Composition for intestinal absorption, agent for treating diabetes, method for producing composition for intestinal absorption, and method for producing agent for treating diabetes

Abstract

The present invention provides a composition for intestinal absorption that allows a medicine containing a poorly soluble compound to be orally administered; an agent for treating diabetes that contains the composition for intestinal absorption; a method for producing the composition for intestinal absorption; and a method for producing the agent for treating diabetes. This composition for intestinal absorption comprises first fine bubbles, a poorly soluble compound, and a physiological solution, wherein the concentration of the first fine bubbles is two billion per mL or more, and at least a part of the poorly soluble compound covers at least a part of the first fine bubbles.

Description

Composition for intestinal absorption, agent for treating diabetes, method for producing the composition for intestinal absorption, and method for producing the agent for treating diabetes

[0001] The present invention relates to a composition for intestinal absorption, a drug for treating diabetes, a method for producing a composition for intestinal absorption, and a method for producing a drug for treating diabetes.

[0002] Generally, there are various methods for administering drugs into the body, such as injections, transdermal agents, and oral preparations. In particular, when a drug contains poorly soluble compounds, these compounds are hardly absorbed by the small intestine, which is responsible for gastrointestinal absorption, so drug administration by injection is employed. For example, Patent Document 1 discloses an injectable antidiabetic agent containing insulin.

[0003] Japanese Patent Publication No. 2022-180537

[0004] However, administering medication by injection is often painful and may require hospital visits, placing a significant physical and mental burden on patients. This is especially true for diabetic patients, who require multiple daily insulin injections for treatment, making the burden particularly heavy. Therefore, there is a need for alternatives to oral medications.

[0005] The present invention addresses the above-mentioned problems and aims to provide: an intestinal absorption composition that allows oral administration of a drug containing a poorly soluble compound; a diabetes treatment agent comprising the composition; a method for producing the intestinal absorption composition; and a method for producing the diabetes treatment agent.

[0006] One aspect of the present invention is an intestinal absorption composition. The intestinal absorption composition contains first microbubbles, a sparingly soluble compound, and a physiological solution, wherein the concentration of the first microbubbles is 2 billion / mL or more, and at least a portion of the sparingly soluble compound coats at least a portion of the first microbubbles.

[0007] In the above embodiment of the intestinal absorption composition, it is preferable that the concentration of the first microbubbles is 15 billion / mL or more.

[0008] In the above embodiment of the intestinal absorption composition, it is preferable that the concentration of the first microbubbles is 30 billion / mL or more.

[0009] In the above embodiment of the intestinal absorption composition, it is preferable that the average bubble diameter of the first microbubbles is less than 1.0 μm.

[0010] In the above embodiment of the intestinal absorption composition, it is preferable that the average bubble diameter of the first microbubbles is 200 nm or less.

[0011] In the above embodiment of the intestinal absorption composition, it is preferable that the first microbubbles contain a gas selected from the group consisting of hydrogen, helium, oxygen, nitrogen, methane, fluorine, neon, carbon dioxide, nitric oxide, oxygen monoxide, ozone, argon, chlorine, ethane, propane, air, and mixed gases containing these.

[0012] In the above embodiment of the intestinal absorption composition, it is preferable that the first microbubbles contain a medical gas.

[0013] In the above embodiment of the intestinal absorption composition, it is preferable that the poorly soluble compound is insulin or a derivative thereof.

[0014] In the above embodiment of the intestinal absorption composition, it is preferable that the physiological solution contains one or more selected from ultrapure water, reverse osmosis water, modified reverse osmosis water, physiological saline, glucose solution, distilled water for injection, purified water, phosphate buffer, and ion-exchanged water.

[0015] In the above embodiment of the intestinal absorption composition, when the concentration of the poorly soluble compound is 120 pM or higher, it is preferable that the absorbance measured at a wavelength of 450 nm and an optical path length of 5.0 mm is 0.05 or higher.

[0016] In the above embodiment of the intestinal absorption composition, it is preferable that the membrane permeability coefficient of the sparingly soluble compound when the concentration of the first microbubbles is 5 billion / mL is greater than the membrane permeability coefficient of the sparingly soluble compound when the concentration of the first microbubbles is 0 billion / mL, and that the membrane permeability coefficient of the sparingly soluble compound when the concentration of the first microbubbles is 10 billion / mL is greater than the membrane permeability coefficient of the sparingly soluble compound when the concentration of the first microbubbles is 5 billion / mL.

[0017] In the above embodiment of the intestinal absorption composition, it is preferable that it be administered orally.

[0018] Another aspect of the present invention is a drug for the treatment of diabetes. This drug for the treatment of diabetes includes the intestinal absorption composition according to the above aspect.

[0019] Another aspect of the present invention is a method for producing an intestinal absorption composition. The method for producing the intestinal absorption composition includes a microbubble group generation step of generating a microbubble group containing first microbubbles and second microbubbles in a physiological solution and synthesizing a physiological solution containing the microbubble group, and a second microbubble removal step of removing the second microbubbles from the physiological solution containing the microbubble group and synthesizing a physiological solution containing microbubbles, wherein the average bubble diameter of the first microbubbles is less than 1.0 μm and the average bubble diameter of the second microbubbles is 1.0 μm or more.

[0020] In the method for producing the intestinal absorption composition according to the above embodiment, it is preferable to further include a first microbubble concentration adjustment step of adjusting the concentration of the first microbubbles in the microbubble-containing physiological solution to 2 billion microbubbles / mL or more, and a mixing step of mixing a sparingly soluble compound with the concentration-adjusted microbubble-containing physiological solution.

[0021] In the method for producing the intestinal absorption composition according to the above embodiment, the first microbubble concentration adjustment step is preferably a step of removing water from the microbubble-containing physiological solution by cross-flow filtration.

[0022] Another aspect of the present invention is a method for producing a drug for treating diabetes. The method for producing the drug for treating diabetes includes a microbubble group generation step of generating a microbubble group containing a first microbubble and a second microbubble in a physiological solution and synthesizing a physiological solution containing the microbubble group, and a second microbubble removal step of removing the second microbubble from the physiological solution containing the microbubble group and synthesizing a physiological solution containing microbubbles, wherein the average bubble diameter of the first microbubbles is less than 1.0 μm and the average bubble diameter of the second microbubbles is 1.0 μm or more.

[0023] In the method for producing a diabetes treatment agent according to the above embodiment, it is preferable to further include a first microbubble concentration adjustment step of adjusting the concentration of the first microbubbles in the microbubble-containing physiological solution to 2 billion bubbles / mL or more, and a mixing step of mixing a poorly soluble compound with the concentration-adjusted microbubble-containing physiological solution.

[0024] In the method for producing a diabetes treatment agent according to the above embodiment, the first microbubble concentration adjustment step is preferably a step of removing water from the microbubble-containing physiological solution by cross-flow filtration.

[0025] According to the present invention, it is possible to provide an intestinal absorption composition that allows oral administration of a drug containing a poorly soluble compound; a diabetes treatment agent containing the composition; a method for producing the intestinal absorption composition; and a method for producing the diabetes treatment agent.

[0026] Figure 1(a) is a transmission electron microscope (TEM) image of the intestinal absorption composition of Example 1. Figure 1(b) is a schematic diagram illustrating the first microbubbles and poorly soluble compounds present in the intestinal absorption composition. Figure 2 is a schematic diagram illustrating part of the manufacturing process of the intestinal absorption composition. Figure 2(a) shows the physiological solution containing microbubbles before the second microbubble removal step, and Figure 2(b) shows the physiological solution containing microbubbles after the second microbubble removal step. Figure 3 is a graph showing the measured absorbance of each comparative example and example sample used in Test Example 1. Figure 4 is a graph showing the relative absorbance of the samples of Examples 1 to 3 to the absorbance of the sample of Comparative Example 1 used in Test Example 1. Figure 5 is a liquid TEM image of the intestinal absorption composition of Example 1 in which undispersed (aggregated) human insulin powder was observed. Figure 6 is a graph showing the relationship between UFB concentration and the amount of undispersed human insulin, as described later. Figure 7 is an explanatory diagram showing the measurement method of the parallel artificial membrane permeability assay (PAMPA) permeability test performed in Test Example 3. Figure 8 is a graph showing the results of comparative measurement 1 in the PAMPA permeability test of Test Example 3. Figure 9 is a graph showing the results of comparative measurement 2 in the PAMPA permeability test of Test Example 3. Figure 10 is a graph showing the results of comparative measurement 3 in the PAMPA permeability test of Test Example 3. Figure 11 is a metallurgical microscope image of the ultrapure water UFB-containing solution in which insulin powder was dissolved. Figure 12 is a schematic diagram of the rat used in the "In situ closed method" of Test Example 4. Figure 13 is a schematic diagram of the small intestine of the rat used in the "In situ closed method" of Test Example 4. Figure 14 is a diagram showing the procedure for administering the insulin administration solutions of the examples and comparative examples to experimental rats. Figure 15 shows the results of measuring plasma insulin concentration when the insulin solution of Comparative Example 3 of Test Example 4 was administered subcutaneously to experimental rats. Figure 16 shows the results of measuring plasma insulin concentration when the insulin solution of Comparative Example 8-1 of Test Example 4 was administered jejunally to experimental rats. Figure 17 shows the results of measuring plasma insulin concentration when the insulin solution of Comparative Example 8-1 of Test Example 4 was administered ileum to experimental rats.Figure 18 shows the results of measuring plasma insulin concentration when the insulin solution of Comparative Example 8-2 of Test Example 4 was administered ileum to experimental rats. Figure 19 is a graph showing the relationship between insulin concentration and aggregate index value in the insulin aggregate formation inhibition confirmation test of Test Example 5. Figure 20 is a graph showing the relationship between insulin concentration and turbidity index value in the insulin aggregate formation inhibition confirmation test of Test Example 5.

[0027] Embodiments of the disclosed technology will be described in detail below. In this specification, unless otherwise specified, the notation "a to b" in the description of numerical ranges means a or greater and b or less.

[0028] In this specification, if multiple upper limits and multiple lower limits are described separately, all numerical ranges that can be freely combined and set using these upper and lower limits are described herein.

[0029] 1. Composition for Intestinal Absorption The intestinal absorption composition of this embodiment contains a first microbubble, a sparingly soluble compound, and a physiological solution. It may also contain other components, including various additives.

[0030] Figure 1(b) is a schematic diagram showing the first microbubbles B1 (hereinafter sometimes referred to as "microbubbles B1") and the sparingly soluble compound A present in the intestinal absorption composition. At least a portion of the sparingly soluble compound A, which will be described later, coats at least a portion of the microbubbles B1 {see Figure 1(b)}. These microbubbles B1 coated with at least a portion of the sparingly soluble compound A are dispersed substantially uniformly in the intestinal absorption composition. As a result, aggregation of the sparingly soluble compound A in the intestinal absorption composition is suppressed, and it is believed that the sparingly soluble compound A in the intestinal absorption composition is absorbed into the body from the intestinal tract. For this reason, the intestinal absorption composition of this embodiment is suitable for oral administration.

[0031] The components included in the intestinal absorption composition of this embodiment will be described in detail below.

[0032] 1-1. First Microbubbles (Average Bubble Diameter) In this embodiment, the first microbubbles B1 preferably have an average bubble diameter of less than 1.0 μm, and more preferably 200 nm or less. By setting the average bubble diameter of microbubbles B1 within the above range, they can remain stable in the liquid for a long period of time, and the absorption rate and absorption rate of the poorly soluble compound A into the intestinal tract can be further improved.

[0033] Among bubbles having an average bubble diameter within the above range, it is more preferable that the microbubbles B1 are "ultrafine bubbles" (registered trademark) (hereinafter sometimes referred to as "UFB"). "Ultrafine bubbles" are defined as bubbles smaller than 1 μm, and in actual application fields, measurement examples using particle characteristic evaluation methods for ultrafine bubbles in water show that many are in the range of 100 to 200 nm (JIS B 8741-1:2019 "Fine bubble technology - General principles relating to the use and measurement of fine bubbles - Part 1: Terminology").

[0034] The average bubble diameter of microbubbles B1 can be calculated from the particle size distribution curve obtained using either a nanotracking particle size analyzer (Malvern Corporation: NanoSight Pro) using the particle measurement method, or a particle size distribution analyzer (Shimadzu Corporation: SLAD-7500nano) using the Mie scattering method. In this embodiment, it is sufficient if the average bubble diameter measured by either of these methods falls within the range described above.

[0035] NanoSight Pro uses nanoparticle tracking analysis (NTA) to visualize particles, and based on that information, it is possible to quickly and easily obtain particle size and particle number concentration information. More specifically, NanoSight Pro emits scattered light from a laser irradiated onto liquid particles in a measurement cell, captures this scattered light as a video with a camera, and can analyze the particle size using the captured video.

[0036] SALD-7500 nano is a measurement method using the Mie scattering method. The measurement method using the Mie scattering method is a method of irradiating a laser beam onto a particle group and obtaining a particle size distribution by calculation from the intensity distribution pattern of the diffracted / scattered light emitted therefrom.

[0037] (Concentration) The lower limit of the concentration of the fine bubbles B1 is preferably 2 billion particles / mL or more, 2.5 billion particles / mL or more, 5 billion particles / mL or more, 10 billion particles / mL or more, 15 billion particles / mL or more, and more preferably 30 billion particles / mL or more. The upper limit of the concentration of the fine bubbles B1 is determined by cubic close packing. Cubic close packing is a concept that means an arrangement in which spheres or the like form a face-centered cubic lattice (FCC) and achieve the maximum packing ratio (about 74%). In actuality, although it may vary depending on the method for measuring the distance of the Brownian motion of the fine bubbles B1, temperature, etc., 500 billion particles / mL or less, 350 billion particles / mL or less, 100 billion particles / mL or less, etc. are preferred. By setting the concentration of the fine bubbles B1 within the above range, the poorly soluble compound A is dispersed substantially uniformly in the composition for intestinal absorption. As a result, aggregation of the poorly soluble compound A in the composition for intestinal absorption is suppressed, and it is considered that the required amount of the poorly soluble compound A is absorbed from the intestinal tract into the living body.

[0038] The concentration of the fine bubbles B1 can be calculated from the particle number concentration information obtained by a nanoparticle tracking particle size measuring device (manufactured by Malvern: NanoSight Pro) using the particle measurement method, or a particle size distribution measuring device (manufactured by Shimadzu Corporation, SALD-7500 nano) using the Mie scattering method. In this embodiment, it is sufficient that the concentration measured by either of these methods is within the range described above. The specific explanations of each device are as described above.

[0039] (Gas contained inside) The gas contained inside the fine bubbles B1 is not particularly limited, and preferably includes a gas selected from the group consisting of, for example, hydrogen, helium, oxygen, nitrogen, methane, fluorine, neon, carbon dioxide, nitric oxide, oxygen monoxide, ozone, argon, chlorine, ethane, propane, air, and a mixed gas containing them.

[0040] As the gas contained inside the microbubble B1, a medical gas may be used. The medical gas refers to a gas or a mixed gas used for treating, diagnosing, preventing patients, and driving surgical instruments. For example, oxygen, nitrous oxide (laughing gas), therapeutic air, carbon dioxide, driving nitrogen, etc. are included.

[0041] The quantitative analysis of the gas contained inside the microbubble B1 can be performed by a gas chromatograph (manufactured by Shimadzu Corporation, GC-2050) using the GC-TCD method, or a gas chromatograph (manufactured by Shimadzu Corporation, GC-2030) using the GC-BID method equipped with a BID (barrier discharge ionization detector).

[0042] By the above device, hydrogen, oxygen, nitrogen, methane, fluorine, carbon dioxide, nitric oxide, oxygen monoxide, argon, chlorine, ethane, propane, air, excluding helium (carrier gas), can be quantitatively measured from the gas vaporized by combustion. In addition, ozone contained inside the microbubble B1 can be quantitatively measured by measuring the absorbance using the color reaction by the KI method (Potassium Iodide Method).

[0043] 1-2. Sparingly soluble compound The sparingly soluble compound A of this embodiment is not particularly limited as long as it is a compound that is difficult to dissolve in water and has been particularly difficult to absorb in the intestinal tract. Examples of the sparingly soluble compound A include low molecular weight compounds, medium molecular weight compounds, high molecular weight compounds, etc.

[0044] The low molecular weight compound is a compound having a molecular weight of less than about 500. For example, low molecular weight pharmaceuticals, indigestible oligosaccharides (with a molecular weight of less than about 300-500), etc. are included.

[0045] The medium molecular weight compound is a compound having a molecular weight of about 500 to several thousand. For example, peptide preparations or their derivatives, indigestible oligosaccharides (with a molecular weight of 500 to several thousand), etc. are included.

[0046] High molecular weight compounds are compounds with molecular weights ranging from several thousand to 30,000. For example, insulin (molecular weight approximately 5,800), alteplase, morteplase, imiglucerase, veraglucerase alpha, agalsidase alpha, agalsidase beta, laronidase, alglucosidase alpha, avalglucidase alpha, idylsulfase, idylsulfase beta, galsulfase, erosulfase alpha, rasburicase, dorunase alpha, asfotase alpha, seberipase alpha, chrysanthaspase, chondroise, elapegademase, cerliponase alpha, glucarpidase, bestronidase alpha, borhyaluronidase alpha, olipudase alpha, andexanet alpha, pegvariase, pegaspargase, eptacog alpha, octocog beta, rurioctocog alpha, rurioctocog alpha pegol, Protein preparations such as turoctocog alfa, turoctocog alfa pegol, effralocdocog alfa, ronoctocog alfa, damoctocog argapegol, simoctocog alfa, nonacog alfa, eftrenonacog alfa, albutrepenonacog alfa, nonacog beta pegol, catridecacog, thrombomodulin alfa, antithrombin gamma, bonicog alfa, efanesoctocog alfa, somatropin, somapsitan, somatrogon, pegvisomant, macermin, carperitide, glucagon, folitropin alfa, folitropin delta, coriogonadotropin alfa, liraglutide, dulaglutide, semaglutide, teduglutide, teriparatide, metreleptin, vosolitide, interferons, erythropoietins, cytokines, toxins, fusion proteins, or derivatives thereof; Rituximab (Rituxan), trastuzumab (Herceptin), basiliximab (Symlect), infliximab (Remicade), palivizumab (Synagis), gemtuzumab ozogamicin (Mylotarg), tocilizumab (Actelam), bevacimab (Avastin), ibritumomab tiuxetan (90Y) (Zevalin), ibritumomab tiuxetan (111In) (Zevalin), cetuximab (Erbitux), adalimumab (Fumira), omalizumab (Xolair),Ranibizumab (Lucentis), eculizumab (Soliris), panitumumab (Vectibix), golimumab (Simponi), uttekinumab (Stelara), canakinumab (Ilaris), certolizumab pegol (Cimzia), mogamulizumab (Poteligeo), denosumab (Prolia), pertuzumab (Perjeta), trastuzumab emtansine (Kadcyla), ofatumab (Arzera), prentuximab vedotin (Adcetris), alemtuzumab (MabCampus), natalizumab (Tysebri), nivolumab (Opdivo), secukinumab Bu (Cosentyx), Ipilimumab (Yervoy), Ramucirumab (Cyramza), Evolocumab (Repatha), Pembrolizumab (Keytruda), Idarucizumab (Prazbind), Mepolizumab (Nucala), Elotuzumab (Empliciti), Ixekizumab (Taltz), Alirocumab (Praluent), Brodalumab (Lumicef), Brimumab (Benlysta), Daratumumab (Darzalex), Avelumab (Bavencio), Bezlotoxumab (Geneprava), Sarilumab (Kevzara), Atezolizumab (Tecentriq), Dupilumab (Dupixent), benralizumab (Fansenra), inotuzumab ozogamicin (Besponsa), guselkumab (Tremfya), emicizumab (Hemlibra), obinutuzumab (Gazyva), durvalumab (Imfinzi), vedolizumab (Entyvio), blinatumomab (Blincyto), romosozumab (Evenity), risankizumab (Skyrizi), ravulizumab (Ultomiris), necitumumab (Portraza), brosumab (Crysvita), brolucizumab (Beovu), trastuzumab deruxtecan (Enhertu) , satralizumab (Enspring), tildrakizumab (Ilumia), isatuximab (Sarclisa), cetuximab santarocan (Acalux), galcanezumab (Elgalti), inebilizumab (Yuplizna), daratumumab / borhyaluronidase alfa (Daraculo), polatuzumab vedotin (Polibee), fremanezumab (Ajovi), erenumab (Aimobig), dinutuximab (Unituxin), cacilibimab / imdevimab (Ronaprib), aniflorumab (Safnero), enfortumab vedotin (Padsev),Examples include antibody drugs such as sotrobimab (Zevudi); polysaccharides such as dextran (dextran 4 with a molecular weight of approximately 4,000, dextran 40 with a molecular weight of approximately 40,000, dextran 70 with a molecular weight of approximately 75,000, etc.) or their derivatives; indigestible oligosaccharides (molecular weight of several thousand to 30,000); and other biopharmaceuticals commonly administered by injection.

[0047] Among the above, the poorly soluble compound A is preferably insulin or a derivative thereof. Insulin or its derivatives include naturally derived substances, semi-artificially produced substances, or artificially produced substances.

[0048] Examples of naturally derived substances include those extracted from humans or other animals. Examples of other animals include mammals, and among these, those derived from pigs are preferred because they have properties similar to human insulin.

[0049] Examples of semi-artificially manufactured insulin include those produced from insulin genes extracted from humans or non-human animals, as mentioned above.

[0050] Examples of artificially manufactured insulin include: 1) insulin expressing an artificially synthesized insulin gene; 2) recombinant insulin expressing a recombinant gene in which a part of a naturally extracted insulin gene has been deleted, substituted, added, and / or inserted (e.g., rapid-acting insulin analog preparations); 3) insulin analogs expressing a gene that has been artificially synthesized from scratch using the recombinant gene sequence from 2); and 4) insulin derivatives in which natural, semi-synthetic, or artificially synthesized insulin has been modified by glycosylation or other means.

[0051] Furthermore, insulin can also exist as a multimer, such as a hexamer, where multiple molecules are aggregated.

[0052] 1-3. Physiological Solutions In this specification, physiological solutions refer collectively to liquids classified as hypotonic or isotonic solutions. A hypotonic solution is a solution with a lower osmotic pressure than a solution in the body (body fluids, blood, etc.). More specifically, it refers to a liquid having an osmotic pressure lower than the osmotic pressure of body fluids (plasma), which is approximately 285 ± 5 mOsm (milliosmoles) / L. The lower limit of the osmotic pressure of a hypotonic solution is not particularly limited, but it is preferably 0 mOsm (milliosmoles) / L or higher.

[0053] An isotonic solution is a solution whose osmotic pressure is approximately the same as that of a living body (body fluid, blood, etc.). More specifically, it refers to a liquid having an osmotic pressure equivalent to that of body fluid (plasma), which is approximately 285 ± 5 mOsm (milliosmoles) / L. In this embodiment, the osmolality can be measured according to the freezing point depression method or the vapor pressure method. For example, it can be measured according to the osmolality measurement method specified in the 18th edition of the Japanese Pharmacopoeia. Alternatively, in this embodiment, the osmotic pressure can be measured using a semipermeable membrane according to van't Hoff's law. The physiological solution according to this embodiment is sufficient if the osmolality or osmotic pressure measured by either of these methods is equal to or less than that of an isotonic solution (i.e., 290 mOsm (milliosmoles) / L or less).

[0054] The physiological solution is not particularly limited as long as it has the above-mentioned osmotic pressure, and preferably contains one or more selected from, for example, ultrapure water, reverse osmosis water, modified reverse osmosis water, physiological saline, glucose solution, distilled water for injection, purified water, phosphate buffer, and ion-exchanged water. Among the above, it is more preferable to include ultrapure water.

[0055] There is no clear definition or international standard for ultrapure water, but examples include ultrapure water used for medical purposes. Because ultrapure water has organic matter, fine particles, dissolved gases, etc. that cannot be removed by the methods used to produce pure water have been removed, it can efficiently generate fine bubbles B1.

[0056] Reverse osmosis water refers to highly purified water obtained by utilizing the reverse osmosis phenomenon, which pushes pure water that has moved to the hypertonic liquid side back to the physiological solution side using a pump or similar device via a semipermeable membrane (reverse osmosis membrane).

[0057] 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 semipermeable membrane (reverse osmosis membrane) with a pore size of 0.1 μm or less.

[0058] 1-4. Other Components The intestinal absorption composition of this embodiment may contain other components, including various additives, in addition to the first microbubbles B1, the poorly soluble compound A, and the physiological solution described above, as long as they do not hinder the effects of the present invention. For example, various additives, including isotonic solutions, hypertonic solutions, other solvents, and viscosity modifiers, may be included, as long as the osmotic pressure of the entire intestinal absorption composition can be maintained at or below approximately 285 ± 5 mOsm (milliosmoles) / L. The upper limit of the osmotic pressure of the entire intestinal absorption composition described above may be 290 mOsm (milliosmoles) / L or less, 285 mOsm (milliosmoles) / L or less, or 280 mOsm (milliosmoles) / L or less. The lower limit of the osmotic pressure of the entire intestinal absorption composition described above is not particularly limited, but is preferably 0 mOsm (milliosmoles) / L or higher, more preferably 50 mOsm (milliosmoles) / L or higher, even more preferably 100 mOsm (milliosmoles) / L or higher, even more preferably 150 mOsm (milliosmoles) / L or higher, and even more preferably 200 mOsm (milliosmoles) / L or higher.

[0059] For example, when administering an intestinal absorption composition orally, it is necessary to manufacture the intestinal absorption composition for encapsulation or enteric coating. When encapsulating, it is preferable to add viscosity modifiers such as glycerin or gelatin to the intestinal absorption composition from the viewpoint of compatibility between the capsule composition and the intestinal absorption composition. When enteric coating is applied, additives such as those disclosed in Japanese Patent No. 7050714 can be added.

[0060] 2. Method for Manufacturing the Intestinal Absorption Composition Figure 2 is a schematic diagram illustrating a part of the manufacturing process of the intestinal absorption composition. The method for manufacturing the intestinal absorption composition of this embodiment includes a microbubble group generation step {see Figure 2(a)} in which a microbubble group B containing a first microbubble B1 and a second microbubble B2 (hereinafter sometimes referred to as "microbubble B2") is generated in a physiological solution C to synthesize a microbubble group-containing physiological solution 10; a microbubble B2 removal step {see Figure 2(b)} in which microbubble B2 is removed from the microbubble group-containing physiological solution 10 to synthesize a microbubble group-containing physiological solution 20; and a mixing step in which a poorly soluble compound A and, if necessary, other components are mixed into the microbubble group-containing physiological solution 20.

[0061] More specifically, using the "microbubble-containing liquid generating apparatus" described in Japanese Patent Application No. 2023-197042 is preferable because it allows for the efficient production of an intestinal absorption composition containing a high concentration (2 billion or more microbubbles / mL) of microbubbles B1. The following describes each step in detail.

[0062] 2-1. Microbubble Group Generation Process In the microbubble group generation process, a microbubble group B containing microbubbles B1 and microbubbles B2, that is, a microbubble group B containing at least microbubbles B1 with an average bubble diameter of less than 1.0 μm, is generated in physiological solution C to synthesize a microbubble group-containing physiological solution 10. In other words, the microbubble group-containing physiological solution 10 contains a mixture of microbubbles B1 with an average bubble diameter of less than 1.0 μm and microbubbles B2 exceeding a specific bubble diameter {see Figure 2(a)}.

[0063] The average bubble diameter of microbubbles B1 is less than 1.0 μm, specifically as described in "1-1. First Microbubbles" above. The average bubble diameter of microbubbles B2 is preferably 1.0 μm or more, and more preferably 1.0 μm or more and less than 100 μm.

[0064] The microbubbles B2 may be bubbles having an average bubble diameter within the above range, and may be specifically "microbubbles" (registered trademark) (microbubble; hereinafter sometimes referred to as "MB"). "Microbubbles" are defined as bubbles (bubbles) of 1 μm or more and less than 100 μm (JIS B 8741-1:2019 "Fine bubble technology - General principles relating to the use and measurement of fine bubbles - Part 1: Terminology").

[0065] Microbubble group B refers to a group of bubbles containing microbubbles B1 and B2 as described above, and the average bubble diameter of microbubble group B is preferably less than 100 μm. Microbubble group B may also be called "fine bubble" (registered trademark). "Fine bubble" is defined as a bubble (bubble) less than 100 μm (JIS B 8741-1:2019 "Fine bubble technology - General principles relating to the use and measurement of fine bubbles - Part 1: Terminology").

[0066] The method for measuring the average bubble diameter of microbubbles B2 and microbubble group B is the same as the measurement method described in "1-1. First Microbubbles" above.

[0067] The method for generating the microbubble group B in the physiological solution C is not particularly limited, and known methods can be used. For example, the following methods, or combinations thereof, can be used.

[0068] (Gas-liquid mixed shear method) This method involves rapidly swirling a gas together with a liquid.

[0069] (Ultrasonic method) This method involves applying shock waves or cavitation to a liquid to further collapse any bubbles that have already formed.

[0070] (Pressurized dissolution method) This method generates bubbles by applying pressure to a gas and a liquid and releasing them all at once.

[0071] (Micropore method) This method involves supplying gas while applying pressure using orifices, etc.

[0072] (Electrolysis method) This method generates gas from electrodes (for example, thin wire-shaped electrodes) immersed in an aqueous solution.

[0073] 2-2. Second Microbubble Removal Step In the second microbubble B2 removal step, microbubbles B2 are removed from the microbubble group-containing physiological solution 10 in which microbubbles B1 and microbubbles B2 are mixed. For example, it is preferable to remove microbubbles B2 that exceed a specific bubble diameter from the microbubble group-containing physiological solution 10.

[0074] One method for removing microbubbles B2 is to filter the entire microbubble-containing physiological solution 10 using a filtration filter. Through the microbubble B2 removal step, a microbubble-containing physiological solution 20 is synthesized from which microbubbles B2 have been removed more effectively.

[0075] The average pore size of the total filtration filter used in the above-described microbubble B2 removal step is preferably 1.00 μm or less, more preferably 0.70 μm or less, even more preferably 0.50 μm or less, even more preferably 0.30 μm or less, and even more preferably 0.22 μm or less, 0.20 μm or less, 0.10 μm or less, etc. The total filtration filter can be used to select the size of the microbubble group B. For this reason, the average bubble diameter of microbubbles B1 can be appropriately adjusted by adjusting the average pore size of the total filtration filter depending on the type of poorly soluble compound A, etc. The lower limit of the average pore size of the total filtration filter is preferably 0.01 μm or more, and more preferably 0.05 μm or more.

[0076] Generally, liquids that pass through a filter with an average pore size of 0.22 μm or less can be used as sterilized liquids. Furthermore, liquids that pass through a filter with an average pore size of 0.10 μm or less can also remove viruses. Therefore, by having a total filtration filter with an average pore size within the above range, the resulting high-concentration physiological solution 20 containing fine bubbles can be used in applications requiring high cleanliness, such as medical applications.

[0077] It is preferable that the ratio of the concentration of microbubbles B1 after the microbubble B2 removal step, relative to the concentration of microbubble group B before the microbubble B2 removal step, be 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, etc. By setting the ratio within the above range, a highly concentrated microbubble-containing physiological solution 20 can be efficiently obtained.

[0078] 2-3. First microbubble concentration adjustment step In the first microbubble B1 concentration adjustment step, the concentration of microbubbles B1 in the microbubble-containing physiological solution 20 from which microbubbles B2 have been removed is adjusted to 2 billion / mL or more.

[0079] After the above microbubble B2 removal step, the concentration of microbubbles B1 in the microbubble-containing physiological solution 20 may be lower than the target concentration. Therefore, if necessary, it is preferable to increase the concentration of microbubbles B1 in the microbubble B1 concentration adjustment step and adjust it to the target concentration of microbubbles B1.

[0080] Methods for adjusting the concentration of microbubbles B1 include removing them by vaporizing water using the boiling point of water, and removing some of the water (including the physiological solution) from the microbubble-containing physiological solution 20 using a cross-flow filtration method.

[0081] The cross-flow filtration method is a method of removing some of the water (including the physiological solution) by filtering the microbubble-containing physiological solution 20 using a cross-flow filter that allows water (including the physiological solution) to pass through but not the microbubbles B1. From the viewpoint of productivity, the method of removing some of the water (including the physiological solution) by cross-flow filtration is preferred.

[0082] The pore size of the cross-flow filter is not particularly limited, as long as it is smaller than the average bubble diameter of the microbubbles B1. By setting the pore size of the cross-flow filter to be smaller than the average bubble diameter of the microbubbles B1, when the microbubble-containing physiological solution 20 is filtered with the cross-flow filter, the microbubbles B1 that cannot pass through the pores of the cross-flow filter remain in the microbubble-containing physiological solution 20, and some water (including the physiological solution) is removed. As a result, the concentration of microbubbles B1 can be increased. Furthermore, from the viewpoint of exhibiting the above-mentioned effects more effectively, the pore size of the cross-flow filter is preferably less than 0.20 μm, more preferably less than 0.10 μm, and even more preferably less than 0.05 μm.

[0083] When the physiological solution is physiological saline or glucose, the pore size of the cross-flow filter may be larger or smaller than the average diameter of the solute dissolved in the physiological solution. In other words, the cross-flow filter may either allow only the water contained in the physiological solution to pass through, or it may allow the physiological solution (including the solute dissolved in water) to pass through, as long as it does not allow the fine bubbles B1 to pass through.

[0084] Furthermore, the microbubble B1 concentration adjustment process may be carried out under reduced pressure. Performing the process under reduced pressure allows for more efficient adjustment to the desired microbubble B1 concentration.

[0085] In the microbubble B1 concentration adjustment step, the microbubble-containing physiological solution 20 may be sterilized as needed. Sterilization makes it possible to use the intestinal absorption composition for medical applications and other uses where cleanliness is required.

[0086] By repeatedly performing the above-described microbubble B2 removal step and B1 concentration adjustment step, the microbubble-containing physiological solution 20 can be efficiently adjusted to the desired microbubble B1 concentration (2 billion bubbles / mL or more).

[0087] 2-4. Mixing Step In the mixing step, the physiological solution 20 containing microbubbles is mixed with the poorly soluble compound A and, if necessary, other components. The mixing step may be performed after the microbubble B2 removal step, or it may be performed during the process of generating the microbubble group B and the microbubble B2 removal step.

[0088] 3. Applications The intestinal absorption composition of this embodiment enables intestinal absorption of poorly soluble compound A and allows for oral administration. Therefore, it can be used as a treatment agent for diseases that were previously difficult to administer orally. For example, when insulin or a derivative thereof is used for poorly soluble compound A, the intestinal absorption composition of this embodiment can be used as a treatment agent for diabetes.

[0089] The diabetes treatment agent of this embodiment includes the above-described intestinal absorption composition. Therefore, it enables intestinal absorption of insulin or its derivatives, and can be administered orally. The diabetes treatment agent is manufactured by processing the above-described intestinal absorption composition into a suitable form for oral administration as a diabetes treatment agent after its manufacture.

[0090] The form of diabetes treatment agents is not particularly limited and can take various forms, including liquid, gel, sol (including colloids), cream, soft capsules, enteric-coated capsules, and other capsule forms. Enteric-coated capsules are capsules in which a drug tablet or capsule is coated with a substance that dissolves after reaching the small intestine.

[0091] The present invention will be described below with reference to examples and comparative examples, but the present invention is not limited to the following. The components used in the comparative examples and examples are as follows.

[0092] (Comparative Example 1: Hypotonic solution) Hypotonic solution (130 mOsm / L): Ultrapure water

[0093] (Comparative Example 2: Isotonic Solution) Isotonic solution: Phosphate-buffered saline (PBS) AccuDia® (D-PBS(-) powder) (manufactured by Shimadzu Diagnostics Corporation) was added to ultrapure water to a concentration of 9.6 g / L and dissolved to synthesize the isotonic solution of Comparative Example 2.

[0094] (Comparative Example 3: Isotonic Solution) Isotonic solution: Sodium chloride (reagent grade, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to ultrapure saline solution to a concentration of 0.9 W / V%, dissolved, and the isotonic solution of Comparative Example 3 was synthesized.

[0095] (Example 1: Hypotonic liquid + UFB) By using the "microbubble-containing liquid (UFB) generation apparatus" described in Japanese Patent Application No. 2023-197042 in the hypotonic liquid of Comparative Example 1, the following UFB was generated to synthesize the UFB-containing hypotonic liquid of Example 1.

[0096] Specifically, in a microbubble generation device, air supplied from a gas supply unit and ultrapure water supplied from a liquid supply tank were mixed to generate a group of microbubbles containing air in the ultrapure water, thereby synthesizing a hypotonic solution containing microbubbles. The synthesized hypotonic solution containing microbubbles was filtered through a filtration filter to remove MB and synthesize a hypotonic solution containing UFB. Some water was removed from the hypotonic solution containing UFB using a cross-flow filter. The above operation was repeated to adjust the UFB concentration to the predetermined concentration shown below, thereby synthesizing the hypotonic solution containing UFB of Example 1.

[0097] NanoSight Pro (Malvern) was used to measure the UFB concentration. • Average bubble diameter: approximately 100 nm • UFB concentration: 15.5 billion cells / mL • Internal gas: air

[0098] (Example 2: Hypotonic liquid + UFB) By using the "microbubble-containing liquid (UFB) generation apparatus" described in Japanese Patent Application No. 2023-197042 in the hypotonic liquid of Comparative Example 1, the following UFB was generated to synthesize the UFB-containing hypotonic liquid of Example 2. Nanosight Pro (Malvern) was used to measure the UFB concentration. Average bubble diameter: approximately 100 nm UFB concentration: 30 billion bubbles / mL Internal gas: air

[0099] (Example 3: Hypotonic liquid + UFB) By using the "microbubble-containing liquid (UFB) generation apparatus" described in Japanese Patent Application No. 2023-197042 in the hypotonic liquid of Comparative Example 1, the following UFB was generated to synthesize the UFB-containing hypotonic liquid of Example 3. Nanosight Pro (Malvern) was used to measure the UFB concentration. Average bubble diameter: approximately 100 nm UFB concentration: 50 billion bubbles / mL Internal gas: air

[0100] (Example 4: Hypotonic liquid + UFB) By using the "microbubble-containing liquid (UFB) generation apparatus" described in Japanese Patent Application No. 2023-197042 in the hypotonic liquid of Comparative Example 1, the following UFB was generated to synthesize the UFB-containing hypotonic liquid of Example 4. Nanosight Pro (Malvern) was used to measure the UFB concentration. Average bubble diameter: approximately 100 nm UFB concentration: 19.2 billion cells / mL Internal gas: air

[0101] (Example 5: Hypotonic liquid + UFB) By using the "microbubble-containing liquid (UFB) generation apparatus" described in Japanese Patent Application No. 2023-197042 in the hypotonic liquid of Comparative Example 1, the following UFB was generated to synthesize the UFB-containing hypotonic liquid of Example 5. Nanosight Pro (Malvern) was used to measure the UFB concentration. Average bubble diameter: approximately 100 nm UFB concentration: 46 billion bubbles / mL Internal gas: air

[0102] (Example 6: Isotonic solution (PBS) + UFB) By using the "microbubble-containing liquid (UFB) generation apparatus" described in Japanese Patent Application No. 2023-197042 in the isotonic solution of Comparative Example 2, the following UFB was generated to synthesize the UFB-containing hypotonic solution of Example 6. Nanosight Pro (Malvern) was used to measure the UFB concentration. Average bubble diameter: approximately 100 nm UFB concentration: 46 billion bubbles / mL Internal gas: air

[0103] (Example 7: Isotonic solution (physiological saline) + UFB) By using the "microbubble-containing liquid (UFB) generation device" described in Japanese Patent Application No. 2023-197042 in the isotonic solution of Comparative Example 3, the following UFB was generated to synthesize the UFB-containing isotonic solution of Example 7. Nanosight Pro (Malvern) was used to measure the UFB concentration. Average bubble diameter: approximately 100 nm UFB concentration: 54 billion bubbles / mL Internal gas: air

[0104] (Example 8: Isotonic solution (physiological saline) + UFB) By using the "microbubble-containing liquid (UFB) generation device" described in Japanese Patent Application No. 2023-197042 in the isotonic solution of Comparative Example 3, the following UFB was generated to synthesize the UFB-containing isotonic solution of Example 8. Nanosight Pro (Malvern) was used to measure the UFB concentration. Average bubble diameter: approximately 100 nm UFB concentration: 13.4 billion cells / mL Internal gas: air

[0105] (Method for producing insulin administration solution) Solid insulin (human recombinant insulin, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dispersed in the UFB-containing hypotonic solutions of Comparative Example 1 and each of Examples 1 to 4 so that the insulin concentration was 500 pM. The obtained UFB-containing hypotonic solutions of Comparative Example 1 and each of Examples 1 to 4 (insulin concentration: 500 pM) were sequentially diluted to prepare intestinal absorption compositions of Comparative Example 1 and each of Examples 1 to 4 with insulin concentrations of 480 pM, 240 pM, 120 pM, 60 pM, 30 pM, 15 pM, and 7.5 pM.

[0106] [Transmission electron microscope (TEM) image of the intestinal absorption composition of Example 1] The intestinal absorption composition of Example 1 (insulin concentration: 500 pM) was sealed in a TEM sample holder for liquid analysis (MA-tek, K-Kit). A TEM image was taken at 200 kV {see Figure 1(a)}. The K-Kit is a TEM sample holder that can transmit electron beams with a SiN film thickness of 30 nm and a window width of 200 nm. Figure 1(a) shows the TEM image of the intestinal absorption composition of Example 1.

[0107] Normally, water and air bubbles do not have enough contrast in TEM images to be distinguishable. In contrast, high molecular weight compounds such as insulin are projected as dark shadows, making their presence identifiable.

[0108] From the TEM image in Figure 1(a), a circular black shadow can be clearly seen, and it can be determined that this circular black shadow is human insulin agent coating the surface of the UFB. From the TEM image in Figure 1(a), it was found that in the intestinal absorption composition of Example 1, the UFB acts as a nucleus, and the human insulin agent coats the surface of the UFB around it.

[0109] This is presumed to be due to the interaction with the negative charge on the surface of the UFB or the force of hydrophobic interaction. The TEM image in Figure 1(a) shows that insulin is present without aggregation.

[0110] [Test Example 1: Absorbance Measurement Test] The UFB-containing hypotonic solutions of Comparative Example 1 and Examples 1 to 3 were sterilized using a pulsed light sterilization device (Iwasaki Electric Co., Ltd., PLS2K02-04). The pulsed light sterilization device releases compressed energy to a xenon lamp in a calculated time of 200 to 300 μs, thereby increasing the radiation intensity per unit time to more than 1000 times that of a low-pressure ultraviolet lamp. This performance can be used to achieve inline sterilization of microorganisms.

[0111] Subsequently, approximately 2 mg of human insulin (11376497001, Lot 79036900) (1.6 mg for ultrapure water, 1.7 mg for UFB water) was measured out into 0.5 mL microresco tubes. Next, human insulin was added to ultrapure water or UFB water to achieve a concentration of 4 mg / mL (150 μL for ultrapure water, 250 μL for UFB water). Assay was performed according to the instructions for the human insulin ELISA kit (manufactured by MRD), and the absorbance was measured.

[0112] Absorbance was measured using the following microplate reader. A 96-well microplate with a well diameter of approximately 5 mm was used. 100 μL (TMB + Stop solution) was filled into each well during measurement. The calculated optical path length A was 5 mm, calculated using the following formula: 0.25 × 0.25 × 3.14 × A = 0.1 (cm). The measured optical path length A was approximately 5.0 mm. ３ )

[0113] <Results> Figure 3 is a graph showing the absorbance measured at 450 / 630 nm and a path length of 5.0 mm for the samples of Comparative Example 1 and each of Examples 1 to 3. Figure 4 is a graph showing the relative absorbance of the samples of Examples 1 to 3 to the absorbance of the sample of Comparative Example 1.

[0114] These results show that in Examples 1 to 3, where UFB was generated in the hypotonic solution, the absorbance was clearly increased compared to Comparative Example 1, where ultrapure water was used as the hypotonic solution. More specifically, in Comparative Example 1, the absorbance was 0.050 or less at all insulin concentrations. In contrast, in Examples 1 to 3, the absorbance was 0.050 or higher when the insulin concentration was 120 pM or higher. This confirmed that the dispersibility of the UFB-containing hypotonic solution with respect to insulin is higher than that of ultrapure water.

[0115] It has been found that the surface of bubbles smaller than nanoscale is negatively charged. It is thought that when insulin is dispersed in water, the positive charge generated on the surface combines electrically with the negative charge of UFB, resulting in behavior similar to that of UFB, which is finely dispersed at 15.5 billion, 30 billion, and 50 billion cells / mL, thus causing the insulin to be uniformly dispersed.

[0116] Based on this principle, it was shown that even high-molecular-weight compounds (insulin), which have previously been difficult to dissolve in hypotonic solutions, could be uniformly dispersed using a UFB-containing hypotonic solution. Considering previous knowledge, this suggests that they can be reliably absorbed into the body via the intestinal tract.

[0117] [Test Example 2: Detection Test for Undispersed Human Insulin Molecules] Approximately 2 mg of human insulin (11376497001, Lot 79036900) (1.6 mg for ultrapure water, 1.7 mg for UFB water) was measured into a 0.5 mL microresco tube. Next, human insulin was added to ultrapure water or UFB water to achieve a concentration of 4 mg / mL (150 μL for ultrapure water, 250 μL for UFB water). Assay was performed according to the instructions for the human Insulin ELISA kit (manufactured by MRD), and the absorbance was measured. From the measured absorbance, the amount of undispersed insulin could be quantified. The specific method is described below.

[0118] The absorbance of the intestinal absorption compositions of Comparative Example 1 and Examples 1 to 4 was measured according to the instructions for the human Insulin ELISA kit (manufactured by MRD). From the measured absorbance, the amount of undispersed human insulin (%) was calculated using the following formula.

[0119] Using the MED ELISA kit, a calibration curve was created from the standard solution included with the kit. The obtained absorbance and known concentration (mU / L) were applied to equation (2), which was derived from the 4-parameter logistic (4-PL) curve equation (equation (1)), and the insulin concentration was calculated from the measured absorbance. y = d + {(a - d) / (1 + (x / c)} ｂ )} (1) x=c×{(a-d) / (y-d)-1} （１／ｂ） (2) a = lower asymptote, d = upper asymptote, b = slope parameter, c = horizontal axis of the midpoint where the vertical axis is (a + b) / 2, y = absorbance, x = insulin concentration (mU / L) The calculated insulin concentration and the theoretically added insulin concentration were applied to equation (3) to calculate the amount (ratio) (%) of undispersed human insulin. Amount (ratio) of undispersed human insulin = 100 - {(measured concentration based on calibration curve / theoretical concentration added) × 100} (3)

[0120] <Results> Figure 5 is a TEM image in liquid of the intestinal absorption composition of Example 1. This directly observes the state of undispersed human insulin molecules in liquid. As shown in Figure 5, it was found that some undispersed human insulin molecules remained in the intestinal absorption composition of Example 1.

[0121] Figure 6 is a graph plotting the obtained insulin concentration values ​​on the vertical axis and the corresponding UFB concentration (bubble count (billions)) of the sample on the horizontal axis. Example 3 yielded the same results as Example 2 and was therefore omitted from Figure 6. As shown in Figure 6, at a UFB concentration of 15.5 billion molecules / mL, 61.9% of undispersed human insulin molecules remained, but at a UFB concentration of 30 billion molecules / mL or higher, the amount of undispersed human insulin could be reduced to 0%. In other words, it was shown that the higher the UFB concentration, the more stably human insulin molecules can be dispersed.

[0122] [Test Example 3: Parallel Artificial Membrane Permeability Assay (PAMPA) Permeability Test] PAMPA permeability was measured based on the method described in M. Kansi et al. (Journal of Medicinal Chemistry, 41, (1998) pp. 1007), with modifications based on S. Bendels et al. (Pharmaceutical Research, 23 (2006) pp. 2525).

[0123] More specifically, Stirwell ＴＭ In a 96-well plate assay using a PAMPA Sandwich plate (Pion), 5 μL of GIT-0 Lipid Solution (Pion) was added to the bottom membrane of the upper plate to create an artificial membrane (PAMPA) (see Figure 7).

[0124] In the donor compartment of the lower plate well (see Figure 7), 200 μL / well of the insulin test solution described later was added. In the acceptor compartment of the upper plate well (see Figure 7), which contained GIT-0 Lipid Solution, 200 μL / well of Acceptor Sink Buffer (ASB: Pion Corporation) was added. After combining the two, the mixture was incubated at 37°C for 4 hours.

[0125] The insulin concentrations in the acceptor and donor compartments were measured by the following method. Using a UV Half Area Plate, 96 Well (manufactured by CORNING), 50 μg / well (optical path length: 0.286 cm) was added in two wells each, and the absorbance spectrum from 200 to 450 nm was measured at every 1 nm using a UV-VIS spectrophotometry {Thermo Scientific Multiskan SkyHigh microplate spectrophotometer (manufactured by Thermo Fisher Scientific)}.

[0126] Then, the absorbance value at 276 nm and the AUC value obtained by summing up the absorbance values across the entire measurement wavelength range were substituted into a four-parameter logistic curve-fit (4-PL) shown in Equation (1) to determine the insulin concentration from the calibration curve thus obtained.

[0127] Equation (4) 4-PL x = c × { (a - d) / (y - d) - 1} （１／ｂ） (4) x: Insulin concentration y: Absorbance AUC at each wavelength a: Lower asymptote (lower limit of the curve: theoretical reaction at zero concentration) b: Slope at c (slope coefficient) c: Midpoint between a and d (value of (a + d) / 2: inflection point) d: Upper asymptote (upper limit of the curve: theoretical response at infinite concentration)

[0128] The insulin concentration obtained by Equation (4) was substituted into the following Equation (5) to calculate the flow rate (FLUX) (μg / cm ２ / min), and then the membrane permeability coefficient (P ｅ ) (cm / s) was calculated from Equation (6).

[0129] Equation (5) Flow rate (FLUX) J = V / A × dc / dt (5) J: Flow rate (FLUX) (μg / cm ２ / min) V: Acceptor volume (mL) A: Membrane area (cm ２ ) dc: Change in concentration in the acceptor (μg / mL) dt: Change in time in the acceptor (min)

[0130] Equation (6) Membrane permeability coefficient (cm / s) P ｅ = J / C ｄｏｎｏｒ(6) P ｅ : Membrane permeability coefficient (cm / s) C ｄｏｎｏｒ : Donor solution concentration (μg / mL) J: Flow rate (FLUX) (μg / cm ２ / min)

[0131] <Comparative Measurement 1: Comparative Example 1 (Ultrapure Water) and Example 5 (Ultrapure Water + UFB)> (Preparation of Insulin Test Solution) (1) 2.0 mg of lyophilized insulin {Merck, Roschhit Recombinant Insulin 11376497001 (insulin dissolved in hydrochloric acid solution at pH 2.3 and then lyophilized)} was added to 500 μL of ultrapure water to which methylcellulose (MC) (methylcellulose 400, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added as an insulin container adsorption inhibitor to a final concentration of 0.001 W / V%, to prepare a 4 mg / mL ultrapure water insulin solution. (2) 70 μL of the ultrapure water insulin solution prepared in (1) above was set aside in a separate container. Then, 490 μL of the hypotonic solution of Comparative Example 1 containing 0.001 W / V% MC was prepared and added to the ultrapure water insulin solution set aside in a separate container to prepare the insulin test solution of Comparative Example 1. The final insulin concentration of the insulin test solution in Comparative Example 1 was 500 μg / mL. (3) 70 μL of the ultrapure water insulin solution prepared in (1) above was set aside in a separate container. Then, a solution was prepared by adding 61 μL of the UFB-containing hypotonic solution of Example 5 to 490 μL of ultrapure water, and this was added to the ultrapure water insulin solution set aside in a separate container to prepare the insulin test solution of Example 5. The final insulin concentration of the insulin test solution of Example 5 was 500 μg / mL, and the final UFB concentration was 5 billion cells / mL. (4) The PAMPA permeability measurements described above were performed on the insulin test solutions of Comparative Example 1 and Example 5.

[0132] <Results> Figure 8 shows the measurement results. Figure 8 is a graph showing the relative values ​​of the membrane permeability coefficient of the absorbance (wavelength 276 nm) and the membrane permeability coefficient of the absorbance (wavelength 276 nm) and the AUC value (hereinafter referred to as the AUC value) of the insulin test solution of Comparative Example 1.

[0133] Figure 8 shows that the insulin test solution of Example 5, which has a UFB concentration of 5 billion cells / mL, exhibits enhanced membrane permeability compared to the insulin test solution of Comparative Example 1, which does not contain UFB.

[0134] <Comparative Measurement 2: Comparative Example 2 (PBS) and Example 6 (PBS + UFB)> (Preparation of Insulin Test Solution) (1) 2.2 mg of lyophilized insulin {Merck, Roschhit recombinant insulin 11376497001 (insulin dissolved in hydrochloric acid solution at pH 2.3 and then lyophilized)} was mixed with 500 μL of PBS to which MC was added as an insulin container adsorption inhibitor to a final concentration of 0.001 W / V%, to prepare a 4 mg / mL PBS insulin solution. (2) 70 μL of the PBS insulin solution prepared in (1) above was separated into a separate container. Then, 490 μL of the isotonic solution of Comparative Example 2 containing 0.001 W / V% MC was prepared and added to the PBS insulin solution separated into the separate container to prepare the insulin test solution of Comparative Example 2. The final insulin concentration of the insulin test solution of Comparative Example 2 was 500 μg / mL. (3) 70 μL of the PBS insulin solution prepared in (1) above was set aside in a separate container. Then, a solution was prepared by adding 30 μL of the UFB-containing isotonic solution of Example 6 to 490 μL of PBS, and this was added to the PBS insulin solution set aside in a separate container to prepare the insulin test solution of Example 6-1. The final insulin concentration of the insulin test solution of Example 6-1 was 500 μg / mL, and the final UFB concentration was 2.5 billion cells / mL. (4) The insulin test solution of Example 6-2 was prepared in the same manner as in (3) above, except that a solution containing 61 μL of the UFB-containing isotonic solution of Example 6 to 490 μL of PBS was added to the PBS insulin solution set aside in a separate container. The final insulin concentration of the insulin test solution of Example 6-2 was 500 μg / mL, and the final UFB concentration was 5 billion cells / mL. (5) The PAMPA permeability of the insulin test solutions of Comparative Example 2, Example 6-1, and Example 6-2 was measured as described above.

[0135] <Results> Figure 9 shows the measurement results. Figure 9 is a graph showing the relative values ​​of the membrane permeability coefficient in absorbance (wavelength 276 nm) and AUC value of the insulin test solution of Example 6-1 and Example 6-2, with respect to the membrane permeability coefficient in absorbance (wavelength 276 nm) and AUC value of the insulin test solution of Comparative Example 2.

[0136] Figure 9 confirms that the insulin test solutions of Example 6-1, with a UFB concentration of 2.5 billion cells / mL, and Example 6-2, with a UFB concentration of 5 billion cells / mL, exhibited enhanced membrane permeability compared to the insulin test solution of Comparative Example 2, which did not contain UFB.

[0137] Furthermore, it was confirmed that the insulin test solution of Example 6-2, which had a UFB concentration of 5 billion cells / mL, exhibited higher membrane permeability than the insulin test solution of Example 6-1, which had a UFB concentration of 2.5 billion cells / mL.

[0138] <Comparative Measurement 3: Comparative Example 3 (Physiological Saline) / Example 7 (Physiological Saline + UFB)> (Preparation of Insulin Test Solution) (1) 3.7 mg of solid insulin (Sigma-Aldrich, human recombinant insulin) was added to 92.5 μL of 0.1 M hydrogen chloride aqueous solution. To this, 500 μL of physiological saline to which MC was added as an insulin container adsorption inhibitor to a final concentration of 0.001 W / V% was added to prepare a 4 mg / mL dilute hydrochloric acid physiological saline insulin solution. (2) 70 μL of the dilute hydrochloric acid physiological saline insulin solution prepared in (1) above was set aside in a separate container. Then, 434 μL of the isotonic solution of Comparative Example 3 containing 0.001 W / V% MC was prepared and added to the dilute hydrochloric acid physiological saline insulin solution set aside in a separate container. Next, 56 μL of 0.1 M sodium hydroxide aqueous solution was added to prepare the insulin test solution of Comparative Example 3. The final insulin concentration of the insulin test solution in Comparative Example 3 was 500 μg / mL. (3) 70 μL of the dilute hydrochloric acid saline insulin solution prepared in (1) above was set aside in a separate container. Then, a solution was prepared by adding 10.4 μL of the UFB-containing isotonic solution of Example 7 to 490 μL of saline, and this was added to the dilute hydrochloric acid saline insulin solution set aside in a separate container. Next, 56 μL of 0.1 M sodium hydroxide aqueous solution was added to prepare the insulin test solution of Example 7-1. The final insulin concentration of the insulin test solution of Example 7-1 was 500 μg / mL, and the final UFB concentration was 1 billion cells / mL. (4) The insulin test solution of Example 7-2 was prepared in the same manner as in (3) above, except that a solution containing 51.9 μL of the UFB-containing isotonic solution of Example 7 to 490 μL of saline was added to the dilute hydrochloric acid saline insulin solution set aside in a separate container. The final insulin concentration of the insulin test solution in Example 7-2 was 500 μg / mL, and the final UFB concentration was 5 billion cells / mL. (5) The insulin test solution of Example 7-3 was prepared in the same manner as in (3) above, except that a solution containing 104 μL of the UFB-containing isotonic solution of Example 7 in 490 μL of physiological saline was added to a dilute hydrochloric acid physiological saline insulin solution that had been separated into a different container. The final insulin concentration of the insulin test solution of Example 7-3 was 500 μg / mL, and the final UFB concentration was 10 billion cells / mL.(6) An insulin test solution for Example 7-4 was prepared in the same manner as in (3) above, except that a solution containing 360 μL of the UFB-containing isotonic solution of Example 7 in 490 μL of physiological saline was added to a dilute hydrochloric acid physiological saline insulin solution that had been separated into a different container. The final insulin concentration of the insulin test solution for Example 7-4 was 500 μg / mL, and the final UFB concentration was 34.7 billion cells / mL. (7) The PAMPA permeability measurements described above were performed on the insulin test solutions of Comparative Example 3 and Examples 7-1 to 7-4.

[0139] <Results> Figure 10 shows the measurement results. Figure 10 is a graph showing the relative values ​​of the membrane permeability coefficient in absorbance (wavelength 276 nm) and AUC value of the insulin test solution of Examples 7-1 to 7-4, with respect to the membrane permeability coefficient in absorbance (wavelength 276 nm) and AUC value of the insulin test solution of Comparative Example 3.

[0140] Figure 10 shows that the insulin test solutions of Example 7-1 (UFB concentration of 1 billion cells / mL), Example 7-2 (UFB concentration of 5 billion cells / mL), Example 7-3 (UFB concentration of 10 billion cells / mL), and Example 7-4 (UFB concentration of 34.7 billion cells / mL) exhibited enhanced membrane permeability compared to the insulin test solution of Comparative Example 3, which did not contain UFB.

[0141] Furthermore, it was confirmed that membrane permeability increased as the UFB concentration rose to 1 billion ions / mL, 5 billion ions / mL, and 10 billion ions / mL.

[0142] PAMPA permeability tests revealed that membrane permeability was enhanced in insulin test solutions with UFB concentrations of 1 to 2.5 billion cells / mL or higher. The PAMPA permeability test is an in vitro test that mimics the actual intestinal membrane by forming an oil layer on a membrane with a gap of 0.45 μm, and is widely used as a gastrointestinal absorption prediction model. Therefore, it is predicted that the inclusion of UFB will enhance insulin absorption in the gastrointestinal tract.

[0143] [Metal Microscope Photograph of Ultrapure Water Containing UFB with Insulin Powder Dissolved] Human insulin powder (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., human recombinant insulin, 099-06473) was dissolved in ultrapure water and ultrapure water containing UFB (UFB concentrations of 500 million ions / mL, 2 billion ions / mL, 5 billion ions / mL, 10 billion ions / mL, 20 billion ions / mL, 30 billion ions / mL, and 50 billion ions / mL) to prepare 4.0 mg / mL solutions. These solutions were then stored in a refrigerator for 6 days, and their appearance was photographed using a metal microscope. Figure 11 shows photographs of the ultrapure water insulin solution and the ultrapure water insulin solution containing UFB, taken with a metal microscope.

[0144] Figure 11 shows that when the UFB concentration exceeds 2 billion cells / mL, the precipitate decreases, and when it exceeds 5 billion cells / mL, the clarity of the supernatant clearly increases. It was found that the inclusion of UFB leads to better dispersion of insulin.

[0145] [Test Example 4: Confirmation Test of the Effect of Energizing Insulin Absorption in the Intestinal Tract Using an Intestinal Absorption Composition] Using the known in-situ closed loop method shown in Figures 12 and 13, the insulin administration solutions (intestinal absorption compositions) of Examples 8-1 and 8-2 below were directly administered to the intestinal tract of the target subjects (rats). Subsequently, the amount absorbed via the intestinal tract was measured by measuring the insulin concentration in the rat plasma. The specific administration procedures for the insulin administration solutions of Examples 8-1 and 8-2 are shown in Figure 14.

[0146] The insulin administration solution (composition for intestinal absorption) of Comparative Example 3 below was administered subcutaneously to the target subject (rat).

[0147] (Preparation of insulin administration solution) ・Insulin administration solution of Comparative Example 3 (1) 3.7 mg of solid insulin (Sigma-Aldrich, human recombinant insulin) was added to 92.5 μL of 0.1 M hydrogen chloride aqueous solution. To this, 500 μL of physiological saline to which MC was added as an insulin container adsorption inhibitor to a final concentration of 0.001 W / V% was added to prepare a 4 mg / mL dilute hydrochloric acid physiological saline insulin solution. (2) 70 μL of the dilute hydrochloric acid physiological saline insulin solution prepared in (1) above was set aside in a separate container. Then, 434 μL of the isotonic solution of Comparative Example 3 containing 0.001 W / V% MC was prepared and added to the dilute hydrochloric acid physiological saline insulin solution set aside in a separate container. Next, 56 μL of 0.1 M sodium hydroxide aqueous solution was added to prepare the insulin administration solution of Comparative Example 3. The final insulin concentration of the insulin administration solution of Comparative Example 3 was 96.25 μg / mL.

[0148] - 4.4 mg of solid insulin (human recombinant insulin, 099-06473, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the insulin administration solution of Example 8-1. 4168.7 μL of physiological saline with added MC to achieve a final concentration of 0.001 W / V% as an insulin container adsorption inhibitor, and 171 μL of the UFB-containing isotonic solution of Example 8 were added. Next, 114 μL of a 0.1 M sodium hydroxide aqueous solution was added to prepare the insulin administration solution of Example 8-1. The final insulin concentration of the insulin administration solution of Example 8-1 was 962.5 μg / mL, and the final UFB concentration was 500 million cells / mL.

[0149] - 4.2 mg of solid insulin (human recombinant insulin, 099-06473, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the insulin administration solution of Example 8-2. 218.6 μL of physiological saline to which MC was added as an insulin container adsorption inhibitor to a final concentration of 0.001 W / V%, and 3930 μL of the UFB-containing isotonic solution of Example 8 were added. Next, 109 μL of a 0.1 M sodium hydroxide aqueous solution was added to prepare the insulin administration solution of Example 8-2. The final insulin concentration of the insulin administration solution of Example 8-2 was 962.5 μg / mL, and the final UFB concentration was 12.1 billion cells / mL.

[0150] <<Experimental Conditions>> (Subjects) SD rats, male, 220-250g (2 rats per group) The above rats were fasted for 24 hours before the test, and no small intestine washing was performed.

[0151] (Insulin Concentration) The insulin concentrations of the insulin administration solutions in each of Examples 8-1, 8-2, and Comparative Example 3 are shown below, converted using the formula 1 mg / mL = 26 IU / mL of human insulin. • Examples 8-1, 8-2 (Intestinal Administration) 962.5 μg / mL ≈ 25 IU / mL • Comparative Example 3 (Subcutaneous Administration) 96.25 μg / mL ≈ 2.5 IU / mL

[0152] (Insulin Dosage) ・Examples 8-1 and 8-2 (Intra-intestinal administration): 0.5 mL / (Rat body weight) 250 g (= 2 mL / kg) ・Comparative Example 3 (Subcutaneous administration): 0.1 mL / (Rat body weight) 250 g (= 0.4 mL / kg)

[0153] Furthermore, since the insulin concentration in the "insulin administration solution (composition for intestinal absorption)" administered intraintestinally is 0.962 mg / mL ≈ 25 IU / mL, administering 2 mL / kg of the "insulin administration solution (composition for intestinal absorption)" is equivalent to an "insulin dosage of 50 IU / kg". When administered subcutaneously, it is equivalent to 1 IU / kg, which is 1 / 50 of that amount.

[0154] (Blood sampling sites after insulin administration) Tail vein, fundus vein, jugular vein (cannulation)

[0155] (Measurement of insulin concentration in rat plasma) The insulin solutions of Example 8-1 and Example 8-2 were administered to the jejunum or ileum loop. The insulin solution of Comparative Example 3 was administered subcutaneously to determine the relative BA ratio, which will be described later.

[0156] Insulin concentrations in rat plasma were measured using a commercially available kit (Insulin ELISA RUO, EIA-2935R, DRG International, Inc., USA) according to the included instructions.

[0157] ≪Results≫ Figure 15 shows the measurement results when the insulin solution of Comparative Example 3 was administered subcutaneously. Figure 16 shows the measurement results when the insulin solution of Example 8-1 was administered jejunally. Figure 17 shows the measurement results of the insulin concentration when the insulin solution of Example 8-1 was administered ileally. Figure 18 shows the measurement changes in insulin concentration when the insulin solution of Example 8-2 was administered ileally.

[0158] Figures 17 and 18 show that in the case of ileal administration, the insulin concentration in the blood peaked approximately 10 minutes after administration of the insulin solution. This indicates that, regardless of the UFB concentration of the insulin solution, insulin absorption from the small intestine into the vein was achieved in a very short time.

[0159] The area under the curve (AUC) of insulin concentration (μIU / mL) in rat plasma obtained over time was determined using the trapezoidal method, and the relative bioavailability (BA) rate was calculated according to the following formula (7). The relative BA rate is the ratio of insulin concentration in rat plasma when the BA rate for subcutaneous administration is set to 100 [%]. The calculated relative BA rates are shown in Table 1. Formula (7): Relative BA rate (%) Relative BA rate (%) = (Insulin concentration AUC value when administered jejunally or ileum) / (Insulin concentration AUC value when administered subcutaneously) × 100 (%)

[0160]

[0161] [Test Example 5: Confirmation Test for Inhibition of Insulin Aggregate Formation by UFB] The confirmation test for inhibition of insulin aggregate formation was conducted based on the information provided by A. Hawe et al. (European Journal of Pharmaceutical Sciences, 38, (2009) pp. 79).

[0162] More specifically, the insulin test solution described later was prepared and added in 2-well portions at a rate of 50 μg / well (optical path length: 0.286 cm) using a UV Half Area Plate, 96 Well (manufactured by CORNING).

[0163] The absorbance spectra from 200 to 450 nm were measured at 1 nm intervals using UV-VIS spectrophotometry with a Thermo Scientific Multiskan SkyHigh microplate spectrophotometer (manufactured by Thermo Fisher Scientific).

[0164] Subsequently, the absorbance values ​​at 214 nm and 350 nm were substituted into equation (8) to calculate the aggregate index. The absorbance values ​​at 214 nm and 900 nm were also substituted into equation (9) to calculate the turbidity index.

[0165] Equation (8) Aggregate Index x = {a / (b - a)} × 100 x: Aggregate Index a: Absorbance value at 350 nm b: Absorbance value at 214 nm

[0166] Equation (9) Turbidity Index y = {c / (b - c)} × 100 y: Turbidity Index c: Absorbance value at 900 nm b: Absorbance value at 214 nm

[0167] <Comparative Measurement: Comparative Example 1 (Urpure Water) and Example 5 (Urpure Water + UFB)> (Preparation of Insulin Test Solution) (1) 3.2 mg of solid insulin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., human recombinant insulin, 099-06473) was mixed with 1,056 μL of ultrapure water to which MC was added as an insulin container adsorption inhibitor to a final concentration of 0.001 W / V%, to prepare a 3 mg / mL ultrapure water insulin solution. (2) 150 μL of the ultrapure water insulin solution prepared in (1) above was separated into a container. Then, an equal amount of the hypotonic solution (ultrapure water) of Comparative Example 1 containing 0.001 W / V% MC was added. The same procedure was carried out to prepare test solutions by sequentially diluting them 2-fold, and insulin test solutions of Comparative Example 1 with different insulin concentrations were obtained. The final insulin concentrations of the insulin test solution in Comparative Example 1 were 0.609 mU / L, 1.22 mU / L, 2.43 mU / L, 4.87 mU / L, 9.74 mU / L, 19.5 mU / L, and 39.0 mU / L. (3) 36.7 μL of 0.1 M aqueous hydrogen chloride solution was added to 1.1 mg of solid insulin used in (1) above. To this, 326 μL of the UFB-containing hypotonic solution from Example 5, to which MC was added as an insulin container adsorption inhibitor to a final concentration of 0.001 W / V%, was added to prepare a 3 mg / mL dilute hydrochloric acid UFB insulin solution. (4) 150 μL of the dilute hydrochloric acid UFB insulin solution prepared in (3) above was taken into a separate container. Then, an equal amount of UFB containing 0.001 W / V% MC was added and mixed. The same procedure was followed to sequentially dilute the test solutions twofold to obtain insulin test solutions of Example 5 with different insulin concentrations. The final insulin concentrations of the insulin test solutions of Example 5 were 0.609 mU / L, 1.22 mU / L, 2.43 mU / L, 4.87 mU / L, 9.74 mU / L, 19.5 mU / L, and 39.0 mU / L. The final UFB concentration for each test solution was 12.1 billion cells / mL.

[0168] <Results> The measurement results are shown in Figures 19 and 20. Figure 19 is a graph plotting the aggregate index value of the insulin test solution of Comparative Example 1 against the insulin concentration and the aggregate index value of the insulin test solution of Example 5 against the insulin concentration. Figure 20 is a graph plotting the turbidity index value of the insulin test solution of Comparative Example 1 against the insulin concentration and the turbidity index of the insulin test solution of Example 5 against the insulin concentration.

[0169] Figure 19 confirms that the presence of UFB during insulin dissolution suppresses aggregate formation.

[0170] Figure 20 shows that when UFB is present during insulin dissolution, insulin-derived turbidity becomes undetectable and is dispersed in the solution.

[0171] [Test Example 6: Confirmation Test of Intestinal Absorption-Promoting Effect of Insulin Using an Intestinal Absorption Composition] Using the known in-situ closed loop method shown in Figures 12 and 13, the insulin administration solutions (intestinal absorption compositions) of Examples 8-3 and 8-4 were directly administered to the intestinal tract of the subjects (rats). Subsequently, the amount absorbed via the intestinal tract was measured by measuring the insulin concentration in the rat plasma. The specific administration procedure for the insulin administration solutions of Examples 8-3 and 8-4 was as shown in Figure 14. This confirmed the absorption-promoting effect of the "intestinal absorption composition" according to this embodiment on high molecular weight compounds (insulin). The insulin administration solution of Comparative Example 3, which was prepared by the same method as in Test Example 4 described above, was subcutaneously administered to the subjects (rats). The insulin administration solutions of Examples 8-3 and 8-4 were prepared as follows.

[0172] - Preparation of the insulin administration solution in Example 8-3: 4.4 mg of solid insulin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., human recombinant insulin, 099-06473) was added to 114 μL of 0.1 M hydrogen chloride aqueous solution. To this, 4168.7 μL of physiological saline to which MC was added as an insulin adsorption inhibitor to a final concentration of 0.001 W / V%, and 171 μL of UFB-containing physiological saline with an average bubble diameter of approximately 100 nm and a UFB concentration of 13.4 billion cells / mL (50 billion cells / mL by UV method) were added. Next, 114 μL of 0.1 M sodium hydroxide aqueous solution was added to prepare the insulin administration solution of Example 8-3. The final insulin concentration of the insulin administration solution of Example 8-3 was 962.5 μg / mL, and the final UFB concentration was 500 million cells / mL (1.87 billion cells / mL by UV method).

[0173] - Preparation of the insulin administration solution in Example 8-4: 4.2 mg of solid insulin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., human recombinant insulin, 099-06473) was added to 109 μL of 0.1 M hydrogen chloride aqueous solution. To this, 218.6 μL of physiological saline to which MC was added as an insulin container adsorption inhibitor to a final concentration of 0.001 W / V%, and 3930 μL of UFB-containing physiological saline with an average bubble diameter of approximately 100 nm and a UFB concentration of 13.4 billion cells / mL (50 billion cells / mL by UV method) were added. Next, 109 μL of 0.1 M sodium hydroxide aqueous solution was added to prepare the insulin administration solution of Example 8-4. The final insulin concentration of the insulin administration solution of Example 8-4 was 962.5 μg / mL, and the final UFB concentration was 12.1 billion cells / mL (45 billion cells / mL by UV method).

[0174] <<Experimental Conditions>> (Subjects) SD rats, male, 220-250g (2 rats per group) The above rats were fasted for 24 hours before the test, and no small intestine washing was performed.

[0175] (Insulin Concentration) The insulin concentrations of the insulin administration solutions in each of Examples 8-3, 8-4, and Comparative Example 3 are shown below, converted using the formula 1 mg / mL = 26 IU / mL of human insulin. • Examples 8-3, 8-4 (Intestinal Administration) 0.962 mg / mL ≈ 25 IU / mL • Comparative Example 3 (Subcutaneous Administration) 0.0962 mg / mL ≈ 2.5 IU / mL

[0176] (Insulin Dosage) ・Examples 8-3, 8-4 (Intra-intestinal administration) 0.5 mL / (Rat body weight) 250 g (= 2 mL / kg) ・Comparative Example 3 (Subcutaneous administration) 0.1 mL / (Rat body weight) 250 g (= 0.4 mL / kg)

[0177] Furthermore, since the insulin concentration in the "insulin administration solution (composition for intestinal absorption)" administered intraintestinally is 0.962 mg / mL ≈ 25 IU / mL, administering 2 mL / kg of the "insulin administration solution (composition for intestinal absorption)" is equivalent to an "insulin dosage of 50 IU / kg". When administered subcutaneously, it is equivalent to 1 IU / kg, which is 1 / 50 of that amount.

[0178] (Blood sampling sites after insulin administration) Tail vein, fundus vein, jugular vein (cannulation)

[0179] (Measurement of insulin concentration in rat plasma) The insulin solutions of Examples 8-3 and 8-4 were administered to the jejunum or ileum loop. To determine the absolute BA ratio described later, the insulin solution of Comparative Example 3 was administered subcutaneously.

[0180] Insulin levels in rat plasma were measured using a commercially available kit (Insulin ELISA RUO, EIA-2935R, DRG International, Inc., USA) according to the included instructions.

[0181] ≪Results≫ Figure 15 shows the measurement results when the insulin solution of Comparative Example 3 was administered subcutaneously. The measurement results when the insulin solution of Example 8-3 was administered jejunally and ileally were the same as the measurement results of Example 8-1 described above, as shown in Figures 16 and 17. The measurement results of the insulin concentration when the insulin solution of Example 8-4 was administered ileally were the same as the measurement results of Example 8-2 described above, as shown in Figure 18.

[0182] The area under the curve (AUC) of insulin concentration (μIU / mL) in rat plasma obtained over time was determined using the trapezoidal method, and the absolute bioavailability (BA) rate was calculated according to the following formula (10). Formula (10): Absolute BA rate (%) = (Insulin concentration AUC value at jejunal or ileal administration / Dose at jejunal or ileal administration (IU / kg)) / (Insulin concentration AUC value at subcutaneous administration / Dose at subcutaneous administration (IU / kg)) × 100 (%)

[0183] The AUC values ​​of insulin concentrations were determined when administered jejunally or ileum in Examples 8-3 and 8-4, and the absolute BA rate was calculated. As a result, the absolute BA rate at a UFB concentration of 500 million cells / mL (Example 8-3) when administered jejunally was 1.2%, and the absolute BA rate at a UFB concentration of 12.1 billion cells / mL (Example 8-4) when administered jejunally was 0.3%. Furthermore, the absolute BA rate at a UFB concentration of 500 million cells / mL (Example 8-3) when administered ileum was 0.5-0.6%, and the absolute BA rate at a UFB concentration of 12.1 billion cells / mL (Example 8-4) when administered ileum was 0.7-1.1%.

[0184] The intestinal absorption composition of the present invention allows for the oral administration of drugs containing poorly soluble compounds, and therefore can be used in the treatment of diabetes and the like. Cross-reference of related applications

[0185] This application claims priority based on Japanese Patent Application No. 2024-178374 filed with the Japan Patent Office on 10 October 2024, Japanese Patent Application No. 2025-019495 filed with the Japan Patent Office on 7 February 2025, and Japanese Patent Application No. 2025-040663 filed with the Japan Patent Office on 13 March 2025, all of which disclosures are incorporated herein by reference in their entirety.

[0186] 10 Physiological solution containing microbubbles 20 (First) Physiological solution containing microbubbles A Sparingly soluble compound B Microbubble group B1 First microbubbles B2 Second microbubbles C Physiological solution

Claims

1. An intestinal absorption composition comprising first microbubbles, a sparingly soluble compound, and a physiological solution, wherein the concentration of the first microbubbles is 2 billion / mL or more, and at least a portion of the sparingly soluble compound coats at least a portion of the first microbubbles.

2. The intestinal absorption composition according to claim 1, wherein the concentration of the first microbubbles is 15 billion / mL or more.

3. The intestinal absorption composition according to claim 1, wherein the concentration of the first microbubbles is 30 billion / mL or more.

4. The intestinal absorption composition according to claim 1, wherein the average bubble diameter of the first microbubbles is less than 1.0 μm.

5. The intestinal absorption composition according to claim 1, wherein the average bubble diameter of the first microbubbles is 200 nm or less.

6. The intestinal absorption composition according to claim 1, wherein the first microbubbles contain a gas selected from the group consisting of hydrogen, helium, oxygen, nitrogen, methane, fluorine, neon, carbon dioxide, nitric oxide, carbon monoxide, ozone, argon, chlorine, ethane, propane, air, and mixed gases containing these.

7. The intestinal absorption composition according to claim 1, wherein the first microbubbles contain a medical gas.

8. The intestinal absorption composition according to claim 1, wherein the poorly soluble compound is insulin or a derivative thereof.

9. The intestinal absorption composition according to claim 1, wherein the physiological solution comprises one or more selected from ultrapure water, reverse osmosis water, modified reverse osmosis water, physiological saline, glucose solution, distilled water for injection, purified water, phosphate buffer, and ion-exchanged water.

10. The intestinal absorption composition according to claim 1, wherein when the concentration of the sparingly soluble compound is 120 pM or more, the absorbance measured at a wavelength of 450 nm and a path length of 5.0 mm is 0.05 or more.

11. The intestinal absorption composition according to claim 1, wherein the membrane permeability coefficient of the sparingly soluble compound when the concentration of the first microbubbles is 5 billion / mL is greater than the membrane permeability coefficient of the sparingly soluble compound when the concentration of the first microbubbles is 0 billion / mL, and the membrane permeability coefficient of the sparingly soluble compound when the concentration of the first microbubbles is 10 billion / mL is greater than the membrane permeability coefficient of the sparingly soluble compound when the concentration of the first microbubbles is 5 billion / mL.

12. The intestinal absorption composition according to claim 1, which is administered orally.

13. A therapeutic agent for diabetes comprising the intestinal absorption composition according to any one of claims 1 to 12.

14. A method for producing an intestinal absorption composition, comprising: a microbubble group generation step of generating a microbubble group containing a first microbubble and a second microbubble in a physiological solution and synthesizing a physiological solution containing the microbubble group; and a second microbubble removal step of removing the second microbubble from the physiological solution containing the microbubble group and synthesizing a physiological solution containing microbubbles, wherein the average bubble diameter of the first microbubbles is less than 1.0 μm and the average bubble diameter of the second microbubbles is 1.0 μm or more.

15. A method for producing an intestinal absorption composition according to claim 14, further comprising: a first microbubble concentration adjustment step of adjusting the concentration of the first microbubbles in the microbubble-containing physiological solution to 2 billion microbubbles / mL or more; and a mixing step of mixing a poorly soluble compound with the concentration-adjusted microbubble-containing physiological solution.

16. The method for producing an intestinal absorption composition according to claim 15, wherein the first microbubble concentration adjustment step is a step of removing water from the microbubble-containing physiological solution by cross-flow filtration.

17. A method for producing a drug for treating diabetes, comprising: a microbubble group generation step of generating a microbubble group containing a first microbubble and a second microbubble in a physiological solution and synthesizing a physiological solution containing the microbubble group; and a second microbubble removal step of removing the second microbubble from the physiological solution containing the microbubble group and synthesizing a physiological solution containing microbubbles, wherein the average bubble diameter of the first microbubbles is less than 1.0 μm and the average bubble diameter of the second microbubbles is 1.0 μm or more.

18. A method for producing a diabetes treatment agent according to claim 17, further comprising: a first microbubble concentration adjustment step of adjusting the concentration of the first microbubbles in the microbubble-containing physiological solution to 2 billion microbubbles / mL or more; and a mixing step of mixing a poorly soluble compound with the concentration-adjusted microbubble-containing physiological solution.

19. The method for producing a diabetes treatment agent according to claim 18, wherein the first microbubble concentration adjustment step is a step of removing water from the microbubble-containing physiological solution by cross-flow filtration.

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