WO2002044218A1 - Compose de derive d'acide hydroxamique et d'acide hyaluronique - Google Patents

Compose de derive d'acide hydroxamique et d'acide hyaluronique Download PDF

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Publication number
WO2002044218A1
WO2002044218A1 PCT/JP2001/010493 JP0110493W WO0244218A1 WO 2002044218 A1 WO2002044218 A1 WO 2002044218A1 JP 0110493 W JP0110493 W JP 0110493W WO 0244218 A1 WO0244218 A1 WO 0244218A1
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group
carbon atoms
conjugate
hyaluronic acid
linear
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PCT/JP2001/010493
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English (en)
Japanese (ja)
Inventor
Hitoshi Ikeya
Tadashi Morikawa
Koichi Takahashi
Akira Okamachi
Tatsuya Tamura
Original Assignee
Chugai Seiyaku Kabushiki Kaisha
Denki Kagaku Kogyo Kabushiki Kaisha
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Priority to AU2002218512A priority Critical patent/AU2002218512A1/en
Publication of WO2002044218A1 publication Critical patent/WO2002044218A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to the binding of a hydroxamic acid derivative to hyaluronic acid, which has an inhibitory activity on matrix meta-oral protease (hereinafter also referred to as MMP) and is useful for treating joint diseases such as osteoarthritis and rheumatoid arthritis.
  • MMP matrix meta-oral protease
  • Articular cartilage is composed of about 70% water, chondrocytes and cartilage matrix.
  • the main components of the cartilage matrix are collagen and proteoglycan, and a network of collagen having a network structure contains proteodarin, which is rich in water retention ability.
  • the cartilage matrix is rich in viscoelasticity and reduces the stimulus load on cartilage, and plays an important role in maintaining the normal shape and function of articular cartilage.
  • OA osteoarthritis
  • RA rheumatoid arthritis
  • Matrix rupture in both diseases is triggered by age-related mechanical stress in OA, hyperproliferation of synovial lining cells, pannus formation, and infiltration of inflammatory cells in RA, all of which are induced by protease induction. It is thought to be triggered by The ability of cartilage matrix to be degraded outside of cells with a neutral pH, and matrix meta-oral proteases that optimize the pH in this region are said to be central players in degradation [Current Opinion m Anti-Inflammatory & Immunomodulatory Inve stigational Drugs, vol. 2, 16-25, (2000)].
  • TIMPs tissue meta-oral protease inhibitors
  • AD AM New MM P like evening with At least 30 types of proteins
  • Some of AD AM and AD AMT S have MMP-like protease activity, for example, ADA M17 (TNF converting enzyme) and ADAMTS-4, -5 (aggliinase-I, -II).
  • ADA M17 TNF converting enzyme
  • ADAMTS-4, -5 aggliinase-I, -II
  • these members of the MMP family are responsible for various functions such as development, angiogenesis, estrous cycle, bone remodeling, and tissue repair.
  • these functions In order for these functions to be properly expressed, the steps of enzyme production, activation, and interaction with the substrate are strictly controlled by in vivo inhibitors such as TIMP.
  • TIMP in vivo inhibitors
  • Hydroxamic acid derivatives that exert MMP inhibitory effects even by oral administration have already been found, and some of them are undergoing clinical trials in cancer patients and joint disease patients.
  • this type of MMPI has, to varying degrees, an inhibitory effect on all MMPs and may also suppress MMPs involved in physiological functions.
  • transient side effects such as bone muscle pain
  • Recently, the development of improved products with increased specificity to specific MMPs has been promoted, but MMPs that are only involved in the disease state have not yet been identified.
  • the discovery of P has not ruled out the possibility of suppressing any physiological effects of MMP during systemic administration.
  • MMPI has excellent pharmacological effects, there are still problems to be solved for clinical application as therapeutic agents for chronic diseases such as OA and RA.
  • hyaluronic acid (hereinafter also referred to as HA) is an in-vivo polylactic acid composed of repeating units of N-acetyldarcosamine and dalcuronic acid. It plays an important role in maintaining the viscoelasticity, load absorbing effect and lubricating effect of the steel. In the cartilage matrix, it binds to cartilage proteodalican to form a polymer called aggrecan, and plays a central role in maintaining water retention and viscoelasticity.
  • HA and its cross-linked product are also collectively referred to as HA preparations. It is widely practiced clinically. Since HA is a component of the extracellular matrix, HA also has a high affinity for cartilage matrix. In addition, because of its high viscoelasticity, it is characterized by being localized in the joint cavity for a long time after being injected into the joint.
  • WO99 / 5963 discloses a conjugate of a hydroxamic acid residue and HA, and the conjugate has MMP inhibitory activity and is used as a therapeutic agent for joint diseases. It is disclosed to obtain. Further improvement of the MMP inhibitory activity and stability of the conjugate specifically disclosed in the publication will lead to the creation of a very excellent conjugate.
  • An object of the present invention is to provide a conjugate of a hydroxamic acid derivative and hyaluronic acid having an MMP inhibitory action.
  • Another object of the present invention is to provide a medicament useful as a therapeutic agent for joint diseases or the like, which contains the above-mentioned conjugate and can localize an MMP inhibitory effect in a joint cavity or on a joint tissue.
  • the present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, a conjugate in which a hyaluronic acid derivative is bound to a specific hydroxamic acid derivative is extremely superior to a conjugate that has been reported so far. They have found that they have MMP inhibitory activity, and have completed the present invention.
  • R 2 is a straight-chain or straight-chain carbon having 1 to 8 carbon atoms which may be substituted by a cycloalkyl group having 3 to 10 carbon atoms or an aryl group having 6 to 14 carbon atoms which may be substituted. Represents a branched alkyl group;
  • R 3 is a linear or branched chain having 1 to 8 carbon atoms which may be substituted by a cycloalkyl group having 3 to 10 carbon atoms or an aryl group having 6 to 14 carbon atoms which may be substituted. Represents an alkyl group in the form of
  • R 4 represents a hydrogen atom or a linear or branched alkyl group having 1 to 4 carbon atoms
  • R 5 represents one R 7 — R 8 — R 9 — where:
  • R 7 represents a linear or branched alkylene group having 1 to 8 carbon atoms
  • R 8 represents a methylene group or an imino group which may be substituted with a linear or branched alkyl group having 1 to 4 carbon atoms, or an oxygen atom;
  • R 9 represents a linear or branched alkylene group having 1 to 10 carbon atoms which may have 1 to 3 oxygen atoms inserted therein;
  • R 6 represents a hydrogen atom or a linear or branched alkyl group having 1 to 4 carbon atoms
  • R 3 may form a ring.
  • a hydroxyl group of hyaluronic acid or a hyaluronic acid derivative or a salt thereof is provided by a carbamate (carbamate) bond.
  • the present invention provides the following formula (8) Reacting a compound represented by the formula, a carbodiimide, and hyaluronic acid to obtain an activated hyaluronic acid;
  • R 14 represents a protecting group.
  • the present invention also provides a medicament comprising the above conjugate.
  • WO99Z59603 discloses a conjugate in which a hydroxamic acid residue is bonded to HA via a spacer, but there is no specific disclosure of the conjugate in the present invention.
  • hydroxamic acid derivative in the present invention includes a substance having a hydroxamic acid skeleton (N-hydroxyamide), and specifically includes, for example, a compound having a group represented by the aforementioned general formula (1). Is included.
  • Examples of the linear or branched alkyl group having 1 to 8 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an i-propyl group, an n-butyl group, a sec-butyl group and an isobutyl group.
  • a methyl group is preferred among the groups including t-butyl group, n-pentyl group, n-hexyl group, n-heptyl group, and n-butyl group.
  • linear or branched alkoxy group having 1 to 8 carbon atoms examples include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy.
  • Examples of the linear or branched alkenyl group having 2 to 8 carbon atoms include a vinyl group, an aryl group, an n-butenyl group, an i-butenyl group, a sec-butenyl group, a pentenyl group, and a hexenyl group. , A heptenyl group, an octenyl group and the like.
  • Ri a hydrogen atom, a hydroxyl group, a methyl group and a methoxy group are particularly preferred.
  • linear or branched alkyl group having 1 to 8 carbon atoms for R 2 methyl Group, ethyl group, n-propyl group, i-propyl group, n-butyl group, sec-butyl group, isobutyl group, t-butyl group, n-pentyl group, n-hexyl group, n-heptyl group , N-octyl group and the like, among which isobutyl group is preferable.
  • a cycloalkyl group having 3 to 10 carbon atoms as a substituent of a linear or branched alkyl group having 1 to 8 carbon atoms of R 2 is preferable.
  • a cycloalkyl group having 5 to 7 carbon atoms is exemplified, and specific examples thereof include a cyclopentyl group, a cyclohexyl group, and a cycloheptyl group.
  • the aryl group having 6 to 14 carbon atoms which may be substituted as a substituent of a linear or branched alkyl group having 1 to 8 carbon atoms of R 2 includes a substituent such as a hydroxyl group or a methoxy group.
  • Examples include an aryl group having 6 to 14 carbon atoms which may have a group, and specific examples include a phenylene group, a p-hydroxyphenylyl group, a p-methoxyphenyl group, and a naphthyl group. .
  • R 2 has the above definition, but R 2 is preferably an isobutyl group.
  • R 3 examples of the linear or branched alkyl group having 1 to 8 carbon atoms for R 3 include a methyl group, an ethyl group, an n-propyl group, an i-propyl group, an n-butyl group, a sec-butyl group, Examples include isobutyl group, t-butyl group, n-pentyl group, n-hexyl group, n-heptyl group, and n-octyl group. Among them, t-butyl group is preferable.
  • Examples of the cycloalkyl group having 3 to 10 carbon atoms as a substituent of the straight-chain or branched-chain alkyl group include a cycloalkyl group having 5 to 7 carbon atoms, and specifically, a cyclopentyl group And a cyclohexyl group or a heptyl group.
  • aryl group having 6 to 14 carbon atoms as a substituent of a linear or branched alkyl group having 1 to 8 carbon atoms of R3, substitution of a hydroxyl group, a methoxy group, etc.
  • examples thereof include an aryl group having 6 to 14 carbon atoms which may have a group, and specific examples include a phenyl group, a P-hydroxyphenyl group, a P-methoxyphenyl group, and a naphthyl group.
  • R 3 has the above definition, but R 3 is preferably a t-butyl group. Is a hydrogen atom, a hydroxyl group, a methyl group, or a methoxy group, R 3 is preferably a t-butyl group.
  • Examples of the linear or branched alkyl group having 1 to 4 carbon atoms for R 4 include a methyl group, an ethyl group, an n-propyl group, an i-propyl group, an n-butyl group, a sec-butyl group, Examples thereof include an isobutyl group and a t-butyl group, and among them, a methyl group is preferable.
  • R is preferably a hydrogen atom or a methyl group, and particularly preferably a hydrogen atom.
  • R 7 is bonded to N of NR 4 and R 9 is bonded to N of NR 6 .
  • Examples of the linear or branched alkylene group having 1 to 8 carbon atoms represented by R 7 include a methylene group, a ethane-1,2-diyl group, a propane-1,3-diyl group, and a butane-1,4-difure.
  • R 7 is preferably an ethane-1,2-diyl group, a propane-1,3-diyl group, or a butane-1,4-diyl group.
  • alkyl group include a methyl group, an ethyl group, an n-propyl group, an i-propyl group, an n-butyl group, a sec-butyl group and a t-butyl group.
  • R 8 a methylene group and an oxygen atom which may be substituted by a linear or branched alkyl group having 1 to 3 carbon atoms are preferable, and a methylene group and an oxygen atom are particularly preferable.
  • R 9 may have 1 to 3 oxygen atoms, and may have 1 to 10 carbon atoms.
  • Non-limiting examples of branched or branched alkylene groups include methylene, ethane-
  • 1,2-diyl group propane-1,3-diyl group, butane-1,4-diyl group, pentane-1,5-diyl group, hexane-1,6-diyl group, heptane-1,7— Diyl group, octane-1,8-diyl group, nonane-1,1,9-diyl group, octane-1,1,10-diyl group, 2-methylpentane-1,3-diyl group, 2-methylbutane-1,1, 4-diyl, 3-methylbutane-1,4-diyl, 3-methylpentane-1,5-diyl, 3-ethylpentane_1,5-diyl, 3-methylhexane-1,6-diyl Group, 4-methylhexane-1,6-diyl group, 4-methylheptane-1,1,7-diyl group, 1-oxapropane
  • R 9 is preferably a 1,2-diyl propane group, a 1,3-diyl propane group, a 1,4-diyl butane group, a 1,6-diyl 3,6-dioxanonane-1,9-diyl group or the like.
  • R 9 is preferably a 1,2-diyl propane group, a 1,3-diyl propane group, a 1,4-diyl butane group, a 1,6-diyl 3,6-dioxanonane-1,9-diyl group or the like.
  • R 5 examples include one (CH 2) 4 -, one (CH 2) 5 -, - (CH 2) 6 -, - (CH 2) 7 _, - (CH 2) 8 -, one (CH 2) 9 one, - (CH 2) 10 - , one (CH 2) u -, - (CH 2) 12 -, - (CH 2) 2 - 0- (CH 2) 2 -, - (CH 2 ) 3 — O-(CH 2 ) 3 —,-(CH 2 ) 4 -0- (CH 2 ) 4 1, 1 (CH 2 ) “O— (CH 2 ) 2 — O— (CH 2 ) 2 -0- (CH 2 ) 3 —, etc.
  • R 5 is — (CH 2 ) 3-0- (CH 2 ) 2 -0- (CH 2 ) 2 —O— (CH 2 ) 3 —
  • Examples of the linear or branched alkyl group having 1 to 4 carbon atoms for R 6 include meth J-group, ethyl group, n-propyl group, i-propyl group, n-butyl group, and sec-butyl group. And isobutyl group and t-butyl group, and among them, a methyl group is preferable.
  • R 6 a methyl group and a hydrogen atom are preferable, and a hydrogen atom is particularly preferable.
  • Non-limiting specific examples of I and L include methylene, ethylene, trimethylene, and tetramethylene, preferably, I includes, for example, trimethylene, and L includes, for example, methylene.
  • K is absent or represents an aromatic ring which may have a substituent, wherein the aromatic ring is phenyl, tolyl, xylyl, naphthyl, biphenyl, anthryl, phenanthryl, pyridyl, indolyl Quinolyl, imidazolyl and the like, and preferably phenyl.
  • I—J—K—L— is, for example, an alcohol derivative or a phenol derivative in which the residue from the 1 ⁇ side has an octalogenated alkyl group, preferably a propylpromide group, and the residue from the R 3 side has a hydroxyl group. It is obtained by condensing a compound which is preferably a parahydroxybenzyl group.
  • Non-limiting examples of -I-JKL- include the general formula (2): And the like.
  • R 2 is preferably an isobutyl group.
  • 1 ⁇ is preferably a hydroxyl group or a methoxy group, and R 3 is preferably a monobutyl group.
  • the group represented by the general formula (1) it is preferable that .1 ⁇ and R 3 are a ring represented by the general formula (2);
  • the group represented by the general formula (1) is preferably a group represented by the following formulas (3) to (7). Included in the invention.
  • hyaluronic acid refers to a disaccharide polymer having a weight average molecular weight of 50,000 to 10,000,000 and consisting of glucuronic acid and N-acetyldarcosamine; And mixtures thereof. From the viewpoint of the viscoelasticity, HA preferably has a weight average molecular weight of 700,000 to 10,000,000, more preferably a weight average molecular weight of 1,000,000 to 10,000,000. preferable.
  • hyaluronic acid derivative means any substance having a hyaluronic acid skeleton derived from hyaluronic acid.
  • Non-limiting specific examples of hyaluronic acid derivatives include;
  • a hyaluronic acid derivative in which the saccharide components, dalc valonic acid and Z or N-acetyltyl darcosamine, have a reducing end;
  • hyaluronic acid derivative in which one or more hydroxyl groups in HA are esterified for example, benzyl esterified HA (Hyaff, registered trademark, Fidia Advanced
  • a derivative obtained by crosslinking a disaccharide polymer composed of glucuronic acid having a weight average molecular weight of 50,000 to 10,000,000 and N-acetyldarcosamine with formaldehyde and further polymerizing the polymer for example, Synvisc (registered trademark, Biomatrix));
  • acetylated HA in which one or more hydroxyl groups in HA are acetylated, or one or more active ingredients of the above HA or HA derivative for example, an anticancer agent (for example, an alkylating agent, an antimetabolite, etc.) Agents, alkyloids, etc.), immunosuppressants, anti-inflammatory agents (including steroids, non-steroidal anti-inflammatory agents, etc.), anti-rheumatic agents, antibacterial agents (] 3-lactam antibiotics , Aminoglycoside antibiotics, macrolide antibiotics, tetracycline antibiotics, new quinoline antibiotics, polypeptide antibiotics, sulfa drugs, etc.) Derivatives obtained by bonding with or without intermediary
  • Non-limiting specific examples of the salt of HA or HA derivative include sodium salt, potassium salt, magnesium salt, calcium salt, aluminum salt and the like.
  • the origin of the HA is not particularly limited, and for example, HA derived from a terrestrial bacterium such as streptococcus, a human, a pig, a chicken, or the like can be used.
  • Non-limiting specific examples of HA, HA derivatives and their salts include, for example, Squel (registered trademark, Nippon Roussel), Alz (registered trademark, Kaken Pharmaceutical), Opegan (registered trademark, Santen Pharmaceutical), Hyalgan ( (Registered trademark, Fidea), Ortobisque (registered trademark, Anika Therapeutics), Hyalon (registered trademark, Pharmacia & Appujon), etc., and various reagent manufacturers such as Wako Pure Chemical Industries, Ltd. HA, HA derivatives and salts thereof described in one catalog can also be mentioned.
  • a group represented by the general formula (1) and a hydroxyl group of N-acetyldarcosamine of HA or an HA derivative or a salt thereof (hereinafter also referred to as “GlcNAc”) are used. Is preferably carbamate-bonded.
  • the binding ratio of the group represented by the general formula (1) to the number of G1cNAc in HA or the HA derivative or a salt thereof is preferably 0.01 to 50%. , 0.1 to 10%.
  • the “binding rate” is represented by the general formula (1) bound to HA relative to the total number of G 1 c NAc contained in HA or HA derivative or a salt thereof constituting the conjugate of the present invention. Is the percentage of the total number of groups.
  • Such a binding ratio can be calculated, for example, by the method described in Example 2 described later.
  • the conjugate of the present invention When the conjugate of the present invention is applied as a medicine, it may be used after being formulated together with a pharmaceutically acceptable excipient or stabilizer.
  • the administration form of the medicament containing the conjugate of the present invention is not particularly limited, and may be oral administration, parenteral administration, systemic administration, or local administration. In general, it is preferable to administer the drug of the present invention parenterally and topically. For example, it can be administered as an injection, intraarticularly, intravenously, intramuscularly or subcutaneously, or a spray, Topical It can be administered transdermally as a rim, lotion, or ointment.
  • the medicament containing the conjugate of the present invention can also be used as a therapeutic drug for joint diseases such as osteoarthritis, rheumatoid arthritis or shoulder periarthritis. It is preferably used in the manufacture of such drugs, as well as in the manufacture of drugs for treating joint diseases.
  • the dose of the medicament of the present invention can be appropriately selected according to the patient's condition, age, sex, etc., but when used as an injection, the dose of the conjugate, which is the active ingredient, is generally 0. 0.1 mg / kg body weight / day to 10 O mgZ body weight kg / day, preferably 0.1 mg / kg body weight / day to 10 mgZ body weight kg / day.
  • the daily dose of the medicament may be administered in several divided doses a day or once a day. Alternatively, it may be administered once every 2 to 28 days.
  • the method for producing the conjugate of the present invention is not particularly limited.
  • the conjugate can be produced by the method shown below, by the method shown in the synthesis examples described below, or by applying the method.
  • carbodiimides and a compound represented by the formula (8) are added to an aqueous solution of HA or an HA derivative or a salt thereof, and the pH of the reaction solution is adjusted to 4 to 4 using an acid or buffer such as hydrochloric acid or phosphoric acid.
  • the reaction is carried out at 0 to 35 ° C for 1 to 96 hours while maintaining at 6. Excess low-molecular substances are removed from the reaction solution by dialysis or the like to obtain an activated HA or HA derivative or a salt thereof.
  • an amine compound represented by the formula (9) is added, and the reaction is carried out using a base such as sodium hydroxide triethylamine.
  • the reaction is carried out at 0-35 ° C for 1-96 hours while maintaining the pH of the solution at 9-12.
  • the amide compound represented by the formula (9) used here has low solubility in water, it may be 1 to 50% of an organic solvent (for example, N, N-dimethylformamide, N-methyl
  • An aqueous solution containing ridone, dioxane, ethanol, pyridine, etc. may be used as the reaction solvent.
  • an organic solvent such as ethanol or acetone is added to the reaction solution to form a precipitate, and the precipitate is subjected to alcohol precipitation, gel filtration, dialysis, ion exchange chromatography or the like.
  • the desired conjugate can be obtained by purification by means. If necessary, before or middle operation deprotecting the R 14 of the purification process (pH 2 6 acid exposure under the, or catalytic reduction) may be added.
  • carbopimides examples include the following compounds
  • Non-limiting specific examples of the substituent in R 1 R 2 R 3 R 4 R 5 and R 6 in the formula (9) are, unless otherwise specified, the non-limiting examples of the substituent in the aforementioned general formula (1). This is the same as the specific example.
  • R 14 protecting group
  • R 14 protecting group
  • 1-Amino-13-benzyloxycarbonylamino-1,4,7,10-trioxatridecane (7.9 g), N- (tert-butoxycarponyl) -L-tert one-port isine (5.1 g), 1 -Hydroxybenzotriazole hydrate (3.4 g), dichloromethane (80 ml) and N, N-dimethylformamide (20m 1) were mixed at 125 ° C with 1-ethyl-3- (3-dimethyl (Aminopropyl) carbodiimide hydrochloride (EDC) (4.3 g) and triethylamine (3.2 ml) were added, and the mixture was stirred at 125 for 30 minutes, at 5 ° C for 1 hour, and further at room temperature for 7 hours.
  • EDC 1-ethyl-3- (3-dimethyl (Aminopropyl) carbodiimide hydrochloride
  • triethylamine 3.2 ml
  • the reaction mixture was concentrated under reduced pressure, dichloromethane was added to the obtained residue, and the mixture was washed with a 5% aqueous solution of citric acid, a saturated aqueous solution of sodium bicarbonate and a saturated aqueous solution of sodium chloride, and then dried over sodium sulfate.
  • Hyaluronic acid sodium salt 600 mg, weight average molecular weight: about 2.2 million was dissolved in distilled water (60 ml), and pyridine (1.2 ml), IN hydrochloric acid (12 ml) and distilled water (46.8 ml) were added to the resulting solution.
  • pyridine 1.2 ml
  • IN hydrochloric acid (12 ml)
  • distilled water 46.8 ml
  • This solution was dialyzed with a dialysis membrane (molecular weight cutoff 12,000 to 14,000, manufactured by Sanko Junyaku Co., Ltd.) at 4 to 6 hours using ion-exchanged water as an external solution.
  • the solution after dialysis was added to the hydroxamic acid derivative N, 1 (13-amino-4,7,10-trioxatridecanyl) 1 N— ( 3S-Hydroxy-41 (N- (1-methoxy-1-methylethoxy) amino)-2R-isobutylsuccinyl)-L-tert-leucinamide in 25 mM aqueous solution (20 ml) was added.
  • the molecular weight of the obtained conjugate was determined by a gel filtration method using hyaluronic acid as a standard substance and found to be about 1.64 million.
  • the concentration of the conjugate aqueous solution was l% (w / V). Further, the obtained conjugate is hydrolyzed with hydrochloric acid, and the amount of t-Leu in the conjugate is measured using an amino acid analyzer.
  • the calculated binding ratio to N-acetyldarcosamine in the salt was 3.8%.
  • conjugate A a conjugate of HA and a hydroxamic acid derivative, hereinafter also referred to as “conjugate A”
  • conjugate A a conjugate of HA and a hydroxamic acid derivative
  • conjugate A a conjugate of HA and a hydroxamic acid derivative
  • Ze SD a hyaluronidase derived from Streptococcus dysgalactiae
  • “Hyal mouth nidase SD” cleaves the N-acetyl-D-darcosaminyl (1 ⁇ 4) darc humic acid bond of HA by elimination reaction, and attaches ⁇ -4,5-glucuron to the non-reducing end. It is an enzyme that produces unsaturated disaccharides having acid residues.
  • Hyaluronidase SD concentration 0.5 U / ml (1 U is p2, the amount of enzyme that releases 1 / x 'mole of unsaturated disaccharide from HA per minute at 37 ° C). 37 ° C, 20 hours.
  • the reaction was stopped by heat-treating the reaction solution (100 ° C, 5 minutes).
  • the resulting heat-treated solution was filtered through a filter (a membrane filter having a pore size of 0.45 m, manufactured by Millipore) to collect a filtrate.
  • the resulting filtrate is fractionated by reversed-phase chromatography under the following conditions to remove oligo-HA (unbound) that passes through the reversed-phase column, and oligo-HA (unsaturated) eluted with acetonitrile gradient is removed.
  • the fraction of the conjugate of the disaccharide) and the hydroxamic acid derivative was collected.
  • LC / MS analysis was performed using the lyophilized product dissolved in 50 methanol as a sample.
  • LC / MS analysis was performed by electrospray ionization (ESI) and sonic spray ionization (SSI) under the following conditions.
  • ESI electrospray ionization
  • SSI sonic spray ionization
  • Sample introduction flow injection method.
  • Ionization method ESI (positive ion mode). Drift 30V, atomizer temperature 250 ° C, pore temperature 120 ° C.
  • the mass is one ionized by adding one sodium ion
  • the mass is one ionized by removing one proton and adding two sodium ions.
  • the conjugate A obtained in Example 2 was hydrolyzed to a hyaluronidase derived from a testicle of a lodge (Sigma, Type V, HA, jS-N-acetyl-D_darcosaminyl (1 ⁇ 4)). The enzyme was then digested into oligo HA (unconjugated) and a conjugate of oligo HA and a hydroxamic acid derivative under the following conditions.
  • Hyaluronidase concentration 10,000 U / ml (Sigma activity).
  • the reaction was stopped by subjecting the reaction solution to heat treatment (100 ° C, 5 minutes).
  • the obtained heat-treated solution was filtered through a filter (a membrane filter with a pore size of 0.45 m, manufactured by Millipore) to collect a filtrate.
  • the filtrate 2501 was applied to a SEP-PAK C18 cartridge (manufactured by Waters), and 5 ml of water was passed through to remove the oligo-HA (unconjugated substance) that passed through. Subsequently, 5 ml of a 20% acetonitrile solution was passed through to obtain a fraction of the eluted conjugate of Oligo HA and the hydroxamic acid derivative. 'The obtained fraction eluted with 20% acetonitrile was freeze-dried and subjected to the following further enzymatic degradation.
  • jS-Darc mouth nidase a product of Sigma Co., Ltd., enzyme that catalyzes the reaction of hydrolyzing type B-10,3-dalcuronide to release D-glucuronic acid.
  • the reaction was stopped by subjecting the reaction solution to heat treatment (100, 5 minutes).
  • the resulting heat-treated solution was filtered through a filter (a membrane filter with a pore size of 0.45 ⁇ , manufactured by Millipore) to collect the filtrate.
  • a conjugate of oligo HA and a hydroxamic acid derivative contained in the filtrate was converted to a chondroitinase derived from Arthrobactor aurescens (trade name “Chondroitinase ACII Arsulo”, manufactured by Seikagaku Corporation), chondroitin sulfate, hyaluronic acid, and the like.
  • the reaction was stopped by heat-treating the reaction solution (100 ° C, 5 minutes).
  • the resulting heat-treated solution was filtered through a filter (a membrane filter with a pore size of 0.45 / xm, manufactured by Millipore), and the filtrate was recovered.
  • the obtained filtrate is subjected to reverse phase chromatography fractionation under the following conditions to remove oligo-HA etc. (non-conjugated substance) which pass through the reverse phase column, and N-acetyl eluted by acetonitrile gradient Fractions of the conjugate of darcosamine and the hydroxamic acid derivative were collected.
  • LC / MS analysis was performed using the lyophilized product dissolved in 50 methanol as a sample.
  • LC / MS analysis was performed by electrospray ionization (ESI) and sonic spray ionization (SSI) under the following conditions.
  • ESI electrospray ionization
  • SSI sonic spray ionization
  • Sample introduction flow injection method.
  • the inhibitory activity of conjugate A synthesized by the method described in Example 2 above on gelatinase A and stromelysin-11 was measured. For comparison, the same measurement was performed for “conjugate 7” described in Example 9 of WO99 / 5963.
  • the inhibitory activity against gelatinase A was determined using a gelase assay kit manufactured by Roche Diagnostics, Inc., according to the attached protocol.
  • the enzyme reaction was measured at 37 ° C. for 1 hour.
  • the enzyme inhibitory activity against stomatis melisin-1 was measured using an activated stromelysin-1 product manufactured by the company using a stomalysin activity measurement kit manufactured by Gagay according to the attached protocol.
  • the enzyme inhibitory activity is expressed by the concentration of each conjugate required to suppress each enzyme activity by 50% when conjugate A and conjugate 7 are not added (IC 5 , the concentration obtained by converting each conjugate to hyaluronic acid) ).
  • IC 5 the concentration obtained by converting each conjugate to hyaluronic acid
  • Table 1 Matrix meta-oral protease inhibitory activity
  • Conjugate A synthesized by the method described in Example 2 above was tested using a modified version of Saito et al.'S method [J. Biochem., 122, 49-54 (1997)]. For comparison, a similar test was performed for “conjugate 7” described in Example 8 of W9999603.
  • a 5-7 week old male marbled knee joint cartilage fragment (about 10 mg) was placed on a culture plate supplemented with 500 ⁇ of 01 ⁇ 6 ( ⁇ 3 modified eagle 'Medium (DMEM) (Fa 1 con Ltd., 4 8 well flat bottom culture plate, in # 3078), in C0 2 incubator one 37 ° C, after replacing the.
  • DMEM modified eagle 'Medium
  • the release rate of hydroxyproline was calculated from the amount of hydroxyproline released from the node piece into the culture supernatant and the amount of hydroxyproline in the residue of joint pieces according to the following formula. 8-hydroxyproline
  • the conjugate of the present invention has an excellent MMP inhibitory action, and can limit the action and prolong the action.
  • the conjugate of the present invention can be used as a drug having improved utility as both a therapeutic agent for joint disease and a drug for HA, for example, a drug having enhanced joint destruction inhibitory activity, which has excellent osteoarthritis and chronic It is expected to be a therapeutic agent for rheumatoid arthritis or shoulder periarthritis.

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Abstract

Composé possédant une activité d'inhibition de MMP et consistant en un composé d'acide hydroxamique représenté par la formule (1) et acide hyaluronique, formule dans laquelle R1 représente hydrogène, hydroxy, alkyle C1-C8; R2 représente alkyle C1-C8; R3 représente alkyle C1-C8; R4 représente hydrogène ou alkyle C1-C4; R5 représente -R7-R8-R9-, dans laquelle R7 représente alkylène C1-C8, R8 représente méthylène, imino, oxygène et R9 représente alkylène C1-C10; R6 représente hydrogène ou alkyle C1-C4, à condition que R1 et R3 en combinaison puissent former un anneau. Ce composé contient un groupe représenté par la formule (1) et acide hyaluronique, un de ses dérivés ou un de ses sels, le premier étant lié à un groupe hydroxyle du dernier par l'intermédiaire d'une liaison carbamate.
PCT/JP2001/010493 2000-11-30 2001-11-30 Compose de derive d'acide hydroxamique et d'acide hyaluronique WO2002044218A1 (fr)

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JP2000-363993 2000-11-30
JP2000363993A JP2004292465A (ja) 2000-11-30 2000-11-30 ヒドロキサム酸誘導体とヒアルロン酸の結合体

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005085294A1 (fr) 2004-03-05 2005-09-15 Denki Kagaku Kogyo Kabushiki Kaisha Composé d’acide hyaluronique/méthotrexate

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5501589B2 (ja) * 2008-08-28 2014-05-21 電気化学工業株式会社 ヒアルロン酸及び／又はその塩の精製法
ITRM20080636A1 (it) * 2008-11-28 2010-05-29 Univ Palermo Procedimento per la produzione di derivati funzionalizzati dell acido ialuronico e relativi idrogeli.

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999059603A1 (fr) * 1998-05-20 1999-11-25 Chugai Seiyaku Kabushiki Kaisha Medicaments contre les affections articulaires lies a l'acide hyaluronique
WO2000046189A1 (fr) * 1999-02-02 2000-08-10 Shionogi & Co., Ltd. Derives de la sulfonamide a structures cycliques

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999059603A1 (fr) * 1998-05-20 1999-11-25 Chugai Seiyaku Kabushiki Kaisha Medicaments contre les affections articulaires lies a l'acide hyaluronique
WO2000046189A1 (fr) * 1999-02-02 2000-08-10 Shionogi & Co., Ltd. Derives de la sulfonamide a structures cycliques

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005085294A1 (fr) 2004-03-05 2005-09-15 Denki Kagaku Kogyo Kabushiki Kaisha Composé d’acide hyaluronique/méthotrexate
US8088916B2 (en) 2004-03-05 2012-01-03 Denki Kagaku Kogyo Kabushiki Kaisha Hyaluronic acid-methotrexate conjugate

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JP2004292465A (ja) 2004-10-21

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