NAB-paclitaxel, un innovativo meccanismo d’azione

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1 NAB-paclitaxel, un innovativo meccanismo d’azione

2 NAB-paclitaxel: la prima nano-chemioterapia target

3 Il mondo in nanoscala Tecnologia nab™ ~130 nm
Capello umano = nm Albumina = 5 nm Emoglobina = 6,5 nm Proteine Globuli rossi GR = 8000 nm Nanodispositivi: Nanopori Dendrimeri Nanotubi Punti quantici Nanogusci Nanoparticelle Tecnologia nab™ ~130 nm Acqua Glucosio Anticorpo Virus Batterio Cellula tumorale Un punto Pallina da tennis Nanometri 3

4 NAB-paclitaxel: limitata dimensione dell’ area di distribuzione
NAB-paclitaxel: dimensione media di 130 nm determinata dalla deviazione del raggio laser dinamico Desai N. et al. The APPS Journal, Vol. 14, No. 2, June 2012

5 NAB-paclitaxel dalla Crio-microscopia elettronica
Le immagini cryo-TEM dimostrano la forma sferica e la dimensione delle nanoparticelle di nab-paclitaxel Desai N. et al. The APPS Journal, Vol. 14, No. 2, June 2012

6 Struttura generale di una nanoparticella
INTERNO: uno o più farmaci alcuni mezzi di contrasto alcuni agenti per il superamento delle biobarriere (es.: permeation enhancer, capaci di forzare le giunzioni endoteliali) SUPERFICIE: Agenti di targeting biomolecolare (es.: anticorpi) alcuni agenti per il superamento delle biobarriere o per l’effetto “stealth” (es.: polietilenglicole) From: M. Ferrari, “Cancer Nanotechnology: opportunities and challenges”, Nat Rev Cancer Mar;5(3):161-71 6

7 NAB-paclitaxel: la prima nano-chemioterapia target veicolata dall‘albumina
molecola singola di NAB-paclitaxel complesso di NAB-paclitaxel dimensioni 4-14 nm albumina paclitaxel nab Technology utilises the endogenous albumin pathways through two mechanisms of actions. Albumin typically shuttles nutrients across endothelial cells from the lumen of the blood vessel to the underlying tissue on the other side of the vessel wall by an active process known as albumin receptor (gp60)-mediated transcytosis. The primary function of the gp60 protein is to transport albumin-bound nutrients across the blood vessel wall. Binding of albumin to gp60 receptors activates another protein, caveolin-1, that creates pockets in the endothelial wall. These pockets, technically known as caveolae, fill up with albumin-bound nutrients and migrate across the cell cytoplasm. Once the caveolae reach the other end of the endothelial cell wall, they deposit their contents into the tumour interstitium. The accumulation of albumin within a tumour appears to be facilitated through the tumour’s secretion of the protein called SPARC (secreted protein acidic and rich in cysteine). SPARC acts like a magnet; it attracts, binds to albumin-bound nutrients, and concentrates them within the tumour interstitium, leading to high concentrations of albumin and thus nab-paclitaxel in the tumour. dimensioni 130 nm Una singola molecola di albumina può legarsi fino a 6 o 7 molecole di paclitaxel5 Desai et al. SABCS [Abstract 1071]. Kratz et al. J Control Release. 2008;132(3): Peters,Jr. Serum albumin. Adv Protein Chem. 1985;37: Desai. Drug Delivery Report. 2008;Winter 2007/2008(16):35-41. Paal et al. Eur J Biochem. 2001;268:

8 La piattaforma nab sfrutta le proprietà naturali dell’albumina
L’albumina è un carrier naturale per il trasporto delle molecole idrofobiche nell’organismo Acidi grassi, ormoni, vitamine ecc. sono trasportati dall’albumina NAB-paclitaxel utilizza le vie di trasporto naturali dell’albumina (gp60 and SPARC) per raggiungere i tumori I tumori hanno un elevato uptake metabolico di albumina Albumin is the most ubiquitous natural protein in the human body, it is an important transport protein known to bind to a variety of endogenous and exogenous compounds. It is a natural carrier for hydrophobic molecules, such as fatty acids, hormones and vitamins, among others. Albumin shuttles macromolecules across the blood vessel wall from the blood space to the underlying tissue on the other side of the vessel wall. This process, which uses natural biological pathways, is known as endothelial transcytosis. nab-Paclitaxel has high binding affinity for human serum albumin and utilises the natural transport pathways of albumin, glycoprotein 60 (gp60) and SPARC, to target tumours, which have a high metabolic uptake of albumin. Purcell et al. Biochim Biophys Acta 2000;1478:61–68 Paal et al. Eur J Biochem 2001;268:2187–2191 Reference Purcell et al. Interaction of taxol with human serum albumin. Biochim Biophys Acta 2000;1478:61–68 Paal et al. High affinity binding of paclitaxel to human serum albumin. Eur J Biochem 2001;268:2187–2191

9 NAB-paclitaxel: meccanismo d’azione unico ed innovativo
2. Legame dei complessi paclitaxel albumina su specifici recettori delle cellule endoteliali, che porta ad un incremento della transcitosi 1. Somministrazione seguita da una rapida dissociazione in singoli complessi di paclitaxel albumina 5. Induzione di apoptosi delle cellule tumorali Summary of the mechanism of action (MoA) of nab®-paclitaxel Upon administration, particles of nab-paclitaxel dissolve. Moving through the bloodstream, paclitaxel exists in 2 main forms: free paclitaxel and albumin-bound paclitaxel. Free paclitaxel is small enough to pass through cells via a passive form of transport. In this way, paclitaxel can be delivered to tumor cells, where it can disrupt microtubule dynamics to induce tumor cell death. Circulating albumin binds to receptors on endothelial cells. This leads to endocytosis of vesicles, which then pass through endothelial cells and are emptied into the subendothelial space. Albumin-bound paclitaxel can pass through endothelial cells in this manner to be delivered to tumors. It is proposed that extracellular matrix albumin-binding proteins, such as secreted protein acidic and rich in cysteine (SPARC), may draw albumin-bound paclitaxel to tumor cells, thus enriching tumoral uptake of the drug. Once inside tumor cells, paclitaxel dissociates from albumin and acts on microtubule dynamics to induce cell death. References Gardner et al. Clin Cancer Res. 2008;14(13): John et al. Am J Physiol Lung Cell Mol Physiol. 2003;284(1):L187-L196. Desai et al. Clin Cancer Res. 2006;12(4): Desai et al. Transl Oncol. 2009;2(2):59-64 3. Transcitosi attiva attraverso le membrane endoteliali nell’interstizio del tumore 4. Legame della proteina SPARC con il complesso paclitaxel-albumina che libera il paclitaxel e lo rende disponibile a diffondersi nella cellula tumorale SPARC, secreted protein acidic and rich in cysteine. Investigation of the functional importance of SPARC with respect to nab-paclitaxel is ongoing.

10 Plasma + conventional paclitaxel
NAB-paclitaxel ha una cinetica lineare e garantisce un’attività antitumorale dose-dipendente Large micelle Cremophor EL, il solvente utilizzato per paclitaxel convenzionale, forma micelle che intrappolano paclitaxel nel plasma L’intrappolamento nelle micelle spiega il profilo farmacocinetico non-lineare e la perdita dell’attività antitumorale dose-dipendente di paclitaxel = Control plasma Plasma + conventional paclitaxel Rispetto a paclitaxel convenzionale, NAB-paclitaxel ha una farmacocinetica lineare nab-Paclitaxel: 30-minute infusion Conv Paclitaxel: 3-hour infusion This slide is an example of entrapment of paclitaxel into micelles under an electron micrograph. Reference 5. Van Zuylen et al. Invest New Drugs. 2001;19(2): Hamad & Moghimi. Expert Opin Drug Deliv. 2008;5:205– Sparreboom et al. Cancer Res. 1999;59(7): Van Tellingen et al. Br J Cancer. 1999;81(2): Winer et al. J Clin Oncol. 2004;22(11): 10

11 NAB-paclitaxel: maggiore biodisponibilità di principio attivo rispetto a paclitaxel convenzionale
nab-Paclitaxel’s 10X higher maximal plasma concentration of free paclitaxel1 may contribute to higher tumor accumulation2 Upon infusion, paclitaxel exits Cremophor micelles slowly compared with the dissolution of nab-paclitaxel This slide focuses more on free paclitaxel than total paclitaxel and is largely based on the data from the Gardner et al (Clin Cancer Res. 2008;14[13]: ) study on the PK of free paclitaxel comparing nab-paclitaxel with conventional paclitaxel. nab-Paclitaxel allows for an ~10-fold increase in the maximum concentration (Cmax) and ~3-fold higher AUC of free paclitaxel compared with conventional paclitaxel (Gardner). The Cmax of free paclitaxel in nab-paclitaxel also occurs sooner than that of conventional paclitaxel, demonstrating that dissolution is more rapid. The image on this slide reflects the higher proportion of free paclitaxel in the nab-paclitaxel formulation after administration. Also represented is paclitaxel entrapped in Cremophor micelles (Sparreboom A et al. Cancer Res. 1999;59[7]: ) in the conventional paclitaxel formulation. Gardner et al. Clin Cancer Res. 2008;14(13): Desai et al. Clin Cancer Res. 2006;12:

12 Nab-paclitaxel sfrutta le proprietà dell’albumina per potenziare la captazione selettiva e l’accumulo di farmaco nel sito tumorale L’ effettiva concentrazione di farmaco rappresenta una priorità terapeutica per le differenti caratteristiche biologiche e patologiche dei tumori Desai N. et al. The APPS Journal, Vol. 14, No. 2, June 2012

13 Tumor AUC nab-Paclitaxel = 1.33 × Conv Paclitaxel P < .0001 ANOVA
NAB-paclitaxel: aumento del 33% della concentrazione intratumorale di farmaco rispetto a paclitaxel convenzionale 8 7 nab-Paclitaxel Conv Paclitaxel 6 Concentration (µg paclitaxel/g) 5 4 Tumor AUC nab-Paclitaxel = 1.33 × Conv Paclitaxel P < ANOVA 3 2 The graph on this slide represents a time course of accumulation of a radioactively labeled form of paclitaxel in MX-1 human breast cancer xenograft tumors in nude mice. A 20 mg/kg dose of either nab-paclitaxel or conventional paclitaxel was administered, and tumors were dissected at the indicated time points for quantification of radioactivity. For each treatment group, 9 mice were evaluated at each time point. The radioactivity over time was then plotted, and AUCs were determined for each group. The nab-paclitaxel group demonstrated a 33% higher AUC compared with the conventional paclitaxel group. Possible explanation: that nab-paclitaxel is taken up by tumors to a greater degree relative to conventional paclitaxel implies that something specific about the physiology or biology of the tumor allows more uptake of the nab-paclitaxel formulation. The reason for this is not entirely clear. Hypothetical explanations center on 3 main ideas. A higher fraction of paclitaxel may be bound to albumin in the nab-paclitaxel formulation. Tumors, including surrounding stromal cells, secrete albumin- binding proteins that would lead to enrichment of albumin-bound drug in tumors. Indeed, tumors are known to take up large quantities of albumin for energy (Kratz F. J Control Release. 2008;132[3]: ). The second explanation centers on increased vascularization and a tumor- specific leakiness of blood vessels (Kratz). The nab-paclitaxel formulation leads to a significantly higher free paclitaxel fraction vs conventional paclitaxel (Gardner ER et al. Clin Cancer Res. 2008;14[13]: ) that could enter the tumors through this vasculature. Thirdly, Desai et al (Clin Cancer Res. 2006;12[4]: ) showed that the nab-paclitaxel formulation allows for higher transport across endothelial cells compared with conventional paclitaxel. This, combined with increased vasculature of tumors, would also explain the greater tumor selectivity of nab-paclitaxel. 0.01 0.1 1 10 100 Hours Dose of both nab-paclitaxel and conventional paclitaxel = 20 mg/kg dose of paclitaxel; experiments in human breast tumor xenografts in nude mice. ANOVA, analysis of variance; AUC, area under the curve; conv, conventional. 1. Desai et al. Clin Cancer Res. 2006;12:

14 Relative concentration (nab-paclitaxel/Conventional paclitaxel)
NAB-paclitaxel vs paclitaxel convenzionale: maggiore selettività tumorale 1.5 NAB-paclitaxel > paclitaxel conv 1.0 NAB-paclitaxel = paclitaxel conv Relative concentration (nab-paclitaxel/Conventional paclitaxel) NAB-paclitaxel < paclitaxel conv 0.5 Normal tissues Tumor Blood Comparative tissue distribution (ratio) of radiolabeled drug in mice bearing human breast tumor xenografts 1 hour after dose.1,2 Hawkins et al. AACR Poster 1189. Scheff . Community Oncol. 2008;5(7 suppl 8):7-13. 14

15 Relative concentration (nab-paclitaxel/Conventional paclitaxel)
NAB-paclitaxel: accumulo preferenziale nei tessuti tumorali rispetto ai tessuti sani nab-Paclitaxel demonstrates greater tumor selectivity compared with conventional paclitaxel in this preclinical model1,2 1.5 NAB-paclitaxel > paclitaxel conv 1.0 = NAB-paclitaxel paclitaxel conv Relative concentration (nab-paclitaxel/Conventional paclitaxel) < NAB-paclitaxel paclitaxel conv 0.5 Muscle Lung Tumor Heart Kidney Spleen Blood Comparative tissue distribution (ratio) of radiolabeled drug in mice bearing human breast tumor xenografts 1 hour after dose.1,2 Hawkins et al. AACR Poster 1189. Scheff . Community Oncol. 2008;5(7 suppl 8):7-13.

16 NAB-paclitaxel vs paclitaxel convenzionale: elevata selettività e maggior accumulo nelle cellule tumorali Consente di ottenere concentrazioni più elevate di paclitaxel libero Trae maggior vantaggio dalla transcitosi per raggiungere elevate concentrazioni nell’interstizio tumorale Interagisce con SPARC attraverso l’albumina, aumentando la concentrazione di NAB-paclitaxel nell’interstizio tumorale SPARC expression is increased in breast cancer tissue compared with normal tissue. SPARC expression was examined by antibody immunostaining of primary breast tumour (n=141), normal human breast tissue (n=115) and other normal human tissues. Immunohistochemistry (IHC) showed that SPARC was over expressed in 46% (65 of 141) of breast carcinoma vs only 1% (1 of 115) of normal breast tissue (P<0.0001). There was no correlation between SPARC staining and T stages, oestrogen receptor (ER) status, or progesterone receptor (PR) status. There was a trend for increased SPARC expression among p53(–) tumours, although this did not reach significance. Desai et al. Clin Cancer Res. 2006;12(4): Scheff. Community Oncol. 2008;5(7 suppl 8):7-13. Gardner et al. Clin Cancer Res. 2008;14(13): Kratz et al. J Control Release. 2008;132(3): Desai et al. Transl Oncol. 2009;2(2):59-64. Reference Trieu et al. SPARC expression in breast tumors may correlate to increased tumor distribution of nanoparticle abulmin-bound pactitaxel (ABI-007) vs taxol. Poster presented at AACR 2005

17 NAB-paclitaxel: un passo avanti nel tumore metastatico della mammella
Utilizza l’albumina come carrier naturale Incrementa la transcitosi attiva attraverso il legame gp60/caveolina-1 Aumenta del 33% la concentrazione intratumorale di paclitaxel

18 Le funzioni della proteina SPARC
SPARC, conosciuta anche come osteonectina o BM40, è una glicoproteina capace di interagire con le proteine della matrice extracellulare1 Le funzioni normali di SPARC includono rimodellamento tessutale, angiogenesi e sviluppo embrionale1 Nei tessuti sani, l’espressione di SPARC è stata rilevata nell’osso e nei tessuti che vanno incontro a sviluppo, rimodellamento e riparazione2 The crystal structure of SPARC3 Watkins et al. Prostaglandins Leukot Essent Fatty Acids. 2005;72(4): Porter et al. J Histochem Cytochem. 1995;43: Protein Database - SPARC. Accessed 16 November 2011. SPARC, secreted protein acidic and rich in cysteine.

19 Ruolo chiave di SPARC nelle metastasi tumorali e nel legame dell’albumina ai tumori
SPARC è regolata con meccanismi da VEGF e TGF-b1 Media il passaggio dell’espressione da E-caderina a N-caderina con il successivo aumento della migrazione cellulare e della capacità invasiva. È implicata nei meccanismi di evasione dei tumori, inibendo la sorveglianza del sistema immunitario, promuovendo l’angiogenesi e l’aggressività delle cellule metastatiche Motamed, K., Intl. J Biochem. Cell Biology. 31, ; Brekken e Sage Matrix Biol. 8, ; Podhajcer et al Cancer Metastasis Rev. 27,

20 SPARC nella linea cellulare di carcinoma mammario umano MX-1
MX-1 Staining for Surface proteins, SPARC is present on MX-1 surface MX-1 Confocal sectioning showing surface membrane associated SPARC Desai et al., SABCS 2004

21 L’aumentata espressione di SPARC è un fattore prognostico negativo in molti tumori
Glioblastoma Overexpression in juxtratumoral perivascular cells but not non-malignant brain vessels1 Meningioma A diagnostic marker for invasive meningiomas regardless of grade8 Tongue Carcinoma A diagnostic marker for invasive meningiomas regardless of grade9 Head & Neck Cancer High/Marker of poor prognosis2 Non-Small Cell Lung Cancer High/Marker of poor prognosis3 Esophageal Cancer High/Marker of poor prognosis10 Multiple Myeloma High levels of SPARC mRNA compared with non-malignant cell lines4 Breast Cancer High/Marker of poor prognosis; shows positive correlation with stage & grade11 Pancreatic Cancer Peritumoral fibroblast expression of SPARC a poor prognostic factor5 Bladder Cancer High/Marker of poor prognosis12 SPARC expression is increased in breast cancer tissue compared with normal tissue. SPARC expression was examined by antibody immunostaining of primary breast tumour (n=141), normal human breast tissue (n=115) and other normal human tissues. Immunohistochemistry (IHC) showed that SPARC was over expressed in 46% (65 of 141) of breast carcinoma vs only 1% (1 of 115) of normal breast tissue (P<0.0001). There was no correlation between SPARC staining and T stages, oestrogen receptor (ER) status, or progesterone receptor (PR) status. There was a trend for increased SPARC expression among p53(–) tumours, although this did not reach significance. Cervical Carcinoma High/Marker of poor prognosis13 Hepatocellular Carcinoma Overexpression by stromal myofibroblasts correlates well with angiogenesis & tumor progression6 Melanoma High/Marker of poor prognosis14 Prostate Carcinoma High levels of SPARC mRNA & protein as a marker of metastatic foci7 Reference Trieu et al. SPARC expression in breast tumors may correlate to increased tumor distribution of nanoparticle abulmin-bound pactitaxel (ABI-007) vs taxol. Poster presented at AACR 2005

22 SPARC, carcinoma mammario, prognosi
Breast Cancer Samples= 120 Normal Breast Tissue= 32 Overall Survival Nottingham Prognostic Index & SPARC expression Watkins G, et al. Prostaglandins, Leukotiens, Essential Fatty Acids. 2005; 72:267-72

23 SPARC e NAB-paclitaxel sono presenti nelle stesse cellule nel microambiente tumorale
L’albumina si accumula nei tumori2 L’accumulo di albumina nei tumori può essere mediato da SPARC1,2 SPARC mostra un’alta affinità per l’albumina3 Nei modelli tumorali, la colorazione per albumina e SPARC mostra la stessa distribuzione1 Fact-check note: the Kratz PDF was not modifiable for fact check. Data on page 181, left column, second paragraph. It is thought that tumor cells and their surrounding stroma secrete SPARC and other extracellular matrix albumin-binding proteins. This secreted SPARC may become bound to tumor cells. In either case, these proteins may encounter nab- paclitaxel in the subendothelial space, possibly leading to enrichment in tumors (Trieu V et al. SKCC [abstract 53] and Desai N et al. Transl Oncol. 2009;2[2]:59-64). The images shown represent immunohistochemical labeling of MX- 1 human breast cancer xenograft tumors in serial sections from nude mice with antibodies against albumin and SPARC. The brown coloring represents positive staining from the antibodies. The substantial similarity in the staining patterns reflect similar distribution. Albumina SPARC Sono in corso studi sull’importanza funzionale di SPARC rispetto a NAB-paclitaxel. 1. Trieu et al. SKCC [Abstract 53]. 2. Kratz et al. J Control Release. 2008;132(3): 3. Sage et al. J Biol Chem. 1994;259(6): SPARC, secreted protein acidic and rich in cysteine.

24 E’stato differenziato il nome del principio attivo!!!
Aggiornamento RCP Vecchia versione Vecchia versione E’stato differenziato il nome del principio attivo!!! Nuova versione

25 Aggiornamento RCP Vecchia versione Proprio per il fatto che sono due farmaci diversi, che uno non vale l’altro, è stato ribadito ANCHE nel paragrafo 4.2 che NON DEVE ESSERE SOSTITUITO con altri taxani Nuova versione

26 Aggiornamento RCP Vecchia versione E’ stato esplicitato che non ci sono rilevanti tossicità, e quindi controindicazioni, nella popolazione anziana!!! Nuova versione