Polymer drug complex

Abstract

To provide a novel drug delivery system.SOLUTION: A polymer drug complex for use in a drug delivery system employing platelets as a drug carrier, the polymer drug complex comprising (A) a cationic polymer and (B) an anti-inflammatory drug, where the (A) cationic polymer and the (B) anti-inflammatory drug are bound to each other.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to polymer drug conjugates. [Background technology]

[0002] Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease of unknown cause. RA is characterized by persistent inflammation in the synovial membrane of joints and progressive destruction of cartilage and bone. With the aging of the population and advances in diagnostic technology, the number of patients diagnosed with RA is increasing year by year. Therefore, there is an urgent need to provide methods for preventing or treating RA.

[0003] Currently, methotrexate (MTX) is known as a treatment for rheumatoid arthritis. Non-Patent Document 1 states that "In patients diagnosed with RA, MTX should be considered as the drug of first choice, taking into account the risk-benefit balance." In Japan, 80% of rheumatoid arthritis patients take MTX.

[0004] On the other hand, methotrexate is usually administered orally, but its distribution throughout the body is known to cause systemic side effects such as bone marrow damage, gastrointestinal damage, and liver damage, and approximately 30% of patients who receive methotrexate are forced to discontinue administration and choose other treatments due to these side effects. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Japan College of Rheumatology, "Guidelines for the Use and Treatment of Methotrexate (MTX) in Rheumatoid Arthritis 2023 Edition," Yodosha Publishing, March 20, 2023 Summary of the Invention [Problem to be solved by the invention]

[0006] The main object of the present invention is to provide a novel drug delivery system, which aims to solve the problem by accumulating drugs in the inflamed area. [Means for solving the problem]

[0007] The present inventors have conducted extensive research to achieve the above-mentioned object. As a result, they have found that a novel drug delivery system can be obtained when a polymer-drug conjugate and platelets are used in combination. Based on this finding, the present inventors have conducted further research and completed the present invention. That is, the present invention includes the following features.

[0008] Item 1. A polymer-drug complex for use in a drug delivery system using platelets as a drug carrier, comprising: The polymer-drug complex comprises (A) a cationic polymer and (B) an anti-inflammatory drug; the (A) cationic polymer and the (B) anti-inflammatory drug are bound to each other; Polymer-drug conjugates. Item 2. The polymer-drug conjugate according to Item 1, wherein the (A) cationic polymer and the (B) anti-inflammatory drug are bonded to each other via a covalent bond. Item 3. The (A) cationic polymer is polylysine, and Item 3. The polymer-drug conjugate according to Item 1 or 2, wherein the anti-inflammatory drug (B) is methotrexate or a derivative thereof. Item 4. A polymer-drug complex and (C) platelets, The polymer-drug complex comprises (A) a cationic polymer and (B) an anti-inflammatory drug; the (A) cationic polymer and the (B) anti-inflammatory drug are bound to each other; Cell preparations. Item 5. The cell preparation according to Item 4, wherein the polymer-drug complex is carried on the platelets (C). Item 6. The cell preparation according to Item 4 or 5, wherein the polymer-drug complex has a solubility in water at 25°C of 100 mg / L or more. Item 7. The cell preparation according to any one of Items 4 to 6, wherein the (A) cationic polymer is at least one polymer selected from the group consisting of polylysine, polyarginine, polyallylamine, polydiallyldimethylammonium chloride, cationized cellulose, cationized guar gum, polyethyleneimine, polyamidoamine, and chitosan. Item 8. The cell preparation according to any one of Items 4 to 6, wherein the (A) cationic polymer is a polypeptide having at least a lysine residue. Item 9-1. The cell preparation according to any one of Items 4 to 8, wherein the anti-inflammatory drug (B) is methotrexate or a derivative thereof. Item 9-2. The cell preparation according to any one of Items 4 to 9-1, wherein the (A) cationic polymer and the (B) anti-inflammatory drug are bound to each other via a covalent bond. Item 9-3. The (A) cationic polymer is polylysine, and Item 9. The cell preparation according to any one of Items 4 to 8, wherein the anti-inflammatory drug (B) is methotrexate or a derivative thereof. Item 9-4. The cell preparation according to any one of Items 4 to 9-3, which is administered directly to an affected area. Item 10. A preventive or therapeutic agent for an inflammatory disease, comprising the cell preparation according to any one of Items 4 to 9-4. Item 11. A preventive or therapeutic agent for rheumatoid arthritis, comprising the cell preparation according to any one of Items 4 to 9-4. Item 12. Use of the polymer-drug conjugate according to any one of Items 1 to 3 or the cell preparation according to any one of Items 4 to 9-4 in a drug delivery system using platelets as a drug carrier. Item 13. A drug delivery system using platelets as a drug carrier, comprising the polymer-drug conjugate according to any one of Items 1 to 3 or the cell preparation according to any one of Items 4 to 9-4. Item 14. A method for producing a cell preparation, comprising: contacting the polymer-drug conjugate with (C) platelets; The polymer-drug complex comprises (A) a cationic polymer and (B) an anti-inflammatory drug; the (A) cationic polymer and the (B) anti-inflammatory drug are bound to each other; Manufacturing method. Item 15-1. The production method according to Item 14, wherein the contacting step is carried out in the presence of citric acid and / or glucose. Item 15-2. The production method according to Item 14 or 15-1, wherein the (A) cationic polymer and the (B) anti-inflammatory drug are bonded to each other via a covalent bond. Item 15-3. The (A) cationic polymer is polylysine, and The method according to any one of Items 14 to 15-2, wherein the anti-inflammatory drug (B) is methotrexate or a derivative thereof. Item 16. A method for treating an inflammatory disease, comprising: administering the cell preparation to a subject in need of treatment for an inflammatory disease; the cell preparation comprises a polymer-drug conjugate and (C) platelets; The polymer-drug complex comprises (A) a cationic polymer and (B) an anti-inflammatory drug; the (A) cationic polymer and the (B) anti-inflammatory drug are bound to each other; Treatment method. Item 17. Collecting the (C) platelets from the subject; contacting the polymer-drug complex with the (C) platelets, Item 17. The method for treating an inflammatory disease according to Item 16. Item 18. The method for treating an inflammatory disease according to Item 16 or 17, wherein the cell preparation is administered directly to the affected area. [Effects of the Invention]

[0009] According to the method of the present invention, a novel drug delivery system can be provided. [Brief explanation of the drawings]

[0010] [Figure 1] 1 shows the 1H-NMR spectrum of Production Example 1 (PLL-F). [Figure 2] 1 shows the 1H-NMR spectrum of Example 1 (PLL-F-MTX). [Figure 3] The results of Test Example 1 are shown below. [Figure 4] The results of Test Example 2 are shown below. [Figure 5] The results of Test Example 3 are shown below. [Figure 6] The results of Test Example 4 are shown below. [Figure 7] 1 shows a scheme of Test Example 5. [Figure 8] The results of Test Example 5 are shown below. [Figure 9] The results of Test Example 6 are shown below. DETAILED DESCRIPTION OF THE INVENTION

[0011] In this specification, the term "containing" is a concept that encompasses all of "comprise," "consist essentially of," and "consist only of."

[0012] In this specification, the expression "A to B" indicating a range of values ​​means "greater than or equal to A and less than or equal to B."

[0013] As used herein, any polymer may have the formula: [ka] [In the formula, Y and Z may be the same or different and represent a structural unit; p and q may be the same or different and represent the number of repetitions of the structural unit.] In the case where the polymer is expressed as above, the structural units Y and Z may each form a block, or may be bonded to each other randomly in the polymer.

[0014] In this specification, when chemical formulas are shown, the description of the substituents present at the polymer terminals may be omitted for convenience, but the substituents are not limited to those apparently present at the polymer terminals.

[0015] (1) Polymer-drug conjugates The polymer-drug conjugate of the present invention (hereinafter sometimes simply referred to as the "conjugate") comprises an (A) cationic polymer and a (B) anti-inflammatory drug, and the (B) anti-inflammatory drug is bound to the (A) cationic polymer. By containing the (B) anti-inflammatory drug bound to the (A) cationic polymer, the conjugate can disperse the (B) anti-inflammatory drug at a high concentration in an aqueous solvent even when the (B) anti-inflammatory drug is poorly water-soluble. Furthermore, by binding the (B) anti-inflammatory drug to the (A) cationic polymer, the conjugate can load the (B) anti-inflammatory drug at a high concentration on platelets without impairing the function of the platelets, which act as drug carriers, even when the (B) anti-inflammatory drug has the effect of activating platelets or impairing platelet function.

[0016] For the above reasons, the complex of the present invention is preferably used in a drug delivery system using platelets as a drug carrier. Alternatively, the complex of the present invention is preferably used in the prevention or treatment of inflammatory diseases using platelets as a drug carrier. Examples of inflammatory diseases include rheumatoid arthritis, seronegative spondyloarthritis (e.g., psoriatic arthritis, ankylosing spondylitis, etc.), polymyalgia rheumatica, vasculitis syndrome, systemic lupus erythematosus, systemic sclerosis, inflammatory myopathy, Behçet's disease, adult Still's disease, sarcoidosis, relapsing polychondritis, inflammatory bowel disease, psoriasis, and other autoimmune diseases. The present invention can also provide a novel drug delivery system using platelets as a drug carrier, comprising the above complex.

[0017] From the viewpoint of delivering a large amount of the anti-inflammatory drug (B) to the affected area, the complex is preferably soluble or dispersible in an aqueous medium. Specifically, the complex preferably has a solubility in water at 25°C of 100 mg / L or more, more preferably 1 g / L or more, and even more preferably 10 g / L or more.

[0018] From the viewpoint of delivering a large amount of the (B) anti-inflammatory drug to the affected area, the complex preferably dissolves or disperses in an aqueous medium without forming particles. The complex may exist in the aqueous medium as aggregates formed by aggregation of a small number of molecules. On the other hand, from the viewpoint of delivering a large amount of the (B) anti-inflammatory drug to the affected area, the complex preferably does not form aggregates, particles, or micelles of 1 μm or larger in the aqueous medium, more preferably does not form aggregates, particles, or micelles of 100 nm or larger, and even more preferably does not form aggregates, particles, or micelles of 10 nm or larger.

[0019] The method for producing the conjugate used in the present invention is not particularly limited, and a wide variety of known reactions for conjugating drugs and polymers can be used. Examples of such reactions include condensation reactions and click reactions. Condensation reactions can be carried out using, for example, a condensing agent.

[0020] (1-1) Cationic polymer The cationic polymer (A) is not particularly limited, and a wide variety of polymers known as cationic polymers can be used. In this specification, the term "cationic polymer" refers to a polymer that has more positive charges than negative charges in an aqueous medium at physiological pH (e.g., pH 7.4).

[0021] (A) The cationic polymer may be, for example, a polymer having an amino group in its main chain or side chain, in which case the amino group may be a primary amino group, a secondary amino group, or a tertiary amino group.

[0022] In the first aspect of the present invention, the cationic polymer (A) is at least one polymer selected from the group consisting of polylysine, polyarginine, polyallylamine, polydiallyldimethylammonium chloride, cationized cellulose, cationized guar gum, polyethyleneimine, polyamidoamine, and chitosan, but is not limited thereto. Among these, polylysine is preferred as the cationic polymer (A) from the viewpoints of solubility, compatibility with platelets, degradability at the affected site, etc.

[0023] The polylysine may be either α-polylysine or ε-polylysine. Of these, α-polylysine is preferred. Furthermore, each amino acid residue constituting the polylysine may be either L- or D-isomer, or may be racemic. In particular, the polylysine is preferably α-poly-L-lysine. By using polylysine as the (A) cationic polymer, the (B) anti-inflammatory drug can be released for a certain period of time at the affected area as the complex is decomposed.

[0024] In the second embodiment of the present invention, the cationic polymer (A) is a polypeptide having at least a lysine residue (Lys), but is not limited thereto.

[0025] The polypeptide may have, in its backbone, a structure represented by the general formula (1): [ka] The structural unit may contain a structural unit represented by the following formula (hereinafter, also referred to as "structural unit C").

[0026] In the above polypeptide, the content of the structural unit represented by general formula (1) (structural unit C) is preferably 10% to 100%, more preferably 50% to 100%, and even more preferably 90% to 100%, of the total number of moles of all structural units constituting the polypeptide, from the viewpoint of binding a larger amount of the (B) anti-inflammatory drug.

[0027] The side chain of the structural unit C, specifically the terminal amino group of the side chain of the lysine residue, can form a bond with (B) an anti-inflammatory drug to provide a polymer-drug conjugate. The bond can be formed by reacting the structural unit C with a reactive group such as isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imide ester, carbodiimide, acid anhydride, or fluoroester.

[0028] When the structural unit C and (B) an anti-inflammatory drug are bonded, the structural unit may be, for example, a structural unit represented by the general formula (1a): [ka] [In the formula, D represents a group derived from (B) an anti-inflammatory drug.] It can be expressed as:

[0029] When the structural unit C and the (B) anti-inflammatory drug are bonded and the (B) anti-inflammatory drug is methotrexate, the structural unit may be, for example, a structural unit represented by the general formula (1b): [ka] It can be expressed as:

[0030] Furthermore, the side chain of the structural unit C can fluorescently label the polymer-drug conjugate by forming a bond with a fluorescent dye molecule, such as fluorescein isothiocyanate (FITC) or rhodamine B (RhoB).

[0031] When the structural unit C and a fluorescent dye molecule are bonded and the fluorescent dye molecule is FITC, the structural unit is, for example, represented by the general formula (1c): [ka] It can be expressed as:

[0032] The polypeptide may have, in addition to the structural unit C, a structural unit represented by the general formula (2): [ka] [In the formula, R represents the side chain of an amino acid residue (excluding lysine residues)] The structural unit may contain a structural unit represented by the following formula (hereinafter, also referred to as "structural unit N").

[0033] In the structural unit N, the side chain of the amino acid residue represented by R is not particularly limited, and the side chain of either a natural amino acid residue or a synthetic amino acid residue can be used. Examples of amino acid residues include glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (IlE), phenylalanine (Phe), serine (Ser), threonine (Thr), 5-hydroxylysine (Hyl), arginine (Arg), aspartic acid (Asp), asparagine (Asn), glutamic acid (Glu), glutamine (Gln), cysteine ​​(CySH), cystine (Cyss), cysteic acid (Cya), methionine (Met), tyrosine (Tyr), thyroxine (Thy), proline (Pro), hydroxyproline (Hyp), tryptophan (Trp), histidine (His), β-alanine, N-methyl-β-alanine, sarcosine, γ-aminobutyric acid, kainic acid, and derivatives thereof. Here, when the amino acid residue is alanine, R represents a methyl group, and when the amino acid residue is glycine, R represents a hydrogen atom.

[0034] In the structural unit N, the side chain of the amino acid residue represented by R may have a protecting group depending on the type of amino acid residue. As the protecting group, any protecting group known to be possessed by the side chain of an amino acid residue can be used, and examples thereof include alkyl-type protecting groups such as tert-butyl (t-Bu) group, benzyl group, allyl group, methyl group, and triphenylmethyl group (Trt); silyl-type protecting groups such as tert-butyldimethylsilyl group (TBS); aryl-type protecting groups such as p-methoxybenzyl group (PMB) and p-methoxyphenyl group (PMP); amide-type protecting groups such as formyl group and acetyl group (Ac); phthalimide-type protecting groups such as phthaloyl group (Phth); benzyloxycarboxamide-type protecting groups such as methyl group (Phth); and methyl group (Phth). Examples of protecting groups include carbamate-type protecting groups such as a methyl group (Cbz), a tert-amyloxycarbonyl group (Aoc), a 9-fluorenylmethoxycarbonyl group (Fmoc), a tert-butyloxycarbonyl group (Boc), an allyloxycarbonyl group (Alloc), and a 2,2,2-triethoxycarbonyl group (Troc); and sulfonamide-type protecting groups such as a 3-nitro-2-pyridinesulfenyl group (Npys), a 2-nitrobenzenesulfonyl group (Ns), and a (2-trimethylsilyl)-ethanesulfonyl group (SES).

[0035] In the structural unit N, the carbon atom to which R is bonded may have a substituent other than R. The substituent is not particularly limited, and examples thereof include alkyl groups having 1 to 10 carbon atoms (e.g., methyl, ethyl, n-propyl, n-butyl, and n-pentyl groups), aryl groups having 6 to 18 carbon atoms (e.g., phenyl, hydroxyphenyl, and naphthyl groups), and alkoxy groups (e.g., methoxy, ethoxy, and propoxy groups).

[0036] In the above polypeptide, the content of the structural unit represented by general formula (2) (structural unit N) is preferably 90% or less, more preferably 50% or less, and even more preferably 10% or less, based on the number of moles of all structural units constituting the polypeptide as 100%, from the viewpoint of adjusting the cationicity of the polypeptide.

[0037] When the polypeptide contains the structural unit C and the structural unit N, it can be represented, for example, by the general formula (3): [ka] [In the formula, R represents a side chain of an amino acid residue, x represents an integer of 1 to 500, and y represents an integer of 1 to 500.] It can be expressed as:

[0038] In general formula (3), x and y represent the number of repetitions of each structural unit. The ratio of the content of structural unit C to the content of structural unit N (x:y; molar ratio) is not particularly limited, but is preferably 99:1 to 1:99, more preferably 99:1 to 50:50, and even more preferably 99:1 to 90:10.

[0039] The structural unit C and the structural unit N may each be used alone or in combination of two or more types.

[0040] When (A) a cationic polymer and (B) an anti-inflammatory drug are bound, the conjugate may be, for example, a compound represented by the general formula (3a): [ka] [In the formula, D represents a group derived from (B) an anti-inflammatory drug. The other symbols are the same as above.] It can be expressed as:

[0041] When (A) a cationic polymer and (B) an anti-inflammatory drug are bound together and the (B) anti-inflammatory drug is methotrexate, the conjugate may be, for example, a conjugate represented by the general formula (3b): [ka] [In the formula, each symbol is the same as defined above.] It can be expressed as:

[0042] Each amino acid residue constituting the above polypeptide may be in either the L-form or the D-form.

[0043] The above polypeptide may have a structural unit other than the structural unit C and the structural unit N. Such a structural unit is not particularly limited, and a wide range of structural units known as structural units contained in polypeptides can be used.

[0044] The cationic polymer (A) is preferably a water-soluble polymer that dissolves or disperses in an aqueous medium. Specifically, the solubility of the cationic polymer (A) in water at 25°C is preferably 100 mg / L or more, more preferably 1 g / L or more, and even more preferably 10 g / L or more.

[0045] The weight average molecular weight of the (A) cationic polymer is not particularly limited, but from the viewpoint of the solubility or dispersibility of the complex, it is preferably 1,000 to 100,000, more preferably 2,000 to 80,000, and even more preferably 5,000 to 50,000.

[0046] The (A) cationic polymer may be used alone or in combination of two or more kinds.

[0047] (1-2) Anti-inflammatory drugs The (B) anti-inflammatory drug is not particularly limited, and a wide variety of drugs known as anti-inflammatory drugs can be used. The (B) anti-inflammatory drug may be any of corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), and biological preparations, but is preferably one having a functional group that easily forms a bond with the functional group of the cationic polymer. When a polymer having an amino group is used as the (A) cationic polymer for the purpose of treating rheumatoid arthritis, methotrexate or a derivative thereof is particularly preferred.

[0048] (B) The anti-inflammatory drug may be used alone or in combination of two or more.

[0049] The solubility of the (B) anti-inflammatory drug is not particularly limited, and it may be either water-soluble or poorly water-soluble. In the present invention, even if the (B) anti-inflammatory drug is poorly water-soluble, a large amount of the (B) anti-inflammatory drug can be loaded onto platelets without impairing platelet function, thereby suppressing side effects caused by the (B) anti-inflammatory drug and enhancing the therapeutic effect of the (B) anti-inflammatory drug. In this specification, "poorly water-soluble" means that the solubility in water at 25°C is 0.1 g / L or less, and includes a drug that is completely insoluble in water at 25°C.

[0050] From the viewpoints of stability and ease of synthesis, the (B) anti-inflammatory drug is preferably bound to the (A) cationic polymer via a covalent bond, more preferably via an amide bond. That is, the (A) cationic polymer and the (B) anti-inflammatory drug are preferably bound to each other via a covalent bond, more preferably via an amide bond. The (B) anti-inflammatory drug can also be bound to the (A) cationic polymer via a bonding mode other than a covalent bond, such as an ionic bond, a coordinate bond, or an intermolecular force.

[0051] The binding amount of (B) anti-inflammatory drug is preferably 0.1 to 50 parts by mass, more preferably 1 to 30 parts by mass, and even more preferably 5 to 15 parts by mass, relative to 100 parts by mass of (A) cationic polymer.

[0052] In the complex, from the viewpoints of solubility, compatibility with platelets, degradability at the affected site, etc., it is preferable that (A) the cationic polymer is polylysine and (B) the anti-inflammatory drug is methotrexate or a derivative thereof.

[0053] (2) Cell preparations The cell preparation of the present invention comprises the above-described complex and (C) platelets. By virtue of the characteristics of the cell preparation of the present invention, the (B) anti-inflammatory drug can be loaded onto the (C) platelets without impairing the function of the (C) platelets, thereby allowing the (B) anti-inflammatory drug to efficiently accumulate at the affected area. Specifically, by utilizing the function of (C) platelets to accumulate at the affected area of ​​cancer, arteriosclerosis, rheumatoid arthritis, and other diseases, the cell preparation of the present invention can efficiently deliver the (B) anti-inflammatory drug to the affected area, thereby suppressing the side effects of the (B) anti-inflammatory drug and enhancing the therapeutic effect of the (B) anti-inflammatory drug.

[0054] For the above reasons, the cell preparation of the present invention is preferably used in a drug delivery system using platelets as a drug carrier. Alternatively, the cell preparation of the present invention is preferably used in the prevention or treatment of inflammatory diseases using platelets as a drug carrier. Examples of inflammatory diseases include rheumatoid arthritis, seronegative spondylitis (e.g., psoriatic arthritis, ankylosing spondylitis, etc.), polymyalgia rheumatica, vasculitis syndrome, systemic lupus erythematosus, systemic sclerosis, inflammatory myopathy, Behçet's disease, adult Still's disease, sarcoidosis, relapsing polychondritis, inflammatory bowel disease, and autoimmune diseases such as psoriasis. The present invention also provides a novel drug delivery system using platelets as a drug carrier, which includes the above-mentioned cell preparation.

[0055] As described above, the cell preparation of the present invention can efficiently deliver an anti-inflammatory drug (B) to an affected area by utilizing the function of (C) platelets, which accumulate at the affected area. The cell preparation of the present invention may be administered directly to the affected area (e.g., the site of inflammation) or indirectly. When administered directly to the affected area, the delivery and accumulation of the anti-inflammatory drug (B) to off-target sites can be further suppressed, thereby further enhancing the effect of suppressing the side effects of the anti-inflammatory drug (B) and further enhancing the anti-inflammatory effect.

[0056] (2-1) Platelets (C) The platelets are not particularly limited, and a wide range of platelets used in known cell preparations can be used.

[0057] (C) Platelets may be platelets derived from living organisms or artificial platelets derived from pluripotent stem cells. Examples of living organisms include mice, rats, dogs, cats, pigs, cows, horses, monkeys, and humans. Examples of pluripotent stem cells include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells).

[0058] (C) Platelets may be used immediately after blood collection, or may be refrigerated or frozen after collection.

[0059] (C) The platelets may be autologous cells derived from the subject, or allogeneic cells derived from a person other than the subject.

[0060] (C) Platelets may be used alone or in combination of two or more types.

[0061] (C) Preferably, the platelets are present in a resting state, i.e., in an unactivated state, prior to administration.

[0062] (C) Whether or not platelets are activated is not particularly limited, and can be evaluated using indicators such as platelet deformation or aggregation, release of intraplatelet granules (e.g., α-granules) or platelet-derived microparticles (PDMPs), and expression of platelet surface markers (e.g., P-selectin (CD62P), CD63, PAC-1, annexin V). Evaluation methods include, for example, microscopy, fluorescence observation, flow cytometry, and ELISA.

[0063] (C) It is preferable that platelets maintain their inherent functions. Examples of such functions include activation ability in response to external stimuli and migration ability to inflammatory sites. The activation ability of platelets can be evaluated, for example, by applying a predetermined stimulus to platelets and then appropriately applying the above-mentioned methods for evaluating activation. The migration ability of platelets can be evaluated, for example, using immunostaining, intravascular imaging, in vivo imaging, etc.

[0064] In the cell preparation of the present invention, from the viewpoint of utilizing the function of platelets, it is preferable that the complex is (C) supported on the surface and / or inside of the platelets, and it is more preferable that the complex is (C) supported inside of the platelets.

[0065] (2-2) Other The cell preparation of the present invention preferably contains an aqueous medium in addition to the above components. A wide variety of buffer solutions used in known cell preparations can be used as the aqueous medium. Examples of buffer solutions include physiological saline, PBS buffer (phosphate-buffered saline), HEPES buffer, and ACD-A solution. The aqueous medium may be blood or platelet-rich plasma. In the cell preparation of the present invention, (C) platelets are preferably dispersed (suspended) in the aqueous medium.

[0066] In the cell preparation of the present invention, (C) the platelet content is 1×10 6 Platelets / mL or more is preferable, 1×10 7 Platelets / mL or more is preferable, and 1×10 8 More preferably, platelets / mL or more.

[0067] In the cell preparation of the present invention, the content of the complex is 1 × 10 per platelet (C). -16 g or more is preferable, and 1×10 -15 g~1×10 -13 g is more preferable, and 1×10 -15 g~1×10 -14 g is more preferred.

[0068] The cell preparation of the present invention preferably contains citric acid and / or glucose from the viewpoints of the encapsulation rate of the complex in platelets, platelet function, and the preventive or therapeutic effect on inflammatory diseases.

[0069] The cell preparation of the present invention may also contain additives used in known cell preparations, such as pH adjusters, isotonicity agents, stabilizers, dispersants, preservatives, antiplatelet drugs, folic acid, albumin, and other proteins.

[0070] (3) Manufacturing method of cell preparation The method for producing the cell preparation of the present invention is not particularly limited, but includes the step of contacting the above complex with (C) platelets.

[0071] The contact method is not particularly limited, and examples thereof include a method of mixing the complex with (C) platelets. Mixing can be carried out in, for example, physiological saline, blood, platelet-rich plasma, ACD-A solution, or other aqueous medium. To inhibit platelet activation, an antiplatelet drug such as prostaglandin E1 can be added to the mixed solvent.

[0072] The step of contacting the complex with (C) platelets is preferably carried out in the presence of citric acid and / or glucose, more preferably in the presence of citric acid and glucose, from the viewpoints of the encapsulation rate of the complex in platelets, platelet function, and the preventive or therapeutic effect on inflammatory diseases.

[0073] In the method for producing the cell preparation of the present invention, the complex is preferably contacted with (C) platelets, and then the platelets are concentrated by, for example, centrifugation or filtration.

[0074] (4) Preventive or therapeutic agent for inflammatory diseases The present invention also provides a preventive or therapeutic agent for inflammatory diseases, comprising the cell preparation.

[0075] Examples of inflammatory diseases include autoimmune diseases such as rheumatoid arthritis, seronegative spondyloarthritis (e.g., psoriatic arthritis, ankylosing spondylitis, etc.), polymyalgia rheumatica, vasculitis syndrome, systemic lupus erythematosus, systemic sclerosis, inflammatory myopathy, Behcet's disease, adult Still's disease, sarcoidosis, relapsing polychondritis, inflammatory bowel disease, and psoriasis.

[0076] The administration method, dosage form, and dosage of the preventive or therapeutic agent for inflammatory diseases of the present invention can be appropriately determined depending on the intended use. For example, the administration form of the preventive or therapeutic agent for inflammatory diseases containing the complex or cell preparation of the present invention as an active ingredient may be oral or parenteral, but is mainly parenteral.

[0077] As described above, the preventive or therapeutic agent for inflammatory diseases of the present invention can efficiently deliver an anti-inflammatory drug (B) to an affected area by utilizing the function of (C) platelets, which accumulate at the affected area. The preventive or therapeutic agent for inflammatory diseases of the present invention may be one for direct administration to an affected area (e.g., an inflamed site) or one for indirect administration. When administered directly to an affected area, the anti-inflammatory drug (B) can be further prevented from being delivered to and accumulated at off-target sites, thereby further enhancing the effect of suppressing the side effects of the anti-inflammatory drug (B) and further enhancing the effect of preventing or treating inflammatory diseases.

[0078] Examples of dosage forms include oral preparations such as tablets, powders, capsules, granules, extracts, and syrups, and parenteral preparations such as injections, drip infusions, nasal drops, eye drops, and suppositories, but injections are the main ones. These preparations can be produced as pharmaceutical compositions containing the complex or cell preparation of the present invention and a pharmaceutically acceptable excipient.

[0079] The effective dose of the agent for preventing or treating inflammatory diseases of the present invention is usually 1×10 10 A dose of at least platelets is appropriate, and this is administered once or multiple times a day. The dosage varies depending on various conditions, and in some cases, a smaller amount than the above range may be sufficient.

[0080] (5) Method for treating inflammatory diseases The method for treating an inflammatory disease of the present invention is not particularly limited, but preferably comprises administering the cell preparation to a subject in need of treatment for an inflammatory disease. To the best of the inventors' knowledge, there have been no reports of actual in vivo administration of a cell preparation containing a polymer-drug conjugate and platelets.

[0081] Examples of inflammatory diseases include autoimmune diseases such as rheumatoid arthritis, seronegative spondyloarthritis (e.g., psoriatic arthritis, ankylosing spondylitis, etc.), polymyalgia rheumatica, vasculitis syndrome, systemic lupus erythematosus, systemic sclerosis, inflammatory myopathy, Behcet's disease, adult Still's disease, sarcoidosis, relapsing polychondritis, inflammatory bowel disease, and psoriasis.

[0082] From the viewpoint of suppressing immune responses, the treatment method of the present invention preferably comprises collecting (C) platelets from a subject and contacting the above-mentioned complex with the (C) platelets. The collection of platelets includes blood collection and / or platelet isolation. Platelet isolation may be completed when platelet-rich plasma is obtained. For blood collection and isolation, methods known in the art can be widely applied. In the treatment method of the present invention, the above-mentioned cell preparation is not limited to an in-house preparation, and allogeneic preparations can also be used.

[0083] The administration is not particularly limited, but can be carried out with reference to "(4) Agents for preventing or treating inflammatory diseases."

[0084] In the treatment method of the present invention, the cell preparation may be administered directly or indirectly to the affected area (e.g., the site of inflammation), although this is not particularly limited. When the cell preparation is administered directly to the affected area, the delivery and accumulation of the (B) anti-inflammatory drug at off-target sites can be further suppressed, thereby further enhancing the effect of suppressing the side effects of the (B) anti-inflammatory drug and further enhancing the effect of preventing or treating inflammatory diseases.

[0085] The subject is primarily a human, but may also be a mouse, rat, dog, cat, pig, cow, horse, monkey, or the like.

[0086] The therapeutic effect of the treatment method of the present invention can be evaluated by administering a certain amount of cell preparation to a subject for a certain period of time, and then measuring, for example, tender joints, swollen joints, inflammatory responses (CRP, ESR), MMP-3, joint ultrasound examination (MRI), serological indicators including inflammatory responses (CRP, ESR, CPK, LDH, MMP-3, etc.), ultrasound examination, X-ray (XP), CT, MRI, etc. [Example]

[0087] The following reference examples and examples will be given to further clarify the features of the present invention, but the present invention is not limited to the following examples.

[0088] To simplify the description in the specification, the following abbreviations may be used: For reagents or solvents, the abbreviations mean: FITC: fluorescein isothiocyanate, MTX: methotrexate, PLL: poly-L-lysine, DMSO: dimethyl sulfoxide, DMT-MM: 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, NMM: N-methylmorpholine, PBS: phosphate buffered saline, ADP: adenosine diphosphate, and PRP: platelet-rich plasma.

[0089] When showing a chemical formula, the description of the substituents present at the polymer terminals may be omitted for convenience, but the substituents are not limited to those apparently present at the polymer terminals.

[0090] ( 1 H-NMR measurement) 2 mg of the obtained powder product was dissolved in 700 μL of DO, transferred to an NMR tube (291-47851, Fujifilm Wako Pure Chemical Industries, Ltd.), and analyzed by NMR (ECZ400s, JEOL). 1 H-NMR spectra were measured (accumulation number = 1024). FID data were analyzed by JEOL Delta.

[0091] (Preparation of platelet suspension) Mice (C57BL / 6N, retired, male; Japan SLC Co., Ltd.) were anesthetized by intraperitoneal administration of 0.3 mL (0.1 mL / 10 g of mouse) of a triple anesthetic mixture (1 mg / mL medetomidine hydrochloride: 5 mg / mL midazolam: 5.0 mg / mL butorphanol tartrate: normal saline = 1.875 mL: 2 mL: 2.5 mL: 18.625 mL). Next, whole blood (10% sodium citrate blood (3.8% sodium citrate solution)) was collected by cardiac blood collection using a 24G needle and a 1 mL syringe (Kansai University Animal Experiment Approval Number: 2315). The collected blood was transferred to a 1.5 mL centrifuge tube and centrifuged (200 g, 10 min) to recover platelet-rich plasma (PRP) as the supernatant. The PRP was centrifuged (800 g, 10 min) to remove the supernatant, and platelets were recovered as the precipitate. 1 mL of PBS was added to the platelet pellet and centrifuged (800 g, 10 min) to wash the platelets. Furthermore, a platelet suspension was prepared using 1 mL of ACD-A solution or PBS as a solvent. Platelet counts were counted using a bacteria counter (bacteria counter hemocytometer, Sunlead Glass Co., Ltd.), and a final platelet count of 1.0 × 10 8 The solution was diluted with PBS to give platelets / mL.

[0092] (Generation of rheumatoid arthritis model mice) Arthritis was induced in mice (SKG / Jcl, 7-week-old, female, CLEA Japan, Inc.) by a single intraperitoneal injection of 20 mg / 0.5 ml of laminarin. Mice aged 11 weeks or older, at which age arthritis develops, were used for platelet administration experiments. All experiments were performed at the Department of Rheumatology and Collagen Diseases, Osaka Medical and Pharmaceutical University.

[0093] [Production Example 1: Synthesis of FITC-labeled poly-L-lysine (PLL-F)] [ka]

[0094] A 4 mg / mL PLL solution was prepared by adding 500 mg of poly-L-lysine hydrochloride (MW = 12,000) to 125 mL of carbonate buffer, and a 1 mg / mL FITC solution was prepared by adding 79.43 mg of FITC to 79.43 mL of DMSO. The FITC solution was added to the PLL solution (ratio of lysine residues to FITC = 1:0.05) and stirred for 8 hours at 4°C in the dark. After the reaction, the pH was adjusted to 3 by dropwise addition of 3 M HCl, and the solution was then dialyzed against 0.1 M HCl for 48 hours (molecular weight cutoff = 1,000 Da). The reaction solution was then concentrated under reduced pressure and lyophilized to obtain a powdered product. 1 As a result of H-NMR measurement, the FITC modification rate was found to be 2.37% (Figure 1).

[0095] Example 1: Synthesis of MTX-conjugated FITC-labeled poly-L-lysine (PLL-F-MTX) [ka]

[0096] 50 mg of PLL-F (Production Example 1) was dissolved in 15 mL of ultrapure water to prepare a PLL-F aqueous solution. The specified amounts of methotrexate (MTX), DMT-MM, and NMM were added to the PLL-F aqueous solution in this order, and the mixture was stirred at room temperature for 4 hours in the dark. The ratios of MTX, DMT-MM, and NMM relative to the lysine residues in PLL are shown in Table 1. After the reaction, the pH of the reaction solution was adjusted to 3 by adding 3 M HCl dropwise. The reaction solution was dialyzed against 0.1 M HCl for 48 hours (molecular weight cutoff: 1000 Da), concentrated under reduced pressure, and lyophilized to obtain a powdered product. When the molar ratio of Lys residues:MTX:DMT-MM:NMM was 1:0.2:0.25:0.25 or 1:0.3:0.35:0.35, the viscosity of the solution increased with increasing MTX content, making it difficult to measure the MTX modification rate. Therefore, in the subsequent experiments, PLL-F-MTX prepared at the molar ratio of Lys residues:MTX:DMT-MM:NMM=1:0.1:0.15:0.15, as shown in Table 1, was used (Example 1). 1As a result of H-NMR measurement, the MTX modification rate of the PLL-F-MTX (Example 1) was 3.29% (FIG. 2).

[0097] [Table 1]

[0098] Example 2: Introduction of PLL-F-MTX into platelets PLL-F and PLL-F-MTX solutions (complex: 0.005 mM, residue: 0.5 mM) were prepared using PBS as a solvent. 100 μL of platelet suspension (1.0 × 10 8 The cells were mixed with 100 μL of PLL-F or PLL-F-MTX solution and incubated at 37° C. for 1 hour.

[0099] [Test Example 1: Comparison of solubility between PLL-F-MTX and MTX] PLL-F-MTX (Example 1) and MTX were dissolved in 1 mL of ultrapure water so that the MTX concentration was 1.1 mM, and the solution was centrifuged (800 g, 10 minutes).

[0100] As shown in Figure 3, MTX did not dissolve in pure water and precipitated at the bottom of the test tube, whereas the complex PLL-F-MTX was completely dissolved in pure water.

[0101] [Test Example 2: Fluorescence observation of PLL-F-MTX in platelets] Platelets contacted with PLL-F (Production Example 1) or PLL-F-MTX (Example 1) were collected by centrifugation (800 g, 10 min) and then washed by adding 1 mL of PBS and centrifugation (1500 g, 10 min). 50 μL of PBS was added to the platelet pellet to suspend the platelets, which were then added dropwise to aminated glass plates (13 mm, Matsunami Glass Industry Co., Ltd.). The aminated glass plates were then placed with the platelet suspension side facing down in each well of a Cell Imaging Plate (Eppendorf, I381901O) containing 10 μL of mounting solution (ProLong™ Diamond Antifade Mountant, ThermoFisher Scientific). The plates were then observed using a confocal laser scanning microscope (C2, Nikon Instruments) (Figure 4).

[0102] 4, the ratio of platelet brightness to background was 0.72 for untreated platelets, whereas it was 2.11 and 2.18 for platelets treated with PLL-F (Production Example 1) and PLL-F-MTX (Example 1), respectively. Since fluorescence was confirmed from the entire platelet, it is believed that the complex of the present invention is supported on the surface and / or inside the platelet.

[0103] [Test Example 3: Quantitation of PLL-F-MTX in platelets] Platelets contacted with PLL-F (Production Example 1) or PLL-F-MTX (Example 1) were collected by centrifugation (800 g, 10 minutes). 1 mL of PBS was added to the platelets, and they were washed by centrifugation (800 g, 10 minutes). 150 μL of 2% Triton-X / PBS was added to the platelet pellet, and the platelets were lysed by incubation (37°C, 1 hour). Fluorescence (excitation wavelength 488 nm, fluorescence wavelength 535 nm) of 150 μL of the resulting solution was measured (FIG. 5).

[0104] It can also be seen from the results shown in FIG. 5 that the complex of the present invention is supported on the surface and / or inside of platelets.

[0105] Test Example 4: Evaluation of the amount of PLL-F-MTX eluted from adenosine diphosphate (ADP)-activated platelets Platelets exposed to PLL-F or PLL-F-MTX were collected by centrifugation (800g, 10 min). The platelet pellet was washed by adding 1 mL of PBS and immediately centrifuging (800g, 10 min). 150 μL of 20 μM ADP / PBS (ADP+) or PBS (ADP-) was added to the platelet pellet and incubated (37°C, protected from light, 20 min). Each sample was centrifuged (800g, 10 min), and the fluorescence of the supernatant was measured (excitation wavelength 488 nm, emission wavelength 535 nm) (Figure 6).

[0106] As shown in FIG. 6, in the cell preparation or drug delivery system of the present invention, platelets are activated in response to external stimuli, and therefore, it is believed that they maintain their innate activation ability.

[0107] Test Example 5: Evaluation of migration of PLL-F-MTX-introduced platelets to sites of arthritis The cell preparation of the present invention was administered to mice as shown in Figure 7. Mice (BALB / C, retired, male, Japan SLC Co., Ltd.) were anesthetized by intraperitoneal administration of 0.3 mL (0.1 mL / kg of mouse) of a triple anesthetic mixture (1 mg / mL medetomidine hydrochloride: 5 mg / mL midazolam: butorphanol tartrate 5.0 mg / mL: physiological saline = 1.875 mL: 2 mL: 2.5 mL: 18.625 mL). Whole blood (10% sodium citrate blood (3.8% sodium citrate solution)) was then collected from the heart using a 24G needle and a 1 mL syringe. The collected blood was centrifuged (200 g, 10 min) to collect platelet-rich plasma (PRP) as the supernatant. The PRP was centrifuged (800 g, 10 min) to remove the supernatant and collect platelets as a precipitate. 1 mL of PBS was added to the platelet pellet, which was then centrifuged (800 g, 10 min) to wash the platelets. 1 mL of PBS was then added to the platelet suspension to prepare a platelet suspension. The platelet count was counted using a bacteria counter (bacteria counter hemocytometer, Sunlead Glass Co., Ltd.) to obtain a platelet count of 6.0 × 10 8The platelet suspension was diluted with PBS to a concentration of platelets / mL. 100 μL of the platelet suspension was mixed with 100 μL of PLL-F-MTX solution (0.005 mM complex, 100 μL) and incubated at 37°C for 1 hour in the dark. Platelets exposed to PLL-F-MTX were collected by centrifugation (800 g, 10 min). The platelet pellet was washed with 100 μL of PBS and centrifuged (800 g, 10 min), and then resuspended in 200 μL of PBS. The PLL-F-MTX-infused platelet suspension or PLL-F-MTX / PBS solution (0.005 mM complex, 200 μL) was administered via the tail vein to rheumatoid arthritis model mice using a 29G needle and a 1 mL syringe. Twenty-four hours after administration, the biodistribution of PLL-F-MTX was evaluated using an in vivo imaging system (IVIS Lumina XR, PerkinElmer) with the anesthetized mice lying face down (Figure 8).

[0108] As shown in Figure 8, no fluorescence was observed at the site of arthritis in the group administered with untreated platelets or the group administered with PLL-F-MTX alone. In contrast, in the group administered with a combination of PLL-F-MTX and platelets, fluorescence was detected at the site of arthritis, and no fluorescence was observed outside the inflammatory site. These results suggest that in the cell preparation or drug delivery system of the present invention, platelets maintain their ability to migrate to the site of inflammation, and this function allows drugs to accumulate at the site of inflammation. To the inventors' knowledge, these results are the first to report that platelets loaded with a polymer-drug conjugate can actually be administered in vivo and accumulate at the affected site while avoiding unwanted activation and thrombosis.

[0109] Example 3: Effect of mixed solution PLL-F-MTX was introduced into platelets in the same manner as in Example 2, except that PBS was replaced with physiological saline, physiological saline containing prostaglandin E1 (1 μM), or ACD-A solution containing citric acid and / or glucose. After introduction, PLL-F-MTX in the platelets was quantified in the same manner as in Test Example 3. The results of the PLL-F-MTX encapsulation rate in platelets are shown in Figure 9.

[0110] 9, the group in which the polymer-drug conjugate was contacted with platelets in the presence of citric acid and / or glucose exhibited a significantly improved encapsulation rate of the conjugate in platelets compared to the group in which the conjugate was contacted in their absence. Therefore, it is believed that contacting the polymer-drug conjugate with platelets in the presence of citric acid and / or glucose can enhance the prophylactic or therapeutic effect of the resulting cell preparation.

Claims

1. 1. A polymer-drug conjugate for use in a drug delivery system using platelets as drug carriers, comprising: The polymer-drug complex comprises (A) a cationic polymer and (B) an anti-inflammatory drug; The (A) cationic polymer and the (B) anti-inflammatory drug are bound to each other. Polymer-drug conjugates.

2. 2. The polymer-drug conjugate of claim 1, wherein the (A) cationic polymer and the (B) anti-inflammatory drug are bonded to each other via a covalent bond.

3. The (A) cationic polymer is polylysine, and The polymer-drug conjugate of claim 1 , wherein the (B) anti-inflammatory drug is methotrexate or a derivative thereof.

4. (C) a polymer-drug conjugate; and (C) platelets, The polymer-drug complex comprises (A) a cationic polymer and (B) an anti-inflammatory drug; The (A) cationic polymer and the (B) anti-inflammatory drug are bound to each other. Cell preparations.

5. The cell preparation according to claim 4 , wherein the polymer-drug complex is carried on the (C) platelets.

6. The cell preparation according to claim 4 or 5, wherein the polymer-drug complex has a solubility in water at 25°C of 100 mg / L or more.

7. The cell preparation according to claim 4 or 5, wherein the (A) cationic polymer is at least one polymer selected from the group consisting of polylysine, polyarginine, polyallylamine, polydiallyldimethylammonium chloride, cationized cellulose, cationized guar gum, polyethyleneimine, polyamidoamine, and chitosan.

8. The cell preparation according to claim 4 or 5, wherein the cationic polymer (A) is a polypeptide having at least a lysine residue.

9. The cell preparation according to claim 4 or 5, wherein the anti-inflammatory drug (B) is methotrexate or a derivative thereof.

10. A preventive or therapeutic agent for an inflammatory disease, comprising the cell preparation according to claim 4 or 5.

11. A preventive or therapeutic agent for rheumatoid arthritis, comprising the cell preparation according to claim 4 or 5.

12. Use of the polymer-drug conjugate according to any one of claims 1 to 3 or the cell preparation according to claim 4 or 5 in a drug delivery system using platelets as a drug carrier.

13. A drug delivery system using platelets as a drug carrier, comprising the polymer-drug conjugate according to any one of claims 1 to 3 or the cell preparation according to claim 4 or 5.

14. A method for producing a cell preparation, comprising: contacting the polymer-drug conjugate with (C) platelets; The polymer-drug complex comprises (A) a cationic polymer and (B) an anti-inflammatory drug; The (A) cationic polymer and the (B) anti-inflammatory drug are bound to each other. Manufacturing method.

15. The method of claim 14 , wherein the contacting step is carried out in the presence of citric acid and / or glucose.

Images