How far from bedside Luciana Mosca a Antonio Francioso a Maria d Erme a Pietro Matricardi b Elita Montanari b Marco Girasole c Simone Dinarelli ID: 1006582
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1. NANOFORMULATIONS FOR POLYPHENOLS How far from bedside? Luciana Moscaa, Antonio Franciosoa, Maria d’Ermea, Pietro Matricardib, Elita Montanarib,Marco Girasolec, Simone Dinarellic, Paola MastromarinodaDepartment of Biochemical Sciences, dDepartment of Public Health and Infectious Diseases, bDepartment of Drug Chemistry and Technologies, Sapienza University of Roma, ITALY;cCNR, Institute of Material Structure, Roma, ITALY
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3. Brown et al (2009) Alcohol Clin Res 33: 1513-23Nakata et al (2012) Biol. Pharm. Bull. 35:273—279Beneficial effects of Polyphenols
4. Conte et al. 2016, Polyphenols Nanoencapsulation for Therapeutic Applications, J Biomol Res Ther , 5:2
5. PolyphenolType of delivery systemBiological testECGCPoly (L-lactide)-poly (ethylene glycol) nanoparticlesIn vitro, PC3 prostate cancer cellsNanolipidic particles (proprietary)In vivo, Bioavailability, RatsCarbohydrate matrix composed ofMaltodextrin/Arabic gumIn vitro, Du145 prostate cancer cellsQUERCETINSolid Lipid Nanoparticles (Soy lecithin, Tween80, PEG)In vivo, Bioavailability, RatsPoly (D,L-lactic-co-glycolic acid) (PLGA) nanocapsulesIn vivo, Rats, oxidative stress2-(hydroxy-propyl)-β-cyclodextrinIn vitro, antioxidant activityCURCUMINLiposomesIn vitro, MCF-7 brest cancerMicellesIn vivo, brest cancer model, miceIn vitro, hepatocellular carcinomaCyclodextrinIn vitro, breast cancer cellaRESVERATROLPro-LiposomesMicelles (Vineatrol30) In vivo, Bioavailability, RatsIn vivo, Bioavailability, HumansPolyester nanoparticlesIn vivo, bioavailability, ratsCyclodextrinsIn vitro, Stability and cytotoxicity Conte et al. 2016, Polyphenols Nanoencapsulation for Therapeutic Applications, J Biomol Res Ther , 5:2
6. Francioso, Mosca (2014) Chemistry stability and bioavailability of ResveratrolMedicinal Chemistry, 10: 237-45 Antioxidant AntiinflammatoryCardioprotectiveNeuroprotectiveChemo-preventiveAntiagingAntiviralIts use as a drug is strongly limited poor solubility scarce bioavailabilityunstability (autoxidation and photosensitivity) ResveratrolPROBLEMStrans-Resveratrol (3,5,4’-trihydroxy-trans-stilbene) is a bioactive polyphenol present in a variety of vegetables, like grapes and peanuts
7. Annual number of publications (PubMed)Smoliga (2011) Mol. Nutr. Food Res. 55, 1129–1141
8. Clinical trials in humans updated 18/12/2018Pharmacokinetic and tolerability studies, Type II diabetes, Obesity, Systemic oxidative stress, Neurodegenerative diseases/cognition/memory, osteoarthritis, cardiovascular diseases, Cancer, Metabolic disorders, NASH, Aging, Upper respiratory tract infections
9. Systemic administration of Resveratrol is unsuccesful due to low bioavailability and rapid metabolism in vivoBioavailability was assessed for food supplement or foods (i.e.red wine)Plasma concentration is in the sub-low microMolar rangeNone of the cinical trials performed to date evidenced significant effects in counteracting human diseases Cottart et al., Resveratrol bioavailability and toxicity in humans, Mol Nutr Food Res 2010
10. Our approach was differentDevelop a new liquid formulation for the topical treatment of viral infections of the upper respiratory tract, i.e. RhinovirusImprove the stability and solubility of resveratrol in aqueous solutions by encapsulation in biopolymers
11. Why Rhinovirus?Rhinoviruses are the etiological agent of about 50-80% of upper respiratory tract infections, and can reach the lower respiratory tract Transmitted mainly via areosol Otpimal temperature replication: 33°C- common cold- otitis media acute sinusitis- COPD exacerbations- asthma exacerbationsNo antivirals or vaccines available
12. The goal is to prevent Rhinovirus infection of the lower respiratory tract
13. Physiology of the nasal cavity and inhaled drugsInhaled drugs exert a local activity; First passage metabolism is avoidedBioavailability and time of retention of inhaled drugs can be improved by bioahesive controlled-release systemsMucoadhesive polymers improve drug permeability, bioavailability, persistance and absorption of the drugsMacromolecular hydrophilic anionic compounds are ideal candidates as mucoadhesive polymers (e.g. carbopol, adragant gum, cellulose derivatives)
14. DRUG
15. Carboxymethylated-β-D-glucan exerts the same biological effects of its precursor (immune modulating activity, antiviral activity, wound healing, structure-forming ability.It shows a high degree of solubility in water due to the presence of the easily ionizable R-CH2COOH groups This feature makes CM-glucan ideal for the development of possible liquid pharmaceutical formulations.Furthermore, it is widely used in drug-delivery and possesses many ionizable anionic groups which make it an ideal mucoadhesive polymerCarboxymethylated-1,3─›1,6-β-glucan
16. Aims of the project To improve the stability of resveratrol in aqueous solutions by the use of various glucan biopolymers (β-Glucan, Carboxymethyl-Glucan), and to study the chemico-physical properties of this association To evaluate the antiviral activity and biochemical effects of Resveratrol and Glucan against Human Rhinovirus 16
17. ResveratrolH2O/tweenCM-Glucanβ-GlucanPreparation of Resveratrol products Extensive vortexing, Sonication
18. Effect of the biopolymers on Resveratrol stability in aqueous solutionsFrancioso et al. (2014) J.Agr. Food Chem /Tween10012080604020
19. Accelerated stability and antioxidant activity of Resveratrol in aqueous solutionsAccelerated stability at 40°CFrancioso et al. (2014) J.Agric Food Chem ResveratrolCMGResv/CMGAged ResveratrolAged Resv/CMGAntioxidant activity
20. FT-IR Spectra of freshly prepared:(b) Resveratrol(a) CM-glucan(d) Resveratrol/CM-glucan association(c) Sample D after 12 months at 25°CCM-glucan confers stability to Resveratrol in aqueous solutionsFrancioso et al. (2014) J.Agric. Food Chem
21. Resveratrol confers structural organization to CM-glucan as demonstrated by Congo red bathochromic shift Francioso et al. (2017) Int. J. Mol Sci.
22. Microcalorimetry confirms that resveratrol confers a high degree of structural organization to CM-glucanThe first transition (Tm1) at 19°C corresponds to a release of the water associated with the triple-stranded helical chain of (1,3)-β-glucans.The thermal transitions in the range from 30 to 80°C are related to different conformational states of CM-glucan in aqueous environment. Francioso et al. (2017) Int. J. Mol Sci.
23. CM-glucan in waterATOMIC FORCE MICROSCOPY STUDIESResveratrol/CM-glucan association in water CM-glucan in alkaline solutionCONCLUSION (I)CM-glucan is able to improve significantly resveratrol stability in aqueous solution by a cooperative structural organization, highlighting that this association may be useful in pharmaceutical preparations Francioso et al. (2017) Int. J. Mol Sci.
24. Aims of the project (II) To improve the stability of resveratrol in aqueous solutions by the use of a natural biopolymer (Glucan), and to study the chemico-physical properties of this association To study antiviral activity and biochemical effects of Resveratrol and Glucan against Human Rhinovirus 16
25. Ex vivo experiments on human nasal epithelia infected with Rhinovirus serotype 16 (HRV-16)
26. Resveratrol inhibition of HRV-16 viral replication in ex vivo explanted human nasal epithelia*p<0.05, **p<0.01, ***p<0.001 vs the respective control.(■, 0.3 MOI, 40 h)(■, 3 MOI, 20 h)
27. Resveratrol and CMG effect on pro-inflammatory cytokines :IL-8IL-8Is a chemokine produced by macrophages and other cell types such as epithelial cells, airway smooth muscle cells and endothelial cells. One of the key factors in inflammatory response. IL-8 induces chemotaxis in target cells, primarily neutrophils but also other granulocytes, causing them to migrate toward the site of infection. It is considered to play a role in bronchiolitisP<0,01P<0,05P<0,01P<0,05Mastromarino et al. (2015) Antiviral research
28. IL-6Can act both as a proinflammatory or antiinflammaotry cytokine. It is secreted by the Tcells and macrophages to stimulate the immune system. It is an important mediator of fever and of the acute phase responseIL-6P<0,01P<0,05P<0,01P<0,05Resveratrol and CMG effect on pro-inflammatory cytokines :Mastromarino et al. (2015) Antiviral research
29. RANTESKnown also as CCL5 chemikine, Chemochina nota anche come CCL5. CCL5 is chemotactic for T cells, eosinophils, and basophils, and plays an active role in recruiting leukocytes into inflammatory sitesRANTESP<0,01P<0,001P<0,01nsResveratrol and CMG effect on pro-inflammatory cytokines :Mastromarino et al. (2015) Antiviral research
30. ICAM-1 ImmunofluorescenceResveratrolo - + CTRL cellsResveratrol inhibits overexpression of ICAM-1 induced by HRV-16 infection in nasal cellsHRV-16 infected cellsMastromarino et al. (2015) Antiviral research
31. Modulation of ICAM-1 and HRV-16 VP2 protein levels by resveratrol in human nasal epithelia uninfected or infected with HRV-16Mastromarino et al. (2015) Antiviral research
32. Conclusions (II)Resveratrol has a strong antiviral activity against HRV-16 and significantly reduces the levels of proinflammatory molecules.CM-glucan does not negatively modulate resveratrol antiviral properties and demonstrated a synergistic antinflammatory effect on IL-6 and -8 production when coadministered with Resveratrol
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34. Clinical trial results
35. Luciana Mosca, Antonio Francioso, Sergio Fanelli, Maria d’Erme, Laura Cervoni, Mario Fontana, Pietro Matricardi, Elita Montanari, Paola Mastromarino, Elena MattiaIst. di Struttura della Materia del CNRMarco Girasole, Simone DinarelliFrancesco MuraRossella Restignoli