Intestinal absorption aids, intestinal absorption compositions, and prophylactic and / or therapeutic agents

Abstract

To provide intestinal absorption aids, intestinal absorption compositions, and prophylactic and / or therapeutic agents for introducing high molecular weight compounds such as insulin into the body via the intestinal tract. [Solution] The intestinal absorption aid of this disclosure comprises (I) and (II) below and is characterized by having an osmotic pressure of less than 280 mOsm / L. The intestinal absorption aid allows the following (III)-iii-a-1 and / or the following (III)-iii-b-1 to be absorbed via the intestinal tract. (I) Solvent containing hypotonic solution (II) Bubbles smaller than nano-size (III)-iii-a-1: Insulin or a derivative thereof, wherein the derivative is an insulin derivative that has been modified by glycosylation. (III)-iii-b-1: Dextran or its derivatives

Description

Technical Field

[0001] The present invention relates to an auxiliary agent for intestinal absorption, and more particularly, to an "auxiliary agent for intestinal absorption" that can be used as a "drug delivery system auxiliary agent" for reliably absorbing a high molecular compound into the living body via the intestine. The present invention also relates to a "composition for intestinal absorption" that can be more reliably absorbed by the intestine. Furthermore, the present invention relates to a "prevention and / or treatment agent" that can more reliably exhibit functions, efficacy, or effects in the living body.

Background Art

[0002] As methods for administering an active substance such as a drug into the living body, various methods such as injections, transdermal absorbents, and oral preparations are used. Among them, injections are used as one of the effective administration methods because the active substance can reach the target site without being decomposed or inactivated by directly administering it to blood vessels, subcutaneous tissue, muscles, etc.

[0003] However, in addition to being painful, injections also have burdens such as the burden of visiting a hospital or the physical and mental burden in the case of self-injection. In particular, when multiple administrations are required per day, the burden is large, so many injections are desired to be replaced with oral preparations.

[0004] However, depending on the type of the active substance, especially when the active substance is a medium or high molecular compound such as a "polysaccharide" or a "protein", it is known that almost no absorption occurs from the small intestine responsible for gastrointestinal absorption, and the development of oral drugs has been difficult.

[0005] On the other hand, regarding the improvement of intestinal absorption, methods such as using an "absorption promoter" such as a membrane-permeable peptide in combination with the active substance have been tried, but a method that does not require such a special "absorption promoter" has been desired.

[0006] Furthermore, the development of devices that generate fine bubbles with a diameter of several tens of micrometers or less, so-called microbubbles, and nanobubbles smaller than 1 μm, is progressing (Patent Document 1), and solutions containing these bubbles are now being used in various fields such as medicine, agriculture, and fisheries and aquaculture.

[0007] Then, the inventors developed a method for using a solution containing these fine bubbles (hereinafter sometimes simply referred to as "nanobubble (water)" or "NB") in "intestinal flora transplantation" (Patent Document 2).

[0008] However, Patent Document 1 primarily concerns sterilization and other purposes using bubbles, and does not relate to a so-called drug delivery system that delivers the target substance to the affected area. Furthermore, Patent Document 2 merely describes a technique for implanting the "target substance (intestinal bacteria group derived from a donor)" near the surface of the recipient's (patient's) intestinal tract, and does not involve absorbing the "target substance (intestinal bacteria)" into the intestinal tract via the intestinal cells of the intestinal wall. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Japanese Patent Publication No. 2012-582 [Patent Document 2] WO2019 / 168034 publication [Overview of the project] [Problems that the invention aims to solve]

[0010] As a result of diligent research, the inventors have surprisingly discovered that a solvent primarily composed of a hypotonic solution, or a solvent containing even finer bubbles in the hypotonic solution, promotes the intestinal absorption of medium- and high-molecular-weight compounds without the use of any other absorption enhancers, leading to the present invention. The objective of this invention is to provide an "intestinal absorption aid" that allows medium- and high-molecular-weight compounds to be absorbed from the intestinal tract without damaging intestinal cells, or an "intestinal absorption composition" or "preventive and / or therapeutic agent" containing these aids and a medium- and high-molecular-weight active substance. [Means for solving the problem]

[0011] One aspect of the present invention is an intestinal absorption aid. This intestinal absorption aid comprises (I) and (II) below and has an osmotic pressure of less than 280 mOsm (milliosmoles) / L, and is an intestinal absorption aid for absorbing (III)-iii-a-1 and / or (III)-iii-b-1 below via the intestinal tract. (I) Solvent containing hypotonic solution (II) Bubbles of nanoscale or smaller (less than 1 μm) (III)-iii-a-1: Insulin (including at least one selected from the group consisting of naturally occurring insulin, insulin produced from an insulin gene extracted from a human or other animal, insulin expressing an artificially synthesized insulin gene, recombinant insulin, insulin analogs, and insulin polymers) or a derivative thereof, wherein the derivative is an insulin derivative that has been modified by glycosylation of the insulin. (III)-iii-b-1: Dextran or its derivatives In the above embodiment of the intestinal absorption aid, it is preferable that the average bubble diameter of (II) is less than 1 μm. In the above embodiment of the intestinal absorption aid, it is preferable that the hypotonic solution in (I) consists of at least one of the following (I)-i to (I)-vii. (I)-i: Ultrapure water (I)-ii: Reverse osmosis water (I)-iii: Modified reverse osmosis water (I)-iv: Hypotonic electrolyte solutions (Solutions 1-4) consisting of physiological saline and glucose solution. (I)-v: Distilled water for injection (I)-vi: Purified water (I)-vii: Ion-exchanged water Another aspect of the present invention is an intestinal absorption composition. This intestinal absorption composition comprises the intestinal absorption aid of the above aspect, along with the following (III)-iii-a-1 and / or (III)-iii-b-1. (III)-iii-a-1: Insulin (including at least one selected from the group consisting of naturally occurring insulin, insulin produced from an insulin gene extracted from a human or other animal, insulin expressing an artificially synthesized insulin gene, recombinant insulin, insulin analogs, and insulin polymers) or a derivative thereof, wherein the derivative is an insulin derivative that has been modified by glycosylation of the insulin. (III)-iii-b-1: Dextran or its derivatives Another aspect of the present invention is a prophylactic and / or therapeutic agent for diseases caused by insulin inactivation or deficiency. The prophylactic and / or therapeutic agent comprises (I) and (II) below, an intestinal absorption adjuvant having an osmotic pressure of less than 280 mOsm (milliosmoles) / L, and (III)-iii-a-1 below. (I) Solvent containing hypotonic solution (II) Bubbles of nanoscale or smaller (less than 1 μm) (III)-iii-a-1: Insulin (including at least one selected from the group consisting of naturally occurring insulin, insulin produced from an insulin gene extracted from a human or other animal, insulin expressing an artificially synthesized insulin gene, recombinant insulin, insulin analogs, and insulin polymers) or a derivative thereof, wherein the derivative is an insulin derivative that has been modified by glycosylation of the insulin. In the above embodiment of the preventive and / or therapeutic agent, it is preferable to administer it by oral administration. In the prophylactic and / or therapeutic agent of the above aspect, it is preferable that the average bubble diameter of (II) is less than 1 μm. In the prophylactic and / or therapeutic agent of the above aspect, it is preferable that the hypotonic solution in (I) is composed of at least one of the following (I)-i to (I)-vii. (I)-i: Ultra-pure water (I)-ii: Reverse osmosis permeated water (I)-iii: Modified reverse osmosis permeated water (I)-iv: "Hypotonic electrolyte infusion solution" (liquids 1 to 4) composed of physiological saline and glucose solution (I)-v: Distilled water for injection (I)-vi: Purified water (I)-vii: Ion-exchanged water Moreover, the above object is achieved by the following First Invention to Tenth Invention.

[0012] <First Invention> An intestinal absorption assisting agent for absorbing an administered target substance via the intestinal tract, characterized by containing the following (I).

[0013] (I) A solvent containing a hypotonic solution

[0014] <Second Invention> The assisting agent according to the First Invention, characterized by containing the following (I) and (II).

[0015] (I) A solvent containing a hypotonic solution (II) Bubbles with a size of nano-scale or less (less than 1 μm)

[0016] <Third Invention> (III) The assisting agent according to the First Invention or the Second Invention, characterized in that the administered target substance is at least one selected from the following (III)-i to (III)-iii.

[0017] (III)-i: Poorly absorbable low molecular weight compounds (III)-ii: Medium molecular weight compounds (III)-iii: High molecular weight compounds

[0018] <The Fourth Invention> (III) The substance to be administered is characterized by being at least one selected from the following: An auxiliary agent according to any one of the first to third inventions.

[0019] (III)-ii-a: Peptides or their derivatives (III)-iii-a: Protein or derivative thereof (III)-iii-b: Polysaccharides or their derivatives

[0020] <The Fifth Invention> (III) An adjuvant according to any one of the first to third inventions, characterized in that the substance to be administered is (III)-iii-a-1 and / or (III)-iii-b-1 below.

[0021] (III)-iii-a-1: Insulin or its derivatives (III)-iii-b-1: Dextran or its derivatives

[0022] <The Sixth Invention> The auxiliary agent according to any one of the first to fifth inventions, characterized in that the hypotonic liquid in (I) consists of at least one of the following (I)-i to (I)-vii.

[0023] (I)-i: Ultrapure water (I)-ii: Reverse osmosis water (I)-iii: Modified reverse osmosis water (I)-iv: Hypotonic electrolyte solutions (Solutions 1-4) consisting of physiological saline and glucose solution. (I)-v: Distilled water for injection (I)-vi: Purified water (I)-vii: Ion-exchanged water

[0024] <The Seventh Invention> The auxiliary agent according to any one of the first to sixth inventions, characterized in that the average bubble diameter of (II) is less than 1 μm.

[0025] <The Eighth Invention> A composition for intestinal absorption, characterized by comprising (III) a substance to be administered, together with an auxiliary agent according to any one of the first to seventh inventions.

[0026] <The Ninth Invention> (III) The intestinal absorption composition according to the eighth invention, characterized in that the substance to be administered is at least one selected from (III)-i to (III)-iii below. (III)-i: Poorly absorbable low molecular weight compounds (III)-ii: Medium molecular compound (III)-iii: High molecular compound

[0027] <The Tenth Invention> Together with the auxiliary agent described in any one of the first to seventh inventions, (III)-iii-a below A preventive and / or therapeutic agent for diseases caused by insulin inactivation or deficiency, characterized by containing -1. (III)-iii-a-1: Insulin or its derivatives [Effects of the Invention]

[0028] The intestinal absorption aid, intestinal absorption composition, and prophylactic and / or therapeutic agent of the present invention have an extremely simple composition, mainly consisting of a hypotonic solution and not requiring the concomitant use of special drugs such as small intestinal permeable peptides. Despite this, they have the advantage of reliably absorbing the target substance, particularly medium- and high-molecular-weight compounds that have previously been extremely difficult to absorb in the intestinal tract, from the intestinal tract. Furthermore, the intestinal absorption aid of the present invention itself does not exhibit cytotoxicity to gastrointestinal cells, and (III) safe intestinal absorption promotion of the target substance is possible. Furthermore, in the case of the intestinal absorption aid of the present invention that further contains nano-sized (less than 1 μm) bubbles, the present invention has been confirmed to have the effect of protecting the target substance from decomposition and inactivation by various digestive enzymes, etc., and it has been found that the substance can fully exert its activity even after intestinal absorption. [Brief explanation of the drawing]

[0029] [Figure 1] Figure 1 shows an overview of the "In situ closed loop method" used in the test example. [Figure 2] Figure 2 shows the procedure for administering the "intestinal absorption composition" of the present invention to experimental rats. [Figure 3] Figure 3 shows the plasma insulin concentrations when the "intestinal absorption compositions" of the examples and comparative examples were administered to the intestinal tract of experimental rats. [Figure 4] Figure 4 shows the plasma concentrations of fluorescently labeled dextran when the "intestinal absorption compositions" of the examples and comparative examples were administered to the intestinal tract of experimental rats. [Figure 5] Figure 5 shows the cytotoxicity (LDH enzyme activity associated with lactate dehydrogenase (LDH) leakage) when the "intestinal absorption aids" of the examples and comparative examples were administered to the intestinal tract of experimental rats. [Figure 6] Figure 6 shows the difference in plasma insulin concentration due to differences in bubble concentration when the "intestinal absorption compositions" of the examples and comparative examples were administered to the intestinal tract of experimental rats. [Figure 7-1] Figure 7-1 shows the effect of nano-sized or smaller bubbles (NBs) on improving the "stability of insulin against digestive enzymes (trypsin)," as measured by the time course of residual insulin concentration. [Figure 7-2] Figure 7-2 shows the effect of nano-sized or smaller bubbles (NBs) on improving the "stability of insulin against digestive enzymes (trypsin)," as demonstrated by the insulin degradation rate constant and half-life. [Modes for carrying out the invention]

[0030] The present invention will be described in detail below.

[0031] [Intestinal absorption aid of the present invention] The "intestinal absorption aid" of the present invention is an aid for (III) the absorption of a substance to be administered via the intestinal tract, and is characterized by comprising the following (I). (I) Solvent containing hypotonic solution

[0032] Furthermore, in this invention, "intestinal absorption" refers to absorption through the intestinal wall. The "intestinal absorption aid" of the present invention refers to an aid that promotes intestinal absorption, regardless of its dosage form or method of administration.

[0033] (I) Solvents containing hypotonic solutions In the present invention, (I) a solvent containing a hypotonic solution means a solvent whose main component is a hypotonic solution, and the "hypotonic solution" used in the solvent in (I) means a solution with a lower osmotic pressure than solutions found in living organisms (body fluids, blood, etc.).

[0034] Specifically, this refers to liquids with an osmotic pressure lower than that of body fluids (plasma), which is approximately 285 ± 5 mOsm (milliosmoles) / L. Examples include, but are not limited to, the following.

[0035] (I)-i: Ultrapure water (There is no clear definition or international standard, but examples include ultrapure water used for medical purposes.) (I)-ii: Reverse osmosis water (RO water: High-purity water obtained by utilizing the reverse osmosis phenomenon, in which pure water that has moved to the hypertonic liquid side is pushed back to the hypotonic liquid side by a pump or other means through a semipermeable membrane (reverse osmosis membrane)). (I)-iii: Modified reverse osmosis water (I)-iv: Hypotonic electrolyte solutions (Solutions 1-4) consisting of physiological saline and glucose solution. (I)-v: Distilled water for injection (I)-vi: Purified water (I)-vii: Ion-exchanged water

[0036] However, among the above, those with low cytotoxicity are preferred.

[0037] (I)-iii's "modified reverse osmosis water" refers to water in which a small portion of the minerals contained in binchotan charcoal has been forcibly passed through, for example, by using a filtration membrane with a pore size of 0.1 μm or less as a semipermeable membrane (reverse osmosis membrane).

[0038] Furthermore, in addition to the hypotonic liquid described above, (I) of the present invention may contain various additives, etc., to the extent that they do not hinder the objectives of the present invention. Specifically, for example, isotonic solutions, hypertonic solutions, other solvents, and various additives can be included within a range that maintains the overall osmotic pressure of the intestinal absorption aid at less than approximately 285 ± 5 mOsm (milliosmoles) / L.

[0039] (II) Bubbles smaller than nanoscale (less than 1 μm) The intestinal absorption aid of the present invention may further contain the following (II).

[0040] (II) Bubbles of nanoscale or smaller (less than 1 μm)

[0041] (Types of gas in bubbles) The "gas component" in the nano-sized or smaller bubbles of (II) used in the present invention is preferably one or more of the following, but is not necessarily limited to these.

[0042] (II)-(i): Atmosphere (II)-(ii): Hydrogen (II)-(iii): Nitrogen (II)-(iv): Ozone (II)-(v): Oxygen (II)-(vi): Carbon dioxide (II)-(vii): Argon

[0043] Using the atmosphere described in (II)-(i) alone is practical and preferable because it eliminates the need to prepare special "gas components" such as (II)-(ii) to (II)-(vii).

[0044] Furthermore, accurately measuring the "sealing ratio of each gas component" in the "bubbles (II) generated in solvent (I)" when "air" or "a mixture of two or more gases" is introduced into solvent (I) is not easy with current technology. This is because, in the process of generating bubbles (II) in solvent (I), the amount and speed at which each gaseous component can dissolve in solvent (I) differs, and it is not easy to accurately determine the type and ratio of gaseous components enclosed in the generated bubbles (II). However, if a gaseous component (let's call it X) is used alone or in combination with air, the enclosed ratio of the gaseous component (X) in the bubbles (II) generated in the solvent (I) should be higher compared to using air alone. This would allow for better utilization of the properties of the gaseous component (X) as described above.

[0045] Furthermore, increasing the hydrogen content in the bubbles compared to when using air alone is preferable because it offers the following advantages.

[0046] 1. The oxidation-reduction potential of the nanobubble water of the present invention using hydrogen (target: -150mV ± 15mV) is as low as that of the oxidation-reduction potential of the intestinal environment (-50mV to -250mV). Therefore, the (III) target substance coated with it is likely to adhere to the intestinal wall, and the concentration of the substance on the surface of intestinal epithelial cells will increase. This will increase the concentration difference between inside and outside the cell, promoting passive diffusion, and thus is thought to have a high effect in promoting intestinal absorption of the target substance.

[0047] 2. When there is inflammation in the intestinal tract, the oxidation-reduction potential tends to be high, and the immune response is enhanced, making it likely that the introduced (III) target substance will be repelled by the recipient's immune system. However, it is thought that the anti-inflammatory effect due to the low oxidation-reduction potential of hydrogen can mitigate this reaction.

[0048] To increase the hydrogen content in bubbles compared to that of the atmosphere, methods include using hydrogen alone, or using a combination of atmosphere and hydrogen.

[0049] In this case, it is possible to simultaneously seal in air and hydrogen gas alone, but other methods include gradually increasing the concentration of hydrogen sealed in with the air, or using air alone initially and then sealing in hydrogen in the later stages. By using these methods, it is thought that hydrogen loss due to dissolution in the solvent can be minimized, and more hydrogen can be sealed in the bubbles.

[0050] When using air and hydrogen simultaneously, there are no particular restrictions on the ratio, but it is preferable, for example, to use at least 10 times the amount of (i) air used in the sealing operation, and more preferably at least 100 times the amount of (i) air used.

[0051] (Size of bubbles) The size of the "(II) nano-sized or smaller (less than 1 micron) bubbles" used in the "intestinal absorption aid" of the present invention is mainly nano-sized or smaller (less than 1 micron), however, the specific size can be flexibly changed depending on the type and size of the (III) substance to be absorbed from the intestinal tract by this aid.

[0052] Specifically, the size of the "(II) bubbles smaller than nanoscale (less than 1 micron)" is preferably around 900 nm or less, and more preferably several hundred nm or less. It is particularly preferable if the size is several tens of nm or less, or even several nm or less, because this may significantly improve the absorption speed and absorption rate of the (III) substance intended for administration to the intestinal tract.

[0053] However, since the effects of this invention can be fully realized even with a bubble size of several hundred nanometers, the bubble size should realistically be determined by balancing it with the manufacturing cost of reducing the bubble size.

[0054] (Percentage of nano-sized or smaller bubbles in the total number of bubbles) Of the total air bubbles in the "intestinal absorption aid," it is not necessary for "(II) nano-sized or smaller bubbles" to account for 100%.

[0055] However, in order for the (III) target substance absorbed into the intestinal tract by the "intestinal absorption aid" of the present invention to fully exert its original function, efficacy, or effect in the body, it is preferable that there be a large number of "(II) nano-sized or smaller bubbles" smaller than the (III) target substance to protect the surface or surroundings of the (III) target substance by coating or the like, or to penetrate into the inside of the (III) target substance through gaps or other openings on its surface or surroundings, thereby more closely protecting the (III) target substance. This is because it is preferable that such bubbles are present in a number sufficient to protect at least the entire surface or surroundings of each individual (III) target substance.

[0056] Furthermore, it is desirable to have as high a proportion as possible of "(II) bubbles smaller than nanosize" in order to minimize the degradation of the auxiliary agent itself due to the collapse of bubbles larger than nanosize.

[0057] Furthermore, the size of the bubbles is not always uniform, and generally there is a certain degree of bubble size distribution.

[0058] Therefore, as a concrete guideline, if the "average diameter" of the bubbles is sufficiently smaller than the overall diameter of the (III) target substance being administered, it is considered that there are many bubbles small enough to protect the (III) target substance. For example, it is preferable to use a solution with an average bubble diameter of less than 1 μm (1000 nm), and more preferably an average bubble diameter of 900 nm or less.

[0059] (Number of air bubbles in the intestinal absorption aid) A higher number of air bubbles is desirable in the "intestinal absorption aid" of the present invention. The specific number varies depending on (III) the type and concentration of the substance to be administered, and cannot be stated definitively, but it has been confirmed that several thousand to several hundred million bubbles / ml, which can generally be generated when using known manufacturing equipment capable of producing "(II) bubbles smaller than nano-size," is sufficient.

[0060] However, a concentration of tens of millions to hundreds of millions of particles per ml is considered more desirable. Furthermore, in terms of preventing the inactivation of the target substance by digestive enzymes in the body, higher concentrations are preferable, for example, several hundred million particles / ml or more is preferable, and more preferably 1 billion particles / ml or more.

[0061] Furthermore, methods for measuring minute bubbles include the Electrical Sensing Zone Method, which utilizes the Coulter principle, and specifically, methods using Beckman Coulter's "Multisizer3," "Multisizer4," and "Multisizer4e." The Coulter principle is a method for measuring the distribution of bubble diameter and number of bubbles by flowing a fixed amount of electrolyte into a cylinder (manometer) with one or more microscopic pores (apertures), placing electrodes inside and outside the manometer, applying a DC voltage (negative inside, positive outside), and measuring the change in electrical resistance between the two electrodes that occurs when particles (bubbles, etc.) pass through a detection zone (aperture-sensitive region).

[0062] Method for manufacturing intestinal absorption aids The "intestinal absorption aid" of the present invention can be manufactured by generating bubbles (II) in solvent (I).

[0063] Examples of methods for generating bubbles (II) in solvent (I) include, but are not limited to, the following methods or combinations thereof.

[0064] Gas-liquid mixed shear method: This method involves rapidly swirling a gas together with a liquid.

[0065] Ultrasonic method: This method involves applying shock waves or cavitation to a liquid to further collapse any bubbles that have already formed.

[0066] Pressurized dissolution method: This method involves applying pressure to a gas and a liquid and releasing them all at once to generate bubbles.

[0067] Micropore method: This method involves supplying gas under pressure using an orifice or similar device.

[0068] Electrolysis method: This method involves generating gas from a thin wire immersed in an aqueous solution.

[0069] Among the above methods, the "gas-liquid mixing shear method" is preferred because it can generate stable nano-sized or smaller bubbles through further shearing of the microbubbles.

[0070] Furthermore, specifically, nanobubbles can be generated by using commercially available devices such as those described in 1) and 2) below in combination. 1) Generation of bubbles with a diameter of 1 micrometer or less using the "Bavitas® HYK-25" micro-nanobubble generator manufactured by Kyowa Kisetsu Co., Ltd. (rotary shear method) 2) Nanobubble formation of micro-nanobubbles using "νG7 (registered trademark)" manufactured by Ahaha Co., Ltd. (stainless steel filter)

[0071] Furthermore, using the "ultrafine bubble (NB) generator" developed by the present inventor and described in the following patent application is particularly preferable because it can efficiently generate nanobubble water with a bubble particle size of less than 1 μm, for example, an average bubble diameter on the order of several nanometers to several hundred nanometers, and a high concentration. Japanese Patent Application No. 2020-57176

[0072] Controlling bubble concentration The concentration of "(II) nano-sized or smaller bubbles" contained in the "intestinal absorption aid" of the present invention can be appropriately controlled according to the following methods, etc., depending on the application. i) Methods to reduce the concentration of "(II) bubbles smaller than nano-size": (I) A method of dilution by first generating bubbles in the solvent, and then adding the solvent from (I) or other solvents. However, in this case, it is preferable to maintain the overall osmotic pressure of the "intestinal absorption aid" at less than approximately 285 ± 5 mOsm (milliosmoles) / L. ii) Methods to increase the concentration of "(II) bubbles smaller than nano-size": ii)-1: A method of increasing the number of rotations, rotation time, pressure, stirring speed, shear time, etc., when preparing a bubble-containing solvent using the apparatus described above. ii)-2: A method of concentrating the prepared foam-containing solvent by filtering it using methods such as ultrafiltration. Specifically, the solution can be concentrated simply and efficiently by placing the bubble-containing solvent (stock solution) into a container such as the Macrosep Advance centrifugal filtration device (Pall Japan Co., Ltd.) and concentrating it by ultrafiltration using a micro-cooled centrifuge (Kubota Shoji Co., Ltd., Model 3740). The concentration ratio varies depending on the concentration of the stock solution and the conditions of the equipment, but for example, it is possible to concentrate it to about 30 times.

[0073] Furthermore, it is believed that including "(II) bubbles smaller than nano-size" will have almost no effect on osmotic pressure.

[0074] Other ingredients Furthermore, in addition to (I) and (II), the "intestinal absorption aid" of the present invention may contain various solvents, additives, etc., to the extent that they do not hinder the objectives of the present invention.

[0075] 《Dosage Form (Dosage Form)》 The form of the "intestinal absorption aid" of the present invention is not particularly limited, and various forms such as liquid, gel, sol (including colloids, etc.) and cream are available. However, if the intestinal absorption aid of the present invention contains air bubbles as described in (II), a form that offers excellent storage stability for the air bubbles is preferred.

[0076] Dosage The dosage of the "intestinal absorption aid" of the present invention can be appropriately selected according to the activity and content of the "(III) target substance for administration" whose intestinal absorption should be promoted by this aid, the specific type of disease, the severity of symptoms, age, sex, weight, and form of administration, and cannot be specified in general terms. However, as an example, a typical adult may be given approximately 0.03 to 1000 mg per day, preferably 0.1 to 500 mg, and more preferably 0.1 to 100 mg, administered in one to several divided doses per day.

[0077] Furthermore, when using the "intestinal absorption aid" of the present invention as an aid to swallowing when taking "(III) oral preparations," an even larger amount of the "intestinal absorption aid" may be used.

[0078] 《Application》 The "intestinal absorption aid" of the present invention can be used to administer into the body almost simultaneously with the "(III) substance to be administered" described later, or with a short time difference (for example, within a few seconds to a few minutes). Furthermore, the "intestinal absorption aid" of the present invention can be used as a constituent material of the "intestinal absorption composition" of the present invention, which will be described later.

[0079] 《Administration Method》 The method of administering the "intestinal absorption aid" of the present invention is not particularly limited as long as the mode of drug absorption is intestinal absorption, and examples include enema catheterization and oral administration, but oral administration is preferred because it places less burden on the patient who is the recipient of the administration.

[0080] (III) Substances to be administered In the present invention, the "(III) substance to be administered," which is the substance to be absorbed from the intestinal tract, can be any compound with a wide range of molecular weights, from low molecular weight compounds to medium and high molecular weight compounds (or derivatives thereof). However, the present invention is particularly useful for "(III)-i: poorly absorbable low molecular weight compounds," "(III)-ii: medium molecular weight compounds," or "(III)-iii: high molecular weight compounds," which have been particularly difficult to absorb in the intestinal tract until now.

[0081] While it is difficult to draw a clear line between low-molecular-weight, medium-molecular-weight, and high-molecular-weight compounds, they are generally classified as follows. However, this is naturally not limited to the examples and definitions listed below.

[0082] Low molecular weight compounds: Small-molecule drugs, or so-called low-molecular-weight drugs, and indigestible oligosaccharides (molecular weight less than 300-500), For example, compounds with a molecular weight of less than approximately 500 are cited as examples, but the scope is not limited to these.

[0083] In particular, "(III)-i: poorly absorbable low molecular weight compounds," which have been considered especially difficult to absorb in the intestinal tract until now, such as low molecular weight compounds belonging to "BCS Class III (highly soluble - low permeability pharmaceuticals)," such as famotidine, or compounds that serve as substrates for efflux transporters, such as vinblastine, are preferred examples because they are highly effective with the adjuvant of the present invention.

[0084] (III)-ii: Medium molecular compounds: Peptide preparations such as cyclosporine A (molecular weight approximately 1202) or its derivatives ((III)-ii-a), indigestible oligosaccharides (molecular weight 500 to several thousand), For example, compounds with molecular weights ranging from approximately 500 to several thousand are examples, but the scope is not limited to these.

[0085] (III)-iii: High molecular compound: Protein preparations such as insulin (molecular weight approximately 5800) or its derivatives ((III)-iii-a-1) ((III)-iii-a), Antibody drugs, Polysaccharides such as dextran (dextran 4 with a molecular weight of approximately 4000, dextran 40 with a molecular weight of approximately 40,000, dextran 70 with a molecular weight of approximately 75,000, etc.) or their derivatives ((III)-iii-b-1), indigestible oligosaccharides (molecular weight of several thousand to 30,000), Other biopharmaceuticals commonly used by injection, For example, compounds with molecular weights ranging from several thousand to around 150,000 are examples, but the scope is not limited to these.

[0086] Furthermore, the term "(III)-iii-a-1: Insulin or its derivatives" used in this invention includes naturally derived substances, artificially produced substances, or semi-artificially produced substances.

[0087] Naturally derived substances include those extracted from humans or other animals. Examples of non-human animals include mammals, and among them, those derived from pigs are preferred because they have properties similar to human insulin.

[0088] Examples of semi-artificially produced insulin include those produced from insulin genes extracted from humans or other animals, as mentioned above.

[0089] As for artificially produced items, 1) Insulin that expresses an artificially synthesized insulin gene. 2) Recombinant insulin (e.g., rapid-acting insulin analog preparations, etc.) expressing recombinant genes in which a portion of the "insulin gene" extracted from nature has been deleted, substituted, added, and / or inserted. 3)2) "Insulin analogs" are created by expressing genes that were artificially synthesized from scratch using the recombinant gene sequences described in 2). 4) "Insulin derivatives" which are natural, semi-synthetic, or artificially synthesized insulin that has been modified by sugar chain addition or other means. These are some examples.

[0090] Furthermore, the insulin used in this invention also includes cases where multiple molecules are aggregated, such as hexamers.

[0091] [The intestinal absorption composition of the present invention] The "intestinal absorption composition" of the present invention is characterized by containing the above-mentioned "intestinal absorption aid" of the present invention along with the (III) substance to be administered.

[0092] The "composition for intestinal absorption" of the present invention means a "composition" or "drug" that is absorbed from the intestinal tract, regardless of its dosage form or method of administration.

[0093] Other ingredients In addition, the "intestinal absorption composition" of the present invention may contain various additives, etc., in addition to (I), (II), and (III), as long as they do not hinder the objectives of the present invention.

[0094] 《Dosage Form (Dosage Form)》 The form of the "intestinal absorption composition" of the present invention is not particularly limited, and various forms include liquid, gel, sol (including colloids, etc.), cream, soft capsules, enteric-coated capsules, and other capsule forms. However, if the intestinal absorption composition of the present invention contains air bubbles as described in (II), a form that offers excellent storage stability for the air bubbles is preferred.

[0095] Enteric-coated capsules are capsules in which a drug tablet or capsule is coated with a substance that dissolves only after reaching the small intestine.

[0096] 《Administration Method》 The method of administering the "intestinal absorption composition" of the present invention is not particularly limited as long as the mode of drug absorption is intestinal absorption, and examples include enema catheterization and oral administration, but oral administration is preferred because it places less burden on the patient who is the recipient of the administration.

[0097] Dosage The dosage of the "intestinal absorption composition" of the present invention can be appropriately selected according to the activity and content of the "(III) substance for administration" contained, the specific type of disease, the severity of symptoms, age, sex, weight, and administration method, and cannot be specified in general terms. However, as an example, a typical adult may be given approximately 0.03 to 1000 mg per day, preferably 0.1 to 500 mg, and more preferably 0.1 to 100 mg, administered in one to several divided doses per day.

[0098] Method for manufacturing the composition The "intestinal absorption composition" of the present invention may be prepared by first manufacturing the above-mentioned "intestinal absorption aid" and then mixing it with "(III) the substance to be administered," or it may be prepared by mixing "(III) the substance to be administered" during the manufacturing process of the "intestinal absorption aid."

[0099] 《Application》 The "intestinal absorption composition" of the present invention can promote the intestinal absorption of high molecular weights such as insulin, and therefore can be used as a "preventive and / or therapeutic agent for diseases caused by insulin inactivation or deficiency" as described below.

[0100] [The present invention provides a preventive and / or therapeutic agent for diseases caused by insulin inactivation or deficiency] The "preventive and / or therapeutic agent for diseases caused by insulin inactivation or deficiency" of the present invention is characterized by containing the above-mentioned "intestinal absorption aid" of the present invention, along with the following (III)-iii-a-1.

[0101] (III)-iii-a-1: Insulin or its derivatives

[0102] The details of insulin or its derivatives in (III)-iii-a-1 are as described above.

[0103] Diseases caused by insulin deficiency include, but are not limited to, the following:

[0104] Type I diabetes Type II diabetes

[0105] Other ingredients Furthermore, the "preventive and / or therapeutic agent" of the present invention may contain various additives, etc., in addition to insulin of type (I), (II), and (III)-iii-a-1 or its derivatives, as long as they do not hinder the objectives of the present invention.

[0106] The administration form (dosage form), method of administration, dosage, manufacturing method, and other aspects of the "preventive and / or therapeutic agent" of the present invention are the same as those of the "composition for intestinal absorption" described above. The "intestinal absorption composition" of the present invention described above can promote the intestinal absorption of high molecular weights such as insulin, and therefore can be used as a "preventive and / or therapeutic agent for diseases caused by insulin inactivation or deficiency." [Examples]

[0107] [Examples 1-5, Comparative Examples 1-2: Intestinal Absorption Adjuvants] The following were prepared as "intestinal absorption aids" for the examples or comparative examples and used as raw materials for the "intestinal absorption composition" of the present invention, which will be described later.

[0108] In the following, nanobubbles (bubbles smaller than nano-size) may be simply referred to as "NB".

[0109] (Comparative example 1: Isotonic solution A) Isotonic solution (282 mOsm / L): PBS (phosphate-buffered saline, pH 7.4)

[0110] (Comparative example 2: Isotonic solution B) The following bubbles were generated in the isotonic solution of Comparative Example 1 by using the "ultrafine bubble (NB) generator" described in Patent 2020-57176. (II) Bubbles smaller than nanoscale (average bubble diameter: approximately 200 nm or less, bubble concentration: several hundred million cells / ml, internal gas: a portion of the atmosphere replaced with 3G pure hydrogen (purity 99.999%))

[0111] (Example 1: Hypotonic solution A) Hypotonic solution (approximately 130mOsm / L or less): Ultrapure water

[0112] (Example 2: Hypotonic solution B) Hypotonic solution (100 mOsm / L or less): (I)-iii above: Modified RO water

[0113] (Example 3: Hypotonic solution B + NB (NB at basic concentration)) In Example 2, the following bubbles were generated in the hypotonic liquid using the "ultrafine bubble (NB) generator" described in Patent 2020-57176.

[0114] (II) Bubbles smaller than nanoscale (average bubble diameter: approximately 200 nm or less, bubble concentration: several hundred million cells / ml, internal gas: a portion of the atmosphere replaced with 3G pure hydrogen (purity 99.999%)) Furthermore, the "Multisizer4e" manufactured by Beckman Coulter, Inc., as mentioned above, was used to measure the bubble concentration.

[0115] (Example 4: Hypotonic solution B + NB (low concentration of NB)) The intestinal absorption aid of "Hypotonic Solution B + NB (NB at basic concentration)" from Example 3 was diluted five times with the aid consisting of hypotonic solution A from Example 1.

[0116] (II) Bubbles smaller than nanoscale (average bubble diameter: approximately 200 nm or less, bubble concentration: several thousand to 100 million bubbles / ml, internal gas: a portion of the atmosphere replaced with 3G pure hydrogen (purity 99.999%))

[0117] (Example 5: Hypotonic solution B + NB (high concentration NB)) In the hypotonic liquid of Example 2, when generating NB using the "Ultra Fine Bubble (NB) Generator" described in Patent 2020-57176, the following bubbles were generated by stirring for four times the time of Example 3.

[0118] (II) Bubbles smaller than nanoscale (average bubble diameter: approximately 200 nm or less, bubble concentration: several hundred million to 1 billion bubbles / ml, internal gas: a portion of the atmosphere replaced with 3G pure hydrogen (purity 99.999%))

[0119] [Examples 6-8, Comparative Examples 3-4: Compositions for intestinal absorption ((III) Target substance for administration: insulin)] Using the "intestinal absorption aid" of the above-mentioned examples or comparative examples, an insulin administration solution was prepared according to the following procedure to obtain the "intestinal absorption composition ((III) target substance for administration: insulin ((III)-iii-a-1))" of the present invention.

[0120] (Method of manufacturing insulin solution) 1) 2.909 mg of solid insulin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., human recombinant insulin) was mixed with 0.1 ml of 0.1 M HCl. 2)1) 3.8 ml each of the above examples or comparative examples of "intestinal absorption aids" containing 0.001 W / V% of the following MC as an insulin container adsorption inhibitor was added to 1). 3)2) 0.1 ml of 0.1 M NaOH was added to prepare the insulin administration solution (composition for intestinal absorption).

[0121] Insulin concentration: 2.909 mg / 4 ml = 0.727 mg / ml ≈ 20 IU / ml

[0122] MC (Methylcellulose): Manufactured by Shin-Etsu Chemical Co., Ltd., Metroze® SM-15 (Molecular weight approximately 60,000)

[0123] (Example 6) Using the "intestinal absorption aid (ultrapure water)" from Example 1, an insulin administration solution (intestinal absorption composition from Example 6) was prepared according to the method described above.

[0124] (Example 7) Using the "intestinal absorption aid (modified RO water)" from Example 2, an insulin administration solution (intestinal absorption composition from Example 7) was prepared according to the method described above.

[0125] (Example 8) Using the "intestinal absorption aid (modified RO water + basic concentration NB)" from Example 3, an insulin administration solution (intestinal absorption composition from Example 8) was prepared according to the method described above.

[0126] (Comparative Example 3) Using the "intestinal absorption aid (PBS)" of Comparative Example 1, an insulin administration solution (intestinal absorption composition of Comparative Example 3) was prepared according to the method described above.

[0127] (Comparative Example 4) Using the "intestinal absorption aid (PBS + basic concentration NB)" of Comparative Example 2, an insulin administration solution (intestinal absorption composition of Comparative Example 4) was prepared according to the method described above.

[0128] [Examples 9-11, Comparative Example 5: Composition for intestinal absorption ((III) Substance to be administered: Dextran)] Using the "intestinal absorption aid" described in the above-mentioned examples or comparative examples, a dextran administration solution was prepared according to the following procedure to obtain the "intestinal absorption composition ((III) target substance for administration: dextran ((III)-iii-b-1))" of the present invention.

[0129] (Method of manufacturing dextran administration solution) In the above-described examples or comparative examples, "FD4 (FITC (Fluorescein isothiocyanate: fluorescently labeled) dextran, molecular weight: approximately 4,000)" was dissolved in the "intestinal absorption aid" to a concentration of 4 mg / ml to prepare a dextran administration solution (intestinal absorption composition).

[0130] (Examples 9-11) Using the "intestinal absorption aids" of Examples 2, 3, and 5, dextran (FD4) administration solutions (intestinal absorption compositions of Examples 9-11) were prepared according to the method described above. Example 9: Example 2 (modified RO water) + FD4 Example 10: Example 3 (modified RO water + basic concentration NB) + FD4 Example 11: Example 5 (modified RO water + high concentration NB) + FD4

[0131] (Comparative Example 5) Using the "intestinal absorption aid (PBS)" of Comparative Example 1, a dextran administration solution (intestinal absorption composition of Comparative Example 5) was prepared according to the method described above.

[0132] [Test Example 1: Test to confirm the effect of a composition for intestinal absorption of insulin on promoting intestinal absorption] Using the known in situ closed-loop method shown in Figure 1, the insulin administration solution (intestinal absorption composition) of the example or comparative example was directly administered to the intestinal tract of the target animal (rat), and then the amount of insulin absorbed via the intestinal tract was measured, thereby confirming the effect of the "intestinal absorption composition" of the present invention on promoting the absorption of high molecular weight compounds (insulin).

[0133] Figure 2 shows the specific administration procedure for the "composition for intestinal absorption."

[0134] The other experimental conditions are as follows:

[0135] Materials and Methods (Target recipients) SD rats, male, 190g (3-10 rats per group)

[0136] (Administered solution) Examples 6-8: "Insulin administration solution (composition for intestinal absorption)" ... Hypotonic solution (+NB) + insulin Comparative Examples 3-4: "Insulin administration solution (composition for intestinal absorption)" ... Isotonic solution (+NB) + insulin

[0137] (Dosage) 0.5 ml / (rat body weight) 200 g (= 2.5 ml / kg)

[0138] Furthermore, as mentioned above, the insulin concentration in the "insulin administration solution (composition for intestinal absorption)" is 0.727 mg / ml ≈ 20 IU / ml. Therefore, administering 2.5 ml / kg of the "insulin administration solution (composition for intestinal absorption)" corresponds to an "insulin dosage of approximately 50 IU / kg".

[0139] (Blood collection site after administration) jugular vein

[0140] (Method for measuring plasma insulin concentration) ELISA (Enzyme-linked immunosorbent assay) (Equipment used) Insulin, Human, ELISA Kit / MRD-10-1113-01-1

[0141] "result" The measurement results are shown in Figure 3. In Figure 3, the unit "μU / ml" for extracted insulin means "μIU / ml". Furthermore, although not shown in the figure, an "insulin administration solution (intestinal absorption composition)" was prepared in the same manner as above using the intestinal absorption aid of Example 4 (low concentration NB) or Example 5 (high concentration NB), and the same test as in Test Example 1 was performed. The results showed that, although there were some differences depending on the concentration of air bubbles (NB), all of them could be reliably absorbed into the body via the intestinal tract.

[0142] 《Consideration》 The results in Figure 3, which show a significant increase in plasma insulin concentration compared to the comparative example, demonstrate that by using a hypotonic solution as the solvent (intestinal absorption aid), even high-molecular-weight compounds (insulin), which have previously been considered extremely difficult to absorb from the intestinal tract, can be reliably absorbed into the body via the intestinal tract. Furthermore, when NB was added to the hypotonic solution, the absorption-promoting effect was slightly reduced compared to the hypotonic solution alone. i) Other verification experiments conducted by the inventors (data not shown) have confirmed that when NB water is used, the storage stability of various active substances in the solvent before administration to a living organism tends to improve. ii) Furthermore, as shown in Test Example 5 below, it was confirmed that using a solution containing NB in ​​addition to hypotonic solution was significantly more effective in protecting insulin from enzymatic degradation in the gastrointestinal tract compared to using hypotonic solution alone (Figures 7-1 and 7-2). As a result, it is possible that the total activity of insulin after intestinal absorption can be maintained at a higher level than with hypotonic solution alone. Considering all these factors, it is thought that a solution containing NB in ​​addition to hypotonic solution may be preferable for intestinal absorption of the target substance compared to a hypotonic solution alone.

[0143] [Test Example 2: Test to confirm the effect of dextran on promoting intestinal absorption using a composition for intestinal absorption] Using the same method as in Test Example 1, the dextran administration solution (intestinal absorption composition) of the example or comparative example was directly administered to the intestinal tract of the target subject (rat), and then the amount of dextran absorbed via the intestinal tract was measured, thereby confirming the effect of the "intestinal absorption composition" of the present invention on promoting the absorption of high molecular weight compounds (dextran).

[0144] The specific administration procedure for the "composition for intestinal absorption" was carried out according to Figure 2. However, blood sampling was performed up to 240 minutes. The other experimental conditions are as follows:

[0145] Materials and Methods (Target recipients) SD rats, male, 190g (3-4 rats per group)

[0146] (Administered solution) Examples 9-11: "Dextran administration solution (composition for intestinal absorption)" ... Hypotonic solution (modified RO water) (+NB) + FD4 Comparative Example 5: "Dextran administration solution (composition for intestinal absorption)" ... Isotonic solution (PBS) + FD4

[0147] (Dosage) 0.5 ml / (rat body weight) 200 g (= 2.5 ml / kg) As mentioned above, the concentration of the dextran solution is 4 mg / ml, so the above dosage corresponds to a fluorescently labeled dextran dosage of 10 mg / (rat body weight) kg.

[0148] (Blood collection site after administration) jugular vein

[0149] (Method for measuring dextran concentration in plasma) The fluorescence intensity was measured and then converted to concentration.

[0150] "result" The measurement results are shown in Figure 4. Note that "means±SE" in the figure means the mean value plus the standard error. Furthermore, N in the diagram represents the number of rats per group.

[0151] 《Consideration》 The results in Figure 4, which show a significant increase in dextran concentration in plasma compared to the comparative example, demonstrate that by using a hypotonic solution as the solvent (intestinal absorption aid), even high-molecular-weight compounds (dextran), which have previously been considered extremely difficult to absorb from the intestinal tract, can be reliably absorbed into the body via the intestinal tract.

[0152] [Test Example 3: Cytotoxicity Confirmation Test of Intestinal Absorption Adjuvants] Lactate dehydrogenase (LDH) is an enzyme present in the cytoplasm of almost all cells. Normally, it does not permeate the cell membrane, but it is known to leak out of the cell if the cell membrane is damaged for any reason. Therefore, measuring the amount of LDH released extracellularly can be used as an indicator of the cytotoxicity of intestinal absorption adjuvants.

[0153] Therefore, using the same in situ closed-loop method as in Test Example 1, each of the "intestinal absorption aids" in the Examples or Comparative Examples was administered to the small intestine of rats. After 1 hour, the administered solution was collected from the intestinal tract, and the amount of LDH contained in the collected solution was measured using the kit described below to determine whether or not cytotoxicity was caused by the intestinal absorption aid.

[0154] Materials and Methods

[0155] (Target recipients) SD rats, male, 190g (3 rats per group)

[0156] (Administered solution) Examples 1-3: "Intestinal absorption aid" ... Hypotonic solution (+NB) Comparative Examples 1-2: "Intestinal Absorption Aid" ... Isotonic solution (+NB)

[0157] Furthermore, the solution shown in Reference Example 1 below was used as an example of a cytotoxic case (positive control).

[0158] (Reference example 1) PBS (isotonic solution) containing 5% Triton X-100... Isotonic solution + Triton X-100

[0159] Triton X-100 is a known surfactant that can solubilize biological membranes without inactivating proteins, and in this test example, it is used as a "positive control" to induce LDH leakage.

[0160] (Dosage) 0.5 ml / (rat body weight) 200 g

[0161] (Equipment used) Dojindo Cytotoxicity Assay Kit (Product name: Cytotoxicity LDH Assay Kit-WST)

[0162] "result" The test results are shown in Figure 5.

[0163] As can be seen in Figure 5, in both the examples and comparative examples, the amount of LDH leakage was extremely low compared to Reference Example 1, which used TritonX-100, a common indicator of cell damage.

[0164] 《Consideration》 In other words, the enhancement of insulin and dextran absorption by the "intestinal absorption aid" of the present invention does not involve forced entry into the intestinal tract after destroying cells, and it has been confirmed that there are no safety issues. came.

[0165] [Example 12: Intestinal absorption aid] Example 3: "Intestinal absorption aid (modified RO water + basic concentration NB)" (hereinafter referred to as NanoGAS) TM (RO)(N:Normal)) is sometimes written as (RO)(N:Normal). 20 mL is placed in a Macrosep Advance centrifugal filtration device (Nippon Pall Co., Ltd.), and concentrated by ultrafiltration using a micro-refrigerated centrifuge (Kubota Shoji Co., Ltd., Model 3740) under centrifugal conditions of 3000xg, 4℃, 15 minutes, to increase the bubble concentration of NB, resulting in "intestinal absorption aid (modified RO water + high concentration NB)" (hereinafter referred to as NanoGAS). TM It may be written as (RO)(C:Conc). 1.5 mL was prepared. Furthermore, a comparison of bubble concentrations using a simplified measurement method confirmed that the high-concentration NB water obtained by this ultrafiltration contained approximately 30 times the amount of bubbles compared to the base concentration.

[0166] [Example 13: Composition for intestinal absorption ((III) Target substance for administration: insulin)] Using the intestinal absorption aid of Example 12, an insulin administration solution (intestinal absorption composition of Example 13) was prepared in accordance with the method described in Example 6, etc. Therefore, the insulin concentration was approximately 20 IU / ml, similar to that in Example 6.

[0167] [Test Example 4: Test to confirm the effect of insulin intestinal absorption-promoting compositions with different bubble concentrations] Except for the experimental conditions described later, the absorption-promoting effect of the "intestinal absorption compositions" of the present invention with different bubble concentrations was confirmed using the in situ closed-loop method, in the same manner as in Test Example 1.

[0168] The experimental conditions are as follows:

[0169] Materials and Methods

[0170] (Target recipients) SD rats, male, 190-230g (3-6 rats per group)

[0171] (Administered solution) "Composition for intestinal absorption in Example 7: Insulin administration solution (Hypotonic solution (RO) + insulin)" "Composition for intestinal absorption in Example 8: Insulin administration solution (Hypotonic solution (RO) + NB (basic concentration) + insulin)" "Composition for intestinal absorption in Example 13: Insulin administration solution (Hypotonic solution (RO) + NB (high concentration) + insulin)" "Comparative Example 3: Composition for intestinal absorption: Insulin administration solution (isotonic solution (PBS) + insulin)" "Comparative Example 4: Composition for intestinal absorption: Insulin administration solution (isotonic solution (PBS) + NB (basic concentration) + insulin)"

[0172] "result" The measurement results are shown in Figure 6.

[0173] Note that "NanoGAS" in Figure 6 TM " refers to NB (Water) (Intestinal Absorption Adjuvant) manufactured by Symbiosis Co., Ltd. Furthermore, in Figure 6, the unit "μU / ml" for extracted insulin means "μIU / ml". As can be seen in Figure 6, by using a hypotonic solution as a solvent (intestinal absorption aid) in the composition, it was found that even high-molecular-weight compounds (insulin), which have previously been considered extremely difficult to absorb from the intestinal tract, can be reliably absorbed into the body via the intestinal tract. Furthermore, in particular, the amount of insulin absorbed in the intestinal tract was especially high in Example 13, where the concentration of nano-sized or smaller bubbles (NBs) was increased.

[0174] 《Consideration》 In addition to the intestinal absorption effect of hypotonic solutions, it was found that the presence of high concentrations of nano-sized or smaller bubbles further improves the gastrointestinal absorption of insulin.

[0175] [Examples 14-16, Comparative Example 6: Composition for intestinal absorption ((III) Target substance for administration: insulin)] Using the "intestinal absorption aids" of Examples 2, 3, 12 or Comparative Example 1 described above, an insulin administration solution was prepared according to the following procedure to obtain the "intestinal absorption composition ((III) target substance for administration: insulin ((III)-iii-a-1))" of the present invention.

[0176] (Method of manufacturing insulin solution) 1) 7.5 mg of solid insulin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., human recombinant insulin) was mixed with 75 μL of 0.1 M HCl. 2)1) 2.85 ml each of the "intestinal absorption aids" from Examples 2, 3, 12 or Comparative Example 1, which contain 0.001 W / V% of the following MC as an insulin container adsorption inhibitor, was added to each of the above samples. 3)2) 75 μL of 0.1 M NaOH was added to obtain the insulin administration solution (composition for intestinal absorption) of Examples 14-16 or Comparative Example 6.

[0177] (Insulin concentration) 7.5 mg / 3 mL = 2.5 mg / mL ≈ 69 IU / mL

[0178] MC (Methylcellulose): Manufactured by Shin-Etsu Chemical Co., Ltd., Metroze® SM-15 (Molecular weight approximately 60,000)

[0179] [Test Example 5: Confirmation test to confirm improved stability of insulin against digestive enzyme (trypsin) degradation by NB] Insulin is actually known to be broken down or otherwise inhibited by various digestive enzymes such as trypsin before it reaches the intestinal tract. Therefore, we investigated the protective effect of NB against the digestive enzyme-mediated degradation of insulin in vitro. Specifically, the protective effect of NB on insulin against digestive enzyme degradation was evaluated by the time course of residual insulin concentration (Figure 7-1), the insulin degradation rate constant, and half-life (Figure 7-2).

[0180] The experimental conditions are as follows:

[0181] Materials and Methods

[0182] (Trypsin concentration) 2.38 mg of trypsin (Sigma-Aldrich, Trypsin derived from porcine pancreas) was administered to 1 mL of each of the "insulin administration solutions (composition for intestinal absorption)" from Examples 14-16 or Comparative Example 6, as listed below, to a final concentration of 10 μM.

[0183] In addition, control subject 1 was not administered trypsin.

[0184] In addition, control subject 2 was administered 1.25 mg / ml of a known STI (soy trypsin inhibitor) along with trypsin.

[0185] (Test liquid) "Insulin administration solution of Example 14 (Hypotonic solution (RO) + insulin)" + Trypsin "Insulin administration solution of Example 15 (Hypotonic solution (RO) + NB (basic concentration) + insulin)" + Trypsin "Insulin administration solution of Example 16 (Hypotonic solution (RO) + NB (high concentration) + insulin)" + Trypsin "Insulin administration solution of Comparative Example 6 (isotonic solution (PBS) + insulin)" + trypsin "Control example 1 (isotonic solution (PBS) + insulin)" ... (without trypsin) "Control example 2 (isotonic solution (PBS) + insulin)" + trypsin + trypsin inhibitor (STI)

[0186] (Test method) Trypsin was added to each test solution to the above concentration, and the samples were stored in a constant temperature bath at 37°C for 90 minutes. As shown in Figure 7-1, the residual insulin concentration was measured periodically by HPLC (Figure 7-1). Furthermore, the results of determining the insulin degradation rate constant and half-life based on these measurement results are shown in Figure 7-2.

[0187] "result" Note that "NanoGAS" is shown in Figures 7-1 and 7-2. TM " refers to NB (Water) (Intestinal Absorption Adjuvant) manufactured by Symbiosis Co., Ltd.

[0188] In Examples 15 and 16, which include NB, the half-life of insulin is: "Control example 1 that does not contain trypsin," "Control case 2: Combination of trypsin with a known trypsin inhibitor (STI)" Not quite to that extent, Compared to the "intestinal absorption composition containing only PBS (Comparative Example 6)," the absorption period was extended by 2 to 2.5 times.

[0189] However, there was not much difference depending on the concentration of NB (Example 15: basic concentration, Example 16: high concentration).

[0190] Furthermore, Example 14 (hypotonic solution only, no NB) did not contribute much to the stability of insulin relative to trypsin.

[0191] 《Consideration》 1) Regarding the usefulness of NB: According to the above-mentioned Test Example 1 (In situ closed-loop method: a test in which the drug is directly administered to the small intestine where trypsin secretion is minimal), Example 8, which contained NB, did not show the same level of intestinal absorption as Examples 6 and 7, which used only hypotonic solution. However, Test Example 5 confirmed that the effect of protecting insulin from trypsin inhibition was higher when NB was present. In other words, the presence of NB is likely important when considering the inhibition of the activity of the target substance by trypsin.

[0192] Furthermore, as the inventors revealed through zeta potential measurements, this is thought to be due to the negative charge on the surface of NB. In other words, since insulin has a negative charge and trypsin has a positive charge in the neutral pH range, it is thought that the negatively charged NB inhibited the interaction between insulin and trypsin.

[0193] 2) Regarding enzymes other than trypsin: Furthermore, the enzymatic degradation experiment in this example 5 was an in vitro experiment using only trypsin as the insulin-degrading enzyme. However, in the body, there are enzymes other than trypsin that break down insulin, and it is thought that Example 16, which has a high concentration of NB, can inhibit a wider variety and larger quantity of enzymes than Example 15, which has the base concentration of NB. This is likely the reason why, in the insulin intestinal absorption experiment of Test Example 4 (Figure 6), Example 16, which contained a higher concentration of NB, showed a more significant enhancing effect than Example 15, which contained the base concentration.

[0194] 3) Examples using only hypotonic solutions Furthermore, although Example 14 (hypotonic solution only, without NB) did not contribute to the stability of insulin against trypsin, considering that in Test Example 1 (Figure 3), "Example 7 with only hypotonic solution," which corresponds to Example 14, showed a higher intestinal absorption effect of insulin than the other examples with NB, it can be considered that in cases where the effect of trypsin on the target substance does not need to be given much consideration, an intestinal absorption aid consisting of a hypotonic solution without NB (or an intestinal absorption composition containing the target substance together with it) is also quite useful. [Industrial applicability]

[0195] The intestinal absorption aid of the present invention has an extremely simple composition, mainly consisting of a hypotonic solution and not requiring the concomitant use of special drugs such as small intestinal permeable peptides. Despite this, it has the advantage of reliably absorbing (III) the target substance, particularly substances that have been extremely difficult to absorb in the intestinal tract until now (for example, (III)-i: poorly absorbable low-molecular-weight compounds, (III)-ii: medium-molecular-weight compounds, or (III)-iii: high-molecular-weight compounds, etc.) from the intestinal tract. Furthermore, the intestinal absorption aid of the present invention itself does not exhibit cytotoxicity to gastrointestinal cells, and (III) safe intestinal absorption promotion of the target substance is possible. Furthermore, in the case of the intestinal absorption aid of the present invention which further contains nano-sized or smaller bubbles (less than 1 μm), (III) the effect of protecting the target substance from decomposition and inactivation by various digestive enzymes, etc., has been confirmed by the present invention, and it has been found that its activity can be fully exercised even after intestinal absorption.

Claims

1. An intestinal absorption aid for absorbing the following (III)-iii-a-1 and / or (III)-iii-b-1 via the intestinal tract, comprising (I) and (II) below and having an osmotic pressure of less than 280 mOsm (milliosmoles) / L. (I) Solvent containing a hypotonic solution (II) Bubbles smaller than nanoscale (less than 1 μm) (III)-iii-a-1: Insulin (including at least one selected from the group consisting of naturally occurring insulin, insulin produced from an insulin gene extracted from a human or other animal, insulin expressing an artificially synthesized insulin gene, recombinant insulin, insulin analogs, and insulin polymers) or a derivative thereof, wherein the derivative is an insulin derivative that has been modified by glycosylation of the insulin. (III)-iii-b-1: Dextran or its derivative

2. The intestinal absorption aid according to claim 1, characterized in that the average bubble diameter of (II) is less than 1 μm.

3. The intestinal absorption aid according to claim 1, characterized in that the hypotonic solution in (I) consists of at least one of the following (I)-i to (I)-vii. (I)-i: Ultrapure water (I)-ii: Reverse osmosis water (I)-iii: Modified reverse osmosis water (I)-iv: Hypotonic electrolyte infusion (Solutions 1-4) consisting of physiological saline and glucose solution. (I)-v: Distilled water for injection (I)-vi: Purified water (I)-vii: Ion-exchanged water

4. An intestinal absorption composition characterized by comprising the following (III)-iii-a-1 and / or (III)-iii-b-1, together with the intestinal absorption aid described in any one of claims 1 to 3. (III)-iii-a-1: Insulin (including at least one selected from the group consisting of naturally occurring insulin, insulin produced from an insulin gene extracted from a human or other animal, insulin expressing an artificially synthesized insulin gene, recombinant insulin, insulin analogs, and insulin polymers) or a derivative thereof, wherein the derivative is an insulin derivative that has been modified by glycosylation of the insulin. (III)-iii-b-1: Dextran or its derivative

5. An intestinal absorption adjuvant comprising (I) and (II) below, having an osmotic pressure of less than 280 mOsm (milliosmoles) / L, The following (III)-iii-a-1 and A preventive and / or therapeutic agent for diseases caused by insulin inactivation or deficiency, characterized by containing the following: (I) Solvent containing a hypotonic solution (II) Bubbles smaller than nanoscale (less than 1 μm) (III)-iii-a-1: Insulin (including at least one selected from the group consisting of naturally occurring insulin, insulin produced from an insulin gene extracted from a human or other animal, insulin expressing an artificially synthesized insulin gene, recombinant insulin, insulin analogs, and insulin polymers) or a derivative thereof, wherein the derivative is an insulin derivative that has been modified by glycosylation of the insulin.

6. The preventive and / or therapeutic agent according to claim 5, administered by oral administration.

7. The preventive and / or therapeutic agent according to claim 5, characterized in that the average bubble diameter of (II) is less than 1 μm.

8. The preventive and / or therapeutic agent according to any one of claims 5 to 7, characterized in that the hypotonic solution in (I) consists of at least one of the following (I)-i to (I)-vii. (I)-i: Ultrapure water (I)-ii: Reverse osmosis water (I)-iii: Modified reverse osmosis water (I)-iv: Hypotonic electrolyte infusion (Solutions 1-4) consisting of physiological saline and glucose solution. (I)-v: Distilled water for injection (I)-vi: Purified water (I)-vii: Ion-exchanged water

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