Platelet aggregators

Abstract

The present invention pertains to a platelet aggregator that contains amorphous polyphosphoric acid as an active ingredient, wherein the polyphosphoric acid is a Ca salt of polyphosphoric acid. The platelet aggregator acts on damaged gastrointestinal mucosa in inflammatory bowel disease and can bring about remission / improvement of the inflammatory bowel disease by exerting a platelet aggregating effect.

Description

[Technical field]

[0001] [Related Applications] This application claims priority to Japanese Patent Application No. 2020-001319 (filed January 8, 2020), the contents of which are incorporated herein by reference. [Technical field] The present invention relates to a platelet aggregating agent containing a calcium salt of polyphosphate as an active ingredient, more specifically to a pharmaceutical agent which acts on damaged gastrointestinal mucosa in inflammatory bowel disease and exerts a platelet aggregating effect, thereby bringing about remission and improvement of the inflammatory bowel disease. [Background technology]

[0002] Anti-inflammatory drugs are used as standard treatments for inflammatory bowel diseases (IBD), such as ulcerative colitis (UC) and Crohn's disease (CD), but there are no drugs that directly induce mucosal healing. While traditional anti-inflammatory drugs have been used as symptomatic treatments for IBD, it has recently been said that the true goal of IBD treatment is "mucosal healing." Probiotics, such as lactic acid bacteria, are known to be highly safe due to long-standing dietary experience and to have a certain intestinal regulating effect, but the mechanism of action of probiotics on intestinal condition remains largely unknown. To elucidate this, identification and analysis of bioactive molecules produced by probiotics is being carried out.

[0003] The inventors identified long-chain polyphosphate derived from malt lactic acid bacteria as a molecule that strengthens the intestinal barrier function. They demonstrated that long-chain polyphosphate improves the deterioration of the intestinal barrier function and intestinal damage caused by DSS treatment (Patent Document 1). They also demonstrated that long-chain polyphosphate induces mucosal healing in patients with refractory ulcerative colitis (Non-Patent Document 1).

[0004] Polyphosphate is known to have wound healing and anti-inflammatory effects, and it is expected that amorphous or nanoparticles composed of calcium polyphosphate will be used for wound dressings and dental materials (Patent Document 2).

[0005] Polyphosphate is also known as a blood coagulation-inducing factor, and when polyphosphate is released from platelets, it activates factor XII protease in the blood, leading to a coagulation reaction. Natural polyphosphate is assumed to be various salts such as Ca salt, Mg salt, Na salt, and K salt, and the existence of amorphous nanoparticles is also known (Non-Patent Documents 2 and 3). Donovan et al. have created nanoparticles from polyphosphates with different chain lengths and reported on the size of polyphosphate and its effect on blood coagulation (Non-Patent Document 4). However, there have been no reports on the effects of amorphous polyphosphate and nanoparticles, particularly on the effects of different salts, on platelet aggregation and inflammatory bowel disease. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2011 / 125619 [Patent Document 2] International Publication No. 2016 / 079006 [Non-patent literature]

[0007] [Non-Patent Document 1] Fujita et al., “Long-Chain Polyphosphate Is a Potential Agent for Inducing Mucosal Healing of the Colon in Ulcerative Colitis.” Clin Pharmacol Ther. 2019 Sep. [Non-Patent Document 2] Feng et al., “Biogenic Polyphosphate Nanoparticles from a Marine Cyanobacterium Synechococcus sp. PCC 7002: Production, Characterization, and Anti-Inflammatory Properties In Vitro.” Mar Drugs. 2018 Sep 10;16(9). [Non-Patent Document 3] Feng et al., “Biogenic Polyphosphate Nanoparticles from Synechococcus sp. PCC 7002 Exhibit Intestinal Protective Potential in Human Intestinal Epithelial Cells In Vitro and Murine Small Intestine Ex Vivo.” J Agric Food Chem. 2018 Aug 1;66(30):8026-8035. [Non-Patent Document 4] Donovan et al., “Size-controlled synthesis of granular polyphosphate nanoparticles at physiologic salt concentrations for blood clotting.” Biomacromolecules. 2014 Nov 10;15(11):3976-84 Summary of the Invention [Problem to be solved by the invention]

[0008] An objective of the present invention is to elucidate the physiological activity of polyphosphate and to carry out an optimal molecular design in order to maximize the activity. [Means for solving the problem]

[0009] The inventors have found that the platelet aggregation effect of polyphosphate (salt) is selective for the calcium (Ca) salt and is not observed in other metal salts such as the sodium (Na) salt, and that the effect of this Ca polyphosphate salt is exerted on damaged gastrointestinal mucosa in inflammatory bowel disease, promoting mucosal healing.

[0010] The present invention is based on the above findings and relates to the following (1) to (9). (1) A platelet aggregating agent comprising amorphous polyphosphoric acid as an active ingredient, wherein the polyphosphoric acid is a Ca salt of polyphosphoric acid. (2) The platelet aggregation agent according to (1), which promotes platelet aggregation in damaged gastrointestinal mucosa. (3) The platelet aggregation agent according to (1) or (2), which is administered to a patient with inflammatory bowel disease. (4) The platelet aggregating agent according to any one of (1) to (3), which is orally administered. (5) The platelet aggregating agent according to any one of (1) to (4), which promotes healing of damaged gastrointestinal mucosa. (6) The platelet aggregating agent according to any one of (1) to (5), wherein the amorphous polyphosphoric acid is in the form of nanoparticles. (7) The platelet aggregating agent according to (6), wherein the zeta potential of the polyphosphoric acid nanoparticles is 0 mV or less. The zeta potential of the polyphosphoric acid nanoparticles is preferably -20 mV or less, more preferably -30 mV or less. (8) The platelet aggregating agent according to (6) or (7), wherein the average particle size of the polyphosphate nanoparticles is 10 nm to 3,000 nm. (9) The platelet aggregating agent according to any one of (1) to (8), wherein the average chain length of polyphosphate is 5 phosphate units or more, preferably 100 phosphate units or more, and more preferably 300 phosphate units or more. Effect of the Invention

[0011] The polyphosphate of the present invention is composed of amorphous or nanoparticles composed of Ca polyphosphate, which is the main component of platelet aggregation. Therefore, it is expected to have a higher platelet aggregation effect than natural polyphosphate. When orally administered, the polyphosphate of the present invention acts on the damaged gastrointestinal mucosa and exerts an excellent platelet aggregation effect, thereby improving and relieving inflammatory bowel disease. [Brief description of the drawings]

[0012] [Figure 1] The intestinal lengths of mice (n=8 for each group) are shown for normal mice (Control), mice with acute enteritis treated with PBS (DSS, PBS), mice with acute enteritis treated with calcium polyphosphate (1 μg / mouse) (DSS, 1 μg PPA-Ca), mice with acute enteritis treated with calcium polyphosphate (5 μg / mouse) (DSS, 5 μg PPA-Ca), and mice with tacrolimus (60 μg / mouse) treatment (DSS, 60 μg TAC). [Diagram 2] Histological severity (n=8 for each group) is shown for normal mice (PBS), PBS-treated mice with acute enteritis (DSS, PBS), calcium polyphosphate (1 μg / mouse)-treated mice with acute enteritis (DSS, 1 μg PPA-Ca), calcium polyphosphate (5 μg / mouse)-treated mice (DSS, 5 μg PPA-Ca), and tacrolimus (60 μg / mouse)-treated mice (DSS, 60 μg TAC). [Diagram 3] The intestinal lengths of mice are shown (normal mice; n=6, acute enteritis mice treated with PBS; n=9, acute enteritis mice treated with sodium polyphosphate; n=7, acute enteritis mice treated with calcium polyphosphate; n=8, tacrolimus; n=5). Normal mice (Control), acute enteritis mice treated with PBS (DSS, PBS), acute enteritis mice treated with sodium polyphosphate (5 μg / mouse) (DSS, PPA-Na), acute enteritis mice treated with calcium polyphosphate (5 μg / mouse) (DSS, PPA-Ca), and tacrolimus (60 μg / mouse) treatment (DSS, TAC). [Figure 4]The results of RT-PCR analysis of inflammatory cytokine (TNFα, IL-1β, IFNγ, IL6) expression (n=6-8) are shown. The graphs show, from the left, normal mice (Control), PBS-treated mice with acute enteritis (DSS, PBS), calcium polyphosphate (1μg / mouse)-treated mice with acute enteritis (DSS, 1μg PPA-Ca), calcium polyphosphate (5μg / mouse)-treated mice (DSS, 5μg PPA-Ca), and tacrolimus (60μg / mouse)-treated mice (DSS, 60μg TAC). [Diagram 5] The results of RT-PCR analysis of inflammatory cytokine (TNFα, IL-1β, IFNγ, IL6) expression (n=5-9) are shown for normal mice (Control), PBS-treated mice with acute enteritis (DSS, PBS), sodium polyphosphate (5μg / mouse)-treated mice with acute enteritis (DSS, PPA-Na), calcium polyphosphate (5μg / mouse)-treated mice (DSS, PPA-Ca), and tacrolimus (60μg / mouse)-treated mice (DSS, TAC). [Figure 6] The CD61 positive area (pixels) is shown (n=7). From the left, the graph shows normal mice (Control), mice with acute enteritis treated with PBS (DSS, PBS), and mice with acute enteritis treated with calcium polyphosphate (5 μg / mouse) (DSS, PPA-Ca). [Figure 7] The graph shows the change in the degree of suspension of the solution over time (n=5). For sodium polyphosphate (PPA-Na), there was almost no difference from the result without addition (negative control). On the other hand, for calcium polyphosphate (PPA-Ca), the degree of suspension rose once immediately after addition, but then decreased to the same extent as for adenosine diphosphate (ADP). [Figure 8] The graph shows the change in wound width over time (n=3). From the top, the graph shows no addition (negative control), sodium polyphosphate (PPA-Na), and calcium polyphosphate (PPA-Ca). [Figure 9]The intestinal lengths of mice (normal mice; n=5, chronic enteritis mouse group treated with PBS, chronic enteritis mouse group treated with calcium polyphosphate, chronic enteritis mouse group treated with sodium polyphosphate; n=7) are shown. Normal mice (Control), chronic enteritis mouse group treated with PBS (DSS, PBS), chronic enteritis mouse group treated with calcium polyphosphate (5 μg / mouse) (DSS, 5 μg PPA-Ca), chronic enteritis mouse group treated with sodium polyphosphate (5 μg / mouse) (DSS, 5 μg PPA-Na). [Figure 10] Histological severity (normal mice; n=5, chronic enteritis mouse group treated with PBS, chronic enteritis mouse group treated with calcium polyphosphate, chronic enteritis mouse group treated with sodium polyphosphate; n=7) is shown. Normal mice (Control), chronic enteritis mouse group treated with PBS (DSS, PBS), chronic enteritis mouse group treated with calcium polyphosphate (5 μg / mouse) (DSS, 5 μg PPA-Ca), chronic enteritis mouse group treated with sodium polyphosphate (5 μg / mouse) (DSS, 5 μg PPA-Na). [Figure 11] The results of RT-PCR analysis of inflammatory cytokine (IL-1β, TNFα, IFNγ) expression (normal mice; n=5, chronic enteritis mouse PBS treatment group, chronic enteritis mouse calcium polyphosphate treatment group, chronic enteritis mouse sodium polyphosphate treatment group; n=7). The graphs show, from the left, normal mice (Control), chronic enteritis mouse PBS treatment group (DSS, PBS), chronic enteritis mouse calcium polyphosphate (5μg / mouse) treatment group (DSS, 5μg PPA-Ca), and chronic enteritis mouse sodium polyphosphate (5μg / mouse) treatment group (DSS, 5μg PPA-Na). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] 1. Polyphosphate

[0014] As used herein, "polyphosphoric acid" refers to a condensed phosphoric acid compound obtained by dehydration condensation of phosphoric acid (H3PO4), and may be linear, cyclic, or branched.

[0015] In this specification, the average chain length of polyphosphate is indicated by the number (n) of "phosphate units" contained in the linear portion of polyphosphate. For example, an average chain length of 10 phosphate units or more (n=10) means that the linear portion of polyphosphate contains 10 or more repeating phosphate units.

[0016] The average chain length of the polyphosphate of the present invention is preferably 5 phosphate units or more, more preferably 100 phosphate units or more, and even more preferably 300 phosphate units or more. The use of a longer-chain polyphosphate improves not only the platelet aggregation effect but also the barrier function strengthening effect, and can more effectively prevent or improve (cure, alleviate) inflammatory bowel disease.

[0017] The polyphosphate is preferably a high molecular weight polyphosphate that does not pass through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa when ultrafiltration is performed. Note that ultrafiltration refers to a method in which a spin column equipped with an ultrafiltration membrane made of PES (polyethersulfone) is used, a sample to be filtered is added to the spin column, and then the sample is centrifuged to filter out molecules in the sample to be filtered.

[0018] Polyphosphate may be chemically synthesized, or may be synthesized in vitro using a biomolecule such as an enzyme, or may be synthesized using a microorganism that produces polyphosphate, etc. When linear polyphosphate is synthesized, it is preferable to synthesize it in vitro using a biomolecule such as an enzyme, or to synthesize it using a microorganism, etc., because linear polyphosphate can be obtained with high efficiency.

[0019] An example of a method for chemically synthesizing polyphosphoric acid is a method in which a reaction solution containing ATP as a raw material is heated to dehydration condensation. The heating temperature can be, for example, 150 to 350°C.

[0020] An example of a method for synthesizing polyphosphate in vitro using biomolecules such as enzymes is a method in which polyphosphate kinase (PPK), an enzyme that synthesizes polyphosphate, is used as the "biomolecule such as enzymes" and polyphosphate is synthesized from ATP by the enzymatic action of PPK. It has been reported that many probiotics, such as strains belonging to Lactobacillus rhamnosus GG and Lactobacillus brevis, possess PPK, and the gene sequence of the enzyme is also published in a database.

[0021] The PPK may be any PPK capable of synthesizing polyphosphate using ATP as a substrate, and may be obtained from any strain expressing PPK, or may be commercially available. For example, the PPK may be derived from Propionibacterium shermanii.

[0022] The enzyme reaction by PPK is reversible, but when a large amount of ADP is present in the reaction solution compared with ATP, the decomposition reaction of polyphosphate becomes dominant so that the ADP / ATP ratio reaches equilibrium. Therefore, in order to efficiently synthesize polyphosphate, it is preferable not to add ADP to the reaction solution. Other conditions such as the composition of the reaction solution, reaction temperature, and reaction time can be appropriately set so as to be optimal for PPK activity and depending on the synthesis scale, etc. As an example, the reaction conditions when synthesizing polyphosphate using PPK derived from Propionibacterium shermanii are shown below. First, the composition of the reaction solution can be 50 mM Tris-HCl (pH 7.4), 40 mM ammonium sulfate, 4 mM MgCl2, 40 mM creatine phosphate, 20 ng / ml creatine kinase, 1 mM ATP (pH 7.2), and 1 U / ml PKK, the reaction temperature can be 37°C, and the reaction time can be 0.5 to 10 hours. The reaction time may be appropriately set depending on the molecular weight and yield of the target polyphosphoric acid. For example, in order to obtain high molecular weight polyphosphoric acid in good yield, the reaction time is preferably 1 to 5 hours.

[0023] Examples of the method of synthesis using microorganisms include a method of culturing a microorganism that produces polyphosphate under appropriate culture conditions to allow the microorganism to produce polyphosphate. Examples of the microorganism that produces polyphosphate include Lactobacillus rhamnosus GG strain, Lactobacillus brevis SBC8803 strain, strains belonging to Lactobacillus, strains belonging to Bifidobacterium, strains belonging to Enterococcus, strains belonging to Lactococcus, strains belonging to Pediococcus, strains belonging to Leuconostoc, strains belonging to Streptococcus, strains belonging to Bacteroides, strains belonging to Eubacterium, and strains belonging to Clostridium.

[0024] Polyphosphate can be synthesized by culturing a microorganism to be used in an appropriate medium under appropriate culture temperature conditions capable of supporting the growth of the microorganism. The synthesized polyphosphate can be recovered from the medium after the culture or by disrupting the microorganism after the culture.

[0025] In the purification step of the synthesized polyphosphate, an appropriate combination of purification methods commonly used in this technical field can be used, such as size exclusion chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), dialysis, salting out, ammonium sulfate precipitation, precipitation, crystallization, etc. The purification method to be used can be appropriately determined depending on the method used in the synthesis step of polyphosphate, the desired degree of purification, the desired yield, etc.

[0026] In the present invention, polyphosphate is amorphous composed of Ca polyphosphate. Amorphous Ca polyphosphate is insoluble in water, and therefore reaches the intestinal mucosa, which is the site of action, in a solid state, and is taken up by epithelial cells by endocytosis, so that a high effect can be expected. In addition, amorphous Ca polyphosphate, which is insoluble in water, simplifies the drug substance production process such as recovery, washing, and drying, and is advantageous in terms of production costs.

[0027] In the present invention, the amorphous Ca polyphosphate is preferably in the form of nanoparticles. In this specification, the term "nanoparticles" refers to particles with a particle diameter on the order of nanometers. The polyphosphate nanoparticles of the present invention are particles with an average particle diameter of 10 nm to 3000 nm, and have a charged zeta potential of 0 mV or less, preferably -20 mV or less, more preferably -30 mV or less. The average particle diameter and zeta potential refer to the average particle diameter and zeta potential determined by dynamic light scattering method / photon correlation method (particle size distribution measurement) and laser Doppler multipoint detection electrophoresis method (zeta potential measurement). Nanoparticles are smooth spheres with a relatively uniform particle diameter, and therefore have favorable physical properties in terms of formulation.

[0028] Nanoparticles composed of calcium salt of polyphosphate can be produced, for example, as follows: sodium salt of polyphosphate is dissolved in water or a buffer solution (e.g., Tris-HCl buffer or carbonate-bicarbonate buffer) and the pH is adjusted (e.g., pH 10). While maintaining the pH with NaOH, CaCl2 is added and stirred to flocculate the calcium salt of polyphosphate, and the precipitate is dried and then sieved through a sieve of an appropriate size.

[0029] As shown in the Examples below, the inventors have found that Ca polyphosphate promotes platelet aggregation in the damaged intestinal mucosa of inflammatory bowel disease, and makes it possible to promote the improvement, remission, and healing of the damaged mucosa. This platelet aggregation promoting effect is not seen in other metal salts of polyphosphate such as sodium polyphosphate, and can be said to be an effect unique to Ca salt.

[0030] 2. Platelet aggregating agents The "platelet aggregating agent" according to the present invention means a substance that has the effect of aggregating platelets. The platelet aggregating agent of the present invention is characterized in that it contains the above-mentioned amorphous Ca salt of polyphosphate as an active ingredient and can aggregate platelets in damaged gastrointestinal mucosa.

[0031] In vivo, polyphosphate is also known as a blood coagulation-inducing factor. Polyphosphate is stored in the dense granules of platelets, and when released in response to a stimulus, it activates factor XII protease in the blood, inducing the coagulation reaction. Natural polyphosphate is composed of various salts such as Ca salt, Mg salt, Na salt, and K salt, and the effects and differences of each salt are not known.

[0032] The inventors have found that by using artificially produced polyphosphate, Ca salt has platelet aggregation effect, whereas other metal salts such as Na salt do not cause platelet aggregation. Furthermore, they have found that an amorphous material composed of Ca salt of polyphosphate induces platelet aggregation in damaged gastrointestinal mucosa by oral administration, thereby promoting the healing of damaged gastrointestinal mucosa.

[0033] The platelet aggregation effect of polyphosphate can be evaluated according to the description in the Examples below. For example, the platelet aggregation effect can be evaluated in vitro by adding a polyphosphate (polyphosphate nanoparticles, etc.) to platelet rich plasma (PRP) and measuring the change in turbidity. In addition, the platelet aggregation effect can be evaluated in vivo by preparing a disease model animal, orally administering a polyphosphate, and observing the localization of CD61 (present on the platelet surface) at the affected site.

[0034] The platelet aggregating agent of the present invention may contain a pharmacologically acceptable carrier or additive, such as, for example, an excipient, a binder, a lubricant, a solvent, a disintegrant, a solubilizing agent, a suspending agent, an emulsifying agent, an isotonicity agent, a stabilizer, a preservative, an antioxidant, a flavoring agent, a coloring agent, a buffer, a flowability promoter, etc., but is not limited thereto, and other commonly used carriers and additives can be appropriately used.

[0035] Specific examples of excipients include organic excipients such as sugars such as lactose, glucose, D-mannitol, starches, celluloses such as crystalline cellulose, and inorganic excipients such as calcium carbonate and kaolin.

[0036] Examples of binders include pregelatinized starch, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, D-mannitol, trehalose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and polyvinyl alcohol.

[0037] Examples of the lubricant include stearic acid, fatty acid salts such as stearates, talc, silicates, and the like.

[0038] Examples of the solvent include purified water, physiological saline, and phosphate buffer.

[0039] Disintegrants include low-substituted hydroxypropyl cellulose, chemically modified celluloses and starches.

[0040] Examples of the solubilizing agent include polyethylene glycol, propylene glycol, trehalose, benzyl benzoate, ethanol, sodium carbonate, sodium citrate, sodium salicylate, and sodium acetate.

[0041] Examples of suspending agents or emulsifying agents include sodium lauryl sulfate, gum arabic, gelatin, lecithin, glycerin monostearate, polyvinyl alcohol, polyvinylpyrrolidone, celluloses such as sodium carboxymethylcellulose, polysorbates, polyoxyethylene hydrogenated castor oil, and the like.

[0042] The isotonicity agent includes sodium chloride, potassium chloride, sugars, glycerin, urea and the like.

[0043] Stabilizers include polyethylene glycol, sodium dextran sulfate, and other amino acids.

[0044] Examples of preservatives include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid.

[0045] Antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; fat-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0046] Examples of the flavoring agent include sweeteners and fragrances that are commonly used in the pharmaceutical field, and examples of the coloring agent include coloring agents that are commonly used in the pharmaceutical field.

[0047] The platelet aggregating agent of the present invention can be safely administered orally or parenterally (e.g., into the oral cavity, esophagus, stomach, small intestine, large intestine, rectum, etc.) by preparing pharmaceutical preparations such as tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, and orally disintegrating tablets), powders, granules, capsules (including soft capsules and microcapsules), liquids, troches, syrups, emulsions, suspensions, injections (e.g., subcutaneous injections, intramuscular injections, intraperitoneal injections, etc.), external preparations (e.g., nasal preparations, transdermal preparations, ointments), suppositories (e.g., rectal suppositories, vaginal suppositories), foams, pellets, nasal preparations, and pulmonary preparations (inhalants). Preferably, the platelet aggregating agent of the present invention can be administered orally, as a suppository, or rectally, with oral administration being particularly preferred.

[0048] The platelet aggregating agent of the present invention may be a controlled release preparation such as a fast release preparation or a sustained release preparation. When it is made into an oral preparation, it may be coated for the purpose of masking, enteric or sustained release, if necessary. Examples of the coating base used for coating include sugar coating base, water-soluble film coating base, enteric film coating base, and sustained release film coating base.

[0049] The platelet aggregating agent of the present invention may be administered to humans or non-human mammals. The dosage and administration method are not particularly limited and can be appropriately determined depending on the condition, age, etc. of the individual to be administered.

[0050] The dose of the platelet aggregating agent of the present invention is appropriately determined depending on the purpose of use, the route of administration, etc. When administered to humans, the dose can be selected, for example, in the range of 0.05 mg / kg to 15 mg / kg, preferably 0.05 mg / kg to 5 mg / kg, more preferably 0.05 mg / kg to 2.5 mg / kg in terms of polyphosphate per day. Alternatively, for example, the dose can be administered in the range of 3 to 900 mg / day, preferably 3 to 300 mg / day, more preferably 3 to 150 mg / day per patient, once a day or in divided doses.

[0051] The polyphosphoric acid of the present invention is composed of an amorphous substance composed of Ca polyphosphate, which is the main component of platelet aggregation, and is therefore expected to have a higher platelet aggregation effect than natural polyphosphoric acid, which is a miscellaneous mixture with other metal salts, etc.

[0052] The platelet aggregating agent of the present invention may be used in combination with other drugs, provided that the object of the present invention is not impaired. Examples of medicines that can be used in combination with the platelet aggregating agent of the present invention include drugs that are commonly used in the treatment of inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, such as 5-aminosalicylic acid (5-ASA) preparations such as mesalazine and salazosulfapyridine; steroid preparations such as prednisolone and methylprednisolone; anti-TNFα preparations such as infliximab and adalimumab; thiopurine preparations such as mercaptopurine and azathioprine; and immunosuppressants such as cyclosporine and tacrolimus. The administration time of the platelet aggregating agent of the present invention and the combined drug is not limited, and they may be administered simultaneously or at a time lag.

[0053] 4. Inflammatory bowel disease As described above, the amorphous polyphosphate composed of Ca salt of polyphosphate exerts a platelet aggregation effect in the damaged gastrointestinal mucosa by oral administration and promotes the healing of the damaged gastrointestinal mucosa. Therefore, the amorphous polyphosphate of the present invention is useful for the prevention or treatment of inflammatory bowel disease (IBD).

[0054] "Inflammatory bowel disease" is a general term for chronic or relapsing / relapsing inflammatory diseases of the intestinal tract, and generally refers to two diseases: ulcerative colitis (UC) and Crohn's disease (CD). Ulcerative colitis is a diffuse, non-specific inflammation of unknown cause that forms erosions and ulcers in the mucous membrane of the large intestine, and often goes into remission and relapse. Crohn's disease is a general term for chronic inflammatory diseases of unknown cause that cause chronic inflammation and ulcers in the mucous membrane of the large and small intestines.

[0055] The inventors have reported that polyphosphate has the effect of recovering intestinal barrier function and preventing or improving inflammatory bowel disease. The amorphous polyphosphate of the present invention has the above-mentioned intestinal barrier function recovery effect in addition to the platelet aggregation effect specific to Ca salts, and therefore more effectively treats damaged gastrointestinal mucosa and induces and maintains remission of inflammatory bowel disease. The present invention also provides a pharmaceutical product for inducing / maintaining remission of inflammatory bowel disease, which contains such a platelet aggregating agent.

[0056] 5.Food and beverages The amorphous polyphosphate of the present invention can be safely ingested orally, and therefore can also be used as an ingredient in foods for specified health uses, foods for special dietary uses, nutritional supplements, health foods, functional foods, foods for the sick, and the like. EXAMPLES

[0057] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0058] Example 1: Preparation of calcium polyphosphate particles 1.2 g of sodium long-chain polyphosphate (average chain length 450-700: Kamui Pharma) was added and dissolved in 60 mL of purified water. While stirring the dissolved aqueous solution of sodium long-chain polyphosphate, the pH of the aqueous solution was measured using a pH meter (HORIBA F-55, pH electrode 9680S-10D), and 1N sodium hydroxide solution (Wako Pure Chemical Industries, Ltd.) was added dropwise to adjust the pH of the aqueous solution to pH 10.

[0059] 0.865 g of calcium chloride hydrate (Japanese Pharmacopoeia "For manufacturing only", manufactured by Wako Pure Chemical Industries, Ltd.) was added and dissolved in 7.5 mL of purified water to prepare an aqueous calcium chloride solution. The entire amount of the prepared aqueous calcium chloride solution was dripped into the aqueous long-chain sodium polyphosphate solution at a drip rate of approximately 0.2 g / min using a peristaltic pump. The pH was constantly checked during the dripping of the aqueous calcium chloride solution, and whenever the pH shifted from pH 10 to the acidic side, 1N sodium hydroxide solution was dripped to maintain the pH at pH 10. After the entire amount of the aqueous calcium chloride solution was dripped, stirring was continued at room temperature for 4 hours.

[0060] The obtained suspension was fractionated in a centrifugal ultrafiltration filter unit (Amicon Ultra-15, nominal molecular weight cutoff: 3 kDa, sample volume: 15 mL) and centrifuged at 4,000 rpm (3,040 × g) and 20 ° C for 3 hours using a centrifuge (KUBOTA Model 5911) to concentrate the solids and simultaneously desalt them. 2 mL of ethanol (99.5%, Wako Pure Chemical Industries) was added to the filter device containing the concentrated precipitate to redisperse it, and then centrifuged at 4,000 rpm (3,040 × g) and 20 ° C for 1 hour to remove the ethanol wash. This washing operation with ethanol was repeated twice.

[0061] The filter device containing the precipitate after washing with ethanol was placed in a vacuum dryer (DP-33, Yamato Scientific), and after vacuuming to reduce pressure, it was dried at 50°C for 12 hours. After drying, the solid content was sieved through a sieve (mesh size: 500 μm) to obtain 1.187 g of calcium polyphosphate particle powder. Assuming that 1 molar equivalent of sodium ions in sodium polyphosphate is 100% replaced by half a molar equivalent of calcium ions, the yield of calcium polyphosphate particles was calculated to be 101.9%.

[0062] The average particle size and zeta potential of the prepared calcium polyphosphate particles were measured using a dynamic light scattering (DLS) particle size distribution analyzer (Zetasizer model Nano-ZS, Marvern Instruments). The average particle size was 1252 nm and the zeta potential was -36.2 mV.

[0063] Example 2: Mouse acute enteritis model 1. Materials and Methods 1.1 Preparation of mouse acute colitis model and treatment test Male BALB / c mice (Charles River Japan) aged 6 to 8 weeks were treated with 2% dextran sulfate sodium (MP Biomedicals) for 5 days by free access. Sodium polyphosphate and calcium polyphosphate were dissolved in phosphate buffer (pH 7.4) and orally administered to the mice once a day from the first day. On the 7th day, the mice were euthanized and samples were taken.

[0064] 1.2 Assessment of bowel length The abdomen of the mouse was pinched with tweezers and opened with dissection scissors, the femur and pelvis joints were removed, and the lower part of the large intestine connected to the anus was cut with scissors. The tip of the large intestine was then carefully pinched with tweezers to separate the large intestine, and the upper part of the cecum was cut with scissors. The excised large intestine was placed on a Kimtowel and the length of the large intestine was measured with a ruler.

[0065] 1.3 RT-PCR The excised intestine was opened with scissors, the colon mucosa was peeled off with a slide glass, and added to 500 μL of Trizol. The tube was centrifuged at 15,000 rpm and 4 °C for 5 minutes, and the supernatant was transferred to a 1.5 mL Eppendorf tube. 0.1 mL of chloroform was added to the tube, mixed with a vortex mixer, and centrifuged at 15,000 rpm and 4 °C for 5 minutes. After centrifugation, 250 μL of the aqueous layer was transferred to a new 1.5 mL Eppendorf tube. 250 μL of isopropanol was added to wash the pellet, and 0.5 mL of 70% ethanol was added to wash the pellet. The pellet was dissolved in 200 μL of sterile water, and RNA was purified using the RNeasy Mini Kit (Qiagen). Reverse transcription reaction was performed using the high-capacity cDNA reverse transcription kit (Applied Biosystems) according to the product documentation. Using the collected cDNA as a template, RT-PCR was performed using the Taqman method with primers specific to various inflammation-related cytokines, and spectrum data was obtained using an Applied Biosystems 7300 Real Time PCR system (Applied Biosystems). The same cDNA was used as a template to obtain the spectrum of 18S rRNA, which was then standardized. The spectrum data was quantified relative to the expression level when the PBS-treated group was set to 1 using the △△Ct method.

[0066] 1.4 Histological severity assessment The excised intestine was opened with scissors to prepare an intestinal roll, and the tissue was fixed in 10% formalin. The fixed tissue was embedded in paraffin, sliced ​​at 4 μm, and stained with hematoxylin and eosin. The histological severity was evaluated based on the evaluation criteria by Berg et al. (Berg DJ.et al.J Clin Invest,1998).

[0067] [Table 1]

[0068] 1.5 CD61 immunohistochemical staining Paraffin-embedded intestinal sections were sliced ​​to 4 μm to prepare tissue slides. The sections were deparaffinized and boiled in citrate buffer (pH 6.0) at 121°C for 20 minutes to activate antigens. Nonspecific reactions were blocked with SuperBlock T20 (PBS) (Thermo Fisher Scientific), and the sections were reacted overnight at 4°C in anti-CD61 antibody (Cell Signaling Technologies). The tissues were washed three times for 5 minutes in PBS, treated with ImmPRESS Universal Reagent Anti-Rabbit IgG (VECTOR Laboratories), and washed three times for 5 minutes in PBS. The tissues were developed with ImmPACT DAB Peroxidase Substrate Kit (VECTOR Laboratories), stained with hematoxylin solution, and then mounted. CD61-positive areas were extracted using the free image analysis software Image J, and quantified by measuring the area of ​​the positive areas.

[0069] 2. Results of mouse acute colitis model 2.1 Evaluation of bowel length and histological severity (Figures 1-3) The intestinal lengths of the PBS-treated group, calcium polyphosphate (PPA-Ca (1 μg / mouse))-treated group, PPA-Ca (5 μg / mouse)-treated group, and tacrolimus (TAC (60 μg / mouse))-treated group are shown in FIG. 1. The histological severity scores of each group are shown in FIG. 2. The PPA-Ca-treated groups (1 μg and 5 μg / mouse) had significantly longer intestinal lengths than the PBS-treated group. Similarly, the histological severity of the PPA-Ca-treated group was significantly lower than that of the PBS-treated group, demonstrating the therapeutic effect of calcium polyphosphate in the mouse acute colitis model.

[0070] The effects of sodium polyphosphate (PPA-Na (5 μg / mouse)) were compared with calcium polyphosphate (PPA-Ca (5 μg / mouse)). The intestinal length of the PPA-Ca treatment group was significantly longer than that of the PBS treatment group, but no significant difference was observed between the PPA-Na treatment group and the PBS treatment group (Figure 3), indicating that calcium polyphosphate has a stronger therapeutic effect than sodium polyphosphate in the mouse acute enteritis model.

[0071] 2.2 Expression of inflammatory cytokines (Figures 4 and 5) The expression of inflammatory cytokines (TNFα, IL-1β, IFNγ, IL6) in each group (PBS treatment group, calcium polyphosphate (PPA-Ca (1 μg / mouse)) treatment group, PPA-Ca (5 μg / mouse) treatment group, and tacrolimus (TAC (60 μg / mouse)) treatment group) was analyzed by RT-PCR and the results are shown in Figure 4. The expression of TNFα, IL-1β, IFNγ, and IL6 was significantly lower in the PPA-Ca treatment groups (1 μg and 5 μg / mouse) than in the PBS treatment group. The inhibitory effect of calcium polyphosphate on the expression of inflammatory cytokines was demonstrated in a mouse acute enteritis model.

[0072] The effects of sodium polyphosphate (PPA-Na (5 μg / mouse)) were compared with those of calcium polyphosphate (PPA-Ca (5 μg / mouse)) (Figure 5). In the PPA-Ca treatment group, the expression of IL-1β and TNFα was significantly lower than in the PBS treatment group, and the expression levels of IFNγ and IL6 were also reduced to the same extent as in the TAC treatment group (60 μg / mouse). On the other hand, in the PPA-Na treatment group, no reduction in expression of any inflammatory cytokines was observed at the same dose, indicating that calcium polyphosphate more strongly suppresses the expression of inflammatory cytokines than sodium polyphosphate in a mouse acute enteritis model.

[0073] 2.3 CD61 positive area (Figure 6) Figure 6 shows a graph comparing the CD61-positive area in the intestinal tissue of a mouse acute enteritis model. The CD61-positive area was significantly enlarged in the DSS-treated group (DSS, PBS) compared to the untreated group (Control), but the CD61-positive area was also significantly enlarged in the PPA-Ca-treated group (DSS, PPA-Ca: 5 μg / mouse) compared to the DSS-treated group (DSS, PBS). Since CD61 is expressed on the platelet surface, the CD61-positive area expands when platelet aggregation occurs. The above results indicate that oral administration of PPA-Ca greatly promotes platelet aggregation on the inflamed intestinal mucosa.

[0074] Example 3: Platelet aggregation assay 1. Materials and Methods After obtaining consent for the study, blood was collected using a blood collection tube containing 3.2% sodium citrate buffer, and platelet rich plasma (PRP) was collected by centrifugation at 800g for 10 minutes. The obtained PRP was then reacted on a 96-well plate with sodium polyphosphate and calcium polyphosphate at 100μg / 100μL PRP, and the absorbance (405nm) was measured every 10 seconds using an EnSpire multimode plate reader (Perkinelmer) based on the platelet aggregation assay by Chan MV et al. (Chan MV et al. Platelets, 2018), and the change in the turbidity of the PRP over time was analyzed. The absorbance at the start of the analysis was set to 1, and the relative value was calculated to analyze the change over time.

[0075] 2. Results (Figure 7) The change in absorbance when sodium polyphosphate (PPA-Na) was added was almost the same as the negative control (no addition). On the other hand, the change in absorbance when calcium polyphosphate (PPA-Ca) was added showed a degree of turbidity comparable to that of the positive control adenosine diphosphate (ADP). It was shown that PPA-Ca exerts a significant platelet aggregation effect, but PPA-Na does not have any platelet aggregation effect.

[0076] Example 4: Wound healing assay 1. Materials and Methods Platelet-rich plasma was collected by the same method as above, sodium polyphosphate and calcium polyphosphate were added at 1 mg / ml each, and the mixture was incubated at room temperature for 20 minutes, after which the mixture was centrifuged at 2,000 g for 10 minutes to remove platelets from the PRP. The collected plasma was added to a Vivaspin 3K column (GE Healthcare) and centrifuged at 3,040 × g to collect relatively low molecular weight plasma components from which platelets and polyphosphate particles had been removed. A linear wound was drawn using the tip of a P200 tip on a human colon epithelial cell line (HCEC-1CT cells) confluently cultured on a 12-well plate, and the relatively low molecular weight plasma components collected above were added. Photographs were taken over time, and the degree of wound healing was evaluated by measuring the width of the linear wound.

[0077] 2. Results (Figure 8) The change in wound width over time is shown in Figure 8. Compared to the negative control (PRP without any addition), plasma components collected from PRP with sodium polyphosphate (PPA-Na) added did not show a significant difference in the degree of wound healing. On the other hand, plasma components collected from PRP with calcium polyphosphate (PPA-Ca) added significantly improved the degree of wound healing. In this assay, platelets and polyphosphate particles were removed from the plasma components added to the cells, and it was demonstrated that humoral factors resulting from platelet aggregation by calcium polyphosphate promote wound healing.

[0078] 3. Discussion It was shown that calcium polyphosphate aggregates platelets in the intestinal mucosa of a mouse colitis model (acute), and that humoral factors resulting from platelet aggregation promote the repair of intestinal epithelial cells. On the other hand, sodium polyphosphate did not show any platelet aggregation effect or any effect of promoting the repair of intestinal epithelial cells based on this.

[0079] Example 5: Chronic enteritis model in mice 1. Materials and Methods 1.1 Creation of a mouse chronic colitis model and treatment test Male C57BL / 6J mice (Charles River Japan) aged 6-8 weeks were treated with 2% dextran sulfate sodium (MP Biomedicals) for 5 days. Distilled water was then allowed to flow freely until the 35th day. Sodium polyphosphate (1 μg / mouse) and calcium polyphosphate (1 μg / mouse) were dissolved in phosphate buffer (pH 7.4) and orally administered to the mice once a day from the 25th day. On the 35th day, the mice were euthanized and samples were taken.

[0080] 1.2 Assessment of bowel length The procedure was carried out according to the method described in Example 2.

[0081] 1.3 RT-PCR The procedure was carried out according to the method described in Example 2.

[0082] 1.4 Histological severity assessment The procedure was carried out according to the method described in Example 2.

[0083] 2.Results 2.1 Evaluation of intestinal length and histological severity (Figures 9 and 10) The intestinal lengths of the PBS-treated group, the calcium polyphosphate (PPA-Ca (5 μg / mouse))-treated group, and the PPA-Na (5 μg / mouse)-treated group are shown in Figure 9. In the chronic colitis model, the PPA-Ca-treated group had a significantly longer intestinal length than the PBS-treated group (p<0.05), and the PPA-Na-treated group also showed a certain tendency toward improvement (p=0.07). The histological severity scores of each group are also shown in Figure 10. Significant improvements in histological severity were observed in both the PPA-Ca-treated group and the PPA-Na-treated group (p<0.05).

[0084] 2.2 Expression of inflammatory cytokines (Figure 11) The results of RT-PCR analysis of the expression of inflammatory cytokines (TNFα, IL-1β, IFNγ) are shown in Figure 11. The expression levels of all inflammatory cytokines in the PPA-Ca-treated group were significantly lower than those in the PBS-treated group (p<0.05). In contrast, the PPA-Na-treated group showed no significant difference from the PBS-treated group, but a certain tendency toward decreased expression of IFNγ was observed (p=0.06).

[0085] 3. Discussion Calcium polyphosphate was considered to be useful for both chronic and acute enteritis. Compared to sodium polyphosphate, calcium polyphosphate was able to promote healing of damaged mucosa even at lower doses, and calcium polyphosphate was particularly superior in acute enteritis. These differences were considered to be due in part to the platelet aggregation effect in damaged mucosa, which was not observed with sodium polyphosphate, and the promotion of mucosal healing by humoral factors resulting from this effect. [Industrial Applicability]

[0086] The present invention can promote platelet aggregation by acting on damaged gastrointestinal mucosa and promote mucosal healing in inflammatory bowel disease, and is therefore useful for the prevention or treatment of inflammatory bowel disease.

[0087] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A platelet aggregating agent comprising amorphous polyphosphate as an active ingredient, said polyphosphate being a Ca salt of polyphosphate, which is administered to a patient suffering from inflammatory bowel disease.

2. The platelet aggregation agent according to claim 1, which promotes platelet aggregation in damaged gastrointestinal mucosa.

3. The platelet aggregation agent according to claim 1 or 2, which is administered orally.

4. The platelet aggregating agent according to any one of claims 1 to 3, which promotes healing of damaged gastrointestinal mucosa.

5. The platelet aggregation agent according to any one of claims 1 to 4, wherein the amorphous polyphosphate is in the form of nanoparticles.

6. The platelet aggregation agent according to claim 5, wherein the zeta potential of the polyphosphate nanoparticles is 0 mV or less.

7. The platelet aggregating agent according to claim 5 or 6, wherein the average particle size of the polyphosphate nanoparticles is 10 nm to 3000 nm.

8. A pharmaceutical for preventing or treating inflammatory bowel disease, comprising amorphous polyphosphate as an active ingredient, said polyphosphate being a calcium salt of polyphosphate, said pharmaceutical being administered to a patient with inflammatory bowel disease.

9. The pharmaceutical agent described in claim 8, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

10. The pharmaceutical product described in claim 8 or 9, wherein the amorphous polyphosphate is in the form of nanoparticles.

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