Method and composition for orally administrating physiological substance to animals

[Detailed Description of the Invention]

The present invention encapsulates and administers a physiologically active substance orally into the polymer which has the disintegration in the living body and living body coexistence nature more than a kind thru/or a copolymer Chinese poem type agent. even if a physiology active agent passes a gastroenteric course -- an effect -- To lose -- the set lymph gland known as "pay Ya Potch° of an animal without things is reached, and it is related with the method and tablet which are made to be absorbed. Conventional technology Microcapsule-ization for Maintenance(ing) so that a sharp physiologically active substance may not be collapsed is a well-known fact. Although a physiologically active substance is generally encapsulated in the protective film substance of polymers, even if the tunic of the substance encapsulated in this case is carried out by the single film of a polymeric mass (it may distribute uniformly in a polymers medium.) The quantity of the physiologically active substance inside a microcapsule can be changed very much from 95% of microcapsule material to a small quantity by hope, and can also change the diameter of a microcapsule arbitrarily from 1 micrometer to 3 m. - It is a lymph gland aggregate to which pay Ya Potch - is located in an ileum, i.e., the near lower part, and is an important portion for defending a human body against bacterial contamination. An antigen is a substance which promotes generation of an antibody and includes a foreign protein or an organization. And all antibodies combine with Ah Si and an antigen in the protein of the kind called an immunoglobulin (1, 9), generate an inactive complex, and make an antigen detoxify. It has a "pay Ya Potch ''IJIA pre B cell, and is .. It increases in the respiratory-organs course of a gastroenteric peculiar part and the upper part, and this is .. It specializes in a mature IJIA composition Brasma cell. And this plasma cell actually secretes antibody molecules. The appearance of antigenic specificity I and 9A Brasma cell by research of Heramans which measured r of the mouse oral-immunization-ization-processed with the antigen, and change of the iFA response, and Bazin, Finally Bad accepted to the peculiar portion of the gastroenteric course continuously after [ one ] mesenteric lymph nodes at first. That is, "pay Ya Potch '' is a source of supply with pre I and abundant GIA cells, and it is clear that those cells follow a circulating route and appear on the long distance membrane surface by antigen stimulus. And it is possible to make the environmental antigen which sends to a membrane part continuously, usual membrane immune system, i.e., stimulated B cell, and originates in an alimentary canal according to this circulating route, and Pathogenesis oppose. The essential importance of the present invention is in the oral immunization capability to induce the antibody for defense. It is already known. When an animal takes in an antigen, an antigenic specificity sIgA antibody appears in a bronchus or a nose washing thing. For example, in the research on one human being, an anti-influenza antibody can be induced to a nose part section by internal use of an influenza vaccine. It sets to the method and tablet of internal use of a pharmacology substance. If it is necessary to make it a pharmacology substance not make it decompose into Removal which passes a gastroenteric course and it is not made, the quantity of a pharmacology ingredient which il+ Reaches "pay Ya Potch" will become unsuitable, and an effective thing will not become. In the state where a physiologically active substance is not protected, a result for which most portion of the physiologically active substance which a lot of things needed to be taken in although the thing of a quantity effective for "pay Ya Potch ° is made to reach, and was prescribed for the patient as a result is not used is brought. If the number of times of internal use is not increased in order to supply "pay Ya Potch ° as Mutually at a long time, increase of such frequency of administration and a given dose is [ failing in / If ] useless, and it is inconvenient. therefore, a membrane immune system is stimulated efficiently and it is a physiologically active substance -- when passing a gastroenteric course toward pay Ya Potch *, while decomposing, the method of solving Purification is required, and especially the useless method of making it reach '' pay Ya Potch "is required, without making Ah Si and an antigen disassemble. That is, the object of the 1 present invention administers a physiologically active substance orally to an animal, makes "pay Ya Potch ° reach f11, and it makes it absorb. It is providing the method of stimulating a membrane immune system, without losing effect, when carrying out the groove of the gastroenteric course of an animal in that case, and a physiologically active substance is used as a core, and it is an allocated type agent for capsules, decomposes in the living body further again, is not harmful to a living body, and is providing the tablet which can be used for internal use. That is, the 1 present invention relates to the method of feeding a physiologically active substance into "pay Ya Potch ° of an animal by internal use, and the tablet for it, Let the physiologically active substance be a micro capsule into the polymer which has biodegradation nature and living body avirulence thru/or a copolymer, It does not decompose in the case of passage of a gastroenteric course, either, or only the minimum decomposition is carried out, but "pay Ya Potch ° is made to reach in an effective quantity, without denaturing a physiologically active substance by it. It defines as a polymerization substance which the living body avirulence as used in the field of the present invention is not harmful to a living body, and there is no carcinogenicity, and does not generate inflammation to a body tissue, the meaning of producing the output easily decomposed by the living body by a vital reaction, and moreover not being accumulated in the living body -- biodegradability -- it is not likely to be -- if it is Leave, there is nothing. this microcapsule -- "pay Ya Potch -- " -- it is a thing of the size which sets and is absorbed alternatively. Therefore, according to the present invention, it is - pay Ya Potch. It is alike and it is solved whether it is during the reaching physiologically active substance and its absorption. The example of real Winding The method of microcapsule-izing trinitro phenyl Kyohall-Rim pet- hemocyanin as an antigen to 50:50 poly(DL-Lactide-co-glycolide) as one example of the 1 present invention below, and medicating a mouse for a long time is described. What should be minded by This \ is also being able to use polymers other than poly(DL-Lactide-co-glycolide), As an example of these polymers, they are poly (glycolic acid) and a mixture copolymer of DL-lactide and glycolide, Although copoly oxalate, polycaprolactone, poly (lactide Co-Power pro lactone), poly (ester amide), poly Ortho ester, poly (beta-hydro-Oxy butyl acid), etc. are raised, these do not mean limitation. Another thing also as a physiologically active substance can be used, and they are an influenza virus, a parainfluenza virus, and a 100-day Cheeks bacillus as those examples. viruses, such as the Lynn Fungus and a pneumonia bacillus, bacteria, and primeval -- there is an antigen for the vaccine which is started by the illness caused by the antigen and microorganism Pathogenesis at the time of receiving sick and carrying out a vaccination in which it is based on an insect etc., and the parasite infection field and which is received sick. 1, microcapsule-izing It was considered as the coloring microcapsule for being a polymer in which biological breakdown is impossible, microcapsule-izing with polystyrene Kumarin who is manufacture (penetrable research) insoluble in water pigment of A and the pigment content microcapsule for "pay Ya Potch", and investigating the permeability of the micro capsule in - pay Ya Potch °. The manufacturing process was as follows. Polystyrene 14.95 # was first dissolved for 1 polymer solution in the good To sake at methylene chloride 255II. Subsequently, Kumarin 0.05 was Heavy addition(ed) in this solution and the churning dissolution was carried out with the electromagnetism swizzle stick. Go 360 g of deionized water into another container. pVAl+oIi is dissolved in this to make 40% of the weight of polyvinyl alcohol (PVA) solution, After adding 6 g of methylene chloride to this bathing liquid and making it saturated with methylene chloride, this was put into lj resin kettle with a 2.5-inch Teflon turbine propeller and a swizzle stick, and it agitated by 3gOrpm. Next, polystyrene / Kumarin mixture was poured into the resin kettle containing this PVA processing medium by the funnel of the diameter of long axis 7■. The stable oil in water emulsion obtained in this way was agitated for about 30 minutes under normal pressure, and the oily particles of the suitable size were obtained. Subsequently, by the aspirator which closed the resin kettle and was connected with Manometer, it decompressed gradually and made pressure power inside a kettle into 520wH and G'. The kettle content evaporated methylene chloride completely in churning under decompression for about 24 hours. After all methylene chloride evaporated, microcapsules were collected by centrifugal separation and it dried at the vacuum room maintained at room temperature for 72 hours. TNP-KLH which is a manufacture water solubility antigen of B and an antigen content microcapsule was encapsulated by Po + (DL-Lactide-co-glycolide) which is biodegradable polyester of living body g toxicity. The manufacturing process of the microcapsule was as follows. First, 50:50 poly (DL-Rakutedo Co glycolide) 0.51 was dissolved in methylene chloride 4.Ol, and polymer solution was manufactured. Subsequently, TNP-KLH (1161gTNP-KLH/ml: after-dialysis floor solution 300mu! was added to poly (DL-Rakutedo Co glycolide) solution, and it processed by Vor tax-Geni e spirally, and made it distribute uniformly) Deionized water 55.2.9 is taken in another container, PVA of 4 and 8 y is dissolved in this, and it takes 81 views for PVA solution of %, After the dissolution of PVA, this was put into the Irritation stick and the loomt resin kettle to which L 5-inch Teflon turbine propeller was attached, and previous polymer solution was added to this PVA processing medium by the funnel of long axis 7 Easy diameter. PVA solution was agitated by about 65 Orpm(s) during addition. moving a resin kettle content into deionized water 3.5.il in 117 beakers, after agitating the obtained oil in water emulsion for about 10 minutes in a resin kettle -- the propeller made from stainless steel (2 inches) -- about -- it agitated by 80 *rpm. The microcapsule obtained in this way was agitated for about 50 minutes in deionized water, and it collected by centrifugal separation, it washed by deionized water, remains PVA were removed, and it freeze-dried. the obtained microcapsule -- about 1~10 micrometers in diameter -- it was spherical. In order to measure TNP-KLHl of the microcapsule containing this antigen, i.e., the core content of this microcapsule, After putting microcapsule 10 Beat into 12 m / centrifugal tube and adding methylene chloride 5.0 ml to this, in order to dissolve poly (Di, - lactide -Co- glycolide), it whirlpool-ized, it ranked next one, deionized water 3. Oml was put into the tube, and it whirlpool-ization-processed for 1 minute still more strongly. The content of the centrifugal tube was divided into the organic layer and the water layer by centrifugal separation, the water layer was moved to the 1017 fixed-quantity flask, and the extract including a water layer was also transferred to this fixed-quantity flask. Subsequently, deionized water was filled to the mark of a flask. It was known in fixed quantity by the protein method at the bottom and the time in the quantity of TNP-KLH in a flask, therefore the amount of TNP-KLHO in a microcapsule that this microcapsule contains TNP-KLH 0 or 2% of the weight. pi, biological research A, a mouse The B A L B /C mouse after the birth [ 8~12 weeks of ] was used for research. B The hemocyanin (KLH) obtained from A IJ nitrophenyl keyhole Rim pet hemocyanin (TNP-KLH) The Keyhole Limpet Megathura arenulata is purchased from Calbfoahem, Trinitro phenyl hub Tint combination was carried out using 21416-IJ nitrobenzene Sulfone acid by the method of Rlttenburg and Amkraut. (TNP-KLH) The replacement ratio was measured spectrophotometrically, 30% of amendment was added to contribution of KLH in this wavelength using molecular extinction coefficient 151100 on the wavelength of 350 m, and having been TNP-KLH was determined. C1 immunization processing What TNP-KLH should microcapsule-ize was made suspended so that it may become the solution consisting of eight copies of filter sterilization tap water, and two copies of sodium bicarbonate (7 or 5% solution) with the antigen of 10 micro ll/ml concentration. Catching of D and living body liquid 1 Serum After collecting and solidifying blood with the capillary tube pipette containing scale Si, serum was collected and centrifuged, and red corpuscles and blood platelets were removed, heated and inactivated, and it stored by -70 degreeC till use. Z intestines secretion It is Lavade solution (25mu Na0j.) to a mouse. 140 m M N a 2 S O4, 10m M K Cj, 20mM NaHOO'415 mM polyethylene 3 * glycol, and osmosis 530 mosM 0.5ml was prescribed for the patient 4 times. A mouse is anesthetized in 15 minutes after the last medication, and also it is 5 from it. 0.1 mg of pyro Calpin were prescribed for the patient by ip injection after the part. Then, an intestines content is discharged over 10~20 minutes. It stimulated. It is 50 mM about this. EDTA It brought together in the What IJ plate which contains solution 3IIIt of the rate of soybean trypsin inhibitor O, and 1 Craftsman / ml in inside, whirlpool-ized strongly, and removed the suspension thing by centrifugal separation. It is moving to the centrifugal tube made from polycarbonate of a circular bottom about top Clear. It is PMSF20micro, after adding PMSF and phenyl methylsulfonyl fluoride 30micro A and becoming clear by high-speed centrifugal separation (27000X, 9.20 minutes, 14degreeC)! aza-[ 1% sodium ] -- adding an id -- FC -- it was considered as the substitution substrate to remains Grotte ase as 10% of solution 8 inside. 5 Saliva A lot of saliva was secreted simultaneously with discharge of an intestines content. bringing together in the Pasteur pipette in this 0.25-m1f capillary tube operation, and preceding clear-ized processing -- aza-[ soybean trypsin inhibitor, PMSF, and / sodium ] -- an id and Fe2 were added 20micro A every. E, an immunization study reagent It is IM to I of the rat by which solid phase adsorption was carried out and by which compatibility processing was carried out. I of the polyclonal goat of singularity and 9 () antibody (a commercial item, the product made from 5 outhern BioteahnologyAssoaiates) were obtained to I, 9C, and IgA, and the singularity about binding capacity with a monoclonal antibody or Melome protein was measured by radioimmunoassay. F, solid phase radioimmunoassay Na carrier free by the Chloramina method The sign of the refining antibodies was carried out by I. 4 degreeC covered the piece of Imron Limbalael measurement (product made from Dynateah) by tmuII/at in B B s overnight as it is also at BSA (cow serum albumin) combined with TNP. As Up\ which does not carry out a Without machine, the piece of standard comparison processed all the specimens under room temperature by BSA 1% among BBS for 2 hours. It diluted so that the antigenic specificity antibody of the iso model in question might serve as 1~l OOO*g/rd, and it was added by washing Replicate well, and inoculation processing of the sample of living body liquid was carried out at room temperature for 6 hours. After washing and + Anti-immunity Glow pudding 100. OOOc p m of the iso model singularity which carried out the sign by 251 was added to each Well, and inoculation processing was carried out by 4 degreeC overnight. it did not join together Well after washing and removing the Knee antibody -- gamma -- radiation measurement was carried out in total (product made from Bekman) 5500 spectrum. To Well which covered measurement with iso model singularity antibody 1microI / well. The standard serum (made by MilesScientifia) containing IF of a known amount was diluted and used twice. A basic program called "Logii-10g" or "Four Parameter Logistia j of the product [ of a strange sample / the measurement curve or interpolation numerical value ] made from BiomedicalComputing Technology Center It used and asked by computer. G, a result By administering orally the polystyrene microcapsule containing penetrable fluorescence pigment Kumarin to "pay Ya Potch * of L pigment content microcapsule to a mouse, it investigated about the absorption to the alimentary canal related lymphatic tissue of a microcapsule, and its size limit. Thing Q which made the fluorescence microcapsule (thing less than 5 micrometers and 8~52 m in diameter) of various sizes suspended by g/of loo shoulders ml to tap water, and 5 rnl were prescribed for the patient into A helmet with the medication needle. 0.5, 1., and 2 hours [ medication and ] after. The mouse was cut open and the small intestine was excised. "pay Ya Potch ° containing an alimentary canal was separated as a 1crIL section, and Intracavity was probed, and it was reversed and froze. The frozen section was investigated about several 1 position 1 size of the microcapsule absorbed by "pay Ya Potch * from Cavity in an alimentary canal under the fluorescence microscope. Although the microcapsule Promotion(ed) between villi became the hindrance of the removal at the time of washing, the penetration to an in-house was not accepted at any places other than "pay Ya Potch °. 2 hours after a microcapsule is accepted in "pay Ya Potch ° of the end piece instead of the central part of a small intestine and a microcapsule moves by wriggle with progress of 1 hour in 0.5 hour after internal use, the gastroenteric whole course sees -- "pay Ya Potch of an ileum -- " -- it accepted. The microcapsule prescribed for the patient has given the impression that it is located in the edge part which is distant from the top part of Do" pay Ya Potch with the Lord, Promotion physically [ while wriggling between the top part and the villus of a periphery ], and it is useful for absorption. Although the particles up to 10 micrometers in diameter were also accepted into "pay Ya Potch", it was known that a microcapsule less than 5 micrometers in diameter will be absorbed in large quantities, and will go into the depths of "pay Ya Potch ° with time progress. these results -- a microcapsule 1~5 micrometers in diameter -- quick -- alternative -- "pay Ya Potch from Cavity in an alimentary canal -- " -- being absorbed is shown. And this has suggested that the microcapsule consisting of biodegradable covering material acts as a leading means for feeding an antigen into alimentary canal-related lymphatic system Braided fiber, and making the membrane surface induce immunity. The protein antigen by a second antigen content biodegradable microcapsule which carried out oral immunization-ized hub ten processing, By having made the microcapsule containing trinitro phenyl Keyhole rim pet hemocyanin (TNP-KLH) into Without machine material, the poly (DL-lactide Cooguri fried) copolymer was separated by Off the coast structure, this microcapsule was separated with the size, and a thing 1~5 micrometers in diameter was chosen. 0*2% of antigen was contained in this microcapsule. The capability to serve as an effective feeding system of an antigen was investigated by medicating A helmet with 0.5rnl (10-micrometer antigen) of carbonate buffer sterile water way 18 ng/ml underwater @ murky solution as Mutually in four days. Two of TNP-KLH by which another mouse group is not encapsulated in order to compare (administered orally by 1"g/m / solution Q, and 5 r!It.) Only the diluent was administered orally to the standard mouse for comparison. Serum, saliva, and a gastrointestinal secretion were obtained from five fast mice in each group 14 days and 28 days after the last medication mouth. These samples were investigated by iso model singularity radioimmunoassay in order to measure the total amount of antibodies of TNP singularity, IJ2JIpG, and an IgA iso model. (1st table) Only I and 9A were almost contained in saliva and a gastrointestinal secretion. This result is what was in agreement with old research, and is proof that the method of collecting these secretions did not bring about contamination of serum. No immunity protocols show a remarkable change of the whole quantity of the immunoglobulin in humors. With the immunization standard mouse, the anti-TNP antibody which exists naturally was seen by IJIM and rga iso model serum, and the 1.GIA iso model was seen by serum and the gastrointestinal secretion. However, microcapsule-ized TNP-KLI for three days (by equivalent medication of 50microy, a secretion has antigenic specificity I which turns around A contemplation remarkably in a top, and 9A antibody, and brought about the appearance in the serum of all the iso models in after-medication 111 days.) (Refer to the 1st table) The amount of antibodies of these further increased in [ of two ] g days. On the other hand, as for an equivalent amount of antigens which were not encapsulated, the effect of singularity antibody induction was not accepted by oral addition, either. EFFECT OF THE INVENTION The effect by the present invention deserves attention in some respects. Since the antigenic specificity antibody hardly induced by the 1st by the conventional Group area immunity method can be remarkably induced in serum or a membrane secretion, it is expected that this method will give the immunity power remarkably strengthened with the membrane as an entrance of the pathology over various kinds of bacteria and virus Pathogenesis. Although the antigen tablet microcapsule-ized by the 2nd can serve as an effective immunity field by administering orally, when not encapsulating, an equivalent amount of things do not become so. Therefore, improvement in epoch-making efficiency is brought about by making this microcapsule-ization reach ' pay Ya Potch *, and making it absorb. And an immune response Continuation [ 5th ] for a long time. the antigen content microcapsule to which the tissue immunity by a protein antigen was administered orally without the adjuvant although the amount of antibodies became a peak on the 7~IIJ day -- 114 days -- a 2g day -- self-teaching -- a high immune response is shown. This shows that the biodegradation of Without machine material and discharge of an antigen cover a long time, and an immune response covers a long time as a result.