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Metabolism-efflux interactions of erythromycin in human cryopreserved hepatocytes. (A) Total metabolic breakdown (cells + medium) of erythromycin was unaffected by addition of 0.5 mM elacridar (metabolic stability assay). (B) Addition of 0.5 mM elacridar did not change the formation rate of the major metabolite N-desmethyl-erythromycin significantly (cells + medium, metabolic stability assay). (C) No change in extracellular erythromycin concentrations could be seen after addition of elacridar to spinning-through-oil experiments. (D) Efflux of the metabolite N-desmethyl-erythromycin was diminished by addition of elacridar (spinning-through-oil experiments). (E) Elacridar (0.5 mM) increased uptake and intracellular concentrations of erythromycin in human cryopreserved hepatocytes (spinning-through-oil experiments). (F) Intracellular levels of the metabolite N-desmethyl-erythromycin increased 2-fold in the presence of elacridar (s-t-o) (mean 6 SD, n = 3; *P , 0.05, **P , 0.01, ***P , 0.001). AU, arbitrary units.
Source publication
Freshly isolated hepatocytes are considered the gold standard for in vitro studies of hepatic drug disposition. To ensure a reliable supply of cells, cryopreserved human hepatocytes are often used. ABC-super family drug efflux transporters are key elements in hepatic drug disposition. These transporters are often considered lost after isolation of...
Contexts in source publication
Context 1
... Figure 8, A and B, the results from a metabolic stability experiment with erythromycin are shown using human cryopreserved hepatocytes in suspension. The addition of 0.5 mM elacridar to the TABLE 1 IVIVE of fexofenadine and erythromycin CL hepatic utilizing in vitro CL int, uptake measurements demonstrating the impact of elacridar CL int, uptake a (ml/min/10 6 cells) ...
Context 2
... change the total (cells + medium) metabolism of erythromycin significantly; neither was the total (cells + medium) formation rate of the main metabolite N-desmethyl-erythromycin affected. The extracellular concentration of erythromycin in spinning- through-oil uptake experiments with human cryopreserved hepato- cytes was not affected by elacridar (Fig. 8C), whereas extracellular levels of N-desmethyl-erythromycin were reduced (Fig. ...
Context 3
... was the total (cells + medium) formation rate of the main metabolite N-desmethyl-erythromycin affected. The extracellular concentration of erythromycin in spinning- through-oil uptake experiments with human cryopreserved hepato- cytes was not affected by elacridar (Fig. 8C), whereas extracellular levels of N-desmethyl-erythromycin were reduced (Fig. ...
Context 4
... of intracellular levels showed that the net uptake of erythromycin was increased by approximately 2-fold after addition of elacridar (Fig. 8E). In parallel with this, intracellular concentrations of N-desmethyl-erythromycin more than doubled in the presence of the inhibitor (Fig. ...
Context 5
... of intracellular levels showed that the net uptake of erythromycin was increased by approximately 2-fold after addition of elacridar (Fig. 8E). In parallel with this, intracellular concentrations of N-desmethyl-erythromycin more than doubled in the presence of the inhibitor (Fig. ...
Similar publications
Studies were conducted to evaluate the impact of time and cryopreservation on aldehyde oxidase (AO) activity in human hepatocytes isolated from ten donor livers, using O6-benzylguanine as a probe substrate. In addition, variability in activity was assessed using cryopreserved hepatocytes from 75 donors. Substantial donor-dependent loss in AO activi...
Citations
... In our previous study, the major metabolite of ERY in chicken liver microsomes was determined to be N-desmethyl-erythromycin A (N-D-ERY) . Reports have indicated that N-D-ERY does not possess antibacterial activity; however, N-D-ERY represents a potential risk to individuals (Lundquist et al., 2014;Sun et al., 2022). Although the use of these antibiotics reduces the mortality of poultry, some antibiotic residues, such as fluoroquinolones, tetracyclines, amphenicols, sulfonamides, MACs, lincosamides, and coccidiostats, still jeopardize the safety of poultry and their products, which needs to be addressed (Chinese Ministry of Agriculture and Rural Affairs, 2024). ...
A simple, rapid and novel method involving ultrahigh-performance liquid chromatography-electrospray ionization tandem triple quadrupole mass spectrometry (UHPLC-ESI–MS/MS) was developed to simultaneously detect erythromycin, its major metabolite and clarithromycin in chicken tissues (muscle, liver and kidney) and eggs (whole egg, albumen and yolk). Samples were extracted using acetonitrile–water (80:20, v/v), and a Cleanert MAS-Q cartridge was used to perform quick, easy, cheap, effective, rugged, and safe (QuEChERS) purification. The average recoveries were 87.78–104.22 %, and the corresponding intraday and interday relative standard deviations were less than 7.10 %. The decision limits and detection capabilities of the chicken tissues and eggs were 2.15–105.21 μg/kg and 2.26–110.42 μg/kg, respectively. For chicken tissues and eggs, the limits of detection and limits of quantification were 0.5 μg/kg and 2.0 μg/kg, respectively. The proposed method was successfully employed to analyse real samples, demonstrating its applicability.
... The uptake experiments show that the MRP/Mrp transporter family plays an important role in the intracellular colistin levels. In suspended hepatocytes, the canalicular efflux transporters (such as MRP/Mrp2) [31] have been shown to undergo partial internalization [32,33]. We therefore hypothesize that the MRP/Mrp3 and 4 most likely mediate colistin efflux. ...
Purpose
Colistin is an antibiotic which is increasingly used as a last-resort therapy in critically-ill patients with multidrug resistant Gram-negative infections. The purpose of this study was to evaluate the mechanisms underlying colistin’s pharmacokinetic (PK) behavior and to characterize its hepatic metabolism.
Methods
In vitro incubations were performed using colistin sulfate with rat liver microsomes (RLM) and with rat and human hepatocytes (RH and HH) in suspension. The uptake of colistin in RH/HH and thefraction of unbound colistin in HH (fu,hep) was determined. In vitro to in vivo extrapolation (IVIVE) was employed to predict the hepatic clearance (CLh) of colistin.
Results
Slow metabolism was detected in RH/HH, with intrinsic clearance (CLint) values of 9.34± 0.50 and 3.25 ± 0.27 mL/min/kg, respectively. Assuming the well-stirred model for hepatic drug elimination, the predicted rat CLh was 3.64± 0.22 mL/min/kg which could explain almost 70% of the reported non-renal in vivo clearance. The predicted human CLh was 91.5 ± 8.83 mL/min, which was within two-fold of the reported plasma clearance in healthy volunteers. When colistin was incubated together with the multidrug resistance-associated protein (MRP/Mrp) inhibitor benzbromarone, the intracellular accumulation of colistin in RH/HH increased significantly.
Conclusion
These findings indicate the major role of hepatic metabolism in the non-renal clearance of colistin, while MRP/Mrp-mediated efflux is involved in the hepatic disposition of colistin. Our data provide detailed quantitative insights into the hereto unknown mechanisms responsible for non-renal elimination of colistin.
... The evidence of the MDR1 protective role in the placenta was extensively reviewed by Joshi et al. [48]. Finally, MDR1 is also present in the membrane of the hepatocytes facing the bile canaliculus (apical) [45,68] and in the apical membrane of the proximal tubule epithelial cells [78]. Thus, MDR1 contributes to the elimination of substances through the bile and also participates in the tubular secretion process. ...
... Here, only MRP2 and MRP3 may participate in the fetus protection from deleterious substances derived from maternal blood. In the liver, MRP2 was identified in the apical membrane of the hepatocytes [55,68,93,119]. On the other hand, MRP3 and MRP4 are present in the basolateral side [36,56,104,151]. In the kidney, MRP2 and MRP4, like MDR1, were shown to be located in the apical side of the proximal tubule epithelial cells [117]. ...
Drug efficacy is dependent on the pharmacokinetics and pharmacodynamics of therapeutic agents. Tight junctions, detoxification enzymes, and drug transporters, due to their localization on epithelial barriers, modulate the absorption, distribution, and the elimination of a drug. The epithelial barriers which control the pharmacokinetic processes are sex steroid hormone targets, and in this way, sex hormones may also control the drug transport across these barriers. Thus, sex steroids contribute to sex differences in drug resistance and have a relevant impact on the sex-related efficacy of many therapeutic drugs. As a consequence, for the further development and optimization of therapeutic strategies, the sex of the individuals must be taken into consideration. Here, we gather and discuss the evidence about the regulation of ATP-binding cassette transporters by sex steroids, and we also describe the signaling pathways by which sex steroids modulate ATP-binding cassette transporters expression, with a focus in the most important ATP-binding cassette transporters involved in multidrug resistance.
... It could therefore be argued to use primary RPTEC cells and establish the kinetics of the overall uptake of a new chemical reporting on an overall apparent Vmax and an overall apparent Km. The scaling factor would then be obtained by quantifying the level of the transporter of interest in both the in vitro model and the in vivo situation by proteomics or western blotting obtaining a REF value (Basit et al. 2019;Bay et al. 2022;Lundquist et al. 2014;Prasad and Unadkat 2014). As described in section 6.3.3 use of a RAF value could also be considered, although not for all transporters verified probe substrates are available. ...
... On the contrary, Li M et. al. and Lundquist P. et. al. showed that bile canalicular efflux transporters including P-gp, BCRP, BSEP and MRP2 are expressed in the plasma membrane of freshly isolated and cryopreserved human hepatocytes (Li et al., 2008;Lundquist et al., 2014). Furthermore, Li M. et. ...
... Published on January 20, 2021as DOI: 10.1124 at ASPET Journals on January 20, 2021 dmd.aspetjournals.org Downloaded from significant portion of transporter proteins in the canalicular plasma membrane of freshly isolated and cryopreserved human hepatocytes (Lundquist et al., 2014). ...
The mechanistic understanding of bile salt disposition is not well established in suspension human hepatocytes (SHH) due to limited information on the expression and function of bile salt export protein (BSEP) in this system. We investigated the transport function of BSEP in SHH using a method involving in situ biosynthesis of bile salts from their precursor bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA). Our data indicated that glycine and taurine-conjugated CA and CDCA were generated efficiently and transported out of hepatocytes in a concentration-and time-dependent manner. We also observed that the membrane protein abundance of BSEP was similar between SHH and sandwich cultured human hepatocytes. Furthermore, known cholestatic agents significantly inhibited G-CA and G-CDCA efflux in SHH. Interestingly, cyclosporine A, troglitazone, itraconazole, loratadine and lovastatin inhibited G-CA efflux more potently than G-CDCA efflux (3-5-fold). Due to these significant differential effects on G-CA and G-CDCA efflux inhibition, we determined the IC50 values of troglitazone for G-CA (9.9 µM) and for G-CDCA (43.1 µM) efflux. The observed discrepancy in the IC50 was attributed to the fact that troglitazone also inhibits organic anion transporting polypeptide (OATP)s and Na+/ taurocholate co-transporting polypeptide (NTCP) in addition to BSEP. The hepatocyte uptake study suggested that both active uptake and passive diffusion contribute to the liver uptake of CA while CDCA primarily undergoes passive diffusion into the liver. In summary, these data demonstrated the expression and function of BSEP and its major role in transport of bile salts in cryopreserved SHH. Significance Statement BSEP transport function and protein abundance was evident in SHH in the present study. The membrane abundance of BSEP protein was similar between SHH and SCHH. The study also illustrated the major role of BSEP relative to basolateral MRP3 and MRP4 in transport of bile salts in SHH. Understanding of BSEP function in SHH may bolster the utility of this platform in mechanistic understanding of bile salt disposition and potentially in the assessment of drugs for BSEP inhibition.
... Therefore, this system provides the possibility of assessing the excretion of drugs and bile components into bile canaliculi [82][83][84]. Hepatocytes in suspension also provide a suitable option for studying drug metabolism and drug transport [85,86]. One major advantage of hepatocyte suspension cultures is their easy, quick and high-yield preparation. ...
The bile salt export pump (BSEP/ABCB11) is responsible for the transport of bile salts from hepatocytes into bile canaliculi. Malfunction of this transporter results in progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2) and intrahepatic cholestasis of pregnancy (ICP). Over the past few years, several small molecular weight compounds have been identified, which hold the potential to treat these genetic diseases (chaperones and potentiators). As the treatment response is mutation-specific, genetic analysis of the patients and their families is required. Furthermore, some of the mutations are refractory to therapy, with the only remaining treatment option being liver transplantation. In this review, we will focus on the molecular structure of ABCB11, reported mutations involved in cholestasis and current treatment options for inherited BSEP deficiencies.
... MRP2, BCRP, P-gp) may be partly maintained in the PHH system. 42,43 Therefore, such ABC transporters may partly contribute to the efflux of drugs in PHH and affect the evaluation of the efflux clearance (PS eff ). ...
Hepatic uptake clearance has been measured in suspended human hepatocytes (SHH). Plated human hepatocytes (PHH) after short-term culturing are increasingly employed to study hepatic transport driven mainly by its higher throughput. To know pros/cons of both systems, the hepatic uptake clearances of several organic anion transporting polypeptide 1B substrates were compared between PHH and SHH by determining the initial uptake velocities or through dynamic model-based analyses. For cerivastatin, pitavastatin and rosuvastatin, initial uptake clearances (PSinf) obtained using PHH were comparable to those using SHH, while cell-to-medium concentration (C/M) ratios were 2.7- to 5.4-fold higher. For pravastatin and dehydropravastatin, hydrophilic compounds with low uptake/cellular binding, their PSinf and C/M ratio in PHH were 1.8- to 3.2-fold lower than those in SHH. These hydrophilic substrates are more prone to wash-off during the uptake study using PHH, which may explain the apparently lower uptake than SHH. The C/M ratios obtained using PHH were stable over an extended time, making PHH suitable to estimate the C/M ratios and hepatocyte-to-medium unbound concentration ratios (Kp,uu). In conclusion, PHH is useful in evaluating hepatic uptake/efflux clearances and Kp,uu of OATP1B substrates in a high-throughput manner, however, a caution is warranted for hydrophilic drugs with low uptake/cellular binding.
... Both OS and OC are ideal drug molecule models with an aliphatic amino group as the fixation site in situ for IHC because a simultaneous comparative study by autoradiography might be almost impossible to perform. Also, as the transport systems responsible for OS or OC uptake or excretion a member of the ATP binding cassette (ABC) transport proteins Mdr1 (P-glycoprotein: P-gp), [19][20][21][22][23] the organic anion transporter (OAT1), 24 the OAT3, 1 multidrug resistance-associated protein 4 (Mrp4/abcc4), [25][26][27] and the peptide transporter1 (Pept1) [28][29][30][31] have been demonstrated by pharmacological studies using knockout mice or cell lines expressing such transporters. However, the in vivo role of transporters in drug disposition, and metabolism, has been scarcely established. ...
... These findings show that OS is excreted via the bile capillary to the bile flows, but OC is virtually not. The bile capillaries' membrane of the hepatocytes are specific sites, where P-gp is highly expressed 22,53,54 strongly suggesting that OS is actually and actively excreted at these sites, possibly through P-gp, since OS is among substrates for P-gp. 21 Previously, we have observed the same bile flow pattern in the IHC for doxorubicin, 32 the amphiphilic cationic drug known as the substrate for P-gp. ...
Among any drugs, no comparative pharmacological study on how prodrug and its active metabolite behave in animal bodies is available. Immunohistochemistry (IHCs) using newly prepared two monoclonal antibodies, AOS-96 and AOC-160, monospecific for oseltamivir (OS) and its metabolite oseltamivir carboxylate (OC) were developed, simultaneously detecting the uptake or excretion of OS and OC in the intestine, liver, and kidney of rats to which OS was orally administered. In the intestine, IHC for OS revealed OS highly distributed to the absorptive epithelia with heavily stained cytoplasmic small granules (CSGs). IHC for OC showed that OC also distributed highly in the epithelia, but without CSGs, suggesting that OS was partly converted to OC in the cells. In the liver, OS distributed in the hepatocytes and on their bile capillaries, as well as on the lumina from the bile capillaries to the interlobular bile ducts. OC distributed in the whole cell of the hepatocytes, but without CSGs nor on any lumina through the interlobular bile ducts. In the kidney, a few levels of OS distributed in the cytoplasm of almost all the renal tubule cells, but they contained numerous CSGs. In contrast, OC distributed highly in the proximal tubules, but very slightly in the lower renal tubules of the nephrons. Thus, it was concluded that the two drugs behave in completely different ways in rat bodies. This paper also discusses a possibility of the correlation of OS or OC levels in tissue cells with their known transporters.
... They demonstrated that unbound drug concentrationbased uptake clearance in SHHs was 2.4-to 64-fold higher in the presence of albumin and provided a better prediction of in vivo CL int,all for OATP1B substrates by considering albumin-mediated hepatic uptake. Although canalicular efflux transporters such as P-gp, MRP2, and BCRP are often considered lost after isolation, Lundquist et al. reported that (1) these efflux transporters were detected on the plasma membrane of SHHs and (2) the uptake of fexofenadine into SHHs increased approximately 1.5-fold in the presence of a typical P-gp inhibitor, elacridar [57]. Another study also suggested that canalicular efflux transporters are not all internalized but are also present in the plasma membrane of SHHs and PHHs [40]. ...
Hepatic uptake mediated by organic anion transporting polypeptide (OATP) 1B1 and 1B3 can serve as a major elimination pathway for various anionic drugs and as a site of drug-drug interactions (DDIs). This article provides an overview of the in vitro approaches used to predict human hepatic clearance (CLh) and the risk of DDIs involving OATP1Bs. On the basis of the so-called extended clearance concept, in vitro-in vivo extrapolation methods using human hepatocytes as in vitro systems have been used to predict the CLh involving OATP1B-mediated hepatic uptake. CLh can be quantitatively predicted using human donor lots possessing adequate OATP1B activities. The contribution of OATP1Bs to hepatic uptake can be estimated by the relative activity factor, the relative expression factor, or selective inhibitor approaches, which offer generally consistent outcomes. In OATP1B1 inhibition assays, substantial substrate dependency was observed. The time-dependent inhibition of OATP1B1 was also noted and may be a mechanism underlying the in vitro-in vivo differences in the inhibition constant of cyclosporine A. Although it is still challenging to quantitatively predict CLh and DDIs involving OATP1Bs from only preclinical data, understanding the utility and limitation of the current in vitro methods will pave the way for better prediction.
... Whether to incorporate BSA into UGT inhibition studies is debatable (Badee et al., 2019;Rowland et al., 2008 (Li et al., 2008;Lundquist et al., 2014). Compounds may passively diffuse into hepatocytes and/or be actively taken up into the cells by transporters such as OATPs, OAT2, OCT1, and NTCP (Shitara et al., 2003). ...
Drug‐drug interactions (DDIs) caused by coadministration of multiple drugs are major safety concerns in the clinic. Several drugs have been withdrawn from the market due to perpetrator or victim DDIs. Strategies have been developed to assess DDI risks early in drug discovery to reduce DDI liabilities. High‐to‐medium throughput assays are available to identify undesirable scaffolds and guide structural modifications to minimize DDIs. Definitive methods are used at later stages of drug discovery and development to provide more accurate measurement of DDI parameters and to enable clinical translations. Physiologically based pharmacokinetic modeling and simulations are powerful tools to accurately predict DDIs and to assess risks in the clinic. Although significant advances have been made over the year, many challenges remain for clinical DDI translations. This includes DDI involving non‐cytochrome P450 enzymes, transporters, enzyme‐transporter interplay, indirect effects from biologics, and pharmacodynamic based DDI. This review focuses on methods that are used to assess hepatic DDIs caused by enzyme inhibition and induction.
