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See all Premium member Presentation Transcript Cell Culture Model for Drug Transport Studies : Cell Culture Model for Drug Transport Studies Wichan Ketjinda Department of Pharmaceutical Technology Pharmaceutical Technology Process of Oral Drug Absorption : Process of Oral Drug Absorption 8/29/2009 Factors affecting absorption : Factors affecting absorption - Solubility - Dissolution rate - Molecular size - Partition coeficient - Chemical degradation - Delivery system 8/29/2009 I. Physical Parameters Factors affecting absorption : Factors affecting absorption - Binding or complexation - Regional pH - Intestinal permeability - Metabolism (Lumen, Hepatic) - Gastric and intestinal transit 8/29/2009 II. Physiological parameters Drug transport pathway : Drug transport pathway 8/29/2009 Slide 6: 8/29/2009 Drug Transport pathway : Drug Transport pathway Transcellular pathway Passive transport Lipophilic compound High efficient route Paracellular pathway Aqueous channel Restricted by tighted junction ( pore ~ 8 Ao ) Low dose and appropriate size allowed passage 8/29/2009 Drug Transport pathway : Drug Transport pathway Carrier-mediated uptake Transporter located at apical and basolateral Apical = proton dependent peptide transporter, sodium dependent sugar and amino acid transporter Basolateral = transporter for organic anion and cation Carrier-mediated Efflux Apical = p-glycoprotien, basolateral = amino acid carrier P-glycoprotien + CP450 limit toxic agent access to circulation 8/29/2009 Slide 9: 8/29/2009 Methods to Assess Drug Absorption from GI Tract : Methods to Assess Drug Absorption from GI Tract I. In vivo animal method - Little predictable - Not suitable for large number of screening studies - Misleading by forcing a correlation for the all of characteristics to human 8/29/2009 Correlation of Oral Bioavailability of Various Compounds : Correlation of Oral Bioavailability of Various Compounds 8/29/2009 Comparison of Bioavailability of Ganciclovir : Comparison of Bioavailability of Ganciclovir 8/29/2009 Methods to Assess Drug Absorption from GI Tract : Methods to Assess Drug Absorption from GI Tract II.In situ animal studies - Advantage of isolating the comparison to the level of the intestine - Measure the rate or extent of uptake drug by tissue (disappear from lumen> drug transport) - Intestinal perfusion method 8/29/2009 Intestinal perfusion model : Intestinal perfusion model 8/29/2009 Intestinal perfusion model : Intestinal perfusion model 8/29/2009 Methods to Assess Drug Absorption from GI Tract : Methods to Assess Drug Absorption from GI Tract III. In Vitro nonbiological Methods Octanol/water distribution - use partition coefficient (log P, log D) to predict drug absorption - determination of a drug extracted into lipid phase of an octanol/water (Lipophilicity) - -3 < log P < 3 good absorption 8/29/2009 Slide 17: 8/29/2009 Methods to Assess Drug Absorption from GI Tract : Methods to Assess Drug Absorption from GI Tract Artificial Membrane (IAM, ILC) - Immobilized artificial membrane (IAM), use chromatography column modified by covalent attachment of phospholipid-like groups to the surface - Immobilized liposome chromatography (ILC),where phospholipids (in the form of multilamella vesicles) are entrapped in the pores of resin beads 8/29/2009 Methods to Assess Drug Absorption from GI Tract : Methods to Assess Drug Absorption from GI Tract Parallel artificial membrane permeation assay(PAMPA), - measured through membrane formed by a mixture of lecithin and inert organic solvent on hydrophobic filter - predict ability of molecule to permeate by passive diffusion 8/29/2009 Methods to Assess Drug Absorption from GI Tract : IV. In Vitro Biological Methods - Brush-Border Membrane Vesicles (BBMV) - Intestinal Rings (Slices) - Everted Intestinal Sac Method -The Ussing Chamber for Excised Intestinal Segments - Cell Culture 8/29/2009 Methods to Assess Drug Absorption from GI Tract Brush-Border Membrane Vesicle : Brush-Border Membrane Vesicle Luminal wall-bound protein and phospholipid from treated cell homogenate are resuspended in buffer Vesicle Vesicle mixed with permeant in buffer and filtered The amount of permeant uptaken by vesicles is determined Only the apical transcellular transport is measured because the extraction method isolates only brush-border component 8/29/2009 BACK Intestinal Rings : Intestinal Rings A section of intestine is isolated immediately, washed to remove debris and enzyme, everted and sliced into rings The segment can be selected from duodenum to rectum Slices are incubated in solution containing the compound After designated time the slices are taken out of the solution for assay process 8/29/2009 everted intestinal rings in solution : everted intestinal rings in solution 8/29/2009 BACK Everted Intestinal Sacs : Everted Intestinal Sacs A section of intestine is derived to prepare the everted sac as described in intestinal ring but without cutting . The everted sac is filled with buffer and put in a flask with buffer containing compound. At the end, the sac is cut and opened at one end, and the serosal fluid is collected to measure the transport. 8/29/2009 BACK Ussing Chamber for Excised Intestinal Segment : Ussing Chamber for Excised Intestinal Segment Side by side diffusion cell Ussing chamber The small sections of intestine are clamped between two glass chamber filled with buffer and nutrition and are gased continuously to maintain the viability of the tissue and mixing. From the appearance in the receptor compartment, the permeabilty of the compound is calculated 8/29/2009 Modified Ussing chamber : Modified Ussing chamber 8/29/2009 BACK Schemetic of ussing chamber : Schemetic of ussing chamber 8/29/2009 Advantage of Cell Culture Method : Advantage of Cell Culture Method Rapid screening for drug absorption & metabolism Ease for study of drug transport Also study of mucosal toxicity caused by drug Provide information of drug formulation No interspecies difference 8/29/2009 Mechanism of Drug Transport Across GI Absorptive Epithelia : Mechanism of Drug Transport Across GI Absorptive Epithelia Transcellular passive diffusion - Propanolol, Testosterone, Ketoprofen Paracellular passive diffusion - Cimetidine, Loperamide, Atenolol, Manitol Carrier-mediated transcellular diffusion - Cyclosporin, Nifedipine, Verapamil,Digoxin Pinocytosis 8/29/2009 Caco - 2 cell line : Caco - 2 cell line Adenocarcinoma cell derived from colon Spontaneous differentiation Brush border membrane on the apical surface Presence of Sucrose-isomaltase, lactase-phlorizin hydrolase, aminopeptidase N Expression of cytochrome P450 Expression of intestinal peptide transporter 8/29/2009 Disadvantage of CACO-2 cell : Disadvantage of CACO-2 cell Narrow tight junction : TEER is higher No producing of mucin 8/29/2009 Slide 32: 8/29/2009 Other Cell Line : Other Cell Line HT29-18C1: differentiates only 10 days, small intestine like resistance, express goblet -secreting mucous cell, express less enzymes T84: differentiate poorly, Collonic crypt cell model, secretes mucous, very high resistance TC-7: subclone of Caco-2 with better array of CP450 metabolising enzyme, high resistance 8/29/2009 Slide 34: 8/29/2009 General factor to consider indeveloping a cell culture : General factor to consider indeveloping a cell culture Cell line Microporous Supporting matrix Culturing condition Condition for conducting transport studies Diffusion apparatus 8/29/2009 Diffusion Apparatus used to study drug transport through cultured cell : Diffusion Apparatus used to study drug transport through cultured cell 8/29/2009 Transport Studies UtilizingCaco-2 cell Monolayer : Transport Studies UtilizingCaco-2 cell Monolayer Caco-2 cells culture to desired passage Growth of cell on microporous filter Characterization of the cell Measurement of drug transport 8/29/2009 Characterization of Caco-2 cell : Characterization of Caco-2 cell Monitor cellular integrity by measuring transepithelium electrical resistance (TEER) Paracellular marker: Mannitol, Lucifer yellow Brush border enzymes :Sucrase-isomaltase, aminopeptidase, alkaline phosphatase 8/29/2009 Instrument for measurement of TEER : Instrument for measurement of TEER Transepithelium Resistance (ohm.cm2) Caco-2 cell 250-600 Small intestine 30-70 Large intestine 100-500 Stomach 100-600 8/29/2009 Slide 40: Instrument for measurement of TEER Slide 41: 8/29/2009 Slide 42: 8/29/2009 Measurement of Drug Transport : Measurement of Drug Transport Rinse cell with Hank’s balance salt solution Add sample in Hank’s upper chamber,incubate 37 o C Remove and replace medium in lower chamber at 15 minutes interval Measure drug content in basal solution Calculate permeability 8/29/2009 Permeability : Permeability Flux rate or permeability coefficient (P) are determined from 8/29/2009 Flux rate = (1/A)(dm/dt) = P(C0-C) Cd Cr Slide 45: 8/29/2009 Cell culture model for corneal epithelium : Cell culture model for corneal epithelium 8/29/2009 EpiDermTM : EpiDermTM Human Skin-Like Structure Norman human-derived epidermal keratinocytes (NHEK) EpiDerm skin model Human Epidermis Mitotically and metabolically active Slide 48: Bioadhesion of drugs Second scan: bound activity Cell associated fluorescence- labelled drug Washing and removal of unbound fluorescence- labelled drug Incubation with fluorescence- labelled lectins 1 h, 37 oC First scan: total activity Confluent Caco-2 cell-monolayers 10 days old 1 2 3 4 Slide 49: Fluorescence binding assay Confluent Caco-2 monolayer Drug-buffer solutions Sample, dilution Cytofluor 1. scan Cell incubation with drug solution Removal of unbound drug by washing Cell-associated drug Addition of solvent Solubilization of bound drug Cytofluor 2. scan You do not have the permission to view this presentation. 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Cell culture in drug transport kwichan Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 989 Category: Science & Tech.. License: All Rights Reserved Like it (6) Dislike it (0) Added: August 29, 2009 This Presentation is Public Favorites: 1 Presentation Description use cell culture as a tool for drug transport evaluation Comments Posting comment... By: xuemei (3 month(s) ago) could you allow me to download it or send it to xuemzhang@hotmail.com? Thank you so much! Saving..... Post Reply Close Saving..... 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See all Premium member Presentation Transcript Cell Culture Model for Drug Transport Studies : Cell Culture Model for Drug Transport Studies Wichan Ketjinda Department of Pharmaceutical Technology Pharmaceutical Technology Process of Oral Drug Absorption : Process of Oral Drug Absorption 8/29/2009 Factors affecting absorption : Factors affecting absorption - Solubility - Dissolution rate - Molecular size - Partition coeficient - Chemical degradation - Delivery system 8/29/2009 I. Physical Parameters Factors affecting absorption : Factors affecting absorption - Binding or complexation - Regional pH - Intestinal permeability - Metabolism (Lumen, Hepatic) - Gastric and intestinal transit 8/29/2009 II. Physiological parameters Drug transport pathway : Drug transport pathway 8/29/2009 Slide 6: 8/29/2009 Drug Transport pathway : Drug Transport pathway Transcellular pathway Passive transport Lipophilic compound High efficient route Paracellular pathway Aqueous channel Restricted by tighted junction ( pore ~ 8 Ao ) Low dose and appropriate size allowed passage 8/29/2009 Drug Transport pathway : Drug Transport pathway Carrier-mediated uptake Transporter located at apical and basolateral Apical = proton dependent peptide transporter, sodium dependent sugar and amino acid transporter Basolateral = transporter for organic anion and cation Carrier-mediated Efflux Apical = p-glycoprotien, basolateral = amino acid carrier P-glycoprotien + CP450 limit toxic agent access to circulation 8/29/2009 Slide 9: 8/29/2009 Methods to Assess Drug Absorption from GI Tract : Methods to Assess Drug Absorption from GI Tract I. In vivo animal method - Little predictable - Not suitable for large number of screening studies - Misleading by forcing a correlation for the all of characteristics to human 8/29/2009 Correlation of Oral Bioavailability of Various Compounds : Correlation of Oral Bioavailability of Various Compounds 8/29/2009 Comparison of Bioavailability of Ganciclovir : Comparison of Bioavailability of Ganciclovir 8/29/2009 Methods to Assess Drug Absorption from GI Tract : Methods to Assess Drug Absorption from GI Tract II.In situ animal studies - Advantage of isolating the comparison to the level of the intestine - Measure the rate or extent of uptake drug by tissue (disappear from lumen> drug transport) - Intestinal perfusion method 8/29/2009 Intestinal perfusion model : Intestinal perfusion model 8/29/2009 Intestinal perfusion model : Intestinal perfusion model 8/29/2009 Methods to Assess Drug Absorption from GI Tract : Methods to Assess Drug Absorption from GI Tract III. In Vitro nonbiological Methods Octanol/water distribution - use partition coefficient (log P, log D) to predict drug absorption - determination of a drug extracted into lipid phase of an octanol/water (Lipophilicity) - -3 < log P < 3 good absorption 8/29/2009 Slide 17: 8/29/2009 Methods to Assess Drug Absorption from GI Tract : Methods to Assess Drug Absorption from GI Tract Artificial Membrane (IAM, ILC) - Immobilized artificial membrane (IAM), use chromatography column modified by covalent attachment of phospholipid-like groups to the surface - Immobilized liposome chromatography (ILC),where phospholipids (in the form of multilamella vesicles) are entrapped in the pores of resin beads 8/29/2009 Methods to Assess Drug Absorption from GI Tract : Methods to Assess Drug Absorption from GI Tract Parallel artificial membrane permeation assay(PAMPA), - measured through membrane formed by a mixture of lecithin and inert organic solvent on hydrophobic filter - predict ability of molecule to permeate by passive diffusion 8/29/2009 Methods to Assess Drug Absorption from GI Tract : IV. In Vitro Biological Methods - Brush-Border Membrane Vesicles (BBMV) - Intestinal Rings (Slices) - Everted Intestinal Sac Method -The Ussing Chamber for Excised Intestinal Segments - Cell Culture 8/29/2009 Methods to Assess Drug Absorption from GI Tract Brush-Border Membrane Vesicle : Brush-Border Membrane Vesicle Luminal wall-bound protein and phospholipid from treated cell homogenate are resuspended in buffer Vesicle Vesicle mixed with permeant in buffer and filtered The amount of permeant uptaken by vesicles is determined Only the apical transcellular transport is measured because the extraction method isolates only brush-border component 8/29/2009 BACK Intestinal Rings : Intestinal Rings A section of intestine is isolated immediately, washed to remove debris and enzyme, everted and sliced into rings The segment can be selected from duodenum to rectum Slices are incubated in solution containing the compound After designated time the slices are taken out of the solution for assay process 8/29/2009 everted intestinal rings in solution : everted intestinal rings in solution 8/29/2009 BACK Everted Intestinal Sacs : Everted Intestinal Sacs A section of intestine is derived to prepare the everted sac as described in intestinal ring but without cutting . The everted sac is filled with buffer and put in a flask with buffer containing compound. At the end, the sac is cut and opened at one end, and the serosal fluid is collected to measure the transport. 8/29/2009 BACK Ussing Chamber for Excised Intestinal Segment : Ussing Chamber for Excised Intestinal Segment Side by side diffusion cell Ussing chamber The small sections of intestine are clamped between two glass chamber filled with buffer and nutrition and are gased continuously to maintain the viability of the tissue and mixing. From the appearance in the receptor compartment, the permeabilty of the compound is calculated 8/29/2009 Modified Ussing chamber : Modified Ussing chamber 8/29/2009 BACK Schemetic of ussing chamber : Schemetic of ussing chamber 8/29/2009 Advantage of Cell Culture Method : Advantage of Cell Culture Method Rapid screening for drug absorption & metabolism Ease for study of drug transport Also study of mucosal toxicity caused by drug Provide information of drug formulation No interspecies difference 8/29/2009 Mechanism of Drug Transport Across GI Absorptive Epithelia : Mechanism of Drug Transport Across GI Absorptive Epithelia Transcellular passive diffusion - Propanolol, Testosterone, Ketoprofen Paracellular passive diffusion - Cimetidine, Loperamide, Atenolol, Manitol Carrier-mediated transcellular diffusion - Cyclosporin, Nifedipine, Verapamil,Digoxin Pinocytosis 8/29/2009 Caco - 2 cell line : Caco - 2 cell line Adenocarcinoma cell derived from colon Spontaneous differentiation Brush border membrane on the apical surface Presence of Sucrose-isomaltase, lactase-phlorizin hydrolase, aminopeptidase N Expression of cytochrome P450 Expression of intestinal peptide transporter 8/29/2009 Disadvantage of CACO-2 cell : Disadvantage of CACO-2 cell Narrow tight junction : TEER is higher No producing of mucin 8/29/2009 Slide 32: 8/29/2009 Other Cell Line : Other Cell Line HT29-18C1: differentiates only 10 days, small intestine like resistance, express goblet -secreting mucous cell, express less enzymes T84: differentiate poorly, Collonic crypt cell model, secretes mucous, very high resistance TC-7: subclone of Caco-2 with better array of CP450 metabolising enzyme, high resistance 8/29/2009 Slide 34: 8/29/2009 General factor to consider indeveloping a cell culture : General factor to consider indeveloping a cell culture Cell line Microporous Supporting matrix Culturing condition Condition for conducting transport studies Diffusion apparatus 8/29/2009 Diffusion Apparatus used to study drug transport through cultured cell : Diffusion Apparatus used to study drug transport through cultured cell 8/29/2009 Transport Studies UtilizingCaco-2 cell Monolayer : Transport Studies UtilizingCaco-2 cell Monolayer Caco-2 cells culture to desired passage Growth of cell on microporous filter Characterization of the cell Measurement of drug transport 8/29/2009 Characterization of Caco-2 cell : Characterization of Caco-2 cell Monitor cellular integrity by measuring transepithelium electrical resistance (TEER) Paracellular marker: Mannitol, Lucifer yellow Brush border enzymes :Sucrase-isomaltase, aminopeptidase, alkaline phosphatase 8/29/2009 Instrument for measurement of TEER : Instrument for measurement of TEER Transepithelium Resistance (ohm.cm2) Caco-2 cell 250-600 Small intestine 30-70 Large intestine 100-500 Stomach 100-600 8/29/2009 Slide 40: Instrument for measurement of TEER Slide 41: 8/29/2009 Slide 42: 8/29/2009 Measurement of Drug Transport : Measurement of Drug Transport Rinse cell with Hank’s balance salt solution Add sample in Hank’s upper chamber,incubate 37 o C Remove and replace medium in lower chamber at 15 minutes interval Measure drug content in basal solution Calculate permeability 8/29/2009 Permeability : Permeability Flux rate or permeability coefficient (P) are determined from 8/29/2009 Flux rate = (1/A)(dm/dt) = P(C0-C) Cd Cr Slide 45: 8/29/2009 Cell culture model for corneal epithelium : Cell culture model for corneal epithelium 8/29/2009 EpiDermTM : EpiDermTM Human Skin-Like Structure Norman human-derived epidermal keratinocytes (NHEK) EpiDerm skin model Human Epidermis Mitotically and metabolically active Slide 48: Bioadhesion of drugs Second scan: bound activity Cell associated fluorescence- labelled drug Washing and removal of unbound fluorescence- labelled drug Incubation with fluorescence- labelled lectins 1 h, 37 oC First scan: total activity Confluent Caco-2 cell-monolayers 10 days old 1 2 3 4 Slide 49: Fluorescence binding assay Confluent Caco-2 monolayer Drug-buffer solutions Sample, dilution Cytofluor 1. scan Cell incubation with drug solution Removal of unbound drug by washing Cell-associated drug Addition of solvent Solubilization of bound drug Cytofluor 2. scan