ITFI20120025A1 - FORMULATION MADE OF SNALP CONTAINING PRE-MIR 34A USEFUL FOR THE TREATMENT OF MULTIPLE MYELOMA. - Google Patents
FORMULATION MADE OF SNALP CONTAINING PRE-MIR 34A USEFUL FOR THE TREATMENT OF MULTIPLE MYELOMA. Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
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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
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Description
Domanda di Brevetto per Invenzione Industriale dal titolo: Patent Application for Industrial Invention entitled:
Formulazione costituita da SNALP contenente pre-miR 34a utile per il trattamento del mieloma multiplo. Formulation consisting of SNALP containing pre-miR 34a useful for the treatment of multiple myeloma.
Campo dell'invenzione Field of the invention
L'invenzione si riferisce al campo dei prodotti ad attività farmacologica in particolare Stato dell'arte The invention relates to the field of products with pharmacological activity, in particular the state of the art
Come è noto il mieloma multiplo (MM) è la seconda malattia ematologica più comune nei Paesi occidentali. Nonostante numerosi progressi nella conoscenza dei meccanismi patobiologici della malattia e lo sviluppo di nuove strategie terapeutiche, i trattamenti oggi disponibili falliscono nell’obiettivo di ottenere una risposta duratura e la maggior parte dei pazienti sperimentano recidiva e esito letale. Numerose anomalie genetiche ed epigenetiche caratterizzano il processo di trasformazione multistep del MM nel midollo osseo dove il microambiente gioca un importante ruolo di supporto alla crescita cellulare e alla resistenza ai terapeutici convenzionali. Tutte le alterazioni acquisite dalle cellule di MM possono deregolare la crescita delle plasmacellule e il network di interazioni molecolari all’interno del microambiente. As is known, multiple myeloma (MM) is the second most common haematological disease in Western countries. Despite numerous advances in the knowledge of the pathbiological mechanisms of the disease and the development of new therapeutic strategies, the treatments available today fail to achieve a lasting response and most patients experience relapse and lethal outcome. Numerous genetic and epigenetic anomalies characterize the multistep transformation process of MM in the bone marrow where the microenvironment plays an important role in supporting cell growth and resistance to conventional therapeutics. All the alterations acquired by MM cells can deregulate the growth of plasma cells and the network of molecular interactions within the microenvironment.
E' altrettanto noto che i microRNA (miRs) sono piccole molecole di RNA non codificanti di circa 20 basi che giocano un ruolo critico nella regolazione post-trascrizionale dell’espressione genica. I miRs sono importanti modulatori di pathways cellulari con ruolo chiave nei processi di proliferazione e sopravvivenza cellulare. I miRs deregolati (down- o upregolati) possono agire come oncogeni (onco-miRs) o soppressori tumorali (TS-miRs) ed hanno perciò suscitato interesse per potenziali strategie terapeutiche. It is also known that microRNAs (miRs) are small non-coding RNA molecules of about 20 bases that play a critical role in the post-transcriptional regulation of gene expression. MiRs are important modulators of cellular pathways with a key role in the processes of cell proliferation and survival. Deregulated (down- or upregulated) miRs can act as oncogenes (onco-miRs) or tumor suppressors (TS-miRs) and have therefore aroused interest in potential therapeutic strategies.
Tra i miRs che risultano frequentemente downregolati in differenti tumori umani, il miR-34a risulta di particolare interesse per le sue potenziali applicazioni terapeutiche. Inoltre, l’attività antitumorale sembra non limitata alle cellule con ridotta espressione endogena di miR34a ma risulta anche efficace in cellule con apparente espressione normale di miR 34a. Among miRs that are frequently downregulated in different human cancers, miR-34a is of particular interest for its potential therapeutic applications. Furthermore, the antitumor activity seems not limited to cells with reduced endogenous expression of miR34a but is also effective in cells with apparent normal expression of miR 34a.
Tuttavia l’utilizzo di miR, macromolecole polianioniche, presenta non poche difficoltà, tra cui degradazione ed inattivazione da parte delle nucleasi presenti nel plasma e nelle cellule (Akhtar et al., 1991), una scarsa penetrazione nelle cellule stesse (Hope et al., 1998; Rojanasakul et al., 1996), una rapida eliminazione piasmatica (Agrawal e Zhang, 1997) nonché una tossicità renale ed emodinamica (Henry et al., 1997). Tutto ciò ne limita fortemente l’impiego pre-clinico e clinico. However, the use of miR, polyanionic macromolecules, presents many difficulties, including degradation and inactivation by the nucleases present in plasma and cells (Akhtar et al., 1991), poor penetration into the cells themselves (Hope et al. , 1998; Rojanasakul et al., 1996), rapid piasmatic elimination (Agrawal and Zhang, 1997) as well as renal and hemodynamic toxicity (Henry et al., 1997). All this severely limits its pre-clinical and clinical use.
Da ciò risulta evidente che l’impiego di miR in terapia, non può prescindere dallo sviluppo di una formulazione che sia in grado di proteggere la molecola dalla degradazione enzimatica, ne consenta l’accumulo preferenzialmente nel sito d’azione e ne promuova la penetrazione all’interno delle cellule bersaglio e le permetta di svolgere la sua azione. E' evidente da quanto detto sopra l'interesse a poter utilizzare un miR quale prodotto farmacologico nel trattamento del mieloma multiplo. From this it is evident that the use of miR in therapy cannot ignore the development of a formulation that is able to protect the molecule from enzymatic degradation, allows its accumulation preferentially in the site of action and promotes its penetration into the inside the target cells and allow it to carry out its action. It is evident from what has been said above the interest in being able to use a miR as a pharmacological product in the treatment of multiple myeloma.
Breve descrizione delle figure Brief description of the figures
La Figura 1 mostra i risultati di test in vivo utilizzando i composti secondo l’invenzione su Descrizione dettagliata dell'invenzione Figure 1 shows the results of in vivo tests using the compounds according to the invention on Detailed description of the invention
E' stato ora sorprendentemente trovato che un particolare miR, miR 34a, wild type o presentante un gruppo -0-CH3in posizione C2’ (miR 34a O-Met ), di sequenza It has now been surprisingly found that a particular miR, miR 34a, wild type or presenting a -0-CH3 group in position C2 '(miR 34a O-Met), of sequence
5’-UGG CAG UGU CUU AGC UGG UUG U-3’ [SEQ ID n°1] 5'-UGG CAG UGU CUU AGC UGG UUG U-3 '[SEQ ID n ° 1]
e un pre-miR34a, avente sequenza and a pre-miR34a, having sequence
5’-(GGC CAG CUG UGA GUG UUU CUU UGG CAG UGU CUU AGC UGG UUG UUG UGA GCA AUA GUA AG G AAG CAA UCA GCA AGU AUA CUG CCC UAG AAG UGC UGC ACG UUG UGG GGC CC)-3’ [SEQ ID n°2] 5 '- (GGC CAG CUG UGA GUG UUU CUU UGG CAG UGU CUU AGC UGG UUG UUG UGA GCA AUA GUA AG G AAG CAA UCA GCA AGU AUA CUG CCC UAG AAG UGC UGC ACG UUG UGG GGC CC) -3' [SEQ ID n ° 2]
possono essere efficacemente utilizzati contro cellule di mieloma multiplo grazie a particolari nanovettori a base lipidica che permettono di superare i problemi suddetti proteggendo il miR 34a o il pre-miR in essi incapsulato dalla degradazione in ambiente biologico, cambiandone la biodistribuzione e favorendo l’interazione di quest’ultimo con cellule bersaglio. can be effectively used against multiple myeloma cells thanks to particular lipid-based nanovectors that allow to overcome the aforementioned problems by protecting the miR 34a or the pre-miR encapsulated in them from degradation in a biological environment, changing their biodistribution and favoring the interaction of the latter with target cells.
Va sottolineato che i pre-miR sono molecole diverse dai miR 34a, sia perché seguono un diverso pathway biochimico, sia per la loro lunghezza; un miR ha una lunghezza di 21 nucleotidi, mentre un pre-miR arriva a 71 nucleotidi. It should be emphasized that pre-miRs are molecules different from miR 34a, both because they follow a different biochemical pathway, and because of their length; a miR has a length of 21 nucleotides, while a pre-miR reaches 71 nucleotides.
In particolare secondo l'invenzione per nanovettori a base lipidica si intendono nanoparticelle, polimeriche o lipidiche, quali nanoparticelle, micelle, liposomi o particelle lipidiche stabilizzate contenenti acidi nucleici (queste ultime anche note come SNALPs) (negli esempi specifici qui di seguito riportati si sono utilizzati questi ultimi come nano vettori). In particular, according to the invention, lipid-based nanoparticles are meant nanoparticles, polymeric or lipidic, such as nanoparticles, micelles, liposomes or stabilized lipid particles containing nucleic acids (the latter also known as SNALPs) (in the specific examples reported below, used as nano vectors).
E' pertanto un oggetto della presente invenzione una formulazione costituita da nanovettori a base lipidica contenente miR 34a o miR 34a O-Met o pre-miR 34a (o loro miscele) utile per il trattamento del mieloma multiplo. Therefore, an object of the present invention is a formulation consisting of lipid-based nanocarriers containing miR 34a or miR 34a O-Met or pre-miR 34a (or mixtures thereof) useful for the treatment of multiple myeloma.
L'invenzione potrà essere meglio compresa alla luce degli esempi qui di seguito riportati. Esempio 1 The invention will be better understood in the light of the following examples. Example 1
Preparazione degli SNALPs caricati con miR 34a o miR 34a O-Met o pre-miR 34a Preparation of SNALPs loaded with miR 34a or miR 34a O-Met or pre-miR 34a
Gli SNALPs utilizzati sono costituiti da una miscela di lipidi (1 ,2-distearoyl-sn-glycero-3-phosphocholine/colesterolo/N-palmitoyl-sphingosine-1 -{succi nyl[methoxy(polyethylene glycol) 2000]}/1 ,2-dioleoyl-3-dimethylammonium-propane (DSPC/CHOL/Cer-PEG/DODAP) (25/45/20/10, mol/mol/mol/mol) o DSPC/DSPE-PEG-Maleinimide(Mal)/CHOL/Cer-PEG/DODAP) e sono preparati mediante la tecnica dell'iniezione di etanolo (Sample et al., 2001 ) modificata. The SNALPs used consist of a mixture of lipids (1, 2-distearoyl-sn-glycero-3-phosphocholine / cholesterol / N-palmitoyl-sphingosine-1 - {succi nyl [methoxy (polyethylene glycol) 2000]} / 1, 2-dioleoyl-3-dimethylammonium-propane (DSPC / CHOL / Cer-PEG / DODAP) (25/45/20/10, mol / mol / mol / mol) or DSPC / DSPE-PEG-Maleinimide (Mal) / CHOL / Cer-PEG / DODAP) and are prepared by the modified ethanol injection technique (Sample et al., 2001).
Quindi una soluzione etanolica (100%) contenente 1 mg di lipidi totali (0,4 mi del volume totale) sono posti in una vial ambrata. Then an ethanolic solution (100%) containing 1 mg of total lipids (0.4 ml of the total volume) are placed in an amber vial.
In un’altra vial, 0,2 mg di miR 34a o miR 34a O-Met o pre-miR 34a (qui di seguito indicati come ON) sono stati solubilizzati in 0,6 mi di acido citrico 20 mM a pH 4,0. In another vial, 0.2 mg of miR 34a or miR 34a O-Met or pre-miR 34a (hereafter referred to as ON) was solubilized in 0.6 ml of 20 mM citric acid at pH 4.0 .
Le due soluzioni sono state riscaldate a 65Ό per 2-3 minuti e successivamente la soluzione etanolica, contenente i lipidi, è stata aggiunta alla soluzione acquosa contente ΓΟΝ, il tutto sotto agitazione. Successivamente le preparazioni sono state tenute a 65° C per circa 1 ora e sottoposte a un processo di estrusione, mediante l’utilizzo di un estrusore termostatato (Northern Lipidi Ine., Canada), a 65Ό. La sos pensione è stata fatta passare 5 volte su membrane di policarbonato con porosità di 0,2 μιτι e 20 volte su membrane con porosità 0,1 μιτι. La sospensione è stata, quindi, dializzata (3,5 kDa cut off) in tampone citrato 20 mM a pH 4,0 per 1 ora circa, per rimuovere l’eccesso di etanolo ed in tampone HBS (20mM HEPES, 145 mM NaCI, pH 7,4) per 18-20 ore per rimuovere il tampone citrato e neutralizzare il DODAP. La sospensione è stata purificata tramite cromatografia a scambio ionico DEAE-Sepharose CL-6B. The two solutions were heated at 65Ό for 2-3 minutes and subsequently the ethanolic solution, containing the lipids, was added to the aqueous solution containing ΓΟΝ, all under stirring. Subsequently, the preparations were kept at 65 ° C for about 1 hour and subjected to an extrusion process, using a thermostated extruder (Northern Lipidi Ine., Canada), at 65Ό. The substrate was passed 5 times on polycarbonate membranes with a porosity of 0.2 μιτι and 20 times on membranes with a porosity of 0.1 μιτι. The suspension was then dialyzed (3.5 kDa cut off) in 20 mM citrate buffer at pH 4.0 for about 1 hour, to remove excess ethanol and in HBS buffer (20mM HEPES, 145 mM NaCI, pH 7.4) for 18-20 hours to remove the citrate buffer and neutralize the DODAP. The suspension was purified by DEAE-Sepharose CL-6B ion exchange chromatography.
Il diametro medio degli SNALP è stato determinato mediante photon correlation spectroscopy (PCS). Ciascun campione è stato diluito con acqua deionizzata e filtrata ed analizzato a 20Ό. Per ciascun campione, il diametro medio e la distribuzione dimensionale sono stati ottenuti dalla media di tre misure. Per ogni formulazione, il diametro medio e ΙΊ.Ρ. sono stati calcolati dalla media di tre differenti lotti. Le formulazioni SNALP incapsulanti miR34a, miR34a O-Met o pre-miR34a, presentano rispettivamente un diametro medio di circa 157, 128 e 208 nm. The mean diameter of the SNALPs was determined by photon correlation spectroscopy (PCS). Each sample was diluted with deionized water and filtered and analyzed at 20Ό. For each sample, the mean diameter and size distribution were obtained from the average of three measurements. For each formulation, the mean diameter is ΙΊ.Ρ. were calculated from the average of three different batches. The encapsulating SNALP formulations miR34a, miR34a O-Met or pre-miR34a, have an average diameter of approximately 157, 128 and 208 nm, respectively.
Il dosaggio dei diversi miRs contenuti all’interno degli SNALPs è stato ottenuto mediante spettroscopia UV (Shimadzu, Giappone): 10 pi di sospensione SNALPs sono stati disciolti in 990 μΙ di metanolo ed analizzati a 260 nm. Il tasso d’incapsulazione dell’ON è stato calcolato come mg di ON/ml di sospensione diviso i mg di lipidi totali. La quantità di DSPC è stata calcolata mediante il saggio di Stewart (vedi Stewart JC. Anal Biochem. 1980). In breve, 10 μΙ di sospensione SNALP sono stati diluiti con 2 mi di acqua contenente ferrotiocianato di ammonio (0,1 N); la soluzione è stata poi addizionata a 2 mi di cloroformio. La concentrazione di DSPC è stata determinata dalla misura dell’assorbanza della fase organica a 485 nm. L’efficienza d’incapsulazione, invece, è stata calcolata come rapporto tra il tasso d’incapsulazione reale e quello teorico per 100. The dosage of the different miRs contained within the SNALPs was obtained by UV spectroscopy (Shimadzu, Japan): 10 µl of SNALPs suspension were dissolved in 990 μΙ of methanol and analyzed at 260 nm. The ON encapsulation rate was calculated as mg of ON / ml of suspension divided by mg of total lipids. The amount of DSPC was calculated using the Stewart assay (see Stewart JC. Anal Biochem. 1980). Briefly, 10 μΙ of SNALP suspension was diluted with 2 ml of water containing ammonium ferrothiocyanate (0.1 N); the solution was then added to 2 ml of chloroform. The concentration of DSPC was determined by measuring the absorbance of the organic phase at 485 nm. The encapsulation efficiency, on the other hand, was calculated as the ratio between the real and theoretical encapsulation rate per 100.
Da tali analisi risulta che l’efficienza d’incapsulazione, per le formulazioni SNALP incapsulanti miR34a risulta essere pari al 82%, mentre per la formulazione miR34a O-Met o pre-miR 34a, risulta essere per entrambe del 100% circa. From these analyzes it appears that the encapsulation efficiency, for the encapsulating SNALP miR34a formulations, is equal to 82%, while for the miR34a O-Met or pre-miR 34a formulation, it is approximately 100% for both.
Esempio 2 Example 2
Esperimenti in vivo. In vivo experiments.
Gli esperimenti in vitro sono stati condotti utilizzando colture di linee cellulari di MM trattate con varie formulazioni preparate secondo l’esempio 1 e sottoposte ad analisi di proliferazione cellulare. Per gli esperimenti in vivo sono stati utilizzati modelli murini xenograft e precisamente topi immunodeficienti (SCID) inoculati sottocute con 5X106 cellule di MM. Quando i tumori raggiungevano una dimensione misurabile venivano trattati per 4 volte a distanza di tre giorni con le diverse formulazioni a base di SNALP. L’attività anti MM del formulato è stata valutata mediante misura della massa tumorale e valutazione della survival degli animali trattati. Gli animali sono stati sacrificati quando i tumori raggiungevano 2 cm di diametro o quando insorgevano eventi che ne compromettevano la qualità della vita. The in vitro experiments were conducted using cultures of MM cell lines treated with various formulations prepared according to example 1 and subjected to cell proliferation analysis. For the in vivo experiments xenograft mouse models were used, namely immunodeficient mice (SCID) inoculated subcutaneously with 5X106 MM cells. When the tumors reached a measurable size they were treated 4 times three days apart with the different formulations based on SNALP. The anti-MM activity of the formulation was evaluated by measuring the tumor mass and evaluating the survival of the treated animals. The animals were sacrificed when the tumors reached 2 cm in diameter or when events occurred that compromised their quality of life.
In Figura 1 , è stata riportata l’inibizione della proliferazione cellulare (asse delle ascisse) rispetto ai giorni di trattamento (asse delle ordinate). In particolare, è possibile osservare che l’attività antitumorale di pre-miRNA 34a nel MM è superiore a quella ottenuta con miR34a e miR34 OMe, entrambi veicolati mediante SNALP. In assenza di nano vettore i miR non mostrano in vivo attività antitumorale nel MM. In Figure 1, the inhibition of cell proliferation (abscissa axis) with respect to the days of treatment (ordinate axis) was reported. In particular, it can be observed that the antitumor activity of pre-miRNA 34a in MM is higher than that obtained with miR34a and miR34 OMe, both conveyed by SNALP. In the absence of a nano vector, miRs do not show antitumor activity in MM in vivo.
Sempre dalla figura 1 si nota come, contrariamente alle attese, la presenza di transferrina sulla superficie degli SNALP non è in grado di aumentare l’efficacia degli SNALP contenenti pre-miR 34 mentre esercita l’effetto atteso sugli SNALP contenenti miR34a. Also from Figure 1 it is noted that, contrary to expectations, the presence of transferrin on the surface of SNALPs is not able to increase the effectiveness of SNALPs containing pre-miR 34 while exerting the expected effect on SNALPs containing miR34a.
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| US20090306194A1 (en) * | 2008-06-06 | 2009-12-10 | Asuragen, Inc. | Novel compositions for the in vivo delivery of rnai agents |
| US20100113560A1 (en) * | 2007-01-26 | 2010-05-06 | Judy Lieberman | Compositions and methods for treating hematopoietic malignancies |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20100113560A1 (en) * | 2007-01-26 | 2010-05-06 | Judy Lieberman | Compositions and methods for treating hematopoietic malignancies |
| US20090306194A1 (en) * | 2008-06-06 | 2009-12-10 | Asuragen, Inc. | Novel compositions for the in vivo delivery of rnai agents |
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| Title |
|---|
| BLOOD, vol. 118, no. 21, November 2011 (2011-11-01), 53RD ANNUAL MEETING AND EXPOSITION OF THE AMERICAN-SOCIETY-OF-HEMATOLOGY (ASH); SAN DIEGO, CA, USA; DECEMBER 10 -13, 2011, pages 1255, ISSN: 0006-4971(print) * |
| DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; November 2011 (2011-11-01), DI MARTINO MARIA TERESA ET AL: "MiR-34a Replacement As a Novel Therapeutic Approach for Multiple Myeloma: Preclinical In Vitro and In Vivo Evidence", XP002682657, Database accession no. PREV201200220190 * |
| DE ANTONELLIS ET AL.: "MiR-34a targeting of notch ligand delta-like 1 impairs CD15+/CD133+ tumor-propagating cells and supports neural differentiation in medulloblastoma.", PLOS ONE, vol. 6, no. 9, September 2011 (2011-09-01), XP002682654, Retrieved from the Internet <URL:www.plosone.org> [retrieved on 20120831] * |
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