ITMI20071321A1 - CYCLODESTRINE-BASED NANOSPUGNE AS A VEHICLE FOR ANTITUMOR DRUGS - Google Patents
CYCLODESTRINE-BASED NANOSPUGNE AS A VEHICLE FOR ANTITUMOR DRUGS Download PDFInfo
- Publication number
- ITMI20071321A1 ITMI20071321A1 IT001321A ITMI20071321A ITMI20071321A1 IT MI20071321 A1 ITMI20071321 A1 IT MI20071321A1 IT 001321 A IT001321 A IT 001321A IT MI20071321 A ITMI20071321 A IT MI20071321A IT MI20071321 A1 ITMI20071321 A1 IT MI20071321A1
- Authority
- IT
- Italy
- Prior art keywords
- paclitaxel
- nanosponges
- drug
- complexes
- cells
- Prior art date
Links
- 239000002246 antineoplastic agent Substances 0.000 title description 4
- 229940041181 antineoplastic drug Drugs 0.000 title description 4
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- 229960001592 paclitaxel Drugs 0.000 claims description 35
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 claims description 35
- 229940079593 drug Drugs 0.000 claims description 23
- 239000003814 drug Substances 0.000 claims description 23
- 229920000858 Cyclodextrin Polymers 0.000 claims description 18
- NKANXQFJJICGDU-QPLCGJKRSA-N Tamoxifen Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 NKANXQFJJICGDU-QPLCGJKRSA-N 0.000 claims description 18
- 239000000203 mixture Substances 0.000 claims description 14
- 238000009472 formulation Methods 0.000 claims description 11
- KLWPJMFMVPTNCC-UHFFFAOYSA-N Camptothecin Natural products CCC1(O)C(=O)OCC2=C1C=C3C4Nc5ccccc5C=C4CN3C2=O KLWPJMFMVPTNCC-UHFFFAOYSA-N 0.000 claims description 9
- 229940127093 camptothecin Drugs 0.000 claims description 9
- VSJKWCGYPAHWDS-FQEVSTJZSA-N camptothecin Chemical compound C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-FQEVSTJZSA-N 0.000 claims description 9
- VSJKWCGYPAHWDS-UHFFFAOYSA-N dl-camptothecin Natural products C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)C5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-UHFFFAOYSA-N 0.000 claims description 9
- 229960001603 tamoxifen Drugs 0.000 claims description 9
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 claims description 7
- 239000001116 FEMA 4028 Substances 0.000 claims description 4
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 claims description 4
- 235000011175 beta-cyclodextrine Nutrition 0.000 claims description 4
- 229960004853 betadex Drugs 0.000 claims description 4
- 239000008194 pharmaceutical composition Substances 0.000 claims description 4
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- ZDZOTLJHXYCWBA-VCVYQWHSSA-N N-debenzoyl-N-(tert-butoxycarbonyl)-10-deacetyltaxol Chemical compound O([C@H]1[C@H]2[C@@](C([C@H](O)C3=C(C)[C@@H](OC(=O)[C@H](O)[C@@H](NC(=O)OC(C)(C)C)C=4C=CC=CC=4)C[C@]1(O)C3(C)C)=O)(C)[C@@H](O)C[C@H]1OC[C@]12OC(=O)C)C(=O)C1=CC=CC=C1 ZDZOTLJHXYCWBA-VCVYQWHSSA-N 0.000 claims description 3
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- 125000003831 tetrazolyl group Chemical group 0.000 description 2
- YZOUYRAONFXZSI-SBHWVFSVSA-N (1S,3R,5R,6R,8R,10R,11R,13R,15R,16R,18R,20R,21R,23R,25R,26R,28R,30R,31S,33R,35R,36R,37S,38R,39S,40R,41S,42R,43S,44R,45S,46R,47S,48R,49S)-5,10,15,20,25,30,35-heptakis(hydroxymethyl)-37,39,40,41,42,43,44,45,46,47,48,49-dodecamethoxy-2,4,7,9,12,14,17,19,22,24,27,29,32,34-tetradecaoxaoctacyclo[31.2.2.23,6.28,11.213,16.218,21.223,26.228,31]nonatetracontane-36,38-diol Chemical compound O([C@@H]([C@H]([C@@H]1OC)OC)O[C@H]2[C@@H](O)[C@@H]([C@@H](O[C@@H]3[C@@H](CO)O[C@@H]([C@H]([C@@H]3O)OC)O[C@@H]3[C@@H](CO)O[C@@H]([C@H]([C@@H]3OC)OC)O[C@@H]3[C@@H](CO)O[C@@H]([C@H]([C@@H]3OC)OC)O[C@@H]3[C@@H](CO)O[C@@H]([C@H]([C@@H]3OC)OC)O3)O[C@@H]2CO)OC)[C@H](CO)[C@H]1O[C@@H]1[C@@H](OC)[C@H](OC)[C@H]3[C@@H](CO)O1 YZOUYRAONFXZSI-SBHWVFSVSA-N 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/13—Amines
- A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
- A61K31/138—Aryloxyalkylamines, e.g. propranolol, tamoxifen, phenoxybenzamine
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/716—Glucans
- A61K31/724—Cyclodextrins
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- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
- A61K47/6951—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
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- C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
- C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
- C08B37/0015—Inclusion compounds, i.e. host-guest compounds, e.g. polyrotaxanes
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
- C08J9/02—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof using blowing gases generated by the reacting monomers or modifying agents during the preparation or modification of macromolecules
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2201/00—Foams characterised by the foaming process
- C08J2201/02—Foams characterised by the foaming process characterised by mechanical pre- or post-treatments
- C08J2201/026—Crosslinking before of after foaming
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Description
Descrizione del brevetto per invenzione industriale avente per titolo: Description of the patent for industrial invention entitled:
“NANOSPUGNE A BASE DI CICLODESTRINE COME VEICOLO PER FARMACI ANTITUMORALI” "CYCLODEXTRIN-BASED NANOSPONGES AS A VEHICLE FOR ANTI-CANCER DRUGS"
La presente invenzione ha per oggetto composizioni farmaceutiche comprendenti nanospugne a base di ciclodestrine come veicolo per farmaci antitumorali insolubili in acqua, in particolare paclitaxel e altri tassani, camptotecina e tamoxifene. The present invention relates to pharmaceutical compositions comprising nanosponges based on cyclodextrins as a vehicle for water-insoluble anticancer drugs, in particular paclitaxel and other taxanes, camptothecin and tamoxifen.
SFONDO DELL’INVENZIONE BACKGROUND OF THE INVENTION
La solubilità di una farmaco è spesso il fattore limitante la sua applicazione in terapia. Pertanto sono in fase di indagine sempre nuove strategie per migliorare la solubilità e la cinetica di rilascio di principi attivi. The solubility of a drug is often the limiting factor for its application in therapy. Therefore, new strategies are always being investigated to improve the solubility and release kinetics of active ingredients.
Le ciclodestrine (CD) sono oligosaccaridi ciclici non riducenti costituiti da 6-8 molecole di glucosio legate con legame 1,4-a-glucosidico, aventi una caratteristica struttura a tronco di cono. La disposizione dei gruppi funzionali delle molecole di glucosio è tale per cui la superficie della molecola è polare mentre la cavità interna risulta essere relativamente lipofila. Cyclodextrins (CD) are non-reducing cyclic oligosaccharides consisting of 6-8 glucose molecules linked with 1,4-a-glucosidic bond, having a characteristic truncated cone structure. The arrangement of the functional groups of the glucose molecules is such that the surface of the molecule is polar while the internal cavity is relatively lipophilic.
La cavità lipofila conferisce alle CD la capacità di formare complessi di inclusione stabili anche in soluzione con molecole organiche di adatta polarità e dimensione. The lipophilic cavity gives the CD the ability to form stable inclusion complexes even in solution with organic molecules of suitable polarity and size.
Per questo motivo le CD sono già state studiate e presentano numerose applicazioni in vari campi della chimica (farmaceutica, analitica, catalisi, cosmetica ecc.) in cui si sfruttano le caratteristiche dei composti di inclusione. In tecnologia farmaceutica tali complessi possono essere adoperati per aumentare la velocità di dissoluzione, la solubilità e la stabilità di farmaci, oppure per mascherare sapori sgradevoli o per convertire sostanze liquide in solidi ecc. For this reason, CDs have already been studied and have numerous applications in various fields of chemistry (pharmaceutical, analytical, catalysis, cosmetics, etc.) in which the characteristics of inclusion compounds are exploited. In pharmaceutical technology such complexes can be used to increase the dissolution rate, solubility and stability of drugs, or to mask unpleasant flavors or to convert liquid substances into solids, etc.
In campo farmaceutico per migliorare la solubilità si adoperano diversi approcci formulativi, quali l’impiego di co-solventi, di tensioattivi, di complessi e di sistemi particolati. In the pharmaceutical field, various formulation approaches are used to improve solubility, such as the use of co-solvents, surfactants, complexes and particulate systems.
Farmaci particolarmente critici sotto il profilo formulativi, in particolar modo a causa della loro insolubilità, sono gli agenti tumorali paclitaxel, docetaxel e in genere i derivati a struttura tassanica, la camptotecina e il tamoxifene. Particularly critical drugs under the formulation profile, especially because of their insolubility, are the tumor agents paclitaxel, docetaxel and in general the derivatives with a taxane structure, camptothecin and tamoxifen.
Il paclitaxel è un importante farmaco antitumorale appartenente alla famiglia dei tassani, prodotti di origine vegetale che svolgono la loro azione antineoplastica inibendo la formazione del fuso mitotico. Paclitaxel is an important anticancer drug belonging to the family of taxanes, products of plant origin that perform their antineoplastic action by inhibiting the formation of the mitotic spindle.
Il paclitaxel è praticamente insolubile in acqua e nei sistemi biologici e pone pertanto problemi per la formulazione di formulazioni iniettabili. Il paclitaxel è attualmente formulato in forma di emulsione lipidica (Cremophor) che ne ha consentito la somministrazione per via endovenosa. I componenti della emulsione, però, sono potenti allergeni che possono causare gravi reazioni di ipersensibilità. Inoltre, i materiali utilizzati per la somministrazione del paclitaxel non possono essere in polivinilcloruro. Paclitaxel is practically insoluble in water and biological systems and therefore poses problems for the formulation of injectable formulations. Paclitaxel is currently formulated in the form of a lipid emulsion (Cremophor) which has allowed it to be administered intravenously. The components of the emulsion, however, are potent allergens that can cause severe hypersensitivity reactions. Also, the materials used for paclitaxel administration cannot be polyvinyl chloride.
La camptotecina è un farmaco di origine vegetale che inibisce la topoisomerasi I, un enzima cruciale nel controllo della crescita cellulare. Sono inoltre stati sviluppati della camptoecina, il topotecan e l’irinotecan per il trattamento di tumori polmonari e colorettali. Camptothecin is a plant-derived drug that inhibits topoisomerase I, an enzyme crucial in controlling cell growth. Camptoecin, topotecan and irinotecan have also been developed for the treatment of lung and colorectal cancers.
Il tamoxifene è un antagonista degli estrogeni utilizzato nel trattamento di carcinomi mammari e nella prevenzione delle recidive. In tal caso, il farmaco viene somministrato per via orale per lunghi periodi di tempo. Tamoxifen is an estrogen antagonist used in the treatment of breast cancers and in the prevention of recurrence. In such a case, the drug is administered orally for long periods of time.
Tutti questi farmaci pongono problemi di biodisponibilità e/o di formulazione a causa della loro scarsa o nulla solubilità in acqua. All these drugs pose bioavailability and / or formulation problems due to their low or no water solubility.
Esiste pertanto la necessità di superare tali limitazioni sviluppando in particolare formulazioni farmaceutiche più semplici e meno rischiose. There is therefore a need to overcome these limitations by developing in particular simpler and less risky pharmaceutical formulations.
Descrizione dell’invenzione Description of the invention
Si è ora trovato che polimeri iper-reticolati di ciclodestrine definiti “nanospugne” per la loro particolare struttura “nanoporosa” sono in grado in grado di veicolare vantaggiosamente farmaci idroinsolubili. It has now been found that hyper-crosslinked cyclodextrin polymers defined as "nanosponges" due to their particular "nanoporous" structure are capable of advantageously delivering hydroinsoluble drugs.
Le nanospugne di ciclodestrine consentono di disperdere a livello molecolare farmaci altrimenti insolubili, stabilizzandone la struttura e proteggendoli da aggressioni di agenti chimici. Ciò può condurre ad un prolungamento dell’ efficacia del farmaco, rispetto alla forma non complessata. The cyclodextrin nanosponges allow to disperse otherwise insoluble drugs at the molecular level, stabilizing their structure and protecting them from the aggression of chemical agents. This can lead to a prolongation of the drug's efficacy, compared to the non-complexed form.
L’invenzione fornisce pertanto in suo primo aspetto complessi di farmaci scelti fra paclitaxel, docetaxel, tamoxifen, camptotecina e loro derivati con nanospugne di ciclodestrine. The invention therefore provides in its first aspect complexes of drugs selected from paclitaxel, docetaxel, tamoxifen, camptothecin and their derivatives with cyclodextrin nanosponges.
L’invenzione riguarda inoltre formulazioni farmaceutiche somministrabili per via orale o parenterale comprendenti come principio attivo detti complessi in miscela con opportuni veicoli o eccipienti. The invention also relates to pharmaceutical formulations that can be administered orally or parenterally comprising as an active ingredient said complexes in mixture with suitable vehicles or excipients.
Le nanospugne di ciclodestrine sono preparate come descritto in EP 1786841 o in WO 03085002 usando un rapporto reticolante/ciclodestrina 2-16 con preferenza 4. Le ciclodestrine possono essere naturali, preferibilmente β-ciclodestrina, o chimicamente parzialmente modificate come metil β-ciclodestrina, alchilossicarbonil ciclodestrine ecc. Le nanospugne possono anche contenere percentuali variabili (5-30% in peso) di destrine lineari. The cyclodextrin nanosponges are prepared as described in EP 1786841 or in WO 03085002 using a crosslinker / cyclodextrin ratio 2-16 with preference 4. The cyclodextrins can be natural, preferably β-cyclodextrin, or chemically partially modified such as methyl β-cyclodextrin, alkyloxycarbonyl cyclodextrins etc. The nanosponges can also contain variable percentages (5-30% by weight) of linear dextrins.
Le nanospugne possono anche essere magnetizzate se preparate in presenza di composti con proprietà magnetiche. Nanosponges can also be magnetized if prepared in the presence of compounds with magnetic properties.
I complessi dell’ invenzione sono preparati per aggiunta di un eccesso di farmaco ad una sospensione acquosa delle nanospugne di ciclodestrine. Dopo agitazione della sospensione per un tempo variabile da 1 a 8 ore, preferibilmente a temperatura ambiente, il complesso viene recuperato filtrando l’eccesso di farmaco non solubilizzato. La formazione del complesso è dimostrata per analisi DSC. The complexes of the invention are prepared by adding an excess of drug to an aqueous suspension of cyclodextrin nanosponges. After stirring the suspension for a time ranging from 1 to 8 hours, preferably at room temperature, the complex is recovered by filtering the excess of unsolubilized drug. The formation of the complex is demonstrated by DSC analysis.
I complessi ottenuti possono essere utilizzati direttamente per la preparazione di formulazioni orali o iniettabili, utilizzando tecniche ed eccipienti convenzionali. Per le formulazioni iniettabili, ad esempio, il complesso potrà essere semplicemente veicolato in acqua sterile, fisiologica o altre soluzioni acquose adatta alla somministrazione parenterale. Per la via orale, i complessi potranno essere dispersi in una matrice di eccipienti, diluenti, lubrificanti, anti-aderenti adatti alla preparazione di capsule e compresse. I dosaggi dipenderanno dal tipo di farmaco complessato e saranno almeno pari o più preferibilmente inferiori a quelli attualmente proposti nella pratica clinica, in virtù della migliorate caratteristiche di biodisponibilità e farmacocinetica. The obtained complexes can be used directly for the preparation of oral or injectable formulations, using conventional techniques and excipients. For the injectable formulations, for example, the complex can be simply conveyed in sterile, physiological water or other aqueous solutions suitable for parenteral administration. For the oral route, the complexes can be dispersed in a matrix of excipients, diluents, lubricants, anti-adhesives suitable for the preparation of capsules and tablets. The dosages will depend on the type of complexed drug and will be at least equal to or more preferably lower than those currently proposed in clinical practice, by virtue of the improved bioavailability and pharmacokinetic characteristics.
La superiorità delle formulazioni dell’invenzione emerge chiaramente confrontando ad esempio l’azione antiproliferativa del paclitaxel in vitro con quella del paclitaxel complessato con le nanospugne (“Complesso”) tramite test di proliferazione cellulare su linee cellulari neoplastiche. The superiority of the formulations of the invention clearly emerges by comparing, for example, the antiproliferative action of paclitaxel in vitro with that of paclitaxel complexed with nanosponges ("Complex") through cell proliferation tests on neoplastic cell lines.
Gli esempi che seguono illustrano l’invenzione in maggior dettaglio. Esempio 1. Preparazione di nanospugne The following examples illustrate the invention in greater detail. Example 1. Preparation of nanosponges
In un pallone da 250 mi si pongono 17,00 g di β-ciclodestrina anidra, 0,93 g di Destrina 20 anidra e 80 g di Difenilcarbonato(DFC) finemente omogeneizzati. Si porta la temperatura gradatamente a 100°C sotto agitazione meccanica e si lascia reagire per 4 h. Al termine la miscela di reazione solidifica e viene lavata numerose volte con acqua distillata e successivamente con acetone per rimuovere il DFC non reagito ed il fenolo presente come sottoprodotto di reazione. Si ottengono 20,2 g di nanospugna. 17.00 g of anhydrous β-cyclodextrin, 0.93 g of anhydrous dextrin 20 and 80 g of finely homogenized diphenylcarbonate (DFC) are placed in a 250 ml flask. The temperature is gradually brought to 100 ° C under mechanical stirring and it is left to react for 4 h. At the end the reaction mixture solidifies and is washed numerous times with distilled water and subsequently with acetone to remove the unreacted DFC and the phenol present as reaction by-product. 20.2 g of nanosponge are obtained.
Esempio 2. Preparazione di nanospugne Example 2. Preparation of nanosponges
In un pallone si pongono 3 g di destrina 20, 20,0 g di beta ciclodestrina e 17,12 g di difenilcarbonato preventivamente finemente miscelati. Si porta il sistema a 100°C in un bagno ad ultrasuoni e si fa reagire per 4 h. Al termine, si pone la massa ottenuta in eccesso di acqua, si filtra e si lava prolungatamente con acqua e successivamente acetone per allontanare DFC non reagito e i sottoprodotti di reazione. Si pone il filtrato in stufa sotto vuoto ad essiccare a 80°C per 2 ore. 3 g of dextrin 20, 20.0 g of beta cyclodextrin and 17.12 g of diphenylcarbonate, previously finely mixed, are placed in a flask. The system is brought to 100 ° C in an ultrasonic bath and reacted for 4 h. At the end, the mass obtained is placed in excess of water, filtered and washed extensively with water and subsequently acetone to remove unreacted DFC and the reaction by-products. The filtrate is placed in a vacuum oven to dry at 80 ° C for 2 hours.
Esempio 2 bis. Preparazione di nanospugne Example 2 bis. Preparation of nanosponges
Come nell’esempio 2, ma con l’aggiunta di 2,0 grammi di polvere di Cobalto. Alla fine si ottengono particelle di nanospugna che mantengono le proprietà complessanti e che risultano essere in grado di legarsi ad un magnete. As in example 2, but with the addition of 2.0 grams of Cobalt powder. In the end, nanosponge particles are obtained which maintain the complexing properties and which are able to bind to a magnet.
Esempio 3. Preparazione del complesso di inclusione Example 3. Preparation of the inclusion complex
Una quantità fissata (5 mg) di nanospugne furono pesate e disperse in acqua distillata in un matraccio di 25 mi sotto agitazione. Un eccesso del farmaco, ad esempio il paclitaxel, fu aggiunto e la sospensione mantenuta sotto costante agitazione per 4 ore. Alla fine la sospensione fu filtrata tramite un filtro per centrifuga. (MICROCON YM 30, Millipore Corporation Bedford MA 01730 U.S.A.) al fine di separare il paclitaxel solubilizzato da quello non solubilizzato. Il filtrato fu analizzato con HPLC per determinare il contenuto di paclitaxel. La formazione del complesso di inclusione fu verificata tramite analisi DSC (Figura 1). A fixed amount (5 mg) of nanosponges were weighed and dispersed in distilled water in a 25 ml flask with stirring. An excess of the drug, for example paclitaxel, was added and the suspension kept under constant stirring for 4 hours. Finally the suspension was filtered through a centrifuge filter. (MICROCON YM 30, Millipore Corporation Bedford MA 01730 U.S.A.) in order to separate the solubilized paclitaxel from the unsolubilized one. The filtrate was analyzed with HPLC to determine the paclitaxel content. The formation of the inclusion complex was verified by DSC analysis (Figure 1).
Esempio 4. Studi di solubilità Example 4. Solubility studies
La determinazione della solubilità è stata condotta in accordo al metodo di Higuchi e Connors. In particolare 10 mg di paclitaxel furono aggiunti a una beuta contenente una soluzione acquosa (10 mi) di diverse percentuali di nanospugne (0,01, 0,02, 0,03, 0,04, 0,05, 0,075, 0,09, 0,1, 0,12, 0,15 e 0,2%). La beuta agitata su uno shaker meccanico a temperatura ambiente. Dopo aver raggiunto l equilibrio (48 ore) la sospensione fu filtrata per centrifugazione utilizzando un filtro di 3000 Dalton di taglio molecolare (MICROCON YM 30, Millipore Corporation Bedford MA 01730 U.S.A.). La soluzione ottenuta fu analizzata per determinare la concentrazione di paclitaxel tramite HPLC a 277 nm usando acetonitrile: Acqua 62:38 v:v come fase mobile previa liofilizzazione del complesso ed estrazione del paclitaxel in quantità note di metanolo. La concentrazione di paclitaxel in mg fu diagrammata contro la concentrazione di nanospugne in percentuale. I dati furono trattati statisticamente utilizzando la regressione lineare. L’analisi di solubilità mostra che il massimo di solubilizzazione è raggiunta con lo 0,5% w/w di sospensione di nanospugne. Oltre questo limite non si osservano ulteriori incrementi di solubilità del paclitaxel. The determination of the solubility was carried out according to the Higuchi and Connors method. In particular 10 mg of paclitaxel were added to a flask containing an aqueous solution (10 ml) of different percentages of nanosponges (0.01, 0.02, 0.03, 0.04, 0.05, 0.075, 0.09 , 0.1, 0.12, 0.15 and 0.2%). The flask shaken on a mechanical shaker at room temperature. After reaching equilibrium (48 hours) the suspension was filtered by centrifugation using a 3000 Dalton molecular cut filter (MICROCON YM 30, Millipore Corporation Bedford MA 01730 U.S.A.). The solution obtained was analyzed to determine the paclitaxel concentration by HPLC at 277 nm using acetonitrile: Water 62:38 v: v as mobile phase after lyophilization of the complex and extraction of paclitaxel in known quantities of methanol. The paclitaxel concentration in mg was plotted against the concentration of nanosponges in percent. The data were statistically processed using linear regression. The solubility analysis shows that the maximum solubilization is reached with 0.5% w / w of nanosponges suspension. Beyond this limit, no further increases in paclitaxel solubility are observed.
In conclusione, 1 mg di farmaco viene solubilizzato da 5 mg di nanospugna. (Figura 2). In conclusion, 1 mg of drug is solubilized by 5 mg of nanosponge. (Figure 2).
Esempio 5: test di attività su cellule cancerose Example 5: activity test on cancerous cells
Il test è basato sull’ incubazione delle cellule per periodi di 24, 48 e 72 h, in presenza di dosi scalari delle due formulazioni avendo cura di somministrare, in ogni caso, la stessa quantità di principio attivo. Gli esperimenti sono stati condotti su varie linee cellulari, e precisamente sulle cellule AT84 di carcinoma squamoso orale spontaneo murino HSC e sulle cellule Cai di carcinoma squamoso orale spontaneo umano. The test is based on cell incubation for periods of 24, 48 and 72 h, in the presence of scaled doses of the two formulations, taking care to administer, in any case, the same amount of active ingredient. The experiments were conducted on various cell lines, namely on the AT84 cells of murine spontaneous oral squamous carcinoma HSC and on the Cai cells of spontaneous human oral squamous carcinoma.
Alla fine del periodo di incubazione alle cellule è stato aggiunto una soluzione di sali di tetrazolio, metabolizzati soltanto dalle cellule vive. Dopo tre ore di incubazione i sali in eccesso sono stati eliminati ed alle cellule è stato aggiunto il DMSO, il quale provoca un viraggio del colore dei suddetti sali ridotti consentendo così una valutazione colorimetrica della quantità del tetrazolio metabolizzato, direttamente proporzionale al 1 numero di cellule vive. I dati presentati (relativi ad una parte degli esperimenti effettuati) sono espressi in percentuale di cellule vive per ogni trattamento rispetto ai controlli, costituiti dalle cellule a cui non è stato aggiunto il paclitaxel nativo né complessato. I dati sono il risultato di esperimenti condotti in ottuplicato. At the end of the incubation period, a solution of tetrazolium salts was added to the cells, metabolized only by living cells. After three hours of incubation, the excess salts were eliminated and DMSO was added to the cells, which causes a change in the color of the aforementioned reduced salts, thus allowing a colorimetric evaluation of the quantity of metabolized tetrazolium, directly proportional to the number of cells. lives. The data presented (relating to a part of the experiments carried out) are expressed as a percentage of live cells for each treatment compared to the controls, consisting of cells to which neither native nor complexed paclitaxel has been added. The data are the result of experiments conducted in octuplicate.
L’intervallo di concentrazioni utilizzate (sempre riferite alla molarità del paclitaxel) è stato definito in base ai dati presenti in letteratura per il solo paclitaxel. The range of concentrations used (always referring to the molarity of paclitaxel) was defined on the basis of the data in the literature for paclitaxel alone.
I dati a 24 h non evidenziano differenze significative tra i due gruppi di trattamento, mentre a 48 e 72 h si evidenzia un effetto antiproliferativo più importante da parte del Complesso. The data at 24 h did not show significant differences between the two treatment groups, while at 48 and 72 h there was a more important antiproliferative effect on the part of the Complex.
Nella figura 3 sono schematizzati i dati dell’ esperimento sulla linea AT84 a 72 h. Da essi risulta evidente una azione antiproliferativa molto più marcata (quasi tre volte) del Complesso rispetto al solo paclitaxel, che si manifesta soprattutto a dosi inferiori. Alle dosi più elevate l’effetto è analogo. Figure 3 summarizes the data of the experiment on the AT84 line at 72 h. From them it is evident a much more pronounced antiproliferative action (almost three times) of the Complex compared to paclitaxel alone, which occurs mainly at lower doses. At higher doses the effect is similar.
Data l’elevata tossicità del paclitaxel, la possibilità di ottenere effetti terapeutici analoghi o superiori a dosi inferiori consente di poter protrarre il trattamento, di incrementarne l’efficacia e di limitare gli effetti collaterali. Inoltre, la nuova formulazione è semplice, dispersa in acqua distillata e non prevede l’utilizzo di altre sostanze organiche neppure come solventi. Given the high toxicity of paclitaxel, the possibility of obtaining similar or higher therapeutic effects at lower doses allows the treatment to be prolonged, to increase its effectiveness and to limit side effects. Furthermore, the new formulation is simple, dispersed in distilled water and does not require the use of other organic substances, not even as solvents.
Risultati analoghi sono stati ottenuti sulle altre cellule, anche se ad intervalli di concentrazione diversi, caratteristici di ogni linea cellulare. Similar results were obtained on the other cells, albeit at different concentration ranges, characteristic of each cell line.
Si è posta l’attenzione in particolare alle concentrazioni più basse, alle quali il paclitaxel non sembrava agire se non in associazione con le nanospugne; in figura 4 e 5 sono riportati i risultati di esperimenti condotti sulle linee cellulari umane a concentrazioni estremamente basse. In entrambi i casi, già a concentrazioni 1 nanomolari, dove il paclitaxel non complessato non è attivo o lo è poco, il Complesso induce una riduzione della proliferazione del 30-50%. Particular attention was paid to the lowest concentrations, at which paclitaxel did not seem to act if not in association with nanosponges; Figures 4 and 5 show the results of experiments conducted on human cell lines at extremely low concentrations. In both cases, already at 1 nanomolar concentrations, where the non-complexed paclitaxel is not active or not very active, the Complex induces a reduction in proliferation of 30-50%.
I controlli interni di ogni esperimento sono rappresentati dalle nanospugne non complessate e da concentrazioni identiche di DMSO, necessario per la solubilizzazione del solo paclitaxel. Nessuna di queste condizioni ha mostrato alcun effetto sulla proliferazione cellulare (la figura 6 mostra i dati relativi all’ esperimento eseguito sulla linea Cai a 48 ore). The internal controls of each experiment are represented by the non-complexed nanosponges and by identical concentrations of DMSO, necessary for the solubilization of paclitaxel alone. None of these conditions showed any effect on cell proliferation (Figure 6 shows the data relating to the experiment performed on the Cai line at 48 hours).
I dati ottenuti con gli esperimenti fin qui condotti su linee cellulari neoplastiche deporrebbero per una azione antiproliferativa molto più accentuata del paclitaxel complessato con le nanospugne rispetto al solo paclitaxel. The data obtained with the experiments conducted up to now on neoplastic cell lines would show a much more pronounced antiproliferative action of paclitaxel complexed with nanosponges than paclitaxel alone.
Questo dato è particolarmente evidente soprattutto a concentrazioni molto basse (circa 1 nM). Inoltre, le sole nanospugne non hanno dimostrato possedere alcun effetto antiproliferativo o citotossico sulle linee cellulari testate. This data is particularly evident especially at very low concentrations (about 1 nM). Furthermore, the nanosponges alone have not been shown to have any antiproliferative or cytotoxic effects on the cell lines tested.
Esempio 6: determinazione della biodisponibilità della formulazione Example 6: determination of the bioavailability of the formulation
Per valutare l’eventuale aumento della biodisponibilità per via orale del paclitaxel complessato con le nanospugne, è stato condotto un esperimento su 20 topi femmine Balb/c di sei settimane. To evaluate the possible increase in the oral bioavailability of paclitaxel complexed with nanosponges, an experiment was conducted on 20 female Balb / c mice of six weeks.
Gli animali sono stati casualmente divisi in due gruppi e, previa anestesia, si è proceduto alla somministrazione tramite sondino oro-gastrico, di 50 mg/kg di paclitaxel con Cremophor, (gruppo di “controllo”) od il corrispettivo equimolare del complesso paclitaxel-nanospugne risospeso in soluzione fisiologica (gruppo “complex”). The animals were randomly divided into two groups and, after anesthesia, the administration of 50 mg / kg of paclitaxel with Cremophor ("control" group) or the equimolar equivalent of the paclitaxel complex was carried out by means of an oral-gastric tube. nanosponges resuspended in physiological solution ("complex" group).
Ad intervalli prestabiliti (30’, 1, 2 e 4 h), si è proceduto al prelievo del sangue rispettivamente a quattro animali per il gruppo complex ed a uno del gruppo di controllo, ed alla successiva separazione del siero ed al suo immediato congelamento per l’analisi in HPLC. At predetermined intervals (30 ', 1, 2 and 4 h), the blood was taken from four animals for the complex group and one from the control group respectively, and the subsequent separation of the serum and its immediate freezing for HPLC analysis.
Per la determinazione quantitativa del paclitaxel nel plasma dei topi dopo somministrazione orale delle due formulazioni, quella commerciale (Taxol) e la sospensione di nanospugne, si è adoperato un metodo cromatografico ad alta pressione (HPLC) a fase inversa. Lo strumento utilizzato per l’analisi è Perkin Elmer (binary pump LC250) collegato ad un rivelatore UV Perkin Elmer (LC-95). Le misure sono state effettuate utilizzando come fase mobile una miscela tampone ammonio acetato 10 mM pH = 5.0:metanolo:acetonitrile (50:10:40, v/v/v) mentre la fase stazionaria è rappresentata da una colonna Varian ODS (250 mm x 4,6 mm). La determinazione analitica si compie con un flusso 1 ml/min e il detector UV alla lunghezza d’onda di 227 nm. For the quantitative determination of paclitaxel in the plasma of mice after oral administration of the two formulations, the commercial one (Taxol) and the suspension of nanosponges, a reverse phase high pressure chromatography (HPLC) method was used. The instrument used for the analysis is Perkin Elmer (binary pump LC250) connected to a Perkin Elmer UV detector (LC-95). The measurements were carried out using a 10 mM ammonium acetate buffer mixture as mobile phase pH = 5.0: methanol: acetonitrile (50:10:40, v / v / v) while the stationary phase is represented by a Varian ODS column (250 mm x 4.6 mm). The analytical determination is carried out with a flow of 1 ml / min and the UV detector at a wavelength of 227 nm.
Preparazione dei campioni biologici: ad un volume di plasma noto (in genere 25 μΐ) sono stati addizionati 200 μΐ di acqua filtrata e 250 μΐ di metanolo. Dopo agitazione su vortex la miscela è estratta tre volte con etere dietilico. Preparation of biological samples: 200 μΐ of filtered water and 250 μΐ of methanol were added to a known volume of plasma (typically 25 μ). After vortexing the mixture is extracted three times with diethyl ether.
Le fasi eteree sono raccolte ed evaporate a secco sotto flusso di azoto. Il residuo viene ripreso con una soluzione acqua:metanolo:acetonitrile, 50:10:40, v/v/v) e dopo agitazione iniettato direttamente nell’HPLC. The ethereal phases are collected and evaporated to dryness under a nitrogen flow. The residue is taken up with a water solution: methanol: acetonitrile, 50:10:40, v / v / v) and after stirring injected directly into the HPLC.
La figura 7 riporta i risultati ottenuti che provano la presenza di elevate concentrazioni di paclitaxel nel sangue dei topi dopo 4 ore dalla somministrazione orale. Figure 7 reports the results obtained that prove the presence of elevated paclitaxel concentrations in the blood of mice 4 hours after oral administration.
Esempio 7 Example 7
L’attività citotossica del tamoxifen veicolato nelle nanospugne è stata determinata sulla linea cellulare MCL-7 (human breast cancer cells) utilizzando come controllo il farmaco non veicolato. I risultati sono mostrati in Ligura 8. The cytotoxic activity of tamoxifen delivered in the nanosponges was determined on the MCL-7 (human breast cancer cells) cell line using the non-delivered drug as a control. The results are shown in Ligura 8.
Il tamoxifen veicolato in nanospugne ha presentato una maggiore attività citotossica rispetto al solo principio attivo dopo una incubazione di 48 ore. In particolare incubando le cellule con il tamoxifen alla concentrazione di 100 ng/ml si ha circa il 40% di cellule sopravvissute, mentre la percentuale di cellule sopravvissute è superiore al 70% con il farmaco non veicolato. Tamoxifen delivered in nanosponges showed greater cytotoxic activity than the active ingredient alone after a 48-hour incubation. In particular, by incubating the cells with tamoxifen at a concentration of 100 ng / ml, approximately 40% of surviving cells are obtained, while the percentage of surviving cells is greater than 70% with the non-conveyed drug.
Esempio 8 Example 8
L’attività citotossica della camptotecina complessata con le nanospugne è stata determinata sulla linea cellulare HCPC1 (hamster cheek pouch carcinoma) utilizzando come controllo il farmaco non complessato. I risultati sono mostrati in Figura 9. La camptotecina veicolata in nanospugne ha presentato una maggiore citotossicità rispetto al solo principio attivo dopo incubazione di 72 ore. In particolare incubando le cellule con la camptotecina alla concentrazione di 64 nM si ha meno del 20% di cellule sopravvissute, mentre la percentuale di cellule sopravvissute è superiore al 90% con il farmaco non veicolato. The cytotoxic activity of camptothecin complexed with nanosponges was determined on the cell line HCPC1 (hamster cheek pouch carcinoma) using the non-complexed drug as a control. The results are shown in Figure 9. Camptothecin delivered in nanosponges showed greater cytotoxicity than the active ingredient alone after 72 hours incubation. In particular, by incubating the cells with camptothecin at a concentration of 64 nM, there are less than 20% of surviving cells, while the percentage of surviving cells is greater than 90% with the drug not delivered.
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IT001321A ITMI20071321A1 (en) | 2007-07-04 | 2007-07-04 | CYCLODESTRINE-BASED NANOSPUGNE AS A VEHICLE FOR ANTITUMOR DRUGS |
JP2010513764A JP2010531827A (en) | 2007-07-04 | 2008-06-27 | Cyclodextrin nanosponges as vehicles for antitumor agents |
CA 2692493 CA2692493A1 (en) | 2007-07-04 | 2008-06-27 | Cyclodextrin-based nanosponges as a vehicle for antitumoral drugs |
EP08784570A EP2175847A1 (en) | 2007-07-04 | 2008-06-27 | Cyclodextrin-based nanosponges as a vehicle for antitumoral drugs |
PCT/EP2008/005290 WO2009003656A1 (en) | 2007-07-04 | 2008-06-27 | Cyclodextrin-based nanosponges as a vehicle for antitumoral drugs |
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