WO2019009434A1

WO2019009434A1 - Drug delivery polymer micelles

Info

Publication number: WO2019009434A1
Application number: PCT/JP2018/026250
Authority: WO
Inventors: 英正勝見; 昌山本
Original assignee: 学校法人京都薬科大学
Priority date: 2017-07-06
Filing date: 2018-07-06
Publication date: 2019-01-10
Also published as: JPWO2019009434A1

Abstract

The present invention relates to: a compound that is formed by linking the α carbonyl group of aspartic acid directly or via a linker to at least 50% of all of the terminal groups of a dendritic polymer that has a plurality of terminal groups by means of a peptide bond or an ester linkage, by linking a hydrophilic polymer segment directly or via a linker to at least 3% of the total of the terminal amino groups from the aspartic acid and the terminal groups of the dendritic polymer not modified with aspartic acid, and linking a hydrophobic segment directly or via a linker to at least 1% of the total of the terminal amino groups from the aspartic acid and the terminal groups of the dendritic polymer not modified with aspartic acid; and a drug delivery carrier that comprises polymer micelles that self-assemble from the compound. The drug delivery carrier forms micelles when mixed with a drug and thereby makes it possible for the drug to be selectively delivered to bones, which makes it possible to provide useful medicine for the treatment of various bone diseases.

Description

Polymeric micelles for drug delivery

The present invention relates to a polymeric micelle for drug delivery that enables osteoporation of a drug in vivo, and a drug comprising the drug encapsulated in the micelle.

Bone is a cancer metastasis-prone organ comparable to lung and liver, and in particular, breast cancer, prostate cancer, thyroid cancer, lung cancer and the like metastasize to bone frequently. Since bone metastasis is accompanied by bone-related events such as intolerable bone pain, pathological fracture, exercise restriction, hypercalcemia, etc., it significantly lowers the QOL of cancer patients and accelerates death. It is coveted.
In conventional bone metastasis treatment, radiation treatment, surgical treatment, administration of analgesics, etc. have been carried out, but all were symptomatic treatment aimed at suppressing pain due to spinal cord compression of cancer that had metastasized to bone. . In addition, since bone has a tissue structure in which drugs do not easily migrate, the bone transposability of the anticancer drug is insufficient, and bone metastasis treatment by conventional chemotherapy is extremely difficult, and the progression of cancer bone metastasis itself is suppressed There was almost no way to do it. In recent years, it has become clear that the growth and metabolism of cancer that has metastasized to bone depend on the growth factor produced from osteoclasts. Zoledronate, a bisphosphonate drug that strongly inhibits the function of cells, is used as a therapeutic agent for bone metastasis. However, zoledronate alone is rarely effective enough, and development of a new therapeutic method by constructing a delivery system for efficiently delivering a drug such as an anticancer drug to bone has been strongly desired.

As a delivery system for efficiently delivering a drug to bone, hitherto, a derivative in which tetracycline as a bone targeting element is directly coupled to a drug (carbonic anhydrase inhibitor) (Non-patent document 1), a bisphosphonate as a bone targeting element There are reports of a derivative (Anti-tumor drug) directly linked to a drug (antitumor drug) (Department 2), and a derivative (patent literature 1) obtained by directly coupling a bisphosphonate to an anticancer drug as a bone targeting element. In addition, it has also been reported that poly (lactic acid-co-glycolic acid) -block-poly (ethylene glycol) (PLGA-PEG) nanoparticles modified with aspartic acid exhibit bone transposition (Non-Patent Document 3) .

However, since derivatization in which a bone targeting element is directly attached to a drug can be applied only to a drug having a functional group to which the targeting element can be attached, there is a concern that pharmacological activity may be reduced due to chemical modification. In addition, conventional bone targeting elements such as tetracycline and bisphosphonates have problems such as pigmentation and aggregate formation in blood, and PLGA-PEG nanoparticles have very slow degradation in vivo. Drug release tends to be delayed. And all the above-mentioned conventional bone targeting techniques had problems, such as migration to organs other than a bone, and the targeting efficiency to a bone being low.

Japanese Patent Publication No. 2010-535701

Under such circumstances, there is an increasing demand for development of a carrier for drug delivery which is excellent in safety and biocompatibility, has a high efficiency of targeting to bone, and can be selectively transferred to bone.

The object of the present invention is to connect bone targeting aspartic acid as a bone targeting element to an end group of a dendritic polymer, and further, for bone targeting type drug delivery containing a hydrophilic polymer segment and a hydrophobic segment The purpose is to provide a polymeric micelle. In addition, the present invention provides a medicine for preventing or treating a bone disease, which comprises various drugs (in particular, hydrophobic drugs) encapsulated in the hydrophobic segment of the above-mentioned polymeric micelle without covalent bond. The purpose is

Under these circumstances, as a result of repeated investigations, the present inventors found that at least 50% of the total number of terminal groups of dendritic polymers having a plurality of terminal groups directly or via a linker, the α-carbonyl group of aspartic acid. Are linked by a peptide bond or an ester bond, and the hydrophilic amino acid is directly or via a linker to at least 3% of the total number of terminal amino groups derived from the aspartic acid and the terminal groups on the non-aspartic acid modified dendritic polymer. A compound in which a combined segment is bound and a hydrophobic segment is bound directly or via a linker to at least 1% of the total number of terminal groups on the terminal amino group and the non-aspartate-modified dendritic polymer ( Hereinafter, the “polymer of the present invention” may be referred to as “polymeric micelle” (hereinafter, may be referred to as “the polymeric micelle of the present invention”). And that it is possible to incorporate a drug into the interior (within the hydrophobic segment) of the polymeric micelle, and the polymeric micelle can achieve high selectivity for bone migration and high targeting efficiency to bone. For the first time to complete the present invention.

That is, the present invention is as follows.
[1] The α-position carbonyl group of aspartic acid is linked by a peptide bond or an ester bond directly or via a linker to at least 50% of the total number of terminal groups of the dendritic polymer having a plurality of terminal groups, The hydrophilic polymer segment is attached directly or via a linker to at least 3% of the total number of terminal groups on the terminal amino group of the terminal and the non-aspartate-modified dendritic polymer, and the terminal amino acid derived from the aspartic acid A compound comprising a hydrophobic segment bound to at least 1% of the total number of end groups on the group and the aspartate-free dendritic polymer directly or via a linker.
[2] The compound according to the above [1], wherein the hydrophilic polymer segment is a group derived from polyethylene glycol.
[3] The compound according to the above [1] or [2], wherein the hydrophobic segment is a residue of a sterol derivative.
[4] The compound according to the above [3], wherein the residue of the sterol derivative is derived from a compound selected from the group consisting of cholesterol, cholestanol, dihydroxycholesterol and cholic acid.
[5] The dendritic polymer is a dendrimer or dendron composed of polyamidoamine, a dendrimer or dendron composed of polylysine, a dendrimer composed of polyethylene glycol and 2,2-bis (hydroxymethyl) propanoic acid And the compound according to any one of the above [1] to [4], which is selected from the group consisting of dendrimers composed of 2,2-bis (hydroxymethyl) propanoic acid or dendrons.
[6] A pharmaceutical composition comprising the compound according to any one of the above [1] to [5], and a drug.
[7] The above-mentioned [6], wherein the drug is at least one selected from the group consisting of an osteoporosis therapeutic drug, a rheumatoid arthritis therapeutic drug, an anticancer drug, an anti-inflammatory drug, an antioxidant, a nucleic acid drug, a radiopharmaceutical and a contrast agent. Pharmaceutical composition as described in-.
[8] A polymeric micelle containing the compound according to any one of the above [1] to [5].
[9] The polymeric micelle of the above-mentioned [8], which further comprises a drug.
[10] The above-mentioned [9], wherein the drug is at least one selected from the group consisting of an osteoporosis therapeutic drug, a rheumatoid arthritis therapeutic drug, an anticancer drug, an anti-inflammatory drug, an antioxidant, a nucleic acid drug, a radiopharmaceutical and a contrast agent. Polymeric micelles described in.
[11] A carrier for drug delivery, comprising the polymeric micelle according to the above-mentioned [8], for selectively delivering a drug to a target tissue in vivo.
[12] The drug delivery carrier according to the above [11], wherein the target tissue is a bone.
[13] A medicament comprising the polymeric micelle according to the above [9] or [10].
[14] The medicine according to the above-mentioned [13], which is an agent for the prophylaxis or treatment of a bone disease.

The compounds of the present invention can form polymeric micelles alone or together with a drug, said polymeric micelles exhibiting high bone migration selectivity and high targeting efficiency to bone, drug release Being excellent in sex, it is extremely useful as a preventive agent (test drug) or a therapeutic agent for various bone diseases. In addition, since the compound and polymer micelle of the present invention are derived from a living body and use aspartic acid having little aggregation in the living body as a bone targeting element, they are excellent in biocompatibility and safety, and It has the advantage of being able to be easily synthesized, etc., and is expected to find wide application as a practical drug delivery carrier.

a shows the pharmacokinetics of labeled PTX-PAMAM micelle ( ¹¹¹ In labeled PAMAM micelle part), b shows the pharmacokinetics of labeled PTX-compound 1a micelle ( ¹¹¹ In labeled compound 1a micelle part). a represents the pharmacokinetics of ³ H-labeled PTX which is contained in the labeled PTX-PAMAM micelles, b shows the pharmacokinetics of ³ H-labeled PTX which is enclosed in labeled PTX- compound 1a micelles. The fluorescence emission site by xylenol orange of a and a 'indicates the bone formation site in bone, respectively, and b and b' indicate fluorescence emission from FITC labeled compound 1a micelle and FITC labeled PAMAM micelle in bone respectively (intrabone C) and c 'show confocal laser scanning micrographs in which a and a' and b and b 'are superimposed. Figure 7 shows the results of treatment experiments for cancer bone metastases of PTX-compound 1a micelles. It shows a SPECT / CT imaging images of the organ distribution after intravenous injection of ¹¹¹ In-labeled body portion excluding the hydrophobic segment of compound 1a. Fig. 7 shows the intraosseous distribution of FITC-labeled Compound 1a micelles and FITC-labeled PAMAM micelles in a bone metastasis model.

The details of the present invention will be described below.

(Definition)

In the present specification, a dendritic polymer in the “dendritic polymer having a plurality of end groups” means a dendrimer or a dendron, and the end group is a functional group present at the end of each branch of the dendrimer or the dendron. And may be a nucleophilic group (eg, an amino group, a hydroxy group, a mercapto group, etc.) or an electrophilic group (eg, a formyl group, a carbonyl group, etc.).
The dendritic polymer is not particularly limited as long as it has a plurality of terminal groups, but preferably, a dendrimer or dendron composed of polyamidoamine, a dendrimer or dendron composed of polylysine, polyethylene glycol and 2, Dendrimers or dendrons composed of 2-bis (hydroxymethyl) propanoic acid dendron, or dendrimers or dendrons composed of 2,2-bis (hydroxymethyl) propanoic acid dendron (eg, commercially available from Sigma-Aldrich) 2, 2-bis (hydroxymethyl) propanoic acid dendron etc.), and what has an amino group, a hydroxyl group, etc. as a several terminal group is mentioned. A more preferred dendritic polymer is a dendrimer consisting of a polyamide amine having an alkyl diamine (eg, ethylene diamine, 1,4-diaminobutane, 1,6-diaminohexane, 1,12-diaminododecane, etc.) as a core (PAMAM) Or a dendrimer consisting of polylysine having an alkyl diamine (eg, 1,6-diaminohexane etc.) as the core (eg, polylysine dendrimer described in M. Ohsaki et al., Bioconjugate Chem., 2002, 13, 510-517 etc. And polylysine (for example, polylysine dendron described in K. L. Chang et al., J. Control. Release. 2011, 156, 195-202, etc.), and as a plurality of terminal groups, amino Group or group It includes those having a proxy group. Among them, PAMAM-NH ₂ dendrimers in which terminal groups are amino groups (eg, first to fifth generation PAMAM-NH ₂ dendrimers commercially available from Sigma-Aldrich) are particularly preferable. In addition, each structural unit (core and branch portion) constituting the above dendrimer or dendron is, for example, a C _1-12 alkyl within a range not adversely affecting the object of the present invention (for example, within a range not reacting with drugs). It may be substituted by a substituent such as a group or a halogen atom (eg, a fluorine atom etc.).
The molecular weight of such a dendritic polymer is not limited as long as it can form a drug-incorporated polymeric micelle, but is about 1000 to 30000 Da, preferably about 3000 to 15000 Da, more preferably about 3000 It is ~ 7000 Da.

In the present specification, "at least 50% of the total number of terminal groups" means at least 50% (50% or more) with respect to the total number of terminal groups of the dendritic polymer (ie, functional groups present at the ends of each branch). The term "end group" means a number of end groups.

In the present specification, “linker” refers to covalently linking (by peptide bond or ester bond) an α-carboxy group of aspartic acid to an end group of a dendritic polymer, or a hydrophilic polymer segment Covalently linking the aspartate-derived terminal amino group of the aspartate-modified moiety on the linear polymer and / or the end group on the non-aspartate-modified dendritic polymer, or A chain of atoms covalently linked to an aspartate-derived terminal amino group of the aspartate-modified moiety on the dendritic macromolecule and / or an end group on the aspartate-free dendritic macromolecule or By means of a bifunctional (homobifunctional or heterobifunctional) or multifunctional chemical moiety comprising a covalent bond.
The linker reagent that forms the bifunctional linker is an electrophilic group that reacts with a nucleophilic group such as an amino group or a hydroxy group present at each end on the aspartate-modified or non-aspartate-modified dendritic polymer. May be included. Examples of such an electrophilic group include, but are not limited to, haloalkyl group, halocarbonyl group, carboxyl group, NHS ester, maleimide group and haloacetamide group.
In another embodiment, the linker reagent has a reactive nucleophile capable of reacting with electrophilic groups (end groups) present at each end on the non-aspartic acid modified dendritic polymer; Form a covalent bond. Such electrophilic groups include, but are not limited to, formyl groups (aldehydes) and carbonyl groups (ketones). Useful nucleophilic groups on the linker include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate and aryl hydrazide.
Linker reagents that form the bifunctional linker are commercially available from various reagent companies. For example, Pierce Inc. And Sigma-Aldrich Co. LLC. Please refer to the catalog etc. Also, bifunctional linkers can be easily introduced by those skilled in the art by methods known per se.
Examples of multifunctional linkers (branched linkers) include chemical moieties derived from pentaerythritol, cysteine, diaminopropionic acid, diaminobutanoic acid, ornithine, lysine, homocysteine, maleimide acid and the like.
Specific ^{linkers, -CO -; - COO -;} - OCO -; - CO-NR 1 -; - NR 1 CO -; - OC-NR 1 -CO- ( wherein, ^{R 1} represents a hydrogen atom Or a C _1-6 alkyl group)); one or more groups selected from the group consisting of -O-, -S-, -COO-, -CO-, -NH- and -CONH- are internally or terminally Alkanediyl (eg, C _1-12 alkanediyl) which may be possessed by arylene, heteroarylene, arylenedicarbonyl (eg, benzene-1,4-dicarbonyl), alkyloxy and alkylamino repeating groups For example, ethylene glycol, triethylene glycol, tetraethylene glycol, propylene glycol, dipropylene glycol, tripropylene glycol, butylene glycol, methyle Glycol, dimethylene glycol, ethylenediamine, 1,3-propylenediamine, 1,6-hexylene diamine, diethylene triamine, triethylene tetramine, Jeffamine (JEFFAMINE (registered trademark)); and, succinate (-OOC-CH ₂ CH ₂ -COO-), succinamide _{_{(-HNOC-CH 2 CH 2 -CONH-}} ), -COO- glutarate _{_{_{(-OOC-CH 2 CH 2 CH}}} 2), adipates _{_{_{(-OOC-CH 2 CH 2 CH}}} 2 CH 2 -COO-), diglycolate _{_{(-OOC-CH 2 -O-CH}} 2 COO-), diacid ester and amides including terephthalate or the like and the like can be mentioned. specific examples of the multifunctional linker, limited in particular But not, for example,

The chemical moiety represented by can be mentioned.

In the present specification, “aspartate-modified” and “aspartate-unmodified” mean that the terminal group of the dendritic polymer and the α-position carboxy group of aspartate are peptides directly or via a linker, respectively. It means a state of being linked by a bond or an ester bond, and a state in which aspartic acid is not linked to an end group of the dendritic polymer.

As used herein, "hydrophilic polymer segment" refers to a water-soluble polymer-derived moiety. The hydrophilic polymer segment is not particularly limited, and examples thereof include segments derived from polyethylene glycol, polyacrylamide, polymethacrylamide and the like, or derivatives thereof. Among them, segments derived from polyethylene glycol or derivatives thereof (ie, groups derived from polyethylene glycol) are preferred. Specific examples of segments derived from polyethylene glycol or derivatives thereof are derived from, for example, amine-reactive PEGylation reagents (eg, PEG-NHS esters such as SUN BRIGHT (registered trademark) ME-020CS, ME-020AS, etc. manufactured by NOF Corporation) Segment having a carbonyl group at one end of polyethylene glycol (-CO- (CH ₂ ) ₂ -CO- (O-CH ₂ -CH ₂ ) _n -OR ² (where n is 30 or more) R ² represents an integer, and R ² represents a hydrogen atom or a C _1-6 alkyl group (preferably, a methyl group)) and the like.
Such a hydrophilic polymer segment may be directly or as a linker to the aspartate-derived terminal amino group of the aspartate-modified moiety on the dendritic polymer and / or the end group on the aspartate-free dendritic polymer. They are linked via the bifunctional linker or multifunctional linker). The terminal amino group of the aspartate-modified portion of the hydrophilic polymer segment on the dendritic polymer and the introduction ratio of the terminal amino group of the hydrophilic polymer segment to the end group on the non-aspartate-modified dendritic polymer are on the dendritic polymer. It is at least 3%, preferably about 3 to 30%, more preferably, based on the total number of terminal amino groups of the aspartic acid-modified moiety and the terminal groups on the non-aspartic acid-modified dendritic polymer. Is about 10 to 15%.
The molecular weight (average molecular weight) of the hydrophilic polymer segment is not particularly limited, but is about 500 to 20000 Da, preferably about 1000 to 5000 Da, and more preferably about 2000 to 5000 Da. .

As used herein, "hydrophobic segment" means a moiety having hydrophobicity. The hydrophobic segment is not particularly limited, and examples thereof include residues of sterol derivatives, C _10-24 hydrocarbyl group, polylactic acid, polylactic acid / glycolic acid copolymer, polyamino acid and the like. Among them, segments consisting of residues of sterol derivatives are preferred. Such sterol derivatives are preferably cholesterol, cholestanol, dihydroxycholesterol or cholic acid, or those cyclopentanone hydrophenanthrene rings are saturated or unsaturated and do not adversely affect the object of the present invention In the scope, the ring may be a compound which may be substituted by a substituent such as a C _1-12 alkyl group, a halogen atom (eg, a fluorine atom etc.), and the residue of the sterol derivative is 3 of the sterol derivative. It is a group from which the hydrogen atom of the hydroxy group is removed. As the hydrophobic segment, particularly preferred is a group from which the hydrogen atom of the 3-hydroxy group of cholesterol or cholic acid has been removed.
Such a hydrophobic segment may be directly or as a linker to the terminal amino group derived from aspartate of the aspartate-modified moiety on the dendritic polymer and / or the end group on the non-aspartate-modified dendritic polymer Preferably, an amino group derived from aspartic acid or a terminal amino group on a dendritic polymer not modified with aspartic acid and a linker (eg, a carbonyl group (for example, a carbonyl group (- CO-), linked via a lysine-derived trifunctional linker _{_{(-NH- (CH 2) 4 CH}} (NH-) CO-) , etc.). The rate of introduction of such a hydrophobic segment into the terminal amino group of the aspartic acid-modified portion on the dendritic polymer and the terminal group on the non-aspartic acid-modified dendritic polymer is the terminal amino group derived from aspartic acid and It is at least 1%, preferably about 1 to 20%, more preferably about 1 to 5%, based on the total number of terminal groups on the non-aspartic acid modified dendritic polymer.

In the present specification, examples of the "drug" include an osteoporosis therapeutic drug, a rheumatoid arthritis therapeutic drug, an anticancer drug, an anti-inflammatory drug, an antioxidant, a nucleic acid drug, a radioactive drug, an imaging agent and the like. The type of drug is not particularly limited, but hydrophobic drugs are preferably used. Specific examples of the drug are shown below, but are not limited to the following specific examples.

As an osteoporosis treatment agent, for example, bisphosphonate, estriol, estradiol, raloxifene, bazedoxifene, denosumab, elcatonin, salmon calcitonin, ipriflavone, teriparatide, calcitriol, alfacalcidol, eldecalcitol, menatetrenone, W9 (bone resorption inhibition peptide) , MG-132 (cathepsin inhibitor) and the like.

As a therapeutic agent for rheumatoid arthritis, for example, bisphosphonate, gold thiomalic acid, auranofin, penicillamine, sulfazosulfapyridine, lobenzarit, actarit, iglatimod, methotrexate, mizoribine, leflunomide, tacrolimus, tofacitinib, infliximab, etanercept, adalimumab, Golimumab, Certolizumab pegol, tocilizumab, apatacept, loxoprofen, celecoxib, prednisolone and the like.

As an anticancer agent, for example, BCG, actinomycin D, asparaginase, acegraton, anastrozole, allopurinol, anthracycline, bicalutamide, antiandrogen, idarubicin, ifosfamide, imatinib, irinotecan, interferon, interferon alpha, interleukin-2 and ubenimex, Exemestane, Estramustine, Estrogen, Etoposide, Enocitabine, Epirubicin, Oxaliplatin, Octreotide, Capeciotabine, Capecitabine, Carbocon, Carboplatin, Carmofur, Cladribine, Clarithromycin, Krestin, Ketoconazole, Gemitinib, Gemcitabine, Gemtuzumab, Cycloserum, Cyclophosphamide Cisplatin, Schizophyllan, Cytarabine, Cipro Putadine, dinostatin stimaramamer, cetuximab, sobuzoxane, tamoxifen, daunorubicin, dacarbazine, daculazine, dactinomycin, thiothepa, tegafur, tegafur uracil, tegafur guimeracil oteracil potassium, dexamethasone, topotecan, trastuzumab, triptreintreme, Doxyfluridine, doxorubicin, docetaxel, nemustine, neocarzinostatin, nedaplatin, paclitaxel, hydroxyurea, hydroxycarbamide, bicalutamide, vinorelbine, vincristine, vindesine, vinblastine, picaribanil, pirarubicin, faurouracil, flutamide, fludarabine, fludrobine, ,Professional Rubazine, progestin, pepromycin, pentostatin, porfimer sodium, mitomycin, mitoxantrone, mitotane, mestane, methotrexate, medroxyprogesterone, mercaptopurine, melphalan, lanimustine, rituximab, leuprolide, lentinan, leucovorin etc. . The above anticancer agents may be used alone or in combination of two or more.

Anti-inflammatory agents include steroidal anti-inflammatory agents and non-steroidal anti-inflammatory agents.
Examples of steroidal anti-inflammatory agents include corticosteroid anti-inflammatory agents such as dexamethasone, triamcinolone acetonide, beclomethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone diacetate, cortisone, cortisol, methazone, triamcinolone, Diflucortrone, difluprednate, diflorazone, flumethasone, fluocinonide, fluocinolone acetonide, alclomethasone, fludrocortisone etc. or salts thereof. More specifically, dexamethasone, triamcinolone acetonide, beclomethasone propionate, hydrocortisone succinate, methylprednisolone succinate, dexamethasone acetate, hydrocortisone acetate, prednisolone acetate, dexamethasone metasulfobenzoic acid, triamcinolone diacetate, butyl acetate prednisolone, Acid dexamethasone, hydrocortisone phosphate, prednisolone phosphate, betamethasone phosphate, prednisolone succinate, cortisone acetate, paramethasone acetate, methylprednisolone acetate, triamcinolone acetate, triamcinolone, hydrocortisone, prednisolone, betamethasone, prednisolone valerate, valeric acid diflucorthrone, , Betamethasone valerate, difluprenate acetate, diflorazone acetate, di Luprednate, betamethasone dipropionate, flumethasone pivalate, fluocinonide, fluocinolone acetonide, fluocinolone acetonide, alclomethasone propionate, beclomethasone propionate, hydrochlorotin butyrate, hydrocortisone butyrate, hydrocortisone butyrate, fludrocortisone butyrate, dexamethasone palmitate, methylprednisolone etc Can be mentioned.

Examples of non-steroidal anti-inflammatory drugs include NSAIDs and COX-2 inhibitors. More specifically, for example, acetylsalicylic acid, alclofenac, aluminoprofen, benoxaprofen, butybufen, buclofenic acid, carprofen, celecoxib, clidenac, diclofenac, diflunisal, etodolac, fenbufen, fenoprofen, fentiacic, fluphenamine Acids, flufenasol, flurbiprofen, flofenac, ibuphenac, ibuprofen, indomethacin, indoprofen, indoprofen, isoxepac, isoxicam, ketoprofen, ketorofen, ketorolac, meclofenamic acid, mefenamic acid, meloxicam, myloprofen, naproxen, oxaprozin, oxyphenbutazone Oxypinac, parecoxib, phenylbutazone, picramilast, piroxicam, pilprofe , Pranoprofen, rofecoxib, sudoxicam, sulindac, suprofen, Teng diclofenac, tiaprofenic acid, tolfenamic acid, tolmetin, tramadol, valdecoxib, zomepirac and the like, or salts thereof.

Examples of the antioxidant include superoxide dismutase, catalase, nitric oxide donor, hydrogen sulfide donor, curcumin, coenzyme Q10, astaxanthin, α-tocopherol, α-tocopherol derivative and the like.

Examples of nucleic acid drugs include siRNA, plasmid DNA, mRNA and the like.

Examples of radioactive agents and contrast agents include yttrium Y-90, gadolinium Ga-67, gadolinium Ga-68, lutetium Lu-177, copper Cu-64, technetium Tc-99, rhenium Re-186 or rhenium Re-188, Radioactive iodine-131 and the like can be mentioned. The radioactive agent and the contrast agent may be used alone or in combination with other drugs.

In the present specification, the “bone disease” may be any disease as long as it develops in association with failure of bone metabolism, acceleration of bone destruction, etc. For example, osteoporosis, periodontal disease, bone metastasis of cancer, chronic Rheumatoid arthritis, osteoarthritis, osteomalacia, hyperparathyroidism, Paget's disease and the like can be mentioned.
In addition, in the present specification, “prevention or treatment” is only required to suppress the occurrence of a symptom associated with failure of bone metabolism, acceleration of bone destruction, etc. or to maintain or suppress the progression, and clearly distinguish between prevention and treatment. It does not have to be done.

In the present specification, “polymer micelle” refers to a molecular assembly formed by mixing a composition of the present invention, or a composition containing the compound of the present invention and a drug in an aqueous medium and self-assembling. The hydrophilic polymer segment and the β-position carboxy group of aspartic acid are disposed outside the micelle (at the interface with the aqueous medium), and the hydrophobic segment and the drug are disposed inside.

As used herein, the term "bone targeting element" refers to a biological recognition function that can specifically bind to bone to form a biological binding pair with the compound of the present invention, polymeric micelle or drug. Means a site having The bone targeting element in the present invention is an aspartic acid modified site (in particular, a β-carboxy group) of the terminal group of the dendritic polymer.

The introduction rate of aspartate to the end groups of the dendritic polymer (aspartate modification rate) required for the compound of the present invention or the medicament of the present invention to exhibit high bone transposition and bone selectivity is the total number of end groups To at least 50%, preferably at least 60%, more preferably at least 70%, particularly preferably at least 90%.

In the present specification, “encapsulating a drug” refers to noncovalently encapsulating a drug molecule within the carrier for drug delivery of the present invention (ie, polymer micelle for drug delivery) by hydrophobic interaction. means. This enables excellent drug release at the target site (bone) in vivo.

The entrapment rate of the drug of the present invention is in the same range as the introduction rate of the hydrophobic segment, and the terminal amino group of the aspartic acid-modified portion of the dendritic polymer and the dendritic high without aspartic acid modification. The total number of end groups on the molecule is at least 1%, preferably about 1 to 20%, more preferably about 1 to 5%.

The following compounds are preferable as the compound of the present invention.
[Compound (A)]
Of at least 50% (preferably at least 60%, more preferably at least 70%, particularly preferably at least 90%) of the total number of end groups of the dendritic polymer having a plurality of end groups directly or via a linker 3-30% (more preferably 10) of the total number of terminal amino groups derived from the aspartic acid and the terminal groups on the non-aspartic acid-modified dendritic polymer, wherein the α-position carbonyl group is linked by a peptide bond or an ester bond ) Or a hydrophilic polymer segment which is a group derived from polyethylene glycol, polyacrylamide or polymethacrylamide is directly or via a linker and is bonded to the aspartic acid-derived terminal amino group and aspartic acid modified No more than 1 to 20% (more preferably 1 to 5%) of the total number of end groups on the dendritic polymer. And the hydrophobic segment is a residue of a sterol derivative formed by bonding, the compounds of the present invention.

[Compound (B)]
Of at least 50% (preferably at least 60%, more preferably at least 70%, particularly preferably at least 90%) of the total number of end groups of the dendritic polymer having a plurality of end groups directly or via a linker 3-30% (more preferably 10) of the total number of terminal amino groups derived from the aspartic acid and the terminal groups on the non-aspartic acid-modified dendritic polymer, wherein the α-position carbonyl group is linked by a peptide bond or an ester bond ~ 15%) directly or via a linker, the formula:

(Here, n represents an integer of 30 or more, R ² represents a hydrogen atom or a C _1-6 alkyl group.) A hydrophilic polymer segment represented by Terminal amino group and 1 to 20% (more preferably 1 to 5%) of the total number of terminal groups on the dendritic polymer not modified with aspartic acid (cholesterol, cholesterol) via a linker (eg, carbonyl group) The compound of the present invention, which comprises a hydrophobic segment attached, which is a residue of a compound selected from the group consisting of stanol, dihydroxycholesterol and cholic acid.

[Compound (C)]
2, a dendrimer composed of polyamidoamine (or dendron), a dendrimer composed of polylysine (or dendron), a dendrimer composed of polyethylene glycol and 2,2-bis (hydroxymethyl) propanoic acid, and 2, At least 50% (preferably 60% or more, more preferably 70% or more) of the total number of terminal groups of the dendritic polymer selected from the group consisting of dendrimers (or dendrons) composed of 2-bis (hydroxymethyl) propanoic acid Particularly preferably 90% or more) directly or via a linker, the α-position carbonyl group of aspartic acid is linked by a peptide bond or an ester bond, and the terminal amino group derived from the aspartic acid and the aspartic acid non-modified dendritic 3 to 30 of the total number of end groups on the polymer (More preferably, 10-15%) to, directly or via a linker, wherein:

[Compound (D)]
At least 50% (preferably 60% or more, more preferably 70% or more) of the total number of terminal groups of the dendritic polymer that is a dendrimer (or dendron) composed of polyamidoamine or a dendrimer (or dendron) composed of polylysine Particularly preferably, the α-position carbonyl group of aspartic acid is linked by peptide bond directly or via a linker to 90% or more), and the terminal amino group derived from the aspartic acid and on the dendritic polymer which is not aspartic acid modified 3-30% (more preferably 10-15%) of the total number of end groups, directly or via a linker, of the formula:

[Compound (E)]
At least 50% (preferably 60% or more, more preferably 70% or more, particularly preferably 90% or more) of the total number of terminal amino groups of the dendritic polymer which is a dendrimer composed of polyamidoamine or a dendrimer composed of polylysine (3-30% of the total number of terminal amino groups derived from the aspartic acid and terminal amino groups on the non-aspartic acid modified dendritic polymer) in which the α-position carbonyl group of aspartic acid is directly linked to the Preferably, 10 to 15%) directly to the formula:

(Here, n represents an integer of 30 or more, R ² represents a hydrogen atom or a C _1-6 alkyl group.) A hydrophilic polymer segment represented by Terminal amino group and 1 to 20% (more preferably 1 to 5%) of the total number of terminal amino groups on the dendritic polymer not modified with aspartic acid (preferably, 1 to 5%), cholesterol via a linker (eg, carbonyl group), A compound of the present invention comprising a hydrophobic segment attached, which is the residue of a compound selected from the group consisting of cholestanol, dihydroxycholesterol and cholic acid.

The following compounds are also suitable as a compound of the present invention.
[Compound (A ')]
3 or more of the total number of terminal amino groups derived from aspartic acid of a compound in which the α-position carbonyl group of aspartic acid is linked by a peptide bond or an ester bond directly or via a linker to terminal groups of dendritic polymers having multiple terminal groups A hydrophilic polymer segment which is a group derived from polyethylene glycol, polyacrylamide or polymethacrylamide is directly or via a linker to ̃30% (more preferably 10 to 15%), and is derived from the aspartic acid The compound of the present invention, wherein a hydrophobic segment which is a residue of a sterol derivative is bonded to 1 to 20% (more preferably 1 to 5%) of the total number of terminal amino groups via a linker.

[Compound (B ')]
3 or more of the total number of terminal amino groups derived from aspartic acid of a compound in which the α-position carbonyl group of aspartic acid is linked by a peptide bond or an ester bond directly or via a linker to terminal groups of dendritic polymers having multiple terminal groups ~ 30% (more preferably 10-15%), directly or via a linker, of the formula:

(Here, n represents an integer of 30 or more, R ² represents a hydrogen atom or a C _1-6 alkyl group.) A hydrophilic polymer segment represented by A compound selected from the group consisting of cholesterol, cholestanol, dihydroxycholesterol and cholic acid via a linker (eg, carbonyl group) in 1 to 20% (more preferably 1 to 5%) of the total number of terminal amino groups The compound of this invention which the hydrophobic segment which is a residue of is combined.

[Compound (C ')]
2, a dendrimer composed of polyamidoamine (or dendron), a dendrimer composed of polylysine (or dendron), a dendrimer composed of polyethylene glycol and 2,2-bis (hydroxymethyl) propanoic acid, and 2, The α-position carbonyl group of aspartic acid is a peptide bond directly or via a linker to the terminal group of a dendritic polymer selected from the group consisting of dendrimers (or dendrons) composed of 2-bis (hydroxymethyl) propanoic acid Or 3 to 30% (more preferably 10 to 15%) of the total number of terminal amino groups derived from aspartic acid of the compounds linked by an ester bond, directly or via a linker,

[Compound (D ')]
The α-position carbonyl group of aspartic acid is linked by a peptide bond directly or via a linker to the terminal group of a dendritic polymer that is a dendrimer (or dendron) composed of polyamidoamine or a dendrimer (or dendron) composed of polylysine 3 to 30% (more preferably 10 to 15%) of the total number of terminal amino groups derived from aspartic acid of the linked compounds, directly or via a linker, to the formula:

[Compound (E ')]
The α-position carbonyl group of aspartic acid is directly linked by a peptide bond to the terminal amino group of the dendritic polymer which is a dendrimer composed of polyamidoamine or a dendrimer composed of polylysine, and the terminal amino group derived from this aspartic acid Directly to the formula: 3-30% (more preferably 10-15%) of the total number

(Here, n represents an integer of 30 or more, and R ² represents a C _1-6 alkyl group.) The hydrophilic polymer segment represented by is bonded, and the terminal amino group derived from the aspartic acid Residue of a compound selected from the group consisting of cholesterol, cholestanol, dihydroxycholesterol and cholic acid via a linker (eg, carbonyl group) in 1 to 20% (more preferably 1 to 5%) of the total number of The compound of this invention which the hydrophobic segment which is is couple | bonded.

The average molecular weight of the compound of the present invention is 4,000 or more, preferably 15,000 or more, and there is no particular upper limit, but 40,000 or less is desirable from the viewpoint of handling ease.

The compounds of the present invention also include those in the form of salts. Examples of the salt of the compound of the present invention include salts with inorganic acids, salts with organic acids, salts with inorganic bases, salts with organic bases, salts with amino acids and the like.

Examples of salts with inorganic acids include salts with hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, perchloric acid and the like.
Examples of salts with organic acids include acetic acid, trifluoroacetic acid, trichloroacetic acid, propionic acid, oxalic acid, maleic acid, citric acid, fumaric acid, lactic acid, malic acid, succinic acid, tartaric acid, gluconic acid, ascorbic acid, Examples thereof include salts with methanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like.
Examples of salts with inorganic bases include sodium salts, potassium salts, calcium salts, magnesium salts, ammonium salts and the like.
As salts with organic bases, for example, methylamine, diethylamine, trimethylamine, triethylamine, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, tris (hydroxymethyl) methylamine, dicyclohexylamine, N, N'-dibenzylethylenediamine, guanidine And salts with pyridine, picoline, choline, cinchonine, meglumine and the like.
Examples of salts with amino acids include salts with lysine, arginine, aspartic acid, glutamic acid and the like.

The compounds of the present invention also include those in the form of solvates. A solvate of the compound of the present invention is a compound of the present invention in which a molecule of a solvent is coordinated, and also includes hydrates. For example, hydrates, ethanolates, dimethyl sulfoxide solvates and the like of the compound of the present invention or a salt thereof can be mentioned.

The compounds of the present invention may be radiopharmaceuticals, contrast agents or isotopes (eg ³ H, ² H (D), ¹⁴ C, ³⁵ S, ⁹⁰ Y, ¹¹¹ In, ⁶⁷ Ga, ⁶⁸ Ga, ¹⁷⁷ Lu, ⁶⁴ Cu, ⁹⁹ Tc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹³¹ I, ¹⁸ F, etc.). As a specific example, for example, a chelate group (eg, diethylenetriamine-N, N, N ', N'',N''-pentaacetic acid (DTPA) group, etc.) is introduced into a part of the terminal group of the compound of the present invention Also, compounds in which ¹¹¹ In is chelate-labeled and compounds labeled with fluorescein isothiocyanate (FITC) are included in the compounds of the present invention.

(Synthesis of the compound of the present invention)
The method for producing the compound of the present invention is not particularly limited, but can be synthesized, for example, through the following reactions.

Starting compounds are readily commercially available as commercially available products unless otherwise noted, or alternatively, methods known per se (eg, Ohsaki, M. et al., Bioconjugate Chem. 2002, 13, 510-517; Tomalia, D. et al. A. et al., Polymer Journal, 1985, 17, 117-132; Chang, K. L. et al., J. Control. Release. 2011, 156, 195-202 etc.) or a method analogous thereto. be able to.

In each of the following steps, the protection or deprotection reaction of a functional group is carried out according to a method known per se, for example, Protective Groups in Organic Synthesis, 4th Ed. , Theodora W. Greene, Peter G. M. It carries out according to the method described in Wuts, Wiley-Interscience (2007) etc., or the method described in the Example of this specification.

In addition, the compound obtained at each process in the following reaction formula can also be used for the next reaction as a reaction liquid or as a crude product. Alternatively, the compound can be isolated from the reaction mixture according to a conventional method, and can be easily purified by a conventional separation means such as recrystallization, distillation, chromatography and the like.

The compounds of the present invention can be produced, for example, by the following production methods 1, 2 and the like.
(Method 1)

(Wherein, L ¹ , L ² and L ³ may be the same or different and each independently represents a linker, R ^a represents an amino group or a hydroxy group, and P and P ′ are the same or And X ¹ and X ² may be the same or different, each independently represents a leaving group, and Z ¹ , Z ² and Z ³ may be different from each other. It may be the same or different and each independently represents an NH or oxygen atom, S ¹ represents a hydrophilic polymer segment, S ² represents a hydrophobic segment, m 1, m 2 and m 3 each represent Independently represent 0 or 1, n2, n3 and n4 each independently represent an integer of 1 or more, n3a, n3b, n4a and n4b each independently represent an integer of 0 or more, and n1 represents , An integer of 8 or more, provided that n1 is equal to or greater than the sum of n2, n3 and n4, an n3 = n3a + n3b, as well as n4 = n4a + n4b.)

Step 1
In this step, after dehydration condensation of the terminal amino group or hydroxy group of the dendritic polymer (1) and the α-position carboxy group of aspartic acid (compound 2) in which the amino group and the β-position carboxy group are protected This is a step of converting into compound 3 by subjecting it to a deprotection reaction.
The reaction is carried out in a solvent which does not affect the reaction, using dehydration condensation reaction conditions known per se and deprotection conditions.

The amount of compound 2 to be used is generally 0.5 to 3 mol, preferably 1 to 1.5 mol, per the total number (1 mol) of the terminal groups of the dendritic polymer (1).

As a condensing agent used for dehydration condensation reaction, for example, 1-ethyl-3- (3′-dimethylaminopropyl) carbodiimide (WSC), dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC), N-ethyl- N'-3-dimethylaminopropyl carbodiimide and its hydrochloride salt (EDC.HCl), hexafluorophosphoric acid (benzotriazol-1-yloxy) tripyrrolidinophosphonium (PyBop), O- (benzotriazol-1-yl)- N, N, N ', N'-tetramethyluronium tetrafluoroborate (TBTU), 1- [bis (dimethylamino) methylene] -5-chloro-1H-benzotriazolium 3-oxide hexafluorophosphate (HCTU ), O-benzotriazole-N, , N ', N'- tetramethyluronium hexafluorophosphate borate (HBTU) and the like.
In the condensation step, if necessary, a condensation additive (eg, 1-hydroxybenzotriazole (HOBt), 1-hydroxy-1H-1,2,3-triazole-5-carboxylic acid ethyl ester (HOCt), It is also possible to add 1-hydroxy-7-azabenzotriazole (HOAt) etc. or a base (eg organic bases such as triethylamine, pyridine, N, N-diisopropylethylamine etc.).
The amount of the condensing agent to be used is generally 0.5 to 3 mol, preferably 1 to 1.5 mol, per the total number (1 mol) of the terminal groups of the dendritic polymer (1).
As the solvent, for example, aromatic hydrocarbons such as toluene and xylene; amide solvents such as N, N-dimethylformamide and N, N-dimethylacetamide; ethers such as diethyl ether, tetrahydrofuran and dioxane; chloroform and dichloromethane And the like, or mixtures thereof. Among these, N, N-dimethylformamide, dichloromethane and the like are preferable.
The reaction temperature is generally −10 to 30 ° C., preferably 0 ° C. to 20 ° C., and the reaction time is generally 1 to 30 hours.

The reaction conditions (reactant, reaction solvent, reaction temperature, reaction time, etc.) in the deprotection reaction vary depending on the kind of protecting group (P and P ′). For example, Protective Groups in Organic Synthesis, 4th Ed. , Theodora W. Greene, Peter G. M. It can carry out in accordance with the method as described in Wuts, Wiley-Interscience (2007) etc., the Example in this specification, or the method according to these.

Step 2
The step is a step of synthesizing a compound 5 by introducing a hydrophilic polymer segment into a part of the terminal amino group derived from aspartic acid of compound 3 and / or the terminal group of dendritic polymer (1).
The reaction is carried out in a solvent that does not affect the reaction.

The amount of compound 4 to be used is generally 0.03 to 0.4 mol, preferably 0.1 to 0.2 mol, per 1 mol of the total number of terminal groups of compound 3.
Although the compound 4 is not particularly limited, it is preferably an amine reactive PEGylation reagent (eg, PEG-NHS ester etc.).

As the solvent, for example, polar solvents such as N, N-dimethylformamide, dimethyl sulfoxide and the like are preferable.
The reaction temperature is generally 10 to 50 ° C., preferably 20 ° C. to 30 ° C., and the reaction time is generally 1 to 30 hours.

Step 3
The step is a step of introducing a hydrophobic segment into a part of the terminal amino group derived from aspartic acid of compound 5 and / or the terminal group of dendritic polymer (1) to synthesize compound 7.
The reaction is carried out in a solvent that does not affect the reaction.

The amount of compound 6 to be used is generally 0.01 to 0.3 mol, preferably 0.01 to 0.1 mol, per 1 mol of the total number of terminal groups of compound 5.
The compound 6 is not particularly limited, but is preferably a sterol derivative, more preferably a compound in which the 3-position hydroxy group of cholesterol is haloformylated (eg, cholesterol chloroformate and the like).

As the solvent, for example, polar solvents such as N, N-dimethylformamide, dimethyl sulfoxide and the like are preferable.
The reaction temperature is generally 10 to 90 ° C., preferably 40 ° C. to 80 ° C., and more preferably 60 ° C. to 80 ° C. The reaction time is usually 2 to 48 hours.

In the above production method, as the compound 4 and the compound 6, one containing a linker (L ² or L ³ ) is used, but the linker does not have to be included in the compound 4 and the compound 6 and It is also possible to react with compound 4 and compound 6 having no linker after introducing a linker to 3 and compound 5.
As the various linkers, those mentioned above can be mentioned, and if necessary, they can be introduced into the compounds 3 to 6 by known methods using known linker reagents.

(Preparation method 2) (when all terminal groups of dendritic polymer (1) are aspartic acid modified)

(Wherein, n5 and n6 each independently represent an integer of 1 or more, and n1 ′ represents an integer of 8 or more, and the other symbols are as defined above, provided that n1 ′ is , Greater than the sum of n5 and n6.)

Step 1
In this step, the terminal amino group or hydroxy group of the dendritic polymer (1 ′) and the α-position carboxy group of aspartic acid (compound 2) in which the amino group and the β-position carboxy group are protected are dehydrated and condensed Thereafter, the compound is converted to compound 8 by being subjected to a deprotection reaction.
The reaction is carried out in a solvent which does not affect the reaction, using dehydration condensation reaction conditions known per se and deprotection conditions.

The amount of compound 2 to be used is generally 1 to 3 mol, preferably 1 to 1.5 mol, per 1 mol of the total number (1 mol) of terminal groups of the dendritic polymer (1 ′).

As a condensing agent used for a dehydration condensation reaction, the thing quoted at the process 1 of the said manufacturing method 1 can be used, for example.
In the condensation step, it is also possible to add the condensation additive and the base mentioned in step 1 of the above-mentioned production method 1 as necessary.
The amount of the condensing agent to be used is generally 1 to 3 moles, preferably 1 to 1.5 moles, relative to the total number (1 mole) of the end groups of the dendritic polymer (1 ′).
As the solvent, for example, the solvents mentioned in step 1 of the above-mentioned production method 1 can be suitably used.
The reaction temperature is generally −10 to 30 ° C., preferably 0 ° C. to 20 ° C., and the reaction time is generally 1 to 30 hours.

Step 2
The step is a step of introducing a hydrophilic polymer segment into a part of the terminal amino group derived from aspartic acid of compound 8 to synthesize compound 9.
The reaction is carried out in a solvent that does not affect the reaction.

The amount of compound 4 to be used is generally 0.03 to 0.4 mol, preferably 0.1 to 0.2 mol, per 1 mol of the total number of terminal amino groups derived from aspartic acid of compound 8 .
Although the compound 4 is not particularly limited, it is preferably an amine reactive PEGylation reagent (eg, PEG-NHS ester etc.).

Step 3
The step is a step of synthesizing a compound 10 by introducing a hydrophobic segment into a part of the terminal amino group derived from aspartic acid of the compound 9.
The reaction is carried out in a solvent that does not affect the reaction.

The amount of compound 6 to be used is generally 0.03 to 0.3 mol, preferably 0.03 to 0.1 mol, per 1 mol of the total number of terminal groups of compound 9.
The compound 6 is not particularly limited, but is preferably a sterol derivative, more preferably a compound in which the 3-position hydroxy group of cholesterol is haloformylated (eg, cholesterol chloroformate and the like).

In the above production method, as the compound 4 and the compound 6, one containing a linker (L ² or L ³ ) is used, but the linker does not have to be included in the compound 4 and the compound 6 and After introducing a linker into 8 and compound 9, it is also possible to react with compound 4 and compound 6 having no linker.
As the various linkers, those mentioned above can be mentioned, and if necessary, they can be introduced into the compounds 4, 6, 8 and 9 by a method known per se using known linker reagents.

(Polymer Micelle Containing the Compound of the Present Invention and Drug of the Present Invention Containing the Polymer Micelle)
The compound of the present invention is mixed alone or with an agent in an aqueous medium (preferably, distilled water, physiological saline, phosphate buffered saline (PBS), etc.), and the O.D. Polymer micelles are formed by self assembly by standing or stirring for 5 to 24 hours. Furthermore, operations such as dialysis, stirring, dilution, concentration, ultrasonic treatment, temperature control, pH control, ionic strength control, addition of an organic solvent and the like can be added as appropriate.
The surface charge and particle size of the polymer micelle can be measured using a particle size analyzer (eg, Zetasizer Nano (manufactured by Malvern Instruments)).

The average particle size of the polymer micelle of the present invention is usually 30 to 200 nm, preferably 30 to 150 nm, more preferably 40 to 100 nm.

When the polymer micelle of the present invention is used as a pharmaceutical, it may include a step of sterilizing and a step of lyophilizing a mixed aqueous solution after sterilization. When the lyophilization step is included, the mixed aqueous solution may contain a lyophilization aid. The freeze-dried preparation thus obtained can be reconstituted to polymeric micelles with distilled water for injection, 5% glucose, physiological saline and the like as needed.

As the lyophilization adjuvant, for example, one or more selected from the group consisting of mannitol, sorbitol, lactic acid, trehalose, and sucrose can be used. Preferably, it is mannitol.

The polymer micelles containing the compound of the present invention are highly bone-migratory and can be used to selectively accumulate bone-selective drugs, so they are useful as medicaments, and excellent as preventive or therapeutic agents for various bone diseases and the like. Can be shown. For example, it is useful for preventing or treating a disease that develops in association with failure of bone metabolism, acceleration of bone destruction, etc. Specific examples of bone disease include osteoporosis, periodontal disease, bone metastasis of cancer, Rheumatoid arthritis, osteoarthritis, osteomalacia, hyperparathyroidism, Paget's disease and the like can be mentioned.

The medicament of the present invention (hereinafter also referred to as "the pharmaceutical composition of the present invention") may contain a pharmaceutically acceptable carrier in addition to the compound of the present invention and the drug described above. As pharmaceutically acceptable carriers, various organic or inorganic carrier substances commonly used as pharmaceutical ingredients are used, and they are compounded as solvents, solubilizers, suspending agents, tonicity agents, buffers, soothing agents, etc. Be done. If necessary, formulation additives such as preservatives, antioxidants and colorants can also be used. Preferred examples of the solvent include, for example, water for injection, alcohol, propylene glycol and the like. Preferred examples of solubilizers include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like. Preferred examples of suspending agents include, for example, surfactants such as stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glycerin monostearate and the like; for example, polyvinyl alcohol Examples thereof include hydrophilic polymers such as alcohol, polyvinyl pyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose and the like. Preferred examples of the tonicity agent include sodium chloride, glycerin, mannitol and the like. Preferred examples of the buffer include buffers such as phosphate, acetate, carbonate, citrate and the like. Preferred examples of the soothing agent include, for example, benzyl alcohol and the like. Preferred examples of preservatives include, for example, p-hydroxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like. Preferred examples of the antioxidant include, for example, sulfite, ascorbic acid and the like.

When the pharmaceutical composition of the present invention is used for treatment of a bone disease and the like, it can be used in combination with an existing drug (eg, a therapeutic agent for a bone disease and the like). When used in combination, the administration order of the pharmaceutical composition of the present invention and the existing drug may be simultaneous or separate. If separate, the pharmaceutical composition of the present invention may be either before or after administration of the existing drug.

Although the administration route of the pharmaceutical composition of the present invention is not particularly limited depending on various situations, for example, it can be administered by oral or parenteral route. The term "parenteral" as used herein includes intravenous, intramuscular, subcutaneous, intranasal, intradermal, instillation, intracerebral, intrarectal, intravaginal and intraperitoneal administration and the like.
When administering the pharmaceutical composition of the present invention, it can be provided in the form of unit dose ampoules or multiple dose containers in the form of an isotonic aqueous solution or suspension for injection. Continuous infusion is also possible, and local administration using a catheter is also desirable. The pharmaceutical composition of the present invention can be administered in a single dose or a plurality of doses, and the administration period and interval thereof are changed according to various situations, and are determined at any time by the judgment of a physician. It is

The dosage of the pharmaceutical composition of the present invention varies depending on the therapeutic purpose, the age to be administered, the route of administration, the frequency of administration, the degree of disease and can be varied widely. The daily dose of the drug contained in the pharmaceutical composition of the present invention can be appropriately determined by those skilled in the art, for example, when administered parenterally to a patient for the purpose of treating bone metastasis of cancer. And 0.01 mg to 100 mg, preferably 0.01 mg to 50 mg, more preferably 0.01 mg to 20 mg per kg body weight, and these can be administered once or several times.

Using the medicine of the present invention, it is confirmed in the following test examples that the drug selectively migrates to bone and is accumulated at a high concentration, and the efficacy in treatment of bone metastasis of cancer is significantly improved. It was done.

EXAMPLES Hereinafter, the present invention will be described in more detail by way of preferred examples and test examples of the present invention. The following examples are provided to exemplarily confirm the effects of the present invention. The present invention is not limited to these, and may be changed without departing from the scope of the present invention.

Hereinafter, the present invention will be described in more detail based on examples, formulation examples and test examples, but the present invention is not limited by these examples and the like, and changes without departing from the scope of the present invention You may
The devices, reagents and the like used in the examples are readily obtainable or prepared according to methods commonly practiced in the art, or commercially available, unless otherwise noted.
"Room temperature" in the following examples usually indicates about 10 ° C to about 25 ° C.

Example 1: Synthesis of the compound of the present invention (compound 1a) Third generation polyamide amine dendrimer (PAMAM; manufactured by Sigma Ardrich; 1.0 equivalent) in N, N-dimethylformamide (DMF) (anhydrous) containing triethylamine And 32.5 equivalents of anhydrous 1-hydroxy-1H-benzotriazole (HOBt) (manufactured by Watanabe Chemical Industries, Ltd.) and 32.5 equivalents of 1- [bis (dimethylamino) methylene] -1H- Add benzotriazolium 3-oxide hexafluorophosphate (HBTU) (manufactured by Merck Millipore), 32.5 equivalents of Boc-Asp (OtBu) -OH (manufactured by Watanabe Chemical Industries, Ltd.), and stir at room temperature for 4 hours In the amino group end of PAMAM for dehydration condensation reaction of Boc-Asp (OtBu) -OH More introduced. The reaction mixture was concentrated under reduced pressure, redissolved in chloroform, and partitioned between 5% sodium carbonate and saturated brine. The organic phase is concentrated under reduced pressure and precipitated with petroleum ether. The precipitate was vacuum dried at room temperature, then dissolved in chloroform and deprotected by treatment with trifluoroacetic acid. After stirring for 1 hour at room temperature, the mixture was concentrated under reduced pressure and precipitated with diethyl ether to obtain aspartic acid modified PAMAM (Asp-PAMAM). The resulting Asp-PAMAM was treated with 5.0 equivalents of amino group-activated activated methoxypolyethylene glycol (PEG-NHS; average molecular weight: 2000) in super dehydrated dimethyl sulfoxide (DMSO) (SUNBRIGHT ME-020CS, NOF Co.). It was made to react with Tokyo, Japan) at room temperature for 24 hours. The pH was adjusted to 8.0 or more by adding triethylamine at the time of reaction to finally obtain PEGylated Asp-PAMAM. The product was washed by ultrafiltration using ultrapure water. PEGylation was identified by MALDI TOF-MS (Microflex; Bruker Daltonics, Bremen, Germany).
Subsequently, the obtained PEGylated Asp-PAMAM is dissolved in DMF, and 1.5 equivalents of cholesterol chloroformate is added, followed by stirring at 80 ° C. for 2 hours, at 60 ° C. for 3 hours, at room temperature for 24 hours Thus, a compound of the present invention (Compound 1a) in which a cholesterol residue was introduced into PEGylated Asp-PAMAM was synthesized by coupling a cholesterol residue to the amino group terminal derived from aspartic acid.

Example 2 Synthesis of Polymeric Micelle Containing Compound 1a and Paclitaxel Compound 1a (1.0 mg) obtained in Example 1 and paclitaxel (PTX) (5.0 mg) (manufactured by Tokyo Chemical Industry Co., Ltd.) 1 mL The mixture is mixed in ultrapure water for 24 hours at room temperature, and filtered using a 0.45 .mu.m membrane filter to form polymeric micelles (PTX) formed by self-assembly of compound 1a in a PTX-encapsulated state Compound 1a micelles were prepared. The surface charge and particle size of the obtained PTX-compound 1a micelles were measured using Zetasizer Nano (Malvern Instruments, Worcestershire, UK).

The measurement results of the particle diameter and surface charge of the obtained PTX-compound 1a micelle are shown in Table 1. The mean particle size was 48.28 ± 21.2 nm and the zeta potential was −13 ± 1.04 mV.

Comparative Example: Synthesis of polymeric micelles derived from non-aspartic acid modified compounds Third generation polyamidoamine dendrimer (PAMAM) (manufactured by Sigma-Aldrich) in N, N-dimethylformamide (DMF) (anhydrous) containing triethylamine (1.0 equivalents) of 5.0 equivalents of amino group-activated activated methoxypolyethylene glycol (PEG-NHS; average molecular weight: 2000) in super dehydrated dimethyl sulfoxide (DMSO) (SUNBRIGHT ME-020CS, NOF Co It was made to react with .Tokyo, Japan) at room temperature for 24 hours.
The pH was adjusted to 8.0 or higher by adding triethylamine during the reaction to obtain PEGylated PAMAM. The product was washed by ultrafiltration using ultrapure water. Subsequently, the obtained PEGylated PAMAM is dissolved in DMF, and 1.5 equivalents of cholesterol chloroformate is added, followed by stirring at 80 ° C. for 2 hours, at 60 ° C. for 3 hours, at room temperature for 24 hours, A non-aspartic acid modified compound was synthesized by coupling a cholesterol residue to the PAMAM-derived amino group terminus. The obtained compound (1.0 mg) and 5.0 mg of paclitaxel (PTX) are mixed in 1 mL of ultrapure water at room temperature for 24 hours, and filtered using a 0.45 μm membrane filter to obtain PTX. Polymeric micelles (PTX-PAMAM micelles) formed by self-assembly of the compound in the state of containing

Test Example 1: Pharmacokinetic evaluation of PTX-compound 1a micelle (test method)
¹¹¹ In-labeled Compound 1a micelles were intravenously administered to ddY mice to evaluate the pharmacokinetics of Compound 1a micelles. That is, micelles formed by the self-assembly of compound 1a (compound 1a micelles) are treated with the bifunctional chelating agent diethylenetriamine-N, N, N ′, N ′ ′, N ′ ′, N ′ ′-pentaacetic acid (DTPA) anhydride Radioactive labeling with ¹¹¹ In was carried out according to the method described by Hnatowich et al. (Int. J. Appl. Radiat. Isot., 12, 327-332 (1982)) using (Dojin Chemical Laboratories). A labeled PTX compound 1a micelle was prepared in which ³ H labeled PTX (made by Moravek) was contained in the obtained ¹¹¹ In labeled compound 1a micelle. Moreover, the labeled PTX-PAMAM micelle which is a comparative example was also prepared by the same method.
The labeled PTX-Compound 1a micelle is intravenously administered to ddY mice at a dose of 10 mg PAMAM / kg, 0.5 mg PTX / kg (6.0 × 10 ⁶ cpm ¹¹¹ In / kg, 30 μCi ³ H / kg), Compound 1a micelle And pharmacokinetics of PTX were evaluated. At appropriate times after intravenous injection, blood was collected from the vena cava under isoflurane anesthesia. Liver, kidney, spleen, heart, and lung tissues were removed along with the bones of the lower extremities, washed with saline and blotted with filter paper to measure the wet weight of the organs. The collected blood was centrifuged at 2000 × g for 5 minutes to obtain plasma. Samples of collected organs and 100 mL of plasma are transferred to a counting tube, and the radioactivity of each sample is subjected to radioactivity of Compound 1a micelle portion using a gamma counter (1480 WizardTM 3 ', Perkin-Elmer, Boston, MA, USA) It was measured. Subsequently, the radioactivity of PTX in the sample was measured by a liquid scintillation counter. Bone radioactivity was calculated as total wet bone weight as 12% of body weight based on the radioactivity measured in the tibia and femur.

The results of the pharmacokinetics of the ¹¹¹ In-labeled Compound 1a portion of the labeled PTX-Compound 1a micelles are shown in FIG.
According to FIG. 1, the bone migration amount of ¹¹¹ In-labeled PAMAM micelles of labeled PTX-PAMAM micelles was 5.6% at 3 hours after administration, and 40% of the dose was transferred to the liver (Figure) 1) a). On the other hand, the bone transfer amount of the ¹¹¹ In-labeled compound 1a micelle portion showed 26% at 3 hours after administration, and almost no transfer to other organs was observed. Therefore, an efficient bone target by aspartate modification was obtained. It has been confirmed that this can be achieved (Fig. 1 b).

The results of pharmacokinetics of ³ H-labeled PTX are shown in FIG.
According to FIG. 2, the bone migration amount of ³ H-labeled PTX by labeled PTX-PAMAM micelles was 7.7% (a in FIG. 2) and was mainly distributed in the liver. On the other hand, the amount of ³ H-labeled PTX transferred to bone by the labeled PTX-compound 1a micelle is 22% (b in FIG. 1), and almost no transfer to other organs is observed, and the body has excellent bone selectivity. It was confirmed to show the dynamics.

Test Example 2 Intraosseous Distribution of Compound 1a Micelle (Test Method)
The intraosseous distribution after intravenous administration of Compound 1a micelles was evaluated. That is, the compound 1a micelle was labeled with fluorescein isothiocyanate (FITC) (manufactured by Sigma-Aldrich) (FITC labeled compound 1a micelle). Further, FITC-labeled PAMAM micelles, which are comparative examples, were also prepared by the same method.
In order to label the site of bone formation in mice, xylenol orange (Wako Pure Chemical Industries, Ltd.) was intravenously administered to ddY mice at 30 mg / kg three days before FITC-labeled Compound 1a micelle administration. FITC-labeled Compound 1a micelle solution was intravenously administered to mice at a dose of 20 mmol FITC / kg. Twenty-four hours after intravenous injection, the lower extremity bones were removed after euthanasia. Histological sections before demineralization from the distal femur and proximal tibia were prepared according to the Kawamoto method (see T. Kawamoto, Arch. Histol. Cytol., 2003, 66, 123-143). The sections were observed using a confocal laser scanning microscope A1R + (Nikon Co., Tokyo Japan).

The results are shown in FIG. The xylenol orange of a and a 'of FIG. 3 shows an osteogenesis site. According to b 'of FIG. 3, almost no fluorescence from FITC-labeled PAMAM micelles was observed, but according to b, green fluorescence from FITC-labeled Compound 1a micelles was strongly observed. Further, according to c of FIG. 3, since the overlap between the fluorescence derived from FITC-labeled Compound 1a micelle and the fluorescence derived from Xylenol Orange was hardly observed, Compound 1a micelle was mainly observed at sites other than osteogenesis sites (osteosis sites). It was shown to be distributed. Since the osteoclastic site is a focus site of osteoporosis and bone metastasis, it was found that it shows an intraosseous distribution that is advantageous for treatment of bone diseases.

Test Example 3: Inhibition of Cancer Bone Metastasis by PTX-Compound 1a Micelle (Test Method)
A bone metastasis model was created by injecting B16-BL6 cells (B16-BL6 / Luc cells, 2 × 10 ⁵ cells) labeled with the firefly luciferase gene into the left ventricle of C57 / BL6 mice. PTX-Compound 1a micelles were injected into the tail vein at a dose of 0.5 mg PTX / kg one and three days after tumor inoculation. Fourteen days after tumor inoculation, 2.5 mg of D-luciferin was intraperitoneally injected into a mouse under anesthesia by intraperitoneal injection of pentobarbital (50 mg / kg) (manufactured by Kyoritsu Pharmaceutical Co., Ltd.), and IVIS Lumina XRMS Series III Multi- Imaging was performed using the Species Optical and X-Ray Imaging System. Then, after euthanizing mice, bones of lower limbs were removed, and luciferase activity in tissues was measured with a luminometer (Lumat LB9507, EG & G Berthold, Bad Wild-bad, Germany). The number of cancer cells in bone was calculated from the obtained luciferase activity using a regression line.

The results are shown in FIG. According to FIG. 4, the administration of PTX-compound 1a micelles significantly suppressed the proliferation of cancer cells in the bones of the lower limbs. In addition, it was confirmed that bone cancer growth was significantly suppressed as compared to the PTX administration group using PTX-PAMAM micelles.

Test Example 4: Organ distribution of the portion excluding the hydrophobic segment of compound 1a by single photon computed computed tomography / computed tomography (SPECT / CT) imaging (test method)
SPECT / CT was performed using NanoSPECT / CT (Bioscan Inc., Washington DC, USA). The ¹¹¹ In labeled form (9.4 MBq / mouse) excluding the hydrophobic segment of compound 1a was intravenously administered to mice. A 60-minute SPECT scan of mice was performed under isoflurane inhalation anesthesia for 6 hours after intravenous administration of the ¹¹¹ In labeled body. Prior to SPECT scan, CT scan of mice was performed under isoflurane inhalation anesthesia for anatomical observation. SPECT images were reconstructed using HiSPECT software (Scivis, Goettingen, Germany). Image analysis was also performed using the Amira 3D data analysis and visualization software package (version 5.1; FEI Company, Hillsboro, OR, USA).

The results are shown in FIG. FIG. 5 shows SPECT / CT imaging images of organ distribution after intravenous injection of the ¹¹¹ In labeled portion of the portion excluding the hydrophobic segment of Compound 1a.
According to FIG. 5, the ¹¹¹ In labeled portion of the portion excluding the hydrophobic segment of compound 1a was partially distributed to the kidney and the bladder (urine excretion), but was selectively accumulated to the bone. In particular, it was observed that bone turnover accumulated specifically at the joints of the shoulders and lower legs where activation was increasing. It has been found that compound 1a micelles are likely to be carriers suitable for diagnostic imaging and treatment of bone metastasis, osteoporosis and the like because bone turnover is activated in disease sites such as bone metastasis and osteoporosis.

Test Example 5 Intraosseous Distribution of PTX-Compound 1a Micelle in a Bone Metastasis Model (Test Method)
A bone metastasis model was created by injecting B16-BL6 cells (B16-BL6 / Luc cells, 2 × 10 ⁵ cells) labeled with the firefly luciferase gene into the left ventricle of C57 / BL6 mice. On day 7 of tumor inoculation, FITC-labeled PTX-compound 1a micelles were intravenously administered, and 24 hours later, lower extremity bones were removed under isoflurane inhalation anesthesia and frozen sections were prepared. After immunostaining of osteoclasts and metastasized cancer cells in the bones of mouse legs, the positional relationship with FITC-labeled PTX-compound 1a micelles was observed with a confocal laser microscope.

The results are shown in FIG. Melan-A (blue fluorescence) indicates a cancer cell. Cathepsin K (red fluorescence) indicates osteoclasts. According to FITC-labeled PAMAM micelles in FIG. 6, fluorescence from FITC-labeled PAMAM micelles was hardly observed, but according to FITC-labeled PTX-Compound 1a micelles, green fluorescence from FITC-labeled PTX-Compound 1a micelles Was strongly observed. Further, according to Merge in FIG. 6, co-localization of fluorescence from FITC-labeled compound 1a micelle, fluorescence from Melan-A (a marker for cancer cells) and fluorescence from Cathepsin K (a marker for osteoclasts) was observed. Thus, it was shown that PTX-compound 1a micelles are mainly distributed in the vicinity of tumor and osteoclasts.
From the above results, it was found that PTX-compound 1a micelles show an intraosseous distribution that is advantageous for bone metastasis treatment.

Formulation example (manufacture of lyophilized formulation)
1) Compound of the present invention (compound 1a) 40 mg
2) mannitol 10 mg
3) Paclitaxel (drug) 3 mg
4) Ultra pure water 1 ml
53 mg / ml in total
After mixing 1), 2), 3) and 4), the mixture was sterilized by filtration using a membrane filter, filled into a container, and lyophilized to obtain a lyophilized preparation.

The compounds of the present invention can form polymeric micelles alone or together with a drug, said polymeric micelles exhibiting high bone migration selectivity and high targeting efficiency to bone, drug release Being excellent in sex, it is extremely useful as a preventive agent (test drug) or a therapeutic agent for various bone diseases. In addition, the compound and polymer micelle of the present invention have the advantage of being excellent in biocompatibility and safety because they use aspartate derived from a living body which has little aggregation in the living body as a bone targeting element. ing. Furthermore, since the compounds and polymer micelles of the present invention can be easily synthesized, they are expected to be applied to a wide range of applications as practical drug delivery carriers and medicaments that are excellent in drug efficacy and have few side effects.

This application is based on patent application No. 2017-132805, the contents of which are incorporated in full herein.

Claims

The α-position carbonyl group of aspartic acid is linked by a peptide bond or an ester bond directly or via a linker to at least 50% of the total number of terminal groups of the dendritic polymer having a plurality of terminal groups, The hydrophilic polymer segment is attached directly or via a linker to at least 3% of the total number of end groups on the group and the aspartate-free dendritic polymer, and the aspartate-derived terminal amino group and asparagine A compound comprising a hydrophobic segment bonded to at least 1% of the total number of end groups on a non-acid-modified dendritic polymer directly or via a linker.
The compound according to claim 1, wherein the hydrophilic polymer segment is a group derived from polyethylene glycol.
The compound according to claim 1 or 2, wherein the hydrophobic segment is a residue of a sterol derivative.
The compound according to claim 3, wherein the residue of the sterol derivative is derived from a compound selected from the group consisting of cholesterol, cholestanol, dihydroxycholesterol and cholic acid.
A dendrimer or dendron composed of polyamidoamine, a dendrimer or dendron composed of polylysine, a dendrimer composed of polyethylene glycol and 2,2-bis (hydroxymethyl) propanoic acid, and 2 5. The compound according to any one of claims 1 to 4, which is selected from the group consisting of dendrimers composed of, 2-bis (hydroxymethyl) propanoic acid or dendrons.
A pharmaceutical composition comprising the compound according to any one of claims 1 to 5 and a drug.
The medicament according to claim 6, wherein the drug is at least one selected from the group consisting of an osteoporosis therapeutic drug, a rheumatoid arthritis therapeutic drug, an anticancer drug, an anti-inflammatory drug, an antioxidant, a nucleic acid drug, a radiopharmaceutical and a contrast agent. Composition.
A polymeric micelle comprising the compound according to any one of claims 1 to 5.
The polymeric micelle according to claim 8, further comprising a drug.
The method according to claim 9, wherein the drug is at least one selected from the group consisting of an osteoporosis therapeutic drug, a rheumatoid arthritis therapeutic drug, an anticancer drug, an anti-inflammatory drug, an antioxidant, a nucleic acid drug, a radiopharmaceutical and a contrast agent. Molecular micelles.
A carrier for drug delivery, comprising the polymeric micelle according to claim 8, for selectively delivering a drug to a target tissue in vivo.
The drug delivery carrier according to claim 11, wherein the target tissue is bone.
A medicament comprising the polymeric micelle according to claim 9 or 10.
The medicament according to claim 13, which is a preventive or therapeutic agent for a bone disease.