Insulin replacement peptide carrier and insulin replacement method

Abstract

The purpose of the present invention is to provide a novel drug capable of replenishing insulin while reducing the burden on patients who need self-injection or the like.　An insulin replacement peptide carrier according to the present invention is characterized by comprising a vesicle of a plant of the Malpighiaceae family, wherein a substance to be carried is an insulin replacement peptide. The insulin replacement peptide includes insulin, an analog of insulin, and an insulin secretion promoting peptide. The insulin secretion promoting peptide is, for example, GLP-1, or an analog of GLP-1. A method for producing an insulin replacement peptide delivery reagent according to the present invention is characterized by comprising a step in which a composite of the vesicle in the insulin replacement peptide carrier and the insulin replacement peptide is formed by causing the insulin replacement peptide carrier according to the present invention comprising the vesicle to coexist in a solvent with the insulin replacement peptide to be delivered.

Description

Carrier for Insulin-Replacing Peptides and Method for Insulin Replacement

[0001] The present invention relates to a carrier for insulin-replacing peptides and a method for insulin replacement.

[0002] Insulin is a peptide hormone that exhibits the effect of lowering blood glucose levels in the body. Patients with type 1 diabetes, which is caused by insulin deficiency due to destruction of pancreatic β-cells, and type 2 diabetes, in which insulin secretion can no longer be promoted by insulin secretion promoters, need to lower blood glucose by supplementing insulin via subcutaneous injection.

[0003] Since insulin injection is performed by self-injection by the patient themselves, it imposes a heavy burden on the patient. For this reason, insulin preparations such as rapid-acting and long-acting insulins have been developed, and development of syringes that are easy to handle and less likely to cause pain is also underway. However, many patients are children or elderly people, and long-term injection may be required in some cases, so reducing the burden on patients and assistants such as their family members still remains a challenge.

[0004] Similarly, the aforementioned insulin secretion promoters also have the same problem because they require injection.

[0005] Therefore, an object of the present invention is to provide a novel drug capable of administering insulin-replacing peptides such as insulin and insulin secretion promoters while reducing the burden on patients such as self-injection.

[0006] In order to achieve the above object, the carrier for insulin-replacing peptides of the present invention comprises vesicles of a plant belonging to the family Plantaginaceae, the substance to be retained is an insulin-replacing peptide, and the insulin-replacing peptide is at least one selected from the group consisting of insulin, insulin analogs, and insulin secretion-promoting peptides.

[0007] The present invention provides a method for producing an insulin delivery reagent, comprising the step of forming a complex between the vesicles of the insulin delivery peptide carrier and the insulin delivery peptide by coexisting the insulin delivery peptide carrier containing the vesicles of the present invention and the insulin delivery peptide in a solvent, wherein the insulin delivery peptide is at least one selected from the group consisting of insulin, insulin analogues, and insulin secretion-promoting peptides.

[0008] The insulin replacement agent, blood glucose control agent, or pharmaceutical for a disease requiring blood glucose control of the present invention each comprises an insulin replacement peptide carrier containing the vesicles of the present invention and an insulin replacement peptide for delivery, wherein the insulin replacement peptide is at least one selected from the group consisting of insulin, insulin analogues, and insulin secretion-promoting peptides.

[0009] The present invention provides an insulin replacement method characterized by including an administration step of administering a complex of an insulin replacement peptide carrier containing the vesicles of the present invention and an insulin replacement peptide for delivery.

[0010] The blood glucose control method of the present invention is characterized by performing the insulin replacement method of the present invention described above.

[0011] The insulin replacement peptide carrier of the present invention contains vesicles from a Malpighiaceae plant such as acerola, and these vesicles can hold the insulin replacement peptide. According to the insulin replacement peptide carrier of the present invention, by administering the complex containing the insulin replacement peptide, for example, orally, it becomes possible to administer insulin or insulin secretion-promoting peptides as an insulin replacement peptide to the body and control blood glucose levels. Thus, according to the present invention, for example, oral administration is possible, enabling easy administration of insulin replacement peptides in a way that reduces the burden on the patient.

[0012] Figure 1 is a graph showing the changes in blood glucose levels of mice in Example 1. Figure 2 is a graph showing the changes in blood glucose levels of mice in Example 2.

[0013] Unless otherwise specified, terms used herein may be used in the sense commonly used in the art.

[0014] [1] A carrier for insulin supplement peptides, comprising vesicles of a Malpighiaceae plant, wherein the retaining object is an insulin supplement peptide, and the insulin supplement peptide is at least one selected from the group consisting of insulin, insulin analogs, and insulin secretion-promoting peptides. [2] The carrier for insulin supplement peptides according to [1], wherein the insulin secretion-promoting peptide is GLP-1 or a GLP-1 analog. [3] The carrier for insulin supplement peptides according to [1] or [2], wherein the vesicles are vesicles of the fruit of a Malpighiaceae plant. [4] The carrier for insulin supplement peptides according to any one of [1] to [3], wherein the Malpighiaceae plant is an acerola species plant. [5] The carrier for insulin supplement peptides according to any one of [1] to [4], wherein the average particle size of the vesicles is 30 to 400 nm. [6] The carrier for insulin supplement peptides according to any one of [1] to [5], wherein the vesicles are extracellular vesicles. [7] The insulin replacement peptide carrier according to any one of [1] to [6], further comprising at least one selected from the group consisting of insulin, insulin analogs, and insulin secretion-promoting peptides. [8] A method for producing an insulin replacement peptide delivery reagent, comprising the step of forming a complex of the vesicles of the insulin replacement peptide carrier and the insulin replacement peptide by coexisting the insulin replacement peptide carrier containing vesicles according to any one of [1] to [6] and an insulin replacement peptide for delivery purposes in a solvent, wherein the insulin replacement peptide is at least one selected from the group consisting of insulin, insulin analogs, and insulin secretion-promoting peptides. [9] A method for producing an insulin replacement peptide delivery reagent according to [8], comprising the step of mixing the insulin replacement peptide carrier and the insulin replacement peptide in the solvent and incubating them.

[10] An insulin supplement comprising an insulin supplement carrier containing vesicles as described in any one of [1] to [6] and an insulin supplement peptide for delivery, wherein the insulin supplement peptide is at least one selected from the group consisting of insulin, insulin analogs, and insulin secretion-promoting peptides.

[11] The insulin supplement according to

[10] , wherein the insulin secretion-promoting peptide is an incretin.

[12] The insulin supplement according to

[11] , wherein the incretin is at least one selected from the group consisting of GLP-1, GLP-1 analogs, GIP, and GIP analogs.

[13] A blood glucose control agent comprising an insulin supplement carrier containing vesicles as described in any one of [1] to [6] and an insulin supplement peptide for delivery.

[14] A pharmaceutical product for a disease requiring blood glucose control, comprising an insulin supplement carrier containing vesicles as described in any one of [1] to [6] and an insulin supplement peptide for delivery.

[15] The pharmaceutical product according to

[14] , wherein the disease is a disease accompanied by symptoms of hyperglycemia.

[16] The pharmaceutical product according to

[14] or

[15] , wherein the disease is diabetes.

[17] The pharmaceutical product according to any one of

[14] to

[16] , wherein the disease is obesity.

[18] An insulin supplementation method characterized by comprising an administration step of administering a complex of an insulin supplement peptide carrier containing vesicles according to any one of [1] to [6] and an insulin supplement peptide for delivery purposes.

[19] The insulin supplementation method according to

[18] , further comprising a complex formation step of forming a complex of the insulin supplement peptide carrier and the insulin supplement peptide prior to the administration step.

[20] The insulin supplementation method according to

[18] or

[19] , wherein the administration step is a step of administering the complex to a living organism.

[21] The insulin supplementation method according to

[19] , wherein the administration method in the administration step is oral administration.

[22] The insulin supplementation method according to

[20] or

[21] , wherein the living organism is a human or a non-human animal.

[23] A blood glucose control method characterized by performing the insulin supplementation method according to any one of

[18] to

[22] .

[0015] (1) Carrier for Insulin Replacement Peptides The carrier for insulin replacement peptides of the present invention is characterized by containing vesicles of Malpighiaceae plants, as described above. The inventors have found that vesicles of Malpighiaceae plants hold the insulin replacement peptide and form a complex, and that blood glucose levels can be controlled by orally administering the complex to a living organism, thus establishing the present invention. The present invention is characterized by containing the above vesicles, and other components and conditions are not particularly limited. Hereinafter, vesicles of Malpighiaceae plants will also be referred to as plant-derived vesicles.

[0016] As described above, the insulin replacement peptide carrier of the present invention can hold the insulin replacement peptide by the vesicles, and by holding it, it can deliver the insulin replacement peptide. For this reason, the insulin replacement peptide carrier of the present invention can also be used as, for example, a reagent for insulin replacement peptide delivery.

[0017] The species of Malpighiaceae plants mentioned above are not particularly limited, and examples include the genus Malpighia, specifically, species such as Malpighia sp., and preferably acerola such as M. emarginata DC., M. glabra, and M. punicifolia.

[0018] The vesicles of the Malpighiaceae plant (plant-derived vesicles) can be prepared from the raw material, for example, using the whole plant (individual) or any part of the plant as the raw material. Examples of the parts include the fruit, fruit peel, pulp, pulp containing seeds, juice, seeds, leaves, etc. The raw material may be, for example, one type of the part, or it may contain two or more types of the parts. The raw material is preferably, for example, fruit, pulp, or juice. The juice can be prepared, for example, by squeezing the fruit or pulp.

[0019] The fruit may be fully ripe, unripe, or a mixture thereof. The fruit may be, for example, fruit stored at room temperature, fruit stored in the refrigerator, or fruit stored frozen. The fruit juice may be, for example, fruit juice stored at room temperature, fruit juice stored in the refrigerator, or fruit juice stored frozen. The vesicles are preferably, for example, vesicle fractions recovered from the fruit juice, as described later.

[0020] The plant-derived vesicles can be prepared, for example, by fractionation from the fruit. The preparation method is not particularly limited, and one example is to recover a liquid fraction containing vesicles from the fruit and fractionate the vesicles from the liquid fraction. The liquid fraction may be, for example, fruit juice extracted from the fruit, crushed fruit, a suspension of the crushed fruit, or an extract from the crushed fruit. The fruit juice can be prepared, for example, by extracting juice from the fruit using a grinding device, a pressing device, etc. The suspension can be prepared, for example, by crushing the fruit and mixing the crushed fruit with a solvent. The extract can be prepared, for example, by mixing the crushed fruit with the solvent and recovering the liquid fraction. The solvent is not particularly limited, and for example, an aqueous solvent can be used. Examples of aqueous solvents include water, buffer solutions, physiological saline, and buffered physiological saline. Examples of buffered physiological saline include phosphate-buffered saline (PBS).

[0021] The method for fractionating vesicles from the liquid fraction is not particularly limited and may include, for example, ultrafiltration, ultracentrifugation, concentration gradient centrifugation, or separation methods using a microliquid system. For the preparation method, commercially available kits may be used, such as the ExoEasy Maxi Kit (trade name, QIAGEN), ExoQuick (trade name, System Bioscience), or Total Exosome Isolation reagent (trade name, Invitrogen).

[0022] The insulin replacement peptide carrier of the present invention can, for example, use a vesicle fraction containing a plurality of the aforementioned vesicles. The size of the vesicles is not particularly limited, and examples of particle sizes include 30-400 nm, 80-300 nm, 150-300 nm, 150-250 nm, 200-250 nm, 100-200 nm, and 80-200 nm. The aforementioned vesicles are also called, for example, microvesicles or nanovesicles. When the vesicle fraction is represented by a particle size distribution, the particle size peaks are not particularly limited and include, for example, 30-400 nm, 80-300 nm, 150-300 nm, 150-250 nm, 200-250 nm, 100-200 nm, 80-200 nm, 200±100 nm, 200±50 nm, 200±30 nm, and 200±20 nm. Furthermore, in the particle size distribution, when all vesicles are considered as 100%, the proportion of vesicles at the aforementioned peaks (for example, 200±50 nm, 200±20 nm) is not particularly limited, with a lower limit of, for example, 30% or more, 50% or more, and 80% or more, and an upper limit of, for example, 70% or less, 80% or less, 90% or less, and 100%. The vesicle fraction is, for example, a fraction extracted from the liquid fraction to have the particle size and particle size distribution described above. When using the vesicle fraction as a carrier for insulin replacement peptides according to the present invention, for example, a fraction extracted from the liquid fraction by an extraction method that results in the particle size and particle size distribution described above can be used. The vesicle fraction may contain, for example, other components derived from the liquid fraction (e.g., the fruit juice or the extract).

[0023] The method for measuring vesicle particle size is not particularly limited and can be performed by, for example, light scattering, Brownian motion-based measurement methods, or electrical resistance methods. Brownian motion-based measurement methods include, for example, nanoparticle tracking analysis, and commercially available nanoparticle analyzers (product name NanoSight, Malvern Corporation) can be used. Similarly, for electrical resistance methods, commercially available nanoparticle multianalyzers (product names qNANO, Exoid) can be used. When using NanoSight, example measurement conditions include a measurement time of 90 seconds and 3 repetitions.

[0024] The plant-derived vesicles may be, for example, extracellular vesicles or intracellular vesicles, with extracellular vesicles being preferred. Examples of such vesicles include exosome-like vesicles. These exosome-like vesicles are, for example, vesicles of a size equivalent to extracellular vesicles (EVs) derived from human cells, and more specifically, vesicles of a size equivalent to small EVs (also called exosomes) derived from human cells. These exosome-like vesicles are, for example, vesicles obtained by the same isolation method as human cell-derived extracellular vesicles, preferably human cell-derived exosomes.

[0025] The extracellular vesicles can also be detected, for example, by plant extracellular vesicle markers. Examples of such markers include TET8, Heat Shock Proteins (HSP60, HSP70, HSP80, HSP90), Aquaporin family Patellines (Patellin 1, Patellin 2, Patellin 3), Syntaxins, Clathrin heavy chain family (Clathrin 1, Clathrin 2), and Sphingolipids. Further information on these markers can be found in, for example, the following publication: Nemati et al., Plant-derived extracellular vesicles: a novel nanomedicine approach with advantages and challenges. Cell Communication and Signaling 2022.

[0026] The object to be held by the insulin replacement peptide carrier of the present invention is an insulin replacement peptide. Examples of the insulin replacement peptide include insulin, insulin analogues, and insulin secretion-promoting peptides. In the case of insulin, insulin itself can be replenished as a substance; in the case of insulin analogues, insulin function can be replenished; and the insulin secretion-promoting peptide can replenish insulin by promoting insulin secretion.

[0027] The insulin may be, for example, insulin purified from an animal, or insulin produced by genetic engineering techniques. In this specification, a peptide identical to the amino acid sequence (original sequence) of animal insulin is referred to as "insulin." In this specification, an amino acid sequence in which a part of the original sequence is altered (mutant sequence) within the range that has the function of blood glucose control of the insulin, or an amino acid sequence modified from the original sequence or the mutant sequence (modified sequence) is referred to as an "insulin analog." Hereinafter, the insulin and the insulin analogs are also referred to as the "insulin group."

[0028] The animals mentioned above are not particularly limited and include, for example, humans and non-human animals, such as mice, rats, rabbits, dogs, monkeys, camels, and cattle. The insulin group to be held in the insulin replacement peptide carrier of the present invention is preferably insulin or insulin analogs based on the amino acid sequence of the same animal species as the target animal species. Specifically, when administered to humans, the amino acid sequence of the insulin is preferably the amino acid sequence of human insulin, and the amino acid sequence of the insulin analog is preferably, for example, a mutant or modified sequence of the amino acid sequence of human insulin.

[0029] The amino acid sequences of the aforementioned insulin include, for example, those registered in databases. Human Insulin: UniProt (Swiss-Prot) P01308 Human insulin mRNA: GenBank NM_000207.3 Human insulin protein: GenBank NP_000198.1 Standard insulin: DrugBank DB00030 Rapid-acting insulin: DrugBank DB01306 Ultra-rapid-acting insulin: DrugBank DB01309 Long-acting insulin: DrugBank DB01308

[0030] Examples of the secretion-promoting peptides include incretins. Examples of incretins include GLP-1, GLP-1 analogs, GIP, GIP analogs, and the like.

[0031] The aforementioned GLP-1 is a glucagon-like peptide-1, a receptor agonist peptide that acts on the GLP-2 receptor. GLP-1 may be, for example, GLP-1 purified from an animal, or GLP-1 produced by genetic engineering techniques. In this specification, a peptide identical to the amino acid sequence (original sequence) of animal GLP-1 is referred to as "GLP-1". In this specification, an amino acid sequence in which a part of the original sequence is altered (mutant sequence) within the range that has the function of promoting insulin secretion of GLP-1, or an amino acid sequence modified from the original sequence or the mutant sequence (modified sequence) is referred to as a "GLP-1 analog". Hereinafter, the aforementioned GLP-1 and the aforementioned GLP-1 analogs are also referred to as the "GLP-1 group".

[0032] The aforementioned GIP is a glucose-dependent insulin secretion-stimulating polypeptide and a receptor agonist peptide that acts on the GIP receptor. GIP may be, for example, GIP purified from an animal, or GIP produced by genetic engineering techniques. In this specification, a peptide identical to the amino acid sequence (original sequence) of animal GIP is referred to as "GIP". In this specification, an amino acid sequence in which a part of the original sequence is altered (mutant sequence) within the range that has the function of promoting insulin secretion of GIP, or an amino acid sequence modified from the original sequence or the mutant sequence (modified sequence) is referred to as a "GIP analog". Hereinafter, the aforementioned GIP and the aforementioned GIP analogs are also referred to as the "GLP-1 group".

[0033] The secretion-promoting peptide may be, for example, a receptor agonist peptide that acts on both the GLP-1 receptor and the GIP receptor. Examples of such peptides include a peptide (chilzepatide) in which GIP is modified to act as an agonist not only on the GIP receptor but also on the GLP-1 receptor.

[0034] The animals mentioned above are not particularly limited and include, for example, humans and non-human animals, such as mice, rats, rabbits, dogs, monkeys, camels, and cattle. The GLP-1 group to be held in the insulin replacement peptide carrier of the present invention is preferably GLP-1 or a GLP-1 analog based on the amino acid sequence of the same animal species as the target animal species. Specifically, when administered to humans, the amino acid sequence of GLP-1 is preferably the amino acid sequence of human GLP-1, and the amino acid sequence of the GLP-1 analog is preferably, for example, a mutant or modified sequence of the amino acid sequence of human GLP-1.

[0035] The amino acid sequences of GLP-1 mentioned above include, for example, those registered in databases. Human GLP-1: DrugBank DB01276 GLP-1: DrugBank DB06655 GLP-1: DrugBank DB09045 GLP-1: DrugBank DB13928

[0036] The insulin replacement peptide carrier of the present invention may or may not contain the insulin replacement peptide at a stage prior to use by the user. When the insulin replacement peptide carrier of the present invention contains the insulin replacement peptide, it is also referred to as an insulin replacement peptide delivery reagent or an insulin replacement peptide DDS reagent.

[0037] As in the former case, if the insulin replacement peptide carrier of the present invention contains the insulin replacement peptide, the user can, for example, use it as an insulin replacement peptide delivery reagent for the administration of the insulin replacement peptide. As in the latter case, if the insulin replacement peptide carrier of the present invention does not contain the insulin replacement peptide, the user can, for example, add the insulin replacement peptide to the insulin replacement peptide carrier of the present invention and use it as an insulin replacement peptide delivery reagent for the administration of the insulin replacement peptide.

[0038] When the carrier for insulin-replacing peptides of the present invention comprises said insulin-replacing peptide, for example, it is preferable that said vesicles and said insulin-replacing peptide form a complex (hereinafter also referred to as "vesicle complex"). The form of said vesicle complex is not particularly limited. For example, it is a state in which said vesicles retain said insulin-replacing peptide; as a specific example, it may be a form in which said insulin-replacing peptide is encapsulated inside said vesicles, or a form in which said insulin-replacing peptide is retained on the outer wall (outer surface) of said vesicles.

[0039] The method for forming said vesicle complex is not particularly limited. A method for forming a vesicle complex that retains said insulin-replacing peptide is illustrated below. The present invention is not limited to these illustrations.

[0040] First, a vesicle complex retaining said insulin-replacing peptide can be formed, for example, by allowing said vesicles of the carrier for insulin-replacing peptides of the present invention and said insulin-replacing peptide to coexist in a solvent.

[0041] Specifically, for example, a complex of said vesicles and said insulin-replacing peptide can be formed by allowing said vesicles and said insulin-replacing peptide to coexist in a solvent and incubating the mixture. The incubation temperature is not particularly limited. For example, it may be in the room temperature range (e.g., 30±10°C) or a temperature range lower than that (e.g., above 0°C and below 20°C), and is preferably a low temperature range (e.g., 4±5°C, 4±3°C, more preferably under ice-cooling conditions (e.g., 1 to 6°C)).

[0042] In said coexistence, the ratio of said vesicles to said insulin-replacing peptide is not particularly limited. For 1 µmol of said insulin-replacing peptide, for example, the amount of vesicles is 5×10 4 to 8×10 4 particles, 2×10 5 to 5×10 5 particles, 2×10 6 to 5×10 6 particles.

[0043] The incubation time is not particularly limited. The lower limit is not particularly limited, and is, for example, 5 minutes or more, or 15 minutes or more; the upper limit is not particularly limited, and for example, a plateau for complex formation can be reached by incubation for about 30 minutes. The solvent is not particularly limited, for example, an aqueous solvent can be used, and specific examples thereof include water, physiological saline, buffer solutions such as PBS, and the like.

[0044] In the coexistence of the vesicles and the insulin-replacing peptide, for example, common introduction methods such as electroporation, lipofection, and the like may also be used. According to the carrier for insulin-replacing peptide of the present invention, for example, the vesicles can be caused to retain the insulin-replacing peptide even only by coexisting the vesicles and the insulin-replacing peptide without using these common introduction methods.

[0045] As described above, according to the carrier for insulin-replacing peptide of the present invention, a complex of the vesicles and the insulin-replacing peptide can be formed, for example, merely by allowing the vesicles and the insulin-replacing peptide to coexist in the solvent. Therefore, the insulin-replacing peptide can be extremely easily caused to be retained in the vesicles. Furthermore, according to the carrier for insulin-replacing peptide of the present invention, for example, even when administered orally, degradation of the retained insulin-replacing peptide in vivo can be suppressed, allowing the insulin-replacing peptide to exert its function. In conventional replacement therapy with the insulin-replacing peptide, the insulin-replacing peptide loses function due to degradation by gastric acid and digestion, so subcutaneous administration and intraperitoneal administration, rather than oral administration, are employed. In contrast, according to the carrier for insulin-replacing peptide of the present invention, the function of the insulin-replacing peptide can be maintained even when administered orally, for example. Therefore, according to the present invention, unlike injection which causes pain and requires special instruments such as a syringe, administration of the insulin-replacing peptide with less burden on the patient can be achieved.

[0046] The recipients of the insulin replacement peptide delivered by the insulin replacement peptide carrier of the present invention are not particularly limited, and include, for example, humans or non-human animals. Examples of non-human animals include mice, rats, rabbits, dogs, monkeys, camels, and cattle.

[0047] (2) Insulin replacement peptide delivery reagent and method for producing the same The insulin replacement peptide delivery reagent of the present invention is characterized by comprising the insulin replacement peptide carrier of the present invention and the insulin replacement peptide. The insulin replacement peptide delivery reagent of the present invention is characterized by comprising vesicles of the Malpighiaceae plant as described above as a carrier for the insulin replacement peptide, and other components and conditions are not particularly limited.

[0048] In the insulin replacement peptide delivery reagent of the present invention, for example, the vesicles hold the insulin replacement peptide. That is, the insulin replacement peptide delivery reagent of the present invention includes, for example, a complex of the vesicles and the insulin replacement peptide (the vesicle complex). The description of the insulin replacement peptide delivery reagent of the present invention can be applied by reference to the description of the insulin replacement peptide carrier of the present invention.

[0049] The insulin replacement peptide delivery reagent of the present invention comprises, for example, a plurality of complexes of the vesicles and the insulin replacement peptide. In the plurality of complexes, the insulin replacement peptide may be, for example, one type or two or more types. Specifically, the insulin replacement peptide may include two or more types of the insulin group, or two or more types of the insulin secretion peptide, or one or two or more types of the insulin group and one or two or more types of the insulin replacement peptide.

[0050] Furthermore, the method for producing the insulin replacement peptide delivery reagent of the present invention is characterized by including a step of forming a complex between the vesicles of the insulin replacement peptide carrier and the insulin replacement peptide by coexisting the insulin replacement peptide carrier (a carrier containing vesicles of the Malpighiaceae plant) and the insulin replacement peptide for delivery purposes in a solvent. The method for producing the present invention is characterized by using the vesicles as the carrier, but other configurations and conditions are not particularly limited.

[0051] The method for producing the insulin replacement peptide delivery reagent of the present invention can be described by reference to the method for forming the vesicle complex in the insulin replacement peptide carrier of the present invention.

[0052] The insulin replacement peptide delivery reagent of the present invention can be used, for example, to supplement insulin in organisms that require blood glucose control, and is therefore also called an insulin replacement agent or a blood glucose control agent.

[0053] Furthermore, the insulin replacement peptide delivery reagent of the present invention can be used, for example, for blood glucose control, and is therefore also referred to as a pharmaceutical product for diseases requiring blood glucose control. The aforementioned diseases include, for example, diseases accompanied by symptoms of hyperglycemia, such as diabetes mellitus. Examples of diabetes mellitus include type 1 diabetes mellitus, type 2 diabetes mellitus, and gestational diabetes mellitus. The application of the pharmaceutical product of the present invention is not limited to diabetes mellitus, but can also be applied to, for example, symptoms of hyperglycemia, severe infections, trauma, surgical procedures, obesity, etc.

[0054] When a patient is a type 1 diabetic with insulin deficiency, a type 2 diabetic whose insulin secretion can no longer be stimulated by the aforementioned insulin secretagogue, or a patient who directly requires insulin function replacement, it is preferable to use, for example, the insulin replacement peptide delivery reagent of the present invention that holds at least one of the aforementioned insulin and the aforementioned insulin analogues.

[0055] Furthermore, in cases where the patient is a type 2 diabetes patient whose insulin secretion can be stimulated with the aforementioned insulin secretion-promoting agent, or a patient whose insulin function can be adequately supplemented by stimulating insulin secretion, it is preferable to use, for example, the insulin replacement peptide delivery reagent of the present invention that holds the aforementioned insulin secretion-promoting peptide for these patients. Specific examples of patients include, in addition to the aforementioned type 2 diabetes patients, obese patients, pre-obese patients, patients at risk of cardiovascular disease, patients with non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), PCOS (polycystic ovary syndrome), and patients with sleep apnea.

[0056] (3) Method for insulin supplementation The insulin supplementation method of the present invention is characterized by including an administration step of administering a complex of an insulin supplement peptide carrier containing the vesicles of the present invention and the insulin supplement peptide for delivery purposes, as described above. The insulin supplementation method of the present invention is characterized by using the vesicles of the Malpighiaceae plant described above, i.e., the insulin supplement peptide carrier of the present invention, as the carrier for delivery, and other configurations and conditions are not particularly limited. Furthermore, in the insulin supplementation method of the present invention, for example, the insulin supplement peptide carrier, the insulin supplement peptide delivery reagent in which the insulin supplement peptide is held on the insulin supplement peptide carrier, and the method for producing the same can be described by reference to the above descriptions of (1) and (2), unless otherwise specified.

[0057] The insulin supplementation method of the present invention may, for example, use the vesicle complex (insulin supplement peptide delivery reagent of the present invention) that has been prepared in advance, or it may further include a step of forming the vesicle complex. That is, the insulin supplementation method of the present invention may, for example, further include a formation step of forming a vesicle complex of the insulin supplement peptide carrier and the insulin supplement peptide prior to the administration step.

[0058] The administration step is, for example, the step of administering the vesicle complex to a living organism. The living organism is not particularly limited and may be a human or a non-human animal, and examples of non-human animals include mice, rats, rabbits, dogs, monkeys, camels, and cattle.

[0059] In the administration step, the preferred method for administering the vesicle complex is, for example, oral administration.

[0060] The insulin replacement method of the present invention can be used, for example, to replace insulin in a body that requires blood glucose control, and therefore is also called a blood glucose control method.

[0061] Furthermore, the insulin replacement method of the present invention can be used, for example, for blood glucose control, and therefore can also be called a treatment method for diseases requiring blood glucose control. The aforementioned diseases include, for example, diseases accompanied by symptoms of hyperglycemia, such as diabetes mellitus. Examples of diabetes mellitus include type 1 diabetes mellitus, type 2 diabetes mellitus, and gestational diabetes mellitus. The application of the present invention is not limited to diabetes mellitus, but can also be applied to, for example, symptoms of hyperglycemia, severe infections, trauma, surgical procedures, obesity, etc.

[0062] The present invention will be described in detail below with reference to examples, etc., but the present invention is not limited to these examples.

[0063] [Example 1] Insulin was administered orally using acerola-derived vesicles, and the effect of suppressing the rise in blood glucose levels was confirmed. The kit, reagents, and equipment were used in accordance with their instruction manuals.

[0064] (1) Preparation of vesicles Using 10 mL of juice from fully ripened acerola fruit from Okinawa, the vesicle fraction containing vesicles was extracted using a commercially available kit (product name Qiagen exoEasy kit, Qiagen). Specifically, the following procedure was performed: The 10 mL of juice was filtered using a membrane filter with a pore size of 0.45 μm (product name Durapore® PVDF Membrane Filter, Millipore). 10 mL of the resulting filtrate was subjected to the kit, and the eluted fraction containing vesicles was separated (isolated) by elution using 400 μL of the kit's Buffer (Buffer XE). The eluted fraction was then subjected to ultracentrifugation (100,000 × g, 49,000 rpm, 70 min, 4°C) to recover the vesicle pellet. This was suspended in 50 μL of PBS to obtain the vesicle fraction. Furthermore, the vesicle fraction was subjected to a nanoparticle analysis system (product name NanoSight, Malvern Corporation) to confirm the particle size distribution of the vesicles contained in the vesicle fraction. The results are shown below. Particle quantity: 2.2 × 10⁻⁶ ８ particles / μL Particle size: Mean 208nm Mode 155nm SD 108nm

[0065] PBS was added to the vesicle fraction, resulting in 4 × 10 ８ The sample was adjusted to particles / mL and designated as an acerola-derived vesicle sample (referred to as the AELN sample).

[0066] (2) Oral glucose tolerance test (OGTT) (Materials) Mice: 8-week-old C57BL, 5 groups (n=3 in each group) Glucose solution: Prepared by dissolving 3g of glucose in 20mL of physiological saline Insulin solution: Sigma Ardrich, IP 100U / mL, PO 280U / μL

[0067] The mice were given free access to water, but were deprived of food from the evening before administration, resulting in a 16-hour fast. At the 16-hour fasting stage, 300 μL of the glucose solution (per mouse) was orally administered to each mouse using a gastric tube. Immediately after glucose administration, the following sample administration procedures were performed on the mice in the five groups.

[0068] (Control group 1) 300 μL of PBS was orally administered to mice using a gastric tube. (Control group 2) 300 μL of a mixture of 50 μL of the ALEN sample and 250 μL of PBS was orally administered to mice using a gastric tube. (Comparative example group) 300 μL of insulin solution (100 U / mL) was orally administered to mice using a gastric tube. (Example group) 300 μL of the insulin solution (100 U / mL) was added to 50 μL of the ALEN sample and allowed to stand on ice for 30 minutes to form a complex of vesicles and insulin. The entire mixture was orally administered to mice using a gastric tube. (Reference example group) 300 μL of insulin solution (100 U / mL) was intraperitoneally administered to mice using a syringe.

[0069] Blood glucose levels were measured in each group of mice by collecting blood samples before glucose administration (0 minutes), and 15 and 30 minutes after sample administration. These results are shown in Figure 1.

[0070] Figure 1 is a graph showing the changes in blood glucose levels in mice, with the vertical axis representing blood glucose concentration and the horizontal axis representing time. As shown in Figure 1, in control group 1 (PBS only) and control group 2 (AELN sample + PBS), an increase in blood glucose levels was observed 15 minutes after glucose administration (glucose load), and no significant change was observed even after 30 minutes. In the comparative example group, where insulin alone was orally administered, despite the administration of insulin, an increase in blood glucose levels similar to that of control groups 1 and 2 was observed after 15 minutes, and no significant decrease in blood glucose levels was observed after 30 minutes. In contrast, in the example group, where a complex of AELN and insulin was orally administered, similar to the reference example group where insulin was administered intraperitoneally, no increase in blood glucose levels was observed after 15 minutes, and a further decrease in blood glucose levels was confirmed after 30 minutes.

[0071] These results show that oral administration of insulin alone does not suppress the rise in blood glucose levels, but oral administration of insulin as a complex with the acerola-derived vesicles allows for the suppression of the rise in blood glucose levels, similar to intraperitoneal administration. Intraperitoneal or subcutaneous injection of insulin places a significant burden on patients, but the present invention allows for oral administration of insulin, thereby greatly reducing the burden on patients.

[0072] [Example 2] GLP-1 was orally administered using acerola-derived vesicles, and the effect of suppressing the rise in blood glucose levels was confirmed.

[0073] OGTT was performed in the same manner as in Example 1, except that GLP-1 was used instead of insulin.

[0074] Specifically, similar to Example 1, fasted mice were orally administered the glucose solution, and immediately afterward, the following sample administration treatment was performed (n=3 in each group).

[0075] (Control group) 300 μL of a mixture of 50 μL of the ALEN sample and 250 μL of PBS was orally administered to mice using a gastric tube. (Comparative example group) 300 μL of GLP-1 solution (3.3 μM) was orally administered to mice using a gastric tube. (Example group) 300 μL of the GLP-1 solution (3.3 μM) was added to 50 μL of the ALEN sample and allowed to stand on ice for 30 minutes to form a complex of the vesicles and GLP-1. The entire mixture was orally administered to mice using a gastric tube. (Reference example group) 300 μL of GLP-1 solution (3.3 μM) was intraperitoneally administered to mice using a syringe.

[0076] Blood glucose levels were measured in each group of mice by collecting blood samples before glucose administration (0 minutes), and 15 and 30 minutes after sample administration. These results are shown in Figure 2.

[0077] Figure 2 is a graph showing the changes in blood glucose levels in mice. The vertical axis represents the relative value (%) of blood glucose concentration, with the concentration before glucose administration (0 minutes) set to 100%. The horizontal axis represents time. As shown in Figure 2, the control group (AELN sample + PBS) showed an increase in blood glucose levels 15 minutes after glucose administration (glucose load), and no significant change was observed even after 30 minutes. In the comparative example group, which received oral administration of GLP-1 alone, despite the administration of GLP-1, a similar increase in blood glucose levels was observed after 15 minutes as in the control group, and no significant decrease in blood glucose levels was observed after 30 minutes. In contrast, the example group, which received oral administration of a complex of AELN and GLP-1, showed no increase in blood glucose levels after 15 minutes, similar to the reference example group that received intraperitoneal administration of GLP-1, and a further decrease in blood glucose levels was confirmed after 30 minutes.

[0078] These results show that oral administration of GLP-1 alone does not suppress the rise in blood glucose levels, but oral administration of GLP-1 as a complex with the acerola-derived vesicles enables the suppression of the rise in blood glucose levels, similar to intraperitoneal administration. Intraperitoneal or subcutaneous injection of GLP-1 places a significant burden on patients, but according to the present invention, oral administration of GLP-1 is possible, thus significantly reducing the burden on patients.

[0079] Although the present invention has been described above with reference to embodiments, the present invention is not limited to the above embodiments. Various modifications to the configuration and details of the present invention can be made that will be understood by those skilled in the art within the scope of the present invention.

[0080] This application claims priority based on Japanese Patent Application No. 2025-49120, filed on 24 March 2025, and incorporates all of its disclosures herein.

[0081] The insulin replacement peptide carrier of the present invention contains vesicles from a Malpighiaceae plant such as acerola, and these vesicles can hold the insulin replacement peptide. According to the insulin replacement peptide carrier of the present invention, the insulin replacement peptide can be administered to the body by means of a complex containing the insulin replacement peptide, for example, by oral administration, thereby enabling control of blood glucose levels. Thus, according to the present invention, for example, oral administration can be employed, making it possible to easily administer the insulin replacement peptide in a way that reduces the burden on the patient.

Claims

1. A carrier for insulin replacement peptides, comprising vesicles of a Malpighiaceae plant, wherein the retained object is an insulin replacement peptide, and the insulin replacement peptide is at least one selected from the group consisting of insulin, insulin analogues, and insulin secretion-promoting peptides.

2. The insulin replacement peptide carrier according to claim 1, wherein the insulin secretion-promoting peptide is GLP-1 or a GLP-1 analog.

3. The carrier for insulin replacement peptide according to claim 1 or 2, wherein the vesicles are vesicles of the fruit of a plant of the family Malpighiaceae.

4. The carrier for insulin replacement peptide according to any one of claims 1 to 3, wherein the Malpighiaceae plant is an acerola species plant.

5. The insulin replacement peptide carrier according to any one of claims 1 to 4, wherein the average particle size of the vesicles is 30 to 400 nm.

6. The carrier for insulin replacement peptide according to any one of claims 1 to 5, wherein the vesicle is an extracellular vesicle.

7. The insulin replacement peptide carrier according to any one of claims 1 to 6, further comprising at least one selected from the group consisting of insulin, insulin analogues, and insulin secretion-promoting peptides.

8. A method for producing an insulin replacement peptide delivery reagent, comprising the step of forming a complex between the vesicles of the insulin replacement peptide carrier and the insulin replacement peptide by coexisting the insulin replacement peptide carrier containing vesicles according to any one of claims 1 to 6 in a solvent, wherein the insulin replacement peptide is at least one selected from the group consisting of insulin, insulin analogues, and insulin secretion-promoting peptides.

9. A method for producing an insulin replacement peptide delivery reagent according to claim 8, comprising the step of mixing the insulin replacement peptide carrier and the insulin replacement peptide in the solvent and incubating them.

10. An insulin replacement agent comprising a carrier for insulin replacement peptide containing vesicles according to any one of claims 1 to 6, and an insulin replacement peptide for delivery, wherein the insulin replacement peptide is at least one selected from the group consisting of insulin, insulin analogues, and insulin secretion-promoting peptides.

11. The insulin supplement according to claim 10, wherein the insulin secretion-promoting peptide is an incretin.

12. The insulin replacement agent according to claim 11, wherein the incretin is at least one selected from the group consisting of GLP-1, GLP-1 analogs, GIP, and GIP analogs.

13. A blood glucose control agent characterized by comprising an insulin replacement peptide carrier containing vesicles as described in any one of claims 1 to 6, and an insulin replacement peptide for delivery purposes.

14. A pharmaceutical product for a disease requiring blood glucose control, characterized by comprising a carrier for insulin replacement peptide containing vesicles as described in any one of claims 1 to 6, and an insulin replacement peptide for delivery purposes.

15. The pharmaceutical product according to claim 14, wherein the disease is a disease accompanied by symptoms of hyperglycemia.

16. The pharmaceutical product according to claim 14 or 15, wherein the disease is diabetes.

17. The pharmaceutical product according to any one of claims 14 to 16, wherein the disease is obesity.

18. An insulin replacement method characterized by comprising an administration step of administering a complex of an insulin replacement peptide carrier containing vesicles according to any one of claims 1 to 6 and an insulin replacement peptide for delivery purposes.

19. The insulin replacement method according to claim 18, further comprising a complex formation step of forming a complex of the insulin replacement peptide carrier and the insulin replacement peptide prior to the administration step.

20. The insulin replacement method according to claim 18 or 19, wherein the administration step is a step of administering the complex to a living organism.

21. The insulin replacement method according to claim 19, wherein the administration method in the administration step is oral administration.

22. The insulin replacement method according to claim 20 or 21, wherein the organism is a human or a non-human animal.

23. A blood glucose control method characterized by performing the insulin replacement method described in any one of claims 18 to 22.

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