Mucoadhesive pharmaceutical dosage forms for the unidirectional release of peptide therapeutic particles

Abstract

Provided herein is a pharmaceutical dosage form for delivering a peptide therapeutic to a mucosal surface. The pharmaceutical dosage form includes a mucoadhesive layer, a peptide-loaded layer, and a water-impermeable layer, the peptide-loaded layer being located between the mucoadhesive layer and the water-impermeable layer, and the water-impermeable layer facilitates unidirectional migration of the encapsulated peptide through the mucoadhesive layer to the target mucosal surface. The pharmaceutical dosage form is suitable for delivering the encapsulated peptide therapeutic to the buccal mucosa, sublingual mucosa, palatal mucosa, and lingual mucosa. The peptide therapeutic may be insulin, an insulin derivative, an insulin analog, pre-insulin, a prodrug of insulin, glucagon-like peptide 1 (GLP-1), a GLP-1 analog, pre-GLP-1, a prodrug of GLP-1, or a combination thereof, and may be suitable for the treatment of diabetes.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 400,863, filed August 25, 2022, entitled "Mucooadhesive Buccal Tablet with Unidirectional Release Behavior Comprising Insulin Particles."

[0002] The present invention relates to a pharmaceutical dosage form for delivering an encapsulated peptide to the oral mucosa, as well as methods and uses of the pharmaceutical dosage form. In particular, provided herein is a pharmaceutical dosage form having a mucoadhesive layer, a peptide-loaded layer, and a water-impermeable layer. The peptide-loaded layer includes an encapsulated peptide. In particular, the encapsulated peptide may be a chitosan-coated peptide particle. Furthermore, the encapsulated peptide may be selected from insulin, insulin derivatives, insulin analogs, pre-insulin, insulin prodrugs, glucagon-like peptide 1 (GLP-1), GLP-1 analogs, pre-GLP-1, GLP-1 prodrugs, or combinations thereof. The pharmaceutical dosage form may be used for the treatment of diabetes or obesity. The pharmaceutical dosage form may be used to deliver an encapsulated peptide to the oral mucosa of a subject. In particular, the oral mucosa may be selected from one or more of the buccal mucosa, sublingual mucosa, palatal mucosa, and lingual mucosa. [Background technology]

[0003] Injectable peptides such as insulin and glucagon-like peptide 1 (GLP-1) and their analogs are increasingly being used to treat diabetes and obesity. Insulin is the mainstay of treatment for insulin-dependent type 1 diabetes. (参照文献1) It is also used to treat non-insulin-dependent type 2 diabetes. (参照文献2)Because insulin tends to be used for long-term treatment, administering it by injection has several drawbacks regarding patient acceptance. For example, non-insulin-dependent patients often delay starting insulin therapy. The perception of insulin injections and anxiety about needles are among the factors associated with this delayed initiation phenomenon, known as psychological insulin resistance. (参照文献3) As a result of these parenteral concerns, many attempts have been made to develop alternative routes of administration, with mixed results, as reported in the literature. In particular, oral administration has been widely studied. (参照文献4-6、42、43) Oral administration of peptide formulations presents many challenges, including low gastric pH, proteolytic enzymes in the upper gastrointestinal tract, and poor absorption and bioavailability. While insulin encapsulation can overcome some of these drawbacks, the way encapsulated insulin is absorbed in the gastrointestinal tract following traditional oral administration is thought to be unpredictable, with different absorption and slower onset of action compared to injection. (参照文献7) Therefore, there is interest in finding alternative routes of administration that offer a rapid onset of action similar to injection, but are more convenient.

[0004] To circumvent the drawbacks of the gastrointestinal tract, alternative routes of administration, such as pulmonary, nasal, transdermal, and buccal administration, have been investigated. (参照文献8、9) Among the various methods of insulin delivery, mucosal administration offers the advantage of non-keratinized epithelium, potentially improving absorption compared to the properties of keratinized epithelium. In particular, delivery via the oral mucosa offers several advantages, including a relatively low enzyme content compared to the gastrointestinal tract, good vascular and lymphatic excretion, potential ease of administration, and a long cell turnover (5–6 days) that may allow the drug to be delivered in a long-lasting, retentive dosage form. (参照文献10) However, drug delivery through the oral mucosa is difficult for several reasons, including the limited absorption area, the barrier properties of tablets and involuntary swallowing, and the continuous dilution of dissolved drugs by saliva. Thin films have been used to increase the absorption area, and mucoadhesive thin films have been used to avoid swallowing the dosage form. (参照文献12、41、46) .

[0005] The application of peptide encapsulation in insulin delivery has also been investigated. The idea of ​​delivering insulin particles through oral tissues has been presented in several studies. (参照文献8、11) Chitosan has been used in mucoadhesive tablets and films for drug delivery. (参照文献44) Furthermore, thiolated chitosan has been investigated for improving mucoadhesion. (参照文献45) However, these methods mainly focus on oral films or patches, and in vivo data are lacking. (参照文献12、13) . Summary of the Invention

[0006] [Problem to be solved by the invention] [Means for solving the problem] The present invention is based, in part, on the surprising discovery that certain mucoadhesive layers are particularly useful for creating pharmaceutical dosage forms suitable for delivering peptides via the oral mucosa. In particular, the mucoadhesive layers of the present invention minimize unwanted loss of peptide to saliva by allowing early release of the peptide while the mucoadhesive layer is still attached to the oral mucosa. Furthermore, the presence of peptide in the chitosan-complexed peptide-loading layer improves bioavailability, since the presence of peptide in the mucoadhesive layer can potentially lead to loss of mucoadhesion. Furthermore, the inclusion of peptide in the mucoadhesive layer results in very rapid peptide release, which can lead to loss of peptide to saliva, and the peptide not being absorbed if cellular tight junctions are not yet open. Alternatively, the presence of peptide in the mucoadhesive layer does not prolong release if tight junctions are open.

[0007] Furthermore, the present invention is based, in part, on the surprising discovery that "encapsulated peptides" with peptide particles coated with mercaptonicotinic acid (MNA)-thioglycolic acid-chitosan (MNA-TG-chitosan) exhibited higher cellular uptake than free peptides or other encapsulated peptides. MNA-TG-chitosan particles exhibited the highest mucosal penetration within 2 hours, followed by peptide-loaded TG-chitosan particles and peptide-loaded chitosan particles.

[0008] Furthermore, the new coating material (MNA-TG-chitosan) was found to be suitable for the oral mucosal delivery of insulin encapsulated in a unidirectional oral mucosal tablet. Compared with existing buccal films, a three-layer oral mucosal tablet was fabricated to achieve unidirectional drug release to the oral mucosa.

[0009] One strategy that could overcome these obstacles is the application of polymeric mucoadhesive buccal tablets, which, due to their small size and ease of use, facilitate patient compliance and are a convenient way to deliver peptides to the oral mucosa.

[0010] Furthermore, it has been discovered fortuitously that it is useful to have a mucoadhesive layer of at least 1 micrometer to obtain suitable peptide release characteristics for insulin, GLP-1, etc. Furthermore, it is preferred for most peptide release dosage forms to have a mucoadhesive layer of 100 μm to 1,500 μm.

[0011] In a first embodiment, a pharmaceutical dosage form is provided, which may comprise: (a) a mucoadhesive layer, the mucoadhesive layer comprising (i) ethylcellulose and an acrylic acid polymer cross-linked with allyl sucrose or allyl pentaerythritol, wherein the acrylic acid polymer cross-linked with allyl sucrose or allyl pentaerythritol is at least 12.5% ​​by weight of the total mucoadhesive layer, or (ii) ethylcellulose, an acrylic acid polymer cross-linked with allyl sucrose or allyl pentaerythritol, wherein the acrylic acid polymer cross-linked with allyl sucrose or allyl pentaerythritol is at least 12.5% ​​by weight of the total mucoadhesive layer, and hydroxypropyl methylcellulose. (b) a peptide-loaded layer, which may include hydroxypropyl cellulose (HPMC); (c) a water-impermeable layer, which may include an encapsulated peptide; and (d) a water-impermeable layer, which may be selected from one or more of ethyl cellulose, polyvinyl chloride, polydimethylsiloxane, hydroxypropyl methylcellulose, hemicellulose, poly(e-caprolactone) (PCL), carboxymethyl cellulose, polyvinyl acetate, propyl cellulose, polymethyl methacrylate, methacrylic acid copolymer, and cellulose acetate phthalate, wherein the peptide-loaded layer is between the mucoadhesive layer and the water-impermeable layer, and the water-impermeable layer facilitates unidirectional migration of the encapsulated peptide through the mucoadhesive layer to the target tissue.

[0012] In a further embodiment, a pharmaceutical dosage form is provided, which may comprise: (a) a mucoadhesive layer, the mucoadhesive layer comprising (i) ethylcellulose and a polymer of acrylic acid cross-linked with allyl sucrose or allyl pentaerythritol, wherein the polymer of acrylic acid cross-linked with allyl sucrose or allyl pentaerythritol is at least 12.5% ​​by weight of the total mucoadhesive layer, or (ii) ethylcellulose, a polymer of acrylic acid cross-linked with allyl sucrose or allyl pentaerythritol, wherein the polymer of acrylic acid cross-linked with allyl sucrose or allyl pentaerythritol is at least 12.5% ​​by weight of the total mucoadhesive layer, and hydroxypropyl methylcellulose (HPMC); or (iii) (b) a peptide-loaded layer, which may comprise one of ethyl cellulose and polyvinylpyrrolidone (PVP); (b) a peptide-loaded layer, which may comprise an encapsulated peptide; and (c) a water-impermeable layer, which is selected from one or more of ethyl cellulose, polyvinyl chloride, polydimethylsiloxane, hydroxypropyl methylcellulose, hemicellulose, poly(e-caprolactone) (PCL), carboxymethyl cellulose, polyvinyl acetate, propyl cellulose, polymethyl methacrylate, methacrylic acid copolymer, and cellulose acetate phthalate, wherein the peptide-loaded layer is between the mucoadhesive layer and the water-impermeable layer, and the water-impermeable layer facilitates unidirectional migration of the encapsulated peptide through the mucoadhesive layer to the target tissue.

[0013] The impermeable layer can form an outer coating while leaving the mucoadhesive surface uncovered to facilitate mucoadhesion. Alternatively, the impermeable layer can form an outer coating in which at least a portion of the mucoadhesive surface is not covered by the impermeable layer. This facilitates mucoadhesion. The ethyl cellulose (i), (ii), or (iii) can comprise at least 50% by weight of the entire mucoadhesive layer, or about 50% to about 75% by weight of the entire mucoadhesive layer. The ethyl cellulose (i), (ii), or (iii) can comprise at least 40% by weight of the entire mucoadhesive layer, or about 40% to about 80% by weight of the entire mucoadhesive layer. The ethyl cellulose (i), (ii), or (iii) can comprise at least 50% by weight of the entire mucoadhesive layer, or about 50% to about 87.5% by weight of the entire mucoadhesive layer. The acrylic acid polymer crosslinked with allyl sucrose or allyl pentaerythritol can comprise about 12.5% ​​to about 50% by weight of the entire mucoadhesive layer. The acrylic acid polymer cross-linked with allyl sucrose or allyl pentaerythritol may comprise about 10% to about 50% by weight of the entire mucoadhesive layer. The acrylic acid polymer cross-linked with allyl sucrose or allyl pentaerythritol may comprise about 10% to about 40% by weight of the entire mucoadhesive layer. The acrylic acid polymer cross-linked with allyl sucrose or allyl pentaerythritol may comprise about 12.5% ​​to about 40% by weight of the entire mucoadhesive layer.

[0014] The acrylic acid polymer crosslinked with allyl sucrose or allyl pentaerythritol may be selected from one or more of the following: Carbopol 934 NF TM , Carbopol 934P NF TM , Carbopol 941 NF TM , Carbopol 942 NF TM , Carbopol 940 NF TM , Carbopol 974 P TM , Carbopol 971 P TM , Noveon AA-1 Polycarbophil TM (Formerly known as Carbopol 976TM ), Carbopol 1342 TM , Carbopol 1382 TM , Carbopol salts TM , Carbopol 981 NF TM , Carbopol 980 NF TM , Carbopol ETD 2050 TM , Carbopol ETD 2020 TM , Carbopol ULTREZ 10 TM , Carbopol 1984 TM , Carbopol 2984 TM , Carbopol 5984 TM , and Carbopol® 71G NF TM The acrylic acid polymer crosslinked with allyl sucrose or allyl pentaerythritol may be selected from one or more of the following: Carbopol 934 NF TM , Carbopol 934P NF TM , Carbopol 941 NF TM , Carbopol 942 NF TM and Carbopol 940NF TM Acrylic acid polymers crosslinked with allyl sucrose or allyl pentaerythritol are available as Carbopol 934P NF TM It could be.

[0015] The thickness of the mucoadhesive layer may be at least 100 μm. The thickness of the mucoadhesive layer may be at least 50 μm. Alternatively, the thickness of the mucoadhesive layer may be at least 1 μm. Preferably, the thickness of the mucoadhesive layer may be 100 μm to 1,500 μm. The thickness of the mucoadhesive layer may be 100 μm to 1,000 μm. The thickness of the mucoadhesive layer may be 100 μm to 2,000 μm. The thickness of the mucoadhesive layer may be 100 μm to 900 μm. The thickness of the mucoadhesive layer may be 100 μm to 800 μm. The thickness of the mucoadhesive layer may be 100 μm to 700 μm. The thickness of the mucoadhesive layer may be 100 μm to 600 μm. The thickness of the mucoadhesive layer may be 100 μm to 500 μm. The thickness of the mucoadhesive layer may be 100 μm to 400 μm. The thickness of the mucoadhesive layer may be 100 μm to 300 μm The thickness of the mucoadhesive layer may be 100 μm to 200 μm.

[0016] The encapsulated peptide may include peptide particles coated with chitosan. The encapsulated peptide may include tripolyphosphate (TPP), chitosan, and a peptide therapeutic agent. The chitosan may be thiolated chitosan. The thiolated chitosan may be selected from MNA-TG-chitosan and TG-chitosan.

[0017] The encapsulated peptide may be a particle having a diameter of about 50 nm to about 10,000 nm. The encapsulated peptide may be a particle having a diameter of about 50 nm to about 9,000 nm. The encapsulated peptide may be a particle having a diameter of about 50 nm to about 8,000 nm. The encapsulated peptide may be a particle having a diameter of about 50 nm to about 7,000 nm. The encapsulated peptide may be a particle having a diameter of about 50 nm to about 6,000 nm. The encapsulated peptide may be a particle having a diameter of about 50 nm to about 5,000 nm. The encapsulated peptide may be a particle having a diameter of about 50 nm to about 4,000 nm. The encapsulated peptide may be a particle having a diameter of about 50 nm to about 3,000 nm. The encapsulated peptide may be a particle having a diameter of about 50 nm to about 2,000 nm. The encapsulated peptide may be a particle having a diameter of about 50 nm to about 1,000 nm. The encapsulated peptide may be a particle having a diameter of about 50 nm to about 900 nm. The encapsulated peptide may be a particle having a diameter of about 50 nm to about 800 nm. The encapsulated peptide may be a particle having a diameter of about 50 nm to about 700 nm. The encapsulated peptide may be a particle having a diameter of about 50 nm to about 6900 nm. The encapsulated peptide may be a particle having a diameter of about 50 nm to about 500 nm. The encapsulated peptide may be a particle having a diameter of about 50 nm to about 400 nm. The encapsulated peptide may be a particle having a diameter of about 100 nm to about 400 nm. The encapsulated peptide may be a particle having a diameter of about 150 nm to about 400 nm.

[0018] The encapsulated peptide may be confined to the peptide loading layer prior to administration and may elute through the mucoadhesive layer after mucoadhesion to the oral mucosa. The encapsulated peptide can be synthesized by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) coupling of chitosan with thioglycolic acid (TGA), followed by thiol-disulfide exchange between thiolated chitosan and 2,2′-disulfanediylnicotinic acid.

[0019] The peptide-loaded layer may further include one or more sweeteners or flavorings. The one or more sweeteners or flavorings may be selected from one or more of lactose, glucose, sucrose, mannitol, xylitol, sorbitol, and trehalose. The sweetener may be mannitol. The peptide-loaded layer may further include a binder. The binder may be sodium alginate.

[0020] The encapsulated peptide can be selected from insulin, insulin derivatives, insulin analogs, pre-insulin, insulin prodrugs, glucagon-like peptide 1 (GLP-1), GLP-1 analogs, pre-GLP-1, GLP-1 prodrugs, or combinations thereof. The insulin analog can be selected from one or more of lispro, aspart, glulisine, glargine, detemirt, detemir, degludec, neutral protamine hagedorn (NPH), and Levemir. The GLP-1 can be selected from one or more of exenatide, liraglutide, dulaglutide, and semaglutide. The target tissue may be the oral mucosa, which may be selected from one or more of the buccal mucosa, sublingual mucosa, palatal mucosa, and lingual mucosa.

[0021] The mucoadhesive layer may have a mucoadhesion of about 1N to about 30N. The mucoadhesive layer may have a mucoadhesion of about 1N to about 25N. The mucoadhesive layer may have a mucoadhesion of about 1N to about 20N. The mucoadhesive layer may have a mucoadhesion of about 1N to about 15N. The mucoadhesive layer may have a mucoadhesion of about 1N to about 10N. The mucoadhesive layer may have a mucoadhesion of about 1N to about 9N. The mucoadhesive layer may have a mucoadhesion of about 1N to about 8N. The mucoadhesive layer may have a mucoadhesion of about 1N to about 7N. The mucoadhesive layer may have a mucoadhesion of about 1N to about 6N. The mucoadhesive layer may have a mucoadhesion of about 1N to about 5N. The mucoadhesive layer may have a mucoadhesion of about 1N to about 4N. The mucoadhesive layer may have a mucoadhesion of about 1N to about 3N. The mucoadhesive layer may have a mucoadhesion of about 1N to about 2N. The pharmaceutical dosage form may be in tablet form. The pharmaceutical dosage form may be for the treatment of diabetes. The pharmaceutical dosage form may be for the treatment of obesity. The pharmaceutical dosage form may be for the treatment of diabetes and obesity.

[0022] In a further embodiment, there is provided a use of the pharmaceutical dosage form described herein for the treatment of diabetes. In a further embodiment, there is provided the use of a pharmaceutical dosage form described herein in the manufacture of a medicament for the treatment of diabetes. In a further embodiment, there is provided the use of the pharmaceutical dosage forms described herein for the treatment of obesity.

[0023] In a further embodiment, there is provided the use of a pharmaceutical dosage form described herein in the manufacture of a medicament for the treatment of obesity. The diabetes can be insulin-dependent diabetes mellitus type 1 (T1D) or type 2 diabetes (T2D). In a further embodiment, a method for treating diabetes is provided, comprising administering to a subject in need thereof a pharmaceutical dosage form described herein.

[0024] In a further embodiment, there is provided a method of treating obesity comprising administering to a subject in need thereof a pharmaceutical dosage form described herein. In a further embodiment, there is provided the use of the pharmaceutical dosage forms described herein for oral mucosal delivery of encapsulated peptides. In a further embodiment, there is provided a method for oral mucosal delivery of a peptide, comprising administering to a subject in need thereof a pharmaceutical dosage form described herein. [Brief explanation of the drawings]

[0025] [Figure 1] Figure 1 shows the characterization of TG-chitosan and pre-activated MNA-TG-chitosan synthesis, where (A) shows the ATR-FTIR of unmodified chitosan, TG-chitosan, and MNA-TG-chitosan, and (B) shows the H-NMR characterization of pre-activated MNA-TG-chitosan. [Figure 2] Figure 2 shows the characteristics of insulin particles coated with various chitosans. Here, (A) shows the size distribution of insulin particles coated with chitosan, TG-chitosan, and MNA-TG-chitosan after reconstitution. (B) shows TEM micrographs of the optimized insulin particles. (C) shows SEM images of spray-dried insulin coated with chitosan, TG-chitosan, and MNA-TG-chitosan. (D) shows ATR-FTIR spectra of free insulin, chitosan, a physical mixture of chitosan / TPP / insulin, and insulin particles dehydrated by various methods. [Figure 3] Figure 3 shows the swelling index of (A) buccal tablets containing pure insulin, (B) buccal tablets containing chitosan-coated insulin particles, (C) buccal tablets containing TG-chitosan-coated insulin particles, and (D) buccal tablets containing MNA-TG-chitosan-coated insulin particles. [Figure 4] Figure 4 shows the permeation behavior of (A) pure insulin buccal tablets, (B) chitosan-coated insulin particle buccal tablets, (C) TG-chitosan-coated insulin particle buccal tablets, (D) MNA-TG-chitosan-coated insulin particle buccal tablets, and (E) the backing layers of the prepared tablets. [Figure 5] Figure 5 shows the uptake of HepG2 cells after 4 hours of incubation with free insulin and insulin particles. (A) Distribution of FITC-insulin taken up by HepG2 cells and (B) geometric mean values ​​of fluorescence intensity from flow cytometry analysis are shown. [Figure 6] Figure 6 shows (A) the effect of chitosan-coated insulin particles on the TEER value of TR-146 cell monolayers, (B) fluorescent images of TR-146 monolayers stained with the tight junction protein ZO-1 after incubation with insulin particles, (C) the effect of particles on epithelial permeation through TR-146 monolayers, and (D) the Papp value of insulin particles through TR-146 cell monolayers. [Figure 7] Figure 7 shows the effect of oral administration of insulin particles and buccal tablets on blood glucose levels in a diabetic rat model. Here, (A) the blood glucose level vs. time profile of diabetic rats and (B) the serum insulin level vs. time profile of normal rats are shown. [Figure 8] FIG. 8 shows the biodistribution of orally administered insulin particles and buccal insulin tablets compared to intraperitoneal injection of insulin. [Figure 9] Figure 9 shows the enzyme activities (U / L) in serum detected to determine whether administration of insulin buccal tablets is safe (alkaline phosphatase (ALP), aspartate transaminase (AST), alanine aminotransferase (ALT)). [Figure 10] FIG. 10 shows the mucoadhesion strength measurement device (a) the setup of the TA.XTplus texture analyzer, and (b) the strength measurements recorded from the mucoadhesion strength measurement device. [Figure 11] FIG. 11 shows the ex-vivo permeation experimental setup for testing tablet dosage forms in porcine mucosal tissue. [Figure 12] FIG. 12 shows a graphic representation of mucoadhesive tablets a) and films b) for oral mucosal delivery of peptides. DETAILED DESCRIPTION OF THE INVENTION

[0026] The following detailed description can be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the drawings show embodiments of the invention. However, the invention is not limited to the precise arrangements, examples, and instrumentalities shown. Terms not directly defined herein are understood to have the general meaning understood in the technical field of the invention.

[0027] <Definition> As used herein, "acrylic acid polymers crosslinked with allyl sucrose or allyl pentaerythritol" are also known as carboxypolymethylene, polyacrylic acid, or poly(1-carboxyethylene), carbomer, or carbopol, and acrylic acid is (C3H4O2) n or may be expressed as shown below, where n = 3 x 10 for Carbomer 934. 6 , 4 × 10 for Carbomer 940 6 , 1 × 10 for Carbomer 941 6 For example, Carbopol 934 NF TM , Carbopol 934P NF TM , Carbopol 941 NF TM , Carbopol 942 NF TM , Carbopol 940 NF TM , Carbopol 974 P TM , Carbopol 971 P TM , Noveon AA-1 Polycarbophil TM (Formerly known as Carbopol 976 TM ), Carbopol 1342 TM , Carbopol 1382 TM , Carbopol salts TM , Carbopol 981 NF TM , Carbopol 980 NF TM , Carbopol ETD 2050 TM , Carbopol ETD 2020TM , Carbopol ULTREZ 10 TM , Carbopol 1984 TM , Carbopol 2984 TM , Carbopol 5984 TM , and Carbopol® 71G NF TM etc.

[0028] [ka]

[0029] As described herein, provided are encapsulated peptide formulations, methods for producing encapsulated peptides using the formulations, and particle compositions for delivering encapsulated peptides to a desired site of action. As used herein, polyvinylpyrrolidone (PVP) is a polymer that can be used in the pharmaceutical dosage forms described herein. In particular, PVP can be used as a substitute for carbomer in the mucoadhesive layer of tablet forms due to its good solubility and mucoadhesive properties.

[0030] As used herein, "sweetener" refers to a sugar or sugar alcohol, such as D-mannitol (CAS No.: 69-65-8) M4125, Sigma Aldrich TM ), lactose (61345, Sigma Aldrich TM ), and trehalose (T9449 D-(+)-trehalose dihydrate, Sigma Aldrich TM) as well. Sweeteners can be used alone or in combination as described herein. As used herein, "sugar" refers to soluble carbohydrates such as monosaccharides, disaccharides, and polysaccharides, and is generally exemplified by glucose and sucrose. As used herein, the sugar is preferably lactose. As used herein, "sugar alcohol" (also called a polyhydric alcohol, polyalcohol, alditol, or glycitol) refers to an organic compound derived from a sugar, containing one hydroxyl group (-OH) attached to each carbon atom. Sugar alcohols are often used as artificial sweeteners, and examples include xylitol and sorbitol. As described herein, the sugar alcohol is preferably mannitol.

[0031] As used herein, "encapsulated peptide" or "encapsulated peptide particle" refers to a particle having a diameter of about 50 nm to about 10,000 nm. More preferably, the particle has a diameter of about 150 nm to about 400 nm. As used herein, "encapsulated peptide" includes peptide particles coated with chitosan or thiolated chitosan, where the thiol groups (-SH) on the thiolated chitosan can form disulfide bonds or other bonds. The thiolated chitosan can be selected from mercaptonicotinic acid (MNA)-thioglycolic acid-chitosan (MNA-TG-chitosan), and thioglycolic acid-chitosan (TG-chitosan), or other chitosans known to those skilled in the art. (参照文献47、48) .

[0032] The peptide particles described herein can be prepared by spray freeze drying, spray drying, or lyophilization. As used herein, "spray freeze drying" refers to a drying process for materials in which a solution is atomized, solidified, and sublimated at low temperatures. The sprayed material is solidified by rapid freezing in a cryogenic fluid, such as liquid nitrogen (LN2), typically along with additional excipients that protect the material's structure, activity, and stability. The mixture is then further dried by freeze-drying in a vacuum chamber to remove residual moisture. As described herein, a formulation for preparing proteins by spray freeze drying is provided. Proteins are typically sensitive to degradation by heat, moisture, or chemical / enzymatic action. This is particularly important when preparing therapeutic proteins, which must preserve their structure and / or biological activity until they are delivered to the desired site of action.

[0033] As used herein, "spray drying" refers to the process of forming a dry powder from a liquid or slurry by rapidly drying with a hot gas. This process typically involves taking the solution or suspension to be dried and atomizing the solution or suspension with an atomizing gas. The atomized solution or suspension is then sprayed into a drying gas, which is heated. The atomized solution or suspension is then dried with the heated drying gas to produce fine particles. "Lyophilization" or "freeze-drying" as used herein is a low-temperature dehydration method. Lyophilization involves freezing a peptide solution at low pressure and removing the ice by sublimation. This contrasts with traditional dehydration methods, which use heat to evaporate water. Heat can denature peptides or proteins.

[0034] The "effective amount" of an active ingredient described herein includes a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" refers to an amount effective, at a dosage and for a duration necessary, to achieve a desired therapeutic result, such as extended peptide delivery, glucose homeostasis, extended lifespan, or extended life expectancy. A therapeutically effective amount of an active ingredient can vary depending on factors such as the subject's disease state, age, sex, and weight, and the ability of the active ingredient to induce a desired response in the subject. Dosage regimens can be adjusted to provide an optimal therapeutic response. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the active ingredient outweigh the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at a dosage and for a duration necessary, to achieve a desired preventative result, such as extended peptide delivery, glucose homeostasis, extended lifespan, extended life expectancy, or prevention of diabetes or obesity. Typically, a prophylactic dose is used in subjects before or at an early stage of disease, and therefore the prophylactically effective amount may be less than the therapeutically effective amount.

[0035] It should be noted that dosages may vary depending on the severity of the symptoms to be alleviated. For a particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the composition. The dosage ranges provided herein are merely exemplary and do not limit the range of dosages that may be selected by a physician. The amount of active ingredient in the composition may vary depending on factors such as the subject's condition, age, sex, and weight. Dosage regimens may be adjusted to obtain the optimal therapeutic response. For example, a single bolus may be administered, or several divided doses may be administered over time, or the dosage may be proportionally reduced or increased depending on the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it may be advantageous to formulate parenteral compositions in dosage unit form. As specifically described herein, most of the particle compositions are adapted for oral mucosal delivery.

[0036] The encapsulated peptides described herein can be administered to a subject. As used herein, a "subject" refers to a human, a non-human primate, a rat, a mouse, a cow, a horse, a pig, a sheep, a goat, a dog, a cat, or the like. The subject may be suspected of or at risk for diabetes, such as type 1 diabetes (T1D) or type 2 diabetes (T2D). Alternatively, the subject may be suspected of or at risk for obesity. Terms not directly defined herein are understood to have the general meaning as understood in the art.

[0037] Various alternative embodiments and examples are described herein, which are illustrative and should not be construed as limiting the scope of the invention. Although various embodiments of the present invention are disclosed herein, many adaptations and modifications can be made within the scope of the present invention according to the general knowledge of those skilled in the art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Various alternative embodiments and examples are described herein. These embodiments and examples are illustrative and should not be construed as limiting the scope of the present invention.

[0038] Materials and Methods <Material> Chitosan (average molecular weight 100 KDa, 75-85% deacetylated), Carbopol 934P TM , hydroxypropyl methylcellulose, and ethyl cellulose were purchased from Sigma-Aldrich TM (Oakville, Ontario, Canada). Sodium tripolyphosphate (TPP) and sodium alginate (low viscosity) were purchased from VWR. TM (Radnor, PA, USA). Recombinant human insulin used in this study was purchased from Fisher Scientific. TM(Purchased from Waltham, Massachusetts, USA). Fluorescein isothiocyanate (FITC)-labeled human insulin and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were from Sigma-Aldrich TM (Purchased from Oakville, Ontario, Canada). The HepG2 and Caco-2 cell lines were obtained from ATCC (Manassas, Virginia, USA), and the TR-146 cell line was from Sigma-Aldrich TM (Purchased from Oakville, Ontario, Canada). All other reagents were of analytical or chromatography grade

[0039] <Synthesis and Purification of MNA-TG-Chitosan> <Synthesis and Purification of Thiolated Chitosan> Thiolated chitosan (TG-chitosan) was synthesized using a previously described method (参照文献14) as shown in the following <Synthesis 1>. Chitosan was first dissolved in 0.1 M HCl and then thioglycolic acid (TGA) was added, and the pH was adjusted to 5.0 with NaOH. 50 mM EDAC was added to activate the carboxyl group of TGA, and the resulting thiolated chitosan was dialyzed to remove unbound TGA

[0040] <Synthesis and Purification of Activated TG-Chitosan (MNA-TG-Chitosan)> Next, TG-chitosan was utilized for the synthesis of pre-activated TG-chitosan with mercaptonicotinic acid (MNA-TG-chitosan). The synthesis route is shown in the following <Synthesis 1>. The pre-activation was performed by dropping the MNA solution into the TG-chitosan solution at a ratio of 1:2. The pH of the reaction mixture was set to 7.5 and continuously stirred at room temperature for 6 h. After the reaction, MNA-TG-chitosan was purified using a dialysis procedure. The purified MNA-TG-chitosan was then frozen, dried, and stored at 4 °C in the dark until use <Synthesis 1: Synthesis Routes of TG-Chitosan and MNA-TG-Chitosan>

[0041] [Chemical Formula]

[0042] <Characterization of TG-chitosan and MNA-TG-chitosan by Ellman's assay, FT-IR, and NMR> As mentioned above (参照文献36) The Ellman assay was used to measure the concentrations of thiol groups and disulfide bonds in lyophilized MNA-TG-chitosan and TG-chitosan. Briefly, for the measurement of thiol groups, TG-chitosan was mixed with phosphate buffer and incubated at room temperature for 30 minutes. Next, Ellman's reagent was added and the mixture was incubated at room temperature in the dark for 90 minutes. After 90 minutes, 100 μL of each mixture was transferred to a 96-well plate and analyzed using a Tecan Infinite M200. TM pro spectrophotometer plate reader (Tecan TM The absorbance at 450 nm was measured using a chromatograph (Mannedorf, Switzerland). For disulfide bond measurements, MNA-TG-chitosan was dissolved in 50 mM Tris buffer. 4% sodium borohydride solution was then added and incubated at 37°C for 60 minutes. After incubation, HCl was added, followed by Ellman's reagent, and the mixture was further incubated at room temperature in the dark for 90 minutes.

[0043] FTIR of TG-chitosan and MNA-TG-chitosan was performed using a universal ATR sampling accessory (PerkinElmer TM , Waltham, Massachusetts, USA) TM FTIR spectrophotometer (PerkinElmer TM The signal was averaged over 4 cm. 2 Resolution of 4000-600cm 2 The results were obtained from 16 scans over a frequency range of . moreover, 1 H-NMR spectra were obtained using a Bruker Avance Cryoprobe TM 600MHz spectrometer (Bruker TM The data were acquired at the University of California, San Diego, California (Cambridge, CA, USA).TM (Mestrelab Research TM , Santiago de Compostela, Spain) were obtained and processed using the 1 H-NMR spectra were obtained using standard acquisition parameters at 298 K (25 °C) from a Bruker TM Obtained by the Pulse program.

[0044] <Insulin particles> <Insulin particle formulation> Insulin particles were (参照文献10) Briefly, cross-linked TPP / insulin solutions were mixed in a 1:2 ratio and homogenized using a Polytron PCU-2-110 high-speed homogenizer (Brinkmann Industries). TM The insulin was added dropwise to the chitosan solution using a syringe while stirring at high speed in a centrifuge (Westbury, NY, USA). After adjusting the pH to 6.1, the mixture was stirred at high speed for an additional 30 minutes. To further homogenize the insulin particles and reduce their particle size, a probe-type ultrasonicator (UP 200ST, Hielscher Ultrasonics) was used. TM The insulin particles were then sonicated for another 30 minutes using a Buchi Mini Spray Dryer (Teltow, Germany). TG-chitosan and MNA-TG-chitosan coated insulin particles were also prepared using the same method. The insulin particles were further processed into buccal tablets. The dried insulin particle powder was then sprayed in a Buchi Mini Spray Dryer B-290 at 90°C with a feed flow rate of 3 L / min and an air flow rate of 4 L / min. TM (BUCHI TM , Flawil, Switzerland).

[0045] <Characteristics of insulin particles> Evaluating the effects of TG-chitosan and MNA-TG-chitosan on insulin particles. The Z-average diameter, polydispersity index (PDI), and zeta potential of all prepared insulin particles were measured using a Litesizer 500 (Anton Paar). TMThe morphology and size distribution were examined by dynamic light scattering (DLS) measurements using a Hitachi H7600. TM Transmission electron microscope (TEM) (Hitachi TM , Tokyo, Japan), and dried insulin particles were characterized using a Helios NanoLab 650 focused ion beam scanning electron microscope. TM (FIB-SEM)(FEI TM , Hillsboro, Oregon, USA) (参照文献37、38) To evaluate the encapsulation efficiency (EE) and loading capacity (LC) of insulin particles, unencapsulated insulin was purified from the ultrafiltration tube and analyzed using an Agilent 1100 series ultrafiltration column. TM HPLC system (Agilent TM , Santa Clara, California, USA) (参照文献39) The percentages of EE and LC were calculated using the following formulas (1) and (2): All dehydrated insulin particles were redissolved in dd water to assess their reconstitution ability. Particle size, PDI, EE, and LC were again tested using the same methods as previously described.

[0046] <Tri-layer buccal tablet containing insulin particles> <Production of tri-layer buccal tablets containing insulin particles> To optimize the insulin release profile along with the tablet's mucoadhesion and simultaneously achieve unidirectional drug release to the buccal mucosa, trilayered buccal tablets containing insulin particles were prepared. As shown in Table 1, the bioadhesive layer was prepared using a 25 mg mixture of carbopol, hydroxypropyl methylcellulose, and ethylcellulose. To optimize the mucoadhesion ability, they were prepared in different ratios (Table 1). The insulin-loaded layer was made of dried insulin particles, mannitol, and sodium alginate (Table 1). The resulting powder mixture was punched in a manual punch tablet press (ZONESUN) equipped with 6 mm round and flat punches. TMThe tablets were directly compressed layer by layer into tablets using a 25 mg ethyl cellulose tablet press (Nanhai Co., Ltd., China). All tablets contained an additional impermeable layer containing 25 mg ethyl cellulose added during the compression step.

[0047] <Physical properties of insulin buccal tablets> The prepared tablets were tested for weight variation, thickness, diameter, uniformity of insulin content, Brinell hardness and friability. Weight variation tests were performed according to the British Pharmacopoeia TM (Commission, 2012) and 20 tablets of Sartorius CP224 S TM Analytical Balance (Sartorius AG TM The thickness and diameter of 10 tablets were determined by measuring with a micrometer. The friability test was performed according to the British Pharmacopoeia (2012). In this study, 10 tablets were accurately weighed and tested using a digital tablet friability tester (BEXCO TM The tablets were placed in a grinding machine (Ambala Cantt, Haryana, India) and rotated at 25 rpm for 4 minutes. The percentage weight loss was then calculated as friability. Uniformity of insulin content was tested by dissolving 10 tablets in 0.1% acetic acid after grinding. Quantitation of insulin was performed using HPLC as described above. Brinell hardness was measured using a TA.XT plus TM Texture analyzer (Stable Micro Systems TM The test was performed using a 3 mm diameter spherical indentation probe mounted on a test tube (Surrey, UK). After the test started, the probe was moved into the sample and, once a force of 50 N was reached, the load was held for a set period of time, after which the probe was completely withdrawn.

[0048] <Surface pH> Prior to the surface pH test, the bioadhesive layer of all tablets was moistened and placed in contact with 1 ml of artificial saliva for 2 hours at room temperature. In this test, the pH of the tablets was measured using a Fisherbrand Accumet AE150 TMThe electrodes of a benchtop pH meter (Waltham, MA, USA) were placed in contact with the tablet surface and then allowed to equilibrate for 1 minute. The surface pH of each tablet was measured three times, and the mean and standard deviation were calculated.

[0049] [Table 1]

[0050] <Swelling index research> This study was conducted to determine the swelling index of each tablet, after which the mean ± SD was calculated. Each insulin buccal tablet was weighed (W0), placed separately on a 2% agar gel plate, and incubated at 37 ± 1°C. During the experiment, the tablets were periodically removed from the Petri dish for up to 8 hours, and the surface moisture was carefully removed with filter paper. After weighing the swollen tablet (W1) a second time, the swelling index was calculated using the following formula (Equation 3): JPEG2025529907000006.jpg14159

[0051] <In vitro mucosal adhesion strength measurement> The mucoadhesion between the oral mucosa and the insulin oral tablet was measured using a TA-XT plus texture analyzer (Stable Micro Systems) equipped with a mucoadhesive device (A / MUC). TM The tissue was evaluated using a peel test using a porcine oral mucosal membrane (UBC, Surrey, UK). Fresh porcine oral tissue was obtained from the UBC Animal Care Centre. The oral tissue was attached to the mucoadhesion test device (Figure 10). To mimic the conditions inside the mouth, the mucoadhesion test device was placed in a beaker filled with artificial saliva (Figure 10). The artificial saliva was at a level just touching the porcine oral tissue (Figure 10). A stirring speed of 150 rpm was applied during the test, and the temperature was set at 37°C. The probe fixed with the insulin tablet was moved 1 mm s toward the surface of the porcine buccal mucosal tissue. -1 The tablet was moved at a constant speed of 0.5 mm s , during which time the tablet was left in contact for 30 seconds without applying any force. After 30 seconds, the tablet-holding probe was moved upward (0.5 mm s ) until contact between the surfaces was broken. -1)It was pulled. The mucoadhesion of the tablets was evaluated by measuring the maximum force required to remove the probe, which directly indicated the mucoadhesion strength.

[0052] The penetration test of insulin buccal tablets was carried out using a mucoadhesion device. Oral tissues were cut to a thickness of 2 mm and attached to the mucoadhesion test device (Figure 11). The setup was the same as that for the mucoadhesion strength measurement (Figure 11). To test the one-way release profile of the tablets, both sides of the tablets were attached to porcine oral tissues, and the penetration behavior was tested. At the time points of 0.5, 1, 2, 3, and 4 hours, 0.5 mL of the liquid in the beaker was withdrawn and its volume was immediately replenished with fresh artificial saliva. The liquid was analyzed for insulin contamination using a human insulin ELISA kit (Abcam TM , Toronto, Ontario, Canada). The insulin that penetrated the oral tissue was calculated from the ratio of the penetrated insulin in the tablet to the total insulin (Equation 4 below). JPEG2025529907000007.jpg17159

[0053] <In vitro cell research - Cell culture> <HepG2 cells> The human hepatocellular carcinoma cell line HepG2 cells were cultured in a 60 mm diameter Nunc TM cell culture Petri dish (Thermo Fisher TM , NY, USA) using Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 100 IU / mL penicillin, and 100 μg / mL streptomycin. The culture was carried out in an environment of 37 °C, 95% relative humidity, and 5% CO2. The medium was changed every 2 - 3 days according to the growth rate. The cells were seeded at 2×10 4 cells / cm 2 and subcultured twice a week using 0.25% trypsin - EDTA.

[0054] <Caco2 cells> Caco2 cells, widely used as a model of the intestinal epithelial barrier, were cultured using Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 100 IU / mL penicillin, and 100 μg / mL streptomycin in a 60 mm diameter Nunc TM cell culture Petri dish (Thermo Fisher TM , NY, USA). The culture was maintained in an environment of 37 °C, 95% relative humidity, and 5% CO2. The medium was changed every 2 - 3 days according to the growth rate. The cells were passaged once a week at a concentration of 2×10 4 cells / cm 2 .

[0055] <TR146 cells> TR146 cells, which are suitable for drug transport research and mimic normal human buccal mucosal epithelium, were cultured using Nutrient Mixture F-12 Ham containing 10% fetal bovine serum, 100 IU / mL penicillin, and 100 μg / mL streptomycin in a 60 mm diameter Nunc TM cell culture Petri dish (Thermo Fisher TM , NY, USA). The culture was carried out in an environment of 37 °C, 95% relative humidity, and 5% CO2. The medium was changed every 2 - 3 days according to the growth rate. The cells were seeded and passaged once a week at 2×10 4 cells / cm 2 .

[0056] <000048​​​​​​​​​​The cells were seeded at a density of 1000 μg / mL. Insulin particles were diluted to various concentrations (50–1000 μg / mL) in the respective culture media and administered to the cells. After 8 hours of incubation, the cells were washed three times with PBS and incubated for an additional 4 hours in medium containing 0.5 mg / mL MTT. Cytotoxicity was assessed using a Tecan Infinite M200. TM pro spectrophotometer plate reader (Tecan TM , Mannedorf, Switzerland) by measuring the enzymatic reduction of the yellow tetrazolium MTT to purple formazan at 570 nm.

[0057] In vitro cellular uptake assay The intracellular uptake efficiency of insulin particles was examined by confocal laser scanning microscopy and flow cytometry analysis. HepG2 cells were cultured at NuncLab-Tek TM 5 x 10 chamber slide system 4 cells / cm 2 Each well of a NuncLab-Tek chamber slide system was treated with free FITC insulin, chitosan-coated FITC insulin particles, TG chitosan, and MNA-TG chitosan at the same concentration of 25 μg / mL and cultured for 4 hours. The cells were then fixed with 4% paraformaldehyde, and the cell nuclei were stained with DAPI. Insulin localization was confirmed using an Olympus FV1000. TM Laser scanning / two-photon confocal microscope (Olympus TM Observations were made using a microscope (Shinjuku-ku, Tokyo).

[0058] For flow cytometry analysis, HepG2 cells were plated in 96-well plates at 5 × 10 4 cells / cm 2 Free FITC insulin, chitosan-coated FITC insulin particles, TG chitosan, and MNA-TG chitosan at a concentration of 10 μg / mL were added to a 96-well plate and cultured for 4 hours. After 4 hours of incubation, the cells were lifted and washed three times with FBS. 5 × 10 cells per sample were used. 4BD LSR II TM Flow cytometer (BD TM , Franklin Lakes, NJ, USA).

[0059] <In vitro penetration test> To assess permeability, Corning TM (Transwell pore size 0.4 μm, growth area 0.33 cm 2 ) inserts were used to test insulin particles. Briefly, 5 × 10 TR146 cells were cultured in a 100-well plate. 4 cells / cm 2 The cells were cultured on the inserts at a density of 100 μg / cm² for 30 days, with the medium replaced every 2 days. Free insulin, chitosan, TG-chitosan, and MNA-TG-chitosan-coated insulin particles were tested in 24-well plates. 0.1 mL samples were taken from the receptor area after 0.5, 1, 2, 3, 4, and 6 hours. The receptor area was immediately replenished with fresh PBS to maintain the sedimentation state.

[0060] <Cell Tight Junction Test - TEER Value Test> The opening of tight junctions was investigated by examining the change in transepithelial electrical resistance (TEER) of epithelial monolayers after insulin particle treatment. This test was performed in a 24-well plate using Corning TM (Transwell pore size 0.4 μm, growth area 0.33 cm 2 ) inserts. TR146 cells were cultured at 5 × 10 4 cells / cm 2 The cells were cultured on the inserts at a density of 100 μg / cm² for 30 days. The cells were cultured with free insulin, chitosan-coated insulin particles, TG-chitosan, and MNA-TG-chitosan, respectively, and the changes in TEER values ​​were measured at various time intervals within 4 hours using Millicell®. TM -Electrical Resistance System TM (Millipore TMAfter incubation, the test samples were removed and the TEER values ​​were monitored for an additional 20 hours.

[0061] <Visualization of tight junctions> The state of tight junctions between TR146 cells after incubation with NP was visualized by immunofluorescence staining of ZO-1 protein. TR146 cells were cultured at Nunc Lab-Tek TM 5 x 10 chamber slide system 4 TR146 cell monolayers were seeded at a density of 1000 cells / mL. Free insulin, chitosan-coated insulin particles, TG-chitosan, and MNA-TG-chitosan were incubated for 4 hours, and then the cells were fixed with 4% paraformaldehyde. The cells were then permeabilized with 0.1% Triton X-100 and blocked with 5% goat serum. Subsequently, the cells were incubated with ZO-1 monoclonal antibody (Thermo Fisher Scientific). TM , New York, USA), followed by goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, FITC (Thermo Fisher Scientific, TM The stained cells were washed three times with PBS, mounted on slides, and analyzed by Olympus FV1000. TM Laser scanning / two-photon confocal microscope (Olympus TM Visualization was performed using the 3D image processing software (Shinjuku-ku, Tokyo).

[0062] In vitro studies <Diabetic rat model> Female Wistar rats weighing 600±30 g were purchased from the University of British Columbia Animal Care Services. Type 1 diabetes was induced by a single intravenous (iv) injection of 55 mg / kg of streptozotocin (STZ) 4 days before the pharmacodynamic study. Body weight and blood glucose levels were measured by tail poke at least every 24 hours after injection. Blood glucose levels were monitored using a OneTouch Verio Reflect™ device. TM Meter (OneTouch TMDiabetes was assessed using test strips purchased from a pharmacy (Surrey, British Columbia, Canada). Rats were considered diabetic if their blood glucose level exceeded 16.0 mM. Prior to the experiment, the rats' insulin resistance was determined to avoid administering incorrect amounts of insulin. Approximately 2 IU / kg of insulin was used to test the rats' insulin resistance.

[0063] <In vivo hypoglycemic effect> Free insulin solution and insulin particles coated with chitosan, TG-chitosan, and MNA-TG-chitosan were administered orally at a dose of 50 IU / kg. In this study, an intraperitoneal injection of 10 IU / kg of insulin was used as a positive control. Another group of diabetic rats was anesthetized and administered insulin and insulin particles coated with chitosan, TG-chitosan, and MNA-TG-chitosan via buccal tablets at a concentration of 50 IU / kg (Figure 9). During the experiment, the rats were fasted and allowed free access to water. Blood glucose levels were measured at designated time points by collecting blood from the tail vein using a glucometer.

[0064] <Biodistribution of orally administered insulin particles and buccal insulin tablets> This study was conducted using female Wistar rats weighing 600 ± 30 grams. The biodistribution of particles was evaluated in accordance with previous studies. (参照文献40) Cy-5-labeled insulin was investigated using Cy-5-labeled insulin prepared according to the method described above. Cy-5-labeled insulin was prepared into particles coated with MNA-TG chitosan and then processed into buccal tablets. The Cy-5-labeled insulin solution was administered by intraperitoneal injection, and the Cy-5-labeled insulin particle solution was administered orally. The buccal tablets containing Cy-5-labeled insulin particles were administered under anesthesia using the same method as described above. The overall distribution of Cy-5-labeled insulin was monitored using an IVIS Lumina II in vivo optical imaging system (PerkinElmer). TMThe results were revealed by near-infrared imaging using a NIRS (Neuron Imaging Laboratory, Waltham, MA, USA). Images were taken 2 hours after insulin administration to all rats. All rats were euthanized and shaved prior to imaging.

[0065] <Pharmacokinetic evaluation> The pharmacokinetics of insulin was evaluated in rats with STZ-induced diabetes as previously described. Free insulin, insulin particles, and insulin buccal tablets were administered using the same method as described above in the "In vitro cytotoxicity test." Serum insulin concentrations were measured using a human insulin ELISA kit (Abcam) according to the manufacturer's instructions. TM The relative bioavailability of insulin was calculated by comparing the area under the curve of the insulin concentration profile in rats receiving oral administration or tablets with that in rats receiving direct intraperitoneal injection. The calculation formula is shown in Equation 5. JPEG2025529907000008.jpg17159AUCoral and AUCip refer to the sum of the areas under the curve of serum insulin concentrations by oral administration or tablet and intraperitoneal injection, respectively. Doseoral and Doseip represent the dose of insulin by oral administration or tablet and intraperitoneal injection, respectively. All intergroup comparison analyses were performed using Graph-Pad Prism 9.4.0. TM for Mac OS (GraphPad Software TM , San Diego, California, USA).

[0066] <In vivo toxicity> Biochemical indicators are available from Abcam TMSerum was tested using alkaline phosphatase (ALP) assay kits (ab83369 or ab83371), aspartate aminotransferase (AST) assay kits (ab105135), and alanine transaminase (ALT) assay kits (ab105134) obtained from Pharmacopoeia, Toronto, Ontario, Canada, according to the manufacturer's instructions. These assays were used to determine whether buccal tablets containing insulin particles coated with chitosan, TG-chitosan, and MNA-TG-chitosan were safe for use. Rats were administered 50 IU / kg insulin buccal tablets daily, euthanized one day later, and serum was used for toxicity analysis.

[0067] <Statistical analysis> All experiments were performed in triplicate, and values ​​are expressed as the mean ± SD. All intergroup comparisons were performed using IBM SPSS Statistics 26 for Mac (IBM TM , Endocott, NY, USA) were evaluated using one-way analysis of variance or t test, and p < 0.05 was considered statistically significant. [Example]

[0068] Example 1: Properties of TG-chitosan and MNA-TG-chitosan The thiol group content of the TG-chitosan conjugate was 218 ± 33 μmol per gram of polymer. The ATR-FTIR spectrum of chitosan (Figure 1A) showed the peaks at 1665 cm and 1666 cm, respectively. -1 and 1622cm -1 This is because chitosan and TG-chitosan (参照文献14) This corresponds to the characteristic amide I band observed in both chitosan and TG-chitosan. -1 and 1512cm -1 The peaks are the amide II bands (CN stretching and NH bending) (参照文献14) This is due to the 3303cm -1 and 3262cm -1 The absorption band at 2531 cm is due to NH stretching.-1 The weak peak indicates the complex of chitosan and TGA. (参照文献14) .

[0069] Furthermore, when the total and free thiol group contents were calculated by Ellman's assay, the preactivated MNA-TG-chitosan showed 138 ± 21 μmol of disulfide bonds per gram of polymer, indicating that more than 50% of the thiol groups were preactivated. (参照文献15) This is consistent with previous results based on the results of the previous study. Furthermore, the preactivated MNA-TG-chitosan was evaluated using FT-IR spectroscopy. Figure 1A shows the peaks at 1653–1619 cm -1 The structure of preactivated MNA-TG-chitosan is recognizable by the NH bend detected at 1712 cm. The presence of an amide bond in MNA-TG-chitosan is confirmed by the NH bend detected at 1712 cm. -1(参照文献15) This was confirmed in contrast to unmodified chitosan. NMR data are shown in Figure 1B, where the coupling reagent disulfanediyl dinicotinic acid was detected. Considering the pure spectrum and the presence of disulfide bonds in MNA-TG-chitosan, this study demonstrated the synthesis of preactivated MNA-TG-chitosan.

[0070] Example 2: Insulin particle characteristics and the effects of TG chitosan and MNA-TG chitosan Chitosan-coated insulin particles were (参照文献10)The optimized insulin particles were prepared using the optimized conditions. The optimized insulin particles had an average particle size of 318 nm, a PDI of 0.18, an encapsulation efficiency of 99.03%, a zeta potential of 9.8 mV, and an insulin loading capacity of 25.19% (m / m) (Table 2A). Transmission electron microscopy (TEM) results showed that the optimized particles were spherical and discrete, with relatively uniform size (Figure 2B). SEM results suggested that spray drying without a bulking agent resulted in a spherical structure with a wrinkled surface (Figure 2C). After reconstitution, the average particle size of the chitosan-coated insulin particles increased to 457 nm, but no significant changes were observed in the PDI, EE, or loading capacity (p<0.05). Using the same optimized preparation conditions, the average particle size of the TG-chitosan-coated insulin particles was reduced compared to the MNA-TG-chitosan-coated insulin particles, while the PDI, EE, and loading capacity remained unchanged (Table 2A). The morphology of dehydrated insulin particles coated with TG-chitosan and MNA-TG-chitosan did not change visibly like that of chitosan-coated insulin particles (Figure 2C). The reconstitution ability of dehydrated insulin particles coated with TG-chitosan and MNA-TG-chitosan also showed smaller particle sizes than insulin particles coated with non-thiolated chitosan (Figure 2A). All these results indicate that MNA-TG-chitosan does not adversely affect the characterization of insulin NPs (nanoparticles). Furthermore, previous studies have demonstrated that TG-chitosan can significantly reduce the average particle size of particles by forming -SS- bonds inside and outside the particles. (参照文献16、17), which is consistent with our results. However, in our case, most of the -SH groups in MNA-TG-chitosan are attached to the MNA groups (Synthesis 1). Therefore, only a portion of them can form -SS- bonds on the outside of the particles, resulting in a slightly smaller average particle size and an increased number of -SH groups, which improves bioadhesive ability. Free insulin, chitosan, a physical mixture of chitosan, TPP, and insulin, and all dehydrated NPs were characterized using ATR-FTIR spectroscopy. Notably, the encapsulated particles freeze-dried with mannitol and the particles spray-dried with or without mannitol exhibited peaks at 1641, 1543, and 1412 cm. -1 An increase in band intensity was observed at 1000 kJ / cm (Figure 2D). These increases in intensity are associated with cross-linking between chitosan, TPP, and insulin. After reconstitution with water, the encapsulation efficiency (EE) of all particles decreased slightly, and a small amount of insulin (approximately 5%) was released during 3 months of storage (Table 2B). However, the average particle size of all particles increased. The particle size of particles spray-dried without mannitol increased to 525 nm, while the particle sizes of spray-dried and freeze-dried particles containing mannitol increased to 872 nm and 921 nm, respectively (Table 2B).

[0071] [Table 2A]

[0072] [Table 2B]

[0073] Example 3: Physical properties of tri-layer buccal tablets All tablets containing insulin particles were tested for content uniformity, weight variation, friability, and Brinell hardness. The mean insulin content of the different formulations ranged from 49.37 ± 0.82 to 50.83 ± 0.49 IU. The mean weight of all tablets prepared ranged from 98.37 ± 1.32 to 101.21 ± 1.41 mg. The friability test showed no physical signs of cracking, tearing, or breakage of the tablets after testing, with a mean weight loss of 0.21 ± 0.12 to 0.29 ± 0.09%. The mean thickness of all tablets ranged from 3.91 ± 0.12 to 4.10 ± 0.14 mm, and the mean diameter was 6.00 ± 0.02 mm. Brinell hardness is an indicator of a material's resistance to wear and deformation and its ability to indent or abrade other materials. (参照文献18) In this study, all tablets had a saturation of approximately 1.8 kg / mm 2 The Brinell hardness of the insulin buccal tablets was 1.07 (data not shown). Based on the results, all formulations of insulin buccal tablets exhibited acceptable content uniformity, weight variation, friability, hardness, and size distribution. Furthermore, the physical properties of the prepared buccal tablets did not change with the use of insulin particles coated with chitosan, TG-chitosan, and MNA-TG-chitosan. Therefore, in other studies, optimized insulin buccal tablets can be obtained using all formulations.

[0074] Example 4: Surface pH studies Because acidic or alkaline pH correlates with irritation to the buccal mucosa in the oral cavity, the surface pH of the tablets was measured. The surface pH of the adhesive layer of all tablets ranged from 5.44 ± 0.20 to 6.12 ± 0.28. Based on these results (data not shown), it can be concluded that all formulations provided acceptable pH levels within the salivary pH range (5.5–7.0) and did not cause local irritation to the mucosal surface upon application. (参照文献19) Furthermore, because the chitosan-, TG-chitosan-, and MNA-TG-chitosan-coated insulin particles were located in the middle layer of the tablets, there was no change in the surface pH of the prepared buccal tablets.

[0075] Example 5: Swelling studies Buccal adhesive tablets must exhibit appropriate swelling behavior to promote sustained drug release and effective adhesion to the mucosa. (参照文献20) As shown in Figure 3, tablets containing insulin-chitosan-encapsulated particles showed a slightly higher swelling index than tablets containing pure insulin. However, tablets containing insulin-loaded TG-chitosan and MNA-TG-chitosan particles did not show a higher swelling index than tablets containing insulin-loaded chitosan particles (Figure 3C and D). The higher swelling rate and extent of the formulations containing the three types of chitosan particles may be due to the large amount of water absorption by these three polymers and their rapid swelling properties. Furthermore, because TG-chitosan and MNA-TG-chitosan did not alter the water absorption properties of chitosan, tablets containing these two materials showed similar swelling properties. Compared to all formulations, F9 showed the highest swelling index at the end of the 8-hour test (Figure 3). Compared to hydroxypropyl methylcellulose, Carbopol 934P TM is highly water soluble (参照文献21) For F3 and F6, the adhesive layer contains Carbopol 934P. TM Although only F9 contains ethyl cellulose, the reduced swelling compared to F9 may be due to the higher content of ethyl cellulose polymer, which is less swellable and insoluble in water.

[0076] Example 6: Ex-vivo evaluation of three-layer buccal tablets containing insulin particles - mucoadhesive strength According to Table 3, different insulin buccal tablets with different adhesive layers exhibited different mucoadhesion strengths. Tablet F9 exhibited the strongest mucoadhesion (1.12 ± 0.16 N), while tablet F1 exhibited the weakest mucoadhesion (0.09 ± 0.01 N). The use of insulin particles coated with chitosan, TG-chitosan, and MNA-TG-chitosan did not increase the mucoadhesion strength of the tablets. This is likely because the insulin particles were located in the middle layer of the buccal tablet and therefore did not contribute as much to mucoadhesion as the mucoadhesive layer.

[0077] Carbopol 934P TM Compared with other formulations with the same ethylcellulose content, F9, F6, and F3 showed the highest mucoadhesion, thus contributing the most to mucoadhesion (Table 3). Analysis of the ratio of ethylcellulose to other materials revealed that the higher the amount of ethylcellulose, the lower the mucoadhesion, so ethylcellulose contributed the least to mucoadhesion. The mucoadhesion of various polymers in this study was significantly higher than that of Carbopol 934P. TM > Hydroxypropyl methylcellulose > Ethyl cellulose. This ranking is consistent with previous research. (参照文献22) It was equivalent to Carbopol 934P TM Since Carbopol 934P has a large number of carboxyl groups, it forms secondary mucoadhesive bonds with mucin through hydrogen bonds, increasing the strength of mucoadhesion. TM When the chains are exposed to high concentrations of mucus, Carbopol 934P TM There was also evidence that other polymers could provide surface mucoadhesion. (参照文献23) .

[0078] The mucoadhesion of hydroxypropyl methylcellulose is comparable to that of Carbopol 934P. TM The reason for its weaker adhesion compared to other materials is thought to be the lack of proton-donating carboxyl groups in its structure, which reduces its ability to form hydrogen bonds. Furthermore, ethyl cellulose was found to contribute the least to adhesion. Increasing the ratio of ethyl cellulose to other materials from 1:1 to 2:1 and then to 3:1 significantly reduced the mucoadhesion (Table 3). This result is consistent with the results of a previous study. (参照文献15) Consistent with this, ethyl cellulose was found to be responsible for the lack of physical integrity of the gel layer formed and the lowest hydration capacity compared to the other materials used in that study.

[0079] [Table 3]

[0080] Example 7: Ex-vivo evaluation of tri-layered buccal tablets containing insulin particles - ex-vivo permeation studies In vitro permeation of insulin from buccal tablets showed that tablets containing insulin particles with various adhesive layers released nearly 100% of their insulin content after 2 hours, with the tablet with the F9 bioadhesive layer showing the highest permeation rate (Figure 4). The permeation rates of the various materials can be ranked in descending order: Carbopol 934P TM >Carbopol 934P TM The order of hydroxypropyl methylcellulose is hydroxypropyl methylcellulose. Of all the materials used in this case, Carbopol 934P TM has a lower gel viscosity than hydroxypropyl methylcellulose, which is thought to contribute to improved mucosal penetration.

[0081] In this study, ethylcellulose generally slowed the release rate of insulin. (参照文献24) This may be related to the formation of a poorly soluble complex of insulin with ethyl cellulose. Ethyl cellulose was also used as a water-impermeable coating layer for the tablets. To test the unidirectional release profile of the tablets, both sides of the tablets were attached to the buccal tissue of pigs and the release behavior was tested. Figure 4E shows that less than 10% of insulin was released over the entire 4-hour test period. This means that when the tablet was applied to a patient, most of the insulin was released from the bioadhesive layer, and a small portion of the insulin leaked from the water-impermeable coating layer, likely due to leakage from the periphery of the tablet.

[0082] With the same bioadhesive layer, tablets containing insulin-loaded particles (encapsulated insulin) resulted in a higher permeation rate than tablets containing free insulin (Figure 4A-D). MNA-TG-chitosan particles showed the highest permeation rate within 2 hours, followed by insulin-loaded TG-chitosan particles and insulin-loaded chitosan particles (Figure 4B, C, and D). Chitosan is thought to be able to open tight junctions in epithelial cells, thereby promoting insulin permeation through oral mucosal tissue.(参照文献25) Furthermore, TG-chitosan and MNA-TG-chitosan may have limited insulin complex formation due to fewer chitosan amino groups available for insulin interaction. As a result, tablets containing insulin particles coated with TG-chitosan and MNA-TG-chitosan released more insulin than tablets containing unmodified chitosan particles. Therefore, to maximize insulin penetration along with the best tablet mucoadhesion and achieve unidirectional insulin release to the buccal mucosa, tablets with a bioadhesive layer of F9 containing insulin particles coated with MNA-TG-chitosan were selected for further study.

[0083] Example 8: In vitro cytotoxicity test The MTT assay was used to investigate the cytotoxicity of all insulin particles coated with chitosan, TG-chitosan, and MNA-TG-chitosan. Because these particles were intended for the preparation of insulin oral tablets, all potential cells in the human body that could come into contact with insulin particles were used in this study. In addition to oral cells (TR-146), the liver is the main organ in which insulin performs its physiological functions. (参照文献26) Hepatocytes (HepG2) were also used in this study. Furthermore, intestinal cells (Caco-2) were also used in this study because ex-vivo penetration tests showed that less than 10% of insulin could leak from the edge of the tablet and directly enter the gastrointestinal tract. It was found that all insulin particles coated with chitosan, TG-chitosan, and MNA-TG-chitosan had no significant effect on cell viability at concentrations between 50 and 1000 μg / ml. This indicates that all insulin particles can be safely used in buccal tablets and can reach the therapeutic range (data not shown).

[0084] Example 9: In vitro cellular uptake assay HepG2 cells are a human liver cancer cell line commonly used as an in vitro hepatocyte uptake model. Cellular uptake was quantified using flow cytometry and visually measured by confocal laser scanning microscopy (CLSM). The intracellular fluorescence intensity of reconstituted spray-dried insulin particles coated with chitosan, TG-chitosan, and MNA-TG-chitosan (Figure 5A) was 2.9, 3.6, and 4.4 times that of the free FITC insulin group, respectively (Figure 5B). These results indicate that encapsulated insulin has higher cellular uptake than free insulin. In addition to the smaller particle size of TG-chitosan- and MNA-TG-chitosan-coated particles, the cellular uptake mechanism is also controlled by the reaction of thiol functional groups on the surface of insulin particles with surface thiols of transmembrane proteins. (参照文献27) This can be done by exchanging disulfide groups on the particle surface with thiol groups on the transmembrane protein, resulting in the formation of new disulfide bonds between the particle and the membrane protein, improving insulin absorption. (参照文献28) Preactivated MNA-TG-chitosan had more free -SH groups, which allowed it to bind more strongly to transmembrane proteins and increase cellular uptake.

[0085] Example 10: Cellular tight junctions and insulin transport Chitosan-based particles are effective in enhancing drug penetration through epithelia, primarily by opening tight junctions that transiently connect epithelial cells. (参照文献29)Chitosan-coated insulin particles, TG-chitosan, and MNA-TG-chitosan significantly reduced the transepithelial electrical resistance (TEER) values ​​of TR146 cells compared with untreated and free insulin-treated cells, suggesting the opening of tight junctions (Figure 6A). Among these results, MNA-TG-chitosan caused the greatest decrease in TEER values, followed by TG-chitosan and chitosan. Furthermore, after removing the test sample and refilling the medium, a gradual recovery of TEER values ​​was observed, suggesting that the opening of tight junctions is a temporary and reversible process. Furthermore, as shown in Figure 6B, the ZO-1 bands in the free insulin-treated group appeared continuous between adjacent cells. After incubation with chitosan-, TG-chitosan-, and MNA-TG-chitosan-coated insulin particles, the ZO-1-stained bands became fragmented and discontinuous, indicating the opening of tight cell junctions. Furthermore, the ZO-1 staining signal was weakest in insulin particles containing MNA-TG-chitosan, followed by TG-chitosan and chitosan, which was consistent with the TEER results. The reason why TG-chitosan and MNA-TG-chitosan can open tight junctions more than chitosan is because the interaction between membrane-bound enzymes and protein thiol groups occurs via different mechanisms. (参照文献30) Thiomers can inhibit protein tyrosine phosphatases via glutathione. Inhibition of this enzyme prevents the dephosphorylation of the tyrosine subunit of occludin, resulting in the formation of tight junctions. (参照文献31) In our case, preactivated MNA-TG-chitosan had more free -SH groups on its surface, and therefore these particles had a higher ability to open cellular tight junctions compared to TG-chitosan.

[0086] The cumulative amount of insulin that crossed the TR-146 monolayer was compared between free insulin and insulin particles coated with chitosan, TG-chitosan, and MNA-TG-chitosan (Figure 6C). The amount of insulin transported in each group within 4 h was as follows: control group (free insulin): 368 ± 32 ng, chitosan-coated insulin particle group: 1863 ± 109 ng, TG-chitosan-coated insulin particle group: 2246 ± 154 ng, and MNA-TG-chitosan-coated insulin particle group: 2593 ± 121 ng. To further compare insulin transport efficiency, Papp values ​​or absolute particle amounts were more appropriate than cumulative insulin amounts. The insulin apparent permeability (Papp) value of MNA-TG-chitosan-coated insulin particles was 3.22 ± 0.23 × 10 -6 cms -1 ) was the highest among all test groups, and TG-chitosan (2.79 ± 0.28 × 10 -6 cms -1 ), chitosan (2.33±0.19×10 -6 cms -1 ), free insulin (1.14 ± 0.11 × 10 -6 cms -1 ) followed by (Figure 6D). Therefore, these results directly demonstrate that insulin transport across a monolayer of TR-146 cells is improved when MNA-TG-chitosan encapsulates insulin. In addition to the fact that TG-chitosan and MNA-TG-chitosan can open cellular tight junctions, they also demonstrated efflux pump inhibitory properties. They inhibit the efflux pump by forming disulfides with cysteine ​​moieties of natural proteins present within the efflux pump channel. (参照文献32、33) It can reversibly inhibit the action of ATP and enhance cell penetration.

[0087] Example 11: In Vivo Study - Insulin Delivery via Buccal Insulin Tablets and Effect on Blood Glucose Levels All rats received a single intravenous injection of 55 mg / kg of STZ into the tail vein under isoflurane anesthesia. Using a blood glucose meter and glucose test strips, hyperglycemia (>16 mmol / L) was detected in tail-tip blood samples from all rats four days later. After confirming that these rats did not develop insulin resistance, a diabetic rat model was established and further testing was performed.

[0088] The insulin delivery efficiency of the particles and buccal tablets was analyzed in rats with established type 1 diabetes. Figure 7A shows the average pharmacodynamic profiles of the various test groups. In the positive control group, which received an intraperitoneal injection of 10 IU / kg insulin solution, blood glucose levels rapidly decreased to approximately 25% of basal levels within 1 hour and remained at this level for the next 1 hour. Oral administration of insulin solution did not significantly alter blood glucose levels (Figure 7A). Oral administration of insulin particles coated with chitosan, TG-chitosan, and MNA-TG-chitosan gradually decreased blood glucose levels to 59%, 52%, and 48% of basal levels, respectively, within 4 hours and maintained this level throughout the test period (Figure 7A). However, the blood glucose-lowering effect only appeared after 2 hours, indicating that it took a considerable amount of time for the particles to reach the small intestine, where insulin is released. Therefore, the traditional oral administration route of insulin is a slow-acting and long-lasting administration method. In the case of insulin buccal tablets, tablets containing particles coated with chitosan, TG-chitosan, and MNA-TG-chitosan rapidly reduced blood glucose levels by 46%, 40%, and 37%, respectively, within 2 hours and maintained this level throughout the test period (Figure 7A). Notably, all rats administered buccal tablets were tested under anesthesia, which may have resulted in a faster response and improved blood glucose-lowering effect for buccal tablets than when the rats were awake. Furthermore, insulin particles coated with MNA-TG-chitosan and buccal tablets containing them showed the highest blood glucose-lowering effect, consistent with the results of in vivo and in vitro tests. All in vivo and in vitro results indicated that buccal tablets containing MNA-TG-chitosan-coated insulin particles have a fast onset of action similar to injections, are more convenient to administer, and have a long-lasting blood glucose-lowering effect.

[0089] Example 12: Pharmacokinetic evaluation of blood insulin concentration To gain a deeper understanding of the effects of different administration forms on blood glucose lowering, serum insulin concentrations were measured by ELISA. In the positive control group, where 10 IU / kg insulin was intraperitoneally injected, blood insulin concentrations rapidly increased to a maximum of 126.3 mIU / L within 1 hour and then rapidly decreased over the next 5 hours (Figure 7B). Oral administration of insulin particles coated with chitosan, TG-chitosan, and MNA-TG-chitosan resulted in a gradual increase in insulin concentrations over the first 4 hours, with maximum concentrations of 41.5, 44.6, and 49.2 mIU / L, respectively, followed by a slow decrease over the next 2 hours (Figure 7B). Administration of buccal tablets containing chitosan-, TG-chitosan-, and MNA-TG-chitosan-coated particles together with insulin resulted in a rapid increase in insulin levels over the first 2 hours, with maximum levels of 47.6, 52.0, and 55.7 mIU / L, respectively, followed by a gradual decrease over the next 4 hours (Figure 7B). The relative insulin bioavailability of orally administered insulin particles and buccal tablets was approximately 13% and 17% compared with intraperitoneal insulin injection, indicating that buccal tablets can further optimize the bioavailability of insulin particles. This pharmacokinetic profile further demonstrated that buccal tablets containing insulin particles had a similar onset of action as injections and a prolonged blood glucose-lowering effect. Furthermore, it can be concluded that buccal tablets containing MNA-TG-chitosan-coated insulin particles had the highest bioavailability compared with other formulations used in this test. Therefore, buccal tablets containing MNA-TG-chitosan-coated insulin particles were the best formulation used in this study because they significantly improved insulin bioavailability.

[0090] Example 13: Biodistribution of orally administered insulin particles and insulin buccal tablets To assess whether oral delivery could bypass the gastrointestinal tract and mimic intraperitoneal delivery, we examined the biodistribution of Cy5-labeled insulin after intraperitoneal injection, oral administration of Cy5-labeled insulin particles, and buccal tablets containing Cy5-labeled insulin particles. As expected, insulin in rats receiving intraperitoneal injections was distributed almost entirely to the liver. (参照文献34) (Figure 8), with some insulin remaining at the injection site. In contrast, insulin distribution in rats administered orally administered insulin particles was primarily in the stomach. In the case of the buccal tablet, it was clear that the biodistribution of insulin was similar to that of those receiving intraperitoneal injection, with insulin present in the liver. This result indicates that the insulin buccal tablet bypassed the gastrointestinal tract and showed a delivery pattern similar to that of intraperitoneal injection.

[0091] Example 14: Toxicity evaluation after oral administration The potential toxicity of oral administration of buccal tablets containing insulin particles coated with chitosan, TG-chitosan, and MNA-TG-chitosan was evaluated by examining liver enzyme activity (Figure 9). (参照文献35) Based on the results of the biodistribution study, most of the insulin released from the buccal tablets accumulated in the liver. No significant differences were observed in alkaline phosphatase (ALP), aspartate transaminase (AST), and alanine aminotransferase (ALT) activities between the groups treated with buccal tablets containing insulin particles coated with chitosan, TG-chitosan, and MNA-TG-chitosan and the control group. In conclusion, there was no toxicity associated with the administration of buccal tablets containing insulin particles.

[0092] Example 15: Dosage form structure of mucoadhesive tablets In general, pharmaceutical dosage forms (e.g., tablets or films) can be composed of different layers to promote mucoadhesion and reduce peptide dissolution in saliva. Thus, a pharmaceutical dosage form can include a peptide-loaded layer, a mucoadhesive layer, and a water-impermeable layer composed of a water-repellent polymer or coating. Combining these three layers in different forms can enable the pharmaceutical dosage form to remain on the oral mucosal surface for extended periods. Figure 12 shows a multilayer dosage form with improved unidirectional release to enhance absorption of the delivered peptide. Using a mucoadhesive polymer, the tablet or film can remain in contact with the oral mucosa for minutes to hours. A water-insoluble polymer can be used as the water-impermeable layer to release the peptide unidirectionally onto the mucosal surface and prevent release of the peptide into saliva. While less than 10% dissolution has been demonstrated in this manner, a tablet design in which the water-impermeable layer surrounds both the peptide-loaded layer and the mucoadhesive layer on the sides is preferred, as this is more likely to reduce dissolution from the sides of the tablet (see the bottom of the middle column in Figure 12). Additionally, Figure 12 also shows a mucoadhesive surface (*) without a water-impermeable layer to facilitate mucoadhesion of the pharmaceutical dosage form to the target mucosal tissue.

[0093] <Conclusion> In this study, we introduced a trilayer oral mucoadhesive tablet containing insulin particles. To make it suitable for oral mucosal delivery, MNA-TG-chitosan was synthesized and found to enhance the mucoadhesive properties of insulin particles while maintaining a small particle size and high encapsulation efficiency. Specific peptide encapsulation was tested, including chitosan / sodium tripolyphosphate / insulin crosslinked particles. These particles were optimized to a particle size of 318 nm, a PDI of 0.18, an encapsulation efficiency of 99.4%, and a loading capacity of 25.01%. MNA-TG-chitosan-coated insulin peptide particles demonstrated the highest cellular uptake and penetration efficiency compared with chitosan- and TG-chitosan-coated insulin particles. To optimize the insulin release profile along with tablet mucoadhesion and simultaneously achieve unidirectional drug release to the oral mucosa, trilayer tablets were prepared. Many factors were considered when evaluating the tablets, including content uniformity, weight variation, thickness, diameter, hardness, friability, swelling index, surface pH, and mucoadhesive strength. The optimized mucoadhesive force of 1.12 ± 0.16 N was achieved by optimizing the materials used for the mucoadhesive layer. Tablets with an optimized mucoadhesive layer containing insulin particles coated with MNA-TG-chitosan showed the highest insulin penetration compared to tablets containing pure chitosan and TG-chitosan-coated insulin particles. In vivo studies demonstrated that insulin peptide tablets can significantly lower blood glucose levels compared to oral administration of free insulin. These insulin peptide tablets also demonstrated a faster onset of hypoglycemic effect compared to oral administration of insulin particles. Blood glucose levels in rats began to decrease 30 minutes after administration of mucoadhesive insulin peptide tablets, whereas oral administration of insulin particles began to decrease blood glucose levels almost 2 hours later. These results indicate that buccal tablets containing insulin particles have a similar fast onset of action to intraperitoneal injection, but are more convenient to administer and provide a more sustained reduction in blood glucose levels.

[0094] Separating the drug layer from the mucosal layer is important because including the therapeutic peptide in the mucoadhesive layer would result in rapid release, which would flood the body with the peptide and shorten the therapeutic effect. Slightly delaying release is useful because it ensures that insulin is released into the tissues when the mucoadhesive layer interacts with the oral mucosa, opening tight junctions and facilitating peptide absorption by the oral mucosa. If the tight junctions are not open, there is a high risk that the released insulin will be lost in the saliva. The average diameter of the encapsulated peptide is 50–10,000 nm, with 150–400 nm being preferable.

[0095] The present disclosure may be further understood by non-limiting examples. Although the description herein includes many specific examples, these should not be construed as limiting the scope of the present disclosure, but merely as illustrative of some of the embodiments of the present disclosure. Thus, for example, the scope of the present disclosure should be determined by the accompanying aspects and their equivalents, rather than by the examples given.

[0096] Many of the molecules disclosed herein may contain one or more ionizable groups (groups that can remove a proton (e.g., —COOH) or add a proton (e.g., amine) or can be quaternized (e.g., amine)). Where appropriate, all possible ionic forms of such molecules and their salts are intended to be individually included in the disclosure herein. With respect to salts of the compounds herein, one of skill in the art can select from a wide variety of available counterions appropriate for preparing the salts of the present disclosure for a given application. For certain applications, the selection of a particular anion or cation to prepare a salt may increase or decrease the solubility of the salt. All formulations or combinations of components described or exemplified herein can be used to practice the present disclosure unless otherwise specified.

[0097] When a range, e.g., a temperature range, a time range, or a composition or concentration range, is presented in the specification, all intermediate ranges and subranges, as well as all individual values ​​within the ranges presented, are intended to be included in the disclosure. It will be understood that any subrange or individual value within a range or subrange presented herein may be excluded from the embodiments herein.

[0098] While various embodiments of the present invention are disclosed herein, many adaptations and modifications may be made within the scope of the present invention according to the general knowledge of one skilled in the art. Such modifications include the substitution of known equivalents for any aspect of the invention to achieve the same result in substantially the same way. Numerical ranges include the numerical values ​​defining the range. The term "comprises" is used herein as an open-ended term substantially equivalent to the phrase "including, but not limited to," and the term "comprises" has a corresponding meaning. As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a thing" includes a plurality of such things. The citation of a reference herein is not an admission that such reference is prior art to embodiments of the present invention. The present invention includes all embodiments and modifications substantially as described above and with reference to the examples and drawings. Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed disclosure pertains.

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Claims

1. A pharmaceutical dosage form is provided, which may include: (a) a mucoadhesive layer, (i) ethylcellulose and an acrylic acid polymer cross-linked with allyl sucrose or allyl pentaerythritol, wherein the acrylic acid polymer cross-linked with the allyl sucrose or the allyl pentaerythritol is at least 12.5% ​​by weight of the total mucoadhesive layer; or (ii) the mucoadhesive layer comprising ethyl cellulose, an acrylic acid polymer cross-linked with allyl sucrose or allyl pentaerythritol, wherein the acrylic acid polymer cross-linked with allyl sucrose or allyl pentaerythritol is at least 12.5% ​​by weight of the total mucoadhesive layer, and hydroxypropyl methylcellulose (HPMC); (b) a peptide-loaded layer comprising an encapsulated peptide; and (c) an impermeable layer selected from one or more of ethyl cellulose, polyvinyl chloride, polydimethylsiloxane, hydroxypropyl methylcellulose, hemicellulose, poly(e-caprolactone) (PCL), carboxymethyl cellulose, polyvinyl acetate, propyl cellulose, polymethyl methacrylate, methacrylic acid copolymer, and cellulose acetate phthalate; The peptide-loaded layer is present between the mucoadhesive layer and the water-impermeable layer, and the water-impermeable layer facilitates unidirectional migration of the encapsulated peptide through the mucoadhesive layer to the target tissue.

2. 2. The pharmaceutical dosage form of claim 1, wherein the water-impermeable layer forms an outer coating, leaving a mucoadhesive surface free of the water-impermeable layer to facilitate mucoadhesion.

3. 3. The pharmaceutical dosage form of claim 1, wherein the ethylcellulose in (i) or (ii) is at least 50% by weight of the total mucoadhesive layer, or about 50% to about 75% by weight of the total mucoadhesive layer.

4. 4. The pharmaceutical dosage form of claim 1, wherein the acrylic acid polymer cross-linked with allyl sucrose or allyl pentaerythritol is about 12.5% ​​to about 50% by weight of the total mucoadhesive layer.

5. The acrylic acid polymer crosslinked with allyl sucrose or allyl pentaerythritol is Carbopol 934 NF ＴＭ , Carbopol 934P NF ＴＭ , Carbopol 941 NF ＴＭ , Carbopol 942 NF ＴＭ , Carbopol 940 NF ＴＭ , Carbopol 974 P ＴＭ , Carbopol 971 P ＴＭ , Noveon AA-1 Polycarbophil ＴＭ (formerly known as Carbopol 976 ＴＭ ), Carbopol 1342 ＴＭ , Carbopol 1382 ＴＭ , Carbopol salts ＴＭ , Carbopol 981 NF ＴＭ , Carbopol 980 NF ＴＭ , Carbopol ETD 2050 ＴＭ , Carbopol ETD 2020 ＴＭ , Carbopol ULTREZ 10 ＴＭ , Carbopol 1984 ＴＭ , Carbopol 2984 ＴＭ , Carbopol 5984 ＴＭ , and Carbopol® 71G NF ＴＭ 5. The pharmaceutical dosage form according to claim 1, wherein the pharmaceutical dosage form is selected from the group consisting of:

6. The acrylic acid polymer crosslinked with allyl sucrose or allyl pentaerythritol is Carbopol 934 NF ＴＭ 6. The pharmaceutical dosage form according to any one of claims 1 to 5, wherein

7. 7. A pharmaceutical dosage form according to any one of claims 1 to 6, wherein the thickness of the mucoadhesive layer is at least 100 μm.

8. 8. The pharmaceutical dosage form of claim 1, wherein the encapsulated peptide comprises chitosan-coated peptide particles.

9. 9. The pharmaceutical dosage form of claim 1, wherein the encapsulated peptide comprises tripolyphosphate (TPP), chitosan, and a peptide therapeutic agent.

10. 10. The pharmaceutical dosage form according to claim 8 or 9, wherein the chitosan is a thiolated chitosan.

11. 11. The pharmaceutical dosage form of claim 10, wherein the thiolated chitosan is selected from mercaptonicotinic acid (MNA)-thioglycolic acid-chitosan (MNA-TG-chitosan) and thioglycolic acid-chitosan (TG-chitosan).

12. 12. The pharmaceutical dosage form of any one of claims 1 to 11, wherein the encapsulated peptide is a particle having a diameter of about 50 nm to about 10,000 nm.

13. 13. The pharmaceutical dosage form according to any one of claims 1 to 12, wherein the encapsulated peptide is in the form of particles having a diameter of about 150 nm to about 400 nm.

14. 14. A pharmaceutical dosage form according to any one of claims 1 to 13, wherein prior to administration, the encapsulated peptide is present only within the peptide-loaded layer.

15. 15. The pharmaceutical dosage form according to any one of claims 1 to 14, wherein the encapsulated peptide is synthesized by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) coupling of chitosan with thioglycolic acid (TGA), followed by thiol-disulfide exchange of thiolated chitosan with 2,2'-disulfanediylnicotinic acid.

16. 16. The pharmaceutical dosage form of any one of claims 1 to 15, wherein the peptide-loaded layer further comprises one or more sweeteners or flavoring agents.

17. 17. The pharmaceutical dosage form of claim 16, wherein the one or more sweeteners or flavoring agents are selected from one or more of lactose, glucose, sucrose, mannitol, xylitol, sorbitol, and trehalose.

18. 18. A pharmaceutical dosage form according to claim 16 or 17, wherein the sweetener is mannitol.

19. 19. The pharmaceutical dosage form of any of claims 1 to 18, wherein the peptide-loaded layer further comprises a binder.

20. 20. A pharmaceutical dosage form according to any one of claims 1 to 19, wherein the binder is sodium alginate.

21. 21. The pharmaceutical dosage form of any of claims 1 to 20, wherein the encapsulated peptide is selected from one or more of insulin, insulin derivatives, insulin analogs, pre-insulin, prodrugs of insulin, glucagon-like peptide 1 (GLP-1), GLP-1 analogs, pre-GLP-1, prodrugs of GLP-1, or combinations thereof.

22. 22. The pharmaceutical dosage form of claim 21, wherein the insulin analog is selected from one or more of lispro, aspart, glulisine, glargine, detemirt, detemir, degludec, neutral protamine hagedorn (NPH), and levemir.

23. 22. The pharmaceutical dosage form of claim 21, wherein the GLP-1 is selected from one or more of exenatide, liraglutide, dulaglutide, and semaglutide.

24. 24. A pharmaceutical dosage form according to any one of claims 1 to 23, wherein the target tissue is the oral mucosa.

25. 25. A pharmaceutical dosage form according to any preceding claim, wherein the oral mucosa is selected from one or more of the buccal mucosa, sublingual mucosa, palatal mucosa, and lingual mucosa.

26. 26. The pharmaceutical dosage form according to any of claims 1 to 25, wherein the mucoadhesive layer has a mucoadhesion of from about 1N to about 30N.

27. 27. A pharmaceutical dosage form according to any one of claims 1 to 26, wherein the pharmaceutical dosage form is a tablet dosage form.

28. 28. A pharmaceutical dosage form according to any one of claims 1 to 27 for the treatment of diabetes.

29. 28. A pharmaceutical dosage form according to any one of claims 1 to 27 for the treatment of obesity.

30. 28. Use of a pharmaceutical dosage form according to any one of claims 1 to 27 for the treatment of diabetes.

31. 28. Use of a pharmaceutical dosage form according to any one of claims 1 to 27 in the manufacture of a medicament for the treatment of diabetes.

32. 28. Use of a pharmaceutical dosage form according to any one of claims 1 to 27 for the treatment of obesity.

33. 28. Use of a pharmaceutical dosage form according to any one of claims 1 to 27 in the manufacture of a medicament for the treatment of obesity.

34. 32. The use according to claim 30 or 31, wherein the diabetes is insulin-dependent diabetes mellitus type 1 (T1D) or type 2 diabetes (T2D).

35. 30. A method for treating diabetes, comprising administering to a subject in need thereof a pharmaceutical dosage form according to any one of claims 1 to 27.

36. 30. A method for treating obesity, comprising administering to a subject in need thereof a pharmaceutical dosage form according to any one of claims 1 to 27.

37. 28. Use of a pharmaceutical dosage form according to any one of claims 1 to 27 for oral mucosal delivery of the encapsulated peptide.

38. 28. A method for oral mucosal delivery of a peptide, comprising administering to a subject in need thereof a pharmaceutical dosage form according to any one of claims 1 to 27.

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