1) The study examined the role of Rho kinase in T cell activation and immune responses.
2) Inhibition of Rho kinase attenuated T cell proliferation, cytokine gene expression, actin polymerization, and aggregation of T cell receptors.
3) Treatment with a Rho kinase inhibitor prolonged survival of allogeneic heart transplants in mice and diminished cytokine mRNA expression in the transplants.
4) Rho kinase promotes structural rearrangements in T cells that are critical for T cell signaling and activation during cellular immune responses.
Evaluation of hepatoprotective agents - Hemant KanaseHemant Kanase
1. Introduction
2. Hepatotoxicity: Mechanism
3. Therapeutic strategies available – their limitations
4. In vivo models of liver damage
- Non-invasive model
a. Chemically induced hepatotoxicity
b. Drug-induced hepatotoxicity
c. Radiation-induced hepatotoxicity
d. Metal-induced hepatotoxicity
e. Diet-induced hepatotoxicity
Models of Acute Hepatitis
Models of chronic hepatitis
Models of fibrosis
Models of cholestasis
Models of steatosis
4. Problems faced with animal studies
5. In vitro models of liver damage
6. Advantages and disadvantages of in vitro models
7. Parameters of evaluation
8. Clinical Assessment
This study investigated the expression and function of PCSK9 in pancreatic islets. The key findings were:
1) PCSK9 was expressed specifically in somatostatin-producing delta cells in human pancreatic islets, but not in alpha or beta cells.
2) PCSK9 deficiency led to increased LDLR protein levels in mouse pancreatic islets, mainly in beta cells. However, PCSK9 deficiency did not alter cholesterol content or insulin secretion from mouse islets.
3) In vivo studies in mice found that glucose tolerance and insulin secretion were similar between PCSK9 deficient and normal mice, suggesting PCSK9 does not impact insulin secretion.
CYP2A6_HPLC_PK_2015 New Simple Method for Coumarin in Liver Cytochrome of RatsWael Ebied
This document describes the development and validation of a new liquid chromatography method for determining coumarin and its 7-hydroxy metabolite as a marker of cytochrome P450 2A6 activity in rats. The method uses a C18 column and isocratic mobile phase to separate coumarin, 7-hydroxycoumarin, and an internal standard. The method was validated and showed good linearity, precision, and accuracy. This validated method was then applied to study the effect of resveratrol, sulforaphane, and thymoquinone on hepatic CYP2A6 activity in spontaneously hypertensive rats.
Metabolism of stability studies and sub cellular fractionsManjunatha D C
This document provides information on metabolic stability studies using cellular and sub-cellular fractions. It discusses hepatocyte and sub-cellular fraction isolation procedures for rats and mice. Metabolic stability is defined as the percentage of parent compound lost over time when exposed to metabolically active systems like hepatocytes, liver microsomes, or liver S9 fractions. Isolated hepatocytes and sub-cellular fractions like cytosol and microsomes are used to study hepatic drug metabolism, uptake, and clearance mediated by drug-metabolizing enzymes and transporters. Detailed methods are provided for isolating hepatocytes and hepatic macrophage from rat and mouse liver, as well as preparing sub-cellular fractions from rat and mouse liver.
This document evaluates several commercial membrane protein extraction kits for their ability to isolate multi-spanning integral membrane proteins from cell and tissue samples for downstream analysis. A sequential detergent extraction method using the Mem-PER Plus kit resulted in increased solubilization and identification of integral membrane proteins compared to other non-detergent or whole cell lysis methods. Specifically, western blot analysis showed higher extraction of proteins like SLC25A6 and AT1A1. Mass spectrometry identification of membrane fractions also revealed more integral membrane proteins annotated from the Uniprot database. Additionally, intact membrane protein complexes like the sodium potassium ATP transporter could be immunoprecipitated after extraction with this sequential detergent method.
Measuring apoptosis in real time with a new luminescent methodMourad FERHAT, PhD
We developed a homogeneous luminogenic annexin V binding assay to detect apoptosis in real time using a multimode plate reader. The detection reagent has two different annexin V fusion proteins engineered to contain complementing domains of a binary luciferase, a substrate for luciferase and a cell impermeable fluorogenic DNA dye to detect necrotic cells. The method allow real-time monitoring of Cellular apoptosis and necrosis in microwell plates without washing steps with a highly sensitive luminescent signal. The AnnexinV luminescent method is amenable to High throughput and is a good alternative to FACS, low-throughput Annexin V-FITC based method.
HepG2 cell model for genotoxicity and steatosis assessmentHCS Pharma
Early detection of toxic events induced by drug cantidats is mandatory in order to avoid late attrition in the process of R&D. Here we present two assays that can be done with the HepG2 human hepatoma cell line: genotoxicity assay (DNA double strand break) and steatosis.
1) The study examined the role of Rho kinase in T cell activation and immune responses.
2) Inhibition of Rho kinase attenuated T cell proliferation, cytokine gene expression, actin polymerization, and aggregation of T cell receptors.
3) Treatment with a Rho kinase inhibitor prolonged survival of allogeneic heart transplants in mice and diminished cytokine mRNA expression in the transplants.
4) Rho kinase promotes structural rearrangements in T cells that are critical for T cell signaling and activation during cellular immune responses.
Evaluation of hepatoprotective agents - Hemant KanaseHemant Kanase
1. Introduction
2. Hepatotoxicity: Mechanism
3. Therapeutic strategies available – their limitations
4. In vivo models of liver damage
- Non-invasive model
a. Chemically induced hepatotoxicity
b. Drug-induced hepatotoxicity
c. Radiation-induced hepatotoxicity
d. Metal-induced hepatotoxicity
e. Diet-induced hepatotoxicity
Models of Acute Hepatitis
Models of chronic hepatitis
Models of fibrosis
Models of cholestasis
Models of steatosis
4. Problems faced with animal studies
5. In vitro models of liver damage
6. Advantages and disadvantages of in vitro models
7. Parameters of evaluation
8. Clinical Assessment
This study investigated the expression and function of PCSK9 in pancreatic islets. The key findings were:
1) PCSK9 was expressed specifically in somatostatin-producing delta cells in human pancreatic islets, but not in alpha or beta cells.
2) PCSK9 deficiency led to increased LDLR protein levels in mouse pancreatic islets, mainly in beta cells. However, PCSK9 deficiency did not alter cholesterol content or insulin secretion from mouse islets.
3) In vivo studies in mice found that glucose tolerance and insulin secretion were similar between PCSK9 deficient and normal mice, suggesting PCSK9 does not impact insulin secretion.
CYP2A6_HPLC_PK_2015 New Simple Method for Coumarin in Liver Cytochrome of RatsWael Ebied
This document describes the development and validation of a new liquid chromatography method for determining coumarin and its 7-hydroxy metabolite as a marker of cytochrome P450 2A6 activity in rats. The method uses a C18 column and isocratic mobile phase to separate coumarin, 7-hydroxycoumarin, and an internal standard. The method was validated and showed good linearity, precision, and accuracy. This validated method was then applied to study the effect of resveratrol, sulforaphane, and thymoquinone on hepatic CYP2A6 activity in spontaneously hypertensive rats.
Metabolism of stability studies and sub cellular fractionsManjunatha D C
This document provides information on metabolic stability studies using cellular and sub-cellular fractions. It discusses hepatocyte and sub-cellular fraction isolation procedures for rats and mice. Metabolic stability is defined as the percentage of parent compound lost over time when exposed to metabolically active systems like hepatocytes, liver microsomes, or liver S9 fractions. Isolated hepatocytes and sub-cellular fractions like cytosol and microsomes are used to study hepatic drug metabolism, uptake, and clearance mediated by drug-metabolizing enzymes and transporters. Detailed methods are provided for isolating hepatocytes and hepatic macrophage from rat and mouse liver, as well as preparing sub-cellular fractions from rat and mouse liver.
This document evaluates several commercial membrane protein extraction kits for their ability to isolate multi-spanning integral membrane proteins from cell and tissue samples for downstream analysis. A sequential detergent extraction method using the Mem-PER Plus kit resulted in increased solubilization and identification of integral membrane proteins compared to other non-detergent or whole cell lysis methods. Specifically, western blot analysis showed higher extraction of proteins like SLC25A6 and AT1A1. Mass spectrometry identification of membrane fractions also revealed more integral membrane proteins annotated from the Uniprot database. Additionally, intact membrane protein complexes like the sodium potassium ATP transporter could be immunoprecipitated after extraction with this sequential detergent method.
Measuring apoptosis in real time with a new luminescent methodMourad FERHAT, PhD
We developed a homogeneous luminogenic annexin V binding assay to detect apoptosis in real time using a multimode plate reader. The detection reagent has two different annexin V fusion proteins engineered to contain complementing domains of a binary luciferase, a substrate for luciferase and a cell impermeable fluorogenic DNA dye to detect necrotic cells. The method allow real-time monitoring of Cellular apoptosis and necrosis in microwell plates without washing steps with a highly sensitive luminescent signal. The AnnexinV luminescent method is amenable to High throughput and is a good alternative to FACS, low-throughput Annexin V-FITC based method.
HepG2 cell model for genotoxicity and steatosis assessmentHCS Pharma
Early detection of toxic events induced by drug cantidats is mandatory in order to avoid late attrition in the process of R&D. Here we present two assays that can be done with the HepG2 human hepatoma cell line: genotoxicity assay (DNA double strand break) and steatosis.
New tools bring greater understanding to cellular metabolism research Mourad FERHAT, PhD
Presentation of Promega Solutions in the field of cellular Metabolism research. Discover new bioluminescent assays for the detection of several metabolites and metabolic process such as : Glucose Uptake, Glucose consumption, Lactate secretion and Glutamine/Glutamate metabolism.
The vitamin E phosphate nucleoside prodrug (VEPNP) platform is designed to bypass two major mechanisms of tumor resistance to nucleosides: nucleoside transport and kinase downregulation. Testing showed that VEPNPs NUC050 and NUC024 were relatively unaffected by the presence of dipyridamole, an inhibitor of nucleoside transport, while the activity of gemcitabine decreased significantly. Further experiments found that NUC050 was able to deliver gemcitabine-monophosphate intracellularly in deoxycytidine kinase-deficient cells, bypassing this mechanism of resistance. A pilot study in mice found that NUC050 had a half-life 13.9 times longer
This document discusses strategies for testing nanomaterials. It begins by introducing Maria Dusinska from the Norwegian Institute for Air Research as the author. It then discusses several key points:
- The importance of characterizing nanomaterials' physicochemical properties which influence biological interactions.
- Recommendations for adapting common toxicity assays like the cytokinesis-blocked micronucleus assay to accurately assess nanomaterials, since they can interfere with assay components.
- Results from the NanoTEST consortium evaluating various nanomaterials in different assays, finding some caused genotoxic effects depending on cell type and dispersion protocol.
- The need to represent in vivo exposure conditions like serum content to understand uptake and effects of nan
This document describes a glucose uptake assay to analyze glucose transport activity in differentiated 3T3 L1 cells. The assay involves treating starved cells with insulin or plant extracts, then exposing the cells to a radioactive cocktail containing tagged glucose. Uptake of the tagged glucose is measured using liquid scintillation counting to analyze the effect of treatments on glucose uptake activity.
This document discusses protein adducts as biomarkers of exposure to alkylating agents. It begins by introducing protein adducts and globin adducts specifically as surrogate biomarkers for DNA adducts. The document then details a research plan to test the hypothesis that globin adducts undergo degradation, producing free amino acid adducts that are excreted in urine and could serve as non-invasive biomarkers of exposure. Initial results are presented demonstrating the successful synthesis of model amino acid adducts and detection of some administered adducts and their metabolites in rat urine. Further research is planned to study the fate of native globin adducts in vivo.
In vitro toxicity testing methods are used to identify potentially hazardous chemicals and confirm lack of toxicity in new substances like drugs and food additives. Cell culture methods screen for toxicity by assessing cell morphology, viability, growth and specialized functions. Both qualitative tests like extraction and direct contact methods, and quantitative assays measuring metabolic activity and membrane integrity are used. While 2D cell cultures have limitations, 3D tissue models better maintain native cell morphology and functions, improving predictions for drug toxicity testing and reducing animal testing and clinical trial failures.
Principle and applications of glucose uptake and calcium influx assay by vivekAnimatedWorld
Principle and applications of glucose uptake assay and calcium influx assay
Diabetes Mellitus and its types
Calcium regulation
Glucose regulation and how it is released in cells.
This document discusses the evaluation of drug toxicity through metabolomic profiling. It describes how metabolomics can be used to detect liver and kidney toxicity induced by various compounds. The study designs involve administering hepatotoxic or nephrotoxic drugs to animals and collecting blood, urine, and tissue samples for analysis. Nuclear magnetic resonance spectroscopy is used to analyze metabolic profiles in biofluids and multivariate statistical analysis is applied to identify biomarkers of toxicity. The document outlines several studies conducted by the National Institute of Food and Drug Safety Evaluation investigating drug toxicity using metabolomics approaches.
This document summarizes a study examining the anti-proliferative effects of Arisaema intermedium lectin (AIL) on human colon cancer cells. AIL was purified from Arisaema intermedium tubers and its effects were tested on HCT-15 colon cancer cells. MTT assays showed AIL inhibited the growth of HCT-15 cells in a dose-dependent manner, with 53% inhibition at 100 μg/mL AIL. Further experiments found AIL treatment led to DNA fragmentation, changes in nucleic acid content, cell morphology changes, and decreased cell viability, indicating AIL induces apoptosis in HCT-15 cells. The results suggest AIL has anti-cancer properties and works through an apoptotic
This document discusses screening methods for anti-cancer agents. It begins with an introduction to cancer, noting that cancer is caused by uncontrolled cell proliferation due to genetic mutations from carcinogens. It then discusses the need for novel anti-cancer drugs due to issues like drug resistance and side effects. The document outlines several in vitro and in vivo screening methods, including membrane integrity assays, functional assays, DNA labeling assays, morphological assays, and reproductive assays. Specific assays discussed in more detail include MTT, LDH, annexin V/PI, trypan blue, and colony forming assays. The document provides details on the principles, advantages and disadvantages of these various screening methods.
ABSTRACT- The anticancer drug arsenic trioxide is effective for acute promyelocytic leukemia. But the clinical trials are
restricted due to its potential side effects. Since the major part of arsenic metabolism and detoxification occurs in liver,
this organ faces the major threat. The hepatic side effects include fatty liver, fibrosis, and inflammation and hepatocyte
degeneration. Our study aimed to evaluate the protective potential of the fatty acid, docosahexaenoic acid, against adversities
of arsenic trioxide in an in vitro model, the Chang liver cells. Two preliminary dose standardization assays, cell
viability and lactate dehydrogenase release assays, were employed. The assays were performed as Pre-treatment,
Co-treatment and Post treatment experiments for a period of 24 hours. Arsenic trioxide at various doses (2.5, 5, 7.5, 10,
12.5 and 15 μM) showed a significant (p≤0.05) dose dependant reduction in cell viability along with a dose dependant
enhancement of lactate dehydrogenase release. However when the cells were treated with a combination of docosahexaenoic
acid at varying concentrations (50, 75, 100, 125 and 150 μM), the above mentioned conditions were found to be
reversed in Pre-treatment and Co-treatment experiments, but not in Post treatment. The most effective combination was
found to be 10 μM arsenic trioxide with 100 μM of docosahexaenoic acid in both Pre-treatment and Co- treatment studies.
Thus the preliminary assays of our study showed that docosahexaenoic acid administration as Pre-treatment or
Co-treatment can aid in reducing arsenic trioxide induced hepatotoxicity. Further studies are required to elucidate the mechanisms
behind the protective effects.
Key Words– Arsenic trioxide, hepatotoxicity, docosahexaenoic acid, cell damage
This study investigated the effects of bile acids on expression of the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene in human hepatocytes. The following key points are summarized:
1) Treatment with chenodeoxycholic acid (CDCA) significantly decreased both PCSK9 mRNA and protein levels in immortalized human hepatocytes.
2) Activation of the farnesoid X receptor (FXR) by its synthetic agonist GW4064 also decreased PCSK9 expression.
3) Coadministration of CDCA counteracted the statin-induced increase in PCSK9 expression, leading to a potentiation of low-density lip
Overcoming Key Challenges of Protein Mass Spectrometry Sample PreparationMourad FERHAT, PhD
Overcoming Key Challenges of Protein Mass Spectrometry Sample Preparation
Bottom-up proteomics is widely accepted as a primary method to characterize proteins. To ensure efficient protein analysis researchers must optimize key steps in the workflow to avoid potential pitfalls such as poor protein sample preparation and inconsistent LC-MS instrument performance. In this presentation, we will:
• Investigate the cause of incomplete trypsin digestion and solution to this problem.
• Discuss the advantage of alternative proteases for mass spec protein analysis.
• Review the impact of mass spec compatible surfactants on protein digestion in gel and protein extraction from animal tissues.
• Detail new reference mass spec protein and peptide materials designed to optimize protein sample preparation steps and monitor key instrument performance parameters.
The presentation should prove valuable to any researcher using bottom-up proteomics, and who is concerned with improving protein mass spec sample preparation and mass spec instrument performance.
Identify Compounds that Rescue Disease Relevant Mutant Membrane ProteinsDiscoverX Corporation
Learn about diseases caused by protein misfolding and how you can screen for compounds, known as pharmacochaperones, that rescue misfolded proteins and could be used as therapeutics.
The PCT MicroTube Adapter Kit, in combination with the PCT SPS, can reliably and reproducibly control the enzymatic digestion of proteins while reducing the time of digestion from hours to minutes with the same or better quality as other, currently available techniques.
Find out more at : www.pressurebiosciences.com
Artursson, Matsson, and Karlgren study in vitro models used for predicting DD...Torben Haagh
In chapter 3 of the book “Transporters in Drug Development” by B. Steffansen and A. S. Grandvuinet, the experts Artursson, Matsson, and Karlgren review in vitro characterization of DDIs with human transport proteins, in particular those that have been shown to have significant clinical effects. Read the full chapter below.
http://bit.ly/SlideshareChapter3
Antihypertensive Peptides; Synthesis, Properties and Application in FoodsAkshay Ramani
1) Hypertension affects over 1 billion people worldwide and is a major cause of death and disability. Oxidative stress and inflammation contribute to hypertension and related conditions like cardiovascular disease.
2) Bioactive peptides derived from food proteins can regulate blood pressure by inhibiting the angiotensin converting enzyme (ACE). ACE is involved in blood pressure regulation and peptide inhibitors of ACE have potential for treating hypertension.
3) Bioactive peptides are released from proteins through enzymatic digestion and fermentation and have been shown to lower blood pressure in animal and human studies by inhibiting ACE.
Metabolomics is the systematic study of small molecule metabolites in biological systems. It aims to identify and quantify all metabolites in a biological sample using analytical techniques like mass spectrometry and nuclear magnetic resonance spectroscopy. The complete set of metabolites present in a cell or organism under a given set of conditions is known as the metabolome. Metabolomics can provide insights into cellular processes and an organism's response to genetic or environmental changes. It has applications in fields like pharmaceutical research, toxicology, nutrition, and agriculture.
This study investigated the role of gut microbiota and bile acid metabolism in the development of non-alcoholic steatohepatitis (NASH)-associated hepatocellular carcinoma (HCC) in mice fed a high-fat diet. The mice developed NASH and HCC within 9 weeks of being fed the steatohepatitis-inducing diet without carcinogens. The gut microbiota was found to promote secondary bile acid production, which activated mTOR signaling in hepatocytes and contributed to oncogenesis. The findings suggest that metabolites produced by gut microbiota like secondary bile acids may mediate HCC development in NASH through mTOR pathway activation in liver cells.
MHY2013 is a novel PPAR pan-agonist that was shown to have beneficial effects on metabolic disorders in mouse models of obesity and diabetes. It activated all PPAR subtypes and increased fatty acid oxidation and energy expenditure in liver, adipose tissue, and skeletal muscle by upregulating genes involved in these pathways. MHY2013 improved insulin sensitivity, lowered blood triglycerides and fatty acids, and reduced hepatic steatosis in obese mice without affecting food intake or body weight. The compound's metabolic effects were mediated through increased levels of the hormones FGF21 and adiponectin.
Microscale culture of human liver cells for drug developmentLuke Lightning
The document describes a new method for culturing human hepatocytes in micropatterned clusters within multi-well plates. This method aims to develop a more effective human liver model for testing drug metabolism and toxicity. The micropatterned hepatocyte clusters demonstrate greater viability and functionality compared to random cell distributions or pure hepatocyte cultures, maintaining liver-specific functions for several weeks. This new model shows potential for high-throughput drug screening and studies of drug-drug interactions and species differences in drug responses.
Quantitative Analysis of Transporter Protein using TripleTOF® 6600 SystemSCIEX
Transport plays an important role in the absorption, distribution, and elimination of a variety of drugs.
In recent years, a large number of transporters, both efflux (ATP-binding cassette (ABC) family) and influx (solute carrier (SLC) family members) have been identified and well characterized in vitro.
However, the abundance of these transporters in the hepatocyte and cell lines as well as in the tissues such as intestine, liver, and kidney has not been accurately quantitated due to technical challenges.
This work aims to build a robust liquid chromatography-mass spectrometry (LC-MS) workflow on the SCIEX TripleTOF® 6600 platform to enable the quantitation of a variety of SLC and ABC drug transporters expressed in the hepatocyte and cell line plasma membranes.
New tools bring greater understanding to cellular metabolism research Mourad FERHAT, PhD
Presentation of Promega Solutions in the field of cellular Metabolism research. Discover new bioluminescent assays for the detection of several metabolites and metabolic process such as : Glucose Uptake, Glucose consumption, Lactate secretion and Glutamine/Glutamate metabolism.
The vitamin E phosphate nucleoside prodrug (VEPNP) platform is designed to bypass two major mechanisms of tumor resistance to nucleosides: nucleoside transport and kinase downregulation. Testing showed that VEPNPs NUC050 and NUC024 were relatively unaffected by the presence of dipyridamole, an inhibitor of nucleoside transport, while the activity of gemcitabine decreased significantly. Further experiments found that NUC050 was able to deliver gemcitabine-monophosphate intracellularly in deoxycytidine kinase-deficient cells, bypassing this mechanism of resistance. A pilot study in mice found that NUC050 had a half-life 13.9 times longer
This document discusses strategies for testing nanomaterials. It begins by introducing Maria Dusinska from the Norwegian Institute for Air Research as the author. It then discusses several key points:
- The importance of characterizing nanomaterials' physicochemical properties which influence biological interactions.
- Recommendations for adapting common toxicity assays like the cytokinesis-blocked micronucleus assay to accurately assess nanomaterials, since they can interfere with assay components.
- Results from the NanoTEST consortium evaluating various nanomaterials in different assays, finding some caused genotoxic effects depending on cell type and dispersion protocol.
- The need to represent in vivo exposure conditions like serum content to understand uptake and effects of nan
This document describes a glucose uptake assay to analyze glucose transport activity in differentiated 3T3 L1 cells. The assay involves treating starved cells with insulin or plant extracts, then exposing the cells to a radioactive cocktail containing tagged glucose. Uptake of the tagged glucose is measured using liquid scintillation counting to analyze the effect of treatments on glucose uptake activity.
This document discusses protein adducts as biomarkers of exposure to alkylating agents. It begins by introducing protein adducts and globin adducts specifically as surrogate biomarkers for DNA adducts. The document then details a research plan to test the hypothesis that globin adducts undergo degradation, producing free amino acid adducts that are excreted in urine and could serve as non-invasive biomarkers of exposure. Initial results are presented demonstrating the successful synthesis of model amino acid adducts and detection of some administered adducts and their metabolites in rat urine. Further research is planned to study the fate of native globin adducts in vivo.
In vitro toxicity testing methods are used to identify potentially hazardous chemicals and confirm lack of toxicity in new substances like drugs and food additives. Cell culture methods screen for toxicity by assessing cell morphology, viability, growth and specialized functions. Both qualitative tests like extraction and direct contact methods, and quantitative assays measuring metabolic activity and membrane integrity are used. While 2D cell cultures have limitations, 3D tissue models better maintain native cell morphology and functions, improving predictions for drug toxicity testing and reducing animal testing and clinical trial failures.
Principle and applications of glucose uptake and calcium influx assay by vivekAnimatedWorld
Principle and applications of glucose uptake assay and calcium influx assay
Diabetes Mellitus and its types
Calcium regulation
Glucose regulation and how it is released in cells.
This document discusses the evaluation of drug toxicity through metabolomic profiling. It describes how metabolomics can be used to detect liver and kidney toxicity induced by various compounds. The study designs involve administering hepatotoxic or nephrotoxic drugs to animals and collecting blood, urine, and tissue samples for analysis. Nuclear magnetic resonance spectroscopy is used to analyze metabolic profiles in biofluids and multivariate statistical analysis is applied to identify biomarkers of toxicity. The document outlines several studies conducted by the National Institute of Food and Drug Safety Evaluation investigating drug toxicity using metabolomics approaches.
This document summarizes a study examining the anti-proliferative effects of Arisaema intermedium lectin (AIL) on human colon cancer cells. AIL was purified from Arisaema intermedium tubers and its effects were tested on HCT-15 colon cancer cells. MTT assays showed AIL inhibited the growth of HCT-15 cells in a dose-dependent manner, with 53% inhibition at 100 μg/mL AIL. Further experiments found AIL treatment led to DNA fragmentation, changes in nucleic acid content, cell morphology changes, and decreased cell viability, indicating AIL induces apoptosis in HCT-15 cells. The results suggest AIL has anti-cancer properties and works through an apoptotic
This document discusses screening methods for anti-cancer agents. It begins with an introduction to cancer, noting that cancer is caused by uncontrolled cell proliferation due to genetic mutations from carcinogens. It then discusses the need for novel anti-cancer drugs due to issues like drug resistance and side effects. The document outlines several in vitro and in vivo screening methods, including membrane integrity assays, functional assays, DNA labeling assays, morphological assays, and reproductive assays. Specific assays discussed in more detail include MTT, LDH, annexin V/PI, trypan blue, and colony forming assays. The document provides details on the principles, advantages and disadvantages of these various screening methods.
ABSTRACT- The anticancer drug arsenic trioxide is effective for acute promyelocytic leukemia. But the clinical trials are
restricted due to its potential side effects. Since the major part of arsenic metabolism and detoxification occurs in liver,
this organ faces the major threat. The hepatic side effects include fatty liver, fibrosis, and inflammation and hepatocyte
degeneration. Our study aimed to evaluate the protective potential of the fatty acid, docosahexaenoic acid, against adversities
of arsenic trioxide in an in vitro model, the Chang liver cells. Two preliminary dose standardization assays, cell
viability and lactate dehydrogenase release assays, were employed. The assays were performed as Pre-treatment,
Co-treatment and Post treatment experiments for a period of 24 hours. Arsenic trioxide at various doses (2.5, 5, 7.5, 10,
12.5 and 15 μM) showed a significant (p≤0.05) dose dependant reduction in cell viability along with a dose dependant
enhancement of lactate dehydrogenase release. However when the cells were treated with a combination of docosahexaenoic
acid at varying concentrations (50, 75, 100, 125 and 150 μM), the above mentioned conditions were found to be
reversed in Pre-treatment and Co-treatment experiments, but not in Post treatment. The most effective combination was
found to be 10 μM arsenic trioxide with 100 μM of docosahexaenoic acid in both Pre-treatment and Co- treatment studies.
Thus the preliminary assays of our study showed that docosahexaenoic acid administration as Pre-treatment or
Co-treatment can aid in reducing arsenic trioxide induced hepatotoxicity. Further studies are required to elucidate the mechanisms
behind the protective effects.
Key Words– Arsenic trioxide, hepatotoxicity, docosahexaenoic acid, cell damage
This study investigated the effects of bile acids on expression of the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene in human hepatocytes. The following key points are summarized:
1) Treatment with chenodeoxycholic acid (CDCA) significantly decreased both PCSK9 mRNA and protein levels in immortalized human hepatocytes.
2) Activation of the farnesoid X receptor (FXR) by its synthetic agonist GW4064 also decreased PCSK9 expression.
3) Coadministration of CDCA counteracted the statin-induced increase in PCSK9 expression, leading to a potentiation of low-density lip
Overcoming Key Challenges of Protein Mass Spectrometry Sample PreparationMourad FERHAT, PhD
Overcoming Key Challenges of Protein Mass Spectrometry Sample Preparation
Bottom-up proteomics is widely accepted as a primary method to characterize proteins. To ensure efficient protein analysis researchers must optimize key steps in the workflow to avoid potential pitfalls such as poor protein sample preparation and inconsistent LC-MS instrument performance. In this presentation, we will:
• Investigate the cause of incomplete trypsin digestion and solution to this problem.
• Discuss the advantage of alternative proteases for mass spec protein analysis.
• Review the impact of mass spec compatible surfactants on protein digestion in gel and protein extraction from animal tissues.
• Detail new reference mass spec protein and peptide materials designed to optimize protein sample preparation steps and monitor key instrument performance parameters.
The presentation should prove valuable to any researcher using bottom-up proteomics, and who is concerned with improving protein mass spec sample preparation and mass spec instrument performance.
Identify Compounds that Rescue Disease Relevant Mutant Membrane ProteinsDiscoverX Corporation
Learn about diseases caused by protein misfolding and how you can screen for compounds, known as pharmacochaperones, that rescue misfolded proteins and could be used as therapeutics.
The PCT MicroTube Adapter Kit, in combination with the PCT SPS, can reliably and reproducibly control the enzymatic digestion of proteins while reducing the time of digestion from hours to minutes with the same or better quality as other, currently available techniques.
Find out more at : www.pressurebiosciences.com
Artursson, Matsson, and Karlgren study in vitro models used for predicting DD...Torben Haagh
In chapter 3 of the book “Transporters in Drug Development” by B. Steffansen and A. S. Grandvuinet, the experts Artursson, Matsson, and Karlgren review in vitro characterization of DDIs with human transport proteins, in particular those that have been shown to have significant clinical effects. Read the full chapter below.
http://bit.ly/SlideshareChapter3
Antihypertensive Peptides; Synthesis, Properties and Application in FoodsAkshay Ramani
1) Hypertension affects over 1 billion people worldwide and is a major cause of death and disability. Oxidative stress and inflammation contribute to hypertension and related conditions like cardiovascular disease.
2) Bioactive peptides derived from food proteins can regulate blood pressure by inhibiting the angiotensin converting enzyme (ACE). ACE is involved in blood pressure regulation and peptide inhibitors of ACE have potential for treating hypertension.
3) Bioactive peptides are released from proteins through enzymatic digestion and fermentation and have been shown to lower blood pressure in animal and human studies by inhibiting ACE.
Metabolomics is the systematic study of small molecule metabolites in biological systems. It aims to identify and quantify all metabolites in a biological sample using analytical techniques like mass spectrometry and nuclear magnetic resonance spectroscopy. The complete set of metabolites present in a cell or organism under a given set of conditions is known as the metabolome. Metabolomics can provide insights into cellular processes and an organism's response to genetic or environmental changes. It has applications in fields like pharmaceutical research, toxicology, nutrition, and agriculture.
This study investigated the role of gut microbiota and bile acid metabolism in the development of non-alcoholic steatohepatitis (NASH)-associated hepatocellular carcinoma (HCC) in mice fed a high-fat diet. The mice developed NASH and HCC within 9 weeks of being fed the steatohepatitis-inducing diet without carcinogens. The gut microbiota was found to promote secondary bile acid production, which activated mTOR signaling in hepatocytes and contributed to oncogenesis. The findings suggest that metabolites produced by gut microbiota like secondary bile acids may mediate HCC development in NASH through mTOR pathway activation in liver cells.
MHY2013 is a novel PPAR pan-agonist that was shown to have beneficial effects on metabolic disorders in mouse models of obesity and diabetes. It activated all PPAR subtypes and increased fatty acid oxidation and energy expenditure in liver, adipose tissue, and skeletal muscle by upregulating genes involved in these pathways. MHY2013 improved insulin sensitivity, lowered blood triglycerides and fatty acids, and reduced hepatic steatosis in obese mice without affecting food intake or body weight. The compound's metabolic effects were mediated through increased levels of the hormones FGF21 and adiponectin.
Microscale culture of human liver cells for drug developmentLuke Lightning
The document describes a new method for culturing human hepatocytes in micropatterned clusters within multi-well plates. This method aims to develop a more effective human liver model for testing drug metabolism and toxicity. The micropatterned hepatocyte clusters demonstrate greater viability and functionality compared to random cell distributions or pure hepatocyte cultures, maintaining liver-specific functions for several weeks. This new model shows potential for high-throughput drug screening and studies of drug-drug interactions and species differences in drug responses.
Quantitative Analysis of Transporter Protein using TripleTOF® 6600 SystemSCIEX
Transport plays an important role in the absorption, distribution, and elimination of a variety of drugs.
In recent years, a large number of transporters, both efflux (ATP-binding cassette (ABC) family) and influx (solute carrier (SLC) family members) have been identified and well characterized in vitro.
However, the abundance of these transporters in the hepatocyte and cell lines as well as in the tissues such as intestine, liver, and kidney has not been accurately quantitated due to technical challenges.
This work aims to build a robust liquid chromatography-mass spectrometry (LC-MS) workflow on the SCIEX TripleTOF® 6600 platform to enable the quantitation of a variety of SLC and ABC drug transporters expressed in the hepatocyte and cell line plasma membranes.
This document outlines a study examining the effects of different cell culture plate coatings on Alpha Mouse Liver 12 (AML12) cells. The coatings tested were collagen, matrigel, and non-coated plates. The study aimed to characterize cell morphology, growth rates, mRNA and protein expression, and sensitivity to acetaminophen toxicity when cultured on different coatings. Results found no significant differences in growth rates, morphology, or toxicity responses between plate coatings. mRNA expression analysis found only actin and albumin expression from cells on collagen plates, and all targets from matrigel plates but not at levels above non-coated plates. Overall, the coatings did not produce the expected enhanced characterization of the AML12
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Assessing gastrointestinal toxicity using human tissues bioptaBiopta Inc.
This document describes the capabilities and research of Biopta, a company that specializes in conducting safety and efficacy tests using human gastrointestinal tissues. Biopta sources tissues from surgical residuals and non-transplantable organs to study drug absorption, metabolism, and safety in human models. Their models can evaluate a range of gastrointestinal toxicity endpoints, including changes in motility, barrier integrity, secretory functions, and drug-metabolizing enzymes. Results from these human tissue models have been shown to translate to clinical outcomes. The models provide a more human-relevant approach for predicting adverse gastrointestinal drug effects compared to animal models.
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Webinar Slide - MetMax Hepatocytes and Enterocytes 09-20-17
1. MetMax™ Hepatocytes and Enterocytes for
the Evaluation of Drug Metabolism
(Patent Pending)
Albert P. Li, Ph. D., President and CEO
In Vitro ADMET Laboratories Inc.
Columbia, MD and Malden, MA
lialbert@invitroadmet.com
2. MetMax™ Hepatocytes and
Enterocytes
• Current hepatocyte and enterocyte
technologies
• MetMax™ human hepatocytes and enterocyte
technologies
– MetMax™ advantage
– Applications
2
3. In Vitro ADMET Laboratories (IVAL)
Columbia, MD and Malden, MA
• Locations: Columbia, MD and Malden, MA
• Date of Incorporation: November, 2004
• Mission: Enhance the efficiency of drug
development with in vitro human and animal
based experimental systems
– accurate assessment of human drug properties
3
4. IVAL Technology
• Primary cells from human/animal organs
– Not cultured to retain organ-specific properties
• Drug metabolism
• Transporter
• Pharmacology
– Cryopreserved
• Ease of use
• Long term storage
5. Key Components of an In Vitro Experimental
System for the Evaluation of Human Toxicants:
The Human MTE Requirement
• Human-specific Metabolism (M)
• Human Targets (T)
• Relevant Endpoints (E)
5
6. Key IVAL Products: Hepatocytes and Enterocytes
Enterocytes: First-pass metabolism of orally-administered drugs
Hepatocytes: First-pass metabolism of absorbed orally-administered drugs
7. Enabling Human Hepatocytes as an
Experimental Tool
• Isolation
• Cryopreservation
• Recovery
• Applications
• Generate scientific acceptance
7
8. Key Milestones
• First demonstration of successful cryopreservation
– Loretz, Li et al. Optimization of cryopreservation procedures for rat and human hepatocytes.
Xenobiotica. 1989 May;19(5):489-98.
• First demonstration of retention of drug metabolizing enzymes after
cryopreservation
– Lu et al. Cryopreserved human hepatocytes: characterization of drug-metabolizing enzyme
activities and applications in higher throughput screening assays for hepatotoxicity, metabolic
stability, and drug-drug interaction potential. Chem Biol Interact. 1999 Jun 1;121(1):17-35.
• First international consensus on utility of cryopreserved human hepatocytes
– Li et al. Present status of the application of cryopreserved hepatocytes in the evaluation of
xenobiotics: consensus of an international expert panel. Chem Biol Interact. 1999 Jun
1;121(1):117-23.
• First demonstration of retention of uptake transporter activities after
cryopreservation
– Shitara, Li et al. Function of uptake transporters for taurocholate and estradiol 17beta-D-
glucuronide in cryopreserved human hepatocytes.
Drug Metab Pharmacokinet. 2003;18(1):33-41.
• First demonstration of effectiveness of CHRM and plateability
– Li. Human hepatocytes: isolation, cryopreservation and applications in drug development.
Chem Biol Interact. 2007 May 20;168(1):16-29.
8
9. Cryopreserved Human Hepatocytes in
Drug Development
• Plateable Cryopreserved Human Hepatocytes
– Drug-drug interactions
• P450 induction
• Reversible and time-dependent inhibition
– Uptake and efflux transport
– Drug toxicity
• Hepatotoxicity screening
• Metabolic activation of protoxicants
9
10. Novel IVAL Product:
Plateable Pooled Donor
Cryopreserved Human Hepatocytes
PHH8015A (10 donor pool; 5 male/5 female)
10
Day 1 Day 7Day 4
IVAL Pool Cryopreserved Hepatocyte Patent Allowed: U. S., China
Patent Pending: European Union
11. Novel IVAL Product:
OnDemand™ Plated Cryopreserved Human Hepatocytes
• Ready to use hepatocytes
– P450 induction studies
– Cytotoxicity studies
– Plated metabolism studies
• Plated from pre-characterized
human hepatocyte lots
• Delivered to laboratories based on
investigator’s schedule – 7 days a
week
11
13. Cryopreserved Human Hepatocytes in
Drug Development
• Suspension:
– Major application: drug metabolism (metabolic
stability screening)
– Drug-drug interactions (P450 inhibition but mainly
done with HLM)
– Uptake transport (now mainly done with plateable
hepatocytes)
13
14. Application of Human Hepatocytes in
Drug Development
• Advantages
– Complete drug metabolizing enzyme activities
• Disadvantages
– Laborious
• Storage in LN2
• Centrifugation and microscopic evaluation of cell viability
and cell concentration
• Sensitive to experimental manipulation (not robot friendly)
• Metabolism may be hindered by drug toxicity (high drug
concentration cannot be used for metabolite profiling)
14
15. MetMax™ Hepatocytes
• Permeabilized, cofactor supplemented
hepatocytes
– An experimental system with the advantages of
hepatocytes and the ease of operation and
robustness of cell free systems
15
19. Ease of use of MetMax™ Hepatocytes
Organelles MetMax™
Intact
Hepatocytes
Microsomes S9
Storage -80 deg. C LN2 -80 deg. C -80 deg. C
Centrifugation No Yes No No
Microscopic
Examination
No Yes No No
Cell Counting No Yes No No
Cofactor
Addition
No No Yes Yes
Thaw and Use Yes No No No
20. MetMax™ Hepatocytes
A Thaw and Use Reagent
20
MetMax™ Hepatocytes
1. Retrieve from -80 deg. freezer
2. Thaw in a 37 deg. water bath
3. Add at equal volume to 2X test article
4. Incubate
Freezer to Incubation:
<5 minutes
Cryopreserved Hepatocytes
1. Retrieve from LN2 freezer
2. Thaw in a 37 deg. water bath
3. Add to recovery medium
4. Centrifuge
5. Microscopic quantification of viability
and cell number
6. Adjust to 2X final cell density
7. Add at equal volume to 2X test article
8. Incubate
Freezer to Incubation:
>30 minutes
21. MetMax™ Human Hepatocytes
• Advantages over human liver S9 and microsomes:
Complete drug metabolism enzyme pathways
• Advantages over intact human hepatocytes for application in
drug metabolism studies:
Ease of use; robustness; maximized enzyme activities
22. Characterization of MetMax™
Human Hepatocytes
Pooled Donor Human Hepatocytes
(PHS9001) vs.
MetMax™ Pooled Human Hepatocytes
(PHHX8011; derived from PHS9001)
22
25. Activity vs. Cell Concentration
• Linear activity vs. cell concentration using cell
concentrations of 0.25, 0.5, 1.0, and 2 million
cells per mL
– 1 million cells per mL chosen as the cell
concentration for subsequent characterization
25
29. Time Course
• Linear time-course for most drug metabolism
enzyme pathways at incubation times of 30,
60, 120, and 240 minutes
• Similar trend of metabolite formation versus
time for intact and MetMax™ human
hepatocytes
• MetMax™ hepatocytes similar or higher than
intact hepatocytes in rates of metabolite
formation
29
30. Sequential Metabolism:
Phase 1 oxidation followed by
Phase 2 conjugation of oxidation
metabolites
A major advantage of hepatocytes
over S9 and HLM
30
36. Intact (PHH) Vs MetMax™ (PHHX) Pooled Donor Human Hepatocytes:
Comparison of 16 Drug Metabolizing Enzyme-Selective Substrates
36
0.100
1.000
10.000
100.000
1000.000
10000.000
Activity(pmol/min/millionhepatocytes)
Drug Metabolizing Enzyme Pathway
PHH PHHX
37. Comparison of Intact and MetMax™
Human Hepatocytes in DME Activities
• MetMax™ human hepatocytes were similar or
higher than intact human hepatocytes in the
metabolism of 17 pathway-selective DME
pathways
• Results suggest that MetMax™ human
hepatocytes can be used for drug metabolism
studies performed routinely with intact
human hepatocytes
37
38. Application of MetMax™ Human
Hepatocytes in the Evaluation of
Intrinsic Hepatic Clearance
38
39. Intrinsic Hepatic Clearance Study
(Collaboration with Karin Brown and Gary Hingorani,
Array Biopharma)
• 18 drug with known in vivo hepatic clearance
evaluated in intact and MetMax™ Pooled
Donor Human Hepatocytes
– Drug concentration: 1 µM
– Hepatocyte: 1 million cells/mL
– Time points: 0, 5, 15, 30, 45
– Endpoint: T1/2
– Prediction: In vivo intrinsic hepatic clearance
39
40. Metabolic Stability Screening:
MetMax ™ Pooled Donor Human Hepatocytes
accurately predict human hepatic clearance in vivo
PHS9001: Intact Human Hepatocytes
PHHX8011: MetMax™ Human Hepatocytes
y = 0.7375x
R² = 0.13
y = 0.7469x
R² = 0.7236
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
0 2 4 6 8 10 12 14 16
InVitroHepaticClearance
In Vivo Systemic Clearance
PHS9001
MetMax PHHX
8001
41. Application of MetMax™ Pooled Donor
Human Hepatocytes in the Evaluation
of Intrinsic Hepatic Clearance
• 18 drugs with known human in vivo hepatic
clearance were evaluated
– Linear correlation with in vivo hepatic clearance
– MetMax™ and intact hepatocytes yielded similar
slopes
– MetMax™ hepatocytes yielded a higher coefficient
of correlation than intact hepatocytes
41
43. Metabolite Profiling:
Limitation of intact human hepatocytes
• Human hepatocytes, with complete DME
pathways, should be ideal for metabolite
profiling studies
• However, due to cytotoxicity, hepatocytes in
general cannot be incubated with high drug
concentrations to allow the production of
adequate quantity of metabolites for
identification
43
47. MetMax™ Human Hepatocytes for
Metabolite Profiling and Identification
• APAP was incubated at noncytotoxic (10 mM) and
cytotoxic (100 and 200 mM) concentrations with intact
and MetMax™ human hepatocytes
– Similar metabolite profiles at the noncytotoxic (10 mM)
concentration (glucuronide, sulfate, GSH conjugate)
– MetMax™ >> intact human hepatocytes in metabolite
formation in at cytotoxic concentrations (100 and 200 mM)
• Results suggest that MetMax™ Human Hepatocytes
can be incubated with high, cytotoxic concentrations
for the generation of metabolites for identification and
profiling
47
49. Cofactor-Directed Pathway Selection
• Intact human hepatocytes allow metabolism
of a drug by all hepatic pathways, selection of
a specific metabolic pathway for evaluation is
not easily accomplished
• In MetMax™ hepatocytes, one can direct
metabolism to specific pathways via selection
of cofactor contents
49
50. Cofactor-Directed Pathway Selection of
Coumarin Metabolism with MetMax™ Human
Hepatocytes
50
7-OH Coumarin
NADPH
UDPGA
Glucuronide
PAPS
Sulfate
51. Cofactor Selection and Expectations for
Coumarin Metabolism by MetMax™ Human
Hepatocytes
• No cofactors: no metabolism
• NADPH only: 7-OH coumarin (7-HC) formation;
no sulfation or glucuronidation
• NADPH + UDPGA: 7-HC and 7-HC-glucuronide;
no 7-HC-sulfate
• NADPH + PAPS: 7-HC and 7-HC-sulfate; no
glucuronide
51
52. Pathway Selection with Cofactors in
MetMax™ Human Hepatocyte:
Coumarin Metabolism
-10.000
10.000
30.000
50.000
70.000
90.000
110.000
7-HC 7-HCS 7-HCG
Activity(pmol/min/millioncells)
Metabolite
No Cofactors
NADPH
UDPGA
PAPS
+UDPGA
No Cofactors
NADPH
+PAPS
53. Cofactor-Directed Coumarin Metabolism
in MetMax™ Human Hepatocytes:
Summary of Results
• No cofactors: no metabolism
• NADPH only
– Mainly 7-OH coumarin (7-HC) formation;
– minimum sulfation and glucuronidation
• NADPH + UDPGA:
– Mainly 7-HC and 7-HC-glucuronide;
– no 7-HC-sulfate
• NADPH + PAPS:
– Mainly 7-HC and 7-HC-sulfate;
– no glucuronide
53
54. MetMax™ Human Hepatocytes as
Exogenous Metabolic Activation
System for the Evaluation of Pro-
toxicants
54
55. Prototoxicant Activation Assay with
MetMax™ Human Hepatocytes
• Target cells: HEK293, devoid of xenobiotic drug
metabolism activities
• Exogenous activation system: MetMax™ human
hepatocytes
• Metabolic negative control: Inactivated (boiled)
MetMax™ human hepatocytes
• Prototoxicants:
– Acetaminophen
– Cyclophosphamide
– Ifosfamide
55
57. MetMax™ Activation of
Acetaminophen
57
With MetMax
Hepatocytes
With Boiled MetMax
Hepatocytes
Without Hepatocytes
Experiment 1:
0.5 million
hepatocytes per mL
Experiment 2:
1.0 million
hepatocytes per mL
59. MetMax™ Activation of Ifosfamide and
Cyclophosphamide
59
With MetMax
Hepatocytes
Without
Hepatocytes
With Boiled MetMax
Hepatocytes
With MetMax
Hepatocytes
Without
Hepatocytes
With Boiled MetMax
Hepatocytes
60. MetMax™ Human Hepatocytes
Activation of Prototoxicants
• Cytotoxicity of acetaminophen,
cyclophosphamide, and ifosphamide towards HEK
293 cells were enhanced by MetMax™ human
hepatocytes
• Activation was inactivated by boiling of the
hepatocytes
• Results suggest that MetMax™ human
hepatocytes can be used as an exogenous
activating system for the evaluation of
prototoxicants
60
62. Why Enterocytes
• Key cell type for oral bioavailability
• First pass metabolism before the liver
• Intestinal DDI with orally co-administered
substances (foods; nutrient supplements;
drugs)
– Intestinal DDI may not occur in the liver due to
lower hepatic exposure (e.g. grapefruit juice)
62
65. As of now, primary enterocytes are
not commercially available for drug
metabolism evaluation
Current commercially available
enterocytes are cultured for multiple
passages with little information on drug
metabolizing enzyme activities
67. Hepatocytes Vs. Enterocytes:
Cellular Protein Contents
y = 1.4478x
R² = 0.9896
y = 0.2629x
R² = 0.7207
0
0.5
1
1.5
2
2.5
3
3.5
0 0.5 1 1.5 2 2.5
Protein(mg)
Cell Number (Millions)
Human Hepatocytes Vs. Human Enterocytes
Cellular Protein Contents
Human Enterocytes
0.26 mg protein/million cells)
Human Hepatocytes
(1.45 mg/million cells)
68. Cryopreservation of Human
Enterocytes at IVAL
• Successful isolation and cryopreservation of
enterocytes with high viability (>75%) and
reproducible yield (1-3 million cells per vial)
68
69. 69
Drug Metab Dispos 45:686–691, June 2017
Human Enterocytes as an In Vitro Model for the Evaluation of
Intestinal Drug Metabolism: Characterization of Drug-Metabolizing
Enzyme Activities of Cryopreserved Human Enterocytes from
Twenty-Four Donors
70. DME Activities of Human Enterocytes
70
Lot
Number
Gender Ethnicity Age CYP2C9 CYP2C19 CYP3A4 UGT SULT 2J2 CES2
HE3005 Male C 23 1.68 0.56 2.7 8.38 8.72 1.20 0.23
HE3006 Female C 44 0.59 0.29 0.13 2.30 2.04 0.73 0.33
HE3007 Male H 43 0.91 0.39 0.99 3.08 4.04 0.57 0.46
HE3008 Male C 18 0.46 0.68 0.87 1.80 1.79 0.33 0.55
HE3009 Female C 44 1.18 0.35 0.72 4.32 7.78 0.25 0.60
HE3010 Male C 47 1.21 0.62 0.46 2.56 3.32 0.99 0.41
HE3011 Female C 50 0.03 0.01 0.09 1.01 1.70 0.33 0.29
HE3013 Female AA 57 NA NA 0.2 NA NA NA NA
HE3014 Male AA 49 0.44 0.11 0.4 3.55 2.66 1.18 0.17
HE3015 Male C 24 2.50 0.49 2.55 7.33 5.23 0.95 0.51
HE3016 Male AA 32 2.05 1.08 1.0 5.71 4.13 0.93 0.34
HE3019 Male C 61 0.24 0.11 0.30 1.47 1.89 0.26 0.30
HE3020 Male C 25 0.31 0.14 0.5 5.83 1.84 0.58 0.59
HE3021 Male AA 60 0.20 0.06 0.17 1.49 1.64 0.19 0.08
HE3027 Female C 53 2.02 0.31 0.7 3.68 2.69 0.76 0.19
HE3028 Male AA 34 0.68 0.21 0.82 3.84 9.02 0.71 0.30
HE3029 Male C 41 0.86 0.12 0.6 6.55 3.65 0.76 0.08
HE3031 Female C 49 0.34 0.09 0.16 1.60 0.79 0.49 0.18
71. New Enterocyte Development
• Preparation and characterization of pooled
multiple-donor cryopreserved enterocytes
• Preparation of MetMax™ Cryopreserved Pooled
Human Enterocytes (patent pending)
– Permeabilized enterocytes supplemented with
cofactors
– Easy to use: Thaw and use – no centrifugation, no cell
counting
– Easy to store: -80 deg. Freezer (Liquid nitrogen not
needed)
– High activity
72. Donors Used for Pooling
10 donors (5 female; 5 male)
20 million cells per donor
Lot No. Gender Race Age (Years)
HE3031 F C 49
HE3032 F C 48
HE3006 F C 44
HE3027 F C 53
HE3011 F C 50
HE3021 M AA 60
HE3019 M C 61
HE3028 M AA 34
HE3033 M H 32
HE3010 M C 47
75. MetMax™ Pooled Donor Human Enterocytes
• MetMax™ human enterocytes were prepared
from Pooled Donor Human Enterocytes and
evaluated for drug metabolizing activities for
multiple pathways
• MetMax™ enterocytes were equal or more
active than Pooled Donor Human Enterocytes
in all pathways evaluated
75
79. 384-well HTS Enteric Herb-Drug Interaction
Assay with MetMax™ Enterocytes
79
0% 1.56% 3.12% 6.25% 12.5% 25% 50% 100%
Black Cohosh Black Cohosh Black Cohosh Black Cohosh Black Cohosh Black Cohosh Black Cohosh Black Cohosh
Black Elderberry Black Elderberry Black Elderberry Black Elderberry Black Elderberry Black Elderberry Black Elderberry Black Elderberry
Cinnamon Cinnamon Cinnamon Cinnamon Cinnamon Cinnamon Cinnamon Cinnamon
Echinacea Echinacea Echinacea Echinacea Echinacea Echinacea Echinacea Echinacea
Garlic Garlic Garlic Garlic Garlic Garlic Garlic Garlic
Ginger Ginger Ginger Ginger Ginger Ginger Ginger Ginger
Ginkgo Ginkgo Ginkgo Ginkgo Ginkgo Ginkgo Ginkgo Ginkgo
Ginseng Ginseng Ginseng Ginseng Ginseng Ginseng Ginseng Ginseng
Grapefruit Juice Grapefruit Juice Grapefruit Juice Grapefruit Juice Grapefruit Juice Grapefruit Juice Grapefruit Juice Horehound
Horehound Horehound Horehound Horehound Horehound Horehound Horehound Horehound
Milk Thistle Milk Thistle Milk Thistle Milk Thistle Milk Thistle AX™Milk Thistle Milk Thistle Milk Thistle
St. John's Wort St. John's Wort St. John's Wort St. John's Wort St. John's Wort St. John's Wort St. John's Wort St. John's Wort
Spirulina Spirulina Spirulina Spirulina Spirulina Spirulina Spirulina Spirulina
Green Tea Green Tea Green Tea Green Tea Green Tea Green Tea Green Tea Green Tea
81. Cryopreservation of Human
Enterocytes
• Successful isolation and cryopreservation of
enterocytes from multiple donors
– viability (>80%)
– yield (1-3 million cells per vial)
• Retention of drug metabolizing enzyme activities
• MetMax™ enhancement of enterocytes DME
activities
• Successful application in the evaluation of food-
drug interactions
– CYP3A4 inhibition by grapefruit juice and herbal
supplements
81
83. MetMax™ Hepatocytes and
Enterocytes
• MetMax™ hepatocytes and enterocytes
represent robust, convenience experimental
system for the evaluation of hepatic and
enteric drug metabolism, respectively
– Metabolic clearance
– Metabolite profiling
– Prototoxicant activation
– Drug-drug interactions
84. Comparison of MetMax™ Hepatocytes/Enterocytes to Intact
Hepatocytes/Enterocytes, S9 and Microsomes: Organelles
Organelles MetMax™
Intact
Hepatocytes/
Enterocytes
Microsomes S9
Endoplasmic
Reticulum
Cytosol
Mitochondria
Lysosomes
Golgi
Plasma
Membranes
Nucleus
85. Ease of use of MetMax™ Hepatocytes/Enterocytes
Organelles MetMax™
Intact
Hepatocytes/
Enterocytes
Microsomes S9
Storage -80 deg. C LN2 -80 deg. C -80 deg. C
Centrifugation No Yes No No
Microscopic
Examination
No Yes No No
Cell Counting No Yes No No
Cofactor
Addition
No No Yes Yes
Thaw and Use Yes No No No
86. MetMax™ Advantage:
Versatility in Metabolism Applications
86
Organelle
Intact
Hepatocytes/
Enterocytes
S9 Microsomes MetMax
Complete Phase 1
and 2 Metabolism
Yes Incomplete Incomplete Yes!!!
Metabolite
Identification at
Cytotoxic Drug
Concentrations
No Yes Yes Yes!!!
Selection of
Pathways by
Cofactors
No Yes Yes Yes!!!
87. MetMax™ Hepatocytes/Enterocytes
Thaw and Use Reagents
87
MetMax™ Hepatocytes/Enterocytes
1. Retrieve from -80 deg. freezer
2. Thaw in a 37 deg. water bath
3. Add at equal volume to 2X test article
4. Incubate
Freezer to Incubation:
<5 minutes
Cryopreserved Hepatocytes/Enterocytes
1. Retrieve from LN2 freezer
2. Thaw in a 37 deg. water bath
3. Add to recovery medium
4. Centrifuge
5. Microscopic quantification of viability
and cell number
6. Adjust to 2X final cell density
7. Add at equal volume to 2X test article
8. Incubate
Freezer to Incubation:
>30 minutes
88. MetMax™
Hepatocytes and Enterocytes
(patent pending)
• Superior in vitro system for the evaluation of
human hepatic (hepatocytes) and enteric
(enterocytes) metabolism with the complete drug
metabolizing enzymes of intact cells and the
efficiency of cell-free systems
– Easy to use: Thaw and use directly*. No centrifugation, no
microscopic examination, no cell counting.
– Maximized phase 1 and phase 2 drug metabolizing enzyme activities
– Robust: Compatible with automated HTS assays*
– Applications include
• Metabolic stability
• Metabolite profiling and identification: can use cytotoxic concentrations*
• Enzyme inhibition
• Metabolic activation of pro-toxicants and pro-mutagens
89. Contact Information
• Albert P. Li, Ph. D., President and CEO:
lialbert@invitroadmet.com
• George Amaral, U. S. Sales and Marketing:
gamaral@invitroadmet.com
• Bez Emadi, European Sales and Marketing:
bemadi@invitroadmet.com
• Deepak Barot, Indian Sales and Marketing:
dbarot@invitroadmet.com
• Nozomi Mizuno, Japanese Sales and Marketing:
n.mizuno@genomembrane.com
89