Validation of 96-well Equilibrium Dialysis with Non-radiolabeled Drug for Definitive Measurement of Protein Binding and Application to Clinical Development of Highly-Bound Drugs

Zamek‐Gliszczynski, Maciej J.; Ruterbories, Kenneth J.; Ajamie, Rose T.; Wickremsinhe, Enaksha; Pothuri, Lavanya; Rao, Mukkavilli V.S.; Basavanakatti, Vinay N.; Pinjari, Jakir; Ramanathan, Vikram; Chaudhary, Archana

doi:10.1002/jps.22452

Cited by 53 publications

(45 citation statements)

References 13 publications

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“…Rat plasma protein binding was determined in vitro using equilibrium dialysis, as described previously (Zamek-Gliszczynski et al, 2011). Rat liver S9 protein binding was determined in vitro following the same protocol and using 0.5 mg/ml liver S9 protein from SD and ZDF rats for the incubation for all compounds, except troglitazone and 7-hydroxycoumarin for which protein concentrations of 0.25 and 0.4 mg/ml were used to mimic assay conditions.…”

Section: Methodsmentioning

confidence: 99%

Difference in the Pharmacokinetics and Hepatic Metabolism of Antidiabetic Drugs in Zucker Diabetic Fatty and Sprague-Dawley Rats

Zhou¹,

Rougée²,

Bedwell³

et al. 2016

Drug Metabolism and Disposition

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The Zucker diabetic fatty (ZDF) rat, an inbred strain of obese Zucker fatty rat, develops early onset of insulin resistance and displays hyperglycemia and hyperlipidemia. The phenotypic changes resemble human type 2 diabetes associated with obesity and therefore the strain is used as a pharmacological model for type 2 diabetes. The aim of the current study was to compare the pharmacokinetics and hepatic metabolism in male ZDF and Sprague-Dawley (SD) rats of five antidiabetic drugs that are known to be cleared via various mechanisms. Among the drugs examined, metformin, cleared through renal excretion, and rosiglitazone, metabolized by hepatic cytochrome P450 2C, did not exhibit differences in the plasma clearance in ZDF and SD rats. In contrast, glibenclamide, metabolized by hepatic CYP3A, canagliflozin, metabolized mainly by UDP-glucuronosyltransferases (UGT), and troglitazone, metabolized by sulfotransferase and UGT, exhibited significantly lower plasma clearance in ZDF than in SD rats after a single intravenous administration. To elucidate the mechanisms for the difference in the drug clearance, studies were performed to characterize the activity of hepatic drug-metabolizing enzymes using liver S9 fractions from the two strains. The results revealed that the activity for CYP3A and UGT was decreased in ZDF rats using the probe substrates, and decreased unbound intrinsic clearance in vitro for glibenclamide, canagliflozin, and troglitazone was consistent with lower plasma clearance in vivo. The difference in pharmacokinetics of these two strains may complicate pharmacokinetic/ pharmacodynamic correlations, given that ZDF is used as a pharmacological model, and SD rat as the pharmacokinetics and toxicology strain.

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Section: Methodsmentioning

confidence: 99%

Difference in the Pharmacokinetics and Hepatic Metabolism of Antidiabetic Drugs in Zucker Diabetic Fatty and Sprague-Dawley Rats

Zhou¹,

Rougée²,

Bedwell³

et al. 2016

Drug Metabolism and Disposition

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“…The determination of unbound compound was performed in human plasma and in plasma protein buffer solution (45 g/L human serum albumin and 0.7 g/L α 1 acid glycoprotein in phosphate buffer, pH 7.4, respectively) using a 96-well format equilibrium dialysis device with a molecular weight cutoff membrane of 12-14 kDa, as described elsewhere (16,17). The derived samples from each dialysis were then quenched with acetonitrile (ratio, 1:2) containing an internal standard, centrifuged, and quantified by LC-MS/MS.…”

Section: Plasma Protein Binding Determination and Binding To Plasma Pmentioning

confidence: 99%

“…[8][9][10][11][12][13][14][15][16]. For that, the fractions unbound in the incubation media and human plasma were experimentally determined and are listed in Table III together with the blood-to-plasma ratios.…”

Section: Ivive Of In Vitro Clearance Assessmentsmentioning

confidence: 99%

Metabolic Profiling of Human Long-Term Liver Models and Hepatic Clearance Predictions from In Vitro Data Using Nonlinear Mixed-Effects Modeling

Kratochwil

Meille

Fowler

et al. 2017

AAPS J

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ABSTRACT.Early prediction of human clearance is often challenging, in particular for the growing number of low-clearance compounds. Long-term in vitro models have been developed which enable sophisticated hepatic drug disposition studies and improved clearance predictions. Here, the cell line HepG2, iPSC-derived hepatocytes (iCell®), the hepatic stem cell line HepaRG™, and human hepatocyte co-cultures (HμREL™ and HepatoPac®) were compared to primary hepatocyte suspension cultures with respect to their key metabolic activities. Similar metabolic activities were found for the long-term models HepaRG™, HμREL™, and HepatoPac® and the short-term suspension cultures when averaged across all 11 enzyme markers, although differences were seen in the activities of CYP2D6 and non-CYP enzymes. For iCell® and HepG2, the metabolic activity was more than tenfold lower. The micropatterned HepatoPac® model was further evaluated with respect to clearance prediction. To assess the in vitro parameters, pharmacokinetic modeling was applied. The determination of intrinsic clearance by nonlinear mixed-effects modeling in a long-term model significantly increased the confidence in the parameter estimation and extended the sensitive range towards 3% of liver blood flow, i.e., >10-fold lower as compared to suspension cultures. For in vitro to in vivo extrapolation, the well-stirred model was used. The micropatterned model gave rise to clearance prediction in man within a twofold error for the majority of low-clearance compounds. Further research is needed to understand whether transporter activity and drug metabolism by non-CYP enzymes, such as UGTs, SULTs, AO, and FMO, is comparable to the in vivo situation in these long-term culture models.KEY WORDS: in vitro clearance; in vitro liver models; IVIVE; nonlinear mixed-effects modeling.

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“…Amyloid ␤ (A␤) plaques consist primarily of assemblies of a small 39 -43 aa peptide arising from the cleavage of APP. The amyloidogenic processing of APP is initiated by BACE1 (also known as ␤-secretase), an aspartyl protease that was cloned by several groups including Sinha et al (1999) and Vassar et al (1999). One of the familial forms of AD is caused by a double mutation, KM¡NL, at amino acids immediately adjacent to the BACE1 cleavage site (Mullan et al, 1992).…”

Section: Introductionmentioning

confidence: 98%

The Potent BACE1 Inhibitor LY2886721 Elicits Robust Central Aβ Pharmacodynamic Responses in Mice, Dogs, and Humans

et al. 2015

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BACE1 is a key protease controlling the formation of amyloid ␤, a peptide hypothesized to play a significant role in the pathogenesis of Alzheimer's disease (AD). Therefore, the development of potent and selective inhibitors of BACE1 has been a focus of many drug discovery efforts in academiaandindustry.Herein,wereportthenonclinicalandearlyclinicaldevelopmentofLY2886721,aBACE1activesiteinhibitorthatreached phase 2 clinical trials in AD. LY2886721 has high selectivity against key off-target proteases, which efficiently translates in vitro activity into robust in vivo amyloid ␤ lowering in nonclinical animal models. Similar potent and persistent amyloid ␤ lowering was observed in plasma and lumbar CSF when single and multiple doses of LY2886721 were administered to healthy human subjects. Collectively, these data add support for BACE1 inhibition as an effective means of amyloid lowering and as an attractive target for potential disease modification therapy in AD.

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Validation of 96-well Equilibrium Dialysis with Non-radiolabeled Drug for Definitive Measurement of Protein Binding and Application to Clinical Development of Highly-Bound Drugs

Cited by 53 publications

References 13 publications

Difference in the Pharmacokinetics and Hepatic Metabolism of Antidiabetic Drugs in Zucker Diabetic Fatty and Sprague-Dawley Rats

Difference in the Pharmacokinetics and Hepatic Metabolism of Antidiabetic Drugs in Zucker Diabetic Fatty and Sprague-Dawley Rats

Metabolic Profiling of Human Long-Term Liver Models and Hepatic Clearance Predictions from In Vitro Data Using Nonlinear Mixed-Effects Modeling

The Potent BACE1 Inhibitor LY2886721 Elicits Robust Central Aβ Pharmacodynamic Responses in Mice, Dogs, and Humans

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