the gastrointestinal tract. It seems to be related to the point that it is not absorbed.

  By the way, in general, in the digestive tract of animals, it is a conventional common sense that oil (fat) is absorbed at the molecular level after being decomposed into fatty acid and glycerin. However, the inventor of the present application has found a surprising oil ball absorption action that a certain minute size of oil balls is taken directly into the body from the intestinal epithelium in the digestive tract of fish and the like. This finding is reported in the following Non-Patent Document 1 and the like. Since then, new additional findings have begun to be obtained that a similar oil-ball absorbing action is observed in some mammals.

“Histological study on egg yolk absorption and nutrient intake in digestive tract of red sea bream and black sea bream larvae” Teruo Miyazaki et al. Bulletin of Faculty of Bioresources, Mie University, pp. 15-27 (1988) If skillful use of the sphere absorption action, it is not possible to construct an oral drug delivery method that allows animals to ingest physiologically active substances such as vaccines, immunostimulatory substances and pharmacologically active substances with a very high efficiency. The present invention has been completed.

  In such an idea, (a) the application target is limited to animals that have been confirmed to absorb oil balls in the gastrointestinal tract, and (b) micro oil balls orally administered to the target animals are oil balls. There are two absolute conditions: having a certain average particle diameter to be the object of absorption. In the conventional techniques such as Patent Document 1 to Patent Document 3 described above, these two conditions are not disclosed at all and are not taken into consideration. Further, the above-mentioned Non-Patent Document 1 relating to the report of the present inventor reports the oil ball absorption action, but does not mention the possibility that this action can be used for oral drug delivery means. The specific implementation method is not considered at all.

(Configuration of the first invention)
The configuration of the first invention of the present application for solving the above-mentioned problem is an oil-in-water type emulsion, and the oil droplet contains a physiologically active substance and is an average subject to oil ball absorption action in the digestive tract of animals. This is a method for administering a physiologically active substance, in which a fine oil sphere having a particle size is prepared and this emulsion is orally administered to a non-human animal in which an action of absorbing oil globules in the digestive tract is recognized.

(Configuration of the second invention)
The configuration of the second invention of the present application for solving the above problem is a method for administering a physiologically active substance, in which the micro oil spheres according to the first invention have an average particle diameter in the range of 1 to 60 μm.

(Configuration of the third invention)
The configuration of the third invention of the present application for solving the above-described problem is that the micro oil sphere according to the first invention or the second invention has a particle structure of any one of (1) to (3) below. This is a method for administering an active substance.
(1) In the fine oil sphere, the bioactive substance forms an extremely fine dispersed phase as it is or with a small amount of water by the action of the oil-soluble emulsifier.
(2) A lipophilic physiologically active substance is dissolved or suspended in the fine oil sphere.
(3) A micro oil sphere has a micelle structure formed by a lipid bilayer, and a physiologically active substance is enclosed therein.

  In the above (1), “by the action of an oil-soluble emulsifier” means that an extremely minute dispersed phase of a physiologically active substance (or a physiologically active substance and a small amount of water) is contained in the inside of a fine oil sphere. The action of the oil-soluble emulsifier means that the oil-soluble emulsifier is stably dispersed in the oil phase as the dispersoid of the fine oil spheres while the oil-soluble emulsifier is held at the interface with the dispersoid in each dispersed phase.

(Configuration of the fourth invention)
The structure of the fourth invention of the present application for solving the above-described problem is that the animal feeds a feed or feed pellet impregnated with the emulsion according to any of the first to third inventions. It is a method of administration of a substance.

(Structure of the fifth invention)
The configuration of the fifth invention of the present application for solving the above-mentioned problem is a method for administering a physiologically active substance, wherein the animal according to any one of the first to fourth inventions is an aquatic animal or a non-human terrestrial mammal. .

(Structure of the sixth invention)
The structure of the sixth invention of the present application for solving the above problem is that the physiologically active substance according to any one of the first to fifth inventions is a vaccine, an immunostimulatory substance, a nutritional substance, a pharmacologically active substance, a natural pigment or a mineral. This is a method for administering a physiologically active substance.

(Structure of the seventh invention)
The structure of the seventh invention of the present application for solving the above-mentioned problem is an oil-in-water emulsion in which fine oil spheres having an average particle size to be subjected to oil sphere absorption action in an animal digestive tract are dispersed, In the micro oil sphere, the bioactive substance-containing emulsification is embedded in the oil phase as it is or by forming an extremely fine dispersed phase with a small amount of water by the action of the oil-soluble emulsifier. It is a liquid. Here, “by the action of the oil-soluble emulsifier” has the same meaning as described in the “Structure of the third invention” column.

(Configuration of the eighth invention)
The configuration of the eighth invention of the present application for solving the above problem is a bioactive substance-containing emulsion in which the fine oil spheres according to the seventh invention have an average particle diameter in the range of 1 to 60 μm.

(Structure of the ninth invention)
The structure of the ninth invention of the present application for solving the above problems is that the physiologically active substance according to the seventh or eighth invention is a vaccine, an immunostimulatory substance, a nutritional substance, a pharmacologically active substance, a natural pigment or a mineral, It is a bioactive substance-containing emulsion.

(Configuration of the tenth invention)
The configuration of the tenth invention of the present application for solving the above problems is a method for producing a physiologically active substance-containing emulsion comprising the following (A) first emulsification step and (B) second emulsification step.
(A) 1st emulsification process: By mixing and emulsifying the bioactive substance or its aqueous solution or water suspension and the oil liquid to which the oil-soluble emulsifier is added, the bioactive substance remains as it is or with a small amount of water. An oily emulsified phase is produced which forms a very fine dispersed phase and is embedded in the oil phase.
(B) Second emulsification step: The oily emulsified phase produced in the first emulsification step and water added with a water-soluble emulsifier are mixed and emulsified, whereby the oily emulsified phase becomes an oil ball in the digestive tract of animals. An oil-in-water emulsion that is dispersed in water as fine oil spheres having an average particle size to be absorbed is produced.

  Here, in the oil phase in the above (A) and in the micro oil sphere in the above (B), an extremely minute dispersed phase of the physiologically active substance (or the physiologically active substance and a small amount of water) is present. In each of the dispersed phases, the oil-soluble emulsifier is held at the interface with the dispersoid, and the oil is stably dispersed in the oil phase or the fine oil spheres as the dispersoid. Further, in the water in the above (B), such fine oil spheres are stable in the water as the dispersoid while holding the water-soluble emulsifier at the interface with the dispersoids (water) in the individual fine oil spheres. Distributed.

(Effect of the first invention)
Since the administration method of the physiologically active substance of the first invention is an oral administration method, it does not give a surgical invasive stress like a vascular injection method to a non-human animal to be administered, for example, fish, shellfish, shellfish Etc. are also easy to apply.

  Next, as the most important feature of the first invention, this method is an oral administration method that effectively utilizes the oil-ball absorbing action in the digestive tract of animals. That is, in the first invention, (b) the application target is limited to animals that have been confirmed to absorb oil balls in the digestive tract, and (b) micro oil balls that are orally administered to the target animals Micro oil spheres satisfying the two conditions of having a certain average particle size to be a target of sphere absorption, and the physiologically active substance as a drug delivery target substance being oil droplets of an oil-in-water emulsion Included in.

  For this reason, in the digestive tract of an orally administered subject animal, the entire micro oil sphere containing a physiologically active substance is directly taken into the body from the intestinal epithelium. As a result, it is possible to avoid the disadvantage that the physiologically active substance is digested before absorption or is not sufficiently absorbed in the digestive tract, which is seen in the conventional oral administration method. Can be taken into the animal body with extremely high efficiency.

(Effect of the second invention)
Since the “average particle size that is the target of oil ball absorption in the digestive tract of animals” in the first invention is actually different depending on the animal species, it is difficult to uniformly define the numerical values. However, it is generally preferable to have an average particle size in the range of 1 to 60 μm if it is defined with numerical values.

(Effect of the third invention)
There are no limitations on the type or particle structure of the “oil ball” that is the target of the oil ball absorption action. For example, those listed in (1) to (3) of the third invention are all stable as an oil ball dispersion. Is representatively exemplified.

  However, when these particle structures (1) to (3) are relatively evaluated, (3) is a vesicle-like particle structure generally called “liposome”, but it is an animal intestinal lipase. When the boundary membrane (lipid bilayer) is collapsed by the above action, the oil ball structure cannot be maintained, and the oil ball absorption function cannot be used. Since the particle structure of (2) is essentially an oil ball, the oil sphere structure is not easily collapsed by the action of lipase, except when an oil-soluble physiologically active substance is dissolved in the oil sphere, In general, it is difficult to finely disperse a physiologically active substance in an oil sphere, and as a result, it is difficult to prepare an oil sphere having a predetermined minute average particle diameter.

  Compared with these, the particle structure of (1) is a particle structure created by the present inventor for the efficient implementation of the first invention, and the details thereof will be described in the seventh invention. The effect of this is that the structure of the oil sphere does not collapse, and there is an advantage that it can be dispersed very finely in the micro oil sphere without considering the affinity of the physiologically active substance.

(Effect of the fourth invention)
The method for oral administration of the emulsion according to the first to third inventions is not limited, and for example, a method of injecting into the oral cavity of fish or the like as the target animal with a syringe or the like is possible, but as in the fourth invention, emulsification Particularly preferred is a method in which an animal is fed with the liquid impregnated in feed or feed pellets. The material of the pellet for feed in this case is not limited, but a porous pellet is particularly preferable.

(Effect of the fifth invention)
The type of animal to be applied in the first invention to the fourth invention is not limited as long as it is a non-human animal in which the action of absorbing oil balls in the digestive tract is recognized, but aquatic animals or non-human terrestrial mammals are particularly preferred. preferable.

(Effect of the sixth invention)
Although the kind of physiologically active substance utilized by 1st invention-5th invention is not limited, A vaccine, an immunostimulatory substance, a nutrient substance, a pharmacological action substance, a natural pigment | dye, a mineral, etc. can be illustrated preferably.

(Effect of the seventh invention)
Since the bioactive substance-containing emulsion of the seventh invention is an oil-in-water emulsion, the dispersion state of the fine oil spheres as oil droplets is stable. In addition, since the micro oil spheres have an average particle size that is the target of the oil ball absorption action in the digestive tract of animals, the oil ball absorption action can be used well in the digestive tract of the target animal, and the target animal can be efficiently used. It can be taken into the body.

  In addition, in this micro oil sphere, the physiologically active substance is embedded in the oil phase as it is or by forming a very fine dispersed phase with a small amount of water by the action of the oil-soluble emulsifier. Therefore, first, when subjected to the action of lipase in the intestinal tract of an animal, even if a part of the surface layer of the microsphere is digested, the particle structure of the microsphere does not collapse like a micelle structure. And is better taken up than the intestinal epithelium. Second, since the physiologically active substance is dispersed in the oil by the action of the oil-soluble emulsifier, the physiologically active substance can be dispersed in an extremely fine dispersion state regardless of its affinity (hydrophilic or lipophilic). It can be embedded in the fine oil spheres, and as a result, it becomes easy to prepare oil globules having a predetermined fine average particle diameter.

(Effect of the eighth invention)
Since the “average particle diameter that is the target of the oil-ball absorbing action in the digestive tract of animals” in the seventh invention is actually different depending on the animal species, it is difficult to uniformly define the numerical values. However, it is generally preferable to have an average particle size in the range of 1 to 60 μm if it is defined with numerical values.

(Effect of the ninth invention)
Although the kind of the physiologically active substance used in the seventh invention or the eighth invention is not limited, a vaccine, an immunostimulatory substance, a nutrient substance, a pharmacological agent, a natural pigment or a mineral can be preferably exemplified.

(Effect of the tenth invention)
The bioactive substance-containing emulsion according to the seventh to ninth inventions can be effectively produced by the production method of the tenth invention.

  In the tenth aspect of the invention, the technical key point in the first emulsification step is to first mix a bioactive substance or an aqueous solution thereof with an oil solution. Second, an oil-soluble emulsifier is added to the oil solution. When these two key points are provided, by using a known appropriate emulsion forming means such as ultrasonic treatment or using a homogenizer, “a physiologically active substance is formed as it is or with a small amount of water to form a very fine dispersed phase. Thus, an “oil-based emulsified phase embedded in the oil phase” can be constituted.

  In the second emulsification step, the above-mentioned oily emulsified phase and water (with a water-soluble emulsifier added) are mixed and emulsified by using a known appropriate emulsion forming means. The bioactive substance-containing emulsion according to the invention to the ninth invention can be produced.

  Next, modes for carrying out the first invention to the tenth invention of the present application will be described including the best mode. In the following, the term “present invention” refers to each invention of the present application collectively.

[Method of administering physiologically active substance]
In the method for administering a physiologically active substance according to the present invention, an oil-in-water type emulsion, wherein the oil droplets contain a physiologically active substance and have an average particle size that is a target for oil ball absorption action in the digestive tract of animals. A fine oil sphere is prepared, and this emulsion is orally administered to a non-human animal in which the action of absorbing oil globules in the digestive tract is recognized.

  In addition, various materials used in the present invention, for example, an oil liquid constituting an oil droplet of an emulsion liquid, a surfactant used for forming an emulsion liquid (an oil-soluble emulsifier and a water-soluble emulsifier used for forming a bioactive substance-containing emulsion liquid described later) It is premised that feed pellets and the like are used that have been confirmed to be non-toxic or physiologically safe for target animals. Furthermore, it is more desirable to use a thing advantageous to the target animal in terms of nutrition and the like.

  In the administration method of the physiologically active substance according to the present invention, the specific method of oral administration of the oil-in-water emulsion is not limited. For example, it can be injected into the oral cavity of the target animal using a syringe or the like, and can be drunk as a liquid for mammals. It can also be mixed with the animal feed. A more preferred method is a method of impregnating the animal feed pellets (particularly preferably, porous feed pellets) and feeding them as they are or after drying. As the feed pellet, various commercially available products can be arbitrarily used.

[Bioactive substances]
The type of the physiologically active substance is not necessarily limited, and can be selected arbitrarily. Preferred examples of the superordinate category include vaccines, immunostimulatory substances, nutritional substances, pharmacologically active substances, natural pigments or minerals.

  More specifically, for example, immunostimulators such as glucan, phycoidan, LPS (lipopolysaccharide), etc., such as pathogenic virus as a vaccine, pulverized product of bacteria or parasite, and the like can be exemplified. Polypeptides having physiological activity such as antibodies can also be exemplified. Nutritional substances such as vitamins can also be exemplified. Examples also include pharmacological agents such as antibiotics, hormones, and interferons. Other examples include natural pigments for imparting a certain color to the target animal. Examples thereof include minerals such as organic or inorganic calcium, iron, and magnesium.

[Target animal]
The animal that is the target of the method for administering a physiologically active substance according to the present invention is not limited as long as it is a non-human animal that is recognized to absorb oil balls in the digestive tract. Particularly preferably, aquatic animals or non-human terrestrial mammals can be exemplified.

  More specifically, as long as an oil-ball absorbing action in the gastrointestinal tract is recognized, for example, non-human mammals such as rabbits, mice, dogs, cats, cows and pigs, birds, amphibians, reptiles, cultured fish and appreciation fish are included. Various kinds of fish, shellfish, oyster shellfish (shrimp, crab, etc.) can be preferably exemplified.

[Emulsions according to the first to sixth inventions]
The emulsions according to the first to sixth inventions are “an oil-in-water type emulsion, wherein the oil droplets contain a physiologically active substance and are targeted for oil ball absorption in the digestive tract of animals. “It is a fine oil sphere having a particle size”.

  Here, the “average particle size that is the target of the oil ball absorption action” in the micro oil spheres is not limited to a uniform size according to the type of the target animal, but for example, the average particle size in the range of 1 to 60 μm An average particle diameter within the range of 1 to 20 μm is more preferable. The particle size distribution of the fine oil spheres (the degree of particle alignment of each fine oil sphere) is not limited, but those having as narrow a particle size distribution as possible within the above average particle size range are preferable.

The specific particle structure of the fine oil sphere is not limited as long as it is an object of oil ball absorption, but the following three types of particle structures (1) to (3) can be preferably exemplified.
(1) In the fine oil sphere, the bioactive substance forms an extremely fine dispersed phase as it is or with a small amount of water by the action of the oil-soluble emulsifier.
(2) A lipophilic physiologically active substance is dissolved or suspended in the fine oil sphere.
(3) A micro oil sphere has a micelle structure formed by a lipid bilayer, and a physiologically active substance is enclosed therein.

  Among the above, (2) is an oil-in-water droplet type wherein a lipophilic physiologically active substance is dissolved or finely suspended in an oil solution, and this oil solution and water are used (preferably further using an emulsifier). It constitutes an emulsion. Further, (3) constitutes a micelle structure (an aqueous solution of a physiologically active substance or the like is enclosed therein) formed by a lipid bilayer in water. The method for preparing the particle structure of (2) and (3) or the method for preparing the emulsion in which the fine oil spheres having these particle structures are dispersed is publicly known or well known. An appropriate method can be selected from the preparation methods and can be easily carried out. The particle structure of (1) and its preparation method will be described in detail in the following section.

[Bioactive substance-containing emulsion]
The biologically active substance-containing emulsion according to the seventh to ninth inventions is an oil-in-water emulsion in which fine oil balls having an average particle size that are targets for oil ball absorption in the digestive tract of animals are dispersed. In the fine oil sphere, the bioactive substance is embedded in the oil phase as it is or with a small amount of water by forming an extremely fine dispersed phase by the action of the oil-soluble emulsifier.

  Here, in the micro oil spheres described above, an extremely minute dispersed phase of a physiologically active substance (or a physiologically active substance and a small amount of water) is oil-soluble at the interface with the dispersoid in each individual dispersed phase. It is stably dispersed in the oil phase of fine oil spheres as a dispersoid while retaining the emulsifier. Such fine oil spheres are stably dispersed in water as a dispersoid while holding a water-soluble emulsifier at the interface with the dispersoid (water) in each of the fine oil spheres.

  The affinity of the physiologically active substance may be any of water solubility, hydrophilicity, oil solubility, and lipophilicity. The preferable average particle size and particle size distribution of the fine oil spheres are the same as those of the above-mentioned “emulsions according to the first to sixth inventions”.

  The characteristic feature of the fine oil spheres contained in the bioactive substance-containing emulsion according to the seventh to ninth inventions is that the bioactive substance forms an extremely fine dispersed phase in the fine oil spheres by the action of the oil-soluble emulsifier. (Embedded in the oil phase). Since the physiologically active substance is finely dispersed as it is or with a small amount of water, the concentration of the physiologically active substance in the minute dispersed phase is 100% or extremely high, and thus the drug delivery effect is extremely high. Such a state of high concentration and fine dispersion of the physiologically active substance can be realized by the method for producing an emulsion described in the next section.

[Method of producing emulsion containing physiologically active substance]
The manufacturing method of the physiologically active substance-containing emulsion according to the tenth invention includes (A) a first emulsification step and (B) a second emulsification step as essential steps, which will be sequentially described below. However, as long as it is necessary or beneficial, an arbitrary pretreatment step, intermediate step or post-treatment step can be added to these steps.

  (A) 1st emulsification process: By mixing and emulsifying the bioactive substance or its aqueous solution or water suspension and the oil liquid to which the oil-soluble emulsifier is added, the bioactive substance remains as it is or with a small amount of water. An oily emulsified phase is produced which forms a very fine dispersed phase and is embedded in the oil phase.

  In this first emulsification step, when the physiologically active substance is used as an aqueous solution or aqueous suspension thereof, the water should be reduced as much as possible, that is, a highly concentrated aqueous solution or a high density aqueous suspension of the physiologically active substance. It is preferable.

  An important technical point in the first emulsification step is to mix and emulsify a bioactive substance or an aqueous solution thereof (phase a) with an oil solution (phase b) added with an oil-soluble emulsifier. Here, the mixing ratio of the a phase and the b phase is not uniformly limited, but is arbitrarily selected in a weight ratio, for example, within a range of about a phase: b phase = 1: 99 to 99: 1.

  When the weight ratio of the a phase and the b phase deviates from the above range, the production of the bioactive substance-embedded micro oil sphere as described above tends to fail. If an oil-soluble emulsifier is not added to the b phase, the production of the bioactive substance-embedded micro oil spheres as described above tends to fail. The amount of the oil-soluble emulsifier added to the b phase is suitably about 1 to 5% by weight.

  “Oil-soluble emulsifier” is not limited in kind as long as there is no problem in toxicity, physiological safety, etc., but its HLB (hydrophilic-lipophilic balance) value is 6 or less, particularly 2 or less. preferable. As the type of emulsifier, a nonionic surfactant is preferable. Examples of preferable oil-soluble emulsifiers that meet the above conditions include linolenic acid hexaglycerin, polyoxyethylene lauryl ether, lecithin, polyoxyethylene oleyl ether, and the like.

  (B) Second emulsification step: The oily emulsified phase produced in the first emulsification step and water added with a water-soluble emulsifier are mixed and emulsified, whereby the oily emulsified phase becomes an oil ball in the digestive tract of animals. An oil-in-water emulsion that is dispersed in water as fine oil spheres having an average particle size to be absorbed is produced.

  The technical point in a 2nd emulsification process is a point which mixes and emulsifies the water (d phase) which added the water-soluble emulsifier with the oil-based emulsification phase (c phase) manufactured at the 1st emulsification process. Here, the mixing ratio of the c phase and the d phase is not uniformly limited, but is arbitrarily selected in a weight ratio, for example, within a range of about c phase: d phase = 2: 1 to 1: 3.

  If the weight ratio of the c phase and the d phase deviates from the above range, the production of the physiologically active substance-containing emulsion according to the present invention tends to fail. Similarly, unless a water-soluble emulsifier is added to the d phase, the production of the bioactive substance-containing emulsion according to the present invention is likely to fail. The amount of the water-soluble emulsifier added to the d phase is suitably about 0.5 to 1.3% by weight.

  The “water-soluble emulsifier” is not limited as long as there is no problem in toxicity, physiological safety, etc., but its HLB (hydrophilic-lipophilic balance) value is 10 or more, particularly 12 or more. preferable. As the type of emulsifier, a nonionic surfactant is preferable. Examples of preferable water-soluble emulsifiers that meet the above conditions include decaglyceryl distearate, glyceryl isostearate, ethylene glycol monoisostearate, ethylene glycol monooleate, and the like.

  The emulsification operation itself in the above (A) first emulsification step and (B) second emulsification step can be appropriately performed using a method using a known ultrasonic wave, a homogenizer, a mixer, or the like. During the emulsification operation, by appropriately selecting the operating conditions of the emulsification means, the particle size of the fine oil spheres in the emulsion containing the bioactive substance and the degree of fine dispersion of the bioactive substance in the fine oil spheres Can be adjusted.

(Example 1)
Using a formalin killed Escherichia coli as a physiologically active substance, an aqueous suspension (first solution) containing the killed bacteria at a high density was prepared. On the other hand, an oil liquid (second liquid) was prepared by adding 5% by weight of an oil-soluble emulsifier (hexaglycerin linolenate: trade name “Sunsoft # 818R”, Taiyo Kagaku Co., Ltd.) to squid liver oil.

  Next, as the first emulsification treatment according to the tenth aspect of the present invention, the first liquid and the second liquid are mixed at a weight ratio of 90:10, and sufficient emulsification treatment is performed with an ultrasonic generator to obtain E. coli formalin. The dense suspension of dead bacteria formed an extremely fine dispersed phase to form an oily emulsified phase (third liquid) embedded in the oil phase.

Next, water (fourth liquid) to which 1% by weight of a water-soluble emulsifier (decaglyceryl distearate: trade name “Sunsoft Q-182S”, manufactured by Taiyo Chemical Co., Ltd.) was added was prepared. Then, as the second emulsification treatment according to the tenth aspect of the invention, the third liquid and the fourth liquid are mixed at a weight ratio of 2: 1 and sufficiently stirred with a mixer, so that the oily emulsified phase is a fine oil sphere. A bioactive substance-containing emulsion dispersed in water was prepared.

  When the particle size of the fine oil spheres contained in this physiologically active substance-containing emulsion was observed, it was about 5 to 10 μm. The prepared emulsion containing the physiologically active substance is stored in the refrigerator for about one week, and then the particle size of the micro oil spheres is observed again. It is about 5 to 10 μm, and the dispersion state of the micro oil spheres is stable. The sex was confirmed.

(Example 2)
In this example, killed formalin of Edwardsiella tarda, a pathogenic bacterium of flounder, was used. Then, an aqueous suspension (first liquid) containing 10 ⁷ individuals / ml of the dead bacteria was prepared, and a physiologically active substance-containing emulsion was prepared in exactly the same manner as in Example 1 except for that point.

  Next, an appropriate amount of this physiologically active substance-containing emulsion was impregnated into a commercially available porous pellet for fish feed. Then, the above-described porous pellets were fed continuously for 15 days to 15 flounder so that the daily emulsion intake per 100 g of fish weight was 10 μl.

  On the 23rd day after the end of the feeding period, blood was collected from 5 tail arteries of the flounder, and the antibody titer of anti-Edwardsiella tarda antibody in the serum was measured as the aggregation titer. On the other hand, as a comparative example, blood was collected from five flounder tail arteries not fed with a physiologically active substance-containing emulsion, and the antibody titer of anti-Edwardsiella tarda antibody in the serum was similarly measured as an agglutination titer.

  The measurement results are shown below. In the following, “administration group” shows 5 fishes according to the example, and “control group” shows 5 fishes according to the comparative example. The numbers in the “Fish” column are specimen numbers of five carp in the administration group and the control group.

Fish Administration group agglutination value Control group agglutination value 1 1024 2
2 1024 4
3 256 2
4 32 16
5 64 16
Apart from the agglutination titer measurement described above, the remaining 10 flounder 23 days after the end of the feeding period according to this example were injected intramuscularly with viable Edwardsiella tarda. (As the amount of attacking bacteria, 0.3 ml of 10 ⁵ individuals / ml of the bacterial solution was injected) and reared for 14 days, and the survival rate was evaluated. On the other hand, as a comparative example, live Edwardsiella tarda was similarly injected into 10 muscles of flounder that were not fed with a biologically active substance-containing emulsion, and were reared for 14 days to evaluate the survival rate.

  The evaluation results are shown below. In the following, the number in the “Elapsed days” column indicates the number of days that have elapsed since the injection of viable bacteria. In addition, “administration group” indicates the flounder according to the example, “control group” indicates the flounder according to the comparative example, and the numbers in each group indicate the number of individuals who died on that day. There were no dead animals until the 6th day in the treatment group and the control group. In the control plot, the cumulative number of dead individuals was 10 (survival rate 0%) at 14 days, whereas in the treated plot, the cumulative dead population was 5 (survival rate 50%) after 14 days. It was.

Elapsed days Administration group Control group 7 0 0
8 0 0
9 0 1
10 0 4
11 1 1
12 1 0
13 3 1
14 0 3
As can be seen from the above results, in the measurement of the antibody titer (aggregation titer) and the survival rate after injection of viable bacteria, the administration group showed significantly superior results as compared with the control group.

(Example 3)
In the present example, the formalin killed by Edwardsiella tarda was also used. Then, while preparing an aqueous suspension (first liquid) containing 10 ⁹ individuals / ml of the dead bacteria, the same second liquid as in Example 1 was prepared.

  Next, as the first emulsification treatment according to the tenth aspect of the present invention, the first liquid and the second liquid are mixed at a weight ratio of 99: 1, and sufficient emulsification treatment is performed with an ultrasonic generator to obtain E. coli formalin. The dense suspension of dead bacteria formed a very fine dispersed phase to form an oily emulsified phase (Group 1 third liquid) embedded in the oil phase. Moreover, said 1st liquid and 2nd liquid were mixed by the weight ratio of 90:10, and the same emulsification process was performed, and the oil-based emulsification phase (3rd liquid of group 2) was formed.

  Next, the 4th liquid of the same content as Example 1 is prepared, and as the 2nd emulsification processing concerning the 10th invention, about the 3rd liquid of group 1 and the 3rd liquid of group 2, respectively, 4 liquids were mixed at a weight ratio of 2: 1, sufficiently stirred with a mixer, and the above-mentioned oily emulsified phase was dispersed in water as fine oil balls ("Group 1 physiologically active substances" Containing emulsions "and" group 2 physiologically active substance-containing emulsions ").

  Next, a commercially available porous pellet for fish feed was impregnated in an appropriate amount with the group 1 physiologically active substance-containing emulsion and the group 2 physiologically active substance-containing emulsion. These porous pellets were allowed to feed freely for 10 days for each 10 flounder (average body weight is about 100 g) so that the daily emulsion intake per 100 g of fish weight was 0.05 ml. It was. The flounder that feeds the group 1 bioactive substance-containing emulsion is called “Group 1”, and the flounder that feeds the group 2 bioactive substance-containing emulsion is called “Group 2”.

  Next, for the flounder related to “Group 1” and “Group 2”, the antibody titer of the anti-Edwardsiella tarda antibody in the serum of the flounder was measured as the aggregation titer on the 23rd day after the end of the feeding period. The results are shown in the column of “Bacteria embedded in oil spheres” in Table 1. The numbers in the “Fish” column of Table 1 are the specimen numbers of 10 flounder samples related to “Group 1” and “Group 2”.

  As can be seen from Table 1, in the flounder of “Group 1” and “Group 2”, a high concentration of anti-Edwardsiella tarda antibody was produced overall.

(Comparative example with respect to Example 3)
The same Edwardsiella tarda formalin killing bacteria as in Example 3, squid liver oil, and an oil-soluble emulsifier were used in the same amount as in “Group 1” in Example 3 to prepare an oily solution.

  Although this oily liquid may be an oily emulsified phase as a result of the first emulsification treatment according to the tenth invention, it has not undergone a process corresponding to the second emulsification treatment, so the physiological activity according to the present invention It is not a substance-containing emulsion.

  The oily liquid was impregnated into porous pellets in the same manner as in Example 3 and administered to 10 flounder in the same manner as in Example 3 to evaluate antibody production. The flounder fed with this oily liquid is defined as the “Group 4” flounder group, and the evaluation results are shown in the “Group-4” column of “Target Area” in Table 1. In addition, 10 groups of flounder that are not normally administered with any formalin killed bacteria or emulsions of Edwardsiella tarda and are kept in a normal manner are designated as “Group 3” flounder groups. In the same manner, antibody production was evaluated in the same manner. The evaluation results are shown in the “Group-3” column of “Target Area” in Table 1.

  As can be seen from Table 1, there is not much difference between the evaluation results of “Group 3” and “Group 4”, and a significant difference between these and the evaluation results of “Group 1” and “Group 2” Is clear.

According to the invention of the present application, for example, various types of fish or aquatic shellfish for aquaculture or appreciation, or for mammals that have been confirmed to absorb oil balls, immunostimulatory substances, pharmacologically active substances, and other various types. The physiologically active substance can be used as an oral drug delivery means for taking in a very high efficiency by a simple means.

Claims (10)

Prepare an oil-in-water emulsion, which is a micro oil sphere having an average particle size that contains a physiologically active substance and is subject to oil sphere absorption action in the digestive tract of animals,
A method for administering a physiologically active substance, wherein the emulsion is orally administered to a non-human animal that is recognized to absorb oil balls in the digestive tract. The method for administering a physiologically active substance according to claim 1, wherein the micro oil spheres have an average particle diameter in the range of 1 to 60 µm. The method for administering a physiologically active substance according to claim 1 or 2, wherein the micro oil sphere has a particle structure of any one of (1) to (3) below.
(1) In the fine oil sphere, the bioactive substance forms an extremely fine dispersed phase as it is or with a small amount of water by the action of the oil-soluble emulsifier.
(2) A lipophilic physiologically active substance is dissolved or suspended in the fine oil sphere.
(3) A micro oil sphere has a micelle structure formed by a lipid bilayer, and a physiologically active substance is enclosed therein. The method for administering a physiologically active substance according to any one of claims 1 to 3, wherein the animal is fed while the emulsion is impregnated with feed or feed pellets. The method for administering a physiologically active substance according to any one of claims 1 to 4, wherein the animal is an aquatic animal or a non-human terrestrial mammal. The method for administering a physiologically active substance according to any one of claims 1 to 5, wherein the physiologically active substance is a vaccine, an immunostimulatory substance, a nutrient substance, a pharmacologically active substance, a natural pigment or a mineral. An oil-in-water emulsion in which fine oil spheres having an average particle size to be absorbed in the animal's gastrointestinal tract are dispersed. In the fine oil spheres, due to the action of an oil-soluble emulsifier, A bioactive substance-containing emulsion characterized in that a bioactive substance is embedded in an oil phase as it is or with a small amount of water to form a very fine dispersed phase. 8. The bioactive substance-containing emulsion according to claim 7, wherein the fine oil spheres have an average particle diameter in the range of 1 to 60 [mu] m. 9. The physiologically active substance-containing emulsion according to claim 7 or 8, wherein the physiologically active substance is a vaccine, an immunostimulatory substance, a nutrient substance, a pharmacologically active substance, a natural pigment or a mineral. The manufacturing method of the bioactive substance containing emulsion characterized by including the following (A) 1st emulsification processes and (B) 2nd emulsification processes.
(A) 1st emulsification process: By mixing and emulsifying the bioactive substance or its aqueous solution or water suspension and the oil liquid to which the oil-soluble emulsifier is added, the bioactive substance remains as it is or with a small amount of water. An oily emulsified phase is produced which forms a very fine dispersed phase and is embedded in the oil phase.
(B) Second emulsification step: The oily emulsified phase produced in the first emulsification step and water added with a water-soluble emulsifier are mixed and emulsified, whereby the oily emulsified phase becomes an oil ball in the digestive tract of animals. An oil-in-water emulsion that is dispersed in water as fine oil spheres having an average particle size to be absorbed is produced.