ITMI930706A1 - GLYCOSAMINOGLICAN WATER SOLUBLE COMPLEXES, THEIR PREPARATION PROCEDURE AND PHARMACEUTICAL COMPOSITIONS THAT CONTAIN THEM - Google Patents
GLYCOSAMINOGLICAN WATER SOLUBLE COMPLEXES, THEIR PREPARATION PROCEDURE AND PHARMACEUTICAL COMPOSITIONS THAT CONTAIN THEM Download PDFInfo
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- ITMI930706A1 ITMI930706A1 IT000706A ITMI930706A ITMI930706A1 IT MI930706 A1 ITMI930706 A1 IT MI930706A1 IT 000706 A IT000706 A IT 000706A IT MI930706 A ITMI930706 A IT MI930706A IT MI930706 A1 ITMI930706 A1 IT MI930706A1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims description 21
- 238000000034 method Methods 0.000 title claims description 11
- 239000008194 pharmaceutical composition Substances 0.000 title claims description 8
- 238000002360 preparation method Methods 0.000 title claims description 6
- 229920002683 Glycosaminoglycan Polymers 0.000 claims description 45
- 229960001231 choline Drugs 0.000 claims description 44
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 claims description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- 102100028085 Glycylpeptide N-tetradecanoyltransferase 1 Human genes 0.000 claims description 18
- 230000000694 effects Effects 0.000 claims description 16
- 239000003146 anticoagulant agent Substances 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 13
- 229940127219 anticoagulant drug Drugs 0.000 claims description 11
- 229920000045 Dermatan sulfate Polymers 0.000 claims description 8
- 101710081880 Glycylpeptide N-tetradecanoyltransferase 1 Proteins 0.000 claims description 8
- 229910052708 sodium Inorganic materials 0.000 claims description 8
- 239000011734 sodium Substances 0.000 claims description 8
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- 239000012466 permeate Substances 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 4
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- 229920003303 ion-exchange polymer Polymers 0.000 claims description 3
- 238000005325 percolation Methods 0.000 claims description 3
- OEANUJAFZLQYOD-CXAZCLJRSA-N (2r,3s,4r,5r,6r)-6-[(2r,3r,4r,5r,6r)-5-acetamido-3-hydroxy-2-(hydroxymethyl)-6-methoxyoxan-4-yl]oxy-4,5-dihydroxy-3-methoxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](OC)O[C@H](CO)[C@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](OC)[C@H](C(O)=O)O1 OEANUJAFZLQYOD-CXAZCLJRSA-N 0.000 claims description 2
- SUJMSBLYCLWFHB-UHFFFAOYSA-L 2-hydroxyethyl(trimethyl)azanium;sulfate Chemical compound [O-]S([O-])(=O)=O.C[N+](C)(C)CCO.C[N+](C)(C)CCO SUJMSBLYCLWFHB-UHFFFAOYSA-L 0.000 claims description 2
- 150000007513 acids Chemical class 0.000 claims description 2
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- 239000003937 drug carrier Substances 0.000 claims 2
- 241000282472 Canis lupus familiaris Species 0.000 claims 1
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- 229920000669 heparin Polymers 0.000 description 12
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 10
- 229960002897 heparin Drugs 0.000 description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- AVJBPWGFOQAPRH-FWMKGIEWSA-L dermatan sulfate Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@H](OS([O-])(=O)=O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](C([O-])=O)O1 AVJBPWGFOQAPRH-FWMKGIEWSA-L 0.000 description 6
- 229940051593 dermatan sulfate Drugs 0.000 description 6
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- 239000001763 2-hydroxyethyl(trimethyl)azanium Substances 0.000 description 3
- 235000019743 Choline chloride Nutrition 0.000 description 3
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 3
- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical compound [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 description 3
- 229960003178 choline chloride Drugs 0.000 description 3
- KXKPYJOVDUMHGS-OSRGNVMNSA-N chondroitin sulfate Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](OS(O)(=O)=O)[C@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](C(O)=O)O1 KXKPYJOVDUMHGS-OSRGNVMNSA-N 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 229940055076 parasympathomimetics choline ester Drugs 0.000 description 3
- 150000003248 quinolines Chemical class 0.000 description 3
- 239000012465 retentate Substances 0.000 description 3
- 229920002971 Heparan sulfate Polymers 0.000 description 2
- 229920000288 Keratan sulfate Polymers 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000002785 anti-thrombosis Effects 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- KBPLFHHGFOOTCA-UHFFFAOYSA-N caprylic alcohol Natural products CCCCCCCCO KBPLFHHGFOOTCA-UHFFFAOYSA-N 0.000 description 2
- 231100000223 dermal penetration Toxicity 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 210000000245 forearm Anatomy 0.000 description 2
- KXCLCNHUUKTANI-RBIYJLQWSA-N keratan Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@H](COS(O)(=O)=O)O[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@H](O[C@@H](O[C@H]3[C@H]([C@@H](COS(O)(=O)=O)O[C@@H](O)[C@@H]3O)O)[C@H](NC(C)=O)[C@H]2O)COS(O)(=O)=O)O[C@H](COS(O)(=O)=O)[C@@H]1O KXCLCNHUUKTANI-RBIYJLQWSA-N 0.000 description 2
- 239000003055 low molecular weight heparin Substances 0.000 description 2
- 229940127215 low-molecular weight heparin Drugs 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- 230000000019 pro-fibrinolytic effect Effects 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
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- 150000003839 salts Chemical class 0.000 description 2
- 231100000274 skin absorption Toxicity 0.000 description 2
- 230000037384 skin absorption Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 159000000000 sodium salts Chemical class 0.000 description 2
- PGOHTUIFYSHAQG-LJSDBVFPSA-N (2S)-6-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-1-[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-4-methylsulfanylbutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-carbamimidamidopentanoyl]amino]propanoyl]pyrrolidine-2-carbonyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-4-methylpentanoyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino]-4-methylpentanoyl]amino]-3-sulfanylpropanoyl]amino]-4-methylsulfanylbutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-hydroxybutanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-4-methylpentanoyl]amino]-3-hydroxybutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-carbamimidamidopentanoyl]amino]-5-oxopentanoyl]amino]-3-hydroxybutanoyl]amino]-3-hydroxypropanoyl]amino]-3-carboxypropanoyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]-5-oxopentanoyl]amino]-3-phenylpropanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-4-oxobutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-4-carboxybutanoyl]amino]-5-oxopentanoyl]amino]hexanoic acid Chemical group CSCC[C@H](N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(O)=O PGOHTUIFYSHAQG-LJSDBVFPSA-N 0.000 description 1
- 208000032928 Dyslipidaemia Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 102100030500 Heparin cofactor 2 Human genes 0.000 description 1
- 101710153650 Heparin cofactor 2 Proteins 0.000 description 1
- 238000004566 IR spectroscopy Methods 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 108010000499 Thromboplastin Proteins 0.000 description 1
- 102000002262 Thromboplastin Human genes 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 230000010933 acylation Effects 0.000 description 1
- 238000005917 acylation reaction Methods 0.000 description 1
- 230000001858 anti-Xa Effects 0.000 description 1
- 229940121363 anti-inflammatory agent Drugs 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- ITHZDDVSAWDQPZ-UHFFFAOYSA-L barium acetate Chemical compound [Ba+2].CC([O-])=O.CC([O-])=O ITHZDDVSAWDQPZ-UHFFFAOYSA-L 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- WOWHHFRSBJGXCM-UHFFFAOYSA-M cetyltrimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+](C)(C)C WOWHHFRSBJGXCM-UHFFFAOYSA-M 0.000 description 1
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- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 description 1
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- TVMXDCGIABBOFY-UHFFFAOYSA-N n-Octanol Natural products CCCCCCCC TVMXDCGIABBOFY-UHFFFAOYSA-N 0.000 description 1
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- 238000005406 washing Methods 0.000 description 1
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Pyrrole Compounds (AREA)
Description
Domanda di brevetto per Invenzione Industriale dal titolo: "Complessi solubili in acqua di glicosaminoglicani, loro procedimento di preparazione e composizioni farmaceutiche che li contengono". Patent application for Industrial Invention entitled: "Water-soluble complexes of glycosaminoglycans, their preparation process and pharmaceutical compositions containing them".
A. CAMPO DELL'INVENZIONE La presente invenzione si riferisce a complessi solubili in acqua di glicosaminoglicani, al loro procedimento di preparazione e a composizioni farmaceutiche che li contengono. I glicosaminoglicani.solfati (GAGS) sono una famiglia di eteropolisaccaridi lineari, in stretta relazione fra loro, che comprendono i seguenti polimeri naturali: eparina (HP), eparansolfato (HS), condroitin-4- e -6-solfato (CS), dermatansolfato (DS), cheratansolfato (KS), frazione Fastmoving (FM).Inoltre sono stati anche preparati alcuni derivati per depolimerizzazione chimica, sovra-solfatazione e acilazione di GAGS naturali. Questi derivati condividono alcune propriet? farmacologiche con i GAGS naturali e possono essere compresi nella famiglia dei GAGS. A. FIELD OF THE INVENTION The present invention relates to water-soluble complexes of glycosaminoglycans, their preparation process and pharmaceutical compositions containing them. Glycosaminoglycans sulphates (GAGS) are a family of linear heteropolysaccharides, closely related to each other, which include the following natural polymers: heparin (HP), heparan sulfate (HS), chondroitin-4- and -6-sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), Fastmoving fraction (FM). In addition, some derivatives have also been prepared for chemical depolymerization, over-sulfation and acylation of natural GAGS. Do these derivatives share some properties? pharmacological with natural GAGS and can be included in the GAGS family.
Le principali attivit? farmacologiche dei GAGS, in base alle quali si sono espansi i loro usi terapeutici, sono: effetti anti- trombotici e pro-fibrinolitici, azione regolatrice nelle iper-dislipidemie, modulazione nella crescita della cellule. Alcuni GAGS posseggono attivit?' anti-coagulante (massima per l'HP) . The main activities pharmacological effects of GAGS, on the basis of which their therapeutic uses have expanded, are: anti-thrombotic and pro-fibrinolytic effects, regulatory action in hyper-dyslipidemias, modulation in cell growth. Do some GAGS own assets? anti-coagulant (maximum for HP).
B. PROBLEMA TECNICO. B. TECHNICAL PROBLEM.
Sfortunatamente, la maggior parte dei GAGS possiede una scarsa biodisponibilita' , sebbene recenti studi farmacocinetici provino la presenza di oligomeri nel flusso sanguigno dopo la loro somministrazione orale. Unfortunately, most GAGS have poor bioavailability, although recent pharmacokinetic studies prove the presence of oligomers in the bloodstream after their oral administration.
C. TECNICA ANTERIORE. C. FRONT TECHNIQUE.
Si sono avanzate varie ipotesi per spiegare la scarsa biodisponibilit? dei GAGS, sia per via orale che per via topica. In particolare: Various hypotheses have been advanced to explain the poor bioavailability? of GAGS, both orally and topically. In particular:
1. i GAGS sono sostanze molto idrofiliche (coefficiente di ripartizione in miscele n-ottanolo/acqua inferiore a 0,01); 2. i GAGS sono composti altamente ionizzabili; 1. GAGS are very hydrophilic substances (partition coefficient in n-octanol / water mixtures less than 0.01); 2. GAGS are highly ionizable compounds;
3 . i pesi molecolari medi dei GAGS sono alti, variando da 10 kD (CS) a 30 kD(DS). Solamente pochi fra gli enzimi presenti nel corpo umano sono capaci di rompere e ricostituire frammenti di GAGS; 3. the average molecular weights of the GAGS are high, ranging from 10 kD (CS) to 30 kD (SD). Only a few of the enzymes present in the human body are capable of breaking up and reconstituting fragments of GAGS;
4. i GAGS hanno una forte affinit?' (capacita' di legame) verso le cellule endoteliali. Per questo motivo vengono velocemente eliminati dal flusso sanguigno, rendendo gli studi farmacocinetici molto imprecisi. 4. Do GAGS have a strong affinity? (binding capacity) towards endothelial cells. For this reason they are quickly cleared from the bloodstream, making pharmacokinetic studies very inaccurate.
Si e' cercato di migliorare la biodisponibilita* dei GAGS mediante una depolimerizzazione controllata. I parametri limitativi di questa tecnica sono costituiti dall'intervallo di pesi molecolari nei quali i GAGS mantengono le loro propriet?' farmaco- terapeutiche e dalla necessita' di mantenere intatte alcune strutture specifiche all'interno della molecola. ;Un miglior tentativo di soluzione del problema e? rappresentato dal modificare chimicamente la molecola originale in modo da abbassare la idrofilicita' e la ionicita' senza influenzare le attivit?? desiderate. ;Questo metodo non esclude una depolimerizzazione preventiva, se applicabile, ed e' stato applicato con successo nella preparazione di N-acil- e N-O-diacil-derivati di GAGS aventi elevate attivit?' anti-trombotica e pro-fibrinolitica, senza rischi di attivit?' emorragica, e con migliorata biodisponibili ta', come dimostrato attraverso somministrazione orale degli stessi e come descritto nel brevetto USA 5.011.919 e nella domanda di brevetto europeo EP-A-90115227.2. ;E? anche noto dalla tecnica anteriore (Domanda di brevetto Europeo EP-A-206 947) che e' possibile migliorare l'assorbimento intestinale di alcuni principi attivi, in particolare antibiotici, agenti antivirali ed agenti antiinflaminatori (ma non vengono citati i GAGS), mescolando detti medicinali con esteri di colina. ;Gli esteri di colina sono stati anche indicati, nel brevetto USA 4,892,737. come capaci di migliorare l'assorbimento dermico di sostanze a carattere cosmetico o anestetico locale quando mescolati in basse proporzioni con dette sostanze. Anche in questo caso non si citano i GAGS fra le sostanze utilizzate. Gli esteri di colina utilizzati nella tecnica anteriore vengono preparati per reazione della colina con acidi grassi aventi da 4 a 20 atomi di carbonio, e, pur aumentando la biodisponibilita' dei medicamenti con i quali vengono mescolati, hanno il difetto di ridurne le attivit?' specifiche e di ostacolare la loro solubilit?' in acqua. ;C. DESCRIZIONE PARTICOLAREGGIATA DELL'INVENZIONE. ;Proseguendo negli studi sui GAGS, la Richiedente ha ora trovato che complessando i GAGS con colina si ottengono prodotti che mantengono inalterate le attivit?' originarie dei GAGS complessati ed al tempo stesso esibiscono significativi miglioramenti nella penetrazione attraverso la pelle. ;Oltre alla migliorata biodisponibilita' rispetto ai GAGS di origine, i GAGS complessati con colina permettono un rilascio controllato del principio attivo, presumibilmente attraverso uno scambio cationico a livello dello strato di delimitazione dell'acqua nelle condizioni fisiologiche della pelle. ;I complessi GAGS-colina secondo l'invenzione sono solubili in acqua ed in miscele acqua/etanolo fino al 30% di etanolo (a differenza dei GAGS di origine) e questa loro propriet?' permette la preparazione di gel etanolici, particolarmente utili per formulare composizioni farmaceutiche di uso topico. ;I complessi GAGS-colina dell'invenzione non sono scindibili per dialisi o per ultrafiltrazione, mentre sono scindibili in soluzioni di cloruro sodico isotoniche e vengono caratterizzati mediante spettrometria IR ed NMR in confronto con i GAGS e la colina separatamente presi. ;Nelle figure allegate sono riportati gli spettrogrammi IR ed NMR del complesso FMF-colina in confronto con l'FMF e la colina separatamente presi . ;In particolare: ;- la Fig. 1 mostra lo spettro IR del complesso FMF-colina; ;- la Fig. 2 mostra lo spettro IR espanso nella regione 2.000-500 cm del complesso FMF-colina; ;- la Fig. 3 mostra lo spettro IR del sale sodico di FMF; ;- la Fig. 4 mostra lo spettro IR del cloruro di colina; ;- la Fig. 5 mostra lo spettro IR espanso nella regione 2.000-500 cm<-1 >del cloruro di colina; ;- la Fig. 6 mostra lo spettro 13C-NMR (50 MHz) del sale sodico di FMF; ;- la Fig. 7 mostra lo spettro 13C-NMR (50 MHz) del complesso FMF-colina; ;- la Fig. 8 mostra lo spettro 13C-NMR (50 MHz) del cloruro di colina. ;I complessi GAGS-colina secondo 1 'invenzione possono quindi essere definiti attraverso le seguenti caratteristiche: ;polveri bianche solubili in acqua ed in miscele acqua/etanolo; ;- attivit?' anticoagulante: compresa fra 10 e 130 IU/mg; ;- contenuto in colina: dal 24 al 38% in peso; ;- colina libera: assente; ;- sodio: NMT 1%; ;- ceneri: NMT 3%; ;- peso molecolare: da 5 kD a 40 kD.? ;I complessi GAGS-colina secondo 1'invenzione hanno un rapporto molare solfato organico/gruppi carbossilici e dati ematochimici (APTT/KCTT, Anti-Xa, HC-II) analoghi a quelli dei rispettivi GAGS originari. ;Per preparare i complessi GAGS-colina dell'invenzione, si frazionano grossolanamente i differenti GAGS che si intendono complessare mediante precipitazione selettiva con quantitativi diversi di cetil-trimetil-ammonio cloruro (QN<+>) {come descritto da Scott - Methods in Carbohydrate Chemistry - R.L. Whistler Ed. - Voi. V - pag. 38 e segg.), scindendo i complessi in soluzione acquosa 2M di NaCl e precipitando i GAGS per aggiunnta di 1 vol/vol di etanolo, separando cosi' l'eparina (HP) dal DS+FM. ;Le frazioni cosi' ottenute costituiscono il materiale di partenza del procedimento di produzione dei complessi GAGS-colina secondo l'invenzione. ;Il procedimento secondo l'invenzione comprende le seguenti fasi fondamentali : ;- i GAGS che si desidera complessare vengono trasformati in acidi liberi mediante percolazione in colonna su resine a scambio ionico cationiche, a temperatura compresa fra 0? e 4?C; - il percolato in uscita dalla colonna viene immediatamente miscelato con una soluzione acquosa di colina in forma basica, detta soluzione essendo stata previamente raffreddata con ghiaccio fondente; ;- la soluzione ottenuta viene quindi ultra-filtrata ad un cutoff da 1.000 a 5-000 Dalton ed infine dia-filtrata per concentrarla; ;- detto concentrato viene ultra-filtrato ad un cut-off da 10.000 a 25.000 D ed i relativi ritenuti e permeati vengono separatamente essiccati a bassa temperatura (freeze-dried). Le resine a scambio ionico utili per la realizzazione del procedimento secondo l'invenzione sono quelle cationiche forti, in forma acida, particolarmente quelle con matrice polistirenica reticolata, come ad esempio la resina IRC-120 della Rohm & Haas. ;L'ultrafiltrazione della soluzione acquosa del complesso GAGS-colina permette di ottenere allo steso tempo la eliminazione dell'eccesso di colina libera e dei sali formatisi in reazione e di separare le frazioni a basso peso molecolare (meno di 6.000 D) che sono costituite da GAGS non aventi le caratteristiche volute. ;E' possibile preparare composizioni farmaceutiche contenenti i complessi GAGS-colina secondo l'invenzione sia per uso orale che per uso topico utilizzando i normali additivi e veicoli usati per detti scopi. ;Si e' trovato che i complessi GAGS-colina dell'invenzione sono particolarmente adatti per preparare composizioni farmaceutiche per uso topico, in particolare per uso transdermico. ;Tipicamente le composizioni farmaceutiche per uso topico secondo l'invenzione contengono un quantitativo di complesso GAGS-colina da 0,1% a 5% in peso. ;I seguenti esempi illustrano alcune forme di realizzazione tipiche del procedimento secondo l?invenzione e dei relativi complessi GAGS-colina ottenuti. ;ESEMPIO 1 ;50 g di eparina grezza, avente una attivit? pari a 100-120 lU/mg vennero disciolti in 500 mi di acqua distillata. La soluzione fu raffreddata a 0?-4?C e percolata in una colonna incamiciata e mantenuta alla stessa temperatura. La colonna avente un'altezza di 290 mm ed un diamtero interno di 20 mm era riempita con resina Amberlite IRC-120 della Rohm & Haas, in forma acida. La velocita? di percolazione fu regolata a 3 ml/min, mantenendo la temperatura a 0?-4?C mediante circolazione di liquido refrigerante nella camicia della colonna.il percolato fu immesso direttamente in un recipiente refrigerato contenente 1.000 mi di una soluzione al 2% di colina in forma basica mantenuta in agitazione. La colonna fu quindi eluita con 500 mi di acqua distillata e l'eluato immesso nello stesso recipiente dove era stato previamente immesso il percolato. La soluzione fu neutralizzata a pH 7 con HC1 6M, agitando e raffreddando, ultrafiltrata a volume costante attraverso membrane con un cut-off a 1.000 D, allo scopo di rimuovere i sali e la colina non reagita, fino a reazione negativa per colina e ioni CI ed infine concentrata per ultrafiltrazione a 300-500 ml. ;La soluzione concentrata fu fatta circolare in un apparecchio di ultrafiltrazione equipaggiato con membrane con cut-off a 10.000 D. Il permeato contiene frazioni epariniche a basso peso molecolare. Il ritenuto viene fatto circolare nell'apparecchiatura di ultra-filtrazione fino a reazione negativa per la colina, quindi immediatamente filtrato su filtri a membrana da 0,22 ?m e essiccato a freddo. ;Si ottengono 40 g di un complesso eparina-colina avente le seguenti caratteristiche: ;- Attivit?' anti-coagulante: 110-130 IU/mg (corrispondente ad un contenuto di eparina di 160-180 IU/mg). ;- elettroforesi (su acetato di cellulosa, con tampone di bario acetato 0,1 N, a pH 5 con acido acetico, soluzione rivelatrice 1% di Alcian blu in acido acetico al 3% e lavaggio con acido acetico al 3%) evidenzia una banda principale corrispondente ad eparina iniettabile RS (frazione slow-moving SMF); ;- sodio: NMT 1%; ;- ceneri: NMT 3%; ;- colina : (saggio di Reineckate): 36 ? 2%; ;- peso molecolare: ?17 ? 3 kD. ;ESEMPIO 2 ;50 g di una frazione di GAGS (ottenuta per frazionamento dei GAGS e separazione dell'eparina) contenente 70% di frazione fast-moving (FMF) e 30%? di dermatan solfato (DS) ed avente un'attivit?' anti -coagulante 80-100 IU/mg vengono sciolti in 500 mi di acqua DI. La soluzione fu raffreddata a 0e-4?C e percolata in una colonna incamiciata e mantenuta alla stessa temperatura come descritto nell'esempio 1. ;La soluzione concentrata fu fatta circolare in un apparecchio di ultrafiltrazione equipaggiato con membrane con cut-off a 25.000 D per 6-8 ore. Il permeato contiene il complesso frazione fast-moving-colina (FMF-colina) mentre il ritenuto contiene il complesso dermatansolfato-colina (DS-colina). ;Sia il filtrato che il ritenuto vengono separatamente filtrati su filtri a membrana da 0,22 pra ed essiccati a freddo. ;Dal permeato si ottengono 30 g di FMF-colina avente le seguenti caratteristiche : ;- Attivit?' anti-coagulante: 60-80 IU/mg; ;elettroforesi (eseguita nelle condizioni indicate nell'esempio 1): evidenzia una banda principale corrispondente alla frazione fast-moving (FMF); ;- sodio: NMT 1%; ;- ceneri: NMT 3%; ;- colina : (saggio di Reineckate): 32 ? 2%; ;- peso molecolare: ?8,5 ? 2 kD. ;Dal ritenuto si ottengono 12 g di DS-colina avente le seguenti caratteristiche : ;- Attivit?' anti-coagulante: NMT 10 IU/mg; ;- elettroforesi (come in esempio 1): evidenzia una banda principale corrispondente a dermatansolfato RS ; ;- sodio: NMT 1%; ;- ceneri: NMT 3%; ;- colina : (saggio di Reineckate): 26 ? 2%; ;- peso molecolare: ?35 ? 5 kD. ;ESEMPIO 3 ;Allo scopo di valutare la velocita' di penetrazione dermica dei complessi GAGS-colina secondo l'invenzione in confronto con i GAGS di partenza, si prepararono dei gel standard contenenti 0,5 g di principio attivo (GAGS originario o complesso GAGS-colina ottenuto) in 100 g di gel. ;I complessi GAGS-colina sottoposti a prova furono FMF-colina come preparato nell'esempio 2 e relativo FMF originario, LMW-HP-colina come preparata nell'esempio 1 e relativa LMW-HP, eparinato di sodio non frazionato (UF). ;Le attivit?' anti-coagulanti dei principi attivi sottoposti a prova sono compendiate nella Tabella 1. ;Le prove di penetrazione dermica furono condotte su volontari, usando la tecnica della "skin window" (J.S. Tan et al. "A method for Measurement of Antibiotics in Human Interstitial Fluid" -J. of Infect. Dis. (1972) 126, pagg. 492/97)? ;Sulla superficie palmare degli avambracci (il sinistro per il GAGS originario ed il destro per il relativo complesso GAGS-colina) furono tracciati con un pennarello due cerchi del diametro di 5 cm. ;I g di gel venne applicato dentro il cerchio segnato e massaggiato per 10 min, quindi l'area venne bagnata, pulita con etanolo al 70% e scarificata. L'area circolare venne quindi coperta con una camera Sykes-More sterile (prodotta dalla Belco Glass Wineland N.J.) dalla quale era stato previamente rimosso il vetro inferiore. La camera venne riempita con una soluzione salina isotonica (ISS) attraverso il tappo in gomma mediante una siringa e venne saldamente fissata all'avambraccio con un nastro trasparente. Una pressione negativa venne indotta nella camera aspirando 0,1 - 0,2 mi di liquido. ;A tempi prefissati, il contenuto della camera fu aspirato con siringa e conservato in tubi di saggio mantenuti in frigorifero a -12?C. ;II procedimento di riempimento con ISS e di successiva aspirazione dello stesso venne ripetuto a tempi diversi. ;I contenuti dei tubi di saggio vennero analizzati per l'attivit?* anti-coagulante (APTT= tempo di tromboplastina per plasma attivato; e 1T=tempo di trombina). An attempt was made to improve the bioavailability * of GAGS by means of a controlled depolymerization. The limiting parameters of this technique are constituted by the range of molecular weights in which the GAGS maintain their properties. pharmaco-therapeutic and the need to keep intact some specific structures within the molecule. ; A better attempt at a solution to the problem and? represented by chemically modifying the original molecule in order to lower the hydrophilicity and ionicity without affecting the activities? you want. ; This method does not exclude a preventive depolymerization, if applicable, and has been successfully applied in the preparation of N-acyl- and N-O-diacyl-derivatives of GAGS having high activity. anti-thrombotic and pro-fibrinolytic, without risk of activity hemorrhagic, and with improved bioavailability, as demonstrated by oral administration of the same and as described in US patent 5,011,919 and in European patent application EP-A-90115227.2. ;AND? also known from the prior art (European patent application EP-A-206 947) that it is possible to improve the intestinal absorption of some active ingredients, in particular antibiotics, antiviral agents and anti-inflammatory agents (but GAGS are not mentioned), by mixing said medicines with choline esters. Choline esters have also been indicated in US patent 4,892,737. as capable of improving the dermal absorption of cosmetic or local anesthetic substances when mixed in low proportions with said substances. Also in this case, GAGS are not mentioned among the substances used. The choline esters used in the prior art are prepared by reaction of choline with fatty acids having from 4 to 20 carbon atoms, and, while increasing the bioavailability of the medicaments with which they are mixed, they have the defect of reducing their activities. specific and to hinder their solubility? ' in water. ; C. DETAILED DESCRIPTION OF THE INVENTION. Continuing with the studies on GAGS, the Applicant has now found that by complexing the GAGS with choline, products are obtained which maintain the activities unaltered. originating from complexed GAGS and at the same time exhibiting significant improvements in penetration through the skin. In addition to the improved bioavailability compared to the original GAGS, the GAGS complexed with choline allow a controlled release of the active principle, presumably through a cationic exchange at the level of the water boundary layer in the physiological conditions of the skin. The GAGS-choline complexes according to the invention are soluble in water and in water / ethanol mixtures up to 30% of ethanol (unlike the original GAGS) and this property of theirs? it allows the preparation of ethanolic gels, particularly useful for formulating pharmaceutical compositions for topical use. The GAGS-choline complexes of the invention are not divisible by dialysis or by ultrafiltration, while they are divisible in isotonic sodium chloride solutions and are characterized by IR and NMR spectrometry in comparison with the separately taken GAGS and choline. ; The attached figures show the IR and NMR spectrograms of the FMF-choline complex in comparison with the FMF and choline separately taken. In particular: - Fig. 1 shows the IR spectrum of the FMF-choline complex; - Fig. 2 shows the expanded IR spectrum in the region 2.000-500 cm of the FMF-choline complex; - Fig. 3 shows the IR spectrum of the sodium salt of FMF; - Fig. 4 shows the IR spectrum of choline chloride; - Fig. 5 shows the expanded IR spectrum in the region 2.000-500 cm <-1> of the choline chloride; - Fig. 6 shows the 13C-NMR spectrum (50 MHz) of the sodium salt of FMF; - Fig. 7 shows the 13C-NMR spectrum (50 MHz) of the FMF-choline complex; ; - Fig. 8 shows the 13C-NMR spectrum (50 MHz) of choline chloride. The GAGS-choline complexes according to the invention can therefore be defined by the following characteristics: white powders soluble in water and in water / ethanol mixtures; ; - activities anticoagulant: between 10 and 130 IU / mg; ; - choline content: from 24 to 38% by weight; ; - free choline: absent; ; - sodium: NMT 1%; ; - ash: NMT 3%; ; - molecular weight: from 5 kD to 40 kD.? The GAGS-choline complexes according to the invention have a molar ratio of organic sulphate / carboxylic groups and haematochemical data (APTT / KCTT, Anti-Xa, HC-II) similar to those of the respective original GAGS. ; To prepare the GAGS-choline complexes of the invention, the different GAGS which are to be complexed by selective precipitation with different quantities of cetyl-trimethyl-ammonium chloride (QN <+>) are roughly fractionated {as described by Scott - Methods in Carbohydrate Chemistry - R.L. Whistler Ed. - Vol. V - pag. 38 et seq.), By splitting the complexes in a 2M aqueous solution of NaCl and precipitating the GAGS by adding 1 vol / vol of ethanol, thus separating the heparin (HP) from the DS + FM. The fractions thus obtained constitute the starting material of the production process of the GAGS-choline complexes according to the invention. The process according to the invention comprises the following fundamental steps: - the GAGS to be complexed are transformed into free acids by percolation in the column on cationic ion exchange resins, at a temperature between 0? and 4? C; - the leachate leaving the column is immediately mixed with an aqueous solution of choline in basic form, said solution having been previously cooled with melting ice; - the solution obtained is then ultra-filtered at a cutoff from 1,000 to 5-000 Dalton and finally dia-filtered to concentrate it; ; - said concentrate is ultra-filtered at a cut-off from 10,000 to 25,000 D and the relative retention and permeate are separately dried at low temperature (freeze-dried). The ion exchange resins useful for carrying out the process according to the invention are the strong cationic ones, in acid form, particularly those with cross-linked polystyrene matrix, such as for example the IRC-120 resin by Rohm &Haas.; The ultrafiltration of the aqueous solution of the GAGS-choline complex allows to obtain at the same time the elimination of the excess of free choline and the salts formed in the reaction and to separate the low molecular weight fractions (less than 6,000 D) which consist by GAGS not having the desired characteristics. It is possible to prepare pharmaceutical compositions containing the GAGS-choline complexes according to the invention for both oral and topical use using the normal additives and vehicles used for said purposes. It has been found that the GAGS-choline complexes of the invention are particularly suitable for preparing pharmaceutical compositions for topical use, in particular for transdermal use. Typically the pharmaceutical compositions for topical use according to the invention contain an amount of GAGS-choline complex from 0.1% to 5% by weight. The following examples illustrate some typical embodiments of the process according to the invention and of the related GAGS-choline complexes obtained. ; EXAMPLE 1; 50 g of crude heparin, having an activity equal to 100-120 lU / mg were dissolved in 500 ml of distilled water. The solution was cooled to 0 ° -4 ° C and percolated into a jacketed column and maintained at the same temperature. The column having a height of 290 mm and an internal diameter of 20 mm was filled with Amberlite IRC-120 resin from Rohm & Haas, in acid form. The speed? of percolation was adjusted to 3 ml / min, maintaining the temperature at 0 ° -4 ° C by circulating coolant in the jacket of the column. the leachate was placed directly into a refrigerated vessel containing 1,000 ml of a 2% solution of choline in basic form kept stirred. The column was then eluted with 500 ml of distilled water and the eluate placed in the same vessel where the leachate had been previously introduced. The solution was neutralized to pH 7 with 6M HCl, stirring and cooling, ultrafiltered at constant volume through membranes with a 1000 D cut-off, in order to remove salts and unreacted choline, up to a negative reaction for choline and ions. CI and finally concentrated by ultrafiltration to 300-500 ml. The concentrated solution was circulated in an ultrafiltration apparatus equipped with membranes with a 10,000 D cut-off. The permeate contains low molecular weight heparin fractions. The retentate is circulated in the ultra-filtration equipment until a negative reaction for the choline, then immediately filtered on 0.22µm membrane filters and cold dried. ; 40 g of a heparin-choline complex are obtained with the following characteristics: anti-coagulant: 110-130 IU / mg (corresponding to a heparin content of 160-180 IU / mg). ; - electrophoresis (on cellulose acetate, with 0.1 N barium acetate buffer, at pH 5 with acetic acid, 1% Alcian blue revealing solution in 3% acetic acid and washing with 3% acetic acid) shows a main band corresponding to injectable heparin RS (slow-moving fraction SMF); ; - sodium: NMT 1%; ; - ash: NMT 3%; ; - choline: (Reineckate's essay): 36? 2%; ; - molecular weight:? 17? 3 kD. ; EXAMPLE 2; 50 g of a GAGS fraction (obtained by fractionation of GAGS and separation of heparin) containing 70% of fast-moving fraction (FMF) and 30%? of dermatan sulfate (DS) and having an activity? anti-coagulant 80-100 IU / mg are dissolved in 500 ml of DI water. The solution was cooled to 0e-4? C and percolated in a jacketed column and maintained at the same temperature as described in Example 1.; The concentrated solution was circulated in an ultrafiltration apparatus equipped with membranes with a 25,000 D cut-off. for 6-8 hours. The permeate contains the fast-moving-choline fraction complex (FMF-choline) while the retentate contains the dermatan sulfate-choline complex (DS-choline). Both the filtrate and the retentate are separately filtered on 0.22 pra membrane filters and cold dried. ; From the permeate, 30 g of FMF-choline are obtained with the following characteristics:; - Activity anti-coagulant: 60-80 IU / mg; electrophoresis (performed under the conditions indicated in example 1): highlights a main band corresponding to the fast-moving fraction (FMF); ; - sodium: NMT 1%; ; - ash: NMT 3%; ; - choline: (Reineckate's essay): 32? 2%; ; - molecular weight:? 8.5? 2 kD. ; From the retained one obtains 12 g of DS-choline having the following characteristics:; - Activity anti-coagulant: NMT 10 IU / mg; ; - electrophoresis (as in example 1): highlights a main band corresponding to dermatan sulfate RS; ; - sodium: NMT 1%; ; - ash: NMT 3%; ; - choline: (Reineckate essay): 26? 2%; ; - molecular weight:? 35? 5 kD. ; EXAMPLE 3; In order to evaluate the speed of dermal penetration of the GAGS-choline complexes according to the invention in comparison with the starting GAGS, standard gels were prepared containing 0.5 g of active principle (original GAGS or GAGS complex -choline obtained) in 100 g of gel. The GAGS-choline complexes tested were FMF-choline as prepared in example 2 and relative original FMF, LMW-HP-choline as prepared in example 1 and relative LMW-HP, unfractionated sodium heparinate (UF). ; Activities anti-coagulants of the active ingredients tested are summarized in Table 1.; The dermal penetration tests were conducted on volunteers, using the "skin window" technique (J.S. Tan et al. "A method for Measurement of Antibiotics in Human Interstitial Fluid "-J. Of Infect. Dis. (1972) 126, pp. 492/97)? On the palmar surface of the forearms (the left for the original GAGS and the right for the related GAGS-choline complex) two circles with a diameter of 5 cm were traced with a felt-tip pen. ; The g of gel was applied inside the marked circle and massaged for 10 min, then the area was wetted, cleaned with 70% ethanol and scarified. The circular area was then covered with a sterile Sykes-More chamber (manufactured by Belco Glass Wineland N.J.) from which the lower glass had been previously removed. The chamber was filled with isotonic saline (ISS) through the rubber stopper using a syringe and was securely attached to the forearm with a clear tape. Negative pressure was induced in the chamber by aspirating 0.1 - 0.2 ml of liquid. At predetermined times, the contents of the chamber were aspirated with a syringe and stored in test tubes kept refrigerated at -12 ° C. The process of filling with ISS and subsequent aspiration of the same was repeated at different times. The contents of the test tubes were analyzed for anti-coagulant activity (APTT = thromboplastin time for activated plasma; and 1T = thrombin time).
I risultati ottenuti sono riportati nelle Tabelle 2 e 3? Come si pu? constatare, i complessi GAGS-colina esibiscono un assorbimento dermico superiore a quello dei GAGS non complessati e della eparina UF. Are the results obtained reported in Tables 2 and 3? How can you? note, the GAGS-choline complexes exhibit a dermal absorption higher than that of the uncomplexed GAGS and the UF heparin.
TABELLA 1 TABLE 1
(1) Frazione fast-moving (1) Fast-moving fraction
(2) Eparina a basso peso molecolare; PM=4.000 ? 500 D (2) Low molecular weight heparin; PM = 4,000? 500 D
(3) Sodio eparinato topico non frazionato. (3) Unfractionated topical sodium heparin.
TABELLA 2. TABLE 2.
TABELLA 3- TABLE 3-
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