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WO2024177082A1 - Oral pharmaceutical composition containing cell membrane-penetrating polycationic isopeptide, polyion complex composite, production method thereof, and oral insulin formulation - Google Patents

Oral pharmaceutical composition containing cell membrane-penetrating polycationic isopeptide, polyion complex composite, production method thereof, and oral insulin formulation Download PDF

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WO2024177082A1
WO2024177082A1 PCT/JP2024/006117 JP2024006117W WO2024177082A1 WO 2024177082 A1 WO2024177082 A1 WO 2024177082A1 JP 2024006117 W JP2024006117 W JP 2024006117W WO 2024177082 A1 WO2024177082 A1 WO 2024177082A1
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Prior art keywords
polyion complex
isopeptide
cell membrane
pharmaceutical composition
pαl
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PCT/JP2024/006117
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French (fr)
Japanese (ja)
Inventor
吉十 濱野
千登勢 丸山
海渡 鈴木
肇 片野
崇志 伊藤
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公立大学法人福井県立大学
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Publication of WO2024177082A1 publication Critical patent/WO2024177082A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to an oral pharmaceutical composition containing a cell membrane-permeable polycation isopeptide, a polyion complex, a method for producing the same, and an oral insulin preparation.
  • Patent Document 1 describes a novel cell membrane-permeable peptide that efficiently penetrates the small intestine, the main tissue for gastrointestinal absorption.
  • the present invention aims to provide an orally administrable pharmaceutical composition containing a physiologically active substance such as a protein or peptide, using a cell membrane-permeable polycation isopeptide, and further to provide a polyion complex that can be used in an orally administrable pharmaceutical composition, a method for producing the same, and an oral insulin preparation.
  • an oral pharmaceutical composition comprising a cell membrane-permeable polycation isopeptide and a physiologically active substance.
  • the oral pharmaceutical composition according to ⁇ 1> further comprising an anionic polymer.
  • the oral pharmaceutical composition according to ⁇ 1> or ⁇ 2> wherein the cell membrane-permeable polycation isopeptide is ⁇ -poly-L- ⁇ -lysine.
  • the anionic polymer is polyphosphoric acid.
  • ⁇ 6> The oral pharmaceutical composition according to ⁇ 2>, comprising the cell membrane-permeable polycationic isopeptide in an amount of 0.01 to 10.0 times by mass relative to the anionic polymer.
  • a polyion complex comprising a cell membrane-permeable polycation isopeptide, a physiologically active substance, and an anionic polymer.
  • the polyion complex according to ⁇ 7>, wherein the cell membrane-permeable polycation isopeptide is ⁇ -poly-L- ⁇ -lysine.
  • ⁇ 9> The polyion complex according to ⁇ 7>, wherein the anionic polymer is polyphosphoric acid.
  • ⁇ 10> The polyion complex according to any one of ⁇ 7> to ⁇ 9>, wherein the solubility in water changes depending on pH.
  • ⁇ 11> The polyion complex according to any one of ⁇ 7> to ⁇ 9>, wherein the cell membrane-permeable polycation isopeptide is contained in an amount of 0.01 to 10.0 times by mass relative to the anionic polymer.
  • ⁇ 12> The method for producing a polyion complex according to any one of ⁇ 7> to ⁇ 9>, wherein the cell membrane-permeable polycation isopeptide and the physiologically active substance are mixed, and then the anionic polymer is mixed therewith.
  • An oral insulin preparation comprising ⁇ -poly-L- ⁇ -lysine and insulin.
  • the present invention provides an orally administrable pharmaceutical composition containing a physiologically active substance such as a protein or peptide, using a cell membrane-permeable polycation isopeptide, and further provides a polyion complex that can be used in the orally administrable pharmaceutical composition, and a method for producing the same.
  • FIG. 1 is a schematic diagram showing the structure of a polyion complex of the present invention.
  • FIG. 1 is a diagram showing a schematic diagram of the behavior of the oral pharmaceutical composition of the present invention in the gastric environment and the intestinal environment.
  • FIG. 2 shows chromatograms obtained by analyzing the supernatants of a bovine serum albumin standard solution and samples 1-1 to 1-8 shown in Table 1 using a high performance liquid chromatography mass spectrometer in the preparation of a bovine serum albumin polyion complex.
  • FIG. 2 is a diagram showing a suspension of bovine serum albumin-polyion complex observed under an optical microscope.
  • FIG. 2 shows chromatograms obtained by analyzing the supernatants of a human insulin standard solution and samples 2-1 to 2-7 shown in Table 2 using a high-performance liquid chromatography mass spectrometer in the preparation of a human insulin polyion complex.
  • FIG. 2 shows a picture of a suspension of human insulin polyion complex observed under an optical microscope.
  • FIG. 13 shows a chromatogram obtained by analyzing the supernatant of Azami Green monomer and the obtained polyion complex using a high-performance liquid chromatography mass spectrometer in the preparation of a polyion complex using Azami Green monomer, a green fluorescent material.
  • FIG. 1 shows chromatograms obtained by analyzing the supernatants of a human insulin standard solution and samples 2-1 to 2-7 shown in Table 2 using a high-performance liquid chromatography mass spectrometer in the preparation of a human insulin polyion complex.
  • FIG. 2 shows a picture of a suspension of human insulin polyion complex observed under an optical microscope.
  • FIG. 13 shows chromatograms obtained by analyzing an IgG standard solution and the supernatant of the obtained polyion complex using a high-performance liquid chromatography mass spectrometer in the preparation of a polyion complex using mouse antibody IgG.
  • FIG. 13 is a diagram showing chromatograms obtained by analyzing an HI standard solution and the supernatant of the obtained polyion complex using a high performance liquid chromatography mass spectrometer in the preparation of a polyion complex using polyacrylic acid.
  • FIG. 13 shows chromatograms obtained by analyzing, by a high-performance liquid chromatography mass spectrometer, supernatants whose pH values were changed to verify the pH-responsive solubility of bovine serum albumin polyion complexes.
  • FIG. 1 is a graph showing the remaining rate of bovine serum albumin after addition of pepsin to verify the biochemical stability of the bovine serum albumin polyion complex.
  • FIG. 13 shows chromatograms obtained by analyzing the supernatant with a high-performance liquid chromatography mass spectrometer after changing the pH to verify the pH-responsive solubility of human insulin polyion complex.
  • 1 is a graph showing the residual rate of human insulin after addition of pepsin to verify the biochemical stability of the human insulin polyion complex.
  • 1 is a graph showing the fluorescence activity before and after the formation of the Azami Green monomer polyion complex.
  • 1 is a graph showing changes in blood glucose levels after intraperitoneal administration (dosage 10 IU/kg) of human insulin polyion complex to healthy mice.
  • 1 is a graph showing changes in blood glucose levels after oral administration of human insulin polyion complex (dosage 100 IU/kg) to healthy mice.
  • 1 is a graph showing changes in blood glucose levels after oral administration of human insulin polyion complex (dosage 300 IU/kg) to healthy mice.
  • 1 is a graph showing changes in blood glucose levels after oral administration of a human insulin polyion complex (dosage 100 IU/kg) to type I diabetic mice.
  • 1 is a graph showing changes in blood glucose levels after oral administration of a human insulin polyion complex (dosage 300 IU/kg) to type I diabetic mice.
  • the oral pharmaceutical composition of this embodiment is characterized by containing a cell membrane-permeable polycation isopeptide and a physiologically active substance.
  • the cell membrane-permeable polycation isopeptide contained in the oral pharmaceutical composition is a type of peptide collectively known as a "cell membrane-permeable peptide.”
  • a cell membrane-permeable peptide a type of peptide collectively known as a "cell membrane-permeable peptide.”
  • multiple amino acids are bound by peptide bonds (-CO-NH-) formed by a condensation polymerization reaction between an amino group bound to the carbon at the 2nd position (the ⁇ -position carbon) of an amino acid and a carboxyl group bound to the carbon at the 2nd position (the ⁇ -position carbon) of another amino acid.
  • an isopeptide is formed by binding multiple amino acids by peptide bonds in which at least one of the amino group and the carboxyl group forming the peptide bond is substituted at a position other than the 2nd position (the ⁇ -position).
  • the cell membrane-permeable polycationic isopeptide of the present invention can be any cationic isopeptide having cell membrane permeability, since it can be easily produced into a polyion complex with an anionic polymer, etc., which will be described later.
  • isopeptides that contain a large amount of basic amino acids, such as lysine ( ⁇ -lysine), ornithine, ⁇ -lysine, diaminobutyric acid, and arginine, and are positively charged can be used.
  • basic amino acids such as lysine ( ⁇ -lysine), ornithine, ⁇ -lysine, diaminobutyric acid, and arginine
  • ⁇ -poly-L- ⁇ -lysine which has a structure in which the carboxyl group of L-lysine and the amino group at the ⁇ -position are linearly linked by an isopeptide bond
  • ⁇ -P ⁇ L ⁇ -poly-L- ⁇ -lysine
  • ⁇ -P ⁇ L natural polycation isopeptide analogues other than ⁇ -P ⁇ L, such as ⁇ -poly-L-diaminobutanoic acid, ⁇ -poly-D-diaminobutanoic acid, ⁇ -poly-L-diaminopropionic acid, etc. can also be used.
  • the structural formula of ⁇ -P ⁇ L is shown in Chemical Formula 1.
  • the structural formula below shows an example in which 25 to 35 L-lysine residues are bonded, but the number of residues varies depending on the bacteria used to produce ⁇ -P ⁇ L.
  • the physiologically active substance contained in the oral pharmaceutical composition according to this embodiment is used to mean both a low molecular weight compound and a high molecular weight substance that exhibits physiological activity when administered to a living body.
  • the low molecular weight compound may include, but is not limited to, low molecular weight compounds contained as active ingredients in pharmaceuticals used for the treatment and/or prevention of various diseases, or low molecular weight compounds having various physiological activities.
  • the polymeric substance include, but are not limited to, proteins, peptides, nucleic acids, and analogs thereof.
  • proteins include proteins having physiological activity, such as proteins used for the treatment and/or prevention of diseases. Examples include, but are not limited to, enzymes, antibodies, transcription factors, or specific parts constituting these.
  • the peptides include physiologically active peptides, such as peptides used for the treatment and/or prevention of diseases, etc.
  • physiologically active peptides such as peptides used for the treatment and/or prevention of diseases, etc.
  • Specific examples include, but are not limited to, insulin, glucagon-like peptide-1, and derivatives thereof for the treatment of diabetes.
  • the physiologically active substance in the oral pharmaceutical composition according to the present embodiment is preferably one or more selected from the group consisting of proteins, peptides, nucleic acids, and enzymes.
  • Drugs that can be preferably used as the physiologically active substance in the oral pharmaceutical composition according to the present embodiment include, but are not limited to, peptide/protein drugs such as insulin and insulin secretion promoters, steroid hormones, nonsteroidal analgesic anti-inflammatory drugs, antihistamines, antiallergic drugs, antiasthmatic drugs, antiparkinsonian drugs, antidementia drugs, psychotropic drugs, antihypertensive drugs, heart disease drugs, circulatory system improving drugs, antiemetics, diuretics, antithrombotic drugs, antirheumatic drugs, and antitumor drugs.
  • peptide/protein drugs such as insulin and insulin secretion promoters, steroid hormones, nonsteroidal analgesic anti-inflammatory drugs, antihistamines, antiallergic drugs, antiasthmatic drugs, anti
  • the physiologically active substance of this embodiment is preferably a protein and/or peptide, since it is easily decomposed by protease in the stomach.
  • insulin such as human insulin can be mentioned as a typical example.
  • the oral pharmaceutical composition according to this embodiment preferably further contains an anionic polymer.
  • the anionic polymer is not limited as long as it is not harmful to the human body, but preferred examples include polyphosphoric acid, anionic polyamino acids such as polyglutamic acid and polyaspartic acid, anionic polysaccharides such as agar, hyaluronan, chondroitin sulfate, dextran sulfate, heparan sulfate, dermatan sulfate, fucoidan, keratan sulfate, heparin, carrageenan, succinoglucan, gum arabic, xanthan gum, alginic acid, pectin, and carboxymethylcellulose, polyacrylic acid, and salts thereof.
  • preferred examples include polyphosphoric acid and salts thereof.
  • anionic polymer commercially available products can be used.
  • the molecular weight of the anionic polymer is not limited, but is preferably 300 to 100,000, more preferably 1,000 to 1,000,000 in terms of number average molecular weight.
  • the amounts of the anionic polymer and the cell membrane-permeable polycationic isopeptide used are appropriately determined depending on the type of each component used.
  • the amount of the cell membrane-permeable polycationic isopeptide is 0.01 to 10.0 times by mass, preferably 0.05 to 2.2 times by mass, relative to the anionic polymer.
  • the method for producing the oral pharmaceutical composition according to this embodiment may be appropriately determined depending on each component used in the composition and the combination thereof.
  • an anionic polymer when used, the cell membrane-permeable polycationic isopeptide and the physiologically active substance are mixed together, and then the anionic polymer is mixed therewith.
  • a preferred production method will now be described in detail.
  • the physiologically active substance is mixed with a buffer solution such as NaH2PO4 - NaHPO4 .
  • the cell membrane-permeable polycationic isopeptide is added as an aqueous solution to the resulting mixture, and mixed to obtain a mixture of the cell membrane-permeable polycationic isopeptide and the physiologically active substance.
  • an anionic polymer is added as an aqueous solution to generate a polyion complex corresponding to the oral pharmaceutical composition in the aqueous solution.
  • the aqueous solution is adjusted to an acidic pH of 3 or less, and the polyion complex is precipitated, and the pharmaceutical composition can be obtained as a suspension.
  • the precipitate can be separated and dried to obtain the desired pharmaceutical composition.
  • the oral pharmaceutical composition according to the present embodiment forms an aggregate consisting of a cell membrane-permeable polycation isopeptide and a physiologically active substance.
  • this aggregate is referred to as a polyion complex, including the case where it further contains other components such as an anionic polymer.
  • the oral pharmaceutical composition further contains an anionic polymer in addition to the cell membrane-permeable polycation isopeptide and the physiologically active substance.
  • the complex formed by containing the anionic polymer is called a polyion complex.
  • the polyion complex means a complex formed by binding or assembling a cationic polymer and an anionic polymer through electrostatic interaction.
  • a polyion complex (40) is formed by mixing a cell membrane-permeable polycation isopeptide (10), which is a cationic polymer, with an anionic polymer (20), and it is considered that the polyion complex (40) contains a physiologically active substance (30) such as a protein.
  • the polyion complex of this embodiment exhibits pH-responsive solubility, in which the solubility in water changes depending on the pH of the water. For example, as described later in the Examples, when the complex forms a polyion complex, it is insoluble in water at an acidic pH of about 3 or less, and is soluble in water at a pH of 6 to 9. From this pH-responsive solubility, as shown in FIG. 2, in a gastric environment (50) with a pH of about 1 to 3, the polyion complex (40) is considered to be poorly soluble in water and therefore does not disintegrate, passing through the stomach and being transported to the intestine.
  • the polyion complex (40) dissolves and separates into a cell membrane-permeable polycation isopeptide (10), an anionic polymer (20), and a physiologically active substance (30), which are considered to pass through the cell membrane and tight junctions of intestinal epithelial cells and are absorbed into the body.
  • Medicines that can be used include peptide/protein medicines such as insulin and insulin secretion promoters, steroid hormones, nonsteroidal analgesics and anti-inflammatory drugs, antihistamines, antiallergy drugs, antiasthma drugs, anti-Parkinson's disease drugs, anti-dementia drugs, psychotropic drugs, antihypertensive drugs, heart disease drugs, circulatory system improvement drugs, antiemetics, diuretics, antithrombotic drugs, antirheumatic drugs, and antitumor drugs.
  • ⁇ -P ⁇ L can be used as the cell membrane-permeable polycationic isopeptide
  • insulin such as human insulin can be used as the physiologically active substance
  • polyphosphate can be used as the anionic polymer.
  • ⁇ -P ⁇ L and polyphosphate have already been used as food additives in the United States, Japan, Korea, and other countries, and their safety to humans has been confirmed.
  • a polyion complex complex consisting of ⁇ -P ⁇ L, polyphosphate, and insulin as components is a mixed preparation formed only by electrostatic interactions, not by covalent bonds, and is therefore not considered a new compound. Therefore, a formulation technology for producing existing medicines such as insulin as polyion complex complexes using ⁇ -P ⁇ L and polyphosphate is expected to be a simple and new oral administration technology that can avoid drug safety tests.
  • the complex obtained in this embodiment can also be used for purposes other than medicine, for example in the form of a polyion complex.
  • possible applications include battery membranes, humidity sensors, antistatic coating membranes, hemodialysis membranes, and membranes for artificial lungs.
  • Example 1 Preparation of bovine serum albumin polyion complex using ⁇ -P ⁇ L and polyphosphate Using ⁇ -P ⁇ L (obtained from Microbuchem LLC) as a cell membrane-permeable polycation isopeptide, polyphosphate (Fujifilm Wako Pure Chemical Industries, Ltd.) as a polyanion, and bovine serum albumin (abbreviated as "BSA”; Nacalai Tesque, Inc.) as a protein, mixing conditions for producing an insoluble polyion complex under acidic conditions were investigated.
  • ⁇ -P ⁇ L obtained from Microbuchem LLC
  • polyphosphate Flujifilm Wako Pure Chemical Industries, Ltd.
  • BSA bovine serum albumin
  • Each reagent was added in the following order in the amounts shown in Table 1, and the final concentration of each reagent in the sample solution is shown in parentheses in Table 1.
  • sample 1-1, 1-2, and 1-3 The pH of samples 1-1, 1-2, and 1-3 was 1.3, 1.9, and 3.2, respectively, and in all cases, a white insoluble precipitate with high crystallinity was precipitated at the bottom of the liquid.
  • the liquid of sample 1-6 (pH 1.5) changed from cloudy to transparent. Since sample 1-6 does not contain BSA, it was found that ⁇ -P ⁇ L and polyphosphate alone do not form a stable insoluble precipitate of polyion complex, and BSA is also essential for the formation of a white insoluble precipitate.
  • the liquid of sample 1-7 separated into two phases, an upper transparent solution and a lower white solution, and no insoluble white precipitate was formed.
  • the liquid of sample 1-8 remained transparent even after standing for 120 minutes. Therefore, the results of Samples 1-7 and 1-8 indicate that both ⁇ -P ⁇ L and polyphosphate, as well as BSA, are essential for the formation of the polyion complex.
  • chromatograms in Figure 3 are total ion current chromatograms (abbreviated as "TIC"), and in the chromatograms, 1 represents ⁇ -P ⁇ L and 2 represents BSA.
  • TIC total ion current chromatograms
  • the suspension of the polyion complex obtained in sample 1-1 was observed under an optical microscope. As shown in FIG. 4, the particles were nonuniform with a diameter of about 5 to 20 ⁇ m, and it was presumed that BSA existed in a state where it was contained within a water-insoluble polyion complex of ⁇ -P ⁇ L and polyphosphate.
  • the order in which each reagent is added is considered to be important. Since the isoelectric point (pI) of BSA is 4.9, it is negatively charged in an aqueous solution of 200 mM NaPB at pH 6.0, and it is considered that the addition of positively charged ⁇ -P ⁇ L causes electrostatic interaction between BSA and ⁇ -P ⁇ L. It was presumed that the addition of polyphosphate, a polyanion, would result in the formation of a polyion complex, which would then form an insoluble white precipitate.
  • pI isoelectric point
  • Example 2 Preparation of human insulin polyion complex using ⁇ -P ⁇ L and polyphosphate Using ⁇ -P ⁇ L as the cell membrane-permeable polycation isopeptide, polyphosphate as the polyanion, and human insulin (abbreviated as "HI”; Nacalai Tesque, Inc.) as the peptide compound, mixing conditions for producing an insoluble polyion complex under acidic conditions were investigated. Each reagent was added in the following order in the amounts shown in Table 2, and the final concentration of each reagent in the sample solution is shown in parentheses in Table 2.
  • Table 2 also shows the state of each sample immediately after the addition of the reagent and after standing for 120 minutes.
  • the pH of samples 2-1, 2-2, and 2-3 was 1.3, 1.8, and 3.0, respectively, and in all cases, a white insoluble precipitate was precipitated at the bottom of the solution.
  • sample 2-6 (pH 1.5) separated into two phases, a clear solution on the top and a white solution on the bottom, and no insoluble white precipitate was formed. Therefore, as in Example 1, it was confirmed that the three components of HI, ⁇ -P ⁇ L, and polyphosphate are essential for the formation of a polyion complex.
  • HFBA heptafluorobutyric acid
  • the polyion complex suspension obtained in Sample 2-1 was observed under an optical microscope. As shown in Figure 6, the polyion complex particles were nonuniform particles with diameters of about 5 to 20 ⁇ m, and it was presumed that HI was present in a water-insoluble polyion complex of ⁇ -P ⁇ L and polyphosphate. As with the BSA polyion complex of Example 1, when preparing the HI polyion complex, the order of adding each reagent is considered to be important. Since the isoelectric point (pI) of HI is 5.3, it is negatively charged in an aqueous solution of 200 mM NaPB at pH 6.0, and it is considered that BSA and ⁇ -P ⁇ L electrostatically interact with each other by adding positively charged ⁇ -P ⁇ L.
  • pI isoelectric point
  • Example 3 Preparation of Polyion Complexes Using Other Proteins
  • Azami Green Monomer A fluorescent protein that emits green fluorescence.
  • the recombinant protein was expressed and purified according to the description in Nature Chemical Biology. 8,791-7. DOI: 10,1038/nchembio. 1040 (2012) and used. (Abbreviated as "mAG”).
  • Normal mouse antibody IgG whole molecule. Fujifilm Wako Pure Chemical Industries, Ltd.
  • IgG insulin receptor gamma gG
  • mAG and IgG the following reagents were added in the following order in the amounts shown in Table 3, and the final concentration of each reagent in the sample solution is shown in parentheses in Table 3.
  • IgG at a concentration of 10 mg/mL (IgG in 10 mM NaPB buffer (pH 7.2) / Dissolved in 0.15M sodium chloride.
  • Aqueous solution of ⁇ -P ⁇ L with a concentration of 100 mg/mL 5) Aqueous solution of polyphosphoric acid with a concentration of 100 mg/mL; 7) Aqueous solution of polyphosphoric acid with a concentration of 125 mg/mL.
  • Fig. 7 and Fig. 8 are all TICs, with the arrow in Fig. 7(a) representing mAG and the arrow in Fig. 8(a) representing IgG. Since no remaining mAG or IgG was detected from Figures 7(b) and 8(b), it was presumed that each sample formed a water-insoluble polyion complex, similar to BSA and HI.
  • Example 4 Preparation of polyion complex using polyacrylic acid It was examined whether a water-insoluble polyion complex of HI could be prepared using polyacrylic acid having an average molecular weight of 5000 (PA5000, Fuji Film Wako Pure Chemical Industries, Ltd.) or polyacrylic acid having an average molecular weight of 25000 (PA25000, Fuji Film Wako Pure Chemical Industries, Ltd.) as a polyanion other than polyphosphate.
  • PA5000 Fuji Film Wako Pure Chemical Industries, Ltd.
  • PA25000 Fuji Film Wako Pure Chemical Industries, Ltd.
  • Example 5 pH-responsive solubility and biochemical stability of BSA polyion complex
  • pH-responsive solubility The same mixed solution as sample 1-1 was prepared in the same manner as in Example 1 and allowed to stand for 120 minutes. Then, an aqueous sodium hydroxide solution was added to adjust the pH of each sample to 3, 5, 7, and 9. As a result, the samples at pH 3 and pH 5 maintained a cloudy state, but the BSA-polyion complexes at pH 7 and pH 9 changed from the cloudy state of sample 1-1 to a transparent solution.
  • the BSA polyion complex was insoluble in water under acidic conditions, and under neutral or higher conditions, the BSA polyion complex was broken down and BSA was released, showing pH-responsive solubility.
  • the acid dissociation constant (pKa) of ⁇ -P ⁇ L is 7.6. Therefore, it is considered that under neutral or weakly alkaline conditions, the polycationicity of ⁇ -P ⁇ L is lost and therefore a polyion complex cannot be formed.
  • Example 6 pH-Responsive Solubility and Biochemical Stability of HI Polyion Complex (1) pH-Responsive Solubility As in Example 2, the same mixed solution as Sample 2-1 was prepared and allowed to stand for 120 minutes. Then, an aqueous sodium hydroxide solution was added to adjust the pH of each sample to 3, 5, 7, and 9, respectively. As a result, the samples at pH 3, pH 5, and pH 7 maintained a cloudy state, but the HI polyion complex at pH 9 changed from the cloudy state of Sample 2-1 to a transparent solution.
  • the samples at each pH were centrifuged at 15,000 rpm for 15 minutes, and the centrifugal supernatant was analyzed by HPLC-ESI-TOF-MS.
  • the chromatograms obtained at each pH are shown in Figure 12. All the chromatograms in Figure 12 are TICs, where 1 is ⁇ -P ⁇ L and 3 is HI.
  • the amount of free HI increased with increasing pH, and free ⁇ -P ⁇ L was detected at pH 7 or higher.
  • the HI polyion complex also exhibited water-insolubility under acidic conditions, and disintegrated and released HI under neutral or higher conditions. In other words, the HI polyion complex exhibited pH-responsive solubility.
  • the horizontal axis of Figure 13 indicates the elapsed time (minutes), and the vertical axis indicates the HI residual rate (%).
  • aqueous solution of HI without polyion complex HI was rapidly decomposed by pepsin (within 5 minutes).
  • no decomposition of HI was observed even after 30 minutes of enzyme treatment, indicating high biochemical stability.
  • Example 7 pH-responsive solubility of mAG polyion complex and IgG polyion complex
  • the mAG polyion complex and IgG polyion complex shown in Table 3 were prepared by the method described in Example 3 and allowed to stand for 120 minutes. Then, an aqueous sodium hydroxide solution was added to adjust the pH to 8 and 9, respectively, and the pH-responsive solubility was examined. It was confirmed that the mAG polyion complex at pH 8 and the IgG polyion complex at pH 9 both disintegrated and dissolved under these alkaline conditions.
  • Example 8 Fluorescence activity after formation of mAG polyion complex A mixture of mAG polyion complexes shown in Table 3 was prepared by the method described in Example 3, and allowed to stand for 120 minutes. Aqueous sodium hydroxide solution was added to adjust the pH to 9, and the fluorescence specific activity of mAG released as the mAG polyion complexes dissolved and disintegrated was measured. Separately, the fluorescence specific activity of mAG just before preparing the mAG polyion complexes was also measured in the same manner. In FIG. 14, the fluorescence activity of mAG before and after the formation of the polyion complexes is shown on the right side, with the activity before the formation (left side) being 100%.
  • the fluorescence specific activity is about 70%.
  • the formation of the polyion complex requires a strong acidic condition by adding polyphosphate, and the final pH of the mAG polyion complex is 1.4.
  • many proteins and peptides are denatured under strong acidic conditions.
  • the green fluorescent protein mAG maintains about 70% of its specific activity even after the formation of the polyion complex. This suggests that the protein/peptide retains some activity even in the strong acidic environment during the formation of the polyion complex, and that the active protein/peptide is released with the collapse of the polyion complex.
  • the polyion complex passes through the stomach environment under acidic conditions without being decomposed by pepsin, and disintegrates in the small intestine and duodenum under neutral or weakly alkaline conditions. Furthermore, it is thought that the proteins and peptides reach the intestine while retaining their physiological activity, and the polyion complex is expected to be an effective oral drug delivery method.
  • mice used in the experiment were inbred C57BL/6J mice (6 weeks old, male, average weight 20g, Ninox Lab Supply Co., Ltd.), which were kept for more than one week after purchase in an environment with a 12-hour light-dark cycle, a room temperature of 22 ⁇ 1°C, and a humidity of 40-70%, with food and water available ad libitum. (The same applies to the mice in the following examples.) After fasting for 3 hours, healthy mice were blood-drawn from the tail vein, and blood glucose levels before drug administration were measured using Niprostat Strip XP3 (Nipro Corporation).
  • Example 2 a suspension containing the same HI polyion complex as sample 2-1 was prepared, appropriately diluted with sterilized 200 mM NaPB buffer (pH 6.0), and the pH of the suspension containing the complex was confirmed to be 2 or less.
  • a 20 mg/mL HI solution dissolved in 10 mM hydrochloric acid
  • a mixture of ⁇ -P ⁇ L/polyphosphate of sample 1-1 (Table 1) were prepared, appropriately diluted with sterilized 200 mM NaPB (pH 6.0), and used in the administration experiment.
  • the doses of HI, ⁇ -P ⁇ L, and polyphosphate were intraperitoneally administered to healthy mice at 10 IU/kg, 0.67 mg/kg, and 6.7 mg/kg, respectively, as shown in Table 5.
  • the HI solution was intraperitoneally administered at a dose of 10 IU/kg, and the ⁇ -P ⁇ L/polyphosphate mixture (Table 1, sample 1-6) was intraperitoneally administered to healthy mice at doses of 0.67 mg/kg and 6.7 mg/kg, respectively (Table 5).
  • Blood was collected from the tail vein 30, 60, and 120 minutes after administration of the HI polyion complex, HI solution, and ⁇ -P ⁇ L/polyphosphate mixture, and the blood glucose level in the blood was measured using Niprostat Strip XP3.
  • FIG. 15 The change in blood glucose level over time (horizontal axis) after administration of each solution is shown in Figure 15, with the blood glucose level before drug administration taken as the standard (100%).
  • (I) indicates the HI polyion complex
  • (II) the HI solution
  • (III) the ⁇ -P ⁇ L/polyphosphate mixed solution.
  • the HI polyion complex (I) was observed to lower blood glucose levels 30, 60, and 120 minutes after administration, as was the HI solution (II) used in the comparative experiment.
  • the ⁇ -P ⁇ L/polyphosphate mixture (III) which is a comparative experiment of a polyion complex that does not contain HI, did not show any blood glucose lowering effect. From the above, it was confirmed that ⁇ -P ⁇ L and polyphosphate have no blood glucose lowering effect, and the blood glucose lowering effect observed in the administration experiment of HI polyion complex (I) is due to the medicinal effect of HI contained in the mixture.
  • HI polyion complex (I) In order for HI polyion complex (I) to show the medicinal effect of blood glucose lowering effect, the water-insoluble HI polyion complex needs to break down and HI is released. Therefore, it was thought that the HI polyion complex showed pH-responsive solubility that breaks down in the neutral environment in the abdominal cavity. In addition, this example revealed that HI released from the HI polyion complex fully maintains its physiological function as a blood glucose lowering hormone.
  • Example 10 Oral administration of HI polyion complex to healthy mice and verification of its effect on blood glucose level (1) Dose: 100 IU/kg After fasting for 3 hours, healthy mice were blood-drawn from the tail vein, and blood glucose levels before drug administration were measured using Niprostat Strip XP3. A suspension containing the same composition of HI polyion complex as sample 2-1 was prepared in the same manner as in Example 2, and appropriately diluted with sterilized 200 mM NaPB buffer (pH 6.0), and the pH of the suspension containing the complex was confirmed to be 2 or less.
  • HI solution dissolved in 10 mM hydrochloric acid
  • a mixture of ⁇ -P ⁇ L/polyphosphate of sample 1-6 (Table 1) were prepared, appropriately diluted with sterilized 200 mM NaPB buffer (pH 6.0), and used in the administration experiment.
  • the doses of HI, ⁇ -P ⁇ L, and polyphosphate were orally administered to healthy mice at 100 IU/kg, 6.7 mg/kg, and 67 mg/kg, respectively, as shown in Table 6.
  • the HI solution was orally administered to healthy mice at a dose of 100 IU/kg, and the ⁇ -P ⁇ L/polyphosphate mixture (Table 1, sample 1-6) was orally administered to healthy mice at doses of 6.7 mg/kg and 67 mg/kg, respectively.
  • blood was collected from the tail vein 30, 60, 120, and 180 minutes later, and the blood glucose level in the blood was measured using Niprostat Strip XP3.
  • HI solution (II) was orally administered to healthy mice.
  • a pH-responsive soluble preparation that is water-insoluble under acidic conditions in the stomach and dissolves under neutral or weakly alkaline conditions in the intestine is desirable.
  • an intestinal absorption promoter it is desirable to use an intestinal absorption promoter in combination to promote absorption of HI in the intestine, and it was expected that the HI polyion complex has both of these characteristics.
  • HI polyion complex (sample 2-1) was orally administered to healthy mice so that the HI dosage was 100 IU/kg, a tendency to lower blood glucose levels was observed compared to administration of HI solution (100 IU/kg), and a significant blood glucose lowering effect was observed 60 minutes and 180 minutes after administration.
  • the HI polyion complex is water-insoluble under acidic conditions in the stomach, and the HI polyion complex disintegrates (dissolves) under neutral or weakly alkaline conditions in the intestinal tract, liberating HI, while ⁇ -P ⁇ L functions as an intestinal absorption promoter, resulting in HI absorption through the intestinal tract.
  • a 20 mg/mL HI solution (dissolved in 10 mM hydrochloric acid), an ⁇ -P ⁇ L/polyphosphate mixture (Table 1, sample 1-6), a polyphosphate/HI mixture (Table 2, sample 2-6), and an ⁇ -P ⁇ L/HI mixture (Table 2, sample 2-7) were prepared, appropriately diluted with sterilized 200 mM NaPB buffer (pH 6.0), and used in the administration experiment.
  • HI, ⁇ -P ⁇ L/polyphosphate mixed solution (Table 1, Samples 1-6), polyphosphate/HI mixed solution (Table 2, Samples 2-6), and ⁇ -P ⁇ L/HI mixed solution (Table 2, Samples 2-7)
  • HI, ⁇ -P ⁇ L, and polyphosphate were orally administered in the amounts shown in Table 7.
  • blood was collected from the tail vein 30, 60, 120, and 180 minutes later, and blood glucose levels were measured using Niprostat Strip XP3.
  • FIG. 17 The time course of blood glucose levels (horizontal axis) following administration of each solution is shown in Figure 17, with the blood glucose level before drug administration taken as the reference (100%).
  • (I) represents the HI polyion complex
  • (II) represents the ⁇ -P ⁇ L/polyphosphate mixed solution
  • (III) represents the ⁇ -P ⁇ L/HI mixed solution
  • (IV) represents the polyphosphate/HI mixed solution
  • (V) represents the HI solution.
  • cell-permeable peptides As mentioned above, in recent years, the use of cell-permeable peptides has been attracting attention as a method for improving the digestive epithelial cell absorbability of biopharmaceuticals. Successful cases have been reported in which cell-permeable peptides were used to improve the digestive epithelial cell absorbability of HI. Among them, polycationic cell-permeable peptides show excellent digestive epithelial cell absorbability and are expected to be put to practical use, but cell-permeable peptides are generally supplied by chemical synthesis, and the problem of their high synthesis costs has not been solved.
  • ⁇ -P ⁇ L is a polycationic isopeptide produced by microorganisms, which shows resistance to various proteolytic enzymes and shows excellent cell membrane permeability.
  • its function as a digestive absorption enhancer has remained unknown.
  • the function of ⁇ -P ⁇ L as a digestive absorption enhancer has been proven, and it has also been revealed that ⁇ -P ⁇ L is important as a polycationic compound that forms a polyion complex with HI together with polyphosphate, and is also important in the pH-responsive solubility of the polyion complex.
  • Example 11 Oral administration of HI polyion complex to streptozotocin-induced type I diabetes mice (1) Preparation of streptozotocin-induced type I diabetes mice (STZ diabetic mice) After fasting for 3 hours, blood was collected from the tail vein, and the blood glucose level before drug administration was measured using Niprostat Strip XP3. Streptozotocin (abbreviated as "STZ”; Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 5 mM sodium citrate buffer (pH 4.5), appropriately diluted, filtered, and sterilized for use in the administration experiment. 100 mg/kg was repeatedly administered intraperitoneally once a day for 3 days (total 300 mg/kg).
  • STZ-administered mice that had been kept for more than one week after STZ administration were fasted for 3 hours, and then blood glucose levels were measured. Mice that showed blood glucose levels of 230 mg/dL or more were used in the experiment as STZ diabetic mice.
  • the doses of HI, ⁇ -P ⁇ L, and polyphosphate were orally administered to STZ diabetic mice at 100 IU/kg, 6.7 mg/kg, and 67 mg/kg, respectively, as shown in Table 8 (HI 100 IU/kg administration group).
  • the doses of HI, ⁇ -P ⁇ L, and polyphosphate were orally administered to STZ diabetic mice at 300 IU/kg, 20 mg/kg, and 200 mg/kg, respectively, as shown in Table 9 (HI 300 IU/kg administration group).
  • the HI dose was orally administered at 100 IU/kg (Table 8) or 300 IU/kg (Table 9). After orally administering each sample solution, blood was collected from the tail vein 30, 60, 120, and 180 minutes later, and blood glucose levels were measured using Niprostat Strip XP3.
  • FIG. 18 The time course of blood glucose levels (horizontal axis) after administration of each solution is shown in FIG. 18 for the HI 100 IU/kg administration group and in FIG. 19 for the HI 300 IU/kg administration group, with the blood glucose level before drug administration taken as the standard (100%).
  • 10 Cell membrane permeable polycation isopeptide
  • 20 Anionic polymer
  • 30 Biologically active substance
  • 40 Polyion complex
  • 50 Gastric environment
  • 60 Intestinal environment

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Abstract

Provided is an oral pharmaceutical composition containing a cell membrane-penetrating polycationic isopeptide and a physiologically active substance. The oral pharmaceutical composition may further contain an anionic polymer. Also provided is a polyion complex composite containing a cell membrane-penetrating polycationic isopeptide, a physiologically active substance and an anionic polymer.

Description

細胞膜透過性ポリカチオンイソペプチドを含む経口医薬組成物、ポリイオンコンプレックス複合体、その製造方法及び経口インスリン製剤Oral pharmaceutical composition containing cell membrane-permeable polycation isopeptide, polyion complex, method for producing same, and oral insulin preparation
　本発明は、細胞膜透過性ポリカチオンイソペプチドを含む経口医薬組成物、ポリイオンコンプレックス複合体、その製造方法及び経口インスリン製剤に関する。 The present invention relates to an oral pharmaceutical composition containing a cell membrane-permeable polycation isopeptide, a polyion complex, a method for producing the same, and an oral insulin preparation.
　特許文献１には、消化管吸収の主要組織である小腸を効率的に透過する新規な細胞膜透過性ペプチドとして、新規な細胞膜透過性ペプチドが記載されている。 Patent Document 1 describes a novel cell membrane-permeable peptide that efficiently penetrates the small intestine, the main tissue for gastrointestinal absorption.
特許第６８５７８７５号公報Patent No. 6857875
　タンパク質やペプチド等の生理活性物質を活性成分とする医薬は、分子標的への特異性が高いため現代医療において重要不可欠な医薬品である。しかし、これらの生理活性物質は胃や腸管などの消化管中で各種タンパク質分解酵素によって分解されてしまうため薬効を示さないことが多く、また、分子量が大きく消化管ではほとんど吸収されないため、その投与法は一般的に注射投与に限られる。注射による投与期間が長期にわたる場合、患者への負担は非常に大きい。タンパク質やペプチド等を有効成分とする医薬について、経口投与が可能になれば、患者の生活の質（ＱＯＬ）を改善することができる。タンパク質／ペプチド医薬の消化管吸収を達成するには、胃内部の酸性条件下で非水溶性を示し、腸管内の中性または弱アルカリ性条件下で溶解する製剤が望ましい。さらに、腸管内にて生体高分子化合物であるタンパク質やペプチドの吸収を促進する腸管吸収促進剤の併用が望ましい。
　本発明は、細胞膜透過性ポリカチオンイソペプチドを用いて、タンパク質やペプチド等の生理活性物質を含む経口投与可能な医薬組成物を提供すること、更に、経口投与可能な医薬組成物に使用可能なポリイオンコンプレックス複合体、その製造方法及び経口インスリン製剤を提供することを目的とする。 Medicines containing biologically active substances such as proteins and peptides as active ingredients are essential medicines in modern medicine because of their high specificity to molecular targets. However, these biologically active substances are often decomposed by various proteolytic enzymes in the digestive tract such as the stomach and intestines, and therefore do not show any medicinal effect. In addition, because the molecular weight is large and they are hardly absorbed in the digestive tract, their administration method is generally limited to injection. If the administration period by injection is long, the burden on the patient is very large. If oral administration of medicines containing proteins, peptides, etc. as active ingredients becomes possible, the quality of life (QOL) of patients can be improved. In order to achieve gastrointestinal absorption of protein/peptide medicines, it is desirable to use a preparation that is insoluble in water under acidic conditions in the stomach and dissolves under neutral or weakly alkaline conditions in the intestine. In addition, it is desirable to use an intestinal absorption promoter that promotes the absorption of biopolymer compounds such as proteins and peptides in the intestine.
The present invention aims to provide an orally administrable pharmaceutical composition containing a physiologically active substance such as a protein or peptide, using a cell membrane-permeable polycation isopeptide, and further to provide a polyion complex that can be used in an orally administrable pharmaceutical composition, a method for producing the same, and an oral insulin preparation.
　かくして本発明の具体的な形態は、下記のとおりである。
＜１＞細胞膜透過性ポリカチオンイソペプチドと、生理活性物質と、を含む経口医薬組成物。
＜２＞さらにアニオン性ポリマーを含む、＜１＞記載の経口医薬組成物。
＜３＞前記細胞膜透過性ポリカチオンイソペプチドがε‐ポリ‐Ｌ‐α‐リジンである、＜１＞又は＜２＞記載の経口医薬組成物。
＜４＞前記アニオン性ポリマーがポリリン酸である、＜２＞記載の経口医薬組成物。
＜５＞ｐＨにより水に対する溶解性が変化する、＜１＞又は＜２＞記載の経口医薬組成物。
＜６＞前記細胞膜透過性ポリカチオンイソペプチドを、前記アニオン性ポリマーに対して０．０１質量倍以上１０．０質量倍以下含む、＜２＞記載の経口医薬組成物。
＜７＞細胞膜透過性ポリカチオンイソペプチドと、生理活性物質と、アニオン性ポリマーと、を含むポリイオンコンプレックス複合体。
＜８＞前記細胞膜透過性ポリカチオンイソペプチドがε‐ポリ‐Ｌ‐α‐リジンである、＜７＞記載のポリイオンコンプレックス複合体。
＜９＞前記アニオン性ポリマーがポリリン酸である、＜７＞記載のポリイオンコンプレックス複合体。
＜１０＞ｐＨにより水に対する溶解性が変化する、＜７＞～＜９＞の何れか記載のポリイオンコンプレックス複合体。
＜１１＞前記細胞膜透過性ポリカチオンイソペプチドを、前記アニオン性ポリマーに対して０．０１質量倍以上１０．０質量倍以下含む、＜７＞～＜９＞の何れか記載のポリイオンコンプレックス複合体。
＜１２＞前記細胞膜透過性ポリカチオンイソペプチドと前記生理活性物質を混合し、次いで前記アニオン性ポリマーを混合する、＜７＞～＜９＞の何れか記載のポリイオンコンプレックス複合体の製造方法。
＜１３＞ε‐ポリ‐Ｌ‐α‐リジンと、インスリンと、を含む経口インスリン製剤。
＜１４＞更にポリリン酸を含む、＜１３＞記載の経口インスリン製剤。
＜１５＞前記生理活性物質は、タンパク質、ペプチド、核酸、および、酵素からなる群から選ばれた１以上である、＜１＞又は＜２＞記載の経口医薬組成物。 Thus, specific embodiments of the present invention are as follows.
<1> An oral pharmaceutical composition comprising a cell membrane-permeable polycation isopeptide and a physiologically active substance.
<2> The oral pharmaceutical composition according to <1>, further comprising an anionic polymer.
<3> The oral pharmaceutical composition according to <1> or <2>, wherein the cell membrane-permeable polycation isopeptide is ε-poly-L-α-lysine.
<4> The oral pharmaceutical composition according to <2>, wherein the anionic polymer is polyphosphoric acid.
<5> The oral pharmaceutical composition according to <1> or <2>, wherein the solubility in water changes depending on pH.
<6> The oral pharmaceutical composition according to <2>, comprising the cell membrane-permeable polycationic isopeptide in an amount of 0.01 to 10.0 times by mass relative to the anionic polymer.
<7> A polyion complex comprising a cell membrane-permeable polycation isopeptide, a physiologically active substance, and an anionic polymer.
<8> The polyion complex according to <7>, wherein the cell membrane-permeable polycation isopeptide is ε-poly-L-α-lysine.
<9> The polyion complex according to <7>, wherein the anionic polymer is polyphosphoric acid.
<10> The polyion complex according to any one of <7> to <9>, wherein the solubility in water changes depending on pH.
<11> The polyion complex according to any one of <7> to <9>, wherein the cell membrane-permeable polycation isopeptide is contained in an amount of 0.01 to 10.0 times by mass relative to the anionic polymer.
<12> The method for producing a polyion complex according to any one of <7> to <9>, wherein the cell membrane-permeable polycation isopeptide and the physiologically active substance are mixed, and then the anionic polymer is mixed therewith.
<13> An oral insulin preparation comprising ε-poly-L-α-lysine and insulin.
<14> The oral insulin preparation according to <13>, further comprising polyphosphate.
<15> The oral pharmaceutical composition according to <1> or <2>, wherein the physiologically active substance is one or more selected from the group consisting of proteins, peptides, nucleic acids, and enzymes.
　本発明によれば、細胞膜透過性ポリカチオンイソペプチドを用いて、タンパク質やペプチドなどの生理活性物質を含む経口投与可能な医薬組成物を提供することができ、更に、当該経口投与可能な医薬組成物に使用可能なポリイオンコンプレックス複合体及びその製造方法を提供することができる。 The present invention provides an orally administrable pharmaceutical composition containing a physiologically active substance such as a protein or peptide, using a cell membrane-permeable polycation isopeptide, and further provides a polyion complex that can be used in the orally administrable pharmaceutical composition, and a method for producing the same.
本発明のポリイオンコンプレックス複合体の構造の模式図を示す図である。FIG. 1 is a schematic diagram showing the structure of a polyion complex of the present invention. 本発明の経口医薬組成物の、胃内環境での挙動および腸内環境での挙動を模式的に示す図である。FIG. 1 is a diagram showing a schematic diagram of the behavior of the oral pharmaceutical composition of the present invention in the gastric environment and the intestinal environment. 牛血清アルブミンポリイオンコンプレックス複合体の調製において、牛血清アルブミン標準液および表１記載の試料１－１～１－８の上清液を高速液体クロマトグラフィー質量分析計で分析した際のクロマトグラムを示す図である。FIG. 2 shows chromatograms obtained by analyzing the supernatants of a bovine serum albumin standard solution and samples 1-1 to 1-8 shown in Table 1 using a high performance liquid chromatography mass spectrometer in the preparation of a bovine serum albumin polyion complex. 牛血清アルブミンポリイオンコンプレックス複合体の懸濁液を光学顕微鏡で観察した図である。FIG. 2 is a diagram showing a suspension of bovine serum albumin-polyion complex observed under an optical microscope. ヒトインスリンポリイオンコンプレックス複合体の調製において、ヒトインスリン標準液および表２記載の試料２－１～２－７の上清液を高速液体クロマトグラフィー質量分析計で分析した際のクロマトグラムを示す図である。FIG. 2 shows chromatograms obtained by analyzing the supernatants of a human insulin standard solution and samples 2-1 to 2-7 shown in Table 2 using a high-performance liquid chromatography mass spectrometer in the preparation of a human insulin polyion complex. ヒトインスリンポリイオンコンプレックス複合体の懸濁液を光学顕微鏡で観察した図である。FIG. 2 shows a picture of a suspension of human insulin polyion complex observed under an optical microscope. 緑色蛍光体であるアザミグリーン単量体を用いたポリイオンコンプレックス複合体の調製において、アザミグリーン単量体および得られたポリイオンコンプレックス複合体の上清液を高速液体クロマトグラフィー質量分析計で分析した際のクロマトグラムを示す図である。FIG. 13 shows a chromatogram obtained by analyzing the supernatant of Azami Green monomer and the obtained polyion complex using a high-performance liquid chromatography mass spectrometer in the preparation of a polyion complex using Azami Green monomer, a green fluorescent material. マウス抗体ＩｇＧを用いたポリイオンコンプレックス複合体の調製において、ＩｇＧ標準液及び得られたポリイオンコンプレックス複合体の上清液を高速液体クロマトグラフィー質量分析計で分析した際のクロマトグラムを示す図である。FIG. 13 shows chromatograms obtained by analyzing an IgG standard solution and the supernatant of the obtained polyion complex using a high-performance liquid chromatography mass spectrometer in the preparation of a polyion complex using mouse antibody IgG. ポリアクリル酸を用いたポリイオンコンプレックス複合体の調製において、ＨＩ標準液及び得られたポリイオンコンプレックス複合体の上清液を高速液体クロマトグラフィー質量分析計で分析した際のクロマトグラムを示す図である。FIG. 13 is a diagram showing chromatograms obtained by analyzing an HI standard solution and the supernatant of the obtained polyion complex using a high performance liquid chromatography mass spectrometer in the preparation of a polyion complex using polyacrylic acid. ウシ血清アルブミンポリイオンコンプレックス複合体のｐＨ応答溶解性検証のためにｐＨを変えた上清液を高速液体クロマトグラフィー質量分析計で分析した際のクロマトグラムを示す図である。FIG. 13 shows chromatograms obtained by analyzing, by a high-performance liquid chromatography mass spectrometer, supernatants whose pH values were changed to verify the pH-responsive solubility of bovine serum albumin polyion complexes. ウシ血清アルブミンポリイオンコンプレックス複合体の生化学的安定性を検証するためペプシン添加後のウシ血清アルブミン残存率を示すグラフである。1 is a graph showing the remaining rate of bovine serum albumin after addition of pepsin to verify the biochemical stability of the bovine serum albumin polyion complex. ヒトインスリンポリイオンコンプレックス複合体のｐＨ応答溶解性検証のためにｐＨを変えた上清液を高速液体クロマトグラフィー質量分析計で分析した際のクロマトグラムを示す図である。FIG. 13 shows chromatograms obtained by analyzing the supernatant with a high-performance liquid chromatography mass spectrometer after changing the pH to verify the pH-responsive solubility of human insulin polyion complex. ヒトインスリンポリイオンコンプレックス複合体の生化学的安定性を検証するためペプシン添加後のヒトインスリン残存率を示すグラフである。1 is a graph showing the residual rate of human insulin after addition of pepsin to verify the biochemical stability of the human insulin polyion complex. アザミグリーン単量体ポリイオンコンプレックス複合体形成前後の蛍光活性を示すグラフである。1 is a graph showing the fluorescence activity before and after the formation of the Azami Green monomer polyion complex. 健常マウスへのヒトインスリンポリイオンコンプレックス複合体の腹腔内投与（投与量１０ＩＵ／ｋｇ）後の血糖値変化を示すグラフである。1 is a graph showing changes in blood glucose levels after intraperitoneal administration (dosage 10 IU/kg) of human insulin polyion complex to healthy mice. 健常マウスへのヒトインスリンポリイオンコンプレックス複合体の経口投与（投与量１００ＩＵ／ｋｇ）後の血糖値変化を示すグラフである。1 is a graph showing changes in blood glucose levels after oral administration of human insulin polyion complex (dosage 100 IU/kg) to healthy mice. 健常マウスへのヒトインスリンポリイオンコンプレックス複合体の経口投与（投与量３００ＩＵ／ｋｇ）後の血糖値変化を示すグラフである。1 is a graph showing changes in blood glucose levels after oral administration of human insulin polyion complex (dosage 300 IU/kg) to healthy mice. Ｉ型糖尿病マウスへのヒトインスリンポリイオンコンプレックス複合体の経口投与（投与量１００ＩＵ／ｋｇ）後の血糖値変化を示すグラフである。1 is a graph showing changes in blood glucose levels after oral administration of a human insulin polyion complex (dosage 100 IU/kg) to type I diabetic mice. Ｉ型糖尿病マウスへのヒトインスリンポリイオンコンプレックス複合体の経口投与（投与量３００ＩＵ／ｋｇ）後の血糖値変化を示すグラフである。1 is a graph showing changes in blood glucose levels after oral administration of a human insulin polyion complex (dosage 300 IU/kg) to type I diabetic mice.
　以下、本発明の実施形態について説明する。これらの説明及び実施例等は実施形態を例示するものであり、発明の範囲を限定するものではない。
　本開示において、数値範囲を表す「〇〇以上〇〇以下」や「〇〇～〇〇」の記載は、特に断りのない限り、記載された上限及び下限を含む数値範囲を意味する。 Hereinafter, embodiments of the present invention will be described. The description and examples are merely illustrative of the embodiments, and are not intended to limit the scope of the invention.
In this disclosure, the expressions "00 to 00" or "00 to 00" representing a numerical range mean a numerical range including the stated upper and lower limits, unless otherwise specified.
　本実施形態にかかる経口医薬組成物は、細胞膜透過性ポリカチオンイソペプチドと、生理活性物質と、を含むことを特徴とする。 The oral pharmaceutical composition of this embodiment is characterized by containing a cell membrane-permeable polycation isopeptide and a physiologically active substance.
＜細胞膜透過性ポリカチオンイソペプチド＞
　経口医薬組成物に含まれる細胞膜透過性ポリカチオンイソペプチドは、「細胞膜透過性ペプチド」として総称されているペプチドの一種である。なお、通常のペプチドは、アミノ酸の２位の炭素（α位の炭素）に結合したアミノ基と、他のアミノ酸の２位の炭素（α位の炭素）に結合したカルボキシル基とが縮重合反応により形成するペプチド結合（‐ＣＯ‐ＮＨ‐）により複数のアミノ酸が結合している。一方、イソペプチドは、ペプチド結合を形成するアミノ基とカルボキシル基の少なくとも一方が、２位（α位）以外に置換しているペプチド結合により複数のアミノ酸が結合して形成される。
　本発明の細胞膜透過性ポリカチオンイソペプチドとしては、細胞膜透過性を有するカチオン性イソペプチドであれば、後述するアニオン性ポリマー等と共にポリイオンコンプレックス複合体を容易に製造できることなどから、特に制限なく使用できる。例えば、リジン（αリジン）、オルニチン、βリジン、ジアミノ酪酸、アルギニンなどの塩基性アミノ酸を多く含み正電荷を帯びているものが使用可能である。
　中でも、Ｌ‐リジンのカルボキシル基とε位のアミノ基がイソペプチド結合により直線状につながった構造を有するε‐ポリ‐Ｌ‐α‐リジン（「ε‐ＰαＬ」と略す。）が、放線菌などの微生物によって生産でき、かつ、厚生労働省告示の既存添加物名簿（三百九十七）に収載され、食品としての安全性が認められ、かつ、消化管吸収促進効果を有していることから、使用に適している。
　ε‐ＰαＬ以外に、γ‐ポリ‐Ｌ‐ジアミノブタン酸、γ‐ポリ‐Ｄ‐ジアミノブタン酸、β‐ポリ‐Ｌ‐ジアミノプロピオン酸等のε‐ＰαＬ以外の天然ポリカチオンイソペプチド類縁体等も使用可能である。
　ε‐ＰαＬの構造式を「化１」に示す。下記構造式では、Ｌ‐リジンが２５～３５残基結合した例を示すが、残基数はε‐ＰαＬの生産に使用する菌によって変化する。 <Cell membrane permeable polycation isopeptide>
The cell membrane-permeable polycation isopeptide contained in the oral pharmaceutical composition is a type of peptide collectively known as a "cell membrane-permeable peptide." In addition, in a normal peptide, multiple amino acids are bound by peptide bonds (-CO-NH-) formed by a condensation polymerization reaction between an amino group bound to the carbon at the 2nd position (the α-position carbon) of an amino acid and a carboxyl group bound to the carbon at the 2nd position (the α-position carbon) of another amino acid. On the other hand, an isopeptide is formed by binding multiple amino acids by peptide bonds in which at least one of the amino group and the carboxyl group forming the peptide bond is substituted at a position other than the 2nd position (the α-position).
The cell membrane-permeable polycationic isopeptide of the present invention can be any cationic isopeptide having cell membrane permeability, since it can be easily produced into a polyion complex with an anionic polymer, etc., which will be described later. For example, isopeptides that contain a large amount of basic amino acids, such as lysine (α-lysine), ornithine, β-lysine, diaminobutyric acid, and arginine, and are positively charged can be used.
Among these, ε-poly-L-α-lysine (abbreviated as “ε-PαL”), which has a structure in which the carboxyl group of L-lysine and the amino group at the ε-position are linearly linked by an isopeptide bond, can be produced by microorganisms such as actinomycetes, is included in the list of existing food additives (397) in the Ministry of Health, Labour and Welfare Notification, is recognized as safe as a food, and has the effect of promoting gastrointestinal absorption, and is therefore suitable for use.
Other than ε-PαL, natural polycation isopeptide analogues other than ε-PαL, such as γ-poly-L-diaminobutanoic acid, γ-poly-D-diaminobutanoic acid, β-poly-L-diaminopropionic acid, etc. can also be used.
The structural formula of ε-PαL is shown in Chemical Formula 1. The structural formula below shows an example in which 25 to 35 L-lysine residues are bonded, but the number of residues varies depending on the bacteria used to produce ε-PαL.
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
　ε‐ＰαＬの製造法としては、特に制限は無いが、自然界から分離されたストレプトマイセス　アルブラス（Ｓｔｒｅｐｔｏｍｙｃｅｓ　ａｌｂｕｌｕｓ）Ｎｏ.３４６－Ｄ株（ＮＢＲＣ１４１４７）を用いた製造法（特公昭５９－２０３５９号公報）が知られ、また、ストレプトマイセス　アルブラス以外の種としては、ストレプトマイセス　ノールセイ（Ｓｔｒｅｐｔｏ ｍｙｃｅｓ　ｎｏｕｒｓｅｉ）（特開平１－１８７０９０号公報）やストレプトマイセスｓｐ．ＳＰ－７２株（Ｓｔｒｅｐｔｏｍｙｃｅｓ　ｓｐ.ＳＰ－７２）（特開２０００－６９９８８号公報）を用いた製造法、ストレプトマイセスｓｐ．ＳＰ－６６株（Ｓｔｒｅ ｐｔｏｍｙｃｅｓ　ｓｐ．ＳＰ－６６）（特開２００１－０１７１５９号公報）を用いた短鎖長ε‐ＰαＬの製造法等が可能である。 There are no particular limitations on the method for producing ε-PαL, but a production method using Streptomyces albulus No. 346-D strain (NBRC14147) isolated from nature (JP Patent Publication 59-20359) is known, and production methods using species other than Streptomyces albulus, such as Streptomyces noursei (JP Patent Publication 1999-187090) and Streptomyces sp. SP-72 (JP Patent Publication 2000-69988), and a method using Streptomyces sp. It is possible to produce short-chain ε-PαL using the SP-66 strain (Streptomyces sp. SP-66) (JP Patent Publication No. 2001-017159).
＜生理活性物質＞
　本実施形態にかかる経口医薬組成物に含まれる生理活性物質とは、生体に投与した場合に、生理的活性を示す低分子量化合物及び高分子物質のいずれも含む意味で用いられる。
　前記低分子量化合物とは、これに限定されないが、種々の疾患の治療及び／又は予防のために用いられる医薬品に有効成分として含まれている低分子化合物、又は種々の生理活性を有する低分子化合物を挙げることができる。
　前記高分子物質としては、これに限定されないが、例えば、タンパク質、ペプチド、核酸、およびこれらの類似体を挙げることができる。
　タンパク質としては、生理活性を有するタンパク質を挙げることができ、疾患の治療及び／又は予防のために用いられるタンパク質等を挙げることができる。例えば、これに限定されないが、酵素、抗体、転写因子、あるいはそれらを構成する特定の部分を挙げることができる。具体的には、アルブミンや抗体を挙げることができる。
　上記ペプチドとしては、生理活性ペプチドを挙げることができ、疾患の治療及び／又は予防のために用いられるペプチド等を挙げることができる。具体的には、これに限定されないが、糖尿病治療に対するインスリン、グルカゴン様ペプチド－１及びその誘導体を挙げることができる。 <Biologically active substances>
The physiologically active substance contained in the oral pharmaceutical composition according to this embodiment is used to mean both a low molecular weight compound and a high molecular weight substance that exhibits physiological activity when administered to a living body.
The low molecular weight compound may include, but is not limited to, low molecular weight compounds contained as active ingredients in pharmaceuticals used for the treatment and/or prevention of various diseases, or low molecular weight compounds having various physiological activities.
Examples of the polymeric substance include, but are not limited to, proteins, peptides, nucleic acids, and analogs thereof.
Examples of proteins include proteins having physiological activity, such as proteins used for the treatment and/or prevention of diseases. Examples include, but are not limited to, enzymes, antibodies, transcription factors, or specific parts constituting these. Specific examples include albumin and antibodies.
The peptides include physiologically active peptides, such as peptides used for the treatment and/or prevention of diseases, etc. Specific examples include, but are not limited to, insulin, glucagon-like peptide-1, and derivatives thereof for the treatment of diabetes.
　総合的に本実施形態にかかる経口医薬組成物における生理活性物質は、タンパク質、ペプチド、核酸、および、酵素からなる群から選ばれた１以上を選択して使用することが好ましい。そして、本実施形態にかかる経口医薬組成物において生理活性物質として好ましく使用できる薬剤は、これに限定されないが、例えば、インスリンおよびインスリン分泌促進剤などのペプチド・タンパク質医薬品、ステロイドホルモン、非ステロイド系鎮痛抗炎症剤、抗ヒスタミン剤、抗アレルギー薬、抗喘息薬、抗パーキンソン病薬、抗痴呆薬、向精神薬、抗高血圧薬、心疾患治療薬、循環器改善薬、制吐剤、利尿剤、抗血栓薬、抗リウマチ薬、抗腫瘍薬を挙げることができる。
　本実施形態の細胞膜透過性ポリカチオンイソペプチドは、腸での透過を促進するものであるので、消化管からの吸収促進に基づいてより有効な薬効を示す物質が本実施形態の生理活性物質として好ましい。胃内でタンパク質分解酵素により分解されやすい点で、生理活性物質はタンパク質及び／又はペプチドが好ましい。特に、ヒトインスリン等のインスリンが典型的な例として挙げることができる。 In general, the physiologically active substance in the oral pharmaceutical composition according to the present embodiment is preferably one or more selected from the group consisting of proteins, peptides, nucleic acids, and enzymes. Drugs that can be preferably used as the physiologically active substance in the oral pharmaceutical composition according to the present embodiment include, but are not limited to, peptide/protein drugs such as insulin and insulin secretion promoters, steroid hormones, nonsteroidal analgesic anti-inflammatory drugs, antihistamines, antiallergic drugs, antiasthmatic drugs, antiparkinsonian drugs, antidementia drugs, psychotropic drugs, antihypertensive drugs, heart disease drugs, circulatory system improving drugs, antiemetics, diuretics, antithrombotic drugs, antirheumatic drugs, and antitumor drugs.
Since the cell membrane-permeable polycation isopeptide of this embodiment promotes intestinal permeation, a substance that exhibits more effective medicinal effects based on the promotion of absorption from the digestive tract is preferred as the physiologically active substance of this embodiment. The physiologically active substance is preferably a protein and/or peptide, since it is easily decomposed by protease in the stomach. In particular, insulin such as human insulin can be mentioned as a typical example.
＜アニオン性ポリマー＞
　本実施形態にかかる経口医薬組成物において、さらにアニオン性ポリマーを含むことが好ましい。アニオン性ポリマーを使用することにより、後述するポリイオンコンプレックス複合体を安定して形成することが可能となる。
　アニオン性ポリマーとしては人体に対して害のない限り制限されることはないが、好適にはポリリン酸；ポリグルタミン酸、ポリアスパラギン酸等のアニオン性ポリアミノ酸；寒天、ヒアルロナン、コンドロイチン硫酸、デキストラン硫酸、ヘパラン硫酸、デルマタン硫酸、フコイダン、ケラタン硫酸、ヘパリン、カラギナン、サクシノグルカン、アラビアゴム、キサンタンガム、アルギン酸、ペクチン、カルボキシメチルセルロース等のアニオン性多糖類；ポリアクリル酸；及び、これらの塩等を挙げることができる。特に、ポリリン酸及びこれらの塩を好適に例示することができる。
　アニオン性ポリマーは、市販品を用いることが可能である。
　アニオン性ポリマーの分子量は制限されないが、数平均分子量として、好ましくは３００～１０００００、より好ましくは１０００～１００００００である。 <Anionic Polymer>
The oral pharmaceutical composition according to this embodiment preferably further contains an anionic polymer. By using the anionic polymer, it becomes possible to stably form a polyion complex, which will be described later.
The anionic polymer is not limited as long as it is not harmful to the human body, but preferred examples include polyphosphoric acid, anionic polyamino acids such as polyglutamic acid and polyaspartic acid, anionic polysaccharides such as agar, hyaluronan, chondroitin sulfate, dextran sulfate, heparan sulfate, dermatan sulfate, fucoidan, keratan sulfate, heparin, carrageenan, succinoglucan, gum arabic, xanthan gum, alginic acid, pectin, and carboxymethylcellulose, polyacrylic acid, and salts thereof. In particular, preferred examples include polyphosphoric acid and salts thereof.
As the anionic polymer, commercially available products can be used.
The molecular weight of the anionic polymer is not limited, but is preferably 300 to 100,000, more preferably 1,000 to 1,000,000 in terms of number average molecular weight.
　アニオン性ポリマーと細胞膜透過性ポリカチオンイソペプチドとの使用量は、使用する各成分の種類等に応じて適宜決定される。例えば、アニオン性ポリマーに対して細胞膜透過性ポリカチオンイソペプチドを０．０１質量倍以上１０．０質量倍以下、好ましくは０．０５質量以上２．２質量倍以下とすることが挙げられる。 The amounts of the anionic polymer and the cell membrane-permeable polycationic isopeptide used are appropriately determined depending on the type of each component used. For example, the amount of the cell membrane-permeable polycationic isopeptide is 0.01 to 10.0 times by mass, preferably 0.05 to 2.2 times by mass, relative to the anionic polymer.
＜経口医薬組成物の製造方法＞
　本実施形態にかかる経口医薬組成物の製造方法は、組成物に用いられる各成分とその組み合わせに応じて適宜決定すればよい。
　例えば、アニオン性ポリマーを用いる場合は、細胞膜透過性ポリカチオンイソペプチドと生理活性物質を混合し、次いでアニオン性ポリマーを混合する。
　以下、好ましい製造方法を詳細に説明する。
　まず、生理活性物質をＮａＨ２ＰＯ４‐ＮａＨＰＯ４等の緩衝液と混合する。得られた混合液に、細胞膜透過性ポリカチオンイソペプチドを水溶液として加えて混合し、細胞膜透過性ポリカチオンイソペプチドと生理活性物質が混合した状態を得る。最後にアニオン性ポリマーを水溶液として加え、水溶液内で経口医薬組成物に相当するポリイオンコンプレックス複合体を生成させる。水溶液をｐＨ３以下の酸性に調製し、ポリイオンコンプレックス複合体を沈殿させそのまま懸濁液として医薬組成物を得ることができる。あるいは、沈殿を分離、乾燥させ、目的とする医薬組成物を得ることができる。 <Method of producing oral pharmaceutical composition>
The method for producing the oral pharmaceutical composition according to this embodiment may be appropriately determined depending on each component used in the composition and the combination thereof.
For example, when an anionic polymer is used, the cell membrane-permeable polycationic isopeptide and the physiologically active substance are mixed together, and then the anionic polymer is mixed therewith.
A preferred production method will now be described in detail.
First, the physiologically active substance is mixed with a buffer solution such as NaH2PO4 - NaHPO4 . The cell membrane-permeable polycationic isopeptide is added as an aqueous solution to the resulting mixture, and mixed to obtain a mixture of the cell membrane-permeable polycationic isopeptide and the physiologically active substance. Finally, an anionic polymer is added as an aqueous solution to generate a polyion complex corresponding to the oral pharmaceutical composition in the aqueous solution. The aqueous solution is adjusted to an acidic pH of 3 or less, and the polyion complex is precipitated, and the pharmaceutical composition can be obtained as a suspension. Alternatively, the precipitate can be separated and dried to obtain the desired pharmaceutical composition.
＜経口医薬組成物＞
　本実施形態にかかる経口医薬組成物は、細胞膜透過性ポリカチオンイソペプチドと生理活性物質からなる集合体を形成する。本明細書では、この集合体を、更にアニオン性ポリマー等の他の成分を含有する場合を含めてポリイオンコンプレックス複合体と称する。
　後述する実施例１０の図１７（ＩＩＩ）のように、細胞膜透過性ポリカチオンイソペプチドと生理活性物質を含む組成物において、生理活性物質が消化管に収促される効果が得られる場合もある。
　経口医薬組成物は、より好ましくは、細胞膜透過性ポリカチオンイソペプチドと生理活性物質に加えて、更にアニオン性ポリマーを含むことが好ましい。アニオン性ポリマーを含んで形成される複合体は、ポリイオンコンプレックスと呼ばれる複合体になる。ポリイオンコンプレックスとは、カチオン性ポリマーとアニオン性ポリマーとが静電相互作用によって結合または集合することにより形成される複合体を意味する。本実施形態では、図１に示すように、カチオン性ポリマーである細胞膜透過性ポリカチオンイソペプチド（１０）とアニオン性ポリマー（２０）とが混合して形成されるポリイオンコンプレックス複合体（４０）が形成され、当該ポリイオンコンプレックス複合体（４０）内にタンパク質等の生理活性物質（３０）が含有されていると考えられる。 <Oral pharmaceutical composition>
The oral pharmaceutical composition according to the present embodiment forms an aggregate consisting of a cell membrane-permeable polycation isopeptide and a physiologically active substance. In this specification, this aggregate is referred to as a polyion complex, including the case where it further contains other components such as an anionic polymer.
As shown in FIG. 17 (III) of Example 10 described later, in a composition containing a cell membrane-permeable polycation isopeptide and a physiologically active substance, the effect of the physiologically active substance being attracted to the digestive tract may also be obtained.
More preferably, the oral pharmaceutical composition further contains an anionic polymer in addition to the cell membrane-permeable polycation isopeptide and the physiologically active substance. The complex formed by containing the anionic polymer is called a polyion complex. The polyion complex means a complex formed by binding or assembling a cationic polymer and an anionic polymer through electrostatic interaction. In this embodiment, as shown in FIG. 1, a polyion complex (40) is formed by mixing a cell membrane-permeable polycation isopeptide (10), which is a cationic polymer, with an anionic polymer (20), and it is considered that the polyion complex (40) contains a physiologically active substance (30) such as a protein.
　本実施形態のポリイオンコンプレックス複合体は、水のｐＨによって、水に対する溶解性が変化するｐＨ応答溶解性を示す。例えば実施例に後述するように複合体がポリイオンコンプレックスを形成している場合、ｐＨ３程度以下の酸性では非水溶性であり、ｐＨ６～９では水溶性を示す。このｐＨ応答溶解性から、図２に示すように、ｐＨが１～３程度である胃内環境（５０）ではポリイオンコンプレックス複合体（４０）は水難溶性であるため崩壊せず、胃を通過して腸へ運ばれると考えられる。そして、ｐＨが６～８程度の腸内環境（６０）ではポリイオンコンプレックス複合体（４０）は溶解して、細胞膜透過性ポリカチオンイソペプチド（１０）、アニオン性ポリマー（２０）及び生理活性物質（３０）に分離し、腸管上皮細胞の細胞膜やタイトジャンクションをそれぞれ通過して体内に吸収されると考えられる。 The polyion complex of this embodiment exhibits pH-responsive solubility, in which the solubility in water changes depending on the pH of the water. For example, as described later in the Examples, when the complex forms a polyion complex, it is insoluble in water at an acidic pH of about 3 or less, and is soluble in water at a pH of 6 to 9. From this pH-responsive solubility, as shown in FIG. 2, in a gastric environment (50) with a pH of about 1 to 3, the polyion complex (40) is considered to be poorly soluble in water and therefore does not disintegrate, passing through the stomach and being transported to the intestine. In an intestinal environment (60) with a pH of about 6 to 8, the polyion complex (40) dissolves and separates into a cell membrane-permeable polycation isopeptide (10), an anionic polymer (20), and a physiologically active substance (30), which are considered to pass through the cell membrane and tight junctions of intestinal epithelial cells and are absorbed into the body.
　従って、従来は注射投与に限られていた医薬品を、経口投与することが可能となる。使用できる医薬品としては、インスリンおよびインスリン分泌促進剤などのペプチド・タンパク質医薬品、ステロイドホルモン、非ステロイド系鎮痛抗炎症剤、抗ヒスタミン剤、抗アレルギー薬、抗喘息薬、抗パーキンソン病薬、抗痴呆薬、向精神薬、抗高血圧薬、心疾患治療薬、循環器改善薬、制吐剤、利尿剤、抗血栓薬、抗リウマチ薬、抗腫瘍薬が挙げられる。
　例えば、経口インスリン製剤とする場合には、細胞膜透過性ポリカチオンイソペプチドとしてε‐ＰαＬを使用し、生理活性物質としてヒトインスリン等のインスリンを使用し、好ましくはさらにアニオン性ポリマーとしてポリリン酸を使用することができる。この組成の場合、ε‐ＰαＬとポリリン酸は、アメリカ、日本、韓国などで既に食品添加物として実利用されており、ヒトへの安全性は確認されている。また、ε‐ＰαＬ、ポリリン酸及びインスリンを構成成分とするポリイオンコンプレックス複合体は、共有結合ではなく静電的な相互作用のみで形成された混合製剤であるため、新規化合物としては見なされない。そのため、ε‐ＰαＬとポリリン酸を用いて既存のインスリンなどの医薬をポリイオンコンプレックス複合体として製造する製剤技術は、薬物の安全性試験を回避できる簡便かつ新しい経口投与技術として期待できる。 Therefore, it is possible to orally administer medicines that were previously only available by injection. Medicines that can be used include peptide/protein medicines such as insulin and insulin secretion promoters, steroid hormones, nonsteroidal analgesics and anti-inflammatory drugs, antihistamines, antiallergy drugs, antiasthma drugs, anti-Parkinson's disease drugs, anti-dementia drugs, psychotropic drugs, antihypertensive drugs, heart disease drugs, circulatory system improvement drugs, antiemetics, diuretics, antithrombotic drugs, antirheumatic drugs, and antitumor drugs.
For example, in the case of an oral insulin preparation, ε-PαL can be used as the cell membrane-permeable polycationic isopeptide, insulin such as human insulin can be used as the physiologically active substance, and preferably polyphosphate can be used as the anionic polymer. In this composition, ε-PαL and polyphosphate have already been used as food additives in the United States, Japan, Korea, and other countries, and their safety to humans has been confirmed. In addition, a polyion complex complex consisting of ε-PαL, polyphosphate, and insulin as components is a mixed preparation formed only by electrostatic interactions, not by covalent bonds, and is therefore not considered a new compound. Therefore, a formulation technology for producing existing medicines such as insulin as polyion complex complexes using ε-PαL and polyphosphate is expected to be a simple and new oral administration technology that can avoid drug safety tests.
　なお、本実施形態で得られた複合体は、例えばポリイオンコンプレックス複合体の形態として、医薬以外への使用も可能である。例えば、可能な用途として、電池用隔膜，湿度センサー，帯電防止被覆膜，血液透析膜，人工肺用膜等が挙げられる。 The complex obtained in this embodiment can also be used for purposes other than medicine, for example in the form of a polyion complex. For example, possible applications include battery membranes, humidity sensors, antistatic coating membranes, hemodialysis membranes, and membranes for artificial lungs.
　以下、実施例により本発明をより具体的に説明するが、発明の実施形態は、これら実施例に何ら限定されるものではない。
〔実施例１〕ε‐ＰαＬ及びポリリン酸を用いた牛血清アルブミンポリイオンコンプレックス複合体の調製
　細胞膜透過性ポリカチオンイソペプチドとしてε‐ＰαＬ（マイクローブケム合同会社より譲り受け）、ポリアニオンとしてポリリン酸（富士フィルム和光純薬（株））、タンパク質として牛血清アルブミン（「ＢＳＡ」と略す。ナカライテスク（株））を使用し、酸性条件下で不溶性のポリイオンコンプレックス複合体を生成する混合条件を検討した。各試薬を次の順に表１記載の量を添加し、各試薬の試料液内での最終濃度を、表１のカッコ内に記載した。
１）水
２）１Ｍ　ＮａＨ２ＰＯ４‐ＮａＨＰＯ４緩衝液（「ＮａＰＢ緩衝液」と略す。）（ｐＨ６．０）
３）濃度２０ｍｇ／ｍＬのＢＳＡ（ｐＨ６．０の２００ｍＭ　ＮａＰＢ緩衝液に溶解）
４）２００ｍＭ　ＮａＰＢ緩衝液 （ｐＨ６．０）
５）濃度１００ｍｇ／ｍＬのε‐ＰαＬ水溶液
６）濃度１００ｍｇ／ｍＬのポリリン酸水溶液 The present invention will be described in more detail below with reference to examples, but the embodiments of the invention are not limited to these examples in any way.
Example 1: Preparation of bovine serum albumin polyion complex using ε-PαL and polyphosphate Using ε-PαL (obtained from Microbuchem LLC) as a cell membrane-permeable polycation isopeptide, polyphosphate (Fujifilm Wako Pure Chemical Industries, Ltd.) as a polyanion, and bovine serum albumin (abbreviated as "BSA"; Nacalai Tesque, Inc.) as a protein, mixing conditions for producing an insoluble polyion complex under acidic conditions were investigated. Each reagent was added in the following order in the amounts shown in Table 1, and the final concentration of each reagent in the sample solution is shown in parentheses in Table 1.
1) Water 2) 1M NaH2PO4 - NaHPO4 buffer solution (abbreviated as "NaPB buffer solution") (pH 6.0)
3) BSA at a concentration of 20 mg/mL (dissolved in 200 mM NaPB buffer at pH 6.0)
4) 200mM NaPB buffer (pH 6.0)
5) Aqueous solution of ε-PαL with a concentration of 100 mg/mL 6) Aqueous solution of polyphosphoric acid with a concentration of 100 mg/mL
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　表１には、各試料の試薬添加直後及び１２０分静置後の状態を併せて記載する。なお、表１中「Ｎ．Ａ．」は、添加しなかったことを示す（後述する他の表でも同様）。
（１）混合直後の状態
　混合後、試料１－１、１－２、１－３、１－６及び１－７の液は速やかに白濁を呈した。一方、試料１－４、１－５及び１－８の液は白濁せずに透明であった。
（２）１２０分静置後の観察
　各試料を１２０分間室温（２５℃）で静置して沈殿形成を観察した。試料１－１、１－２及び１－３のｐＨは、それぞれ、１．３、１．９、３．２であり、いずれも、液の下側に、白色かつ結晶性の高い良好な不溶性沈殿が沈降した。試料１－６の液（ｐＨ１．５）は、白濁から透明に変化した。試料１－６にはＢＳＡが含まれていないことから、ε‐ＰαＬとポリリン酸とだけでは安定なポリイオンコンプレックス複合体の不溶性沈殿は形成されず、白色の不溶性沈殿の生成にはＢＳＡも必須であることが判明した。また、試料１－７の液は、上側の透明溶液と下側の白色溶液の２相に分離し、不溶性の白色沈殿は形成されなかった。なお、試料１－８の液は１２０分静置後も透明なままであった。
　従って、試料１－７及び１－８の結果は、ポリイオンコンプレックス複合体の形成には、ＢＳＡと共に、ε‐ＰαＬとポリリン酸の両者が必須であることを示している。 The state of each sample immediately after the addition of the reagent and after standing for 120 minutes are also shown in Table 1. In Table 1, "NA" indicates that no reagent was added (this also applies to other tables described later).
(1) State immediately after mixing After mixing, the solutions of Samples 1-1, 1-2, 1-3, 1-6 and 1-7 quickly became cloudy, whereas the solutions of Samples 1-4, 1-5 and 1-8 were transparent and not cloudy.
(2) Observation after 120 minutes of standing Each sample was left standing at room temperature (25°C) for 120 minutes, and the formation of precipitate was observed. The pH of samples 1-1, 1-2, and 1-3 was 1.3, 1.9, and 3.2, respectively, and in all cases, a white insoluble precipitate with high crystallinity was precipitated at the bottom of the liquid. The liquid of sample 1-6 (pH 1.5) changed from cloudy to transparent. Since sample 1-6 does not contain BSA, it was found that ε-PαL and polyphosphate alone do not form a stable insoluble precipitate of polyion complex, and BSA is also essential for the formation of a white insoluble precipitate. In addition, the liquid of sample 1-7 separated into two phases, an upper transparent solution and a lower white solution, and no insoluble white precipitate was formed. The liquid of sample 1-8 remained transparent even after standing for 120 minutes.
Therefore, the results of Samples 1-7 and 1-8 indicate that both ε-PαL and polyphosphate, as well as BSA, are essential for the formation of the polyion complex.
（３）上清の分析及び沈殿の観察（ブルカージャパン株式会社製：Ｂｒｕｋｅｒ　ｍａＸｉｓ　Ｐｌｕｓシステム）
　１２０分間室温で静置した各試料を１５，０００ｒｐｍで１５分間遠心分離し、上清を高速液体クロマトグラフィーエレクトロスプレーイオン化飛行時間型質量分析機（ＨＰＬＣ－ＥＳＴ－ＴＯＦ－ＭＳ。ブルカージャパン株式会社製）で分析し、上清に残存するε‐ＰαＬとＢＳＡを確認した。図３に（ａ）３ｍｇ／ｍＬ ＢＳＡ標準液及び（ｂ）試料１－１～１－８について、得られたクロマトグラムを、縦軸を強度、横軸を時間（分）として示す。図３のクロマトグラムは全てトータルイオンカレントクロマトグラム（「ＴＩＣと略す。」）であり、クロマトグラム中、１はε‐ＰαＬ、２はＢＳＡを示す。
　ε‐ＰαＬ及びタンパク質であるＢＳＡの分析は次の条件で行った。
・Ｂｒｕｋｅｒ　ｍａＸｉｓ　Ｐｌｕｓシステム
　充填剤：Ｓｕｎｓｈｅｌｌ　Ｃ８－３０ＨＴ　３．４μｍ、２．１ｍｍ　ＩＤ×１５０ｍｍ；７０℃；移動相Ａ　０．１％トリフルオロ酢酸（ＴＦＡ）水溶液；移動相Ｂ　０．１％ＴＦＡのアセトニトリル（ＡＣＮ）溶液；流速０．３ｍＬ／分；移動相のグラジエント, ０－３０分３１％―３７％移動相Ｂ，３０－３０．１分３７％－９５％移動相Ｂ，３０．１－４０分９５％移動相Ｂ。
　試料１－１（ｐＨ１．３）では残存するε‐ＰαＬとＢＳＡが検出されなかった。このことから、ε‐ＰαＬとＢＳＡは不溶性の白色沈殿（ポリイオンコンプレックス複合体）として存在することが確認された。試料１－１で得られたポリイオンコンプレックス複合体の懸濁液を光学顕微鏡下で観察した。図４に示すように、その粒子は直径５～２０μｍ程度の不均一な粒子であり、ε‐ＰαＬとポリリン酸の非水溶性ポリイオンコンプレックス複合体内にＢＳＡが含有される状態で存在していると推測された。 (3) Analysis of Supernatant and Observation of Precipitation (Bruker Japan: Bruker maxis Plus System)
Each sample was left to stand at room temperature for 120 minutes and centrifuged at 15,000 rpm for 15 minutes, and the supernatant was analyzed by a high performance liquid chromatography electrospray ionization time-of-flight mass spectrometer (HPLC-EST-TOF-MS, manufactured by Bruker Japan Co., Ltd.) to confirm the ε-PαL and BSA remaining in the supernatant. Figure 3 shows the chromatograms obtained for (a) a 3 mg/mL BSA standard solution and (b) samples 1-1 to 1-8, with the ordinate representing intensity and the abscissa representing time (min). All chromatograms in Figure 3 are total ion current chromatograms (abbreviated as "TIC"), and in the chromatograms, 1 represents ε-PαL and 2 represents BSA.
The analysis of ε-PαL and the protein BSA was carried out under the following conditions.
Bruker maxis Plus system Packing: Sunshell C8-30HT 3.4 μm, 2.1 mm ID x 150 mm; 70° C.; Mobile phase A 0.1% trifluoroacetic acid (TFA) in water; Mobile phase B 0.1% TFA in acetonitrile (ACN); flow rate 0.3 mL/min; Mobile phase gradient: 0-30 min 31%-37% mobile phase B, 30-30.1 min 37%-95% mobile phase B, 30.1-40 min 95% mobile phase B.
In sample 1-1 (pH 1.3), no residual ε-PαL or BSA was detected. This confirmed that ε-PαL and BSA existed as an insoluble white precipitate (polyion complex). The suspension of the polyion complex obtained in sample 1-1 was observed under an optical microscope. As shown in FIG. 4, the particles were nonuniform with a diameter of about 5 to 20 μm, and it was presumed that BSA existed in a state where it was contained within a water-insoluble polyion complex of ε-PαL and polyphosphate.
　ＢＳＡポリイオンコンプレックス複合体を調製する際、各試薬を添加する順番が重要であると考えられる。ＢＳＡの等電点（ｐＩ）は４．９であるため、ｐＨ６．０の２００ｍＭ　ＮａＰＢの水溶液中では負に帯電しており、正に帯電しているε‐ＰαＬを加えることにより、ＢＳＡとε‐ＰαＬは静電的に相互作用をしていると考えられる。そこに、ポリアニオンであるポリリン酸の添加により、ポリイオンコンプレックス複合体が形成され、不溶性の白色沈殿が形成されると推測された。また、この沈殿の形成にはポリリン酸、ε‐ＰαＬ、及びＢＳＡの三者の静電的な相互作用が重要であるが、ポリリン酸の濃度比率が低い試料１－４と１－５は透明であり（表１）、ポリイオンコンプレックス複合体が全く形成されない。これらの事実から、ポリイオンコンプレックス複合体の形成にはポリアニオンであるポリリン酸の濃度が重要であることが判明した。 When preparing the BSA polyion complex, the order in which each reagent is added is considered to be important. Since the isoelectric point (pI) of BSA is 4.9, it is negatively charged in an aqueous solution of 200 mM NaPB at pH 6.0, and it is considered that the addition of positively charged ε-PαL causes electrostatic interaction between BSA and ε-PαL. It was presumed that the addition of polyphosphate, a polyanion, would result in the formation of a polyion complex, which would then form an insoluble white precipitate. Furthermore, the electrostatic interaction between polyphosphate, ε-PαL, and BSA is important for the formation of this precipitate, but samples 1-4 and 1-5, which have a low concentration ratio of polyphosphate, are transparent (Table 1), and no polyion complex is formed at all. From these facts, it was found that the concentration of polyphosphate, a polyanion, is important for the formation of a polyion complex.
〔実施例２〕ε‐ＰαＬ及びポリリン酸を用いたヒトインスリンポリイオンコンプレックス複合体の調製
　細胞膜透過性ポリカチオンイソペプチドとしてε‐ＰαＬ、ポリアニオンとしてポリリン酸、ペプチド化合物としてヒトインスリン（「ＨＩ」と略す。ナカライテスク（株））を使用し、酸性条件下で不溶性のポリイオンコンプレックス複合体を生成する混合条件を検討した。各試薬を次の順に表２記載の量を添加し、各試薬の試料液内での最終濃度を、表２のカッコ内に記載した。
１）水
２）１Ｍ　ＮａＰＢ緩衝液（ｐＨ６．０）
３）濃度２０ｍｇ／ｍＬのＨＩ（１０ｍＭ 塩酸に溶解させた）
４）濃度１００ｍｇ／ｍＬのε‐ＰαＬ水溶液
５）濃度１００ｍｇ／ｍＬのポリリン酸水溶液 [Example 2] Preparation of human insulin polyion complex using ε-PαL and polyphosphate Using ε-PαL as the cell membrane-permeable polycation isopeptide, polyphosphate as the polyanion, and human insulin (abbreviated as "HI"; Nacalai Tesque, Inc.) as the peptide compound, mixing conditions for producing an insoluble polyion complex under acidic conditions were investigated. Each reagent was added in the following order in the amounts shown in Table 2, and the final concentration of each reagent in the sample solution is shown in parentheses in Table 2.
1) Water 2) 1M NaPB buffer (pH 6.0)
3) HI at a concentration of 20 mg/mL (dissolved in 10 mM hydrochloric acid)
4) ε-PαL aqueous solution with a concentration of 100 mg/mL 5) Polyphosphoric acid aqueous solution with a concentration of 100 mg/mL
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　表２に、各試料の試薬添加直後及び１２０分静置後の状態を併せて記載する。
（１）混合直後の状態
　混合後、試料２－１、２－２、２－３及び２－６の液は速やかに白濁を呈した。一方、試料２－４、２－５及び２－７の液は白濁せずに透明であった。
（２）１２０分静置後の観察
　各試料を１２０分間室温（２５℃）で静置して沈殿形成を観察した。試料２－１、２－２及び２－３のｐＨは、それぞれ、１．３、１．８、３．０であり、いずれも、液の下側に、白色の不溶性沈殿が沈降した。試料２－６の液（ｐＨ１．５）は、上側の透明溶液と下側の白色溶液の２相に分離し、不溶性の白色沈殿は形成されなかった。従って、実施例１と同様に、ポリイオンコンプレックス複合体の形成には、ＨＩ、ε‐ＰαＬ及びポリリン酸の３者が必須であることが確認できた。 Table 2 also shows the state of each sample immediately after the addition of the reagent and after standing for 120 minutes.
(1) State immediately after mixing After mixing, the solutions of Samples 2-1, 2-2, 2-3 and 2-6 quickly became cloudy, whereas the solutions of Samples 2-4, 2-5 and 2-7 were transparent and not cloudy.
(2) Observation after standing for 120 minutes Each sample was left standing at room temperature (25°C) for 120 minutes, and the formation of precipitate was observed. The pH of samples 2-1, 2-2, and 2-3 was 1.3, 1.8, and 3.0, respectively, and in all cases, a white insoluble precipitate was precipitated at the bottom of the solution. The solution of sample 2-6 (pH 1.5) separated into two phases, a clear solution on the top and a white solution on the bottom, and no insoluble white precipitate was formed. Therefore, as in Example 1, it was confirmed that the three components of HI, ε-PαL, and polyphosphate are essential for the formation of a polyion complex.
（３）上清の分析及び沈殿の観察
　１２０分間室温で静置した各試料を１５，０００ｒｐｍで１５分間遠心分離し、上清をＨＰＬＣ－ＥＳＴ－ＴＯＦ－ＭＳで分析し、上清に残存するε‐ＰαＬとＨＩを確認した。得られたクロマトグラムを、図５（ａ）３ｍｇ／ｍＬのＨＩ標準液及び（ｂ）試料２－１～２－７に、縦軸を強度、横軸を時間（分）として示す。図５のクロマトグラムは全てＴＩＣであり、クロマトグラム中、３はＨＩ、１はε‐ＰαＬを示す。
　ε‐ＰαＬ及びペプチド化合物であるＨＩの分析は次の条件で行った。
・Ｂｒｕｋｅｒ　ｍａＸｉｓ　Ｐｌｕｓシステム
　充填剤　ＳｕｎＳｈｅｌｌ　ＨＦＣ　１８－１６，２．６μｍ，２．１ｍｍ　ＩＤ×　１５０ｍｍ；４０℃；移動相Ａ　０．１％ヘプタフルオロ酪酸（ＨＦＢＡ）水溶液及び移動相Ｂ０．１％ＨＦＢＡのＡＣＮ溶液；流速０．３ｍＬ／分；移動相のグラジエント, ０―５分５％―１５％移動相Ｂ，５－２２分２５％－６０％移動相Ｂ，２２－２７分６０％－９５％移動相Ｂ，２７－３０分９５％移動相Ｂ。
　試料２－１（ｐＨ１．３）と２－２（ｐＨ１．８）では、遊離のε‐ＰαＬは全く検出されず、微量のＨＩが検出された。従って、ε‐ＰαＬと大部分のＨＩが不溶性の白色沈殿（ポリイオンコンプレックス複合体）として存在することが確認された。 (3) Analysis of the Supernatant and Observation of Precipitation Each sample was left to stand at room temperature for 120 minutes and then centrifuged at 15,000 rpm for 15 minutes. The supernatant was analyzed by HPLC-EST-TOF-MS to confirm the ε-PαL and HI remaining in the supernatant. The obtained chromatograms are shown in Figure 5 (a) 3 mg/mL HI standard solution and (b) Samples 2-1 to 2-7, with the vertical axis representing intensity and the horizontal axis representing time (min). All chromatograms in Figure 5 are TICs, and in the chromatograms, 3 represents HI and 1 represents ε-PαL.
The analysis of ε-PαL and the peptide compound HI was carried out under the following conditions.
Bruker maxis Plus system Packing material SunShell HFC 18-16, 2.6 μm, 2.1 mm ID x 150 mm; 40° C.; mobile phase A 0.1% heptafluorobutyric acid (HFBA) in water and mobile phase B 0.1% HFBA in ACN; flow rate 0.3 mL/min; mobile phase gradient: 0-5 min 5%-15% mobile phase B, 5-22 min 25%-60% mobile phase B, 22-27 min 60%-95% mobile phase B, 27-30 min 95% mobile phase B.
In samples 2-1 (pH 1.3) and 2-2 (pH 1.8), no free ε-PαL was detected, but only trace amounts of HI were detected, confirming that ε-PαL and most of HI existed as an insoluble white precipitate (polyion complex).
　試料２－１で得られたポリイオンコンプレックス複合体の懸濁液を光学顕微鏡下で観察した。図６に示すように、ポリイオンコンプレックス複合体の粒子は直径５～２０μｍ程度の不均一な粒子であり、ε‐ＰαＬとポリリン酸の非水溶性ポリイオンコンプレックス複合体内にＨＩが含有された状態で存在していると推測された。
　実施例１のＢＳＡポリイオンコンプレックス複合体と同様に、ＨＩポリイオンコンプレックス複合体を調製する際、各試薬を添加する順番が重要であると考えられる。ＨＩの等電点（ｐＩ）は５．３であるため、ｐＨ６．０の２００ｍＭ　ＮａＰＢの水溶液中では負に帯電しており、正に帯電しているε‐ＰαＬを加えることにより、ＢＳＡとε‐ＰαＬは静電的に相互作用をしていると考えられる。更に、そこに、ポリアニオンであるポリリン酸を添加することにより、ポリイオンコンプレックス複合体が形成され、不溶性の白色沈殿が形成されると推測された。また、この沈殿の形成にはポリリン酸、ε‐ＰαＬ、及びＨＩの三者の静電的な相互作用が重要であるが、ポリリン酸の濃度比率が低い試料２－４と２－５は透明であり、ポリイオンコンプレックス複合体が全く形成されていない（表２）。これらの事実から、ポリイオンコンプレックス複合体の形成にはポリアニオンであるポリリン酸の濃度が重要であることが判明した。 The polyion complex suspension obtained in Sample 2-1 was observed under an optical microscope. As shown in Figure 6, the polyion complex particles were nonuniform particles with diameters of about 5 to 20 μm, and it was presumed that HI was present in a water-insoluble polyion complex of ε-PαL and polyphosphate.
As with the BSA polyion complex of Example 1, when preparing the HI polyion complex, the order of adding each reagent is considered to be important. Since the isoelectric point (pI) of HI is 5.3, it is negatively charged in an aqueous solution of 200 mM NaPB at pH 6.0, and it is considered that BSA and ε-PαL electrostatically interact with each other by adding positively charged ε-PαL. Furthermore, it was presumed that a polyion complex is formed by adding polyphosphate, which is a polyanion, and an insoluble white precipitate is formed. In addition, the electrostatic interaction between polyphosphate, ε-PαL, and HI is important for the formation of this precipitate, but samples 2-4 and 2-5, which have a low concentration ratio of polyphosphate, are transparent and no polyion complex is formed at all (Table 2). From these facts, it was found that the concentration of polyphosphate, which is a polyanion, is important for the formation of a polyion complex.
〔実施例３〕他のタンパク質を用いたポリイオンコンプレックス複合体の調製
　実施例１及び２のポリイオンコンプレックス複合体の調製方法が次のタンパク質に適用できるか検討するため、実験を行った。
・アザミグリーン単量体：緑色蛍光を発する蛍光タンパク質。Ｎａｔｕｒｅ　Ｃｈｅｍｉｃａｌ　Ｂｉｏｌｏｇｙ．８，７９１－７．ＤＯＩ：１０，１０３８／ｎｃｈｅｍｂｉｏ．１０４０（２０１２）の記載に従い、組み換えタンパク質を発現させ、精製して使用した。（「ｍＡＧ」と略す。）
・正常マウス抗体ＩｇＧ：全分子。富士フィルム和光純薬（株）（「ＩｇＧ」と略す。）　これら２つのタンパク質ｍＡＧ及びＩｇＧについて、次の各試薬を次の順に表３記載の量を添加し、各試薬の試料液内での最終濃度を、表３のカッコ内に記載した。
１）水
２）１Ｍ　ＮａＰＢ緩衝液 （ｐＨ６．０）
３）濃度２０ｍｇ／ｍＬのｍＡＧ（ｍＡＧを１０ｍＭ　炭酸水素ナトリウム水溶液に溶解させた。）
４）濃度１０ｍｇ／ｍＬのＩｇＧ（ＩｇＧを１０ｍＭ　ＮａＰＢ緩衝液（ｐＨ７．２）／
０．１５Ｍ 塩化ナトリウムに溶解させた。）
５）濃度１００ｍｇ／ｍＬのε‐ＰαＬ水溶液
６）濃度１００ｍｇ／ｍＬのポリリン酸水溶液
７）濃度１２５ｍｇ／ｍＬのポリリン酸水溶液 Example 3 Preparation of Polyion Complexes Using Other Proteins Experiments were carried out to examine whether the methods for preparing polyion complexes in Examples 1 and 2 can be applied to the following proteins.
Azami Green Monomer: A fluorescent protein that emits green fluorescence. The recombinant protein was expressed and purified according to the description in Nature Chemical Biology. 8,791-7. DOI: 10,1038/nchembio. 1040 (2012) and used. (Abbreviated as "mAG").
Normal mouse antibody IgG: whole molecule. Fujifilm Wako Pure Chemical Industries, Ltd. (abbreviated as "IgG") For these two proteins mAG and IgG, the following reagents were added in the following order in the amounts shown in Table 3, and the final concentration of each reagent in the sample solution is shown in parentheses in Table 3.
1) Water 2) 1M NaPB buffer (pH 6.0)
3) mAG at a concentration of 20 mg/mL (mAG was dissolved in a 10 mM aqueous sodium bicarbonate solution).
4) IgG at a concentration of 10 mg/mL (IgG in 10 mM NaPB buffer (pH 7.2) /
Dissolved in 0.15M sodium chloride.
5) Aqueous solution of ε-PαL with a concentration of 100 mg/mL; 6) Aqueous solution of polyphosphoric acid with a concentration of 100 mg/mL; 7) Aqueous solution of polyphosphoric acid with a concentration of 125 mg/mL.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
（１）１２０分静置後の観察
　各試料を１２０分間室温（２５℃）で静置して沈殿形成を観察した。ｍＡＧポリイオンコンプレックス複合体は緑色沈殿、ＩｇＧポリイオンコンプレックス複合体は白色沈殿を生じた。
（２）上清の分析及び沈殿の観察
　１２０分間室温で静置した各試料を１５，０００ｒｐｍで１５分間遠心分離し、上清をＨＰＬＣ－ＥＳＴ－ＴＯＦ－ＭＳで分析し、上清を分析した。得られたクロマトグラムを、縦軸を強度、横軸を時間（分）として、図７（ａ）０．４ｍｇ／ｍＬのｍＡＧ標準液、（ｂ）ｍＡＧポリイオンコンプレックス複合体の調製液、そして、図８（ａ）２．５ｍｇ／ｍＬのＩｇＧ標準液及び（ｂ）ＩｇＧポリイオンコンプレックス複合体の調製液に示す。図７と図８のクロマトグラムは全てＴＩＣであり、図７（ａ）の矢印はｍＡＧ、図８（ａ）の矢印はＩｇＧを示す。
　図７（ｂ）及び図８（ｂ）から残存するｍＡＧ及びＩｇＧは検出されなかったことから、ＢＳＡ及びＨＩと同様に、各試料は非水溶性のポリイオンコンプレックス複合体を形成していると推測された。 (1) Observation after standing for 120 minutes Each sample was stood for 120 minutes at room temperature (25° C.) and observed for precipitate formation. The mAG polyion complex formed a green precipitate, and the IgG polyion complex formed a white precipitate.
(2) Analysis of the Supernatant and Observation of Precipitation Each sample was left to stand at room temperature for 120 minutes, and then centrifuged at 15,000 rpm for 15 minutes. The supernatant was analyzed by HPLC-EST-TOF-MS. The obtained chromatograms are shown in Fig. 7(a) for 0.4 mg/mL mAG standard solution, (b) for mAG polyion complex preparation, and in Fig. 8(a) for 2.5 mg/mL IgG standard solution, and (b) for IgG polyion complex preparation, with the vertical axis representing intensity and the horizontal axis representing time (min). The chromatograms in Fig. 7 and Fig. 8 are all TICs, with the arrow in Fig. 7(a) representing mAG and the arrow in Fig. 8(a) representing IgG.
Since no remaining mAG or IgG was detected from Figures 7(b) and 8(b), it was presumed that each sample formed a water-insoluble polyion complex, similar to BSA and HI.
〔実施例４〕ポリアクリル酸を用いたポリイオンコンプレックス複合体の調製
　ポリリン酸以外のポリアニオンとして平均分子量５０００のポリアクリル酸（ＰＡ５０００．富士フィルム和光純薬（株））または平均分子量２５０００のポリアクリル酸（ＰＡ２５０００．富士フィルム和光純薬（株））を用いて、ＨＩの非水溶性ポリイオンコンプレックス複合体が調製できるか否かを検証した。
　表４に記載した各試薬を記載した順に、記載した量ずつ添加した。また、各試薬の試料液内での最終濃度を、表４のカッコ内に記載した。
１）水
２）１Ｍ　ＮａＰＢ緩衝液 （ｐＨ６．０）
３）濃度２０ｍｇ／ｍＬのＨＩ(１０ｍＭ塩酸に溶解)
４）濃度１００ｍｇ／ｍＬのε‐ＰαＬ水溶液
５）濃度１００ｍｇ／ｍＬのＰＡ５０００水溶液又はＰＡ２５０００水溶液 Example 4 Preparation of polyion complex using polyacrylic acid It was examined whether a water-insoluble polyion complex of HI could be prepared using polyacrylic acid having an average molecular weight of 5000 (PA5000, Fuji Film Wako Pure Chemical Industries, Ltd.) or polyacrylic acid having an average molecular weight of 25000 (PA25000, Fuji Film Wako Pure Chemical Industries, Ltd.) as a polyanion other than polyphosphate.
Each reagent shown in Table 4 was added in the order shown and in the amount shown. The final concentration of each reagent in the sample solution is shown in parentheses in Table 4.
1) Water 2) 1M NaPB buffer (pH 6.0)
3) HI at a concentration of 20 mg/mL (dissolved in 10 mM hydrochloric acid)
4) An aqueous solution of ε-PαL having a concentration of 100 mg/mL. 5) An aqueous solution of PA5000 or PA25000 having a concentration of 100 mg/mL.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　ＰＡ５０００を用いた試料では、得られた混合液は白濁した。しかし、１２０分静置したところ、白色で粘着性の油滴状物質としてポリイオンコンプレックス複合体が得られた。また、ＰＡ２５０００を用いた試料では、得られた混合液は僅かに白濁し、１２０分静置したところ、薄白色で粘着性の油滴状物質としてポリイオンコンプレックス複合体が得られた。
　これら混合液を１５，０００ｒｐｍで１５分間遠心分離した後、その上清をＨＰＬＣ－ＥＳＴ－ＴＯＦ－ＭＳで分析した。クロマトグラムを、図９に（ａ）３ｍｇ／ｍＬのＨＩ標準液と共に、（ｂ）ＰＡ５０００ポリイオンコンプレックス複合体及び（ｃ）ＰＡ２５０００ポリイオンコンプレックス複合体を示す。なお、図９のクロマトグラムは全てＴＩＣであり、（ａ）の矢印はＨＩであり、（ｃ）の＊印はＰＡ２５０００に含有される夾雑物である。
　図９から明らかなように残存のＨＩは検出されなかったことから、ポリリン酸を用いた場合と同じく、ポリアクリル酸においても非水溶性のポリイオンコンプレックス複合体が形成されていると推測された。しかしながら、ポリイオンコンプレックス複合体は他の実施例と異なり結晶性物質とはならず油滴状物質を形成することから、ポリイオンコンプレックス複合体の使用法、用途等は限定されると考えられた。 In the sample using PA5000, the mixture became cloudy. However, after standing for 120 minutes, a polyion complex was obtained as a white, sticky oil droplet-like substance. In the sample using PA25000, the mixture became slightly cloudy, but after standing for 120 minutes, a polyion complex was obtained as a pale white, sticky oil droplet-like substance.
The mixture was centrifuged at 15,000 rpm for 15 minutes, and the supernatant was analyzed by HPLC-EST-TOF-MS. Chromatograms are shown in FIG. 9 for (a) 3 mg/mL HI standard solution, (b) PA5000 polyion complex, and (c) PA25000 polyion complex. All chromatograms in FIG. 9 are TIC, the arrow in (a) indicates HI, and the * mark in (c) indicates impurities contained in PA25000.
As is clear from Fig. 9, no residual HI was detected, and therefore it was presumed that a water-insoluble polyion complex was formed in polyacrylic acid as in the case of using polyphosphoric acid. However, unlike the other examples, the polyion complex did not become a crystalline substance but formed an oily substance, and therefore the usage and uses of the polyion complex were considered to be limited.
〔実施例５〕ＢＳＡポリイオンコンプレックス複合体のｐＨ応答溶解性と生化学的安定性（１）ｐＨ応答溶解性　
　実施例１と同様に試料１－１と同じ混合液を作成し、１２０分間静置した。その後、水酸化ナトリウム水溶液を添加し、各試料のｐＨを３、５、７及び９にそれぞれ調製した。その結果、ｐＨ３とｐＨ５は白濁状態を維持したが、ｐＨ７及びｐＨ９のＢＳＡポリイオンコンプレックス複合体は、試料１－１の白濁から透明溶液に変化した。
　次いで、ＢＳＡポリイオンコンプレックス複合体のｐＨ応答溶解性を検証するために、各ｐＨの試料を１５０００ｒｐｍで１５分間遠心分離し、遠心上清をＨＰＬＣ－ＥＳＩ―ＴＯＦ－ＭＳを用いて分析し、ｐＨの上昇に伴うＢＳＡポリイオンコンプレックス複合体の崩壊によって放出される遊離のＢＳＡを検出した。各ｐＨで得られたクロマトグラムを図１０に示す。図１０のクロマトグラムは全てＴＩＣであり、図１０の１はε‐ＰαＬ、２はＢＳＡである。
　図１０から明らかなように、ｐＨ５以上の条件で遊離のε‐ＰαＬとＢＳＡが検出された。従って、ＢＳＡポリイオンコンプレックス複合体は、酸性条件下で非水溶性を示し、中性以上の条件ではＢＳＡポリイオンコンプレックス複合体が崩壊しＢＳＡが遊離され、ｐＨ応答溶解性を示した。ε‐ＰαＬの酸解離定数（ｐＫａ）は７．６である。よって、中性・弱アルカリ性条件では、ε‐ＰαＬのポリカチオン性が失われるためポリイオンコンプレックス複合体を形成できないと考えられる。 [Example 5] pH-responsive solubility and biochemical stability of BSA polyion complex (1) pH-responsive solubility
The same mixed solution as sample 1-1 was prepared in the same manner as in Example 1 and allowed to stand for 120 minutes. Then, an aqueous sodium hydroxide solution was added to adjust the pH of each sample to 3, 5, 7, and 9. As a result, the samples at pH 3 and pH 5 maintained a cloudy state, but the BSA-polyion complexes at pH 7 and pH 9 changed from the cloudy state of sample 1-1 to a transparent solution.
Next, to verify the pH-responsive solubility of the BSA polyion complex, the samples at each pH were centrifuged at 15,000 rpm for 15 minutes, and the centrifugal supernatant was analyzed by HPLC-ESI-TOF-MS to detect free BSA released by the collapse of the BSA polyion complex with increasing pH. The chromatograms obtained at each pH are shown in Figure 10. All chromatograms in Figure 10 are TICs, with 1 in Figure 10 being ε-PαL and 2 being BSA.
As is clear from Figure 10, free ε-PαL and BSA were detected under conditions of pH 5 or higher. Therefore, the BSA polyion complex was insoluble in water under acidic conditions, and under neutral or higher conditions, the BSA polyion complex was broken down and BSA was released, showing pH-responsive solubility. The acid dissociation constant (pKa) of ε-PαL is 7.6. Therefore, it is considered that under neutral or weakly alkaline conditions, the polycationicity of ε-PαL is lost and therefore a polyion complex cannot be formed.
（２）生化学的安定性
　ＢＳＡポリイオンコンプレックス複合体にタンパク質分解酵素であるペプシンを添加し、ＢＳＡポリイオンコンプレックス複合体の生化学安定性を検証した。
　具体的には、試料１－１に、ペプシン（富士フィルム和光純薬（株））を１００ｍＭ　塩酸に溶解し、ペプシンの最終濃度１５０ｕｎｉｔ／ｍＬになるように添加し、３７℃で保温した。０、５、１５及び３０分後に試料をサンプリングし、水酸化ナトリウム水溶液を添加して混合液のｐＨを９に調製することで酵素反応を停止すると同時に、ＢＳＡポリイオンコンプレックス複合体を崩壊させ、遊離したＢＳＡの濃度をブラッドフォード法（Ｂｕｌｌ．　ｏｆ　Ｙａｍａｇａｔａ　Ｕｎｉｖ．，　Ｅｄｕｃ．　Ｓｃｉ．　１６，４（２０１７））により定量した。また、比較実験として、３ｍｇ／ｍＬ ＢＳＡ水溶液を同様にペプシンで処理して、時間毎に残存するＢＳＡをブラッドフォード法で定量した。
　結果を、図１１にａとして試料１－１を、ｂとして比較実験を示す。なお、図１１の横軸は経過時間（分）、縦軸はＢＳＡ残存率（％）である。図１１のデータは平均±標準偏差（ｎ＝３）を示し、アスタリスク（＊）は、統計的有意差（Ｓｔｕｄｅｎｔ‘ｓ　Ｔ－ｔｅｓｔ）を次の基準で示す。
　　＊ｐ＜０．０５、＊＊ｐ＜０．０１、＊＊＊ｐ＜０．００１
　ポリイオンコンプレックス複合体を形成していないＢＳＡ水溶液（ｂ）においては、速やか（５分以内）にペプシンによって分解された。一方、ＢＳＡポリイオンコンプレックス複合体（ａ）は３０分間の酵素処理をしてもＢＳＡの分解は観察されず、高い生化学的安定性を示した。 (2) Biochemical Stability The biochemical stability of the BSA polyion complex was examined by adding pepsin, a protease, to the BSA polyion complex.
Specifically, pepsin (Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 100 mM hydrochloric acid and added to sample 1-1 so that the final concentration of pepsin was 150 units/mL, and the mixture was incubated at 37°C. Samples were sampled after 0, 5, 15 and 30 minutes, and the pH of the mixture was adjusted to 9 by adding an aqueous sodium hydroxide solution to stop the enzyme reaction. At the same time, the BSA polyion complex was disrupted, and the concentration of the liberated BSA was quantified by the Bradford method (Bull. of Yamagata Univ., Educ. Sci. 16,4 (2017)). In addition, as a comparative experiment, a 3 mg/mL BSA aqueous solution was similarly treated with pepsin, and the remaining BSA was quantified by the Bradford method every hour.
The results are shown in Figure 11, with Sample 1-1 as a and the comparative experiment as b. The horizontal axis of Figure 11 is the elapsed time (minutes), and the vertical axis is the BSA remaining rate (%). The data in Figure 11 are shown as average ± standard deviation (n = 3), and an asterisk (*) indicates statistically significant difference (Student's T-test) according to the following criteria.
*p<0.05, **p<0.01, ***p<0.001
In the aqueous solution of BSA without polyion complex (b), BSA was rapidly decomposed by pepsin (within 5 minutes). On the other hand, in the aqueous solution of BSA with polyion complex (a), no decomposition of BSA was observed even after 30 minutes of enzyme treatment, indicating high biochemical stability.
〔実施例６〕ＨＩポリイオンコンプレックス複合体のｐＨ応答溶解性と生化学的安定性
（１）ｐＨ応答溶解性
　実施例２と同様に試料２－１と同じ混合液を作成し、１２０分間静置した。その後、水酸化ナトリウム水溶液を添加し、各試料のｐＨを３、５、７及び９にそれぞれ調製した。その結果、ｐＨ３、ｐＨ５及びｐＨ７は白濁状態を維持したが、ｐＨ９のＨＩポリイオンコンプレックス複合体は、試料２－１の白濁から透明溶液に変化した。
　次いで、ＨＩポリイオンコンプレックス複合体のｐＨ応答溶解性を検証するために、各ｐＨの試料を１５０００ｒｐｍで１５分間遠心分離し、遠心上清をＨＰＬＣ－ＥＳＩ―ＴＯＦ－ＭＳを用いて分析した。各ｐＨで得られたクロマトグラムを図１２に示す。図１２のクロマトグラムは全てＴＩＣであり、図１２において、１はε‐ＰαＬ、３はＨＩである。
　ｐＨの上昇とともに遊離のＨＩ量が増加し、ｐＨ７以上で遊離のε‐ＰαＬが検出された。従って、ＨＩポリイオンコンプレックス複合体においても、酸性条件下で非水溶性を示し、中性以上の条件ではＨＩポリイオンコンプレックス複合体が崩壊しＨＩが遊離する。すなわち、ＨＩポリイオンコンプレックス複合体はｐＨ応答溶解性を示した。 Example 6 pH-Responsive Solubility and Biochemical Stability of HI Polyion Complex (1) pH-Responsive Solubility As in Example 2, the same mixed solution as Sample 2-1 was prepared and allowed to stand for 120 minutes. Then, an aqueous sodium hydroxide solution was added to adjust the pH of each sample to 3, 5, 7, and 9, respectively. As a result, the samples at pH 3, pH 5, and pH 7 maintained a cloudy state, but the HI polyion complex at pH 9 changed from the cloudy state of Sample 2-1 to a transparent solution.
Next, to verify the pH-responsive solubility of the HI polyion complex, the samples at each pH were centrifuged at 15,000 rpm for 15 minutes, and the centrifugal supernatant was analyzed by HPLC-ESI-TOF-MS. The chromatograms obtained at each pH are shown in Figure 12. All the chromatograms in Figure 12 are TICs, where 1 is ε-PαL and 3 is HI.
The amount of free HI increased with increasing pH, and free ε-PαL was detected at pH 7 or higher. Thus, the HI polyion complex also exhibited water-insolubility under acidic conditions, and disintegrated and released HI under neutral or higher conditions. In other words, the HI polyion complex exhibited pH-responsive solubility.
（２）生化学的安定性
　ＨＩポリイオンコンプレックス複合体にタンパク質分解酵素であるペプシンを添加し、ＨＩポリイオンコンプレックス複合体の生化学安定性を検証した。
　具体的には、試料２－１に、タンパク質分解酵素であるペプシンを１００ｍＭ 塩酸に溶解し、ペプシンの最終濃度１ｕｎｉｔ／ｍＬになるように添加し、３７℃で保温した。０、５、１５及び３０分後に試料をサンプリングし、水酸化ナトリウム水溶液を添加して混合液のｐＨを９に調製することで酵素反応を停止すると同時に、ＨＩポリイオンコンプレックス複合体を崩壊させた。さらにクロロホルムを当量添加し攪拌した後、１５，０００ｒｐｍ、１０分遠心分離し、上清を回収することでペプシンを除去した。遠心上清中の遊離したＨＩの濃度をＨＰＬＣ－ＥＳＴ－ＴＯＦ－ＭＳを用いて定量分析した。また、比較実験として、３ｍｇ／ｍＬ ＨＩ水溶液を同様にペプシンで処理して、時間毎に残存するＨＩをＨＰＬＣ－ＥＳＴ－ＴＯＦ－ＭＳを用いて定量分析した。
　結果を、図１３にａとして試料２－１を、ｂとしてＨＩ水溶液を示す。なお、図１３の横軸は経過時間（分）、縦軸はＨＩ残存率（％）を示す。図１３のデータは平均±標準偏差（ｎ＝３）を示し、アスタリスク（＊）は、統計的有意差（Ｓｔｕｄｅｎｔ‘ｓ　Ｔ－ｔｅｓｔ）を次の基準で示す。
　　＊ｐ＜０．０５、＊＊ｐ＜０．０１、＊＊＊ｐ＜０．００１
　ポリイオンコンプレックス複合体を形成していないＨＩ水溶液（ｂ）においては、ＨＩは速やか（５分以内）にペプシンによって分解された。一方、ＨＩポリイオンコンプレックス複合体（ａ）は３０分間の酵素処理をしてもＨＩの分解は観察されず、高い生化学的安定性を示した。 (2) Biochemical Stability The biochemical stability of the HI polyion complex was examined by adding pepsin, a protease, to the HI polyion complex.
Specifically, pepsin, a protease, was dissolved in 100 mM hydrochloric acid and added to sample 2-1 so that the final concentration of pepsin was 1 unit/mL, and the mixture was incubated at 37°C. After 0, 5, 15, and 30 minutes, the sample was sampled, and an aqueous sodium hydroxide solution was added to adjust the pH of the mixture to 9 to stop the enzyme reaction and simultaneously disintegrate the HI polyion complex. Furthermore, an equivalent amount of chloroform was added and stirred, and then the mixture was centrifuged at 15,000 rpm for 10 minutes, and the supernatant was collected to remove pepsin. The concentration of free HI in the centrifugal supernatant was quantitatively analyzed using HPLC-EST-TOF-MS. In addition, as a comparative experiment, a 3 mg/mL HI aqueous solution was similarly treated with pepsin, and the remaining HI was quantitatively analyzed at each time using HPLC-EST-TOF-MS.
The results are shown in Figure 13, with Sample 2-1 as a and the HI aqueous solution as b. The horizontal axis of Figure 13 indicates the elapsed time (minutes), and the vertical axis indicates the HI residual rate (%). The data in Figure 13 are shown as average ± standard deviation (n = 3), and an asterisk (*) indicates statistically significant difference (Student's T-test) according to the following criteria.
*p<0.05, **p<0.01, ***p<0.001
In the aqueous solution of HI without polyion complex (b), HI was rapidly decomposed by pepsin (within 5 minutes). On the other hand, in the aqueous solution of HI with no polyion complex (a), no decomposition of HI was observed even after 30 minutes of enzyme treatment, indicating high biochemical stability.
〔実施例７〕ｍＡＧポリイオンコンプレックス複合体及びＩｇＧポリイオンコンプレックス複合体のｐＨ応答溶解性
　実施例３に記載する方法にて表３に記載するｍＡＧポリイオンコンプレックス複合体及びＩｇＧポリイオンコンプレックス複合体を調製し、１２０分間静置した。次いで、水酸化ナトリウム水溶液を添加し、ｐＨをそれぞれ８と９に調製し、ｐＨ応答溶解性を検証した。
　ｐＨ８におけるｍＡＧポリイオンコンプレックス複合体、及び、ｐＨ９におけるＩｇＧポリイオンコンプレックス複合体は、いずれもこれらアルカリ性条件にてこれらポリイオンコンプレックス複合体は崩壊し、溶解することが確認された。 [Example 7] pH-responsive solubility of mAG polyion complex and IgG polyion complex The mAG polyion complex and IgG polyion complex shown in Table 3 were prepared by the method described in Example 3 and allowed to stand for 120 minutes. Then, an aqueous sodium hydroxide solution was added to adjust the pH to 8 and 9, respectively, and the pH-responsive solubility was examined.
It was confirmed that the mAG polyion complex at pH 8 and the IgG polyion complex at pH 9 both disintegrated and dissolved under these alkaline conditions.
〔実施例８〕ｍＡＧポリイオンコンプレックス複合体形成後の蛍光活性
　実施例３に記載する方法にて表３に記載するｍＡＧポリイオンコンプレックス複合体の混合液を調製し、１２０分間静置した。水酸化ナトリウム水溶液を加えｐＨを９に調製し、ｍＡＧポリイオンコンプレックス複合体が溶解して崩壊するのに伴って遊離するｍＡＧの蛍光比活性を測定した。また、別途、ｍＡＧポリイオンコンプレックス複合体を調製する直前のｍＡＧの蛍光比活性も同様に測定した。図１４に、ポリイオンコンプレックス複合体形成前後のｍＡＧの蛍光活性を、形成前の活性（左側）を１００％として、形成後の比活性を右側に記載する。データは平均±標準偏差（ｎ＝３）を表す。ポリイオンコンプレックス複合体形成後も約７割の蛍光比活性を示している。
　ポリイオンコンプレックス複合体の形成には、ポリリン酸の添加による強酸性条件が必要であり、ｍＡＧポリイオンコンプレックス複合体の最終ｐＨは１．４である。一般的に、タンパク質やペプチドの多くは強酸性条件下で変性する。緑色蛍光タンパクであるｍＡＧでは、ポリイオンコンプレックス複合体形成後でも約７割程度の比活性を維持している。従って、ポリイオンコンプレックス複合体形成時の強酸環境であってもタンパク質／ペプチドは活性をある程度保つことが示唆され、ポリイオンコンプレックス複合体の崩壊に伴い、活性を保ったタンパク質／ペプチドがリリースされると考えられる。
　以上のことから、ポリイオンコンプレックス複合体は、酸性条件下である胃内環境においてペプシンによる分解を受けることなく通過し、中性・弱塩基性条件である小腸・十二指腸において、そのポリイオンコンプレックス複合体は崩壊することが期待される。さらに、タンパク質やペプチドの生理活性を保ったまま腸内に到達すると考えられ、ポリイオンコンプレックス複合体は有効な経口ドラッグデリバリー法として期待できる。 [Example 8] Fluorescence activity after formation of mAG polyion complex A mixture of mAG polyion complexes shown in Table 3 was prepared by the method described in Example 3, and allowed to stand for 120 minutes. Aqueous sodium hydroxide solution was added to adjust the pH to 9, and the fluorescence specific activity of mAG released as the mAG polyion complexes dissolved and disintegrated was measured. Separately, the fluorescence specific activity of mAG just before preparing the mAG polyion complexes was also measured in the same manner. In FIG. 14, the fluorescence activity of mAG before and after the formation of the polyion complexes is shown on the right side, with the activity before the formation (left side) being 100%. The data show the average ± standard deviation (n = 3). After the formation of the polyion complexes, the fluorescence specific activity is about 70%.
The formation of the polyion complex requires a strong acidic condition by adding polyphosphate, and the final pH of the mAG polyion complex is 1.4. Generally, many proteins and peptides are denatured under strong acidic conditions. The green fluorescent protein mAG maintains about 70% of its specific activity even after the formation of the polyion complex. This suggests that the protein/peptide retains some activity even in the strong acidic environment during the formation of the polyion complex, and that the active protein/peptide is released with the collapse of the polyion complex.
From the above, it is expected that the polyion complex passes through the stomach environment under acidic conditions without being decomposed by pepsin, and disintegrates in the small intestine and duodenum under neutral or weakly alkaline conditions. Furthermore, it is thought that the proteins and peptides reach the intestine while retaining their physiological activity, and the polyion complex is expected to be an effective oral drug delivery method.
〔実施例９〕健常マウスへのＨＩポリイオンコンプレックス複合体の腹腔内投与と血糖値効果作用検証
　マウスは近交系Ｃ５７ＢＬ／６Ｊ（６週齢、オス、平均体重２０ｇ。ニノックスラボサプライ（株））を、１２時間毎の明暗周期、２２±１℃の室温、４０―７０％湿度の条件下、餌と水を自由に摂取可能な環境で購入から１週間以上予備飼育し、実験に使用した。（以降の実施例のマウスも同様である。）
　健常マウスを３時間絶食させた後、尻尾の静脈から採血し、ニプロスタットストリップＸＰ３（ニプロ（株））を用いて薬物投与前の血糖値を測定した。実施例２と同様に試料２－１と同じＨＩポリイオンコンプレックス複合体を含む懸濁液を調製し、滅菌した２００ｍＭ　ＮａＰＢ緩衝液（ｐＨ６．０）で適宜希釈した後、複合体を含む懸濁液のｐＨが２以下であることを確認した。また、２０ｍｇ／ｍＬ　ＨＩ溶液（１０ｍＭ 塩酸に溶解）と試料１－１（表１）のε‐ＰαＬ／ポリリン酸混合液を調製し、滅菌した２００ｍＭ　ＮａＰＢ（ｐＨ６．０）で適宜希釈し投与実験に用いた。 [Example 9] Intraperitoneal administration of HI polyion complex to healthy mice and verification of its effect on blood glucose level The mice used in the experiment were inbred C57BL/6J mice (6 weeks old, male, average weight 20g, Ninox Lab Supply Co., Ltd.), which were kept for more than one week after purchase in an environment with a 12-hour light-dark cycle, a room temperature of 22±1°C, and a humidity of 40-70%, with food and water available ad libitum. (The same applies to the mice in the following examples.)
After fasting for 3 hours, healthy mice were blood-drawn from the tail vein, and blood glucose levels before drug administration were measured using Niprostat Strip XP3 (Nipro Corporation). As in Example 2, a suspension containing the same HI polyion complex as sample 2-1 was prepared, appropriately diluted with sterilized 200 mM NaPB buffer (pH 6.0), and the pH of the suspension containing the complex was confirmed to be 2 or less. In addition, a 20 mg/mL HI solution (dissolved in 10 mM hydrochloric acid) and a mixture of ε-PαL/polyphosphate of sample 1-1 (Table 1) were prepared, appropriately diluted with sterilized 200 mM NaPB (pH 6.0), and used in the administration experiment.
　ＨＩポリイオンコンプレックス複合体（試料２－１）の投与においては、表５に記載するように、ＨＩ、ε‐ＰαＬ及びポリリン酸の投与量がそれぞれ１０ＩＵ／ｋｇ、０．６７ｍｇ／ｋｇ、６．７ｍｇ／ｋｇになるように健常マウスに腹腔内投与した。ＨＩ溶液は、ＨＩ投与量が１０ＩＵ／ｋｇになるように腹腔内投与し、ε‐ＰαＬ／ポリリン酸混合液（表１。試料１－６）はε‐ＰαＬとポリリン酸の投与量がそれぞれ０．６７ｍｇ／ｋｇ、６．７ｍｇ／ｋｇになるように健常マウスに腹腔内投与した（表５）。ＨＩポリイオンコンプレックス複合体、ＨＩ溶液、ε‐ＰαＬ／ポリリン酸混合液の投与から３０，６０及び１２０分後に尻尾の静脈から採血し、ニプロスタットストリップＸＰ３を用いて血液内の血糖値を測定した。 In the administration of the HI polyion complex (sample 2-1), the doses of HI, ε-PαL, and polyphosphate were intraperitoneally administered to healthy mice at 10 IU/kg, 0.67 mg/kg, and 6.7 mg/kg, respectively, as shown in Table 5. The HI solution was intraperitoneally administered at a dose of 10 IU/kg, and the ε-PαL/polyphosphate mixture (Table 1, sample 1-6) was intraperitoneally administered to healthy mice at doses of 0.67 mg/kg and 6.7 mg/kg, respectively (Table 5). Blood was collected from the tail vein 30, 60, and 120 minutes after administration of the HI polyion complex, HI solution, and ε-PαL/polyphosphate mixture, and the blood glucose level in the blood was measured using Niprostat Strip XP3.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　各液の投与における血糖値の時間変化（横軸）を、薬物投与前の血糖値を基準（１００％）として図１５に示す。図１５において、（Ｉ）はＨＩポリイオンコンプレックス複合体、（ＩＩ）はＨＩ溶液、（ＩＩＩ）はε‐ＰαＬ／ポリリン酸混合液を示す。データは平均±標準偏差（ｎ＝４）を示し、アスタリスク（＊）は、ε‐ＰαＬ／ポリリン酸混合液（ＩＩＩ）投与との統計的有意差（Ｏｎｅ－ｗａｙ　ＡＮＯＶＡ、Ｔｕｋｅｙ‘ｓ　ｔｅｓｔ）を次の基準で示す。
　　＊ｐ＜０．０５、＊＊ｐ＜０．０１、＊＊＊ｐ＜０．００１
　結果として、比較実験として用いたＨＩ溶液（ＩＩ）と同じく、ＨＩポリイオンコンプレックス複合体（Ｉ）は投与後３０分、６０分、１２０分で血糖値降下を観察した。またＨＩが含まれないポリイオンコンプレックス複合体の比較実験であるε‐ＰαＬ／ポリリン酸混合液（ＩＩＩ）は、血糖値降下作用を示さなかった。
　以上のことから、ε‐ＰαＬとポリリン酸に血糖値降下作用は無く、ＨＩポリイオンコンプレックス複合体（Ｉ）の投与実験で観察された血糖値降下作用は、その混合液に含まれるＨＩの薬効によるものであることが確認された。ＨＩポリイオンコンプレックス複合体（Ｉ）が血糖値降下作用の薬効を示すためには、非水溶性のＨＩポリイオンコンプレックス複合体が崩壊しＨＩが遊離する必要がある。従って、ＨＩポリイオンコンプレックス複合体は腹腔内の中性環境で崩壊するｐＨ応答溶解性を示したと考えられた。また、本実施例によって、ＨＩポリイオンコンプレックス複合体から遊離したＨＩは、血糖値降下ホルモンとしての生理機能を十分に維持していることが判明した。 The change in blood glucose level over time (horizontal axis) after administration of each solution is shown in Figure 15, with the blood glucose level before drug administration taken as the standard (100%). In Figure 15, (I) indicates the HI polyion complex, (II) the HI solution, and (III) the ε-PαL/polyphosphate mixed solution. Data are shown as mean ± standard deviation (n = 4), and an asterisk (*) indicates a statistically significant difference (One-way ANOVA, Tukey's test) from administration of the ε-PαL/polyphosphate mixed solution (III) according to the following criteria.
*p<0.05, **p<0.01, ***p<0.001
As a result, the HI polyion complex (I) was observed to lower blood glucose levels 30, 60, and 120 minutes after administration, as was the HI solution (II) used in the comparative experiment. In addition, the ε-PαL/polyphosphate mixture (III), which is a comparative experiment of a polyion complex that does not contain HI, did not show any blood glucose lowering effect.
From the above, it was confirmed that ε-PαL and polyphosphate have no blood glucose lowering effect, and the blood glucose lowering effect observed in the administration experiment of HI polyion complex (I) is due to the medicinal effect of HI contained in the mixture. In order for HI polyion complex (I) to show the medicinal effect of blood glucose lowering effect, the water-insoluble HI polyion complex needs to break down and HI is released. Therefore, it was thought that the HI polyion complex showed pH-responsive solubility that breaks down in the neutral environment in the abdominal cavity. In addition, this example revealed that HI released from the HI polyion complex fully maintains its physiological function as a blood glucose lowering hormone.
〔実施例１０〕健常マウスへのＨＩポリイオンコンプレックス複合体の経口投与と血糖値効果作用検証
（１）投与量１００ＩＵ／ｋｇ
　健常マウスを３時間絶食させた後、尻尾の静脈から採血し、ニプロスタットストリップＸＰ３を用いて薬物投与前の血糖値を測定した。実施例２と同様にして試料２－１と同じ組成のＨＩポリイオンコンプレックス複合体を含む懸濁液を調製し、滅菌した２００ｍＭ　ＮａＰＢ緩衝液（ｐＨ６．０）で適宜希釈した後、複合体を含む懸濁液のｐＨが２以下であることを確認した。また、２０ｍｇ／ｍＬ　ＨＩ溶液（１０ｍＭ 塩酸に溶解）と試料１－６（表１）のε‐ＰαＬ／ポリリン酸混合液を調製し、滅菌した２００ｍＭ　ＮａＰＢ緩衝液（ｐＨ６．０）で適宜希釈し投与実験に用いた。
　ＨＩポリイオンコンプレックス複合体（試料２－１）の投与においては、表６に記載するようにＨＩ、ε‐ＰαＬ及びポリリン酸の投与量がそれぞれ１００ＩＵ／ｋｇ、６．７ｍｇ／ｋｇ、６７ｍｇ／ｋｇになるように健常マウスに経口投与した。ＨＩ溶液は、ＨＩ投与量が１００ＩＵ／ｋｇになるように経口投与し、ε‐ＰαＬ／ポリリン酸混合液（表１。試料１－６）はε‐ＰαＬとポリリン酸の投与量がそれぞれ６．７ｍｇ／ｋｇ、６７ｍｇ／ｋｇになるように健常マウスに経口投与した。各溶液を経口投与した後、３０，６０、１２０及び１８０分後に尻尾の静脈から採血し、ニプロスタットストリップＸＰ３を用いて血液内の血糖値を測定した。 [Example 10] Oral administration of HI polyion complex to healthy mice and verification of its effect on blood glucose level (1) Dose: 100 IU/kg
After fasting for 3 hours, healthy mice were blood-drawn from the tail vein, and blood glucose levels before drug administration were measured using Niprostat Strip XP3. A suspension containing the same composition of HI polyion complex as sample 2-1 was prepared in the same manner as in Example 2, and appropriately diluted with sterilized 200 mM NaPB buffer (pH 6.0), and the pH of the suspension containing the complex was confirmed to be 2 or less. In addition, a 20 mg/mL HI solution (dissolved in 10 mM hydrochloric acid) and a mixture of ε-PαL/polyphosphate of sample 1-6 (Table 1) were prepared, appropriately diluted with sterilized 200 mM NaPB buffer (pH 6.0), and used in the administration experiment.
In the administration of the HI polyion complex (sample 2-1), the doses of HI, ε-PαL, and polyphosphate were orally administered to healthy mice at 100 IU/kg, 6.7 mg/kg, and 67 mg/kg, respectively, as shown in Table 6. The HI solution was orally administered to healthy mice at a dose of 100 IU/kg, and the ε-PαL/polyphosphate mixture (Table 1, sample 1-6) was orally administered to healthy mice at doses of 6.7 mg/kg and 67 mg/kg, respectively. After the oral administration of each solution, blood was collected from the tail vein 30, 60, 120, and 180 minutes later, and the blood glucose level in the blood was measured using Niprostat Strip XP3.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
　各液の投与における血糖値の時間変化（横軸）を、薬物投与前の血糖値を基準（１００％）として図１６に示す。図１６において、（Ｉ）はＨＩポリイオンコンプレックス複合体、（ＩＩ）はＨＩ溶液、（ＩＩＩ）はε‐ＰαＬ／ポリリン酸混合液を示す。データは平均±標準偏差（ｎ＝６）を示し、アスタリスク（＊）は、ＨＩ溶液投与との統計的有意差（Ｏｎｅ－ｗａｙ　ＡＮＯＶＡ、Ｔｕｋｅｙ‘ｓ　ｔｅｓｔ）を次の基準で示す。
　　＊ｐ＜０．０５、＊＊ｐ＜０．０１、＊＊＊ｐ＜０．００１
　ＨＩはペプチドホルモンである。従って、消化管内の各種タンパク質分解酵素によって分解されるため、経口投与では薬効を示さない。実際に、本実施例においても健常マウスにＨＩ溶液（ＩＩ）を経口投与しても血糖値降下作用は観察されなかった。ＨＩの消化管吸収を達成するには、胃内部の酸性条件下で非水溶性を示し、腸管内の中性または弱アルカリ性条件下で溶解するｐＨ応答溶解性の製剤が望ましい。さらに、腸管内にてＨＩの吸収を促進する腸管吸収促進剤の併用が望ましく、ＨＩポリイオンコンプレックス複合体は、その特徴を兼ね備えていると期待された。そこで、ＨＩ投与量として１００ＩＵ／ｋｇとなるようにＨＩポリイオンコンプレックス複合体（試料２－１）を健常マウスに経口投与したところ、ＨＩ溶液（１００ＩＵ／ｋｇ）の投与と比較して血糖値の降下傾向が観察され、投与後６０分と１８０分においては有意な血糖値降下作用を認めた。これは、ＨＩポリイオンコンプレックス複合体が胃内部の酸性条件下で非水溶性を示し、腸管内の中性または弱アルカリ性条件下でＨＩポリイオンコンプレックス複合体が崩壊（溶解）し、ＨＩが遊離するとともにε‐ＰαＬが腸管吸収促進剤として機能し、ＨＩが腸管吸収されたと考えられた。他方、ε‐ＰαＬ／ポリリン酸混合液（試料１－６）（ＩＩＩ）の経口投与では血糖値降下作用が全く観察されなかったことから、ε‐ＰαＬとポリリン酸は、経口投与においても血糖値降下作用の薬効を示さないことが判明した。 The change in blood glucose level over time (horizontal axis) after administration of each solution is shown in Figure 16, with the blood glucose level before drug administration taken as the standard (100%). In Figure 16, (I) indicates the HI polyion complex, (II) the HI solution, and (III) the ε-PαL/polyphosphate mixture. Data are shown as mean ± standard deviation (n = 6), and asterisks (*) indicate statistically significant differences from administration of the HI solution (One-way ANOVA, Tukey's test) according to the following criteria.
*p<0.05, **p<0.01, ***p<0.001
HI is a peptide hormone. Therefore, since it is decomposed by various proteolytic enzymes in the digestive tract, oral administration does not show any medicinal effect. In fact, even in this example, no blood glucose lowering effect was observed when HI solution (II) was orally administered to healthy mice. In order to achieve digestive absorption of HI, a pH-responsive soluble preparation that is water-insoluble under acidic conditions in the stomach and dissolves under neutral or weakly alkaline conditions in the intestine is desirable. Furthermore, it is desirable to use an intestinal absorption promoter in combination to promote absorption of HI in the intestine, and it was expected that the HI polyion complex has both of these characteristics. Therefore, when HI polyion complex (sample 2-1) was orally administered to healthy mice so that the HI dosage was 100 IU/kg, a tendency to lower blood glucose levels was observed compared to administration of HI solution (100 IU/kg), and a significant blood glucose lowering effect was observed 60 minutes and 180 minutes after administration. This is believed to be because the HI polyion complex is water-insoluble under acidic conditions in the stomach, and the HI polyion complex disintegrates (dissolves) under neutral or weakly alkaline conditions in the intestinal tract, liberating HI, while ε-PαL functions as an intestinal absorption promoter, resulting in HI absorption through the intestinal tract. On the other hand, no blood glucose lowering effect was observed when the ε-PαL/polyphosphate mixture (sample 1-6) (III) was orally administered, demonstrating that ε-PαL and polyphosphate do not exhibit any medicinal effect of lowering blood glucose levels, even when administered orally.
　ＨＩの経口投与製剤は実用化に至っていないが、これまでに、消化酵素の阻害剤の併用、腸溶性カプセル剤、ナノ粒子（ミセル）などが開発されている。いずれの手法も、ＨＩの腹腔内投与と比較してより多くの投与量を必要とする（通常、３０から６０ＩＵ／ｋｇ程度）。ＨＩポリイオンコンプレックス複合体（試料２－１）においても、ＨＩ投与量として１００ＩＵ／ｋｇで血糖値の降下傾向が観察したことから、より有意な血糖値降下作用を観察するには１００ＩＵ／ｋｇ以上の投与量が必要であると推測された。 Although oral administration formulations of HI have not yet been put to practical use, combination use with digestive enzyme inhibitors, enteric capsules, nanoparticles (micelles), etc. have been developed. All of these methods require a larger dosage compared to intraperitoneal administration of HI (usually around 30 to 60 IU/kg). A tendency for blood glucose levels to decrease was also observed with the HI polyion complex (sample 2-1) at an HI dosage of 100 IU/kg, so it was inferred that a dosage of 100 IU/kg or more would be necessary to observe a more significant blood glucose lowering effect.
（２）投与量３００ＩＵ／ｋｇ
　ＨＩポリイオンコンプレックス複合体において、より有意な血糖値降下作用を観察するために、ＨＩ投与量として３００ＩＵ／ｋｇになるように健常マウスに経口投与した。
　具体的には、健常マウスを３時間絶食させた後、尻尾の静脈から採血し、ニプロスタットストリップＸＰ３を用いて薬物投与前の血糖値を測定した。実施例２と同様にして試料２－１と同じ組成のＨＩポリイオンコンプレックス複合体を含む懸濁液を調製し、滅菌した２００ｍＭ　ＮａＰＢ緩衝液（ｐＨ６．０）で適宜希釈した後、複合体を含む懸濁液のｐＨが２以下であることを確認した。また、２０ｍｇ／ｍＬ　ＨＩ溶液（１０ｍＭ 塩酸に溶解）とε‐ＰαＬ／ポリリン酸混合液（表１。試料１－６）、ポリリン酸／ＨＩ混合溶液（表２。試料２－６）及びε‐ＰαＬ／ＨＩ混合液（表２。試料２－７）を作成し、滅菌した２００ｍＭ　ＮａＰＢ緩衝液（ｐＨ６．０）で適宜希釈し投与実験に用いた。
　ＨＩポリイオンコンプレックス複合体、ＨＩ溶液、ε‐ＰαＬ／ポリリン酸混合液（表１。試料１－６）、ポリリン酸／ＨＩ混合溶液（表２。試料２－６）及びε‐ＰαＬ／ＨＩ混合液（表２。試料２－７）の投与においては、ＨＩ、ε‐ＰαＬ、ポリリン酸の投与量がそれぞれ表７に記載する量となるように経口投与した。各サンプル溶液を経口投与した後、３０、６０、１２０、及び１８０分後に尻尾の静脈から採血し、ニプロスタットストリップＸＰ３を用いて血糖値を測定した。 (2) Dosage: 300 IU/kg
In order to observe a more significant blood glucose lowering effect of the HI polyion complex, the HI was orally administered to healthy mice at a dose of 300 IU/kg.
Specifically, healthy mice were fasted for 3 hours, blood was collected from the tail vein, and blood glucose levels before drug administration were measured using Niprostat Strip XP3. A suspension containing an HI polyion complex having the same composition as sample 2-1 was prepared in the same manner as in Example 2, and appropriately diluted with sterilized 200 mM NaPB buffer (pH 6.0), and the pH of the suspension containing the complex was confirmed to be 2 or less. In addition, a 20 mg/mL HI solution (dissolved in 10 mM hydrochloric acid), an ε-PαL/polyphosphate mixture (Table 1, sample 1-6), a polyphosphate/HI mixture (Table 2, sample 2-6), and an ε-PαL/HI mixture (Table 2, sample 2-7) were prepared, appropriately diluted with sterilized 200 mM NaPB buffer (pH 6.0), and used in the administration experiment.
In administering the HI polyion complex, HI solution, ε-PαL/polyphosphate mixed solution (Table 1, Samples 1-6), polyphosphate/HI mixed solution (Table 2, Samples 2-6), and ε-PαL/HI mixed solution (Table 2, Samples 2-7), HI, ε-PαL, and polyphosphate were orally administered in the amounts shown in Table 7. After oral administration of each sample solution, blood was collected from the tail vein 30, 60, 120, and 180 minutes later, and blood glucose levels were measured using Niprostat Strip XP3.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　各液の投与における血糖値の時間変化（横軸）を、薬物投与前の血糖値を基準（１００％）として図１７に示す。図１７において、（Ｉ）はＨＩポリイオンコンプレックス複合体、（ＩＩ）はε‐ＰαＬ／ポリリン酸混合液、（ＩＩＩ）はε‐ＰαＬ／ＨＩ混合液、（ＩＶ）はポリリン酸／ＨＩ混合液及び（Ｖ）はＨＩ溶液を示す。データは平均±標準偏差（ｎ＝６）を示し、アスタリスク（＊）は、ＨＩ溶液（Ｖ）投与との統計的有意差（Ｏｎｅ－ｗａｙ　ＡＮＯＶＡ、Ｔｕｋｅｙ‘ｓ　ｔｅｓｔ）を次の基準で示す。
　　＊ｐ＜０．０５、＊＊ｐ＜０．０１、＊＊＊ｐ＜０．００１
　図１７から次のことが観察された。ＨＩポリイオンコンプレックス複合体（Ｉ）では、投与後３０～１２０分後において顕著かつ有意な血糖値降下作用を観察した。他方、比較実験に用いたε‐ＰαＬ／ポリリン酸混合液（表１の試料１－６）（ＩＩ）、ポリリン酸／ＨＩ混合溶液（表２の試料２－６）（ＩＶ）及びＨＩ溶液（Ｖ）には、血糖値降下作用は観察されなかった。しかし、ε‐ＰαＬ／ＨＩ混合液（表２の試料２－７）（ＩＩＩ）の経口投与は血糖値の降下傾向が観察され、投与後６０分では有意な血糖値降下作用を認めた。以上のことから、ε‐ＰαＬがＨＩの腸管吸収性に重要であることが判明した。 The time course of blood glucose levels (horizontal axis) following administration of each solution is shown in Figure 17, with the blood glucose level before drug administration taken as the reference (100%). In Figure 17, (I) represents the HI polyion complex, (II) represents the ε-PαL/polyphosphate mixed solution, (III) represents the ε-PαL/HI mixed solution, (IV) represents the polyphosphate/HI mixed solution, and (V) represents the HI solution. Data are shown as mean ± standard deviation (n = 6), and an asterisk (*) indicates a statistically significant difference (One-way ANOVA, Tukey's test) from administration of HI solution (V) according to the following criteria.
*p<0.05, **p<0.01, ***p<0.001
The following was observed from FIG. 17. In the case of the HI polyion complex (I), a remarkable and significant blood glucose level lowering effect was observed 30 to 120 minutes after administration. On the other hand, in the ε-PαL/polyphosphate mixed solution (sample 1-6 in Table 1) (II), the polyphosphate/HI mixed solution (sample 2-6 in Table 2) (IV) and the HI solution (V) used in the comparative experiment, no blood glucose level lowering effect was observed. However, in the case of the oral administration of the ε-PαL/HI mixed solution (sample 2-7 in Table 2) (III), a tendency to lower blood glucose level was observed, and a significant blood glucose level lowering effect was observed 60 minutes after administration. From the above, it was found that ε-PαL is important for the intestinal absorption of HI.
　前述したように、近年、バイオ医薬の消化管上皮細胞吸収性を改善させる手法として細胞透過性ペプチドの利用が注目を集めている。細胞透過性ペプチドを用いてＨＩの消化管上皮細胞吸収性を向上させた成功例も報告されている。中でも、ポリカチオン性の細胞透過性ペプチドは優れた消化管上皮細胞吸収性を示し、その実用化が期待されているが、細胞透過性ペプチドは一般的に化学合成で供給され、その高い合成コストの問題は解決されていない。他方、ε‐ＰαＬは微生物によって生産されるポリカチオン性のイソペプチドであり、各種タンパク質分解酵素にも耐性を示し、かつ、優れた細胞膜透過性を示す。しかし、消化管吸収促進剤としての機能については不明のままであった。本発明では、ε‐ＰαＬの消化管吸収促進剤としての機能が証明され、また、ε‐ＰαＬがポリリン酸とともにＨＩとポリイオンコンプレックス複合体を形成するポリカチオン化合物として重要であり、また、そのポリイオンコンプレックス複合体のｐＨ応答溶解性においても重要であることが明らかになった。 As mentioned above, in recent years, the use of cell-permeable peptides has been attracting attention as a method for improving the digestive epithelial cell absorbability of biopharmaceuticals. Successful cases have been reported in which cell-permeable peptides were used to improve the digestive epithelial cell absorbability of HI. Among them, polycationic cell-permeable peptides show excellent digestive epithelial cell absorbability and are expected to be put to practical use, but cell-permeable peptides are generally supplied by chemical synthesis, and the problem of their high synthesis costs has not been solved. On the other hand, ε-PαL is a polycationic isopeptide produced by microorganisms, which shows resistance to various proteolytic enzymes and shows excellent cell membrane permeability. However, its function as a digestive absorption enhancer has remained unknown. In the present invention, the function of ε-PαL as a digestive absorption enhancer has been proven, and it has also been revealed that ε-PαL is important as a polycationic compound that forms a polyion complex with HI together with polyphosphate, and is also important in the pH-responsive solubility of the polyion complex.
〔実施例１１〕ストレプトゾトシン誘発性Ｉ型糖尿病マウスへのＨＩポリイオンコンプレックス複合体の経口投与
（１）ストレプトゾトシン誘発性Ｉ型糖尿病マウス（ＳＴＺ糖尿病マウス）の作製
　健常マウスを３時間絶食させた後、尻尾の静脈から採血し、ニプロスタットストリップＸＰ３を用いて薬物投与前の血糖値を測定した。ストレプトゾトシン（「ＳＴＺ」と略す。富士フィルム和光純薬（株））を５ｍＭクエン酸ナトリム緩衝液（ｐＨ４．５）に溶解し、適宜希釈した後、濾過除菌して投与実験に用いた。１日に１回１００ｍｇ／ｋｇを３日間、腹腔内に反復投与した（合計３００ｍｇ／ｋｇ）。ＳＴＺ投与から１週間以上飼育したＳＴＺ投与マウスを３時間絶食させた後、血糖値を測定し、２３０ｍｇ／ｄＬ以上の血糖値を示したマウスをＳＴＺ糖尿病マウスとして実験に使用した。 [Example 11] Oral administration of HI polyion complex to streptozotocin-induced type I diabetes mice (1) Preparation of streptozotocin-induced type I diabetes mice (STZ diabetic mice) After fasting for 3 hours, blood was collected from the tail vein, and the blood glucose level before drug administration was measured using Niprostat Strip XP3. Streptozotocin (abbreviated as "STZ"; Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 5 mM sodium citrate buffer (pH 4.5), appropriately diluted, filtered, and sterilized for use in the administration experiment. 100 mg/kg was repeatedly administered intraperitoneally once a day for 3 days (total 300 mg/kg). STZ-administered mice that had been kept for more than one week after STZ administration were fasted for 3 hours, and then blood glucose levels were measured. Mice that showed blood glucose levels of 230 mg/dL or more were used in the experiment as STZ diabetic mice.
（２）ＳＴＺ糖尿病マウスへのＨＩポリイオンコンプレックス複合体の経口投与
　ＳＴＺ糖尿病マウスを３時間絶食させた後、尻尾の静脈から採血し、ニプロスタットストリップＸＰ３を用いて薬物投与前の血糖値を測定した。実施例２と同様にして試料２－zのＨＩポリイオンコンプレックス複合体を含む懸濁液を調製し、滅菌した２００ｍＭ　ＮａＰＢ緩衝液（ｐＨ６．０）で適宜希釈した後、複合体を含む懸濁液のｐＨが２以下であることを確認した。また、２０ｍｇ／ｍＬ　ＨＩ溶液（１０ｍＭ 塩酸に溶解）を調製し、滅菌した２００ｍＭ　ＮａＰＢ緩衝液（ｐＨ６．０）で適宜希釈し投与実験に用いた。
　ＨＩポリイオンコンプレックス複合体の投与においては、表８に記載するようにＨＩ、ε‐ＰαＬ、ポリリン酸の投与量がそれぞれ１００ＩＵ／ｋｇ、６．７ｍｇ／ｋｇ、６７ｍｇ／ｋｇになるようにＳＴＺ糖尿病マウスに経口投与した（ＨＩ１００ＩＵ／ｋｇ投与群）。また同様に、表９に記載するように、ＨＩ、ε‐ＰαＬ、ポリリン酸の投与量がそれぞれ３００ＩＵ／ｋｇ、２０ｍｇ／ｋｇと２００ｍｇ／ｋｇになるようにＳＴＺ糖尿病マウスに経口投与した（ＨＩ３００ＩＵ／ｋｇ投与群）。ＨＩ溶液の投与においては、ＨＩ投与量が１００ＩＵ／ｋｇ（表８）あるいは３００ＩＵ／ｋｇ（表９）になるように経口投与した。各サンプル溶液を経口投与した後、３０、６０、１２０、１８０分後に尻尾の静脈から採血し、ニプロスタットストリップＸＰ３を用いて血糖値を測定した。 (2) Oral administration of HI polyion complex to STZ diabetic mice After fasting for 3 hours, blood was collected from the tail vein, and the blood glucose level before drug administration was measured using Niprostat Strip XP3. A suspension containing the HI polyion complex of sample 2-z was prepared in the same manner as in Example 2, and appropriately diluted with sterilized 200 mM NaPB buffer (pH 6.0), and the pH of the suspension containing the complex was confirmed to be 2 or less. In addition, a 20 mg/mL HI solution (dissolved in 10 mM hydrochloric acid) was prepared, and appropriately diluted with sterilized 200 mM NaPB buffer (pH 6.0) and used in the administration experiment.
In the administration of the HI polyion complex, the doses of HI, ε-PαL, and polyphosphate were orally administered to STZ diabetic mice at 100 IU/kg, 6.7 mg/kg, and 67 mg/kg, respectively, as shown in Table 8 (HI 100 IU/kg administration group). Similarly, the doses of HI, ε-PαL, and polyphosphate were orally administered to STZ diabetic mice at 300 IU/kg, 20 mg/kg, and 200 mg/kg, respectively, as shown in Table 9 (HI 300 IU/kg administration group). In the administration of the HI solution, the HI dose was orally administered at 100 IU/kg (Table 8) or 300 IU/kg (Table 9). After orally administering each sample solution, blood was collected from the tail vein 30, 60, 120, and 180 minutes later, and blood glucose levels were measured using Niprostat Strip XP3.
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
　各液の投与における血糖値の時間変化（横軸）を、薬物投与前の血糖値を基準（１００％）としてＨＩ１００ＩＵ／ｋｇ投与群を図１８に、ＨＩ３００ＩＵ／ｋｇ投与群を図１９に示す。なお、図１８及び図１９において、（Ｉ）はＨＩポリイオンコンプレックス複合体、（ＩＩ）はＨＩ溶液を示す。データは平均±標準偏差（図１８はｎ＝６、図１９はｎ＝１１）を示し、アスタリスク（＊）は、ＨＩ溶液投与との統計的有意差（Ｓｔｕｄｅｎｔ‘ｓ　ｔ－ｔｅｓｔ）を次の基準で示す。
　　＊ｐ＜０．０５、＊＊ｐ＜０．０１、＊＊＊ｐ＜０．００１
　ＳＴＺ糖尿病マウスにＨＩ溶液（ＩＩ）を経口投与したところ、どちらの投与群も健常マウスと同じく、血糖値降下作用は観察されなかった。しかし、ＨＩポリイオンコンプレックス複合体をＨＩ投与量として１００ＩＵ／ｋｇで経口投与した場合は投与後６０分で有意な血糖値降下作用を認め、３００ＩＵ／ｋｇで経口投与した場合は、投与後３０、６０、１２０、１８０分で顕著かつ有意な血糖値降下作用を示した。
　ＳＴＺ糖尿病マウスはＨＩ分泌能が低下している。従って、ＨＩポリイオンコンプレックス複合体の投与によってマウス本来のインスリン分泌が促進されたのではなく、ＨＩポリイオンコンプレックス複合体から遊離したＨＩが直接作用し、マウスの血糖値を降下させたと考えられた。 The time course of blood glucose levels (horizontal axis) after administration of each solution is shown in FIG. 18 for the HI 100 IU/kg administration group and in FIG. 19 for the HI 300 IU/kg administration group, with the blood glucose level before drug administration taken as the standard (100%). In FIG. 18 and FIG. 19, (I) indicates the HI polyion complex, and (II) indicates the HI solution. Data are shown as mean ± standard deviation (n=6 in FIG. 18, n=11 in FIG. 19), and an asterisk (*) indicates a statistically significant difference (Student's t-test) from administration of the HI solution according to the following criteria.
*p<0.05, **p<0.01, ***p<0.001
When STZ-diabetic mice were orally administered HI solution (II), no blood glucose lowering effect was observed in either administration group, as in healthy mice. However, when HI polyion complex was orally administered at a dose of 100 IU/kg, a significant blood glucose lowering effect was observed 60 minutes after administration, and when HI polyion complex was orally administered at a dose of 300 IU/kg, a remarkable and significant blood glucose lowering effect was observed 30, 60, 120, and 180 minutes after administration.
STZ-induced diabetic mice have a reduced ability to secrete HI. Therefore, it was believed that the administration of the HI polyion complex did not promote the inherent insulin secretion of the mice, but that the HI released from the HI polyion complex acted directly to lower the blood glucose level of the mice.
１０：細胞膜透過性ポリカチオンイソペプチド、２０：アニオン性ポリマー、３０：生理活性物質、４０：ポリイオンコンプレックス複合体、５０：胃内環境、６０：腸内環境 10: Cell membrane permeable polycation isopeptide, 20: Anionic polymer, 30: Biologically active substance, 40: Polyion complex, 50: Gastric environment, 60: Intestinal environment

Claims (15)

  1. 　細胞膜透過性ポリカチオンイソペプチドと、
    　生理活性物質と、
    を含む経口医薬組成物。     A cell membrane-permeable polycationic isopeptide;
    A biologically active substance;
    13. An oral pharmaceutical composition comprising:
  2. 　さらにアニオン性ポリマーを含む、請求項１記載の経口医薬組成物。 The oral pharmaceutical composition of claim 1, further comprising an anionic polymer.
  3. 　前記細胞膜透過性ポリカチオンイソペプチドがε‐ポリ‐Ｌ‐α‐リジンである、請求項１又は２記載の経口医薬組成物。 The oral pharmaceutical composition according to claim 1 or 2, wherein the cell membrane-permeable polycationic isopeptide is ε-poly-L-α-lysine.
  4. 　前記アニオン性ポリマーがポリリン酸である、請求項２記載の経口医薬組成物。 The oral pharmaceutical composition of claim 2, wherein the anionic polymer is polyphosphoric acid.
  5. 　ｐＨにより水に対する溶解性が変化する、請求項１又は２記載の経口医薬組成物。 The oral pharmaceutical composition according to claim 1 or 2, whose solubility in water changes depending on pH.
  6. 　前記細胞膜透過性ポリカチオンイソペプチドを、前記アニオン性ポリマーに対して０．０１質量倍以上１０．０質量倍以下含む、請求項２記載の経口医薬組成物。 The oral pharmaceutical composition according to claim 2, comprising 0.01 to 10.0 times by mass of the cell membrane-permeable polycationic isopeptide relative to the anionic polymer.
  7. 　細胞膜透過性ポリカチオンイソペプチドと、
    　生理活性物質と、
    　アニオン性ポリマーと、
    を含むポリイオンコンプレックス複合体。     A cell membrane-permeable polycationic isopeptide;
    A biologically active substance;
    an anionic polymer;
    A polyion complex comprising:
  8. 　前記細胞膜透過性ポリカチオンイソペプチドがε‐ポリ‐Ｌ‐α‐リジンである、請求項７記載のポリイオンコンプレックス複合体。 The polyion complex of claim 7, wherein the cell membrane-permeable polycation isopeptide is ε-poly-L-α-lysine.
  9. 　前記アニオン性ポリマーがポリリン酸である、請求項７記載のポリイオンコンプレックス複合体。 The polyion complex of claim 7, wherein the anionic polymer is polyphosphoric acid.
  10. 　ｐＨにより水に対する溶解性が変化する、請求項７～９の何れか１項記載のポリイオンコンプレックス複合体。 The polyion complex according to any one of claims 7 to 9, the solubility of which in water changes depending on the pH.
  11. 　前記細胞膜透過性ポリカチオンイソペプチドを、前記アニオン性ポリマーに対して０．０１質量倍以上１０．０質量倍以下含む、請求項７～９の何れか１項記載のポリイオンコンプレックス複合体。 The polyion complex according to any one of claims 7 to 9, comprising 0.01 to 10.0 times by mass of the cell membrane-permeable polycation isopeptide relative to the anionic polymer.
  12. 　前記細胞膜透過性ポリカチオンイソペプチドと前記生理活性物質を混合し、次いで前記アニオン性ポリマーを混合する、請求項７～９の何れか１項記載のポリイオンコンプレックス複合体の製造方法。 The method for producing a polyion complex according to any one of claims 7 to 9, comprising mixing the cell membrane-permeable polycationic isopeptide with the physiologically active substance, and then mixing the anionic polymer.
  13. 　ε‐ポリ‐Ｌ‐α‐リジンと、
    　インスリンと、
    を含む経口インスリン製剤。     ε-poly-L-α-lysine,
    Insulin and
    13. An oral insulin preparation comprising:
  14. 　更にポリリン酸を含む、請求項１３記載の経口インスリン製剤。 The oral insulin preparation of claim 13, further comprising polyphosphate.
  15. 　前記生理活性物質は、タンパク質、ペプチド、核酸、および、酵素からなる群から選ばれた１以上である、請求項１又は２記載の経口医薬組成物。 The oral pharmaceutical composition according to claim 1 or 2, wherein the physiologically active substance is one or more selected from the group consisting of proteins, peptides, nucleic acids, and enzymes.
PCT/JP2024/006117 2023-02-22 2024-02-20 Oral pharmaceutical composition containing cell membrane-penetrating polycationic isopeptide, polyion complex composite, production method thereof, and oral insulin formulation WO2024177082A1 (en)

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