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WO2024177082A1 - Composition pharmaceutique orale contenant un isopeptide polycationique pénétrant la membrane cellulaire, composite de complexe polyionique, son procédé de production et formulation d'insuline orale - Google Patents

Composition pharmaceutique orale contenant un isopeptide polycationique pénétrant la membrane cellulaire, composite de complexe polyionique, son procédé de production et formulation d'insuline orale 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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polyion complex
isopeptide
cell membrane
pharmaceutical composition
pαl
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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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Fukui Prefectural University
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Fukui Prefectural University
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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

L'invention concerne une composition pharmaceutique orale contenant un isopeptide polycationique pénétrant la membrane cellulaire et une substance physiologiquement active. La composition pharmaceutique orale peut en outre contenir un polymère anionique. L'invention concerne également un composite de complexe polyionique contenant un isopeptide polycationique pénétrant la membrane cellulaire, une substance physiologiquement active et un polymère anionique.
PCT/JP2024/006117 2023-02-22 2024-02-20 Composition pharmaceutique orale contenant un isopeptide polycationique pénétrant la membrane cellulaire, composite de complexe polyionique, son procédé de production et formulation d'insuline orale Ceased WO2024177082A1 (fr)

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