caco2 final ppt
TRANSCRIPT
A SEMINAR ON
TRANSPORT ACROSS CACO-2 MONOLAYER
GUIDED BY: BY: PATEL AKSHAY
DR.MANISH PATEL M.PHARM- 1(ph’ceutics) (head of department) ROLL NO-05 Department of Pharmaceutics NOOTAN PHARMACY COLLEGE,VISNAGAR
content
• Introduction • Intestinal absorption• Intestinal absorption models• Caco-2 monolayer• Caco-2 cell culture• Characterization of cell culture• Permeability assay• Permeability assay validation• Biological and analytical consideration• References
INTRODUCTION PART
• What is Caco2 cell line? The Caco-2 cell line is a continuous line of
heterogeneous human epithelial colorectal adenocarcinoma cells in intenstine.
it is developed by the Sloan-Kettering Institute for Cancer Research
Caco2 cell line is differentiates spontanously into enterocyte cell line.
Intestinal Absorption: Biology
Enterocite biology:
Absorptive cells of intestine, function is terminal digestion and absorption of water and nutrients from the intestinal lumen.
Polarised monolayer :
-Apical: microvilli face the interior of gut and increase the surface available for absorption by>1000 fold
-Basal: faces away from gut in contact with extracellular matrix.
Intestinal transport mechanism: Major types
• Para cellular: For hydrophilic drugs with MW < 200
• Transcellular: For most lipophilic drugs. This route
involves either passive diffusion, carrier mediated or
receptor mediated endocytosis
• A: passive trans- and paracellular diffusion; • B: carrier mediated absorption at apical and basolateral
membranes; • C: active efflux transporter on apical membrane, acting during
absorption;• D: active efflux transporter on apical membrane, offering an
additional route for drug clearance from the circulation; • E: intracellular metabolising enzymes localized inside the
enterocytes, possibly combined with an active efflux transporter on apical and basolateral membranes.
Intestinal absorption models1 Animal studies (Rat) - Very low throughput
2 In situ intestinal models - Very low throughput, expensive
Human/rat primary intestinal cells -Short functional life, lose differentiation characteristics
3 Intestinal Epithelial Barrier Models
MDCK cell line: Madin-Darby Canine kidney cell line, varied transporter expression , in vitro model for BBB
HT 29 Cells: Colon carcinoma, cultured with galactose, express mucus producing goblet cells differenciation
Caco-2 Monolayer: Human colorectal adenocarcinoma Cell monolayer
Caco-2 cell culture
• The Caco-2 cell line is an immortalized line of heterogeneous human epithelial colorectal adenocarcinoma cells, developed by the Sloan-Kettering Institute for Cancer Research.
• Caco-2 cell monolayers spontaneously differentiate to express morphological and functional characteristics of mature small-intestinal enterocytes. The differentiated monolayers are polarized, with microvilli on the apical side.
Advantage of Caco-2 monolayer
• Spontaneously differentiate to express morphological (polarized columnar cells) and functional characteristics of mature small-intestinal enterocytes
• Four times higher in transepithelial resistance compared to HT 29-cell monolayer
• It expresses various drug metabolizing enzymes like, aminopeptidase, esterase, and sulfatase.
Limitation of CaCO-2 monolayer
• Tissue in the villus contains more than one cell type• Dose not produce the mucus and unstirred water Observed in
the intestine• No P-450 drug metabolizing enzyme activity has been
reported• Expensive method• Time consuming as 21 days required for full cell
differentiation• The necessity of LC / MS or HPLC for quantitation• Influence of P-gp is difficult to estimate
Characteristics of parenteral Caco-2 cells
Origin Human colorectal adenocarcinoma
Growth in culture Monolayer epithelial cells
Differentiation 21 days after confluence in standard culture medium
Morphology Polarised cells, with tight junctions, apical, brush border
Electrical parameters High electrical resistance
Digestive enzymes Typical membranous peptidases and disaccharidases of the small intestine
Active transport Amino acids, sugars, vitamins, hormones
Membrane ionic transport
Na+/K+ ATPase, H+/K+ ATPase, Na+/H+ exchange, apical Cl- channels
Membrane non-ionic transporters
Permeability-glycoprotein, multidrug resistant associated protein, lung cancer associated resistance protein
Receptors Vitamin B12, vitamin D3, epidermal growth factor, sugar transporters (GLUT1, GLUT3, GLUT5, GLUT2, SGLT1)
Ingredients Quantity
Dulbecco's modified Eagle medium (DMEM) (with l-glutamine,4500mg/l D-glucose, without sodium pyruvate)
10% HIFBS (Heat Inactivated Fetal Bovine Serum, inactivation at +56 °C for 30 min with NaHCO3)
10%(V/V)
Non essential amino acids(NEAA) 1% (V/V)
L-glutamine 0.876g/l
penicillin 100 mg/ml
Streptomycin 100 mg/ml
CaCO2 Culture media
Transport media
Ingredients Quantity
Dulbecco’s modified Eagle’s medium (DMEM) base (without glucose, lglutamine, phenol red, sodium pyruvate and sodium bicarbonate)
HEPES (4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid) 4.76 g/l
NaCl 1.987 g/l
D-glucose, without sodium pyruvate 4500mg/l
l-glutamine 0.876g/l
Characterization of cell monolayer
1. Cellular Architecture (Microvilli, Junctional Complexes)
2. Barrier Function ( Lucifer Yellow Permeability, TEER)
3. Differentiation Markers (Alkaline Phosphate, Brush-Border
Peptidases)
4. Transporters (P-Glycoprotein, PepT1, Amino acid
Transporter, glucose)
1.CELLULAR ARCHITECTURE
3 Days 21 Days
2.BARRIER FUNCTION
• A. Trans Epithelial Electrical Resistance (TEER) measurements:
1. Measurement of the integrity of the caco-2 cell monolayer
2. After washing the cell monolayer with 37°C tempered D-PBS (with Ca2+, Mg2+) (Dulbecco's Phosphate Buffered Saline), 1600 μl transport medium was added into the apical and 2800 μl transport medium was added into the basal compartment.
3. Allow for equilibrium for 60 min in the cell culture incubator.
4. The measurement chamber was tempered to 37°C with transport medium before the measurement. For the post-experimental TEER measurement, the withdrawn volume in the apical compartment was replaced with transport medium before TEER was measured.
5.Caco-2 monolayer with TEER values exceeding 250
Ωcm2 were used for transport experiments. 6. Background TEER may be recorded in wells without
cell monolayers, and can be subtracted from the raw TEER values with cells.
B. Lucifer yellow (LY) rejection:
Rinse the monolayer three times with 300 µL HBSS ( Hank’s buffer saline solution) in the apical wells and 28
mL in the feeder tray.
Add 300 µL of LY solution to each well in the filter plate (Apical Template).
Add 600 µL HBSS to each well of a 24-well receiver tray (Basolateral Template).
Assemble the filter plate and 24-well receiver plate and incubate for 1–2 hours at 37°C.
Remove the filter plate from the receiver plate and place the receiver plate into a fluorescent plate reader. Determine the LY fluorescence using an excitation wavelength of 425-430 nm and an emission wavelength of 515-520 nm, 540 nm.
Calculate the percent of LY rejection across the cell monolayer
by measuring fluorescence in the receiver plate as compared to
an ‘equilibrium’ standard.
The standard plate should consist of 4 wells with 600 µL HBSS
(blank) and 4 wells with 200 µL LY (100 µg/mL) + 400 µL
HBSS (equilibrium samples)
Calculate the LY rejection using the following equation:
LY Rejection=100%-%LY Passage
Caco-2 Permeability AssayProcedure:1. After the desired cell growth period, remove the plate from the
incubator and determine the electrical resistance for each well (as described above). Wash the monolayer, exchanging the volume three times using sterile HBSS, pH 7.4. After washing, remove the buffer from the filter plate and feeder tray.
2. Transfer the filter plate to a 24-well transport analysis plate. 3. To determine the rate of drug transport in the apical to basolateral
direction, add 300 µL of the test compounds to the filter well. Drug concentrations typically ranging from 10 µm to 200 µm may be used (achieve desired concentration using HBSS, pH 7.4 or an alternative buffer of desired pH).
4. Fill the wells of the 24-well receiver plate with 600 µL buffer. 5. To determine the rate of drug transport in the basolateral to apical
direction, add 600 µL of the test compounds to the 24-well receiver plate.
6. Fill the filter wells (apical compartment) with 300 µL of buffer.7. Join the filter and receiver plates once all drugs and buffer have
been added. Begin timing the experiment. 8. Incubate at 37°C shaking at 60 rpm on a rotary shaker. Typical
incubation times are 1 to 2 hours.
For LC/MS analysis: At the end of the incubation, remove a fixed volume (typically 50–100 µL) directly from the apical and basolateral wells (using the basolateral access holes) or by disassembling the plates. Transfer the volume to a clean plate.
CALCULATION OF APPEARENT PERMEABILITY:
• Papp=Area * time *
[Drug]acceptor
[Drug]initial,donor
Where, VA= volume in ml in the acceptor well area =the surface area of the membrane time= total transport time in seconds
VA
Caco-2 assay permeability validation
• According to FDA guidelines, validation of Caco-2 permeation assay was performed using reference drugs known as low, moderate or high absorbers.
• 10 Reference Chemicals
HIGH ABSORPTION INTERMEDIATE ABSORPTION LOW ABSORPTIONIsopropanol (reference) Cimetidine (reference) Atenolol (reference)Ethylene glycol (chemical)
Paraquat (pesticide) Cupric sulphate (chemical)
Sodium Valproate (drug)
Paracetamol (drug) Colchicine (drug)
Acetylsalicylic acid (drug)
Applications Of Caco-2 Cell Model To Drug Absorption And Transport Studies
Application ExampleExamine factors affecting the transport of drugs
Investigation of pH,glucose, temperature, inhibitors on the tranport of a-methyldopaExamine the effect of mannitol concenttration on transportInvestigate the effect of temperature on various transport mechanismExamine the influence of peptide structure on tranport across the epitelial cell; explore the relationship of permeability coefficient across the Caco-2 monolayer and the number of potential hydrogrn-bonding sites in the solute
study drug transport mediated by various carriers
Characterize vitamin D receptor
Identify bile acid carrier
Investigate the tranporter mediated mechanism for glucose
Examine the role of gamma glutamine transpepitidase in amino acid transport
Explore the carrier mediated transport for large neutral amino acids like phenylalanine and proline
Identify a dipeptide pore tarnsport system involved in the efflux of cephalosporin across the basolateral membrane
Investigate metabolism reactions
Phase II metabolism :Sulphation of para nitrophenol and dopamine,Glucuronidation of para nitrophenol
Investigate vitamin A metabolism
Study the metabolism of oxygenated derivatives of arachidonic acid
Investigate the activity of acyl coenzyme A cholesterole activity in the presence of PD 128042
Investigate fatty acid uptake and metabolism
Biological pharmaceutical and analytical consideration
ASSAY DEVELOPMENT
APPLICATION OF Caco-2 MODEL
• 1) In Drug Discovery: To test the absorption profiles of the new molecular entities in the lead optimization state.
• 2) In pre- clinical drug development: US FDA recognizes Caco-2 to measure permeability as part of the bioequivalence
waiver process.
• 3) To evaluate effect of pharmaceutical excipients.
• 4) To study transport mechanism fro many compounds
• 5) In drug metabolism & toxicity effects.
• 6) Others like study of CFTR; regulation of protein expression; genetics study.
CACO-2: PHARMACEUTICAL CONSIDERATIONS
Biopharmaceutical classification system
In vitro/in vivo correlation
Other model co-related with caco2
PAMPA VERSUS Caco-2 MODEL
• PAMPA & caco-2 should not be considered as competing permeability methods.
• Good correlation between PAMPA & caco-2 data for a compound indicates a predominance of passive diffusion in its permeation.
• Lack of correlation indicates • absorptive (active, paracellular ,gradient effect for
acids) or• ecretarys (efflux, gradient effect for bases)
permeation mechanism
PAMPA MODEL CACO-2 CELL MODEL
no cell culture involved, so not required long planning
based on cell culture, so required planning
High throughput & low cost For HT, 96- well plats & compatible detection system
Useful only for passive transcellular permeability
For paracellular & transcellular permeability
Useful tool in early drug discovery to assess the permeability potential of large no. of compounds.
Method is more suitable during lead optimization or preclinical development stages, where true transepithelial permeability is needed.
The Madin-Darby canine kidney (MDCK) cell model
• one of the commonly used cell monolayer systems to assess the human intestine barrier.
• MDCK cell lines can reach full differentiation in 3-7days and are therefore relatively easy for cell
culturing and assay maintenance. • DISADVANTAGE• MDCK cell lines originate from dog kidney. • The expression of transporters is quite different
from human intestine.
BACK UP J Pharm Sci 92:1545-1558, 2003
J Pharm Sci 93:1440-1453, 2004J Pharm Sci 90:1776-1786, 2001J Pharm Sci 90:1593-1598, 2001
David Werner Blaser, Determination of drug absorption parameters in Caco-2 cell monolayers with a mathematical model encompassing passive diffusion, carrier mediated efflux, non-specific binding and phase II metabolism, Ph.D Thesis, University of Basel, 2007.
Marcel Schneider, Investigation of the Transport of Lipophilic Drugs in Structurally Diverse Lipid Formulations through Caco-2 Cell Monolayer Using Mathematical Modeling, Ph.D Thesis, University of Basel, 2008.
THANKS TO ALL