caco-2 cell permeability assay for intestinal absorption .pptx

The Caco-2 cell permeability assay is a widely used in vitro model for evaluating the intestinal absorption and permeability of drug candidates during the early stages of Pharmaceutical development. It is a Valuable tool for studying transport and absorption.
This presentation focused on the CaCO-2 cell line permeability assay protocol, conditions along with the factors affecting, advantages, limitations, and considerations during the protocol with a wide range of application and future aspects.

1. Department of Pharmacology and Toxicology Caco-2 cell permeability assay & COMET assay Presented by Dheeraj M.S. Pharm./2023-25/PT/14

2. Table of Contents 3 5 6 7 8 9 10 Introduction & Importance Principle Experimental setup and cell culture conditions Procedure for measurement of permeability Data analysis and interpretation Factor affecting Applications Introduction & History Types of COMET assay Procedure Staining and visualization Parameters Advantages, Disadvantages & considerations Applications 13 14 15 16 17 18 19 Caco-2 cell permeability assay COMET Assay

3. Caco-2 Cell Permeability Assay: Introduction The Caco-2 cell permeability assay is a widely used in vitro model for evaluating the intestinal absorption and permeability of drug candidates during the early stages of Pharmaceutical development. Isolated in 1974 and Used for the first time in 1980. Caco-2 cells are human epithelial colorectal adenocarcinoma cells that, when cultured under appropriate conditions, differentiate into a monolayer resembling the intestinal epithelium. Valuable tool for studying the transport and absorption. Important insights into the potential oral bioavailability of drug candidates. Helps inform medicinal chemistry efforts and guide the selection of promising drug leads for further development. Da Cancerous cells

4. Importance of Caco-2 cells in drug development Studying intestinal drug transport mechanisms Predict the oral bioavailability of compounds Evaluating the absorption and permeability of drug candidates in drug discovery Crucial decisions drug development pipeline, a higher rate of success in clinical trials Mechanisms underlying drug absorption, such as passive diffusion etc Optimized physicochemical properties and improved bioavailability Evaluate the potential interactions

5. Principles of Caco-2 Cell Permeability Assay Cultivation of Caco-2 cells differentiates and develops tight junctions, and apical microvilli, and expresses a variety of transporters and enzymes found in the human intestine which mimic the human intestinal epithelium. The key principles underlying the Caco-2 cell permeability assay include: Permeability measurement. Directionality Barrier integrity Correlation with in vivo absorption Mechanistic insights

6. Experimental Setup and Cell Culture Conditions 1 Cell Seeding and Differentiation The Caco-2 cell permeability assay begins by seeding the Caco-2 cells onto specialized permeable membrane supports, such as Transwell plates. The cells are allowed to grow and differentiate over the course of 21-28 days, during which time they form a polarized monolayer that closely resembles the intestinal epithelium. Crucial step for the formation of tight junctions, microvilli, and others. 2 Culture Media and Conditions Caco-2 cells are typically cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum, non-essential amino acids, and antibiotics. The cells are maintained in a humidified incubator at 37°C with 5% CO 2 to mimic physiological conditions. The culture medium is replaced every 2-3 days to provide the cells with fresh nutrients and maintain optimal growth. 3 Transepithelial Electrical Resistance (TEER) To ensure the integrity of the Caco-2 cell monolayer, the transepithelial electrical resistance (TEER) is regularly measured using specialized equipment. TEER provides a quantitative assessment of the tightness of the cell-cell junctions and the overall barrier function of the monolayer. Only Caco-2 cell cultures with a TEER value above a predetermined threshold are used for permeability experiments, ensuring the reliability of the data.

7. Procedure for measuring the Permeability of test compound Cell culture Compound preparation Apical and basolateral compartment setup Replace the media after forming the monolayer, and add the solution HBSS prepared. The apical compartment simulates the luminal side (intestinal lumen), while the basolateral compartment simulates the blood side. Incubation period Sample collection Calculation of permeability Data analysis Incubate the cells at 37°C for a specified period (commonly 1-2 hours) to allow compound permeation across the monolayer. Collection of samples from the apical and basolateral compartments and analysis of the samples using appropriate methods (e.g., HPLC, LC-MS) to quantify the amount of compound that has crossed the monolayer. Calculate the apparent permeability coefficient (Papp) using the formula: Papp = Vr *dc/dt*1/ ACo Compare the test and control compound permeability and interpret the data using the compound's potential for intestinal absorption. Growing on the semipermeable polycarbonate surface, subculturing, mono layer formation of Caco2 cells Weig h the test and control compound, and mix in the appropriate solution like ethanol, DMSO, etc.

8. Data Analysis and Interpretation Permeability Parameters Key permeability parameters measured are the apparent permeability coefficient (Papp), the efflux ratio, and the fractional absorption. Higher Papp generally correlates with better intestinal absorption. The efflux ratio helps identify whether the compound is a substrate for efflux transporters, which could limit its absorption, while the fractional absorption estimation offers a more direct prediction of in vivo absorption. Data Interpretation 10 -6 <P app good permeability and absorption potential 10 -7 >P app poor permeability and absorption potential An efflux ratio greater than 2 suggests the involvement of efflux transporters, which may need to be addressed through medicinal chemistry efforts or the use of efflux inhibitors. The fractional absorption estimate can be used to further refine the assessment of a compound's oral bioavailability potential. Comparison to In Vivo Data Studies reported a strong positive correlation between Caco-2 permeability parameters and the fraction of the dose absorbed in humans. This correlation allows researchers to use the Caco-2 assay as a predictive tool for estimating the oral bioavailability of drug candidates, which is crucial for selecting the most promising compounds to advance in the drug development pipeline. Limitations and Considerations Factors such as cell culture conditions, passage number, and the specific experimental setup can impact the assay results. Additionally, the Caco-2 model may not fully capture all the complexities of the in vivo intestinal environment, such as the presence of mucus, gut microbiota, and other physiological factors. Careful interpretation of the data, in the context of these limitations, is essential for making informed decisions about the absorption potential of drug candidates.

9. Factors Affecting Caco-2 Cell Permeability Cell Culture Conditions The permeability of Caco-2 cells can be significantly influenced by the culture conditions, such as The growth medium Seeding density Duration of differentiation. Factors like the pH, osmolarity, and the presence of specific nutrients or supplements in the culture medium can also impact the expression of transporters and the overall barrier properties of the caco-2 cells. Tight Junction Formation Determines the permeability of the monolayer. Regulate the paracellular transport of compounds and maintain the polarized nature of the epithelial layer. Factors that disrupt the tight junction integrity, such as the presence of surfactants or certain pharmaceuticals, can alter the permeability of the Caco-2 cell monolayer and lead to inaccurate predictions of drug absorption. Transporter Expression Transporters, including efflux pumps like P-glycoprotein (P- gp ) and influx transporters, which significantly impact the permeability of test compounds. Transporters can be influenced by factors such as cell passage number, culture conditions, and the presence of specific inducers or inhibitors. Transporter profile used in the permeability assay is crucial for interpreting the results and identifying potential issues with drug-transporter interactions. Compound Properties The physicochemical properties of the test compound, influence its permeability across the Caco-2 cell monolayer. Highly lipophilic compounds may exhibit greater passive diffusion, while charged molecules or those that are substrates for active transporters may show more complex transport behavior. Understanding the characteristics of the test compound is essential for designing the Caco-2 cell permeability assay and correctly interpreting the results.

10. Prediction of Oral Bioavailability The strong correlation between Caco-2 permeability parameters and in vivo intestinal absorption data allows researchers to use the assay as a reliable tool for estimating the fraction of a dose that will be absorbed in humans. Formulation Development Using C aco-2 cells, researchers can evaluate the impact of different excipients, delivery systems, and manufacturing processes on the compound's absorption potential. This information is used for designing effective oral dosage forms Drug discovery and development Evaluating the absorption potential of drug candidates, the assay helps researchers identify promising compounds with favorable pharmacokinetic profiles and a higher likelihood of success in clinical trials. Investigation of Drug-Drug Interactions By measuring the bidirectional transport of a drug candidate in the presence of known inhibitors or substrates of these transporters, researchers can identify potential issues with co-administration of the drug and develop strategies to mitigate the risk of reduced oral bioavailability or altered pharmacokinetics. Applications

11. References Van Breemen , Richard B., and Yongmei Li. "Caco-2 cell permeability assays to measure drug absorption."  Expert opinion on drug metabolism & toxicology  1.2 (2005): 175-185. Uchida, Masashi, et al. "A modified fast (4 day) 96-well plate Caco-2 permeability assay."  Journal of pharmacological and toxicological methods  59.1 (2009): 39-43. Hubatsch , Ina, Eva GE Ragnarsson , and Per Artursson . "Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers."  Nature Protocols  2.9 (2007): 2111-2119. Press, Barry, and Deanna Di Grandi . "Permeability for intestinal absorption: Caco-2 assay and related issues."  Current drug metabolism  9.9 (2008): 893-900. Sun, Huadong, et al. "The Caco-2 cell monolayer: usefulness and limitations."  Expert opinion on drug metabolism & toxicology  4.4 (2008): 395-411. Fredlund , Linda, Susanne Winiwarter , and Constanze Hilgendorf . "In vitro intrinsic permeability: a transporter-independent measure of Caco-2 cell permeability in drug design and development."  Molecular pharmaceutics  14.5 (2017): 1601-1609.