MÉTODOS IN VITRO DE DETERMINACIÓN DE LA PERMEABILIDAD. CULTIVOS CELULARES Y PAMPA

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1 MÉTODOS IN VITRO DE DETERMINACIÓN DE LA PERMEABILIDAD. CULTIVOS CELULARES Y PAMPA Virginia Merino (Virginia.Merino@uv.es) Departamento de Farmacia y Tecnología Farmacéutica. Universitat de València. España Red-Biofarma. Red para el desarrollo de metodologías biofarmacéuticas racionales que incrementen la competencia y el impacto social de las Industrias Farmacéuticas Locales (DCI-ALA/ /10/21526/ /ALFA 111(2010)29) 1

2 PAMPA: Parallel Artificial Membrane Permeability Assay Caco-2 cell lines 2

3 Ar9ficial Membranes to simulate biological ones: Gastrointes9nal Skin BBB PAMPA: Parallel Artificial Membrane Permeability Assay Useful to predict passive transcellular transport through membranes Rapid- 96 well plate automated system Depending on the lipids selected Cheaper and easier to standardize than Caco- 2 cells 3

transcellular transport through membranes Rapid- 96 well plate automated system

4 log D vs PAMPA Par99on coefficient Equilibrium D = C C s o PAMPA Transport ΔQ P e = Δ S C t Aqueous Donor Organic solvent Donor Lipid mixture Acceptor 4

5 GENERAL PAMPA ASSAY CONDITIONS From Faller B, Current Drug Metab, 2008, 9: IMPORTANT PARAMETERS: Lipid composition Filter material Donor and acceptor composition: ph and solubility Effect of UWL: agitation rate 5

Lipid composition Filter material Donor and acceptor

6 LIPID COMPOSITION Intes9nal membrane of rodents: Phospha9dylcholine and Phospha9dylethanolamine: 60-70% Fluidity controlled by the degree of unsatura9on of acyl chains and % of cholesterol From Faller B, Current Drug Metab, 2008, 9:

Fluidity controlled by the degree of unsatura9on of acyl

7 PAMPA and ph From Kerns EH et al, J Pharm Sci, 2004, 93 (6):

8 Effect of UWL Donor ABL Lipid Barrier Acceptor 1 P e = P 1 UWL + P 1 LipidBarrier DEPENDS ON THE IONISED FRACTION 8

9 Taller de Capacitación en Metodologías Biofarmacéu9cas Permeabilidad in vitro: PAMPA, Caco- 2 Effect of UWL Corrected UWL Thickness UWL rat~ 100µm UWL Caco- 2~ 800 µm From Bermejo et al. Eur. J. Pharm. Sci, 2004, 21(4): Departamento Farmacia y Tecnología Farmacéu@ca V Merino 9

100µm UWL Caco- 2~ 800 µm From Bermejo et al. Eur. J. Pharm.

10 PAMPA vs Intestinal Permeability Depends on different factors: Membrane composi9on selected: i.e. 1% lecithin PAMPA predicts beler than 10% lecithin PAMPA (higher membrane reten9on) ABL thickness: i.e. If data are not corrected for UWL the permeability of high lipophilic compounds is underes9mated Mechanism of absorp9on 10

ected: i.e. 1% lecithin PAMPA predicts beler than 10% lecithin PAMPA (higher

11 PAMPA vs Intestinal Permeability From Faller B, Current Drug Metab, 2008, 9:

12 IN VITRO ALTERNATIVES FOR ASSESING PERMEABILITY UPTAKE MODELS: BBMV Rings Isolated cells Cultured cells CELL CULTURES TRANSPORT MODELS: Everted sacs Side- to- side diffusion cells Cultured cells Inmortalizad cells derived from normal cells, induced tumors or from human colonic cancer Caco- 2 line (from human colonic cancer) are the most widely used. 12

cells Cultured cells Inmortalizad cells derived from normal cells, induced tumors or

13 MAIN CHARACTERISTICS OF CACO-2 Grow on a porous support Polarized and confluent monolayers, Brush border membrane With 9ght juc9ons Phase I and II metabolism enzimes Ac9ve transporters: entrace and efflux High TEER ( Ω*cm2) higher than SI (25-40 Ω*cm2) NOT EXACTLY AT THE SAME LEVEL THAN IN VIVO 13

metabolism enzimes Ac9ve transporters: entrace and efflux High TEER (150-450

14 HOW ARE CELLS USED? LINE MANTENANCE Culture media: DMEM Penicillin 10% FBS Streptomycin 1% NEAA Glutamine 37ºC, 5% de CO 2 y 95% RH Asepsia CONDITIONING FOR TRANSPORT EXPERIMENTS Transfer the number of cell required to a porous transwell Wait until confluence in said conditions 14

NEAA Glutamine 37ºC, 5% de CO 2 y 95% RH Asepsia CONDITIONING FOR

15 HOW ARE CELLS USED? TRANSPORT EXPERIMENTS Orbital shaker Donor: HBSS+ DRUG Receptor: HBSS ph SINK or NON SINK UWL thickness RESULTS ΔQ P t eff = Δ S C P ab Drug solu@on P ba 0,5 ml 0,5 ml 1,5 ml 1,5 ml 15

16 VALIDATION OF CELL LINE LUCIFER YELLOW Paracelular Permeability P ap < cm/s METOPROLOL Transcelular Permeability P ap > cm/s RODAMINE P-glicoprotein activity P ap < cm/s TEER Confluence TEER > 200 Ω cm 2 (100 more than insert without cells) 16

10-6 cm/s RODAMINE P-glicoprotein activity P ap < 20 10-6 cm/s

17 TEER Manitol Flux From Dresman JB and Lennernas H Ed, Oral Drug Absorp9on. Predic9on and assessment. Marcel Dekker Inc, New York,

18 CACO-2 vs INTESTINAL TISSUES Good relation with passive absorbed compounds Papp> cm/s: completely absorbed Papp< cm/s: incompletely absorbed HIGHER DIFFERENCES FOR DRUGS POORLY AND INCOMPLETELY ABSORBED 18

completely absorbed Papp<1 10-7 cm/s: incompletely

19 CACO-2 vs INTESTINAL TISSUES Lower permeability than in vivo Tight junc9ons Surface available LOW Perm HIGH Perm In vivo Caco- 2 19

20 CACO-2 vs INTESTINAL TISSUES For ac9vely transported substances depends on the transporter expression QUALITATIVE ALTHOUGH NOT ALWAYS P Artursson et al. Adv Drug Deliv Rev 46 (2001) :

21 VALIDATION OF CELL LINE It is difficult to compare lab- lab data Proper9es of the cell can vary between labs Proper9es of cells in one lab change with 9me: Passage number Time in culture Cell medium Filter support QUALITATIVE MORE THAN QUANTITATIVE P Artursson et al. Adv Drug Deliv Rev 46 (2001) :

22 PAMPA-Caco-2 Correlation Caco- 2 Papp (cm/s) Paracellular Ac9ve uptake Ac9ve efflux PAMPA Papp (cm/s) From Kerns EH et al, J Pharm Sci, 2004, 93 (6):

23 Passive diffusion Mechanistic information Confirmation of the mechanisms of absorption Frequency of use PAMPA Caco- 2 Animal Exploratory Discovery Pre-development Development 23

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