Application of substrate depletion assay for early prediction of nonlinear pharmacokinetics in drug discovery: Assessment of nonlinearity of metoprolol, timolol, and propranolol

Kono, Hiroshi; Kawase, Atsushi; Iwaki, Masaya

doi:10.1002/jps.20490

Cited by 15 publications

(14 citation statements)

References 28 publications

(36 reference statements)

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“…To date, although numerous studies have been conducted to investigate the inhibitory effect of parent NSAIDs on OATs, RFC-1, MRPs and BCRP-mediated MTX transport, 10,14,15,17,28) unbound plasma concentration of NSAIDs were too low to cause an interaction between MTX and NSAIDs at clinical doses. 15,33,34) Our recent study suggested that NSAIDs-Glu inhibit MRP2 and MRP4-mediated MTX efflux more strongly than parent NSAIDs.…”

Section: Resultsmentioning

confidence: 99%

“…Preparation of NSAIDs-Glu Rat liver microsomes were prepared according to the method previously reported. 28,29) β-1-O-glucuronides of NSAIDs were prepared biosynthetically in vitro from respective NSAIDs using rat liver microsomes according to the published method. 25,30) The purity of the glucuronides obtained was determined by HPLC at a UV wavelength of 254 nm, with the remaining fraction consisting of polar impurities that did not yield the respective parent drugs.…”

Section: Methodsmentioning

confidence: 99%

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Inhibition of Methotrexate Uptake <i>via</i> Organic Anion Transporters OAT1 and OAT3 by Glucuronides of Nonsteroidal Anti-inflammatory Drugs

Iwaki

Shimada

Irino

et al. 2017

Biological & Pharmaceutical Bulletin

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Combination therapy of non-steroidal anti-inflammatory drugs (NSAIDs) and methotrexate (MTX) sometimes triggers adverse effects, such as liver injury, renal failure, gastrointestinal disorders, and myelosuppression, owing to the reduction of MTX clearance. Previous reports have suggested that NSAIDs inhibit renal MTX uptake via organic anion transporters (OATs) and reduced folate transporter (RFC)-1 and efflux via multidrug resistance-associated proteins (MRPs). Recently, our laboratory found inhibitory effects of NSAIDs-glucuronide (NSAIDs-Glu), a major metabolite of NSAIDs, on MRP-mediated MTX transport as a new site of interaction between MTX and NSAIDs. However, it remains unclear that whether NSAIDsGlu inhibit renal uptake of MTX. Therefore, the present study aimed to evaluate inhibitory effects of several NSAIDs-Glu (diclofenac, R-and S-ibuprofen, R-and S-flurbiprofen, and R-and S-naproxen) on human OAT1 and OAT3-mediated MTX transport. In this study, [ 3 H]MTX uptake was observed by using human OAT1 and OAT3-overexpressing HEK293 cells in the presence or absence of NSAIDs-Glu. All examined NSAIDs-Glu exhibited concentration-dependent inhibitory effects on MTX uptake via OAT1 and OAT3. Our results indicated that NSAIDs-Glu are more potent (5-to 15-fold) inhibitors of OAT3 than OAT1. Moreover, stereoselective inhibitory effects of NSAIDs-Glu on OATs-mediated MTX uptake were not observed, unlike on MRPs-mediated transport. These findings suggest that inhibition of OAT1 and OAT3-mediated renal uptake of MTX by plasma NSAIDs-Glu may be one of the competitive sites underlying complex drug interaction between MTX and NSAIDs.Key words drug interaction; methotrexate; non-steroidal anti-inflammatory drug; glucuronide; organic anion transporter Although methotrexate (MTX) is often co-administered with non-steroidal anti-inflammatory drugs (NSAIDs) to patients suffering from rheumatoid arthritis and neoplastic diseases, several NSAIDs, such as ketoprofen, indomethacin, naproxen, and diclofenac, reduce MTX clearance. Thus, combined administration of MTX and NSAIDs can result in elevated plasma MTX concentrations and severe adverse effects, such as liver injury, renal failure, gastrointestinal disorders, and myelosuppression. [1][2][3][4][5] In fact, the MTX prescribers' information states that combined use of MTX and NSAIDs may increase plasma MTX concentrations and adverse effects.MTX is primarily excreted in urine via glomerular filtration and active tubular secretion in its unchanged form.6) At a clinical plasma concentration of MTX (0.2-10 µM), approximately 25% of renal clearance is excreted by glomerular filtration and the remaining 75% is excreted by active tubular secretion. 7)In the tubular secretory process, organic anion transporters (OAT) 1 and 3 and reduced folate carrier (RFC)-1 transport MTX from the blood across the basolateral membrane. [8][9][10][11] Multidrug resistance-related proteins (MRP) 2 and 4 and breast cancer-resistant protein (BCRP) transport MTX across the apical membrane into the urine w...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Inhibition of Methotrexate Uptake <i>via</i> Organic Anion Transporters OAT1 and OAT3 by Glucuronides of Nonsteroidal Anti-inflammatory Drugs

Iwaki

Shimada

Irino

et al. 2017

Biological & Pharmaceutical Bulletin

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“…Measurement of intrinsic clearance by substrate depletion is favored over metabolite formation kinetics for routine assay, reflecting a need for pathway inclusivity as well as expediency (Obach and Reed-Hagen 2002; Komura et al 2005; Mohutsky et al 2006; Soars et al 2007a). Although this approach seems appropriate for the primary hepatocyte model, the validity of the underlying assumptions, particularly regarding the combined impact of uptake clearance with metabolic clearance, remains under debate (Jones and Houston 2004, 2012).…”

Section: Use Of In Vitro Systems For Predicting Metabolism and Drug Imentioning

confidence: 99%

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME

Hewitt²,

et al. 2013

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This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell–derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-013-1078-5) contains supplementary material, which is available to authorized users.

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“…Fifty microlitres of samples were injected onto an end‐capped C18 column (Symmetry Shield ™ RP18, 5 µm, 250 × 4.6 mm; Waters Corp, Milford, MA, USA) and separated under isocratic elution with a mobile phase consisting of 53% of 80 mM ammonium acetate (pH: 2.5) and 47% of acetonitrile and methanol (1:1). Substrate depletion was monitored in incubate and control samples to determine the isoform of cytochrome P450 enzyme responsible for the metabolism of vinblastine …”

Section: Methodsmentioning

confidence: 99%

Reaction phenotyping of vinblastine metabolism in dogs

Achanta

Maxwell

2014

Vet Comparative Oncology

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Vinblastine is a vinca alkaloid used either as a single agent or in combination therapy for the treatment of canine mast cell tumours and lymphomas. The objective of this study was to determine which isoform of cytochrome P450 enzyme is responsible for the majority of vinblastine metabolism in dogs. A panel of eight recombinant canine cytochrome P450 enzymes (CYP1A1, CYP1A2, CYP3A12, CYP3A26, CYP2B11, CYP2C41, CYP2C21 and CYP2D15) were incubated in vitro with vinblastine. Findings were confirmed by the use of canine polyclonal antibodies of cytochrome P450 enzymes (CYP1A1, CYP3A12, CYP2B11 and CYP2C21) that were pre-incubated with individual and pooled hepatic microsomes that were purified from canine liver. Substrate depletion was observed in the presence of recombinant CYP3A12, whereas depletion did not substantially occur when microsomes were pre-incubated with polyclonal antibodies against CYP3A12. These findings confirmed that CYP3A12 is the major cytochrome P450 isoform responsible for the metabolism of vinblastine in dogs.

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Application of substrate depletion assay for early prediction of nonlinear pharmacokinetics in drug discovery: Assessment of nonlinearity of metoprolol, timolol, and propranolol

Cited by 15 publications

References 28 publications

Inhibition of Methotrexate Uptake <i>via</i> Organic Anion Transporters OAT1 and OAT3 by Glucuronides of Nonsteroidal Anti-inflammatory Drugs

Inhibition of Methotrexate Uptake <i>via</i> Organic Anion Transporters OAT1 and OAT3 by Glucuronides of Nonsteroidal Anti-inflammatory Drugs

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME

Reaction phenotyping of vinblastine metabolism in dogs

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