ITFI20010243A1 - SOLUBLE AND BIOCOMPATIBLE GELS OF CROSS-LINKED HYALURONIC ACID WITH L-AMINO ACIDS OR B-FUNCTIONAL L-AMINOESTERS - Google Patents
SOLUBLE AND BIOCOMPATIBLE GELS OF CROSS-LINKED HYALURONIC ACID WITH L-AMINO ACIDS OR B-FUNCTIONAL L-AMINOESTERS Download PDFInfo
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- ITFI20010243A1 ITFI20010243A1 ITFI20010243A ITFI20010243A1 IT FI20010243 A1 ITFI20010243 A1 IT FI20010243A1 IT FI20010243 A ITFI20010243 A IT FI20010243A IT FI20010243 A1 ITFI20010243 A1 IT FI20010243A1
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- 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 title claims description 34
- 229920002674 hyaluronan Polymers 0.000 title claims description 34
- 229960003160 hyaluronic acid Drugs 0.000 title claims description 33
- 150000008575 L-amino acids Chemical class 0.000 title claims description 7
- 239000000499 gel Substances 0.000 claims description 25
- 238000000034 method Methods 0.000 claims description 21
- 239000000243 solution Substances 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 230000008569 process Effects 0.000 claims description 17
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 238000006243 chemical reaction Methods 0.000 claims description 14
- 230000001588 bifunctional effect Effects 0.000 claims description 10
- 239000012528 membrane Substances 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 8
- 239000003431 cross linking reagent Substances 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 7
- 239000012190 activator Substances 0.000 claims description 6
- 210000001519 tissue Anatomy 0.000 claims description 5
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 4
- 230000003213 activating effect Effects 0.000 claims description 4
- 238000011026 diafiltration Methods 0.000 claims description 4
- DZIYAIZKJOHVQC-KLXURFKVSA-N ethyl (2s)-2,6-diaminohexanoate;dihydrochloride Chemical compound Cl.Cl.CCOC(=O)[C@@H](N)CCCCN DZIYAIZKJOHVQC-KLXURFKVSA-N 0.000 claims description 4
- NDBQJIBNNUJNHA-DFWYDOINSA-N methyl (2s)-2-amino-3-hydroxypropanoate;hydrochloride Chemical compound Cl.COC(=O)[C@@H](N)CO NDBQJIBNNUJNHA-DFWYDOINSA-N 0.000 claims description 4
- 239000000178 monomer Substances 0.000 claims description 4
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical group Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 239000003960 organic solvent Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 210000001789 adipocyte Anatomy 0.000 claims description 2
- 210000000988 bone and bone Anatomy 0.000 claims description 2
- 210000004207 dermis Anatomy 0.000 claims description 2
- 238000000502 dialysis Methods 0.000 claims description 2
- 210000002615 epidermis Anatomy 0.000 claims description 2
- JZJQCLZQSHLSFB-WCCKRBBISA-N ethyl (2s)-2-amino-3-hydroxypropanoate;hydrochloride Chemical compound Cl.CCOC(=O)[C@@H](N)CO JZJQCLZQSHLSFB-WCCKRBBISA-N 0.000 claims description 2
- 238000001704 evaporation Methods 0.000 claims description 2
- 230000008020 evaporation Effects 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- SXZCBVCQHOJXDR-ILKKLZGPSA-N hydron;methyl (2s)-2,6-diaminohexanoate;dichloride Chemical compound Cl.Cl.COC(=O)[C@@H](N)CCCCN SXZCBVCQHOJXDR-ILKKLZGPSA-N 0.000 claims description 2
- 201000008482 osteoarthritis Diseases 0.000 claims description 2
- 238000001556 precipitation Methods 0.000 claims description 2
- 229960001153 serine Drugs 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 239000000758 substrate Substances 0.000 claims description 2
- WPLOVIFNBMNBPD-ATHMIXSHSA-N subtilin Chemical compound CC1SCC(NC2=O)C(=O)NC(CC(N)=O)C(=O)NC(C(=O)NC(CCCCN)C(=O)NC(C(C)CC)C(=O)NC(=C)C(=O)NC(CCCCN)C(O)=O)CSC(C)C2NC(=O)C(CC(C)C)NC(=O)C1NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C1NC(=O)C(=C/C)/NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)CNC(=O)C(NC(=O)C(NC(=O)C2NC(=O)CNC(=O)C3CCCN3C(=O)C(NC(=O)C3NC(=O)C(CC(C)C)NC(=O)C(=C)NC(=O)C(CCC(O)=O)NC(=O)C(NC(=O)C(CCCCN)NC(=O)C(N)CC=4C5=CC=CC=C5NC=4)CSC3)C(C)SC2)C(C)C)C(C)SC1)CC1=CC=CC=C1 WPLOVIFNBMNBPD-ATHMIXSHSA-N 0.000 claims description 2
- 238000000108 ultra-filtration Methods 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 3
- 239000004971 Cross linker Substances 0.000 claims 2
- 230000035484 reaction time Effects 0.000 claims 1
- 239000000047 product Substances 0.000 description 18
- KIUKXJAPPMFGSW-YXBJCWEESA-N (2s,4s,5r,6s)-6-[(2s,3r,5s,6r)-3-acetamido-2-[(3s,4r,5r,6r)-6-[(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]1C(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(O[C@@H]3[C@@H]([C@@H](O)C(O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)C(C(O)=O)O1 KIUKXJAPPMFGSW-YXBJCWEESA-N 0.000 description 6
- 229940024606 amino acid Drugs 0.000 description 5
- 235000001014 amino acid Nutrition 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- 210000003954 umbilical cord Anatomy 0.000 description 2
- NOWKCMXCCJGMRR-UHFFFAOYSA-N Aziridine Chemical compound C1CN1 NOWKCMXCCJGMRR-UHFFFAOYSA-N 0.000 description 1
- 241000561734 Celosia cristata Species 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 235000019766 L-Lysine Nutrition 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound 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
- 241000906034 Orthops Species 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 235000010633 broth Nutrition 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- NCEXYHBECQHGNR-UHFFFAOYSA-N chembl421 Chemical compound C1=C(O)C(C(=O)O)=CC(N=NC=2C=CC(=CC=2)S(=O)(=O)NC=2N=CC=CC=2)=C1 NCEXYHBECQHGNR-UHFFFAOYSA-N 0.000 description 1
- 210000001520 comb Anatomy 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- AFOSIXZFDONLBT-UHFFFAOYSA-N divinyl sulfone Chemical compound C=CS(=O)(=O)C=C AFOSIXZFDONLBT-UHFFFAOYSA-N 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- CZEPJJXZASVXQF-ZETCQYMHSA-N ethyl (2s)-2,6-diaminohexanoate Chemical compound CCOC(=O)[C@@H](N)CCCCN CZEPJJXZASVXQF-ZETCQYMHSA-N 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000010408 film Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- KIUKXJAPPMFGSW-MNSSHETKSA-N hyaluronan Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)C1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H](C(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-MNSSHETKSA-N 0.000 description 1
- 229940099552 hyaluronan Drugs 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 230000001050 lubricating effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000010907 mechanical stirring Methods 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000013379 physicochemical characterization Methods 0.000 description 1
- -1 preferred Chemical class 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000012265 solid product Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 210000001179 synovial fluid Anatomy 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000010409 thin film Substances 0.000 description 1
- 210000004127 vitreous body Anatomy 0.000 description 1
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- Materials For Medical Uses (AREA)
Description
Descrizione della Domanda di Brevetto per Invenzione Industriale dal titolo: Gel solubili e biocompatibili di Acido laluronico cross-linked con L-amminoacidi o L-amminoesteri bifunzionali Description of the Patent Application for Industrial Invention entitled: Soluble and biocompatible gels of cross-linked hyaluronic acid with L-amino acids or bifunctional L-aminoesters
Campo dell’invenzione Field of the invention
La presente invenzione si riferisce a gel solubili e biocompatibili costituiti da Acido laluronico cross-linked con L-amminoacidi o L-amminoesteri bifunzionali, o loro miscele, ad un processo per la loro preparazione ed al loro uso in campo farmaceutico, cosmetico, chirurgico e medico in generale. The present invention refers to soluble and biocompatible gels consisting of cross-linked hyaluronic acid with L-amino acids or bifunctional L-aminoesters, or their mixtures, to a process for their preparation and their use in the pharmaceutical, cosmetic, surgical and doctor in general.
Stato dell’arte: State of the art:
L’acido laluronico è un mucopolisaccaride costituito da unità alternate di Acido D-Glucoronico e N-Acetil-D-Glucosammina, legati insieme attraverso legami β 1-3 e β1-4. Hyaluronic acid is a mucopolysaccharide consisting of alternating units of D-Glucoronic acid and N-Acetyl-D-Glucosamine, linked together through β 1-3 and β1-4 bonds.
In natura l’Acido laluronico si trova nel fluido sinoviale dei giunti articolari, nell’umore vitreo degli occhi, nel cordone ombelicale e nel tessuto connettivo. L’Acido laluronico viene ottenuto per estrazione da tessuti animali come le creste di gallo o i cordoni ombelicali, oppure recuperato da brodi di fermentazione di particolari ceppi di Streptococchi. In nature, hyaluronic acid is found in the synovial fluid of the joint joints, in the vitreous humor of the eyes, in the umbilical cord and in the connective tissue. Hyaluronic acid is obtained by extraction from animal tissues such as cockscombs or umbilical cords, or recovered from fermentation broths of particular strains of Streptococci.
Il rapido sviluppo delle biotecnologie ha portato ad una ottimizzazione e potenziamento di quest’ultima strategia di produzione dell'Acido Ialuronico, risultando oggi la più semplice e conveniente. The rapid development of biotechnologies has led to an optimization and enhancement of this last Hyaluronic Acid production strategy, making it the simplest and most convenient today.
Le applicazioni dell’Acido Ialuronico, che vanno dall’uso in chirurgia a quello farmacologico e in genere biomedico, sono state ampiamente descritte in letteratura, vedi ad esempio: Balazs et al."Hyaluronan Biomaterials: Medicai Applications”, Handbook of Biomaterials and Applications, ed. DL Wise et al., 1995, 2719-2741; US -5,559,104,1996; Pape, Balazs, Ophthalmology, 87, No. 7, 1980; Iwata, Clin. Orthop., 289, 285-291 ; 1993; US - 5,128,326; US - 4,500,676; US - 5,840,046; US - 5,795,584; US - 6,010,692; US - 5, 658, 331. The applications of Hyaluronic Acid, ranging from use in surgery to pharmacological and generally biomedical use, have been widely described in the literature, see for example: Balazs et al. "Hyaluronan Biomaterials: Medicai Applications", Handbook of Biomaterials and Applications , ed. DL Wise et al., 1995, 2719-2741; US -5,559,104,1996; Pape, Balazs, Ophthalmology, 87, No. 7, 1980; Iwata, Clin. Orthop., 289, 285-291; 1993; US - 5,128,326; US - 4,500,676; US - 5,840,046; US - 5,795,584; US - 6,010,692; US - 5, 658, 331.
Poiché l’importante ruolo svolto dall’Acido laluronico neH’organismo umano è determinato dalle particolari caratteristiche manifestate dalle sue soluzioni acquose come le proprietà viscoelastiche, lubrificanti e idrofiliche, i ricercatori si sono da tempo orientati verso la sintesi di nuovi derivati dell’acido ialuronico che migliorino o modulino dette caratteristiche in modo da ottenere i prodotti più adatti per i vari impieghi. Since the important role played by hyaluronic acid in the human organism is determined by the particular characteristics manifested by its aqueous solutions such as viscoelastic, lubricating and hydrophilic properties, researchers have long been oriented towards the synthesis of new hyaluronic acid derivatives. that improve or modulate said characteristics in order to obtain the most suitable products for the various uses.
Una vasta letteratura riporta vari prodotti di cross-link dell’ Acido laluronico con, ad esempio, formaldeide (Balazs, U.S. Pat. 4,713,448, 1987), divinil sulfone (Balazs, U.S. Pat. 4,582,865, 1986), aziridine, alcoli (Della Valle, U.S. Pat. 4,851,521, 1989) e amminoacidi monofunzionali (Hamilton, U.S. Pat. 4,937,270, 1990). A vast literature reports various cross-link products of Hyaluronic acid with, for example, formaldehyde (Balazs, U.S. Pat. 4,713,448, 1987), divinyl sulfone (Balazs, U.S. Pat. 4,582,865, 1986), aziridine, alcohols (Della Valle , U.S. Pat. 4,851,521, 1989) and monofunctional amino acids (Hamilton, U.S. Pat. 4,937,270, 1990).
In particolare Miller et al., U.S. Pat. 5,760,200, 1998 descriveva già i metodi preparazione di derivati insolubili dell’Acido laluronico, ad esempio con L-lisina etilestere, in presenza di una carbodiimmide solubile in acqua; la reazione veniva condotta in forte eccesso di attivante e amminoacido allo scopo di ottenere un gel insolubile. In particular Miller et al., U.S. Pat. 5,760,200, 1998 already described the methods of preparing insoluble derivatives of hyaluronic acid, for example with L-lysine ethyl ester, in the presence of a water-soluble carbodiimide; the reaction was carried out in a strong excess of activator and amino acid in order to obtain an insoluble gel.
Inoltre nel brevetto (WO 01/58961), a nome della stessa Richiedente, sono descritti derivati di Acido laluronico cross-linked con L-amminoacidi o L-amminoesteri bifunzionali, e loro processi di preparazione sia in acqua che in solventi organici, tuttavia i prodotti ivi descritti e rivendicati, pur nella loro validità, sono però suscettibili di ulteriori miglioramenti delle loro caratteristiche di viscoelasticità, biocompatibilità e purezza in vista degli impieghi farmacologici, medici o chirurgici cui questi prodotti sono normalmente destinati. Furthermore in the patent (WO 01/58961), in the name of the same Applicant, derivatives of cross-linked hyaluronic acid with L-amino acids or bifunctional L-aminoesters are described, and their preparation processes both in water and in organic solvents, however the the products described and claimed therein, while valid, are however susceptible of further improvements in their characteristics of viscoelasticity, biocompatibility and purity in view of the pharmacological, medical or surgical uses for which these products are normally intended.
E’ evidente in questa ottica l’interesse a disporre di sempre nuovi prodotti in modo da allargare e migliorare l'impiego dell'Acido laluronico per gli usi di interesse o per nuove applicazioni. From this perspective, the interest in always having new products in order to expand and improve the use of Hyaluronic Acid for uses of interest or for new applications is evident.
Descrizione dettagliata dell’invenzione Detailed description of the invention
La presente invenzione consente di rispondere alle esigenze suddette rendendo disponibili gel solubili in acqua e biocompatibili derivati dal cross-link dell’Acido laluronico con L-amminoacidi o L-amminoesteri bifunzionali o loro miscele. The present invention allows to meet the aforementioned needs by making available water-soluble and biocompatible gels derived from the cross-link of hyaluronic acid with L-amino acids or bifunctional L-aminoesters or their mixtures.
Dette caratteristiche rendono i prodotti secondo l’invenzione ideali per l’impiego in numerosi campi, dal farmaceutico al medico. These characteristics make the products according to the invention ideal for use in numerous fields, from pharmaceuticals to doctors.
Il prodotto, sotto forma di gel o film, può essere ad esempio utilizzato come materiale anti-aderenziale in campo chirurgico (addominale, spinale, ecc.); in questo caso il materiale forma una barriera di separazione tra i tessuti lesionati e viene riassorbito dall’organismo dopo un periodo di tempo sufficiente alla ricostruzione dei tessuti. The product, in the form of gel or film, can be used, for example, as an anti-adhesion material in the surgical field (abdominal, spinal, etc.); in this case the material forms a separation barrier between the injured tissues and is reabsorbed by the body after a period of time sufficient for the reconstruction of the tissues.
I prodotti secondo l'invenzione possono essere inoltre impiegati come substrato per l’ingegneria dei tessuti (derma, epidermide, ossa, cellule adipose, ecc.); infine può trovare applicazione in campo oftalmico, dermatologico, nel campo delle osteoartriti, ecc. The products according to the invention can also be used as a substrate for tissue engineering (dermis, epidermis, bones, fat cells, etc.); finally it can find application in the ophthalmic, dermatological field, in the field of osteoarthritis, etc.
I prodotti secondo l'invenzione si preparano in acqua oppure in miscele acqua e solventi organici come DMF o DMSO, a seconda delle applicazioni. In tutti i casi la reazione avviene in due passaggi successivi: il primo consiste nell'attivazione dell'Acido laluronico e il secondo nella formazione dei legami tra Acido laluronico e agente cross-linkante. L'attivazione dell'Acido laluronico avviene ad opera di una carbodiimmide solubile in acqua, secondo uno schema noto (vedi ad esempio Tomihata, J. Biomed. Mater. Res., 1997, 37(2), 243-251; Danishefsky, Carbohydrate Res., 1971, 16, 199-205). Tra le diverse carbodiimmidi solubili in acqua, particolarmente preferita, secondo l'invenzione, è la N-3-dimetilammino-propiletilcarbodiimmide cloridrato. Gli agenti cross-linkanti utilizzati secondo l’invenzione sono a L-amminoacidi bifunzionali, aventi cioè un secondo gruppo funzionale oltre a quello amminoacidico, o i loro esteri o miscele. Particolarmente preferiti sono la L-Lisina, L-Serina, L-Lisina etilestere dicloridrato, L-Lisina metilestere dicloridrato, L-Serina metilestere cloridrato, L-Serina etilestere cloridrato o loro miscele. L’utilizzo degli amminoesteri al posto degli amminoacidi assicura la protezione delle funzioni carbossiliche degli amminoacidi nei confronti di un’eventuale attivazione e coinvolgimento in reazioni collaterali. The products according to the invention are prepared in water or in mixtures of water and organic solvents such as DMF or DMSO, depending on the applications. In all cases the reaction takes place in two successive steps: the first consists in the activation of the hyaluronic acid and the second in the formation of the bonds between hyaluronic acid and the cross-linking agent. The activation of hyaluronic acid occurs by a water-soluble carbodiimide, according to a known scheme (see for example Tomihata, J. Biomed. Mater. Res., 1997, 37 (2), 243-251; Danishefsky, Carbohydrate Res., 1971, 16, 199-205). Among the various water-soluble carbodiimides, particularly preferred, according to the invention, is N-3-dimethylamino-propylethylcarbodiimide hydrochloride. The cross-linking agents used according to the invention are bifunctional L-amino acids, i.e. having a second functional group in addition to the amino acid one, or their esters or mixtures. Particularly preferred are L-Lysine, L-Serine, L-Lysine ethyl ester dihydrochloride, L-Lysine methyl ester dihydrochloride, L-Serine methyl ester hydrochloride, L-Serine ethyl ester hydrochloride or their mixtures. The use of amino esters instead of amino acids ensures the protection of the carboxylic functions of amino acids against any activation and involvement in side reactions.
La reazione viene condotta all'interno di un reattore termostatato e munito di agitazione meccanica. L’Acido laluronico sale sodico viene disciolto in acqua in concentrazione compresa tra 0.5-2.5% a seconda delle caratteristiche che si vogliono ottenere nel prodotto finale; la concentrazione preferita è tra 1-1.5%. The reaction is carried out inside a thermostated reactor equipped with mechanical stirring. Hyaluronic acid sodium salt is dissolved in water in a concentration between 0.5-2.5% depending on the characteristics to be obtained in the final product; the preferred concentration is between 1-1.5%.
La temperatura di reazione è uno dei fattori determinanti per ottenere i prodotti secondo l’invenzione e deve essere compresa tra 0-25°C, preferibilmente tra 0° -10°C. The reaction temperature is one of the determining factors for obtaining the products according to the invention and must be between 0-25 ° C, preferably between 0 ° -10 ° C.
Il pH di reazione viene portato a valori compresi tra 3 - 6, preferibilmente tra 4 - 5, ad esempio aggiungendo una soluzione di HCI diluito. The reaction pH is brought to values between 3 - 6, preferably between 4 - 5, for example by adding a solution of diluted HCI.
Un altro fattore determinante per ottenere i prodotti secondo l’invenzione è il rapporto tra attivante e Acido laluronico sale sodico di partenza; l’attivante viene aggiunto in quantità comprese tra 0.05-0.5 equivalenti per equivalente monomerico di Acido laluronico, preferibilmente tra 0.1 e 0.2 equivalenti. Utilizzare quantità di attivante superiori rispetto a quelle riportate sopra comporta la formazione prevalente di legami irreversibili tra Acido laluronico e attivante a scapito di quelli tra Acido laluronico e agente cross-linkante il che si traduce nell’ottenimento di prodotti indesiderati, insolubili o parzialmente insolubili in acqua e con caratteristiche di biocompatibilità e purezza diverse rispetto a quelle dei prodotti descritti nella presente invenzione. Another determining factor for obtaining the products according to the invention is the ratio between activator and Hyaluronic acid starting sodium salt; the activator is added in quantities ranging from 0.05-0.5 equivalents per monomeric equivalent of hyaluronic acid, preferably between 0.1 and 0.2 equivalents. Using higher quantities of activator than those indicated above leads to the prevailing formation of irreversible bonds between Hyaluronic Acid and activator to the detriment of those between Hyaluronic Acid and cross-linking agent which results in obtaining unwanted, insoluble or partially insoluble products in water and with characteristics of biocompatibility and purity different from those of the products described in the present invention.
L’Agente cross-linkante, infine, viene aggiunto in quantità comprese tra 0.1 - 1 equivalenti per equivalente di unità monomerica di Acido laluronico di partenza. Finally, the cross-linking agent is added in quantities between 0.1 - 1 equivalent per equivalent of the starting hyaluronic acid monomer unit.
Terminate le aggiunte suddette la miscela di reazione è mantenuta a temperatura e in agitazione per un periodo di tempo compreso tra 15 minuti - 4 ore, preferibilmente tra 30 minuti e 2 ore. At the end of the above additions, the reaction mixture is kept at temperature and under stirring for a period of time comprised between 15 minutes - 4 hours, preferably between 30 minutes and 2 hours.
Si aggiunge quindi una soluzione di NaCI 1 M e una soluzione tampone a pH 7 - 8 (preferibilmente 7.5) quindi si procede ad una purificazione secondo i metodi già conosciuti tra cui dialisi e/o diafiltrazione su membrana di ultrafiltrazione e/o precipitazione con solvente organico e/o evaporazione sotto vuoto e/o liofilizzazione. I metodi preferiti sono la diafiltrazione su membrana a cutoff di 30 KDa e la liofilizzazione. Il prodotto solido può essere ridisciolto in acqua o in soluzione fisiologica, in diverse concentrazioni per ottenere soluzioni viscose o gel trasparenti; è possibile ottenere anche film sottili, membrane, a seconda del tipo di applicazione a cui viene destinato. A 1 M NaCl solution and a buffer solution at pH 7 - 8 (preferably 7.5) are then added, then a purification is carried out according to the already known methods including dialysis and / or diafiltration on membrane of ultrafiltration and / or precipitation with solvent organic and / or evaporation under vacuum and / or lyophilization. Preferred methods are membrane diafiltration at 30 KDa cutoff and freeze drying. The solid product can be redissolved in water or physiological solution, in different concentrations to obtain viscous solutions or transparent gels; it is also possible to obtain thin films, membranes, depending on the type of application it is intended for.
Il prodotto può infine essere sterilizzato, ad esempio per filtrazione a 0.2 μ· Finally, the product can be sterilized, for example by filtration at 0.2 μ
Le quantità reciproche di Acido laluronico/attivante/agente crosslinkante influenzano il grado di cross-link del prodotto e quindi verranno adottate in funzione delle proprietà visco-elastiche che si intendono ottenere. The reciprocal quantities of hyaluronic acid / activating / cross-linking agent influence the degree of cross-linking of the product and therefore will be adopted according to the visco-elastic properties to be obtained.
Ovviamente le caratteristiche del prodotto finale saranno anche influenzate dal tipo di Acido laluronico utilizzato come prodotto di partenza per la reazione. E’ infatti evidente che a parità di altre condizioni Acido laluronico avente peso molecolare più alto consentirà di ottenere un gel più viscoso e compatto rispetto a quello ottenibile a partire da Acido laluronico di peso molecolare inferiore. Obviously the characteristics of the final product will also be influenced by the type of hyaluronic acid used as the starting product for the reaction. It is in fact evident that other conditions being equal Hyaluronic acid having a higher molecular weight will make it possible to obtain a more viscous and compact gel than that obtainable from hyaluronic acid with a lower molecular weight.
Preferibilmente secondo l’invenzione si utilizza Acido laluronico avente . peso molecolare compreso tra 100.000 e 2.000.000 ed i prodotti finali ottenuti hanno peso molecolare compreso tra 200.000 e 2.500.000. Preferably, according to the invention, hyaluronic acid is used having. molecular weight between 100,000 and 2,000,000 and the final products obtained have a molecular weight between 200,000 and 2,500,000.
I prodotti finali presentano un grado di cross-link compreso tra 2-40 %. L’invenzione sarà meglio compresa alla luce degli esempi qui di seguito riportati. The final products have a degree of cross-link between 2-40%. The invention will be better understood in the light of the examples given below.
ESEMPIO 1 EXAMPLE 1
1.0 g di Acido laluronico sale sodico (PM 1.550.000) (2.5 mmoli) vengono disciolti in 100 mi di acqua demineralizzata. La temperatura di reazione viene mantenuta a 5°C e il pH corretto a 4.5 aggiungendo una soluzione di HCI diluito. Vengono aggiunti 0.048 g (0.1 eq.) di N-3-dimetilammino-propiletilcarbodiimmide cloridrato e 0.31 g (0.5 eq.) di L-Lisina etilestere dicloridrato. 1.0 g of Hyaluronic acid sodium salt (MW 1,550,000) (2.5 mmoles) are dissolved in 100 ml of demineralized water. The reaction temperature is maintained at 5 ° C and the pH corrected at 4.5 by adding a solution of diluted HCI. 0.048 g (0.1 eq.) Of N-3-dimethylamino-propylethylcarbodiimide hydrochloride and 0.31 g (0.5 eq.) Of L-Lysine ethyl ester dihydrochloride are added.
Dopo 1 ora vengono aggiunti 10 mi di soluzione di NaCI 1M e 10 mi di soluzione tampone a pH 7.5; il gel viene diafiltrato con 10 volumi di acqua demineralizzata su membrana a cutoff di 30 KDa e infine liofilizzato. After 1 hour, 10 ml of 1M NaCl solution and 10 ml of buffer solution at pH 7.5 are added; the gel is diafiltered with 10 volumes of demineralized water on a 30 KDa cutoff membrane and finally lyophilized.
ESEMPIO 2 EXAMPLE 2
1.0 g di Acido laluronico sale sodico (PM1.550.000) (2.5 mmoli) vengono disciolti in 100 mi di acqua demineralizzata. La temperatura di reazione viene mantenuta a 5°C e il pH corretto a 4.5 aggiungendo una soluzione di HCI diluito. Vengono aggiunti 0.096 g (0.2 eq.) di N-3dimetilammino-propiletilcarbodiimmide cloridrato e 0.31 g (0.5 eq.) di L-Lisina etilestere dicloridrato. 1.0 g of Hyaluronic acid sodium salt (PM1.550.000) (2.5 mmoles) are dissolved in 100 ml of demineralized water. The reaction temperature is maintained at 5 ° C and the pH corrected at 4.5 by adding a solution of diluted HCI. 0.096 g (0.2 eq.) Of N-3dimethylamino-propylethylcarbodiimide hydrochloride and 0.31 g (0.5 eq.) Of L-Lysine ethyl ester dihydrochloride are added.
Dopo 1 ora vengono aggiunti 10 mi di soluzione di NaCI 1M e 10 mi di soluzione tampone a pH 7.5; il gel viene diafiltrato con 10 volumi di acqua demineralizzata su membrana a cutoff di 30 KDa e infine liofilizzato. After 1 hour, 10 ml of 1M NaCl solution and 10 ml of buffer solution at pH 7.5 are added; the gel is diafiltered with 10 volumes of demineralized water on a 30 KDa cutoff membrane and finally lyophilized.
ESEMPIO 3 EXAMPLE 3
1.0 g di Acido laluronico sale sodico (PM 750.000) (2.5 mmoli) vengono disciolti in 100 mi di acqua deinineralizzata. La temperatura di reazione viene mantenuta a 5°C e il pH corretto a 4.5 aggiungendo una soluzione di HCI diluito. Vengono aggiunti 0.048 g (0.1 eq.) di N-3-dimetilammino-propiletilcarbodiimmide cloridrato e 0.20 g (0.5 eq.) di L-Serina metilestere cloridrato. 1.0 g of Hyaluronic acid sodium salt (MW 750,000) (2.5 mmoles) are dissolved in 100 ml of deinineralized water. The reaction temperature is maintained at 5 ° C and the pH corrected at 4.5 by adding a solution of diluted HCI. 0.048 g (0.1 eq.) Of N-3-dimethylamino-propylethylcarbodiimide hydrochloride and 0.20 g (0.5 eq.) Of L-Serine methylester hydrochloride are added.
Dopo 1 ora vengono aggiunti 10 mi di soluzione di NaCI 1M e 10 mi di soluzione tampone a pH 7.5; il gel viene diafiltrato con 10 volumi di acqua demineralizzata su membrana a cutoff di 30 KDa e infine liofilizzato. After 1 hour, 10 ml of 1M NaCl solution and 10 ml of buffer solution at pH 7.5 are added; the gel is diafiltered with 10 volumes of demineralized water on a 30 KDa cutoff membrane and finally lyophilized.
ESEMPIO 4 EXAMPLE 4
1.0 g di Acido laluronico sale sodico (PM 750.000) (2.5 mmoli) vengono disciolti in 100 mi di acqua demineralizzata. La temperatura di reazione viene mantenuta a 5°C e il pH corretto a 4.5 aggiungendo una soluzione di HCI diluito. Vengono aggiunti 0.096 g (0.2 eq.) di N-3-dimetilammino-propiletilcarbodiimmide cloridrato e 0.20 g (0.5 eq.) di L-Serina metilestere cloridrato. 1.0 g of Hyaluronic acid sodium salt (MW 750,000) (2.5 mmoles) are dissolved in 100 ml of demineralized water. The reaction temperature is maintained at 5 ° C and the pH corrected at 4.5 by adding a solution of diluted HCI. 0.096 g (0.2 eq.) Of N-3-dimethylamino-propylethylcarbodiimide hydrochloride and 0.20 g (0.5 eq.) Of L-Serine methylester hydrochloride are added.
Dopo 1 ora vengono aggiunti 10 mi di soluzione di NaCI 1M e 10 mi di soluzione tampone a pH 7.5; il gel viene diafiltrato con 10 volumi di acqua demineralizzata su membrana a cutoff di 30 KDa e infine liofilizzato. After 1 hour, 10 ml of 1M NaCl solution and 10 ml of buffer solution at pH 7.5 are added; the gel is diafiltered with 10 volumes of demineralized water on a 30 KDa cutoff membrane and finally lyophilized.
ESEMPIO 5 EXAMPLE 5
1.0 g di Acido laluronico sale sodico (PM 1.600.000) (2.5 mmoli) vengono disciolti in 100 mi di acqua demineralizzata. La temperatura di reazione viene mantenuta a 5°C e il pH corretto a 4.5 aggiungendo una soluzione di HCI diluito. Vengono aggiunti 0.048 g (0.1 eq.) di N-3-dimetilammino-propiletilcarbodiimmide cloridrato e 0.18 g (0.5 eq.) di L-Lisina. 1.0 g of Hyaluronic acid sodium salt (MW 1,600,000) (2.5 mmoles) are dissolved in 100 ml of demineralized water. The reaction temperature is maintained at 5 ° C and the pH corrected at 4.5 by adding a solution of diluted HCI. 0.048 g (0.1 eq.) Of N-3-dimethylamino-propylethylcarbodiimide hydrochloride and 0.18 g (0.5 eq.) Of L-Lysine are added.
Dopo 1 ora vengono aggiunti 10 mi di soluzione di NaCI 1M e 10 mi di soluzione tampone a pH 7.5; il gel viene diafiltrato con 10 volumi di acqua demineralizzata su membrana a cutoff di 30 KDa e infine liofilizzato. After 1 hour, 10 ml of 1M NaCl solution and 10 ml of buffer solution at pH 7.5 are added; the gel is diafiltered with 10 volumes of demineralized water on a 30 KDa cutoff membrane and finally lyophilized.
ESEMPIO 6 EXAMPLE 6
Il solido ottenuto dall'esempio 1 viene ridisciolto in acqua in concentrazione di 3-4 g/l e filtrato a 0.2 micron, in condizioni asettiche. Il gel filtrato risulta sterile ed ha un contenuto di endotossine inferiore a 0.2 EU/mg (ss). The solid obtained from example 1 is redissolved in water in a concentration of 3-4 g / l and filtered at 0.2 micron, under aseptic conditions. The filtered gel is sterile and has an endotoxin content lower than 0.2 EU / mg (ss).
La caratterizzazione chimico-fisica ha evidenziato un grado di cross-link del 10%, una perdita all'essiccamento del 18-20% e i seguenti segnali The physico-chemical characterization showed a cross-link degree of 10%, a loss on drying of 18-20% and the following signals
Claims (21)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ITFI20010243 ITFI20010243A1 (en) | 2001-12-19 | 2001-12-19 | SOLUBLE AND BIOCOMPATIBLE GELS OF CROSS-LINKED HYALURONIC ACID WITH L-AMINO ACIDS OR B-FUNCTIONAL L-AMINOESTERS |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ITFI20010243 ITFI20010243A1 (en) | 2001-12-19 | 2001-12-19 | SOLUBLE AND BIOCOMPATIBLE GELS OF CROSS-LINKED HYALURONIC ACID WITH L-AMINO ACIDS OR B-FUNCTIONAL L-AMINOESTERS |
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|---|---|
| ITFI20010243A1 true ITFI20010243A1 (en) | 2003-06-19 |
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| Application Number | Title | Priority Date | Filing Date |
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| ITFI20010243 ITFI20010243A1 (en) | 2001-12-19 | 2001-12-19 | SOLUBLE AND BIOCOMPATIBLE GELS OF CROSS-LINKED HYALURONIC ACID WITH L-AMINO ACIDS OR B-FUNCTIONAL L-AMINOESTERS |
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| IT (1) | ITFI20010243A1 (en) |
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2001
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