Kenneth Brouwer - Academia.edu (original) (raw)

Papers by Kenneth Brouwer

Research paper thumbnail of P126 - Observed vs. predicted effects of protein binding: Improving in vivo predictions of hepatic clearance, intracellular concentration and IC50 values

Drug Metabolism and Pharmacokinetics, 2020

Research paper thumbnail of 120918 , a P-Glycoprotein Modulator , Increases the Concentration of Unbound Amprenavir in the Central Nervous System in Rats

GF120918, a P-Glycoprotein Modulator, Increases the Concentration of Unbound Amprenavir in the Ce... more GF120918, a P-Glycoprotein Modulator, Increases the Concentration of Unbound Amprenavir in the Central Nervous System in Rats Jeffrey E. Edwards, Kenneth R. Brouwer, and Patrick J. McNamara* Graduate Center for Toxicology and Department of Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky, and Bioanalysis and Drug Metabolism, Glaxo Wellcome, Inc., Research Triangle Park, North Carolina

Research paper thumbnail of Methodes et compositions destinees a accroitre la penetration des inhibiteurs de la protease du vih

L'invention concerne des methodes destinees a accroitre la penetration de composes inhibant l... more L'invention concerne des methodes destinees a accroitre la penetration de composes inhibant la protease du VIH dans les tissus exprimant la glycoproteine-P.

Research paper thumbnail of Use of Ca 2 1 Modulation to Evaluate Biliary Excretion in Sandwich-Cultured Rat Hepatocytes 1

Previous work in our laboratory has indicated that biliary excretion of a substrate in sandwich-c... more Previous work in our laboratory has indicated that biliary excretion of a substrate in sandwich-cultured hepatocytes can be quantitated by measurement of substrate accumulation in the presence and absence of extracellular Ca. The present study was designed to examine the effects of Ca on taurocholate accumulation and tight junction integrity in cultured hepatocytes. Kinetic modeling was used to characterize taurocholate disposition in the hepatocyte monolayers in the presence and absence of extracellular Ca. The accumulation of taurocholate in freshly isolated hepatocytes, which lack an intact canalicular network, was the same in the presence and absence of extracellular Ca. Electron microscopy studies showed that Ca depletion increased the permeability of the tight junctions to ruthenium red, demonstrating that tight junctions were the major diffusional barrier between the canalicular lumen and the extracellular space. Cell morphology and substrate accumulation studies in the monol...

Research paper thumbnail of Abstract 2943: Severe hepatotoxicity of mithramycin therapy caused by altering expression of hepatocellular bile transporters

Experimental and Molecular Therapeutics

Research paper thumbnail of Cholestatic Drug Induced Liver Injury: A Function of Bile Salt Export Pump Inhibition and Farnesoid X Receptor Antagonism

Applied In Vitro Toxicology

Introduction: In vitro bile salt export pump (BSEP) inhibition alone does not accurately predict ... more Introduction: In vitro bile salt export pump (BSEP) inhibition alone does not accurately predict in vivo drug-induced liver injury in humans, suggesting that other mechanisms may be involved. Inhibition of BSEP has been shown to increase the hepatocellular concentrations of bile acids (BAs), and hepatobiliary disposition of BAs is tightly regulated by the farnesoid X receptor (FXR). Activation of FXR leads to decreased synthesis of BAs and increased expression of BA efflux transporters, BSEP, and organic solute and steroid transporter (OSTa/b). The link between BSEP inhibition and activation of the BA compensatory mechanism has not been clearly demonstrated. Materials and Methods: Therefore, we utilized BSEP inhibitors, cyclosporine A (CsA) and troglitazone (Trog), to explore this ''triggering'' event using Transporter CertifiedÔ sandwich-cultured human hepatocytes (SCHH). Results: Biliary excretion of glycine-cholic acid (GCA) as a percentage of total GCA accumulation was reduced in SCHH treated with either CsA or Trog. Within 12 hours, CsA treatment concomitantly increased intracellular concentrations (ICCs) of GCA and FGF19 mRNA content, an FXR-target gene. Separately, a synergistic 76.6fold increase of OSTb mRNA was observed following CsA and chenodeoxycholic acid co-exposure. In contrast, Trog exposure prevented the synergistic increase of OSTb, a component of the basolateral BA efflux transporter, under the same conditions. Finally, BA toxicity potency was increased in SCHH exposed to Trog but not CsA. Discussion and Conclusions: Overall, these results suggested that BSEP inhibition activates the BA compensatory mechanism reducing ICCs of BAs. However, Trog possesses both BSEP inhibition and FXR antagonist properties blocking the activation of the compensatory mechanism resulting in BA-induced hepatotoxicity.

Research paper thumbnail of An Evaluation of Potential Inhibition of CYP3A4/5 and CYP2C9 Enzymatic Activity by Boswellia serrata Extract

Applied In Vitro Toxicology

Abstract Introduction: As the use of dietary supplements increases, botanical–drug interaction (B... more Abstract Introduction: As the use of dietary supplements increases, botanical–drug interaction (BDI) potentials should be evaluated. Boswellia serrata extract (BSE) is traditionally used as an anti...

Research paper thumbnail of An integrated in vitro screen using sandwich-cultured human hepatocytes for prediction of cholestatic hepatotoxicity

Drug Metabolism and Pharmacokinetics

Research paper thumbnail of Can BSEP Inhibition Testing In Drug Discovery And Development Reduce Liver Injury Risk? - An International Transporter Consortium Perspective

Clinical pharmacology and therapeutics, Jan 23, 2018

Bile Salt Export Pump (BSEP) inhibition has emerged as an important mechanism that may contribute... more Bile Salt Export Pump (BSEP) inhibition has emerged as an important mechanism that may contribute to the initiation of human drug-induced liver injury (DILI). Proactive evaluation and understanding of BSEP inhibition is recommended in drug discovery and development, to aid internal decision making on DILI risk. BSEP inhibition can be quantified using in vitro assays. When interpreting assay data, it is important to consider in vivo drug exposure. Currently, this can be undertaken most effectively by consideration of total C . However, since total drug concentrations are not predictive of pharmacological effect, the relationship between total exposure and BSEP inhibition is not causal. Various follow up studies can aid interpretation of in vitro BSEP inhibition data and may be undertaken on a case by case basis. BSEP inhibition is one of several mechanisms by which drugs may cause DILI, therefore should be considered alongside other mechanisms when evaluating possible DILI risk. This...

Research paper thumbnail of Farnesoid X Receptor Agonists Obeticholic Acid and Chenodeoxycholic Acid Increase Bile Acid Efflux in Sandwich-Cultured Human Hepatocytes: Functional Evidence and Mechanisms

The Journal of pharmacology and experimental therapeutics, 2018

The farnesoid X receptor (FXR) is a nuclear receptor that regulates genes involved in bile acid h... more The farnesoid X receptor (FXR) is a nuclear receptor that regulates genes involved in bile acid homeostasis. FXR agonists, obeticholic acid (OCA) and chenodeoxycholic acid (CDCA), increase mRNA expression of efflux transporters in sandwich-cultured human hepatocytes (SCHH). This study evaluated the effects of OCA and CDCA treatment on the uptake, basolateral efflux, and biliary excretion of a model bile acid, taurocholate (TCA), in SCHH. In addition, changes in the protein expression of TCA uptake and efflux transporters were investigated. SCHH were treated with 1 M OCA, 100 M CDCA, or vehicle control for 72 hours followed by quantification of deuterated TCA uptake and efflux over time in Ca-containing and Ca-free conditions ( = 3 donors). A mechanistic pharmacokinetic model was fit to the TCA mass-time data to obtain estimates for total uptake clearance (CL), total intrinsic basolateral efflux clearance (CL), and total intrinsic biliary clearance (CL). Modeling results revealed tha...

Research paper thumbnail of Transient Changes in Hepatic Physiology That Alter Bilirubin and Bile Acid Transport May Explain Elevations in Liver Chemistries Observed in Clinical Trials of GGF2 (Cimaglermin alfa)

Toxicological sciences : an official journal of the Society of Toxicology, Jan 23, 2017

GGF2 is a recombinant human neuregulin-1β in development for chronic heart failure. Phase 1 clini... more GGF2 is a recombinant human neuregulin-1β in development for chronic heart failure. Phase 1 clinical trials of GGF2 were put on hold when transient elevations in serum aminotransferases and total bilirubin were observed in 2 of 43 subjects who received single doses of GGF2 at 1.5 or 0.378 mg/kg. However, aminotransferase elevations were modest and not typical of liver injury sufficient to result in elevated serum bilirubin. Cynomolgus monkeys administered a single 15 mg/kg dose of GGF2 had similar transient elevations in serum aminotransferases and bilirubin as well as transient elevations in serum bile acids. However, no hepatocellular necrosis was observed in liver biopsies obtained during peak elevations. When sandwich-cultured human hepatocytes were treated with GGF2 for up to 72 h at concentrations ∼0.8-fold average plasma C max for the 0.378 mg/kg dose, no cytotoxicity was observed. Gene expression profiling identified ∼50% reductions in mRNAs coding for bilirubin transporters...

Research paper thumbnail of Evidence of FXR Activation with Obeticholic Acid in an in Vitro Intestinal Model

Research paper thumbnail of Basolateral Efflux Transporters: A Potentially Important Pathway for the Prevention of Cholestatic Hepatotoxicity

Applied In Vitro Toxicology

The hepatocyte adaptive response has been postulated to play a protective role in cholestatic dis... more The hepatocyte adaptive response has been postulated to play a protective role in cholestatic disease states, including primary biliary cirrhosis where bile acid biliary efflux is reduced. Regulation of bile acid synthesis by farnesoid X receptor (FXR) has been well defined; however, bile acid transport has not. Utilizing sandwichcultured human hepatocytes (SCHH) and B-CLEAR Ò technology, we demonstrated that basolateral efflux and not canalicular efflux (bile salt export pump [BSEP]) was the primary driver of bile acid intracellular accumulation. Following 72 hours of exposure to CDCA, decreases of total endogenous bile acid mass and CYP7A1 mRNA content were observed in SCHH consistent with FXR activation. No marked changes were observed in mRNA content of bile acid uptake transporters, however, induction of bile acid efflux transporters OSTa (3.1-6.8 •), OSTb (21-187 •), and BSEP (2.2-7.5 •) mRNA content was observed. While decreases of d8-TCA biliary clearance were inconsistent with the increases in BSEP mRNA content, decreases in the intracellular concentrations of the model bile acid, d 8-TCA, were observed in SCHH following CDCA exposure. Overall, these data suggest that basolateral efflux of bile acids via OSTa/b is a potentially important compensatory mechanism to prevent cholestatic hepatotoxicity.

Research paper thumbnail of The use of cryopreserved and fresh human hepatocytes and human liver microsomes for the in vitro evaluation of Boswellia serrata extract (BSE) as an inhibitor of CYP450

Research paper thumbnail of Prediction of Hepatic Efflux Transporter-Mediated Drug Interactions: When Is it Optimal to Measure Intracellular Unbound Fraction of Inhibitors?

Journal of Pharmaceutical Sciences

The intracellular unbound inhibitor concentration ([I] unbound,cell) is the most relevant concent... more The intracellular unbound inhibitor concentration ([I] unbound,cell) is the most relevant concentration for predicting the inhibition of hepatic efflux transporters. However, the intracellular unbound fraction of inhibitor in hepatocytes (f u,cell,inhibitor) is not routinely determined. Studies are needed to evaluate the benefit of measuring f u,cell,inhibitor and using [I] unbound,cell versus intracellular total inhibitor concentration ([I] total,cell) when predicting inhibitory effects. This study examined the benefit of using [I] unbound,cell to predict hepatocellular bile acid disposition. Cellular total concentrations of taurocholate ([TCA] total,cell), a prototypical bile acid, were simulated using pharmacokinetic parameters estimated from sandwichcultured human hepatocytes. The effect of various theoretical inhibitors was simulated by varying ([I] total,cell / half maximal inhibitory concentration [IC 50 ]) values. In addition, the fold change was calculated as the simulated [TCA] total,cell when f u,cell,inhibitor ¼ 1 divided by the simulated [TCA] total,cell when f u,cell,inhibitor ¼ 0.5-0.01. The lowest ([I] total,cell /IC 50) value leading to a >2-fold change in [TCA] total,cell was chosen as a cutoff, and a framework was developed to categorize risk inhibitors for which the measurement of f u,cell,inhibitor is optimal. Fifteen compounds were categorized, 5 of which were compared with experimental observations. Future work is needed to evaluate this framework based on additional experimental data. In conclusion, the benefit of measuring f u,cell,inhibitor to predict hepatic efflux transporteremediated drug-bile acid interactions can be determined a priori.

Research paper thumbnail of Obeticholic acid, a selective farnesoid X receptor agonist, regulates bile acid homeostasis in sandwich-cultured human hepatocytes

Pharmacology research & perspectives, 2017

Farnesoid X receptor (FXR) is a master regulator of bile acid homeostasis through transcriptional... more Farnesoid X receptor (FXR) is a master regulator of bile acid homeostasis through transcriptional regulation of genes involved in bile acid synthesis and cellular membrane transport. Impairment of bile acid efflux due to cholangiopathies results in chronic cholestasis leading to abnormal elevation of intrahepatic and systemic bile acid levels. Obeticholic acid (OCA) is a potent and selective FXR agonist that is 100-fold more potent than the endogenous ligand chenodeoxycholic acid (CDCA). The effects of OCA on genes involved in bile acid homeostasis were investigated using sandwich-cultured human hepatocytes. Gene expression was determined by measuring mRNA levels. OCA dose-dependently increased fibroblast growth factor-19 (FGF-19) and small heterodimer partner (SHP) which, in turn, suppress mRNA levels of cholesterol 7-alpha-hydroxylase (CYP7A1), the rate-limiting enzyme for de novo synthesis of bile acids. Consistent with CYP7A1 suppression, total bile acid content was decreased by...

Research paper thumbnail of Prediction of Clinically Relevant Herb-Drug Clearance Interactions Using a Whole Cell Approach: Schisandra sphenanthera case study

Drug metabolism and disposition: the biological fate of chemicals, Sep 11, 2017

The Schisandraceae family is reported to have a range of pharmacological activities, including an... more The Schisandraceae family is reported to have a range of pharmacological activities, including anti-inflammatory effects. As with all herbal preparations, extracts of Schisandra species are mixtures composed of >50 lignans including schizandrins and deoxyschizandrins. In China, Schisandra sphenanthera extract (SSE) is often co-administered with immunosuppressant treatment of transplant recipients. In cases of co-administration, the potential for herb-drug interactions (HDI) increases. Clinical studies have been employed to assess HDI of extracts including SSE. Clinical results demonstrated that chronic SSE administration reduced midazolam (MDZ) clearance by 55.4% in healthy volunteers. While clinical studies are definitive and considered the "gold standard", these studies are impractical for routine HDI assessments. Alternatively, in vitro strategies can be utilized to reduce the need for clinical studies. Transporter Certified™ human hepatocytes in sandwich-culture (SC...

Research paper thumbnail of Assessing Potential Herb-Drug Interactions Using a Common Framework Approach: Boswellia serrata as a Case Study

Research paper thumbnail of d 8 -Taurocholate (d 8 -TCA) biliary efflux (A) and clearance (B) in sandwich-cultured human hepatocytes exposed to ambrisentan, bosentan, and macitentan

Research paper thumbnail of Increased oral bioavailability of paclitaxel by GF120918 in mice through selective modulation of P-glycoprotein

Clinical Cancer Research an Official Journal of the American Association For Cancer Research, Nov 1, 2000

Previous studies in mice with disrupted mdr1a P-glycoprotein genes have shown that the oral bioav... more Previous studies in mice with disrupted mdr1a P-glycoprotein genes have shown that the oral bioavailability of paclitaxel is very low because of the presence of this drugtransporting protein in the intestinal wall. Additional studies with cyclosporin A have shown that this P-glycoproteininhibiting agent is able to increase the bioavailability of paclitaxel in mouse models and in patients. However, the potential immune-suppressive side effects of cyclosporin A renders this compound less suitable for chronic use in cancer patients. In this paper we present the results obtained with GF120918, an experimental P-glycoprotein inhibitor, on the oral bioavailability of paclitaxel in both wild-type and mdr1ab knockout mice. GF120918 (25 mg/kg) was administered p.o. by gavage 15 min or 2 h before oral or i.v. dosing of paclitaxel, respectively. Paclitaxel plasma levels were quantified by high-performance liquid chromatography. GF120918 increased the plasma values for areas under the concentration-time curve of oral paclitaxel in wild-type mice by 6.6-fold from 408 to 2701 ng ⅐ ml ؊1 ⅐ h. Calculated relative to their respective values for area under the concentrationtime curve after i.v. administration, GF120918 increased the oral bioavailability of paclitaxel in wild-type mice from 8.5 to 40.2%. The plasma pharmacokinetics of paclitaxel in mdr1ab knockout mice was not altered by GF120918, whereas the pharmacokinetics of paclitaxel in wild-type mice receiving GF120918 became comparable with mdr1ab knockout mice. This result indicates that GF120918 at this dose-level selectively and completely blocks P-glycoprotein in the intestines and does not notably interfere in the elimination of paclitaxel by metabolism or other transporters. On the basis of this result, GF120918 has been selected for additional study in humans.

Research paper thumbnail of P126 - Observed vs. predicted effects of protein binding: Improving in vivo predictions of hepatic clearance, intracellular concentration and IC50 values

Drug Metabolism and Pharmacokinetics, 2020

Research paper thumbnail of 120918 , a P-Glycoprotein Modulator , Increases the Concentration of Unbound Amprenavir in the Central Nervous System in Rats

GF120918, a P-Glycoprotein Modulator, Increases the Concentration of Unbound Amprenavir in the Ce... more GF120918, a P-Glycoprotein Modulator, Increases the Concentration of Unbound Amprenavir in the Central Nervous System in Rats Jeffrey E. Edwards, Kenneth R. Brouwer, and Patrick J. McNamara* Graduate Center for Toxicology and Department of Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky, and Bioanalysis and Drug Metabolism, Glaxo Wellcome, Inc., Research Triangle Park, North Carolina

Research paper thumbnail of Methodes et compositions destinees a accroitre la penetration des inhibiteurs de la protease du vih

L'invention concerne des methodes destinees a accroitre la penetration de composes inhibant l... more L'invention concerne des methodes destinees a accroitre la penetration de composes inhibant la protease du VIH dans les tissus exprimant la glycoproteine-P.

Research paper thumbnail of Use of Ca 2 1 Modulation to Evaluate Biliary Excretion in Sandwich-Cultured Rat Hepatocytes 1

Previous work in our laboratory has indicated that biliary excretion of a substrate in sandwich-c... more Previous work in our laboratory has indicated that biliary excretion of a substrate in sandwich-cultured hepatocytes can be quantitated by measurement of substrate accumulation in the presence and absence of extracellular Ca. The present study was designed to examine the effects of Ca on taurocholate accumulation and tight junction integrity in cultured hepatocytes. Kinetic modeling was used to characterize taurocholate disposition in the hepatocyte monolayers in the presence and absence of extracellular Ca. The accumulation of taurocholate in freshly isolated hepatocytes, which lack an intact canalicular network, was the same in the presence and absence of extracellular Ca. Electron microscopy studies showed that Ca depletion increased the permeability of the tight junctions to ruthenium red, demonstrating that tight junctions were the major diffusional barrier between the canalicular lumen and the extracellular space. Cell morphology and substrate accumulation studies in the monol...

Research paper thumbnail of Abstract 2943: Severe hepatotoxicity of mithramycin therapy caused by altering expression of hepatocellular bile transporters

Experimental and Molecular Therapeutics

Research paper thumbnail of Cholestatic Drug Induced Liver Injury: A Function of Bile Salt Export Pump Inhibition and Farnesoid X Receptor Antagonism

Applied In Vitro Toxicology

Introduction: In vitro bile salt export pump (BSEP) inhibition alone does not accurately predict ... more Introduction: In vitro bile salt export pump (BSEP) inhibition alone does not accurately predict in vivo drug-induced liver injury in humans, suggesting that other mechanisms may be involved. Inhibition of BSEP has been shown to increase the hepatocellular concentrations of bile acids (BAs), and hepatobiliary disposition of BAs is tightly regulated by the farnesoid X receptor (FXR). Activation of FXR leads to decreased synthesis of BAs and increased expression of BA efflux transporters, BSEP, and organic solute and steroid transporter (OSTa/b). The link between BSEP inhibition and activation of the BA compensatory mechanism has not been clearly demonstrated. Materials and Methods: Therefore, we utilized BSEP inhibitors, cyclosporine A (CsA) and troglitazone (Trog), to explore this ''triggering'' event using Transporter CertifiedÔ sandwich-cultured human hepatocytes (SCHH). Results: Biliary excretion of glycine-cholic acid (GCA) as a percentage of total GCA accumulation was reduced in SCHH treated with either CsA or Trog. Within 12 hours, CsA treatment concomitantly increased intracellular concentrations (ICCs) of GCA and FGF19 mRNA content, an FXR-target gene. Separately, a synergistic 76.6fold increase of OSTb mRNA was observed following CsA and chenodeoxycholic acid co-exposure. In contrast, Trog exposure prevented the synergistic increase of OSTb, a component of the basolateral BA efflux transporter, under the same conditions. Finally, BA toxicity potency was increased in SCHH exposed to Trog but not CsA. Discussion and Conclusions: Overall, these results suggested that BSEP inhibition activates the BA compensatory mechanism reducing ICCs of BAs. However, Trog possesses both BSEP inhibition and FXR antagonist properties blocking the activation of the compensatory mechanism resulting in BA-induced hepatotoxicity.

Research paper thumbnail of An Evaluation of Potential Inhibition of CYP3A4/5 and CYP2C9 Enzymatic Activity by Boswellia serrata Extract

Applied In Vitro Toxicology

Abstract Introduction: As the use of dietary supplements increases, botanical–drug interaction (B... more Abstract Introduction: As the use of dietary supplements increases, botanical–drug interaction (BDI) potentials should be evaluated. Boswellia serrata extract (BSE) is traditionally used as an anti...

Research paper thumbnail of An integrated in vitro screen using sandwich-cultured human hepatocytes for prediction of cholestatic hepatotoxicity

Drug Metabolism and Pharmacokinetics

Research paper thumbnail of Can BSEP Inhibition Testing In Drug Discovery And Development Reduce Liver Injury Risk? - An International Transporter Consortium Perspective

Clinical pharmacology and therapeutics, Jan 23, 2018

Bile Salt Export Pump (BSEP) inhibition has emerged as an important mechanism that may contribute... more Bile Salt Export Pump (BSEP) inhibition has emerged as an important mechanism that may contribute to the initiation of human drug-induced liver injury (DILI). Proactive evaluation and understanding of BSEP inhibition is recommended in drug discovery and development, to aid internal decision making on DILI risk. BSEP inhibition can be quantified using in vitro assays. When interpreting assay data, it is important to consider in vivo drug exposure. Currently, this can be undertaken most effectively by consideration of total C . However, since total drug concentrations are not predictive of pharmacological effect, the relationship between total exposure and BSEP inhibition is not causal. Various follow up studies can aid interpretation of in vitro BSEP inhibition data and may be undertaken on a case by case basis. BSEP inhibition is one of several mechanisms by which drugs may cause DILI, therefore should be considered alongside other mechanisms when evaluating possible DILI risk. This...

Research paper thumbnail of Farnesoid X Receptor Agonists Obeticholic Acid and Chenodeoxycholic Acid Increase Bile Acid Efflux in Sandwich-Cultured Human Hepatocytes: Functional Evidence and Mechanisms

The Journal of pharmacology and experimental therapeutics, 2018

The farnesoid X receptor (FXR) is a nuclear receptor that regulates genes involved in bile acid h... more The farnesoid X receptor (FXR) is a nuclear receptor that regulates genes involved in bile acid homeostasis. FXR agonists, obeticholic acid (OCA) and chenodeoxycholic acid (CDCA), increase mRNA expression of efflux transporters in sandwich-cultured human hepatocytes (SCHH). This study evaluated the effects of OCA and CDCA treatment on the uptake, basolateral efflux, and biliary excretion of a model bile acid, taurocholate (TCA), in SCHH. In addition, changes in the protein expression of TCA uptake and efflux transporters were investigated. SCHH were treated with 1 M OCA, 100 M CDCA, or vehicle control for 72 hours followed by quantification of deuterated TCA uptake and efflux over time in Ca-containing and Ca-free conditions ( = 3 donors). A mechanistic pharmacokinetic model was fit to the TCA mass-time data to obtain estimates for total uptake clearance (CL), total intrinsic basolateral efflux clearance (CL), and total intrinsic biliary clearance (CL). Modeling results revealed tha...

Research paper thumbnail of Transient Changes in Hepatic Physiology That Alter Bilirubin and Bile Acid Transport May Explain Elevations in Liver Chemistries Observed in Clinical Trials of GGF2 (Cimaglermin alfa)

Toxicological sciences : an official journal of the Society of Toxicology, Jan 23, 2017

GGF2 is a recombinant human neuregulin-1β in development for chronic heart failure. Phase 1 clini... more GGF2 is a recombinant human neuregulin-1β in development for chronic heart failure. Phase 1 clinical trials of GGF2 were put on hold when transient elevations in serum aminotransferases and total bilirubin were observed in 2 of 43 subjects who received single doses of GGF2 at 1.5 or 0.378 mg/kg. However, aminotransferase elevations were modest and not typical of liver injury sufficient to result in elevated serum bilirubin. Cynomolgus monkeys administered a single 15 mg/kg dose of GGF2 had similar transient elevations in serum aminotransferases and bilirubin as well as transient elevations in serum bile acids. However, no hepatocellular necrosis was observed in liver biopsies obtained during peak elevations. When sandwich-cultured human hepatocytes were treated with GGF2 for up to 72 h at concentrations ∼0.8-fold average plasma C max for the 0.378 mg/kg dose, no cytotoxicity was observed. Gene expression profiling identified ∼50% reductions in mRNAs coding for bilirubin transporters...

Research paper thumbnail of Evidence of FXR Activation with Obeticholic Acid in an in Vitro Intestinal Model

Research paper thumbnail of Basolateral Efflux Transporters: A Potentially Important Pathway for the Prevention of Cholestatic Hepatotoxicity

Applied In Vitro Toxicology

The hepatocyte adaptive response has been postulated to play a protective role in cholestatic dis... more The hepatocyte adaptive response has been postulated to play a protective role in cholestatic disease states, including primary biliary cirrhosis where bile acid biliary efflux is reduced. Regulation of bile acid synthesis by farnesoid X receptor (FXR) has been well defined; however, bile acid transport has not. Utilizing sandwichcultured human hepatocytes (SCHH) and B-CLEAR Ò technology, we demonstrated that basolateral efflux and not canalicular efflux (bile salt export pump [BSEP]) was the primary driver of bile acid intracellular accumulation. Following 72 hours of exposure to CDCA, decreases of total endogenous bile acid mass and CYP7A1 mRNA content were observed in SCHH consistent with FXR activation. No marked changes were observed in mRNA content of bile acid uptake transporters, however, induction of bile acid efflux transporters OSTa (3.1-6.8 •), OSTb (21-187 •), and BSEP (2.2-7.5 •) mRNA content was observed. While decreases of d8-TCA biliary clearance were inconsistent with the increases in BSEP mRNA content, decreases in the intracellular concentrations of the model bile acid, d 8-TCA, were observed in SCHH following CDCA exposure. Overall, these data suggest that basolateral efflux of bile acids via OSTa/b is a potentially important compensatory mechanism to prevent cholestatic hepatotoxicity.

Research paper thumbnail of The use of cryopreserved and fresh human hepatocytes and human liver microsomes for the in vitro evaluation of Boswellia serrata extract (BSE) as an inhibitor of CYP450

Research paper thumbnail of Prediction of Hepatic Efflux Transporter-Mediated Drug Interactions: When Is it Optimal to Measure Intracellular Unbound Fraction of Inhibitors?

Journal of Pharmaceutical Sciences

The intracellular unbound inhibitor concentration ([I] unbound,cell) is the most relevant concent... more The intracellular unbound inhibitor concentration ([I] unbound,cell) is the most relevant concentration for predicting the inhibition of hepatic efflux transporters. However, the intracellular unbound fraction of inhibitor in hepatocytes (f u,cell,inhibitor) is not routinely determined. Studies are needed to evaluate the benefit of measuring f u,cell,inhibitor and using [I] unbound,cell versus intracellular total inhibitor concentration ([I] total,cell) when predicting inhibitory effects. This study examined the benefit of using [I] unbound,cell to predict hepatocellular bile acid disposition. Cellular total concentrations of taurocholate ([TCA] total,cell), a prototypical bile acid, were simulated using pharmacokinetic parameters estimated from sandwichcultured human hepatocytes. The effect of various theoretical inhibitors was simulated by varying ([I] total,cell / half maximal inhibitory concentration [IC 50 ]) values. In addition, the fold change was calculated as the simulated [TCA] total,cell when f u,cell,inhibitor ¼ 1 divided by the simulated [TCA] total,cell when f u,cell,inhibitor ¼ 0.5-0.01. The lowest ([I] total,cell /IC 50) value leading to a >2-fold change in [TCA] total,cell was chosen as a cutoff, and a framework was developed to categorize risk inhibitors for which the measurement of f u,cell,inhibitor is optimal. Fifteen compounds were categorized, 5 of which were compared with experimental observations. Future work is needed to evaluate this framework based on additional experimental data. In conclusion, the benefit of measuring f u,cell,inhibitor to predict hepatic efflux transporteremediated drug-bile acid interactions can be determined a priori.

Research paper thumbnail of Obeticholic acid, a selective farnesoid X receptor agonist, regulates bile acid homeostasis in sandwich-cultured human hepatocytes

Pharmacology research & perspectives, 2017

Farnesoid X receptor (FXR) is a master regulator of bile acid homeostasis through transcriptional... more Farnesoid X receptor (FXR) is a master regulator of bile acid homeostasis through transcriptional regulation of genes involved in bile acid synthesis and cellular membrane transport. Impairment of bile acid efflux due to cholangiopathies results in chronic cholestasis leading to abnormal elevation of intrahepatic and systemic bile acid levels. Obeticholic acid (OCA) is a potent and selective FXR agonist that is 100-fold more potent than the endogenous ligand chenodeoxycholic acid (CDCA). The effects of OCA on genes involved in bile acid homeostasis were investigated using sandwich-cultured human hepatocytes. Gene expression was determined by measuring mRNA levels. OCA dose-dependently increased fibroblast growth factor-19 (FGF-19) and small heterodimer partner (SHP) which, in turn, suppress mRNA levels of cholesterol 7-alpha-hydroxylase (CYP7A1), the rate-limiting enzyme for de novo synthesis of bile acids. Consistent with CYP7A1 suppression, total bile acid content was decreased by...

Research paper thumbnail of Prediction of Clinically Relevant Herb-Drug Clearance Interactions Using a Whole Cell Approach: Schisandra sphenanthera case study

Drug metabolism and disposition: the biological fate of chemicals, Sep 11, 2017

The Schisandraceae family is reported to have a range of pharmacological activities, including an... more The Schisandraceae family is reported to have a range of pharmacological activities, including anti-inflammatory effects. As with all herbal preparations, extracts of Schisandra species are mixtures composed of >50 lignans including schizandrins and deoxyschizandrins. In China, Schisandra sphenanthera extract (SSE) is often co-administered with immunosuppressant treatment of transplant recipients. In cases of co-administration, the potential for herb-drug interactions (HDI) increases. Clinical studies have been employed to assess HDI of extracts including SSE. Clinical results demonstrated that chronic SSE administration reduced midazolam (MDZ) clearance by 55.4% in healthy volunteers. While clinical studies are definitive and considered the "gold standard", these studies are impractical for routine HDI assessments. Alternatively, in vitro strategies can be utilized to reduce the need for clinical studies. Transporter Certified™ human hepatocytes in sandwich-culture (SC...

Research paper thumbnail of Assessing Potential Herb-Drug Interactions Using a Common Framework Approach: Boswellia serrata as a Case Study

Research paper thumbnail of d 8 -Taurocholate (d 8 -TCA) biliary efflux (A) and clearance (B) in sandwich-cultured human hepatocytes exposed to ambrisentan, bosentan, and macitentan

Research paper thumbnail of Increased oral bioavailability of paclitaxel by GF120918 in mice through selective modulation of P-glycoprotein

Clinical Cancer Research an Official Journal of the American Association For Cancer Research, Nov 1, 2000

Previous studies in mice with disrupted mdr1a P-glycoprotein genes have shown that the oral bioav... more Previous studies in mice with disrupted mdr1a P-glycoprotein genes have shown that the oral bioavailability of paclitaxel is very low because of the presence of this drugtransporting protein in the intestinal wall. Additional studies with cyclosporin A have shown that this P-glycoproteininhibiting agent is able to increase the bioavailability of paclitaxel in mouse models and in patients. However, the potential immune-suppressive side effects of cyclosporin A renders this compound less suitable for chronic use in cancer patients. In this paper we present the results obtained with GF120918, an experimental P-glycoprotein inhibitor, on the oral bioavailability of paclitaxel in both wild-type and mdr1ab knockout mice. GF120918 (25 mg/kg) was administered p.o. by gavage 15 min or 2 h before oral or i.v. dosing of paclitaxel, respectively. Paclitaxel plasma levels were quantified by high-performance liquid chromatography. GF120918 increased the plasma values for areas under the concentration-time curve of oral paclitaxel in wild-type mice by 6.6-fold from 408 to 2701 ng ⅐ ml ؊1 ⅐ h. Calculated relative to their respective values for area under the concentrationtime curve after i.v. administration, GF120918 increased the oral bioavailability of paclitaxel in wild-type mice from 8.5 to 40.2%. The plasma pharmacokinetics of paclitaxel in mdr1ab knockout mice was not altered by GF120918, whereas the pharmacokinetics of paclitaxel in wild-type mice receiving GF120918 became comparable with mdr1ab knockout mice. This result indicates that GF120918 at this dose-level selectively and completely blocks P-glycoprotein in the intestines and does not notably interfere in the elimination of paclitaxel by metabolism or other transporters. On the basis of this result, GF120918 has been selected for additional study in humans.