Inhibition of Organic Anion Transporting Polypeptide 1B1 and 1B3 by Betulinic Acid: Effects of Preincubation and Albumin in the Media

Oh, Yunseok; Jeong, Yoo-Seong; Kim, Min-Soo; Min, Jee Sun; Ryoo, Gongmi; Park, Ji Eun; Jun, Yearin; Song, YoungGoo; Chun, Se-Eun; Han, Songhee; Bae, Soo Kyung; Chung, Suk‐Jae; Lee, Wooin

doi:10.1016/j.xphs.2018.02.010

Cited by 11 publications

(6 citation statements)

References 45 publications

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“…The IC 50 values after preincubation are closer to the in vivo transporter inhibition rate constant estimated by fitting clinical OATP1B DDI data to a physiologically based pharmacokinetic model (Gertz et al, 2013;Shitara and Sugiyama, 2017). Furthermore, the inhibitory effect of CsA against OATP1B1 and OATP1B3 persisted for at least 90 minutes after CsA was removed from the incubation buffer (Oh et al, 2018). In agreement, CsA exhibited a lasting organic anion transporting polypeptide inhibitory effect in vivo.…”

Section: Introductionsupporting

confidence: 71%

Enhanced and Persistent Inhibition of Organic Cation Transporter 1 Activity by Preincubation of Cyclosporine A

et al. 2019

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Recent pharmacogenetic evidence indicates that hepatic organic cation transporter (OCT) 1 can serve as the locus of drug-drug interactions (DDIs) with significant pharmacokinetic and pharmacodynamic consequences. We examined the impact of preincubation on the extent of OCT1 inhibition in transfected human embryonic kidney 293 (HEK293) cells. Following 30-minute preincubation with an inhibitor, approximately 50-fold higher inhibition potency was observed for cyclosporine A (CsA) against OCT1mediated uptake of metformin compared with coincubation, with IC 50 values of 0.43 6 0.12 and 21.6 6 4.5 mM, respectively. By comparison, only small shifts (£2-fold) in preincubation IC 50 versus coincubation were observed for quinidine, pyrimethamine, ritonavir, and trimethoprim. The shift in CsA OCT1 IC 50 was substrate dependent since it ranged from >1.2to 50.2-fold using different experimental substrates. The inhibition potential of CsA toward OCT1 was confirmed by fenoterol hepatocyte uptake experiment. Furthermore, no shift in CsA IC 50 was observed with HEK293 cells transfected with OCT2 and organic anion transporter (OAT) 1 and OAT3. Short exposure (30 minutes) to 10 mM CsA produced longlasting inhibition (at least 120 minutes) of the OCT1-mediated uptake of metformin in OCT1-HEK293 cells, which was likely attributable to the retention of CsA in the cells, as shown by the fact that inhibitory cellular concentrations of CsA were maintained long after the removal of the compound from the incubation buffer. The potent and persistent inhibitory effect after exposure to CsA warrants careful consideration in the design and interpretation of clinical OCT1 DDI studies. SIGNIFICANCE STATEMENT Preincubation of OATP1B1 and OATP1B3 with their inhibitor may result in the enhancement of the inhibitory potency in a cell-based assay. However, limited data are available on potentiation of OCT1 inhibition by preincubation, which is a clinically relevant drug transporter. For the first time, we observed a 50-fold increase in CsA inhibitory potency against OCT1-mediated transport of metformin following a preincubation step. The CsA preincubation effect on OCT1 inhibition is substrate dependent. Moreover, the inhibition potential of CsA toward OCT1 is confirmed by hepatocyte uptake experiment. This study delivers clear evidences about the potent and persistent inhibitory effect on OCT1 after exposure to CsA. Further studies are needed to assess the effect of CsA on OCT1 drug substrates in vivo. This study was supported by the Bristol-Myers Squibb Company.

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Section: Introductionsupporting

confidence: 71%

Enhanced and Persistent Inhibition of Organic Cation Transporter 1 Activity by Preincubation of Cyclosporine A

et al. 2019

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“…The latter medium conditions potentially alter free drug concentrations due to protein binding. Lowering of the inhibitory potency of betulinic acid ( 4 ) due to protein binding has already been identified in a previous study, where this compound was used as inhibitor for the organic anion transporting polypeptide OATP1B3 50 . A further factor could be the different stoichiometry of the single preS1 peptide molecule and the multitude of preS1 domains of the large envelope proteins of HBV/HDV virions.…”

Section: Discussionmentioning

confidence: 74%

Selective hepatitis B and D virus entry inhibitors from the group of pentacyclic lupane-type betulin-derived triterpenoids

Kirstgen

Lowjaga

Müller

et al. 2020

Sci Rep

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Current treatment options against hepatitis B and D virus (HBV/HDV) infections have only limited curative effects. Identification of Na+/taurocholate co-transporting polypeptide (NTCP) as the high-affinity hepatic receptor for both viruses in 2012 enables target-based development of HBV/HDV cell-entry inhibitors. Many studies already identified appropriate NTCP inhibitors. However, most of them interfere with NTCP’s physiological function as a hepatic bile acid transporter. To overcome this drawback, the present study aimed to find compounds that specifically block HBV/HDV binding to NTCP without affecting its transporter function. A novel assay was conceptualized to screen for both in parallel; virus binding to NTCP (measured via binding of a preS1-derived peptide of the large HBV/HDV envelope protein) and bile acid transport via NTCP. Hits were subsequently validated by in vitro HDV infection studies using NTCP-HepG2 cells. Derivatives of the birch-derived pentacyclic lupane-type triterpenoid betulin revealed clear NTCP inhibitory potency and selectivity for the virus receptor function of NTCP. Best performing compounds in both aspects were 2, 6, 19, and 25. In conclusion, betulin derivatives show clear structure–activity relationships for potent and selective inhibition of the HBV/HDV virus receptor function of NTCP without tackling its physiological bile acid transport function and therefore are promising drug candidates.

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“…Here, we evaluated HEK293 cells overexpressing OATP1B1 and OATP1B3 in protein-free buffer and 100% plasma incubations as an alternative system. Research groups have recently begun adding protein to incubations with overexpressing cell lines using, for instance, 2% human serum albumin (Fukuchi et al, 2017) or 10% fetal bovine serum (Oh et al, 2018). In this investigation, using 100% human plasma with HEK293 cells was also demonstrated to be a reasonable option since the cells maintained high viability throughout the study.…”

Section: Discussionmentioning

confidence: 94%

Changes in Organic Anion Transporting Polypeptide Uptake in HEK293 Overexpressing Cells in the Presence and Absence of Human Plasma

Bowman¹,

Chen²,

Chen³

et al. 2019

Drug Metab Dispos

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Generating accurate in vitro data is crucial for in vitro to in vivo extrapolation and pharmacokinetic predictions. The use of human embryonic kidney (HEK) 293 cells overexpressing organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 in protein-free buffer and 100% human plasma incubations was explored for the uptake of four OATP substrates: pravastatin, rosuvastatin, repaglinide, and pitavastatin. Differences were observed for each parameter [unbound Michaelis constant (K m,u ), V max , intrinsic clearance (CL int ), and unbound passive diffusion P dif,u ] obtained from the buffer and plasma incubations in both cells, and the fold differences were greatest for the highly protein bound compounds. The fold change in K m,u values ranged from 1.91 to 619, and the fold change in V max values ranged from 1.22 to 97.4. As a result, in both cells, the CL int values generated in the plasma incubations were higher by 0.762-to 31.7-fold than the values generated in the protein-free buffer. The passive diffusion was also higher in the plasma incubations for all four compounds, with a fold difference range of 1.73-23.4. These shifts in the presence and absence of human plasma suggest that plasma proteins may play a role in both the active uptake and passive diffusion processes. The results also support the idea of a transporter-induced protein-binding shift, where high protein binding may not limit the uptake of compounds that have high affinity for transporters. The addition of plasma to incubations leading to higher CL int values for transporter substrates helps mitigate the underprediction commonly noted with in vitro to in vivo extrapolation. SIGNIFICANCE STATEMENTThe current investigation brings a new perspective on how to mitigate the underprediction commonly noted with in vitro to in vivo extrapolation for OATP substrates by using HEK293 cells overexpressing OATP1B1 and OATP1B3. It also supports the idea of a transporter-induced protein-binding shift, where high protein binding may not limit the uptake of compounds that have high affinity for transporters.

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Inhibition of Organic Anion Transporting Polypeptide 1B1 and 1B3 by Betulinic Acid: Effects of Preincubation and Albumin in the Media

Cited by 11 publications

References 45 publications

Enhanced and Persistent Inhibition of Organic Cation Transporter 1 Activity by Preincubation of Cyclosporine A

Enhanced and Persistent Inhibition of Organic Cation Transporter 1 Activity by Preincubation of Cyclosporine A

Selective hepatitis B and D virus entry inhibitors from the group of pentacyclic lupane-type betulin-derived triterpenoids

Changes in Organic Anion Transporting Polypeptide Uptake in HEK293 Overexpressing Cells in the Presence and Absence of Human Plasma

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