Lipid nanocapsules: a tool for improving - AAPS

WaterOilConductivityPhase Inversion Temperature (PIT)Soft energy methodO/WA.I.PIZW/OTemperature Easy Fast Not expansive Solvent-free No heavy equipmentpswc2010 PatentMicroemulsionLNC

Encapsulation of lipophilic drugs‣ Amiodarone (S = 2 µg/mL)‣ Vitamins A, E,…‣ Ibuprofen (S = 56 µg/mL)‣ Antiseptic agents: chlorhexidine,…‣ Anticancer drugs‣ Etoposide (S = 0.25 µg/mL)‣ Paclitaxel (S = 0.25 µg/mL)‣ Docetaxel (S = 6-7 µg/mL)‣ Derivatives of Camptothecin, Sn38‣ BCNU‣ Derivatives of 4-hydroxy Tamoxifen,…‣ DNA and RNA lipid complexes‣ But, also hydrophilic drugs by playing with formulation variablespswc2010

Routes of administrationPulmonaryrouteLNCpswc2010

Development strategy of an oralLNC formulation‣ Proof of concept with a PK study with PTX-LNC‣ Set-up of specific evaluation tools for absorptionmechanism study:‣ Single Pass Intestinal Perfusion‣ Stability in biomimetic media‣ Caco-2 cell monolayers‣ Encapsulation of other drug candidates : Ibuprofen,Sn38,…pswc2010

First PK Study with PTX-LNCS. Peltier et al., Pharm. Res.,23 (2006) 1243-1250pswc2010

Conclusions of the PK study‣ C max and AUC were increased threefold‣ F was enhanced from 6.5% to 20% in the LNC group‣ 6/8 rats reached the PTX therapeutic level in the LNC groupvs 2/6 in the reference group (Taxol®): sufficient amounts ofPTX were given with this formulation‣ P-gp inhibition by the LNCs‣ The LNCs increase PTX absoption but do not solve the interindividual variabilitypswc2010

Development strategy of an oralLNC formulation‣ Proof of concept with a PK study with PTX-LNC‣ Set-up of specific evaluation tools for absorptionmechanism study:‣ Single Pass Intestinal Perfusion‣ Stability in biomimetic media‣ Caco-2 cell monolayers‣ Encapsulation of other drug candidates : Ibuprofen,Sn38,…pswc2010

Stability in gastro-intestinal mediaStability is determined by monitoring :size & drug content versus time aftercontact with biomimetic mediaSGFFassif V2Fessif V2pswc2010Fassif V2E. Roger et al. Int J Pharm 379 (2009) 360-365

Methodology used with Caco-2 cells‣ Determination of intracellular and basal PTXby LC-MS/MS‣ Nile red encapsulation for intracellularvisualisation by confocal microscopy &quantification by FACS‣ Transport experiments at 4°C or 37°C‣ TEER determination for tight junction openingtesting‣ Inhibitions of different receptor-mediatedendocytosis pathwayspswc2010

Uptake and permeability studies1- Nile red LNCMerged2- PTX-loaded LNCE.Roger et al., J Contr Rel, 140 (2009) 174-181pswc2010

Mechanistic studypswc2010 E.Roger et al. Nanomedicine 5(2) (2010) 287-306

LNC uptake - Identification of receptor-mediatedendocytosis pathwaysMethylβCD/Lovastatin: ✖lipid raftsFilipin: ✖caveolae-mediat. endocytosisChlorpromazin: ✖clathrin-mediat.endocytosisFilipin has the most importantimpact on LNC uptake but theother inhibitors also decrease it.intracellular trafficking?pswc2010

PTX-LNCs : where are we ?‣ Enhancement of permeability and bioavailability bya factor 3 after nano-encapsulation of PTX‣ Passive diffusion and energy-dependent transport‣ Several endocytosis pathways‣ No transport in lymph or via the paracellular route‣ Interaction with P-gp‣ Intracellular processing remains undefinedpswc2010

Development strategy of an oralLNC formulation‣ Proof of concept with a PK study with PTX-LNC‣ Set-up of specific evaluation tools for absorptionmechanism study:‣ Single Pass Intestinal Perfusion‣ Stability in biomimetic media‣ Caco-2 cell monolayers‣ Encapsulation of other drug candidates : Ibuprofen,Sn38,…pswc2010

Sn38-LNC‣ Poorly soluble anticancer drug.‣ Active metabolite of camptothecin‣ Encapsulation in LNCs/presence of Transcutol®‣ Stability in gastro-intestinal media‣ Caco-2 experimentspswc2010

Stability of Sn38-loaded LNCsSGFpswc2010

Permeability across Caco-2 cellsFree Sn38Sn38 loaded LNCspswc2010

Cytotoxicity on HT-29 cellsCell survival curves were not different between Sn38 & Sn38LNCs, showing a total drug activity recovery after encapsulation.pswc2010

Nano-encapsulation of Sn 38‣ Sn 38-loaded lipid nanocapsules:‣ Stability in gastro-intestinal fluids‣ Enhancement of transepithelial transport‣ Similar cytotoxicity to free Sn 38‣ Ongoing PK studiespswc2010

Lipid nanocapsules : a goodformulation candidate for the oralroute‣ Adjustable size between 20 to 130 nm‣ Stability in simulated media‣ Permeability enhancement demonstrated fordifferent drugs‣ Absolute bioavailability enhancement‣ Proof of concept showed with PTXpswc2010

Perspectives‣ Assessment on cell monolayers producing mucus(HT29 MTX)‣ Surface modification (chitosan)OHOHOHONH 2OHOO HONH 2nO HOOHR 1ONHOHLNCØ 50 nm, ζ -3 mVR 1 =OLipochitosan37°C, 1h(C 18 )+++ +++ ++ ++++++Lipochitosan-modified LNCØ 55 nm, ζ +30 mVHirsjärvi et al. Eur. J. Pharm. Biopharm. (2010) 76, 200pswc2010

AcknowledgementsINSERM U646 – AngersF. Lagarce, E. Garcion,S. Peltier, E. Roger, N. Lautram,P. Legras…Financial support:Ligue contre le Cancer 49Ethypharm:P. Oury, C. Herry

LNC transport through the Caco-2 monolayer• TEMControlIn presence of LNCsApical sideBasolateral sidepswc2010Presence of nano-objects on the BL side when LNC are introduced onthe apical side

Interaction with PgP‣ Rather complex : Cross interaction‣ Observed in different passage of Caco-2 cells‣ Verified on glioma cell lines‣ LNCs inhibit PgP (role of solutol Hs15)‣ PgP has also a role in decreasing LNCs Uptakepswc2010