CN117530921A - Hyaluronic acid-rebamipide conjugate, preparation method thereof and eye drops - Google Patents
Hyaluronic acid-rebamipide conjugate, preparation method thereof and eye drops Download PDFInfo
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- CN117530921A CN117530921A CN202311695404.2A CN202311695404A CN117530921A CN 117530921 A CN117530921 A CN 117530921A CN 202311695404 A CN202311695404 A CN 202311695404A CN 117530921 A CN117530921 A CN 117530921A
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- 229950004535 rebamipide Drugs 0.000 title claims abstract description 115
- 239000003889 eye drop Substances 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 229940012356 eye drops Drugs 0.000 title description 20
- ALLWOAVDORUJLA-UHFFFAOYSA-N Rebamipida Chemical compound C=1C(=O)NC2=CC=CC=C2C=1CC(C(=O)O)NC(=O)C1=CC=C(Cl)C=C1 ALLWOAVDORUJLA-UHFFFAOYSA-N 0.000 claims abstract description 76
- 229920002674 hyaluronan Polymers 0.000 claims abstract description 54
- 229960003160 hyaluronic acid Drugs 0.000 claims abstract description 54
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims abstract description 44
- 208000003556 Dry Eye Syndromes Diseases 0.000 claims abstract description 19
- 206010013774 Dry eye Diseases 0.000 claims abstract description 19
- 238000006243 chemical reaction Methods 0.000 claims description 36
- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 7
- 230000008569 process Effects 0.000 claims description 5
- 239000000022 bacteriostatic agent Substances 0.000 claims description 2
- 230000003204 osmotic effect Effects 0.000 claims description 2
- 238000006467 substitution reaction Methods 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 19
- 229940079593 drug Drugs 0.000 abstract description 13
- 102000015728 Mucins Human genes 0.000 abstract description 5
- 108010063954 Mucins Proteins 0.000 abstract description 5
- 210000004400 mucous membrane Anatomy 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 229920002521 macromolecule Polymers 0.000 abstract description 3
- 230000003020 moisturizing effect Effects 0.000 abstract description 3
- 230000028327 secretion Effects 0.000 abstract description 3
- 108090000371 Esterases Proteins 0.000 abstract description 2
- 230000009471 action Effects 0.000 abstract description 2
- 230000007246 mechanism Effects 0.000 abstract description 2
- 230000001737 promoting effect Effects 0.000 abstract description 2
- 208000024891 symptom Diseases 0.000 abstract description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 32
- 238000005119 centrifugation Methods 0.000 description 20
- 238000012869 ethanol precipitation Methods 0.000 description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 17
- 239000000243 solution Substances 0.000 description 13
- 239000000047 product Substances 0.000 description 12
- 239000008213 purified water Substances 0.000 description 12
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 10
- 238000005481 NMR spectroscopy Methods 0.000 description 10
- 238000004090 dissolution Methods 0.000 description 10
- 229910052739 hydrogen Inorganic materials 0.000 description 10
- 239000001257 hydrogen Substances 0.000 description 10
- 239000002244 precipitate Substances 0.000 description 10
- 238000001556 precipitation Methods 0.000 description 10
- 238000001228 spectrum Methods 0.000 description 10
- 239000000725 suspension Substances 0.000 description 6
- 238000012512 characterization method Methods 0.000 description 4
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 4
- 208000005494 xerophthalmia Diseases 0.000 description 4
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 208000007882 Gastritis Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229920002385 Sodium hyaluronate Polymers 0.000 description 2
- 241000204066 Tsukamurella Species 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 230000001050 lubricating effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 229940010747 sodium hyaluronate Drugs 0.000 description 2
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 1
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 1
- 108010036949 Cyclosporine Proteins 0.000 description 1
- 229940123457 Free radical scavenger Drugs 0.000 description 1
- 208000022873 Ocular disease Diseases 0.000 description 1
- 208000007107 Stomach Ulcer Diseases 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000000607 artificial tear Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229960002152 chlorhexidine acetate Drugs 0.000 description 1
- 208000023652 chronic gastritis Diseases 0.000 description 1
- 229960001265 ciclosporin Drugs 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 210000000795 conjunctiva Anatomy 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 229930182912 cyclosporin Natural products 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 208000000718 duodenal ulcer Diseases 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 201000005917 gastric ulcer Diseases 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 229940051875 mucins Drugs 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- MCSINKKTEDDPNK-UHFFFAOYSA-N propyl propionate Chemical compound CCCOC(=O)CC MCSINKKTEDDPNK-UHFFFAOYSA-N 0.000 description 1
- 150000003180 prostaglandins Chemical class 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- BBMHARZCALWXSL-UHFFFAOYSA-M sodium dihydrogenphosphate monohydrate Chemical compound O.[Na+].OP(O)([O-])=O BBMHARZCALWXSL-UHFFFAOYSA-M 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4704—2-Quinolinones, e.g. carbostyril
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/728—Hyaluronic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0048—Eye, e.g. artificial tears
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- 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
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- Chemical & Material Sciences (AREA)
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- General Health & Medical Sciences (AREA)
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- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Ophthalmology & Optometry (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Dermatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Polymers & Plastics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention belongs to the technical field of medicines, and in particular relates to a hyaluronic acid-rebamipide conjugate, a preparation method thereof and an ophthalmic eye drop. The hyaluronic acid-rebamipide conjugate has a structure as shown in formula (I):in the formula (I), x is not 0 and is less than n. The invention couples the rebamipide with the hyaluronic acid, which can greatly improve the solubility of the rebamipide, reduce the dosage of the medicine, and the hyaluronic acid has the functions of moisturizing and lubricatingTo rapidly alleviate symptoms of dry eye. The conjugate combines molecules with two different action mechanisms, the hyaluronic acid of the macromolecule plays a role in immediately relieving dry eye, and the rebamipide of the micromolecule can be released after the ocular surface is hydrolyzed by esterase, so that the effect of repairing ocular surface mucous membrane cells and promoting mucin secretion is achieved.
Description
Technical Field
The invention belongs to the technical field of medicines, and in particular relates to a hyaluronic acid-rebamipide conjugate, a preparation method thereof and an ophthalmic eye drop.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Dry eye, a tear dyscrasia ocular disease that manifests as dry eyes. Many factors that form dry eye, such as abnormal regulation of ocular nerves, inflammatory response, abnormal expression of ocular surface mucin, apoptosis, etc. At present, 16 medicines are clinically used for xerophthalmia, 11 medicines belong to artificial tears, and sodium hyaluronate eye drops are most commonly used. The sodium hyaluronate dilute solution is similar to tears, has non-Newtonian fluid characteristics, is similar to tears, and has good biocompatibility.
The hyaluronic acid eye drops can be used as a therapeutic drug for treating mild dry eye, but the dry eye is difficult to radically treat aiming at the pathogenesis of the dry eye, and at present, only a small amount of drugs for treating moderate and severe dry eye exist, wherein the cyclosporine eye drops have better therapeutic effects on the moderate and severe dry eye, but the drugs have slow effects and have adverse reactions such as burning feeling and the like when being used. Rebamipide is a drug for protecting mucous membrane, and its mechanism of action is to enhance mucous membrane protection by stimulating mucin and prostaglandin production. Rebamipide is a drug for gastric and duodenal ulcer lesions and acute and chronic gastritis developed by the tsukamurella pharmaceutical in the last 90 th century, and a suspension eye drop of rebamipide was developed by the tsukamurella pharmaceutical in 2012 for the treatment of dry eye.
Rebamipide is a powerful antioxidant and free radical scavenger, and plays a role in treating dry eye by enhancing tear secretion and increasing the level of mucins covering conjunctiva and cornea surfaces. Rebamipide is hardly soluble in water, only in dimethyl sulfoxide or dimethylformamide. Therefore, the medicine is difficult to prepare into solution type eye drops, the rebamipide eye drops on the market are 2% suspension eye drops, and the rebamipide bulk drug is required to be subjected to micronization treatment to reach a certain particle size requirement, so that the preparation cost is high. The rebamipide eye drops are suspension formulations, have large dosage, poor patient compliance, and low rebamipide solubility, resulting in low bioavailability.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a hyaluronic acid-rebamipide conjugate, a preparation method thereof and an ophthalmic eye drop. The invention couples the rebamipide which is difficult to dissolve in water with the hyaluronic acid, thereby improving the solubility of the rebamipide in water, and the hyaluronic acid has the functions of moisturizing and lubricating, and is also a medicine for treating xerophthalmia. The combination of the two drugs for treating xerophthalmia can not only improve the solubility of rebamipide but also solve the problem of short duration of drug effect of hyaluronic acid eye drops.
In order to achieve the above object, the present invention is realized by the following technical scheme:
in a first aspect, the present invention provides a hyaluronic acid-rebamipide conjugate having a structure as shown in formula (I):
in the formula (I), x is not 0 and is less than n.
The coupling of the rebamipide and the hyaluronic acid can improve the water solubility of the rebamipide, is convenient for preparing water-soluble eye drops, and can avoid micronization process used in the preparation process of the rebamipide suspension eye drops. In addition, the hyaluronic acid is used as a macromolecule, has certain viscosity, and can prolong the retention time of the rebamipide on the ocular surface so as to increase the bioavailability of the rebamipide, thereby reducing the dosage and the administration frequency of the rebamipide. Hyaluronic acid and rebamipide conjugate can combine the advantages of the two drugs for treating dry eye, and improve the disadvantages of each of the two drugs. The coupling of the rebamipide and the hyaluronic acid can greatly improve the solubility of the rebamipide, reduce the dosage of the medicine, and in addition, the hyaluronic acid has the functions of moisturizing and lubricating, so that the symptoms of dry eyes can be quickly relieved.
Preferably, the hyaluronic acid has a number average molecular weight of 50-500kDa.
Preferably, the degree of substitution of rebamipide is 2% to 30%.
In a second aspect, the present invention provides a method for preparing a hyaluronic acid-rebamipide conjugate according to the first aspect, comprising the steps of:
reacting rebamipide with carbonyl diimidazole to obtain an activated intermediate, and reacting the intermediate with hyaluronic acid to obtain the hyaluronic acid-rebamipide conjugate.
Preferably, the molar ratio of rebamipide to carbonyldiimidazole is 1:0.9-1.1.
Preferably, the reaction temperature of rebamipide and carbonyldiimidazole is room temperature, and the time is 20-40min.
Preferably, the molar ratio of rebamipide to hyaluronic acid is from 0.2:1 to 5:1.
Preferably, the reaction temperature of the intermediate and the hyaluronic acid is 30-70 ℃ and the reaction time is 20-28h.
In a third aspect, the present invention provides the use of a hyaluronic acid-rebamipide conjugate according to the first aspect in the preparation of a dry eye treatment product.
In a fourth aspect, the present invention provides an eye drop comprising a hyaluronic acid-rebamipide conjugate according to the first aspect, an osmotic pressure regulator, a pH regulator and a bacteriostatic agent.
Preferably, the concentration of hyaluronic acid-rebamipide conjugate is 0.05% to 1% W/V.
The rebamipide eye drops developed in the large scale of the tsukamur pharmacy are suspension eye drops, so that the rebamipide bulk drug needs to be subjected to micronization treatment to reach a certain particle size requirement. The hyaluronic acid-rebamipide conjugate disclosed by the invention belongs to water-soluble molecules, and can be directly dissolved in water to prepare aqueous eye drops. Compared with the original ground product, the hyaluronic acid-rebamipide conjugate eye drop has simpler preparation process and better dosage form, and combines the advantages of hyaluronic acid and rebamipide in dry eye treatment.
The beneficial effects obtained by one or more of the technical schemes of the invention are as follows:
the hyaluronic acid-rebamipide conjugate prepared by the invention has the advantages of simple synthesis method, controllable amount of conjugated rebamipide, and capability of coupling together the macromolecular medicament hyaluronic acid for treating xerophthalmia and the micromolecular medicament rebamipide without introducing other bridging molecules. The conjugate combines molecules with two different action mechanisms, the hyaluronic acid of the macromolecule plays a role in immediately relieving dry eye, and the rebamipide of the micromolecule can be released after the ocular surface is hydrolyzed by esterase, so that the effect of repairing ocular surface mucous membrane cells and promoting mucin secretion is achieved.
Compared with the rebamipide suspension eye drops on the market, the conjugate disclosed by the invention is water-soluble eye drops, has a simple prescription, combines the therapeutic effect of hyaluronic acid and rebamipide on dry eye, and has rapid and lasting effect. In addition, the preparation method is simple, the sources of the used raw materials are wide, the price is low, and the preparation method has good application prospect.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a nuclear magnetic resonance spectrum of a hyaluronic acid-rebamipide conjugate in example 1 of the invention;
FIG. 2 is a nuclear magnetic resonance spectrum of a hyaluronic acid-rebamipide conjugate according to example 4 of the invention;
FIG. 3 is a nuclear magnetic resonance spectrum of a hyaluronic acid-rebamipide conjugate according to example 5 of the invention;
FIG. 4 is a nuclear magnetic resonance spectrum of a hyaluronic acid-rebamipide conjugate according to example 8 of the invention;
FIG. 5 is the UV-visible spectrum of the hyaluronic acid-rebamipide conjugate of example 2 of the invention.
Detailed Description
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail below with reference to specific examples and comparative examples.
Example 1
Rebamipide (0.074 g,0.2 mmol) was weighed and placed in a two-necked round bottom flask, anhydrous DMSO (2 mL) was added for dissolution, carbonyldiimidazole (0.032 g,0.2 mmol) was then weighed and added to the flask, the reaction system was placed at 30℃for reaction for 30min, and hyaluronic acid (0.3831 g,1mmol, molecular weight 50000 Da) was then added thereto; the reaction was carried out at 40℃for 24 hours. After the reaction, ethanol precipitation and centrifugation are carried out, finally, precipitation is collected, a purified water solution product is used, ethanol precipitation is used again, and after centrifugation, the precipitate is dried in vacuum to obtain the rebamipide-hyaluronic acid conjugate (0.462 g, yield 82.9%), the structure of the hyaluronic acid rebamipide conjugate is determined by nuclear magnetic resonance hydrogen spectrum, and the rebamipide content in the conjugate is calculated by ultraviolet.
Nuclear magnetic characterization of hyaluronic acid-rebamipide conjugates (400 MHz, D 2 O) is shown in fig. 1.
Example 2
Rebamipide (0.185 g,0.5 mmol) was weighed and placed in a two-necked round bottom flask, anhydrous DMSO (2 mL) was added for dissolution, carbonyldiimidazole (0.081 g,0.5 mmol) was then weighed and added to the flask, the reaction system was placed at 25℃for 30min, and hyaluronic acid (0.381 g,1mmol, molecular weight: 50000 Da) was then added thereto; the reaction was carried out at 40℃for 28h. After the reaction, ethanol precipitation and centrifugation are carried out, finally, precipitation is collected, a purified water solution product is used, ethanol precipitation is used again, and after centrifugation, the precipitate is dried in vacuum to obtain the rebamipide-hyaluronic acid conjugate (0.507 g, yield is 85.2%), the structure of the hyaluronic acid rebamipide conjugate is determined by nuclear magnetic resonance hydrogen spectrum, and the rebamipide content in the conjugate is calculated by ultraviolet.
The uv-vis spectrum of the hyaluronic acid-rebamipide conjugate is shown in fig. 5.
Example 3
Rebamipide (0.370 g,1 mmol) was weighed into a two-necked round bottom flask, anhydrous DMSO (4 mL) was added for dissolution, carbonyldiimidazole (0.162 g,1 mmol) was then weighed into the flask, the reaction system was allowed to react at 25℃for 30min, and hyaluronic acid (0.381 g,1mmol, molecular weight 50000 Da) was then added thereto; the reaction was carried out at 70℃for 24 hours. After the reaction, ethanol precipitation and centrifugation are carried out, finally, precipitation is collected, a purified water solution product is used, ethanol precipitation is used again, and after centrifugation, the precipitate is dried in vacuum to obtain the rebamipide-hyaluronic acid conjugate (0.507 g, yield 91.0%), the structure of the hyaluronic acid rebamipide conjugate is determined by nuclear magnetic resonance hydrogen spectrum, and the rebamipide content in the conjugate is calculated by ultraviolet.
Example 4
Rebamipide (0.740 g,1 mmol) was weighed into a two-necked round bottom flask, anhydrous DMSO (8 mL) was added for dissolution, carbonyldiimidazole (0.324 g,1 mmol) was then weighed into the flask, the reaction system was allowed to react at 25℃for 40min, and hyaluronic acid (0.3831 g,1mmol, molecular weight 50000 Da) was then added thereto; the reaction was carried out at 40℃for 20h. After the reaction, ethanol precipitation and centrifugation are carried out, finally, precipitation is collected, a purified water solution product is used, ethanol precipitation is used again, after centrifugation, the precipitate is dried in vacuum, thus obtaining the rebamipide-hyaluronic acid conjugate (0.425 g, yield 76.0%), the structure of the hyaluronic acid rebamipide conjugate is determined by nuclear magnetic resonance hydrogen spectrum, and the rebamipide content in the conjugate is calculated by ultraviolet.
Nuclear magnetic characterization of hyaluronic acid-rebamipide conjugates (400 MHz, D 2 O) is shown in fig. 2.
Example 5
Rebamipide (0.074 g,0.2 mmol) was weighed and placed in a two-necked round bottom flask, anhydrous DMSO (2 mL) was added for dissolution, carbonyldiimidazole (0.032 g,0.2 mmol) was then weighed and added to the flask, the reaction system was placed at 25℃for 30min, and hyaluronic acid (0.3831 g,1mmol, molecular weight 500000 Da) was then added thereto; the reaction was carried out at 50℃for 24 hours. After the reaction, ethanol precipitation and centrifugation are carried out, finally, precipitation is collected, a purified water solution product is used, ethanol precipitation is used again, and after centrifugation, the precipitate is dried in vacuum to obtain the rebamipide-hyaluronic acid conjugate (0.405 g, yield 72.6%), the structure of the hyaluronic acid rebamipide conjugate is determined by nuclear magnetic resonance hydrogen spectrum, and the rebamipide content in the conjugate is calculated by ultraviolet.
Nuclear magnetic characterization of hyaluronic acid-rebamipide conjugates (400 MHz, D 2 O) is shown in fig. 3.
Example 6
Rebamipide (0.185 g,0.5 mmol) was weighed and placed in a two-necked round bottom flask, anhydrous DMSO (2 mL) was added for dissolution, carbonyldiimidazole (0.081 g,0.5 mmol) was then weighed and added to the flask, the reaction system was placed at 25℃for 30min, and hyaluronic acid (0.3831 g,1mmol, molecular weight 500000 Da) was then added thereto; the reaction was carried out at 40℃for 24 hours. After the reaction, ethanol precipitation and centrifugation are carried out, finally, precipitation is collected, a purified water solution product is used, ethanol precipitation is used again, and after centrifugation, the precipitate is dried in vacuum to obtain the rebamipide-hyaluronic acid conjugate (0.493 g, yield 88.3%), the structure of the hyaluronic acid rebamipide conjugate is determined by nuclear magnetic resonance hydrogen spectrum, and the rebamipide content in the conjugate is calculated by ultraviolet.
Example 7
Rebamipide (0.370 g,1 mmol) was weighed into a two-necked round bottom flask, anhydrous DMSO (4 mL) was added for dissolution, carbonyldiimidazole (0.162 g,1 mmol) was then weighed into the flask, the reaction system was allowed to react at 25℃for 30min, and hyaluronic acid (0.381 g,1mmol, molecular weight 500000 Da) was then added thereto; the reaction was carried out at 40℃for 24 hours. After the reaction, ethanol precipitation and centrifugation are carried out, finally, precipitation is collected, a purified water solution product is used, ethanol precipitation is used again, and after centrifugation, the precipitate is dried in vacuum to obtain the rebamipide-hyaluronic acid conjugate (0.512 g, yield 91.7%), the structure of the hyaluronic acid rebamipide conjugate is determined by nuclear magnetic resonance hydrogen spectrum, and the rebamipide content in the conjugate is calculated by ultraviolet.
Example 8
Rebamipide (0.740 g,2 mmol) was weighed into a two-necked round bottom flask, anhydrous DMSO (8 mL) was added for dissolution, carbonyldiimidazole (0.324 g,2 mmol) was then weighed into the flask, the reaction system was placed at 25℃for 30min, and hyaluronic acid (0.3831 g,1mmol, molecular weight 500000 Da) was then added; the reaction was carried out at 70℃for 24 hours. After the reaction, ethanol precipitation and centrifugation are carried out, finally, precipitation is collected, a purified water solution product is used, ethanol precipitation is used again, and after centrifugation, the precipitate is dried in vacuum to obtain the rebamipide-hyaluronic acid conjugate (0.466 g, yield 83.5%), the structure of the hyaluronic acid rebamipide conjugate is determined by nuclear magnetic resonance hydrogen spectrum, and the rebamipide content in the conjugate is calculated by ultraviolet.
Nuclear magnetic characterization of hyaluronic acid-rebamipide conjugates (400 MHz, D 2 O) is shown in fig. 4.
The rebamipide content and rebamipide solubility of the hyaluronic acid-rebamipide conjugates prepared in examples 1 to 8 are shown in table 1.
TABLE 1 content and solubility of rebamipide in hyaluronic acid-rebamipide conjugate
As shown in table 1, the solubility of rebamipide in the hyaluronic acid-rebamipide conjugate was improved.
Example 9
The formulations of hyaluronic acid-rebamipide conjugate eye drops are shown in table 2.
0.1g of the hyaluronic acid-rebamipide conjugate synthesized in example 1, 0.2g of polyvinylpyrrolidone, 1.62g of anhydrous disodium hydrogen phosphate, 0.24g of sodium dihydrogen phosphate monohydrate, 0.8g of sodium chloride and 0.02g of chlorhexidine acetate are dissolved in 100mL of purified water, and the solution is fixed to a volume of 200mL with purified water to obtain 0.05% hyaluronic acid-rebamipide conjugate eye drops. The osmolality of the hyaluronic acid-rebamipide conjugate eye drops was measured to be 282mOsmol/kg and the pH was measured to be 6.82.
Example 10
Rebamipide (0.074 g,0.2 mmol) was weighed and placed in a two-necked round bottom flask, anhydrous DMSO (2 mL) was added for dissolution, carbonyldiimidazole (0.028 g,0.18 mmol) was then weighed and added to the flask, the reaction system was placed at 30℃for reaction for 30min, and hyaluronic acid (0.3831 g,1mmol, molecular weight 50000 Da) was then added thereto; the reaction was carried out at 40℃for 24 hours. After the reaction, ethanol precipitation and centrifugation are carried out, finally, precipitation is collected, a purified water solution product is used, ethanol precipitation is used again, and after centrifugation, the precipitate is dried in vacuum to obtain the rebamipide-hyaluronic acid conjugate (0.462 g, yield 82.9%), the structure of the hyaluronic acid rebamipide conjugate is determined by nuclear magnetic resonance hydrogen spectrum, and the rebamipide content in the conjugate is calculated by ultraviolet.
Example 11
Rebamipide (0.074 g,0.2 mmol) was weighed and placed in a two-necked round bottom flask, anhydrous DMSO (2 mL) was added for dissolution, carbonyldiimidazole (0.035 g,0.22 mmol) was then weighed and added to the flask, the reaction system was placed at 30℃for reaction for 30min, and hyaluronic acid (0.3831 g,1mmol, molecular weight 50000 Da) was then added thereto; the reaction was carried out at 40℃for 24 hours. After the reaction, ethanol precipitation and centrifugation are carried out, finally, precipitation is collected, a purified water solution product is used, ethanol precipitation is used again, and after centrifugation, the precipitate is dried in vacuum to obtain the rebamipide-hyaluronic acid conjugate (0.462 g, yield 82.9%), the structure of the hyaluronic acid rebamipide conjugate is determined by nuclear magnetic resonance hydrogen spectrum, and the rebamipide content in the conjugate is calculated by ultraviolet.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A hyaluronic acid-rebamipide conjugate, characterized by having a structure as shown in formula (I):
in the formula (I), x is not 0 and is less than n.
2. The hyaluronic acid-rebamipide conjugate according to claim 1, wherein the number average molecular weight of hyaluronic acid is 50-500kDa.
3. The hyaluronic acid-rebamipide conjugate according to claim 1, wherein the degree of substitution of rebamipide is 2% to 30%.
4. A process for the preparation of a hyaluronic acid-rebamipide conjugate according to any one of claims 1-3, comprising the steps of:
reacting rebamipide with carbonyl diimidazole to obtain an activated intermediate, and reacting the intermediate with hyaluronic acid to obtain the hyaluronic acid-rebamipide conjugate.
5. The process of claim 4, wherein the molar ratio of rebamipide to carbonyldiimidazole is 1:0.9-1.1.
6. The process of claim 4, wherein the reaction temperature of rebamipide with carbonyldiimidazole is in the range of 20 to 40 minutes.
7. The method of claim 4, wherein the molar ratio of rebamipide to hyaluronic acid is from 0.2:1 to 5:1.
8. The process of claim 4, wherein the intermediate is reacted with hyaluronic acid at a temperature of 30-70 ℃ for a period of 20-28 hours.
9. Use of a hyaluronic acid-rebamipide conjugate according to any one of claims 1-3 in the preparation of a dry eye treatment product.
10. An eye drop comprising the hyaluronic acid-rebamipide conjugate of any one of claims 1-3, an osmotic pressure regulator, a pH regulator, and a bacteriostatic agent.
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