Kinetic characterization of bile salt transport by human NTCP (SLC10A1)

Jani, Márton; Beéry, Erzsébet; Heslop, Teresa; Töth, Beáta; Jagota, Bhavana; Kis, Emese; Park, B. Kevin; Krajcsi, Péter; Weaver, Richard J.

doi:10.1016/j.tiv.2017.10.012

Cited by 19 publications

(23 citation statements)

References 24 publications

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“…Examination of human sodium/taurocholate cotransporting polypeptide (NTCP, encoded by SLC10A1) was determined based on uptake of tritium labeled TCA in CHO cells and human multidrug resistance‐associated protein 3/4 (MRP3/4, encoded by ABCC3/4) transporter inhibition in vesicles obtained from HEK293 cells using tritium labeled estradiol 17 β‐D‐glucuronide and dehydroepiandrosterone sulfate as substrates, respectively. Both assays were conducted at Solvo Biotechnology (Hungary) according to their protocols . Calculations for the Heuman index for hydrophobicity of bile acids in serum were based on the sums of bile acid hydrophobicity for each measured bile acid (AUC 0‐12 h) where larger values are considered a more hydrophobic environment than smaller values…”

Section: Methodsmentioning

confidence: 99%

“…Both assays were conducted at Solvo Biotechnology (Hungary) according to their protocols. 23,24 Calculations for the Heuman index for hydrophobicity of bile acids in serum were based on the sums of bile acid hydrophobicity for each measured bile acid (AUC 0-12 h) where larger values are considered a more hydrophobic environment than smaller values. 25…”

Section: Summary Methods For In Vitro Investigative Workmentioning

confidence: 99%

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Phase I study of PF‐04895162, a Kv7 channel opener, reveals unexpected hepatotoxicity in healthy subjects, but not rats or monkeys: clinical evidence of disrupted bile acid homeostasis

Aleo

Aubrecht

Bonin

et al. 2019

Pharmacology Res & Perspec

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During a randomized Phase 1 clinical trial the drug candidate, PF‐04895162 (ICA‐105665), caused transaminase elevations (≥grade 1) in six of eight healthy subjects treated at 300 mg twice daily for 2‐weeks (NCT01691274). This was unexpected since studies in rats (<6 months) and cynomolgus monkeys (<9 months) treated up to 100 mg/kg/day did not identify the liver as a target organ. Mechanistic studies showed PF‐04895162 had low cytotoxic potential in human hepatocytes, but inhibited liver mitochondrial function and bile salt export protein (BSEP) transport. Clinical relevance of these postulated mechanisms of liver injury was explored in three treated subjects that consented to analysis of residual pharmacokinetic plasma samples. Compared to a nonresponder, two subjects with transaminase elevations displayed higher levels of miRNA122 and total/conjugated bile acid species, whereas one demonstrated impaired postprandial clearance of systemic bile acids. Elevated taurine and glycine conjugated to unconjugated bile acid ratios were observed in two subjects, one before the onset of elevated transaminases. Based on the affinity of conjugated bile acid species for transport by BSEP, the profile of plasma conjugated/unconjugated bile acid species was consistent with inhibition of BSEP. These data collectively suggest that the human liver injury by PF‐04895162 was due to alterations in bile acid handling driven by dual BSEP/mitochondrial inhibition, two important risk factors associated with drug‐induced liver injury in humans. Alterations in systemic bile acid composition were more important than total bile acids in the manifestation of clinical liver injury and may be a very early biomarker of BSEP inhibition.

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Section: Methodsmentioning

confidence: 99%

Section: Summary Methods For In Vitro Investigative Workmentioning

confidence: 99%

Phase I study of PF‐04895162, a Kv7 channel opener, reveals unexpected hepatotoxicity in healthy subjects, but not rats or monkeys: clinical evidence of disrupted bile acid homeostasis

Aleo

Aubrecht

Bonin

et al. 2019

Pharmacology Res & Perspec

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“…22 There are some reports that CDCA underwent transcellular transport across Sodium Taurocholate Cotransporting Polypeptide-Madin-Darby Canine Kideney cells (NTCP-MDCK) and Sodium Taurocholate Cotransporting Polypeptide-Lily Laboratories Cells Porcine Kidney (NTCP-LLC-PK1) monolayers, [36][37][38][39][40] although others reported no uptake using CHO cells expressing NTCP. 25 These studies were done using LC/MS for detection of CDCA. Addition of a fluorescent moiety can significantly change the properties of a molecular probe, as it changes the size and physicochemical properties compared to the parent compound.…”

Section: Discussionmentioning

confidence: 99%

“…While trying to optimize the signal-to-noise ratio, it was found that by changing either the cell density or the substrate concentration we could change the sensitivity of the measurements (uptake ratio ranging from 8 to almost 30; Figure 2G). Another documented 25 way to increase the signal is to increase protein expression using the histone deacetylase inhibitor sodium butyrate. As shown in Figure 2H, treatment with butyrate increases both total (immunoblot) and surface (cytometry) expression of the protein, with a corresponding increase in uptake of CDCA-NBD.…”

Section: Ntcp-mediated Cdca-nbd Transportmentioning

confidence: 99%

Assessment of Statin Interactions With the Human NTCP Transporter Using a Novel Fluorescence Assay

Wang

Wilkerson

et al. 2020

Int J Toxicol

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Sodium taurocholate cotransporting polypeptide (NTCP), which is highly expressed in the sinusoidal membrane of hepatocytes, maintains bile acid homeostasis and participates in the hepatic disposition of a variety of endogenous substances as well as xenobiotics. Manifested by the involvement of organic anion-transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3) in the hepatic uptake of statin drugs, sinusoidal membrane transporters play an important role in the pharmacokinetics and pharmacodynamics of these agents. It has been speculated that NTCP may function as an alternative pathway for statin hepatic uptake, complementary to OATP1B1 and OATP1B3. In the current study, we produced stable NTCP-expressing human embryonic kidney 293 (HEK293) cells and developed a fluorescence-based assay using flow cytometry for measuring NTCP transport with chenodeoxycholyl-(Nε-7-nitrobenz-2-oxa-1,3-diazole)-lysine (CDCA-NBD) as the substrate. NTCP-mediated CDCA-NBD transport was time-dependent and exhibited typical Michaelis–Menten kinetics, with a K m of 6.12 µM. Compounds known to interact with NTCP, including chenodeoxycholic acid and taurocholic acid, displayed concentration-dependent inhibition of NTCP-mediated CDCA-NBD transport. We report here a systematic evaluation of the interaction between statins and the NTCP transporter. Utilizing this system, several statins were either found to inhibit NTCP-dependent transport or act as substrates. We find a good correlation between the reported lipophilicity of statins and their ability to inhibit NTCP. The objective was to develop a higher-throughput system to evaluate potential inhibitors such as the statins. The in vitro assays using CDCA-NBD as fluorescent substrate are convenient, rapid, and have utility in screening drug candidates for potential drug–NTCP interactions.

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“…Tritiated taurocholate is used almost exclusively as the probe substrate in BSEP inhibition assays. Taurocholate, however, is of limited relevance to humans as it is neither the most abundant nor the most toxic bile salt in our species [70]. Glycochenodeoxycholate (GCDC), the most abundant bile salt in humans, should therefore be preferred as a probe substrate if clinical relevance is paramount.…”

Section: In Vitro Testing Of Bilirubin Metabolism and Transportmentioning

confidence: 99%

Prediction of Drug-Induced Hyperbilirubinemia by In Vitro Testing

Tátrai

Krajcsi

2020

Pharmaceutics

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Bilirubin, the end product of heme catabolism, is produced continuously in the body and may reach toxic levels if accumulates in the serum and tissues; therefore, a highly efficient mechanism evolved for its disposition. Normally, unconjugated bilirubin enters hepatocytes through the uptake transporters organic anion transporting polypeptide (OATP) 1B1 and 1B3, undergoes glucuronidation by the Phase II enzyme UDP glucuronosyltransferase 1A1 (UGT1A1), and conjugated forms are excreted into the bile by the canalicular export pump multidrug resistance protein 2 (MRP2). Any remaining conjugated bilirubin is transported back to the blood by MRP3 and passed on for uptake and excretion by downstream hepatocytes or the kidney. The bile salt export pump BSEP as the main motor of bile flow is indirectly involved in bilirubin disposition. Genetic mutations and xenobiotics that interfere with this machinery may impede bilirubin disposition and cause hyperbilirubinemia. Several pharmaceutical compounds are known to cause hyperbilirubinemia via inhibition of OATP1Bs, UGT1A1, or BSEP. Herein we briefly review the in vitro prediction methods that serve to identify drugs with a potential to induce hyperbilirubinemia. In vitro assays can be deployed early in drug development and may help to minimize late-stage attrition. Based on current evidence, drugs that behave as mono- or multispecific inhibitors of OATP1B1, UGT1A1, and BSEP in vitro are at risk of causing clinically significant hyperbilirubinemia. By integrating inhibition data from in vitro assays, drug serum concentrations, and clinical reports of hyperbilirubinemia, predictor cut-off values have been established and are provisionally suggested in this review. Further validation of in vitro readouts to clinical outcomes is expected to enhance the predictive power of these assays.

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Kinetic characterization of bile salt transport by human NTCP (SLC10A1)

Cited by 19 publications

References 24 publications

Phase I study of PF‐04895162, a Kv7 channel opener, reveals unexpected hepatotoxicity in healthy subjects, but not rats or monkeys: clinical evidence of disrupted bile acid homeostasis

Phase I study of PF‐04895162, a Kv7 channel opener, reveals unexpected hepatotoxicity in healthy subjects, but not rats or monkeys: clinical evidence of disrupted bile acid homeostasis

Assessment of Statin Interactions With the Human NTCP Transporter Using a Novel Fluorescence Assay

Prediction of Drug-Induced Hyperbilirubinemia by In Vitro Testing

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