Induction of human hepatic cytochrome P-450 3A4 expression by antifungal succinate dehydrogenase inhibitors.

Succinate dehydrogenase inhibitors (SDHIs) are widely-used fungicides, to which humans are exposed and for which putative health risks are of concern. In order to identify human molecular targets for these agrochemicals, the interactions of 15 SDHIs with expression and activity of human cytochrome P-450 3A4 (CYP3A4), a major hepatic drug metabolizing enzyme, were investigated in vitro. 12/15 SDHIs, i.e., bixafen, boscalid, fluopyram, flutolanil, fluxapyroxad, furametpyr, isofetamid, isopyrazam, penflufen, penthiopyrad, pydiflumetofen and sedaxane, were found to enhance CYP3A4 mRNA expression in human hepatic HepaRG cells and primary human hepatocytes exposed for 48 h to 10 µM SDHIs, whereas 3/15 SDHIs, i.e., benzovindiflupyr, carboxin and thifluzamide, were without effect. The inducing effects were concentrations-dependent for boscalid (EC50=22.5 µM), fluopyram (EC50=4.8 µM) and flutolanil (EC50=53.6 µM). They were fully prevented by SPA70, an antagonist of the Pregnane X Receptor, thus underlining the implication of this xenobiotic-sensing receptor. Increase in CYP3A4 mRNA in response to SDHIs paralleled enhanced CYP3A4 protein expression for most of SDHIs. With respect to CYP3A4 activity, it was directly inhibited by some SDHIs, including bixafen, fluopyram, fluxapyroxad, isofetamid, isopyrazam, penthiopyrad and sedaxane, which therefore appears as dual regulators of CYP3A4, being both inducer of its expression and inhibitor of its activity. The inducing effect nevertheless predominates for these SDHIs, except for isopyrazam and sedaxane, whereas boscalid and flutolanil were pure inducers of CYP3A4 expression and activity. Most of SDHIs appear therefore as in vitro inducers of CYP3A4 expression in cultured hepatic cells, when, however, used at concentrations rather higher than those expected in humans in response to environmental or dietary exposure to these agrochemicals.

Combined in silico and in vitro approaches to identify P-glycoprotein-inhibiting pesticides.

The P-glycoprotein (P-gp) efflux pump plays a major role in xenobiotic detoxification. The inhibition of its activity by environmental contaminants remains however rather little characterised. The present study was designed to develop a combination of different approaches to identify P-gp inhibitors among a large number of pesticides using in silico and in vitro models. First, the prediction performance of four web tools was evaluated alone or in combination using a set of recently marketed drugs. The best combination of web tools-AdmetSAR2.0/PgpRules/pkCSM-was next used to predict P-gp activity inhibition by 762 pesticides. Among the 187 pesticides predicted to be P-gp inhibitors, 11 were tested in vitro for their ability to inhibit the efflux of reference substrates (rhodamine 123 and Hoechst 33342) in P-gp overexpressing MCF7R cells and to inhibit the efflux of the reference substrate rhodamine 123 in the Caco-2 cell monolayer. In MCF7R cell assays, ivermectin B1a, emamectin B1 benzoate, spinosad, dimethomorph and tralkoxydim inhibited P-gp activity; ivermectin B1a, emamectin B1 benzoate and spinosad were determined to be stronger inhibitors (half-maximal inhibitory concentration [IC50 ] of 3 ± 1, 5 ± 1 and 7 ± 1 µM, respectively) than dimethomorph and tralkoxydim (IC50 of 102 ± 7 and 88 ± 7 µM, respectively). Ivermectin B1a, emamectin B1 benzoate, spinosad and dimethomorph also inhibited P-gp activity in Caco-2 cell monolayer assays, with dimethomorph being a weaker P-gp inhibitor. These combined approaches could be used to identify P-gp inhibitors among food contaminants, but need to be optimised and adapted for high-throughput screening.

High-resolution mass spectrometry identifies delayed biomarkers for improved precision in acetaminophen/paracetamol human biomonitoring.

Paracetamol/acetaminophen (N-acetyl-p-aminophenol, APAP) is a top selling analgesic used in more than 600 prescription and non-prescription pharmaceuticals. To study efficiently some of the potential undesirable effects associated with increasing APAP consumption (e.g., developmental disorders, drug-induced liver injury), there is a need to improve current APAP biomonitoring methods that are limited by APAP short half-life. Here, we demonstrate using high-resolution mass spectrometry (HRMS) in several human studies that APAP thiomethyl metabolite conjugates (S-methyl-3-thioacetaminophen sulfate and S-methyl-3-thioacetaminophen sulphoxide sulfate) are stable biomarkers with delayed excretion rates compared to conventional APAP metabolites, that could provide a more reliable history of APAP ingestion in epidemiological studies. We also show that these biomarkers could serve as relevant clinical markers to diagnose APAP acute intoxication in overdosed patients, when free APAP have nearly disappeared from blood. Using in vitro liver models (HepaRG cells and primary human hepatocytes), we then confirm that these thiomethyl metabolites are directly linked to the toxic N-acetyl-p-benzoquinone imine (NAPQI) elimination, and produced via an overlooked pathway called the thiomethyl shunt pathway. Further studies will be needed to determine whether the production of the reactive hepatotoxic NAPQI metabolites is currently underestimated in human. Nevertheless, these biomarkers could already serve to improve APAP human biomonitoring, and investigate, for instance, inter-individual variability in NAPQI production to study underlying causes involved in APAP-induced hepatotoxicity. Overall, our findings demonstrate the potential of exposomics-based HRMS approach to advance towards a better precision for human biomonitoring.

Interactions of organophosphate flame retardants with human drug transporters.

Organophosphate flame retardants (OPFRs) are environmental pollutants of increasing interest, widely distributed in the environment and exerting possible deleterious effects towards the human health. The present study investigates in vitro their possible interactions with human drug transporters, which are targets for environmental chemicals and actors of their toxicokinetics. Some OPFRs, i.e., tris(2-butoxyethyl) phosphate (TBOEP), tris(1,3-dichloroisopropyl) phosphate (TDCPP), tri-o-cresyl phosphate (TOCP) and triphenyl phosphate (TPHP), were found to inhibit activities of some transporters, such as organic anion transporter 3 (OAT3), organic anion transporting polypeptide (OATP) 1B1, OATP1B3, organic cation transporter 2 (OCT2) or breast cancer resistance protein (BCRP). These effects were concentration-dependent, with IC50 values ranging from 6.1 µM (for TDCPP-mediated inhibition of OCT2) to 51.4 µM (for TOCP-mediated inhibition of BCRP). OPFRs also blocked the transporter-dependent membrane passage of endogenous substrates, notably that of hormones. OAT3 however failed to transport TBOEP and TPHP. OPFRs additionally repressed mRNA expressions of some transporters in cultured human hepatic HepaRG cells, especially those of OAT2 and OCT1 in response to TOCP, with IC50 values of 2.3 µM and 2.5 µM, respectively. These data therefore add OPFRs to the expanding list of pollutants interacting with drug transporters, even if OPFR concentrations required to impact transporters, in the 2-50 µM range, are rather higher than those observed in humans environmentally or dietarily exposed to these chemicals.

Interactions of human drug transporters with chemical additives present in plastics: Potential consequences for toxicokinetics and health.

Human membrane drug transporters are recognized as major actors of pharmacokinetics; they also handle endogenous compounds, including hormones and metabolites. Chemical additives present in plastics interact with human drug transporters, which may have consequences for the toxicokinetics and toxicity of these widely-distributed environmental and/or dietary pollutants, to which humans are highly exposed. The present review summarizes key findings about this topic. In vitro assays have demonstrated that various plastic additives, including bisphenols, phthalates, brominated flame retardants, poly-alkyl phenols and per- and poly-fluoroalkyl substances, can inhibit the activities of solute carrier uptake transporters and/or ATP-binding cassette efflux pumps. Some are substrates for transporters or can regulate their expression. The relatively low human concentration of plastic additives from environmental or dietary exposure is a key parameter to consider to appreciate the in vivo relevance of plasticizer-transporter interactions and their consequences for human toxicokinetics and toxicity of plastic additives, although even low concentrations of pollutants (in the nM range) may have clinical effects. Existing data about interactions of plastic additives with drug transporters remain somewhat sparse and incomplete. A more systematic characterization of plasticizer-transporter relationships is needed. The potential effects of chemical additive mixtures towards transporter activities and the identification of transporter substrates among plasticizers, as well as their interactions with transporters of emerging relevance deserve particular attention. A better understanding of the human toxicokinetics of plastic additives may help to fully integrate the possible contribution of transporters to the absorption, distribution, metabolism and excretion of plastics-related chemicals, as well as to their deleterious effects towards human health.

Drug transporter expression and activity in cryopreserved human hepatocytes isolated from chimeric TK-NOG mice with humanized livers.

Chimeric mice with humanized liver are thought to represent a sustainable source of isolated human hepatocytes for in vitro studying detoxification of drugs in humans. Because drug transporters are now recognized as key-actors of the hepatic detoxifying process, the present study was designed to characterize mRNA expression and activity of main hepatic drug transporters in cryopreserved human hepatocytes isolated from chimeric TK-NOG mice and termed HepaSH cells. Such cells after thawing were shown to exhibit a profile of hepatic solute carrier (SLC) and ATP-binding cassette (ABC) drug transporter mRNA levels well correlated to those found in cryopreserved primary human hepatocytes or human livers. HepaSH cells used either as suspensions or as 24 h-cultures additionally displayed notable activities of uptake SLCs, including organic anion transporting polypeptides (OATPs), organic anion transporter 2 (OAT2) or sodium-taurocholate co-transporting polypeptide (NTCP). SLC transporter mRNA expression, as well as SLC activities, nevertheless fell in HepaSH cells cultured for 120 h, which may reflect a partial dedifferentiation of these cells with time in culture in the conventional monolayer culture conditions used in the study. These data therefore support the use of cryopreserved HepaSH cells as either suspensions or short-term cultures for drug transport studies.

Determination of glyphosate and AMPA in indoor settled dust by hydrophilic interaction liquid chromatography with tandem mass spectrometry and implications for human exposure.

The widespread application of glyphosate leads to significant contamination of outdoor environmental compartments, notably air and soil, which can contaminate indoor air and dust. This study assessed the contamination of indoor household dust for the first time in France and potential exposure to glyphosate through the inadvertent ingestion of dust. A specific and new analytical method was developed using HILIC MS/MS (hydrophilic interaction liquid chromatography with tandem mass spectrometry) to measure polar pesticides, such as glyphosate, aminomethylphosphonic acid, and glufosinate, in indoor dust, with a low quantification limit (25 ng/g). The dust from vacuum cleaner bags of 60 rural and urban households (Brittany, France) was analyzed. All samples contained glyphosate (median 1675 ng/g for rural dwellings (n = 29), 457 ng/g for urban dwellings (n = 31)), more than 90 % contained aminomethylphosphonic acid, and none contained glufosinate. Concentrations were influenced by the rural or urban setting, the proximity of crops, and the use of weed killers on driveways or lawns. Glyphosate exposure via indoor dust ingestion was < 1 % of both acceptable daily intake and dietary intake. However, the high quantification limit of the glyphosate concentration in the food analysis method probably leads to overestimation of the dose from food.

Involvement of Transporters in Intestinal Drug-Drug Interactions of Oral Targeted Anticancer Drugs Assessed by Changes in Drug Absorption Time.

(1) Background: Oral targeted anticancer drugs are victims of presystemic pharmacokinetic drug−drug interactions (DDI). Identification of the nature of these DDIs, i.e., enzyme-based or/and transporter-based, is challenging, since most of these drugs are substrates of intestinal and/or hepatic cytochrome P-450 enzymes and of intestinal membrane transporters. (2) Methods: Variations in mean absorption time (MAT) between DDIs and control period (MAT ratios < 0.77 or >1.30) have been proposed to implicate transporters in DDIs at the intestinal level. This methodology has been applied to a large set of oral targeted anticancer drugs (n = 54, involved in 77 DDI studies), from DDI studies available either in the international literature and/or in publicly accessible FDA files. (3) Results: Significant variations in MAT were evidenced in 33 DDI studies, 12 of which could be explained by modulation of an efflux transporter. In 21 DDI studies, modulation of efflux transporters could not explain the MAT variation, suggesting a possible relevant role of influx transporters in the intestinal absorption. (4) Conclusions: This methodology allows one to suggest the involvement of intestinal transporters in DDIs, and should be used in conjunction with in vitro methodologies to help understanding the origin of DDIs.

Inhibition of canalicular and sinusoidal taurocholate efflux by cholestatic drugs in human hepatoma HepaRG cells.

HepaRG cells are highly-differentiated human hepatoma cells, which are increasingly recognized as a convenient cellular model for in vitro evaluation of hepatic metabolism, transport, and/or toxicity of drugs. The present study was designed to evaluate whether HepaRG cells can also be useful for studying drug-mediated inhibition of canalicular and/or sinusoidal hepatic efflux of bile acids, which constitutes a major mechanism of drug-induced liver toxicity. For this purpose, HepaRG cells, initially loaded with the bile acid taurocholate (TC), were reincubated in TC-free transport assay medium, in the presence or absence of calcium or drugs, before analysis of TC retention. This method allowed us to objectivize and quantitatively measure biliary and sinusoidal efflux of TC from HepaRG cells, through distinguishing cellular and canalicular compartments. In particular, time-course analysis of the TC-free reincubation period of HepaRG cells, that is, the efflux period, indicated that a 20 min-efflux period allowed reaching biliary and sinusoidal excretion indexes for TC around 80% and 60%, respectively. Addition of the prototypical cholestatic drugs bosentan, cyclosporin A, glibenclamide, or troglitazone during the TC-free efflux phase period was demonstrated to markedly inhibit canalicular and sinusoidal secretion of TC, whereas, by contrast, incubation with the noncholestatic compounds salicylic acid or flumazenil was without effect. Such data therefore support the use of human HepaRG cells for in vitro predicting drug-induced liver toxicity (DILI) due to the inhibition of hepatic bile acid secretion, using a biphasic TC loading/efflux assay.

Drug transporters are implicated in the diffusion of tacrolimus into the T lymphocyte in kidney and liver transplant recipients: Genetic, mRNA, protein expression, and functionality.

Because of a narrow therapeutic index and a wide inter- and intra-patient variability, therapeutic drug monitoring of the immunosuppressant drug tacrolimus (TAC) based on whole-blood concentrations (Cblood) is mandatory in solid organ transplant recipients. Using peripheral blood mononuclear cells concentrations (CPBMC) could improve patient outcomes. The poor correlation between Cblood and CPBMC makes hypothesize that drug transporters are implicated in the intracellular accumulation of TAC. The aim of this work was therefore to clinically study: i) the role of genetic variants and ii) the effect of mRNA and protein expression of 4 drug transporters on the TAC CPBMC/blood ratio. In addition, functional in vitro experiments were performed to mechanistically validate the clinical observations. Genetic variants of ABCB1/P-gp and SLC28A3/CNT3 did not influence TAC CPBMC in liver transplant recipients (LTR). ABCC2/MRP2 at the mRNA level; ABCB1/P-gp, SLC28A3/CNT3 and SLC29A1/ENT1 at the protein level; correlated with the CPBMC/blood in kidney and LTR. In vitro results suing transporter-expressing cells confirmed that TAC is substrate of P-gp but not MRP2, whereas experiments remained inconclusive for CNT3 and ENT1. In conclusion, the genetic-transcription-protein-functional approach presented in this work provides new insights in the understanding of TAC transport at the T lymphocyte plasma membrane.

Interactions of organophosphorus pesticides with ATP-binding cassette (ABC) drug transporters.

Although pharmaceutical companies have to study drug-transporter interaction, environmental contaminant interactions with these transporters are not well characterised. In this study, we demonstrated using in vitro transfected cell line that some organophosphorus pesticides are able to interact with drug efflux transporters like P-glycoprotein, BCRP and MRPs.According to our results, dibrom was found to inhibit only Hoechst binding site of P-gp with an IC50 closed to 77 µM, phosmet inhibited BCRP efflux with an IC50 of 42 µM and only profenofos was able to inhibit BCRP, MRPs and P-gp at two binding sites. As profenofos appeared to be a potent ABC transporter inhibitor, we studied its potential substrate property towards P-gp.Using a docking approach, we developed an in silico tool to study pesticide properties to be a probe or inhibitor of P-gp transporter. From both in silico and in vitro results, profenofos was not considered as a P-gp substrate.Combining both in vitro and docking methods appears to be an attractive approach to select pesticides that would not pass into the blood systemic circulation.

Contribution of Humanized Liver Chimeric Mice to the Study of Human Hepatic Drug Transporters: State of the Art and Perspectives.

Chimeric mice with humanized livers constitute an attractive emergent experimental model for investigating human metabolism and disposition of drugs. The present review was designed to summarize key findings about the use of this model for studying human hepatic drug transporters, which are now recognized as important players in pharmacokinetics and consequently have to be considered from a regulatory perspective during pharmaceutical drug development. The reviewed data indicate that chimeric mice with humanized livers have been successfully used for analysing the implications of human hepatic drug transporters for drug hepatobiliary elimination, drug-drug interactions and drug-induced cholestasis. Such transporter studies have been performed in vivo with chimeric mice and/or in vitro with human hepatocytes isolated from humanized liver and used either in suspension or in culture. The residual presence of mouse hepatocytes and the potential morphological/histological alterations of the humanized liver, as well as its immunodeficient mouse environment, have, however, to be considered when using chimeric mice with humanized livers for transporter studies. Finally, if the proof of concept of applying chimeric mice with humanized livers to hepatic drug transport is established, more experimental data on this topic, including from standardization approaches, are likely required to completely and accurately demonstrate the robustness, convenience and added value of this chimeric mouse model for drug transporter studies.

Pharmacokinetic Imaging Using 99mTc-Mebrofenin to Untangle the Pattern of Hepatocyte Transporter Disruptions Induced by Endotoxemia in Rats.

Endotoxemia-induced inflammation may impact the activity of hepatocyte transporters, which control the hepatobiliary elimination of drugs and bile acids. 99mTc-mebrofenin is a non-metabolized substrate of transporters expressed at the different poles of hepatocytes. 99mTc-mebrofenin imaging was performed in rats after the injection of lipopolysaccharide (LPS). Changes in transporter expression were assessed using quantitative polymerase chain reaction of resected liver samples. Moreover, the particular impact of pharmacokinetic drug-drug interactions in the context of endotoxemia was investigated using rifampicin (40 mg/kg), a potent inhibitor of hepatocyte transporters. LPS increased 99mTc-mebrofenin exposure in the liver (1.7 ± 0.4-fold). Kinetic modeling revealed that endotoxemia did not impact the blood-to-liver uptake of 99mTc-mebrofenin, which is mediated by organic anion-transporting polypeptide (Oatp) transporters. However, liver-to-bile and liver-to-blood efflux rates were dramatically decreased, leading to liver accumulation. The transcriptomic profile of hepatocyte transporters consistently showed a downregulation of multidrug resistance-associated proteins 2 and 3 (Mrp2 and Mrp3), which mediate the canalicular and sinusoidal efflux of 99mTc-mebrofenin in hepatocytes, respectively. Rifampicin effectively blocked both the Oatp-mediated influx and the Mrp2/3-related efflux of 99mTc-mebrofenin. The additive impact of endotoxemia and rifampicin led to a 3.0 ± 1.3-fold increase in blood exposure compared with healthy non-treated animals. 99mTc-mebrofenin imaging is useful to investigate disease-associated change in hepatocyte transporter function.

Substrate-Dependent Trans-Stimulation of Organic Cation Transporter 2 Activity.

The search of substrates for solute carriers (SLCs) constitutes a major issue, owing notably to the role played by some SLCs, such as the renal electrogenic organic cation transporter (OCT) 2 (SLC22A2), in pharmacokinetics, drug-drug interactions and drug toxicity. For this purpose, substrates have been proposed to be identified by their cis-inhibition and trans-stimulation properties towards transporter activity. To get insights on the sensitivity of this approach for identifying SLC substrates, 15 various exogenous and endogenous OCT2 substrates were analysed in the present study, using 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (DiASP) as a fluorescent OCT2 tracer substrate. All OCT2 substrates cis-inhibited DiASP uptake in OCT2-overexpressing HEK293 cells, with IC50 values ranging from 0.24 µM (for ipratropium) to 2.39 mM (for dopamine). By contrast, only 4/15 substrates, i.e., acetylcholine, agmatine, choline and metformin, trans-stimulated DiASP uptake, with a full suppression of the trans-stimulating effect of metformin by the reference OCT2 inhibitor amitriptyline. An analysis of molecular descriptors next indicated that trans-stimulating OCT2 substrates exhibit lower molecular weight, volume, polarizability and lipophilicity than non-trans-stimulating counterparts. Overall, these data indicated a rather low sensitivity (26.7%) of the trans-stimulation assay for identifying OCT2 substrates, and caution with respect to the use of such assay may therefore be considered.

Differential Inhibition of Equilibrative Nucleoside Transporter 1 (ENT1) Activity by Tyrosine Kinase Inhibitors.

BACKGROUND AND OBJECTIVES: Equilibrative nucleoside transporter (ENT) 1 is a widely-expressed drug transporter, handling nucleoside analogues as well as endogenous nucleosides. ENT1 has been postulated to be inhibited by some marketed tyrosine kinase inhibitors (TKIs). To obtain insights into this point, the interactions of 24 TKIs with ENT1 activity have been analyzed. METHODS: Inhibition of ENT1 activity was investigated in vitro through quantifying the decrease of [3H]-uridine uptake caused by TKIs in HAP1 ENT2-knockout cells, exhibiting selective ENT1 expression. TKI effects towards ENT1-mediated transport were additionally characterized in terms of their in vivo relevance and of their relationship to TKI molecular descriptors. Putative transport of the TKI lorlatinib by ENT1/ENT2 was analyzed by LC-MS/MS. RESULTS: Of 24 TKIs, 12 of them, each used at 10 µM, were found to behave as moderate or strong inhibitors of ENT1, i.e., they decreased ENT1 activity by at least 35%. This inhibition was concentration-dependent for at least the strongest ones (IC50 less than 10 µM) and was correlated with some molecular descriptors, especially with atom-type E-state indices. Lorlatinib was notably a potent in vitro inhibitor of ENT1/ENT2 (IC50 values around 1.0-2.5 µM) and was predicted to inhibit these nucleoside transporters at relevant clinical concentrations, without, however, being a substrate for them. CONCLUSION: Our data unambiguously add ENT1 to the list of drug transporters inhibited by TKIs, especially by lorlatinib. This point likely merits attention in terms of possible drug-drug interactions, notably for nucleoside analogues, whose ENT1-mediated uptake into their target cells may be hampered by co-administrated TKIs such as lorlatinib.

Comparative in silico prediction of P-glycoprotein-mediated transport for 2010-2020 US FDA-approved drugs using six Web-tools.

P-glycoprotein (P-gp) is an efflux pump implicated in pharmacokinetics and drug-drug interactions. The identification of its substrates is consequently an important issue, notably for drugs under development. For such a purpose, various in silico methods have been developed, but their relevance remains to be fully established. The present study was designed to get insight about this point, through determining the performance values of six freely accessible Web-tools (ADMETlab, AdmetSAR2.0, PgpRules, pkCSM, SwissADME and vNN-ADMET), computationally predicting P-gp-mediated transport. Using an external test set of 231 marketed drugs, approved over the 2010-2020 period by the US Food and Drug Administration and fully in vitro characterized for their P-gp substrate status, various performance parameters (including sensitivity, specificity, accuracy, Matthews correlation coefficient and area under the receiver operating characteristics curve) were determined. They were found to rather poorly meet criteria commonly required for acceptable prediction, whatever the Web-tools were used alone or in combination. Predictions of being P-gp substrate or non-substrate by these online in silico methods may therefore be considered with caution.

Drivers of absolute systemic bioavailability after oral pulmonary inhalation in humans.

There are few studies in humans dealing with the relationship between physico-chemical properties of drugs and their systemic bioavailability after administration via oral inhalation route (Fpulm). Getting further insight in the determinants of Fpulm after oral pulmonary inhalation could be of value for drugs considered for a systemic delivery as a result of poor oral bioavailability, as well as for drugs considered for a local delivery to anticipate their undesirable systemic effects. To better delineate the parameters influencing the systemic delivery after oral pulmonary inhalation in humans, we studied the influence of physico-chemical and permeability properties obtained in silico on the rate and extent of Fpulm in a series of 77 compounds with or without marketing approval for pulmonary delivery, and intended either for local or for systemic delivery. Principal component analysis (PCA) showed mainly that Fpulm was positively correlated with Papp and negatively correlated with %TPSA, without a significant influence of solubility and ionization fraction, and no apparent link with lipophilicity and drug size parameters. As a result of the small sample set, the performance of the different models as predictive of Fpulm were quite average with random forest algorithm displaying the best performance. As a whole, the different models captured between 50 and 60% of the variability with a prediction error of less than 20%. Tmax data suggested a significant positive influence of lipophilicity on absorption rate while charge apparently had no influence. A significant linear relationship between Cmax and dose (R2 = "0.79) highlighted that Cmax was primarily dependent on dose and absorption rate and could be used to estimate Cmax in humans for new inhaled drugs.

Inhibition of organic cation transporter 3 activity by tyrosine kinase inhibitors.

Organic cation transporter (OCT) 3 (SLC22A3) is a widely expressed drug transporter, handling notably metformin and platinum derivatives, as well as endogenous compounds like monoamine neurotransmitters. OCT3 has been shown to be inhibited by a few marketed tyrosine kinase inhibitors (TKIs). The present study was designed to determine whether additional TKIs may interact with OCT3. For this purpose, the effects of 25 TKIs toward OCT3 activity were analyzed using OCT3-overexpressing HEK293 cells. 13/25 TKIs, each used at 10 µM, were found to behave as moderate or strong inhibitors of OCT3 activity, that is, they decreased OCT3-mediated uptake of the fluorescent dye 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide by at least 50% or 80%, respectively. This OCT3 inhibition was correlated to some molecular descriptors of TKIs, such as the percentage of H atoms and that of cationic forms at pH = 7.4. It was concentration-dependent, notably for brigatinib, ceritinib, and crizotinib, which exhibited low half maximal inhibitory concentration (IC50 ) values in the 28-106 nM range. Clinical concentrations of these three marketed TKIs, as well as those of pacritinib, were next predicted to inhibit in vivo OCT3 activity according to regulatory criteria. Cellular TKI accumulation experiments as well as trans-stimulation assays, however, demonstrated that OCT3 does not transport brigatinib, ceritinib, crizotinib, and pacritinib, thus discarding any implication of OCT3 in the pharmacokinetics of these TKIs. Taken together, these data suggest that some TKIs may act as potent inhibitors of OCT3 activity, which may have consequences in terms of drug-drug interactions and toxicity.

Differential in vitro interactions of the Janus kinase inhibitor ruxolitinib with human SLC drug transporters.

Interactions of the Janus kinase (JAK) inhibitor ruxolitinib with solute carriers (SLCs) remain incompletely characterised. The present study was therefore designed to investigate this issue.The interactions of ruxolitinib with SLCs were analysed using transporter-overexpressing human embryonic kidney HEK293 cells. Substrate accumulation was detected by spectrofluorimetry, liquid chromatography coupled to tandem mass spectrometry or scintillation counting.Ruxolitinib was found to potently inhibit the activities of organic anion transporter 3 (OAT3), organic cation transporter 2 (OCT2), multidrug and toxin extrusion 1 (MATE1) and MATE2-K (half maximal inhibitory concentration (IC50) < 10 µM). It blocked OAT1, OAT4, OATP1B1, OATP1B3, OATP2B1 and OCT3, but in a weaker manner (IC50 > 10 µM), whereas OCT1 was not impacted. No time-dependent inhibition was highlighted. When applying the US Food and Drug Administration (FDA) criteria for transporters-related drug-drug interaction risk, OCT2 and MATE2-K, unlike MATE1 and OAT3, were predicted to be in vivo inhibited by ruxolitinib. Cellular uptake studies additionally indicated that ruxolitinib is a substrate for MATE1 and MATE2-K, but not for OAT3 and OCT2.Ruxolitinib in vitro blocked activities of most of SLC transporters. Only OCT2 and MATE-2K may be however clinically inhibited by the JAK inhibitor, with the caution for OCT2 that in vitro inhibition data were generated with an FDA-non recommended fluorescent substrate. Ruxolitinib MATEs-mediated transport may additionally deserve attention for its possible pharmacological consequences in MATE-positive cells.

Interactions of janus kinase inhibitors with drug transporters and consequences for pharmacokinetics and toxicity.

Introduction: Janus kinase inhibitors (JAKinibs) constitute an emerging and promising pharmacological class of anti-inflammatory or anti-cancer drugs, used notably for the treatment of rheumatoid arthritis and some myeloproliferative neoplasms.Areas covered: This review provides an overview of the interactions between marketed JAKinibs and major uptake and efflux drug transporters. Consequences regarding pharmacokinetics, drug-drug interactions and toxicity are summarized.Expert opinion: JAKinibs interact in vitro with transporters in various ways, as inhibitors or as substrates of transporters or as regulators of transporter expression. This may theoretically result in drug-drug interactions (DDIs), with JAKinibs acting as perpetrators or as victims, or in toxicity, via impairment of thiamine transport. Clinical significance in terms of DDIs for JAKinib-transporter interactions remains however poorly documented. In this context, the in vivo unbound concentration of JAKinibs is likely a key parameter to consider for evaluating the clinical relevance of JAKinibs-mediated transporter inhibition. Additionally, the interplay with drug metabolism as well as possible interactions with transporters of emerging importance and time-dependent inhibition have to be taken into account. The role drug transporters may play in controlling cellular JAKinib concentrations and efficacy in target cells is also an issue of interest.