1. A SEMINAR ONTRANSPORT 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

3. Intestinal absorption

4. Intestinal absorption models

5. Caco-2 monolayer

6. Caco-2 cell culture

7. Characterization of cell culture

8. Permeability assay

9. Permeability assay validation

10. Biological and analytical consideration

12. 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.

15. B: carrier mediated absorption at apical and basolateral membranes;

16. C: active efflux transporter on apical membrane, acting during absorption;

17. D: active efflux transporter on apical membrane, offering an additional route for drug clearance from the circulation;

20. HT 29 Cells: Colon carcinoma, cultured with galactose, express mucus producing goblet cells differenciation

23. Four times higher in transepithelial resistance compared to HT 29-cell monolayer

25. Dose not produce the mucus and unstirred water Observed in the intestine

26. No P-450 drug metabolizing enzyme activity has been reported

27. Expensive method

28. Time consuming as 21 days required for full cell differentiation

29. The necessity of LC / MS or HPLC for quantitation

31. CaCO2 Culture media

32. Transport media

33. Characterization of cell monolayer Cellular Architecture (Microvilli, Junctional Complexes) Barrier Function ( Lucifer Yellow Permeability, TEER) Differentiation Markers (Alkaline Phosphate, Brush-Border Peptidases) Transporters (P-Glycoprotein, PepT1, Amino acid Transporter, glucose)

34. 1.CELLULAR ARCHITECTURE 3 Days 21 Days

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 For the post-experimental TEER measurement, the withdrawn volume in the apical compartment was replaced with transport medium before TEER was measured. 5.Caco-2 monolayer with TEER values exceeding 250 Ωcm2 were used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.

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.

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

39. Caco-2 Permeability Assay Procedure: 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. Transfer the filter plate to a 24-well transport analysis plate. 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). Fill the wells of the 24-well receiver plate with 600 µL buffer. 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.

40. Fill the filter wells (apical compartment) with 300 µL of buffer. Join the filter and receiver plates once all drugs and buffer have been added. Begin timing the experiment. 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.

45. Biological pharmaceutical and analytical consideration

46. ASSAY DEVELOPMENT

50. 4) To study transport mechanism fro many compounds

51. 5) In drug metabolism & toxicity effects.

56. Good correlationbetween PAMPA & caco-2 data for a compound indicates a predominance ofpassive diffusion in its permeation.

57. Lack of correlationindicates

58. absorptive (active, paracellular ,gradient effect for acids) or

62. MDCK cell lines originate from dogkidney.