MXPA06008467A - Esters of hyaluronic acid with rhein, process for their preparation and compositions comprising the same - Google Patents
Esters of hyaluronic acid with rhein, process for their preparation and compositions comprising the sameInfo
- Publication number
- MXPA06008467A MXPA06008467A MXPA/A/2006/008467A MXPA06008467A MXPA06008467A MX PA06008467 A MXPA06008467 A MX PA06008467A MX PA06008467 A MXPA06008467 A MX PA06008467A MX PA06008467 A MXPA06008467 A MX PA06008467A
- Authority
- MX
- Mexico
- Prior art keywords
- queen
- compound
- hyaluronic acid
- pharmaceutical composition
- process according
- Prior art date
Links
- 229920002674 hyaluronan Polymers 0.000 title claims abstract description 73
- 229960003160 hyaluronic acid Drugs 0.000 title claims abstract description 73
- MAKUBRYLFHZREJ-JWBQXVCJSA-M sodium;(2S,3S,4R,5R,6R)-3-[(2S,3R,5S,6R)-3-acetamido-5-hydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4,5,6-trihydroxyoxane-2-carboxylate Chemical compound [Na+].CC(=O)N[C@@H]1C[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](C([O-])=O)O[C@@H](O)[C@H](O)[C@H]1O MAKUBRYLFHZREJ-JWBQXVCJSA-M 0.000 title claims abstract description 71
- 238000000034 method Methods 0.000 title claims description 25
- 239000000203 mixture Substances 0.000 title claims description 15
- 150000002148 esters Chemical class 0.000 title abstract description 6
- 238000002360 preparation method Methods 0.000 title description 5
- 150000001875 compounds Chemical class 0.000 claims abstract description 85
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 19
- 125000003158 alcohol group Chemical group 0.000 claims abstract description 15
- 238000004519 manufacturing process Methods 0.000 claims abstract description 7
- 239000002253 acid Substances 0.000 claims description 21
- VEXZGXHMUGYJMC-UHFFFAOYSA-M chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 20
- 239000011780 sodium chloride Substances 0.000 claims description 19
- 238000006243 chemical reaction Methods 0.000 claims description 18
- 150000003839 salts Chemical class 0.000 claims description 18
- 238000000502 dialysis Methods 0.000 claims description 15
- 238000010992 reflux Methods 0.000 claims description 12
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- GPRLSGONYQIRFK-UHFFFAOYSA-N hydron Chemical compound [H+] GPRLSGONYQIRFK-UHFFFAOYSA-N 0.000 claims description 4
- 239000003085 diluting agent Substances 0.000 claims description 3
- 238000001764 infiltration Methods 0.000 claims description 3
- 159000000000 sodium salts Chemical class 0.000 claims description 3
- CPBZARXQRZTYGI-UHFFFAOYSA-N 3-cyclopentylpropylcyclohexane Chemical compound C1CCCCC1CCCC1CCCC1 CPBZARXQRZTYGI-UHFFFAOYSA-N 0.000 claims description 2
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- YMWUJEATGCHHMB-UHFFFAOYSA-N methylene dichloride Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 8
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- YXFVVABEGXRONW-UHFFFAOYSA-N toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- 102100006425 GAPDH Human genes 0.000 description 5
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- XDTMQSROBMDMFD-UHFFFAOYSA-N cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
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- 108060005980 Collagenase family Proteins 0.000 description 3
- 102000020504 Collagenase family Human genes 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 3
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- 238000000692 Student's t-test Methods 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N acetic acid ethyl ester Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 229960002424 collagenase Drugs 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- BDAGIHXWWSANSR-UHFFFAOYSA-N formic acid Chemical compound OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 3
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- FYSNRJHAOHDILO-UHFFFAOYSA-N Thionyl chloride Chemical compound ClS(Cl)=O FYSNRJHAOHDILO-UHFFFAOYSA-N 0.000 description 2
- 239000000370 acceptor Substances 0.000 description 2
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 2
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- 239000002299 complementary DNA Substances 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 125000004185 ester group Chemical group 0.000 description 2
- 238000005227 gel permeation chromatography Methods 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
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- 239000000463 material Substances 0.000 description 2
- 229920002106 messenger RNA Polymers 0.000 description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 2
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
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- 239000008363 phosphate buffer Substances 0.000 description 2
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- 239000003755 preservative agent Substances 0.000 description 2
- 230000001681 protective Effects 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 230000001954 sterilising Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000002194 synthesizing Effects 0.000 description 2
- 230000017423 tissue regeneration Effects 0.000 description 2
- HPYYIBPNNXXEAA-IRBJBBIRSA-N (2S,3S,4S,5R)-2,3,4,5-tetrahydroxy-6-oxohexanoic acid;N-[(3R,4R,5S,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)oxan-3-yl]acetamide Chemical compound O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O.CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O HPYYIBPNNXXEAA-IRBJBBIRSA-N 0.000 description 1
- FCDLCPWAQCPTKC-UHFFFAOYSA-N 4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carboxylic acid Chemical group C1=CC=C2C(=O)C3=CC(C(=O)O)=CC(O)=C3C(=O)C2=C1O FCDLCPWAQCPTKC-UHFFFAOYSA-N 0.000 description 1
- RZVHIXYEVGDQDX-UHFFFAOYSA-N Anthraquinone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3C(=O)C2=C1 RZVHIXYEVGDQDX-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
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- 241000561734 Celosia cristata Species 0.000 description 1
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- 206010012735 Diarrhoea Diseases 0.000 description 1
- 229940012356 Eye Drops Drugs 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- -1 HA compound Chemical class 0.000 description 1
- 210000001624 Hip Anatomy 0.000 description 1
- 102000001974 Hyaluronidase Human genes 0.000 description 1
- 108010074224 Hyaluronoglucosaminidase Proteins 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 208000009883 Joint Disease Diseases 0.000 description 1
- 102100014894 MMP2 Human genes 0.000 description 1
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- 102000000422 Matrix Metalloproteinase 3 Human genes 0.000 description 1
- 102000005741 Metalloproteases Human genes 0.000 description 1
- 108010006035 Metalloproteases Proteins 0.000 description 1
- 210000003205 Muscles Anatomy 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-Acetylglucosamine Chemical group CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- 229950006780 N-Acetylglucosamine Drugs 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 206010039073 Rheumatoid arthritis Diseases 0.000 description 1
- 229910006124 SOCl2 Inorganic materials 0.000 description 1
- 241000735631 Senna pendula Species 0.000 description 1
- 206010049514 Traumatic fracture Diseases 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
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- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Vitamin C Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 1
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- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical group O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 229930013930 alkaloids Natural products 0.000 description 1
- 230000001195 anabolic Effects 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
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- 125000004063 butyryl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
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- 229960002773 hyaluronidase Drugs 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
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- 238000002329 infrared spectrum Methods 0.000 description 1
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- 159000000003 magnesium salts Chemical class 0.000 description 1
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- 229940111202 pepsin Drugs 0.000 description 1
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- 159000000001 potassium salts Chemical class 0.000 description 1
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- 239000002244 precipitate Substances 0.000 description 1
- 150000003138 primary alcohols Chemical class 0.000 description 1
- 125000001501 propionyl group Chemical group O=C([*])C([H])([H])C([H])([H])[H] 0.000 description 1
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
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Abstract
The present invention relates to esters of hyaluronic acid with rhein, more particularly to a compound based on hyaluronic acid,wherein alcohol groups of hyaluronic acid are esterified with rhein, to a process for preparing said ester and to a pharmaceutical composition comprising said ester.
Description
STERES OF HYALURONIC ACID WITH QUEEN. PROCESS FOR YOUR PREPARATION AND COMPOSITIONS THAT INCLUDE THE SAME
FIELD OF THE INVENTION The present invention relates to esters of hyaluronic acid (HA) with queen, more particularly to a compound based on hyaluronic acid, wherein the alcohol groups of hyaluronic acid are esterified with queen, in a process for preparing compound and to a pharmaceutical composition comprising such compound.
PREVIOUS TECHNIQUE The queen is an alkaloid derived from senna which has anti-inflammatory and tissue protection properties. The queen, whose chemical name is 4,5-dihydroxy-9,10-dihydro-9,10-dioxo-2-anthracene carboxylic acid, has the following general formula (I)
wherein R is H. This substance is administered through the oral route, usually as diacetylreine, a derivative of the above general formula (I) wherein each of the R groups is an acetyl group, which has a greater bodily availability and is mainly used to treat inflammation of the joints. However, the queen and diacetylreine present the disadvantage of having a considerable laxative action, which can equal to lead to diarrhea and in this way make use of it is not advisable for elderly or debilitated patients. In addition, as a result of the insolubility of the queen and diacetylreine in water, this side effect can not be eliminated by administering these active ingredients through the parenteral or intraarticular route. Hyaluronic acid is a natural mucopolysaccharide that is formed by alternating units of D-glucuronic acid and N-acetylglucosamine, as depicted in a general manner immediately.
N-acetyl glucosamine glucuronic acid to form a linear chain having a molecular weight greater than 13 x 10 Daltons. Hyaluronic acid occurs in all the soft tissues of the body and in many physiological fluids such as, for example, the synovial fluid of the joints and the vitreous humor of the eyes. Hyaluronic acid is used in many clinical applications in its acid or saline form. In particular, it is used with great success in the inflammation of the joints, where it is administered by infiltration directly into the joint and acts by means of a dual mechanism: On the one hand reduces inflammation of the joint and on the other increases the synovial fluid viscosity, thereby benefiting the cartilage, which is more lubricated as a result. It is also applicable in ophthalmology, where it is used for its protective and anti-inflammatory properties and tissue repair, by virtue of its anabolic-reconstructive action in cartilage and skin. However, hyaluronic acid is known to suffer from degradation. It has been reported that the degradation of hyaluronic acid is caused by hydrolysis, depending on the conditions of pH and concentration of cations [compare for example, Uchiyama H. et al. J. Biol. Chem. 1990; 265: 7753-7759; Tokita Y. and Okamoto A., Polymer Degr. and Stab. nineteen ninety five; 48: 269-273; Hawkins C.L. and Davies M.J. Free Rad. Biol. Med. 1998; 24: 1396-1410; Schiller J. et al. Current Med. Chem.
2003; 10: 2123-2145]. After extensive studies, the present invention has found that hyaluronic acid having esterified ester groups with queen have surprisingly greater stability than hyaluronic acid and furthermore has an improved pharmacological activity compared to that observed with respect to hyaluronic acid and queen. used separately. In addition, the present invention has found that hyaluronic acid having ester groups esterified with queen can be advantageously used for local administration, thereby avoiding the drawbacks associated with oral administration of the queen. The present invention was achieved based on these results.
COMPENDIUM OF THE INVENTION According to a first aspect, the present invention relates to a compound based on hyaluronic acid, wherein the alcohol groups of hyaluronic acid are esterified with the queen, as such or in derivative form, or a salt thereof, which not only has greater stability that the hyaluronic acid, but also an improved pharmacological activity compared to that observed with respect to the hyaluronic acid and queen used separately, and in addition which can be used for local administration, in this way avoiding the drawbacks associated with oral administration of the Queen . According to a second aspect, the present invention relates to a process for preparing the compound or a salt thereof according to the first aspect, which comprises reacting queen acid chloride, as such or in derivative form, with hyaluronic acid. According to a third aspect, the present invention relates to a pharmaceutical composition comprising the compound or a salt thereof according to the first aspect in combination with suitable excipients and / or diluents. According to other aspects, the present invention relates to a medicinal product or a medical device for human or veterinary use, formed by a composition according to the third aspect, and the use of a compound or a salt thereof according to the first aspect for preparing a medicament for the treatment of inflammatory diseases, or for tissue repair, or for preparing biomaterials. Other advantages of the present invention will arise in the following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the "" H-NMR spectrum of the queen obtained by saponification of the HA-Re compound of the present invention, Figure 2 shows the IR spectrum of the queen obtained by saponification of the HA compound -Re of the present invention, Figure 3 shows the HPLC-MS analysis of the queen obtained by saponification of the compound HA-Re of the present invention, Figure 4 reports the results obtained in RT-PCR experiments comparing the effect of acid hyaluronic concentration in the pharmacological concentration with the effect of the HA-Re compound of the invention at the same concentrations Figure 5 reports the results of RT-PCR experiments in which the effect of the queen at pharmacological doses is compared with the dose effect of the compound HA-Re of the present invention Figure 6 shows the 13C-NMR spectrum of the HA-Re compound according to the present invention Figure 7 shows the spectr or 1H-NMR of the compound HA-Re according to the present invention.
DETAILED DESCRIPTION OF THE INVENTION The present invention will now be described in a more detailed manner. The present invention provides a compound based on hyaluronic acid, wherein the alcohol groups of hyaluronic acid are esterified with queen, as such or in derivative form, or a salt thereof. In the present description, the compound of the present invention will also be referred to as "HA-Re compound." It should be noted that in the present description and claims, the term "queen" means queen as such or in derivative form. Salts of the HA-Re compound according to the present invention preferably include pharmaceutically acceptable salts, for example, a sodium salt, a potassium salt, a magnesium salt, a calcium salt, or other conventional pharmaceutically acceptable salts, more preferably the sodium salt. According to the present invention, the term
"derived form" of the queen includes any derivative of the queen which is pharmacologically active in vivo and in which the queen's acid group is available to form ester bonds with the hydroxyl groups of hyaluronic acid. Preference is given to queen derivatives which make the anthraquinone available in vivo. Examples of queen in derivative form according to the present invention include the queen as depicted in the following formula
wherein R is independently any suitable hydroxy-protecting group, preferably an acyl group, for example, an acetyl, propionyl, butyryl or pivaloyl group, without being limited thereto. In a preferred embodiment of the present invention the queen is in derivative form, and more preferably is diacetylreine. According to the present invention, the queen preferably is esterified at least 5% of the esterifiable alcohol groups of hyaluronic acid, more preferably between 5 and 50%, and even more preferably between 5 and 50%.
twenty%. Particular preference is given to a compound wherein the queen esterifies 10% of the esterifiable alcohol groups of hyaluronic acid.
The compound HA-Re can be prepared by a process according to the invention which comprises reacting queen acid chloride with hyaluronic acid, preferably in an amount such that a percentage ratio between the mmol of the acid chloride of queen and the meq of the esterifiable alcohol units of hyaluronic acid are greater than 5%, more preferably between 5% and 50%, even more preferably between 5% and 20%, and according to a particularly preferred embodiment 10%. The selected process takes due account of the choice of solvent depending on the acceptability of its residues (ICH - International Conference of Concordance of Technical Requirements for the Registration of Drugs for Human Use). Preferably, the aforementioned process according to the present invention comprises the following steps: a) preparing a suspension of hyaluronic acid in a non-polar aprotic solvent, b) add dissolved queen acid chloride in a minimum amount of a non-polar aprotic solvent and a hydrogen ion acceptor, c) leave the mixture under stirring at reflux for a time which is sufficient for the esterification reaction to take place , and d) evaporate the solvent. Examples of non-polar aprotic solvents which may be used in step a) include cyclohexane, tetrahydrofuran, toluene, dichloromethane, n-hexane, most preferably cyclohexane. The non-polar aprotic solvents which can be used to dissolve the queen acid chloride are not limited, but can preferably be selected to be the same as those used in step a). Examples of the hydrogen ion acceptors which can be added in step b) include pyridine, triethylamine, most preferably Et3N; The time during which the reaction is allowed to reflux is not limited, but may be preferably for at least 20 hours. The hyaluronic acid which can be used to prepare the HA-Re compound according to the present invention preferably has a molecular weight of 500,000 to 3,000,000 Da, more preferably about 600,000 Da. The molecular weight of the hyaluronic acid can be determined according to a conventional manner, such as, for example, gel permeation chromatography (GPC). The hyaluronic acid which is used to prepare the HA-Re compound according to the present invention can be commercially available (for example, from Fidia Farmaceutici SpA-Abano T. PD) or it can be prepared for example, through extraction of cockscomb, through the fermentation of bacteria carrying a mucin layer or through other conventional ways [compare Proteoglycan Protocols, Humana Press, RV lozzo Ed Totowa, 2001]. The acid chloride of queen which can be used in the process of preparation of the compound HA-Re according to the present invention can be obtained by means of a process comprising the following steps: a ') preparing a queen suspension in a non-polar aprotic solvent; b ') add an amount of SOCl2 to obtain a molar ratio between S0C12 and queen of greater than 10; c ') leave the reaction under stirring at reflux under an inert atmosphere for a sufficient time to form the queen acid chloride; and d ') removing the solvent and the excess of S0C12 that did not react by distillation. Examples of non-polar aprotic solvents which may be used in step a ') include cyclohexane, tetrahydrofuran, toluene, dichloromethane, n-hexane, preferably a chloride solvent, and more preferably Ch 2 Cl 2. The time during which the reaction is allowed to reflux in step c ') is not limited, it may preferably be at least 3 hours. The queen, as such or in derivative form, which may be used in step a ') to prepare the acid chloride of queen may be commercially available (for example, from
Aldrich) or it can be synthesized according to conventional processes [compare Nawa H et al. J. Org. Chem. 1961; 26
979-981 and references reported herein; Smith C. et al. Tetrahedron Lett. 1993; 34: 7447-7450; Gallagher P.T. et al. Tetrahedron Lett. 1994; 35: 289-292]. According to a particularly preferred application, the HA-Re compound of the present invention which is obtained using the process according to the present invention is purified. This purification is preferably carried out using a dialysis membrane. In this case, as will be written in the examples that follow, preference is given to the use of the dialysis membrane which is commercially available under the trade name "Slide-A-Lyzer 3.5K" (Pierce, Rockford, IL USA), following the manufacturer's instructions. As previously discussed, the HA-Re compound of the present invention has an advantageously high stability, being stable for at least 36 months at a temperature of 4 ° C ± 0.5 ° C in aqueous solution, preferably buffered to pH 7.4, such as, for example, a phosphate-buffered saline solution according to the Official Italian Pharmacopoeia, XI edition. The HA-Re compound according to the present invention has inflammatory, healing, reconstructive and anabolic properties in the skin and cartilage. The present invention therefore also relates to a pharmaceutical composition comprising an HA-Re compound according to the present invention in combination with suitable excipients and / or diluents. In particular, the pharmaceutical composition according to the present invention can be a medical device and / or a medicinal product for human and veterinary use. The pharmaceutical composition according to the present invention preferably has a formulation suitable for local-regional administration. A particularly preferred pharmaceutical composition according to the present invention is a composition suitable for use through intra-articular infiltration, through ophthalmic administration, for example, eye drops and ophthalmic ointments, and through topical administration. Preferably, the composition of the invention is in the form of an aqueous dispersion.
This dispersion is preferably a buffered solution having a physiological pH, most preferably a pH of 7.4, for example, a phosphate buffered saline solution prepared according to the Official Italian Pharmacopoeia, XI edition. According to a particularly preferred application, in the pharmaceutical composition of the present invention, the HA-Re compound or a salt thereof according to the present invention is present in a concentration ranging from 0.5% to 2% w / v, preferably at a concentration of 1% w / v. Another object of the present invention is the use of the compound HA-Re or a salt thereof according to the present invention for preparing a medicament for the treatment of inflammatory diseases, preferably including inflammatory diseases of the joints, in particular osteoarthritis and arthritis rheumatoid A further object of the present invention is also the use of the HA-Re compound or a salt thereof according to the present invention for preparing a medicament for repairing tissue, in which the tissue is cartilage or skin. In addition, the HA-Re compound or a salt thereof according to the present invention can be used to prepare biomaterials, for example, gauzes for the treatment of wounds or burns and matrices for cell growth that are to be used in the treatment of burns and implantology. The present invention will be better illustrated by the following experimental Examples as well as Figures.
EXAMPLES Example 1 Preparation of queen acid chloride The queen (provided by Aldrich) (21.5 mg, 0.075 mmol) was placed in a circular 50 ml flask and CH2Cl2 (15 ml) was added thereto. The suspension turned orange. Then S0C12 (0.5 ml, 6.9 mmol) was added to the suspension. The reaction was carried out with stirring at reflux (50 ° C) in an inert atmosphere (N2). The reaction mixture was allowed to reflux for 3 hours and turned a light orange-yellow color. In order to remove in CH2C12 and the excess of S0C12 that did not react, toluene was added
(approximately 5 ml) and the mixture was distilled at 500 mmHg, corresponding to 6.6 x 104 Pa, at least 4 times to obtain 23 mg of crude queen acid chloride (production: quantitative). The product was identified by TLC, ethyl acetate.
Example 2 Separation of compound HA-Re Hyaluronic acid (provided by Fidia Farmaceutici SpA-Abano T. PD, average molecular weight of approximately 600,000 Da) (277.3 mg, 4.6 x 104 mmol, corresponding to 0.75 meq. primary alcohol) was suspended in cyclohexane (20 ml). The queen acid chloride prepared as described in Example 1 (21.5 mg, 0.075 mmol) dissolved in a minimum amount of CH2C12 was added. After Et3N (3 ml) was added the suspension turned red. The reaction was carried out with stirring at reflux (70 ° C) in an inert atmosphere (N2). After a short time, the suspension turned a red-orange color, which became darker after about three hours. After 20 hours, the reaction was stopped and the solvent was evaporated under reduced pressure (650 mmHg, corresponding to 8.7 x 104 Pa) to dryness, resulting in the HA-Re compound in the form of a light yellow precipitate.
Example 3 Purification of compound HA-Re a) sample preparation. A phosphate buffered saline solution was added
(5 ml) At pH 7.4 to the compound HA-Re (0.1019 g) which was obtained as described in example 2. A two-phase system was obtained and the solution turned orange-yellow while the residue was represented by a yellow-brown mass of gelatinous consistency. After a wait of at least 24 hours, a viscous colloidal system of brown color was obtained, b) purification A dialysis membrane "Slide-A-Lyzer® 3.5k" (Pierce, Rockford, IL USA) was allowed to hydrate wood appropriate with phosphate buffered saline at pH 7.4. Following the manufacturer's instructions, an adequate amount of the HA-Re compound was introduced to be purified. The dialysis was carried out for 2 hours against phosphate buffer pH 7.4 (after only 20 minutes the buffered solution appeared to obtain a slightly yellow color). The operation was repeated at least three times until the buffered solution remained colorless, testing it for the absence of absorption in the visible spectrum. The HA-Re compound was recovered from the membrane by dialysis. The purity of the HA-Re compound obtained in this way was 99.8%
Example 4 Analysis of the HA-Re compound of the present invention obtained. 1) Test using a UV-VIS spectrophotometer The concentration of the queen in the HA-Re purified compound obtained as described in Example 3 was evaluated by taking a spectrophotometric reading at 430 nm based on the "consistent" calibration in the range 10"5-10-3 (R2 = 0.9999) This wavelength was selected because the hyaluronic acid absorbs in the ultraviolet range, which therefore gives difficulty for the quantitative determination of queen. spectrophotometric, the production of the esterification reaction based on the queen was found to be 58% Considering example 2, the amount of queen used in the reaction was selected to esterify a maximum of 10% of the esterifiable alcohol groups primary of hyaluronic acid Because the production of the esterification reaction based on the queen was found to be 58%, it can be estimated that 5.8% of the esterified alcohol groups icables of hyaluronic acid were esterified.
2) XH-NMR analysis A 1 H-NMR spectrum of the HA-Re compound obtained in Example 2 was carried out in an adulterated buffered solution using a Varian VRX300 spectrometer. However, problems were found when this spectrum was interpreted since the percentage of queen that reacts with hyaluronic acid does not make it possible to identify the aromatic ring. Taking into account the deficient solubility of the queen in aqueous environments and considering that the esterification is a reversible reaction, the saponification, for example, based on hydrolysis, was therefore carried out until obtaining the queen of the compound HA-Re. The saponification compound obtained was precipitated. A spectrum of ""? -NMR of the saponification compound thus obtained was carried out in dimethylsulfoxide (DMSO) using the Varian VRX300 spectrometer. The spectrum obtained, which is shown in Figure 1, completely matches that of the queen.
3) Analysis I.R A spectrum I.R. was carried out in Nujol for the compound obtained from the saponification of the HA-Re compound of the present invention (using the Spectrum BX FT-IR System, Perkin Elmer). As shown in Figure 2, the spectrum obtained coincides with that of the pure queen.
4) HPLC-MS An HPLC-MS analysis was carried out using (using the Agilent 1100 kit, LC / MSD series) in the compound obtained from the saponification obtained by saponification of the HA-Re compound of the present invention, using as the mobile phase an 80:20 methanol / water mixture containing 2.5% formic acid, at a flow rate of 0.8 ml / min. As can be seen in Figure 3, the mass of the compound corresponds to that of the queen.
Example 5 Evaluation of the technological characteristics of the compound
HA-Re of the invention 1) Evaluation of hydrolytic stability The HA-Re compound of the invention as obtained in Example 2 and not purified by dialysis was stored for more than six months in the dry state in flasks, in the dark and at room temperature (22 ° C). This sample was then purified by dialysis as described in Example 3 and the concentration of the queen was evaluated using a UV-VIS spectrophotometer. It was found that the concentration is equal to that obtained for the synthesized product immediately. The experimental results therefore show that, in the dry state, no degradation is observed. In addition, the HA-Re compound of the invention produced as described in Example 2 and purified by dialysis as described in Example 3 was stored for six months in a 2% solution in phosphate buffer pH 7.4, in flasks. , in the dark and at a temperature of 4 ° C. The presence of foreign bodies was found in the sample, due to the use of non-sterile material and the fact that no preservatives were used. Once the sample was again subjected to dialysis using the same method as described in Example 3, subjecting it to dialysis for at least four days. By means of a UV-VIS test it was not possible to find some queen released within the different buffer solutions used for dialysis. In this case also, therefore, the compound was shown to be chemically stable because no release of queen of the HA-Re compound was found at least in demonstrable terms using current analytical techniques. Therefore, even in solution, hydrolytic degradation was not observed. The results obtained make it possible to establish that the HA-Re compound according to the invention is stable for at least 24 months under refrigerated conditions (at 4 ° C ± 0.5 ° C) in aqueous solution. This affirmation comes from the nce of any perceptible hydrolytic degradation. In addition, in order to rule out the possibility of the hydrolytic degradation of hyaluronic acid occurring during the esterification reaction. with queen, a white test of the reaction was carried out. In particular, the same conditions were used as in the reaction link of the queen to hyaluronic acid, but in the nce of queen. In particular, the hyaluronic acid (100 mg) was added to a mixture of cyclohexane (10 ml) dichloromethane (1 ml) and triethylamine (1 ml) and the reaction was carried out under reflux (70 ° C) under an inert atmosphere (N2) for 24 hours; after that time, the solvents were removed from the reaction in a nitrogen atmosphere. The compound obtained was much less soluble in water than hyaluronic acid in the native state and was characterized by a non-determinable viscosity after 24 hours of dispersion. This means that the depolymerization can be discarded, since it would have to result in a solubility in water and therefore a reduction in viscosity. Finally, a verification of the hydrolytic stability of the purified HA-Re compound of the invention obtained in Example 3 was made after sterilization. As discussed previously, it was found that the use of non-sterile material and the fact that no preservatives are used, led to the presence of foreign bodies in the samples of the compound of the invention. In particular a 1% solution of the HA-Re compound of the invention, purified using dialysis membrane, in a phosphate buffered saline solution at pH 7.4 was prepared. Given the experimental evidence showing that hyaluronic acid is a heat sensitive molecule (Biomaterials 23 (2002) 4503-4513), the sample obtained in this way was sterilized using saturated steam pressurized in an autoclave for 20 minutes at 121 ° C. The sample was then subjected to dialysis again on a dialysis membrane in order to evaluate some presence of queen in the dialysis fluid; No small portions of queen were found. These results allow to draw the conclusion that the sample is hydrolytically stable after heat sterilization. 2) Analysis of the rheological characteristics and of the injectability The injectability of the compound HA-Re of the invention was analyzed in comparison to the hyaluronic acid both high and low molecular weight. In particular, the following three samples were prepared: a) A 1% w / v solution of high molecular weight hyaluronic acid (approximate average molecular weight)
1 '200' 000) in phosphate buffered saline, pH 7.4 b) A 1% w / v solution of low molecular weight hyaluronic acid (average molecular weight approximately 600,000 in phosphate buffered saline pH 7.4 c) A 1% w / v solution of the HA-Re compound of the invention in phosphate buffered saline, pH 7.4 The viscosity of the samples was measured using a VISCOMATE MODEL VM-10A (glass bottle: 3 ml; conditions of agitation, temperature 20 ± 0.2 ° C), with the following values obtained: a) 77 = 78 .4 mPa 'sb) 77 = 64. 8 mPa 's c) 77 = 47. 9 mPa 's The results obtained show that the compound
HA-Re of the invention has better injectability than hyaluronic acid in native high and low molecular weight state. This is because a 1% w / v solution of the HA-Re compound of the invention has a lower viscosity than a 1% w / v solution of low molecular weight hyaluronic acid, which in turn has a viscosity lower than a 1% w / v solution of high molecular weight hyaluronic acid. The reduction in viscosity that was observed, once demonstrated that there is no depolymerization reaction of the hyaluronic acid after the esterification reaction, can be attributed to the covalent interaction between the queen and the hyaluronic acid.
Example 6 Evaluation of the pharmacological activity of the compound HA-Re of the invention Normal cartilage biopsies were obtained from 5 individuals (3 male and 2 female, average age: 59.3 ± 5.1 years) during a hip or femur surgery as Traumatic fracture result. The subjects chosen for the study did not present biochemical or clinical signs of inflammatory or joint diseases, and presented normal cartilage at both the macroscopic and microscopic levels. The cartilage was collected under sterile conditions and immediately processed for chondrocyte isolation. The samples were first cleaned of any muscle adherent, connective or subchondral bone tissues, then minced into fragments of 1-3 mm3 fragment and rinsed in phosphate buffered saline pH 7.2 (PBS). The individual chondrocytes were then released by repeated enzymatic digestions of 60-75 min. 37 ° C with 0.25% trypsin, 400U / ml collagenase 1, lOOOU / ml collagenase II and one mg / ml hyaluronidase. Cells were pooled, washed extensively in PBS and seeded at high density in 35 mm dishes (45 x 10 3 cells / cm 2). Culture medium was Coon 's modified Ham's F12 supplemented with 10% FCS (Mascia Brunelli, Milano, Italy). Chondrocyte genotype maintenance was estimated by the detection of type II collagenase after digestion with pepsin from the culture supernatant. Cell viability was evaluated by the trypan blue exclusion test. Cell duplication was determined at regular intervals by culture trypsinization and cell number quantification. The stimulation experiments were performed when the primary cultures reached cofluence (step 0).
The cells were then cultured for two days in the presence of ascorbic acid (50 μg / ml) and incubated from here for 20 hours in the absence or presence of IL-lβ (hr) (5 ng / ml), with or without addition of different concentrations of hyaluronic acid (HA), compound of the invention (HA-Re) or queen. Both hyaluronic acid and queen have been reported to have beneficial effects on osteoarthritis due to their ability to inhibit the activity of metalloproteinases (MMPs) involved in cartilage catabolism. In order to study the effect of several compounds on the expression of MMP in human chondrocytes, Real Time PCR assays were performed. The total RNA of cultured human chondrocytes was brought using Trizol
(Gibco BRL) according to the manufacturer's instructions. The cDNA strands were sintered using a Superscript First-Strand synthesis set (Gibco BRL) with 1 μg total RNA. The primers were as follows: MMP-1 (collagenase) coding strand: 5'- CTGAAGGTGATGAAGCAGCC-3 'Non-coding strand: 5' -AGTCCAAGAGAATGGCCGAG-3
(fragment size 428 bp); MMP-3 (stromelysin) coding strand: 5'-CCTCTGATGGCCCAGAATTGA-3 ', Non-coding strand: 5' -GAAATTGGCCACTCCCTGGGT-3 '(fragment size 440 bp); Glyceraldehyde 3 phosphate dehydrogenase (GAPDH), Coding strand: 5 '-CCACCCATGGCAAATTCCATGGCA-3'; Non-coding strand: 5 '-TCTAGACGGCAGGTCAGGTCCA (fragment size 598 bp). The amplification was performed at 60-64 ° C for 45 cycles in an iCycler Thermal Cycler (Bio-Rad Hercules, CA), and the data was analyzed using the iCycler iQ Optical System Software. The relative expression in each sample was calculated by a mathematical method based on the real-time PCR efficiencies using the GAPDH mRNA as references. All samples were analyzed in triplicate. After 45 cycles of amplification, the threshold cycle values were automatically calculated, and the femtograms of the start cDNA were calculated from a standard curve covering a limit of four orders of magnitude. The standard curves of MMPs and GAPDH range from 1 to 1000 femtograms per 25-μl reaction. The proportions to the start amount of the MMPs to the GAPDH were calculated. We analyzed the statistical differences of the results between different relevant controls and experimental variables using the Student's t-test. Figures 4 and 5 reported the results obtained in the RT-PCR experiments. All samples were analyzed in triplicate. In each experiment, the change in MMP mRNA expression was expressed as increasing to ten fold when compared to untreated cells. The mean and the standard deviation of three experiments are shown. The t-test for paired student data was used to determine the meaning of the effects of various treatments. The statistical difference between the treatment and control groups is also reported: * = p < 0.01; ** = p < 0.001 (Student t test). Treatment with IL-1 led to a dramatic increase in the expression of both MMP-1 and MMP-3, consistent with data from the literature. In the experiments reported in Figure 4, the effect of HA on the pharmacological concentration commonly reported in the literature (1 mg / ml) was compared with the effect of HA-Re (compound of the invention) at the same concentrations (1 mg / l). ml). Similar results were obtained in a range of 0.1-1.5 HA concentrations. The exposure of human chondrocytes
HA at pharmacological doses (1 mg / ml) was able to significantly prevent the induction of MMP1 and MMP2 by IL1
(Figure 4 A and B). Surprisingly, similar doses of the HA-Re compound led to a more dramatic protective effect even, with the expression of MMPs brought back to basal level despite exposure to IL1. Figure 5 reports the results of the experiments in which the effect of the queen at pharmacological doses (10 μM) is compared with the effect of a similar dose of HA-Re. It also appears that the compound of the invention HA-Re is more potent than Re alone in achieving a down-regulation of the MMP expression induced by IL1. Example 7 A further synthesis of HA-Re according to the present invention was performed as in example 2, except that 20 mg of hyaluronic acid and 23 mg of queen acid chloride were used, corresponding to stoichiometric concentrations of queen and acid hyaluronic acid based on primary alcoholic groups of hyaluronic acid. Figure 6 shows the 13C-NMR spectrum of the HA-Re compound obtained in this manner according to the present invention, wherein the characteristic peak at 175 ppm, specific for ester functions, clearly appears. Figure 7 shows the XH-NMR spectrum of the HA-Re compound thus obtained from the present invention, where the characteristic peak between 7 and 8 ppm specific for aromatic queen rings clearly appears. Therefore, it clearly appears from these two NMR spectra that in the HA-Re compound according to the present invention, the queen esterifies hyaluronic acid alcohol groups.
Claims (36)
- CLAIMS 1. A compound based on hyaluronic acid, wherein the alcohol groups of hyaluronic acid are esterified with queen, as such or in derivative form or a salt thereof.
- 2. The compound according to claim 1, wherein the queen esterifies at least 5% of the esterifiable alcohol groups of hyaluronic acid.
- 3. The compound according to claim 2, wherein the queen esterifies from 5% to 50% of the esterifiable alcohol groups of hyaluronic acid.
- 4. The compound according to claim 3, wherein the queen esterifies from 5% to 20% of the esterifiable alcohol groups of hyaluronic acid.
- 5. The compound according to claim 4, wherein the queen esterifies 10% of the esterifiable alcohol groups of hyaluronic acid.
- 6. Sodium salts of the compound according to any one of Claims 1 to 5.
- 7. A process for preparing a compound or salt thereof according to any of Claims 1 to 6, which comprises reacting reigns as such or in a derivative form with hyaluronic acid.
- 8. The process according to claim 7, wherein the queen acid chloride and the hyaluronic acid are in an amount such that a percentage ratio between the mmol of acid chloride of queen and the meq. of the esterifiable alcohol units of hyaluronic acid is at least 5%.
- 9. The process according to claim 8, wherein the proportion of percentages varies from 5% to 50%.
- 10. The process according to claim 9, wherein the proportion of percentages varies from 5% to 20%.
- 11. The process according to claim 10, wherein the proportion of percentages is 10%.
- The process according to any of Claims 7 to 11, which comprises the following steps: a) preparing a suspension of hyaluronic acid in a non-polar aprotic solvent; b) add dissolved queen acid chloride in a non-polar aprotic solvent and a hydrogen ion acceptor; c) leaving the mixture under stirring at reflux for a time which is sufficient for the esterification reaction to take place; and d) evaporate the solvent.
- 13. The process according to claim 12, wherein the non-polar aprotic solvent of step a) is cyclohexane.
- 14- The process according to claims 12 or 13, wherein step b), the hydrogen ion acceptor is Net3.
- 15. The process according to any of claims 12 to 14, wherein step c), the reaction is allowed to reflux for at least 20 hours.
- 16. The process according to any of Claims 7 to 15, wherein the queen acid chloride is obtained by a process means comprising the following steps: a ') preparing a queen suspension in an aprotic solvent not polar; b ') add an amount of S0C12 until obtaining a molar ratio between S0C12 and the queen of greater than 10; c ') leave the reaction under stirring at reflux in an inert atmosphere for a time sufficient for the queen acid chloride to form; and d ') remove the solvent and excess S0C12 without reaction by distillation.
- 17. The process according to claim 16, wherein the non-polar aprotic solvent from step a ') is a chloride solvent.
- 18. The process according to Claim 17 wherein the chloride solvent is CH C12 19.
- The process according to any of Claims 16 to 18, wherein step c ') the reaction is allowed to reflux for at least 3 hours.
- The process according to any of Claims 7 to 19, which further comprise a final step of purification.
- 21. The process according to Claim 20, wherein the purification step is carried out using a dialysis membrane.
- 22. A pharmaceutical composition comprising the compound or a salt thereof according to any of the Claims 1 to 6 in combination with suitable excipients and / or diluents.
- 23. The pharmaceutical composition according to Claim 22, which has a suitable formulation for local-regional administration.
- 24. The pharmaceutical composition according to Claim 23, which is suitable for administration through intra-articular infiltration.
- 25. The pharmaceutical composition according to Claim 23, which is suitable for ophthalmic administration.
- 26. The pharmaceutical composition according to Claim 23, which is suitable for topical administration.
- 27. The pharmaceutical composition according to any of Claims 22 to 26, in the form of an aqueous dispersion.
- 28. The pharmaceutical composition according to Claim 27, wherein the dispersion is a buffered solution having a pH of 7.4.
- 29. The pharmaceutical composition according to Claim 27 or 28, wherein the compound in a concentration ranges from 0.1% to 2% w / v.
- 30. The pharmaceutical composition according to claim 29, wherein the compound is in a concentration of 1% w / v.
- 31. A medicinal product for human or veterinary use, formed by a pharmaceutical composition according to any of Claims 22 to 30.
- 32. A medical device for human or veterinary use, formed by a pharmaceutical composition according to any of the claims. 22 to 30.
- 33. A use of a compound or a salt thereof according to any of Claims 1 to 6 for preparing a medicament for the treatment of inflammatory diseases.
- 34. The use according to claim 33, wherein the inflammatory diseases are inflammatory diseases of the joints.
- 35. A use of a compound or a salt thereof according to any of Claims 1 to 6 for preparing a medicament for repairing tissue, in which the tissue is cartilage or skin.
- 36. A use of a compound or a salt thereof according to any of Claims 1 to 6 for preparing biomaterials.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| MIMI2004A000347 | 2004-02-26 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| MXPA06008467A true MXPA06008467A (en) | 2006-12-13 |
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