In Vitro Investigation of Amyloid-<i>β</i> Hepatobiliary Disposition in Sandwich-Cultured Primary Rat Hepatocytes

Mohamed, Loqman A.; Kaddoumi, Amal

doi:10.1124/dmd.113.052514

Cited by 21 publications

(29 citation statements)

References 51 publications

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“…Hence, inhibition of P-gp efflux function by valspodar resulted in more intracellular accumulation of tacrine compared to untreated control. The inhibitory effect of valspodar on P-gp function in SCHs is consistent with our previous report (23). A previous work, however, by Doan and colleagues demonstrated that tacrine is not a P-gp substrate based on permeability screening studies for 93 compounds performed on MDR1-transfected MDCK type II cells (38).…”

Section: Discussionsupporting

confidence: 91%

“…Rats age were around 2-3 months with average body weight between 260 to 340 g. Animals were allowed to easy access for water and standard food and maintained at 22 Isolation of primary rat hepatocytes and preparation of sandwich culture The isolation of hepatocytes from animals' livers were performed as previously described (23). After isolation of hepatocytes by several steps of centrifugation, cells were resuspended in serum-free medium and viability was determined using trypan blue method.…”

Section: Animalsmentioning

confidence: 99%

“…Time and concentration-dependent studies of tacrine were determined in day 1 of SCHs (no canalicular spaces were formed thus only uptake process can be investigated) (23). To determine the optimum time for tacrine uptake, SCHs were first gently washed with standard HBSS twice and then incubated with 1 µM tacrine for designated time points (2, 5, 10 and 30 min).…”

Section: Time and Concentration-dependent Uptake Of Tacrine In Schsmentioning

confidence: 99%

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Tacrine Sinusoidal Uptake and Biliary Excretion in Sandwich-Cultured Primary Rat Hepatocytes

Mohamed

Kaddoumi

2014

J Pharm Pharm Sci

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-PURPOSE. The knowledge of hepatic disposition kinetics of tacrine, a first cholinesterase inhibitor was approved by FDA for the treatment of Alzheimer's disease (AD), would help to understand its hepatotoxicity, its therapeutic effect, and improve the management of patients with AD. The current study aims to characterize tacrine hepatic transport kinetics and study the role of organic cation transporters (OCTs), Pglycoprotein (P-gp) and multidrug resistance-associated protein (MRP2) in tacrine sinusoidal uptake and biliary excretion. METHODS. Modulation of tacrine hepatic uptake and efflux, biliary excretion index (BEI%), were performed in sandwich-cultured primary rat hepatocytes (SCHs) using transporters inhibitors. Conformation of the integrity of SCHs model was established by capturing images with light-contrast and fluorescence microscopy. RESULTS. Tacrine uptake in SCHs was carrier-mediated process and saturable with apparent K m of 31.5±9.6 µM and V max of 908±72 pmol/min/mg protein. Tetraethyl ammonium (TEA), cimetidine and verapamil significantly reduced tacrine uptake with more pronounced effect observed with verapamil which caused 3-fold reduction in tacrine uptake, indicating role for OCTs. Tacrine has a biliary excretion in SCHs with maximum BEI% value of 22.9±1.9% at 10 min of incubation. Addition of MK571 and valspodar decreased the BEI% of tacrine by 40 and 60% suggesting roles for canalicular MRP2 and P-gp, respectively. CONCLUSIONS. Our results show that in addition to metabolism, tacrine hepatic disposition is carrier-mediated process mediated by sinusoidal OCTs, and canalicular MRP2 and P-gp.

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Section: Discussionsupporting

confidence: 91%

Section: Animalsmentioning

confidence: 99%

Section: Time and Concentration-dependent Uptake Of Tacrine In Schsmentioning

confidence: 99%

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Tacrine Sinusoidal Uptake and Biliary Excretion in Sandwich-Cultured Primary Rat Hepatocytes

Mohamed

Kaddoumi

2014

J Pharm Pharm Sci

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“…gp, and MRP2, respectively (Wolf et al, 2008 ± 8%, respectively, which is in the accepted range for hepatocytes with functionally active bile canalicular efflux transporters (Wolf et al, 2008;Mohamed and Kaddoumi, 2013).…”

Section: Biliary Clearance In Rat Schmentioning

confidence: 84%

Hepatobiliary Disposition of Atovaquone: A Case of Mechanistically Unusual Biliary Clearance

Patel¹,

Johnson²,

Sychterz³

et al. 2018

J Pharmacol Exp Ther

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“…Cryopreserved primary human hepatocytes were revived according to the protocol and seeded in 96-well collagen I-coated plates (BD Gentest) at a density of 3 Â 10 5 cells/ml. Primary rat hepatocytes were prepared with a previously published two-step collagenase perfusion technique with the following modifications: the perfusion buffer and digestion buffer flow rates were set to a constant 17.5 and 15.0 ml/min, respectively; collagenase type II was used instead of type I (Seglen, 1976;Mohamed and Kaddoumi, 2013;Shukla et al, 2013). The fresh primary rat hepatocytes were also seeded in the 96-well collagen I-coated plates at a density of 3 Â 10 5 cells/ml.…”

Section: Methodsmentioning

confidence: 99%

Bioactivation of 3-n-Butylphthalide via Sulfation of Its Major Metabolite 3-Hydroxy-NBP: Mediated Mainly by Sulfotransferase 1A1

et al. 2014

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3-n-Butylphthalide (NBP) [(6)-3-butyl-1(3H)-isobenzofuranone] is an anti-cerebral-ischemia drug. Moderate hepatotoxicity has been observed in clinical applications. One of the major metabolites, 3-N-acetylcysteine-NBP, has been detected in human urine, indicating the formation of a reactive metabolite. We elucidated the formation mechanism of the reactive metabolite and its association with the hepatotoxicity of NBP. The in vitro incubations revealed that 3-glutathione-NBP (3-GSH-NBP) was observed only in fresh rat liver homogenate rather than in liver microsomes, liver cytosol, or liver 9,000g supernatant supplemented with NADPH and GSH. We also detected 3-GSH-NBP when 39-phosphoadenosine-59-phosphosulfate was added in GSH-fortified human liver cytosol (HLC). The formation of 3-GSH-NBP was 39.3-fold higher using 3-hydroxy-NBP (3-OH-NBP) as the substrate than NBP. The sulfotransferase (SULT) inhibitors DCNP (2,6-dichloro-4-nitrophenol) and quercetin suppressed 3-GSH-NBP formation in HLC by 75 and 82%, respectively, suggesting that 3-OH-NBP sulfation was involved in 3-GSH-NBP formation. Further SULT phenotyping revealed that SULT1A1 is the major isoform responsible for the sulfation. Dose-dependent toxicity was observed in primary rat hepatocytes exposed to 3-OH-NBP, with an IC 50 of approximately 168 mM. Addition of DCNP and quercetin significantly increased cell viability, whereas L-buthionine-sulfoximine (a GSH depleter) decreased cell viability. Overall, our study revealed the underlying mechanism for the bioactivation of NBP is as follows. NBP is first oxidized to 3-OH-NBP and further undergoes sulfation to form 3-OH-NBP sulfate. The sulfate spontaneously cleaves off, generating highly reactive electrophilic cations, which can bind either to GSH to detoxify or to hepatocellular proteins to cause undesirable side effects.

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In Vitro Investigation of Amyloid-β Hepatobiliary Disposition in Sandwich-Cultured Primary Rat Hepatocytes

Cited by 21 publications

References 51 publications

Tacrine Sinusoidal Uptake and Biliary Excretion in Sandwich-Cultured Primary Rat Hepatocytes

Tacrine Sinusoidal Uptake and Biliary Excretion in Sandwich-Cultured Primary Rat Hepatocytes

Hepatobiliary Disposition of Atovaquone: A Case of Mechanistically Unusual Biliary Clearance

Bioactivation of 3-n-Butylphthalide via Sulfation of Its Major Metabolite 3-Hydroxy-NBP: Mediated Mainly by Sulfotransferase 1A1

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