Hepatic metabolic stability is a key pharmacokinetic parameter in drug discovery. Metabolic stabi... more Hepatic metabolic stability is a key pharmacokinetic parameter in drug discovery. Metabolic stability is usually assessed in microsomal fractions and only the best compounds progress in the drug discovery process. A high-throughput single time point substrate depletion assay in rat liver microsomes (RLM) is employed at the National Center for Advancing Translational Sciences. Between 2012 and 2020, RLM stability data was generated for ~ 24,000 compounds from more than 250 projects that cover a wide range of pharmacological targets and cellular pathways. Although a crucial endpoint, little or no data exists in the public domain. In this study, computational models were developed for predicting RLM stability using different machine learning methods. In addition, a retrospective time-split validation was performed, and local models were built for projects that performed poorly with global models. Further analysis revealed inherent medicinal chemistry knowledge potentially useful to che...
Journal of pharmaceutical and biomedical analysis, Jan 31, 2017
An accurate, rapid and selective method was developed to quantify cyclocreatine in mouse and rat ... more An accurate, rapid and selective method was developed to quantify cyclocreatine in mouse and rat plasma using hydrophilic interaction (HILIC) ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The plasma samples were prepared by protein precipitation with acetonitrile:methanol (70:30). Chromatographic separation was performed on a HILIC BEH amide column (2.1mm×50mm, 1.7μm) with a 3min gradient elution at a flow rate of 0.5mL/min. For mass spectrometric detection, selected reaction monitoring (SRM) was used; the SRM transitions were m/z 144→98 and m/z 144→56 for cyclocreatine and m/z 148→102 for the internal standard (D4-cyclocreatine) in the positive ionization mode. No endogenous components interfered with the analysis of cyclocreatine and the internal standard in mouse and rat plasma. Plasma calibration curves were constructed in the range of 0.01-25μM. The correlation coefficient of the calibration curves was greater than 0.99. The mean intraday assay ...
Drug metabolism and disposition: the biological fate of chemicals, Oct 14, 2016
Advancement of in silico tools would be enabled by availability of data for metabolic reaction ra... more Advancement of in silico tools would be enabled by availability of data for metabolic reaction rates and intrinsic clearance (CLint) of a diverse compound structure dataset by specific metabolic enzymes. Our goal is to measure CLint for a large set of compounds with each major human cytochrome P450 (CYP) isozyme. In order to achieve our goal, it is of utmost importance to develop an automated, robust, sensitive, high-throughput metabolic stability assay that can efficiently handle large volume of compound sets. The substrate depletion method (in vitro half-life (t1/2) method) was chosen to determine CLint The assay (384-well format) consisted of three parts: a robotic system for incubation and sample clean up; two different, integrated, ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) platforms to determine the percent remaining of parent compound, and an automated data analysis system. The CYP3A4 assay was evaluated using two long-t1/2 compounds, carbamazepine an...
International Journal of Pharmaceutics, Apr 20, 2007
The disparity of IC(50)s from CYP450 inhibition assays used to assess drug-drug interaction poten... more The disparity of IC(50)s from CYP450 inhibition assays used to assess drug-drug interaction potential was investigated, in order to have evidence for selecting a reliable in vitro CYP450 inhibition assay to support drug discovery. Three assays were studied: individual rhCYP isozymes and corresponding coumarin derivative-probe substrates with fluorescent detection, human liver microsomes (HLM) and cocktail drug-probe substrates with LC-MS detection, and double cocktail rhCYP isozymes mix and drug-probe mix with LC-MS detection. Data comparisons showed that the rhCYP-fluorescent assay and the cocktail assay with HLM-LC-MS had weak correlation. Detection method and probe substrates were shown to not be the major cause of the disparity in IC(50)s. However, the enzyme source and composition (HLM versus, rhCYP) caused disparity in IC(50)s. Specifically, the high concentrations of CYP isozymes often used with HLM-based assays produced high probe substrate conversion and test compound metabolism, which should both contribute to artificially higher IC(50)s. Non-specific binding of substrate to higher concentration proteins and lipids in the HLM-based assays should also contribute to higher IC(50)s. The modified double cocktail assay was found to overcome limitations of the other two assays. It uses an rhCYP isozymes mix, drug-probe substrate mix, low protein concentration, and LC-MS detection. The double cocktail assay is sensitive, selective, and high throughout for use in drug discovery to provide an early alert to potential toxicity with regard to drug-drug interaction, prioritize chemical series, and guide structural modification to circumvent CYP450 inhibition.
The solubility of a compound depends on its structure and solution conditions. Structure determin... more The solubility of a compound depends on its structure and solution conditions. Structure determines the lipophilicity, hydrogen bonding, molecular volume, crystal energy and ionizability, which determine solubility. Solution conditions are affected by pH, co-solvents, additives, ionic strength, time and temperature. Many drug discovery experiments are conducted under "kinetic" solubility conditions. In drug discovery, solubility has a major impact on bioassays, formulation for in vivo dosing, and intestinal absorption. A good goal for the solubility of drug discovery compounds is >60 ug/mL. Equilibrium solubility assays can be conducted in moderate throughput, by incubating excess solid with buffer and agitating for several days, prior to filtration and HPLC quantitation. Kinetic solubility assays are performed in high throughput with shorter incubation times and high throughput analyses using plate readers. The most frequently used of these are the nephelometric assay and direct UV assay, which begin by adding a small volume of DMSO stock solution of each test compound to buffer. In nephelometry, this solution is serially diluted across a microtitre plate and undissolved particles are detected via light scattering. In direct UV, undissolved particles are separated by filtration, after which the dissolved material is quantitated using UV absorption. Equilibrium solubility is useful for preformulation. Kinetic solubility is useful for rapid compound assessment, guiding optimization via structure modification, and diagnosing bioassays. It is often useful to customize solubility experiments using conditions that answer specific research questions of drug discovery teams, such as compound selection and vehicle development for pharmacology and PK studies.
Metabolic stability plays an important role in the success of drug candidates. First-pass metabol... more Metabolic stability plays an important role in the success of drug candidates. First-pass metabolism is one of the major causes of poor oral bioavailability and short half-life. Traditionally, metabolic stability was evaluated at a later stage of drug discovery and required laborious manual manipulations. With the advance of high-throughput screening, combinatorial chemistry, and early profiling of drug-like properties, automated and rapid stability assays are needed to meet the increasing demand of throughput, speed, and reproducibility at earlier stages of drug discovery. The authors describe optimization of a simple, robust, high-throughput microsomal stability assay developed in a 96-well format. The assay consists of 2 automated components: robotic sample preparation for incubation and cleanup and rapid liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analysis to determine percent remaining of the parent compound. The reagent solutions and procedural steps were optimized for automation. Variables affecting assay results were investigated. The variability introduced by microsome preparations from different sources (various vendors and batches) was studied and indicates the need for careful control. Quality control and normalization of the stability results are critical when applying the screening data, generated at different times or research sites, to discovery projects.
The pharmaceutical industry is facing an ever increasing challenge to deliver safer and more effe... more The pharmaceutical industry is facing an ever increasing challenge to deliver safer and more effective medicines. Traditionally, drug discovery programs were driven solely by potency, regardless of the properties. As a result, the development of non-drug-like molecules was costly, had high risk and low success rate. To meet the challenges, the bar has been rising higher for drug candidates. They not only need to be active, but also drug-like to be advanced to clinical development. Drug-like properties, such as solubility, permeability, metabolic stability and transporter effects are of critical importance for the success of drug candidates. They affect oral bioavailability, metabolism, clearance, toxicity, as well as in vitro pharmacology. Insoluble and impermeable compounds can result in erroneous biological data and unreliable SAR in enzyme and cell-based assays. Rapid metabolism by enzymes and high efflux by transporters can lead to high clearance, short half-life, low systemic exposure and inadequate efficacy. Early property information helps teams make informed decisions and avoids wasting precious resources. Structure-property relationships are essential to guide structural modification to improve properties. High throughput ADME/TOX assays have been implemented and are being widely used to drive drug discovery projects in parallel with activity screening. Property design has become an integrated and inseparable part of the modern drug discovery paradigm. The approach has been proven to be a winning strategy.
Journal of Chemical Information and Modeling, Jun 28, 2010
Due to the high attrition rate of central nervous system drug candidates during clinical trials, ... more Due to the high attrition rate of central nervous system drug candidates during clinical trials, the assessment of blood-brain barrier (BBB) penetration in early research is particularly important. A genetic approximation (GA)-based regression model was developed for predicting in vivo blood-brain partitioning data, expressed as logBB (log[brain]/[blood]). The model was built using an in-house data set of 193 compounds assembled from 22 different therapeutic projects. The final model (cross-validated r(2) = 0.72) with five molecular descriptors was selected based on validation using several large internal and external test sets. We demonstrate the potential utility of the model by applying it to a set of literature reported secretase inhibitors. In addition, we describe a rule-based approach for rapid assessment of brain penetration with several simple molecular descriptors.
A successful drug must combine both potency and drug-like properties. Traditionally, pharmaceutic... more A successful drug must combine both potency and drug-like properties. Traditionally, pharmaceutical companies have focused on activity optimization, based on in vitro and in vivo biological assays. Nowadays, many of the pharmaceutical property assays are implemented early in drug discovery, so that properties can be optimized in parallel with activity (Smith, 2002; Kerns, 2001; Kerns and Di, 2003; Di and Kerns, 2003).
Physicochemical screens are increasingly used in drug discovery to provide an early understanding... more Physicochemical screens are increasingly used in drug discovery to provide an early understanding of key properties that affect ADME. They also insure that discovery experiments are planned and interpreted correctly. Key physicochemical properties discussed are: integrity, pKa, lipophilicity, solubility and permeability. Various groups might implement literature methods or develop their own. Commercial instruments are also available for these properties to allow groups to rapidly provide data for their organizations.:
The principles and screening strategies for brain penetration in drug discovery are important in ... more The principles and screening strategies for brain penetration in drug discovery are important in identifying drug candidates with desirable CNS properties. Define key variables and assays that are essential for determining brain penetration. This review covers issues, methods, and strategies for assessing brain penetration of small molecules in drug discovery. Brain penetration is assessed using both initial rate and extent at steady-state. Unbound drug is the active species that exerts pharmacological effects. Low brain penetration can be due to low blood-brain barrier (BBB) permeability, P-glycoprotein (Pgp) efflux, or high plasma protein binding. Successful methods include: parallel artificial membrane permeability assay (PAMPA)-BBB permeability, MDR1-MDCKII for Pgp efflux, B-P dialysis for fraction unbound, and in vivo B/P ratio to extrapolate unbound brain drug concentration.
Current opinion in drug discovery & development, 2005
Pharmaceutical profiling assays provide an early assessment of drug-like properties, such as solu... more Pharmaceutical profiling assays provide an early assessment of drug-like properties, such as solubility, permeability, metabolism, stability and drug-drug interactions. This information can be used to alert project teams to potential property issues, predict and diagnose in vivo assay results, guide structure-property relationships, provide insight into structure modification, and help drug discovery teams to make informed decisions. Successful drugs can be developed when biological activities of interest and pharmaceutical properties are optimized in parallel.
Hepatic metabolic stability is a key pharmacokinetic parameter in drug discovery. Metabolic stabi... more Hepatic metabolic stability is a key pharmacokinetic parameter in drug discovery. Metabolic stability is usually assessed in microsomal fractions and only the best compounds progress in the drug discovery process. A high-throughput single time point substrate depletion assay in rat liver microsomes (RLM) is employed at the National Center for Advancing Translational Sciences. Between 2012 and 2020, RLM stability data was generated for ~ 24,000 compounds from more than 250 projects that cover a wide range of pharmacological targets and cellular pathways. Although a crucial endpoint, little or no data exists in the public domain. In this study, computational models were developed for predicting RLM stability using different machine learning methods. In addition, a retrospective time-split validation was performed, and local models were built for projects that performed poorly with global models. Further analysis revealed inherent medicinal chemistry knowledge potentially useful to che...
Journal of pharmaceutical and biomedical analysis, Jan 31, 2017
An accurate, rapid and selective method was developed to quantify cyclocreatine in mouse and rat ... more An accurate, rapid and selective method was developed to quantify cyclocreatine in mouse and rat plasma using hydrophilic interaction (HILIC) ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The plasma samples were prepared by protein precipitation with acetonitrile:methanol (70:30). Chromatographic separation was performed on a HILIC BEH amide column (2.1mm×50mm, 1.7μm) with a 3min gradient elution at a flow rate of 0.5mL/min. For mass spectrometric detection, selected reaction monitoring (SRM) was used; the SRM transitions were m/z 144→98 and m/z 144→56 for cyclocreatine and m/z 148→102 for the internal standard (D4-cyclocreatine) in the positive ionization mode. No endogenous components interfered with the analysis of cyclocreatine and the internal standard in mouse and rat plasma. Plasma calibration curves were constructed in the range of 0.01-25μM. The correlation coefficient of the calibration curves was greater than 0.99. The mean intraday assay ...
Drug metabolism and disposition: the biological fate of chemicals, Oct 14, 2016
Advancement of in silico tools would be enabled by availability of data for metabolic reaction ra... more Advancement of in silico tools would be enabled by availability of data for metabolic reaction rates and intrinsic clearance (CLint) of a diverse compound structure dataset by specific metabolic enzymes. Our goal is to measure CLint for a large set of compounds with each major human cytochrome P450 (CYP) isozyme. In order to achieve our goal, it is of utmost importance to develop an automated, robust, sensitive, high-throughput metabolic stability assay that can efficiently handle large volume of compound sets. The substrate depletion method (in vitro half-life (t1/2) method) was chosen to determine CLint The assay (384-well format) consisted of three parts: a robotic system for incubation and sample clean up; two different, integrated, ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) platforms to determine the percent remaining of parent compound, and an automated data analysis system. The CYP3A4 assay was evaluated using two long-t1/2 compounds, carbamazepine an...
International Journal of Pharmaceutics, Apr 20, 2007
The disparity of IC(50)s from CYP450 inhibition assays used to assess drug-drug interaction poten... more The disparity of IC(50)s from CYP450 inhibition assays used to assess drug-drug interaction potential was investigated, in order to have evidence for selecting a reliable in vitro CYP450 inhibition assay to support drug discovery. Three assays were studied: individual rhCYP isozymes and corresponding coumarin derivative-probe substrates with fluorescent detection, human liver microsomes (HLM) and cocktail drug-probe substrates with LC-MS detection, and double cocktail rhCYP isozymes mix and drug-probe mix with LC-MS detection. Data comparisons showed that the rhCYP-fluorescent assay and the cocktail assay with HLM-LC-MS had weak correlation. Detection method and probe substrates were shown to not be the major cause of the disparity in IC(50)s. However, the enzyme source and composition (HLM versus, rhCYP) caused disparity in IC(50)s. Specifically, the high concentrations of CYP isozymes often used with HLM-based assays produced high probe substrate conversion and test compound metabolism, which should both contribute to artificially higher IC(50)s. Non-specific binding of substrate to higher concentration proteins and lipids in the HLM-based assays should also contribute to higher IC(50)s. The modified double cocktail assay was found to overcome limitations of the other two assays. It uses an rhCYP isozymes mix, drug-probe substrate mix, low protein concentration, and LC-MS detection. The double cocktail assay is sensitive, selective, and high throughout for use in drug discovery to provide an early alert to potential toxicity with regard to drug-drug interaction, prioritize chemical series, and guide structural modification to circumvent CYP450 inhibition.
The solubility of a compound depends on its structure and solution conditions. Structure determin... more The solubility of a compound depends on its structure and solution conditions. Structure determines the lipophilicity, hydrogen bonding, molecular volume, crystal energy and ionizability, which determine solubility. Solution conditions are affected by pH, co-solvents, additives, ionic strength, time and temperature. Many drug discovery experiments are conducted under "kinetic" solubility conditions. In drug discovery, solubility has a major impact on bioassays, formulation for in vivo dosing, and intestinal absorption. A good goal for the solubility of drug discovery compounds is >60 ug/mL. Equilibrium solubility assays can be conducted in moderate throughput, by incubating excess solid with buffer and agitating for several days, prior to filtration and HPLC quantitation. Kinetic solubility assays are performed in high throughput with shorter incubation times and high throughput analyses using plate readers. The most frequently used of these are the nephelometric assay and direct UV assay, which begin by adding a small volume of DMSO stock solution of each test compound to buffer. In nephelometry, this solution is serially diluted across a microtitre plate and undissolved particles are detected via light scattering. In direct UV, undissolved particles are separated by filtration, after which the dissolved material is quantitated using UV absorption. Equilibrium solubility is useful for preformulation. Kinetic solubility is useful for rapid compound assessment, guiding optimization via structure modification, and diagnosing bioassays. It is often useful to customize solubility experiments using conditions that answer specific research questions of drug discovery teams, such as compound selection and vehicle development for pharmacology and PK studies.
Metabolic stability plays an important role in the success of drug candidates. First-pass metabol... more Metabolic stability plays an important role in the success of drug candidates. First-pass metabolism is one of the major causes of poor oral bioavailability and short half-life. Traditionally, metabolic stability was evaluated at a later stage of drug discovery and required laborious manual manipulations. With the advance of high-throughput screening, combinatorial chemistry, and early profiling of drug-like properties, automated and rapid stability assays are needed to meet the increasing demand of throughput, speed, and reproducibility at earlier stages of drug discovery. The authors describe optimization of a simple, robust, high-throughput microsomal stability assay developed in a 96-well format. The assay consists of 2 automated components: robotic sample preparation for incubation and cleanup and rapid liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analysis to determine percent remaining of the parent compound. The reagent solutions and procedural steps were optimized for automation. Variables affecting assay results were investigated. The variability introduced by microsome preparations from different sources (various vendors and batches) was studied and indicates the need for careful control. Quality control and normalization of the stability results are critical when applying the screening data, generated at different times or research sites, to discovery projects.
The pharmaceutical industry is facing an ever increasing challenge to deliver safer and more effe... more The pharmaceutical industry is facing an ever increasing challenge to deliver safer and more effective medicines. Traditionally, drug discovery programs were driven solely by potency, regardless of the properties. As a result, the development of non-drug-like molecules was costly, had high risk and low success rate. To meet the challenges, the bar has been rising higher for drug candidates. They not only need to be active, but also drug-like to be advanced to clinical development. Drug-like properties, such as solubility, permeability, metabolic stability and transporter effects are of critical importance for the success of drug candidates. They affect oral bioavailability, metabolism, clearance, toxicity, as well as in vitro pharmacology. Insoluble and impermeable compounds can result in erroneous biological data and unreliable SAR in enzyme and cell-based assays. Rapid metabolism by enzymes and high efflux by transporters can lead to high clearance, short half-life, low systemic exposure and inadequate efficacy. Early property information helps teams make informed decisions and avoids wasting precious resources. Structure-property relationships are essential to guide structural modification to improve properties. High throughput ADME/TOX assays have been implemented and are being widely used to drive drug discovery projects in parallel with activity screening. Property design has become an integrated and inseparable part of the modern drug discovery paradigm. The approach has been proven to be a winning strategy.
Journal of Chemical Information and Modeling, Jun 28, 2010
Due to the high attrition rate of central nervous system drug candidates during clinical trials, ... more Due to the high attrition rate of central nervous system drug candidates during clinical trials, the assessment of blood-brain barrier (BBB) penetration in early research is particularly important. A genetic approximation (GA)-based regression model was developed for predicting in vivo blood-brain partitioning data, expressed as logBB (log[brain]/[blood]). The model was built using an in-house data set of 193 compounds assembled from 22 different therapeutic projects. The final model (cross-validated r(2) = 0.72) with five molecular descriptors was selected based on validation using several large internal and external test sets. We demonstrate the potential utility of the model by applying it to a set of literature reported secretase inhibitors. In addition, we describe a rule-based approach for rapid assessment of brain penetration with several simple molecular descriptors.
A successful drug must combine both potency and drug-like properties. Traditionally, pharmaceutic... more A successful drug must combine both potency and drug-like properties. Traditionally, pharmaceutical companies have focused on activity optimization, based on in vitro and in vivo biological assays. Nowadays, many of the pharmaceutical property assays are implemented early in drug discovery, so that properties can be optimized in parallel with activity (Smith, 2002; Kerns, 2001; Kerns and Di, 2003; Di and Kerns, 2003).
Physicochemical screens are increasingly used in drug discovery to provide an early understanding... more Physicochemical screens are increasingly used in drug discovery to provide an early understanding of key properties that affect ADME. They also insure that discovery experiments are planned and interpreted correctly. Key physicochemical properties discussed are: integrity, pKa, lipophilicity, solubility and permeability. Various groups might implement literature methods or develop their own. Commercial instruments are also available for these properties to allow groups to rapidly provide data for their organizations.:
The principles and screening strategies for brain penetration in drug discovery are important in ... more The principles and screening strategies for brain penetration in drug discovery are important in identifying drug candidates with desirable CNS properties. Define key variables and assays that are essential for determining brain penetration. This review covers issues, methods, and strategies for assessing brain penetration of small molecules in drug discovery. Brain penetration is assessed using both initial rate and extent at steady-state. Unbound drug is the active species that exerts pharmacological effects. Low brain penetration can be due to low blood-brain barrier (BBB) permeability, P-glycoprotein (Pgp) efflux, or high plasma protein binding. Successful methods include: parallel artificial membrane permeability assay (PAMPA)-BBB permeability, MDR1-MDCKII for Pgp efflux, B-P dialysis for fraction unbound, and in vivo B/P ratio to extrapolate unbound brain drug concentration.
Current opinion in drug discovery & development, 2005
Pharmaceutical profiling assays provide an early assessment of drug-like properties, such as solu... more Pharmaceutical profiling assays provide an early assessment of drug-like properties, such as solubility, permeability, metabolism, stability and drug-drug interactions. This information can be used to alert project teams to potential property issues, predict and diagnose in vivo assay results, guide structure-property relationships, provide insight into structure modification, and help drug discovery teams to make informed decisions. Successful drugs can be developed when biological activities of interest and pharmaceutical properties are optimized in parallel.
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Papers by Edward Kerns