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