Caco2 final ppt.


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Caco2 final ppt.

  1. 1. A SEMINAR ONTRANSPORT ACROSS CACO-2 MONOLAYER<br />GUIDED BY:BY: PATEL AKSHAY<br />DR.MANISH PATEL M.PHARM- 1(ph’ceutics)<br /> (head of department) ROLL NO-05<br /> Department of Pharmaceutics<br />NOOTAN PHARMACY COLLEGE,VISNAGAR<br />
  2. 2. content<br /><ul><li>Introduction
  3. 3. Intestinal absorption
  4. 4. Intestinal absorption models
  5. 5. Caco-2 monolayer
  6. 6. Caco-2 cell culture
  7. 7. Characterization of cell culture
  8. 8. Permeability assay
  9. 9. Permeability assay validation
  10. 10. Biological and analytical consideration
  11. 11. References</li></li></ul><li>INTRODUCTION PART<br />What is Caco2 cell line?<br />The Caco-2 cell line is a continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells in intenstine.<br /> it is developed by the Sloan-Kettering Institute for Cancer Research<br /> Caco2 cell line is differentiates spontanously into enterocyte cell line. <br />
  12. 12. Intestinal Absorption: Biology<br />Enterocite biology:<br />Absorptive cells of intestine, function is terminal digestion and absorption of water and nutrients from the intestinal lumen.<br />Polarised monolayer :<br /> -Apical: microvilli face the interior of gut and increase the surface available for absorption by>1000 fold<br /> -Basal: faces away from gut in contact with extracellular matrix.<br />
  13. 13. Intestinal transport mechanism: Major types<br /><ul><li>Para cellular: For hydrophilic drugs with MW < 200
  14. 14. Transcellular: For most lipophilic drugs. This route involves either passive diffusion, carrier mediated or receptor mediated endocytosis</li></li></ul><li><ul><li>A: passive trans- and paracellular diffusion;
  15. 15. B: carrier mediated absorption at apical and basolateral membranes;
  16. 16. C: active efflux transporter on apical membrane, acting during absorption;
  17. 17. D: active efflux transporter on apical membrane, offering an additional route for drug clearance from the circulation;
  18. 18. E: intracellular metabolising enzymes localized inside the enterocytes, possibly combined with an active efflux transporter on apical and basolateral membranes. </li></li></ul><li>
  19. 19. Intestinal absorption models<br />1Animal studies (Rat) - Very low throughput<br />2 Insitu intestinal models - Very low throughput, expensive <br /> Human/rat primary intestinal cells -Short functional life, lose differentiation characteristics<br />3 Intestinal Epithelial Barrier Models<br /><ul><li>MDCK cell line: Madin-Darby Canine kidney cell line, varied transporter expression , in vitro model for BBB
  20. 20. HT 29 Cells: Colon carcinoma, cultured with galactose, express mucus producing goblet cells differenciation
  21. 21. Caco-2 Monolayer: Human colorectal adenocarcinoma Cell monolayer</li></li></ul><li>Caco-2 cell culture<br /><ul><li>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.
  22. 22. 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.</li></li></ul><li>Advantage of Caco-2 monolayer<br /><ul><li>Spontaneously differentiate to express morphological (polarized columnar cells) and functional characteristics of mature small-intestinal enterocytes
  23. 23. Four times higher in transepithelial resistance compared to HT 29-cell monolayer
  24. 24. It expresses various drug metabolizing enzymes like, aminopeptidase, esterase, and sulfatase.</li></li></ul><li>Limitation of CaCO-2 monolayer<br /><ul><li>Tissue in the villus contains more than one cell type
  25. 25. Dose not produce the mucus and unstirred water Observed in the intestine
  26. 26. No P-450 drug metabolizing enzyme activity has been reported
  27. 27. Expensive method
  28. 28. Time consuming as 21 days required for full cell differentiation
  29. 29. The necessity of LC / MS or HPLC for quantitation
  30. 30. Influence of P-gp is difficult to estimate</li></li></ul><li>Characteristics of parenteral Caco-2 cells<br />
  31. 31. CaCO2 Culture media<br />
  32. 32. Transport media<br />
  33. 33. Characterization of cell monolayer<br />Cellular Architecture (Microvilli, Junctional Complexes)<br />Barrier Function ( Lucifer Yellow Permeability, TEER)<br />Differentiation Markers (Alkaline Phosphate, Brush-Border Peptidases)<br />Transporters (P-Glycoprotein, PepT1, Amino acid Transporter, glucose)<br />
  34. 34. 1.CELLULAR ARCHITECTURE<br />3 Days<br />21 Days<br />
  35. 35. 2.BARRIER FUNCTION<br /><ul><li>A. Trans Epithelial Electrical Resistance (TEER) measurements:</li></ul>Measurement of the integrity of the caco-2 cell monolayer<br />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. <br />Allow for equilibrium for 60 min in the cell culture incubator.<br />
  36. 36. 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.<br />5.Caco-2 monolayer with TEER values exceeding 250 Ωcm2 were used for transport experiments.<br />6. Background TEER may be recorded in wells without cell monolayers, and can be subtracted from the raw TEER values with cells.<br />
  37. 37. 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. <br />
  38. 38. 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<br />
  39. 39. Caco-2 Permeability Assay<br />Procedure:<br />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. <br />Transfer the filter plate to a 24-well transport analysis plate. <br />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). <br />Fill the wells of the 24-well receiver plate with 600 µL buffer. <br />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. <br />
  40. 40. Fill the filter wells (apical compartment) with 300 µL of buffer.<br />Join the filter and receiver plates once all drugs and buffer have been added. Begin timing the experiment. <br />Incubate at 37°C shaking at 60 rpm on a rotary shaker. Typical incubation times are 1 to 2 hours. <br />For LC/MS analysis:<br /> 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. <br />
  41. 41. CALCULATION OF APPEARENT PERMEABILITY:<br /><ul><li>Papp=</li></ul>[Drug]acceptor<br />VA<br />*<br />Area * time<br />[Drug]initial,donor<br />Where, <br />VA= volume in ml in the acceptor well<br />area =the surface area of the membrane<br />time= total transport time in seconds<br />
  42. 42. Caco-2 assay permeability validation<br /><ul><li>According to FDA guidelines, validation of Caco-2 permeation assay was performed using reference drugs known as low, moderate or high absorbers.
  43. 43. 10 Reference Chemicals</li></li></ul><li>Applications Of Caco-2 Cell Model To Drug Absorption And Transport Studies<br />
  44. 44.
  45. 45. Biological pharmaceutical and analytical consideration<br />
  46. 46. ASSAY DEVELOPMENT<br />
  47. 47.
  48. 48. APPLICATION OF Caco-2 MODEL<br /><ul><li>1) In Drug Discovery: To test the absorption profiles of the new molecular entities in the lead optimization state.
  49. 49. 2) In pre- clinical drug development:</li></ul> US FDA recognizes Caco-2 to measure permeability as part of the bioequivalence waiver process.<br /><ul><li>3) To evaluate effect of pharmaceutical excipients.
  50. 50. 4) To study transport mechanism fro many compounds
  51. 51. 5) In drug metabolism & toxicity effects.
  52. 52. 6) Others like study of CFTR; regulation of protein expression; genetics study.</li></li></ul><li>CACO-2: PHARMACEUTICAL CONSIDERATIONS<br />Biopharmaceutical <br />classification system<br />In vitro/in vivo correlation<br />
  53. 53. <ul><li> Other model co-related</li></ul> with caco2<br />
  54. 54.
  55. 55. PAMPA VERSUS Caco-2 MODEL<br /><ul><li>PAMPA & caco-2 should not be considered as competing permeability methods.
  56. 56. Good correlationbetween PAMPA & caco-2 data for a compound indicates a predominance ofpassive diffusion in its permeation.
  57. 57. Lack of correlationindicates
  58. 58. absorptive (active, paracellular ,gradient effect for acids) or
  59. 59. ecretarys(efflux, gradient effect for bases) permeation mechanism</li></li></ul><li>
  60. 60. The Madin-Darby canine kidney (MDCK) cell model<br /><ul><li>one of the commonly used cell monolayer systems to assess the human intestine barrier.
  61. 61. MDCK cell lines can reach full differentiation in</li></ul> 3-7days and are therefore relatively easy for cell culturing and assay maintenance. <br /><ul><li>DISADVANTAGE
  62. 62. MDCK cell lines originate from dogkidney.
  63. 63. The expression of transporters is quite different from human intestine. </li></li></ul><li>BACK UP<br />J PharmSci 92:1545-1558, 2003<br /> J PharmSci 93:1440-1453, 2004<br /> J PharmSci 90:1776-1786, 2001<br /> J PharmSci 90:1593-1598, 2001 <br /><ul><li>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.
  64. 64. 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.</li></li></ul><li>THANKS TO ALL<br />