Impact of hypoxia and reoxygenation on the extra/intracellular metabolome and on transporter expression in a human kidney proximal tubular cell line.

INTRODUCTION: Ischemia-reperfusion injury (IRI) induces several perturbations that alter immediate kidney graft function after transplantation and may affect long-term graft outcomes. Given the IRI-dependent metabolic disturbances previously reported, we hypothesized that proximal transporters handling endo/exogenous substrates may be victims of such lesions. OBJECTIVES: This study aimed to determine the impact of hypoxia/reoxygenation on the human proximal transport system through two semi-targeted omics analyses. METHODS: Human proximal tubular cells were cultured in hypoxia (6 or 24 h), each followed by 2, 24 or 48-h reoxygenation. We investigated the transcriptomic modulation of transporters. Using semi-targeted LC-MS/MS profiling, we characterized the extra/intracellular metabolome. Statistical modelling was used to identify significant metabolic variations. RESULTS: The expression profile of transporters was impacted during hypoxia (y + LAT1 and OCTN2), reoxygenation (MRP2, PEPT1/2, rBAT, and OATP4C1), or in both conditions (P-gp and GLUT1). The P-gp and GLUT1 transcripts increased (FC (fold change) = 2.93 and 4.11, respectively) after 2-h reoxygenation preceded by 24-h hypoxia. We observed a downregulation (FC = 0.42) of y+LAT1 after 24-h hypoxia, and of PEPT2 after 24-h hypoxia followed by 2-h reoxygenation (FC = 0.40). Metabolomics showed that hypoxia altered the energetic pathways. However, intracellular metabolic homeostasis and cellular exchanges were promptly restored after reoxygenation. CONCLUSION: This study provides insight into the transcriptomic response of the tubular transporters to hypoxia/reoxygenation. No correlation was found between the expression of transporters and the metabolic variations observed. Given the complexity of studying the global tubular transport systems, we propose that further studies focus on targeted transporters.

A single Bayesian estimator for iohexol clearance estimation in ICU, liver failure and renal transplant patients.

AIMS: Iohexol clearance has been proposed to estimate the glomerular filtration rate (GFR). A population pharmacokinetics (popPK) model was developed from heterogeneous patients. A Bayesian estimator (MAP-BE) based on a limited sampling strategy (LSS) was derived and evaluated in external patients. METHODS: Full pharmacokinetic data (7-12 samples) from 172 patients receiving iohexol for measurement of their GFR (unstable and stable ICU patients, liver failure patients and kidney transplant patients) were split into development (n = 136) and validation (n = 36) datasets. A PopPK model was developed in Monolix and was used to develop MAP-BE based on LSS. Its performance for GFR estimation was evaluated in the validation set. RESULTS: A two-compartment model with first-order elimination best described the data. The final model included the type of patients on volume of distribution (Vd), clearance and intercompartmental constants, serum creatinine on clearance and body weight on Vd. The best LSS included samples at 0.1-1-9 h exhibiting a relative mean prediction error (MPE) (RMSE) = -3.7% (14.3%) and better performance than the Bröchner-Mortensen formula (-3.0%/17%). Split by type of patients, the highest interindividual variability and imprecision was observed in unstable ICU patients (MPE (RMSE) = 3.7% (18.8%)) while the best performances were obtained for renal transplant patients (MPE (RMSE) = 1.0% (5.8%)). All LSS that included samples before 9 hours for the third sample were associated with an increased imprecision. CONCLUSION: A single MAP-BE of iohexol based on a three-sample LSS for four heterogeneous populations was developed and allowed accurate estimation of GFR in kidney transplant patients, slightly biased in stable ICU patients and slightly imprecise in unstable ICU patients.

Kinetic Glomerular Filtration Rate Equations in Patients With Shock: Comparison With the Iohexol-Based Gold-Standard Method.

OBJECTIVES: Static glomerular filtration rate formulas are not suitable for critically ill patients because of nonsteady state glomerular filtration rate and variation in the volume of distribution. Kinetic glomerular filtration rate formulas remain to be evaluated against a gold standard. We assessed the most accurate kinetic glomerular filtration rate formula as compared to iohexol clearance among patients with shock. DESIGN: Retrospective multicentric study. SETTING: Three French ICUs in tertiary teaching hospitals. PATIENTS: Fifty-seven patients within the first 12 hours of shock. MEASUREMENTS AND MAIN RESULTS: On day 1, we compared kinetic glomerular filtration rate formulas with iohexol clearance, with or without creatinine concentration correction according to changes in volume of distribution and ideal body weight. We analyzed three static glomerular filtration rate formulas (Cockcroft and Gault, modification of diet in renal disease, and Chronic Kidney Disease-Epidemiology Collaboration), urinary creatinine clearance, and seven kinetic glomerular filtration rate formulas (Jelliffe, Chen, Chiou and Hsu, Moran and Myers, Yashiro, Seelhammer, and Brater). We evaluated 33 variants of these formulas after applying corrective factors. The bias ranged from 12 to 47 mL/min/1.73 m2. Only the Yashiro equation had a lower bias than urinary creatinine clearance before applying corrective factors (15 vs 20 mL/min/1.73 m2). The corrected Moran and Myers formula had the best mean bias, 12 mL/min/1.73 m2, but wide limits of agreement (-50 to 73). The corrected Moran and Myers value was within 30% of iohexol-clearance-measured glomerular filtration rate for 27 patients (47.4%) and was within 10% for nine patients (15.8%); other formulas showed even worse accuracy. CONCLUSIONS: Kinetic glomerular filtration rate equations are not accurate enough for glomerular filtration rate estimation in the first hours of shock, when glomerular filtration rate is greatly decreased. They can both under- or overestimate glomerular filtration rate, with a trend to overestimation. Applying corrective factors to creatinine concentration or volume of distribution did not improve accuracy sufficiently to make these formulas reliable. Clinicians should not use kinetic glomerular filtration rate equations to estimate glomerular filtration rate in patients with shock.

Hypercalcemia is associated with a poor prognosis in lymphoma a retrospective monocentric matched-control study and extensive review of published reported cases.

The prognostic significance of hypercalcemia in lymphoma has only been studied on small series to date. We conducted a retrospective, monocentric, matched-control study that aimed to compare the outcome of patients diagnosed with any histological subtype of lymphoma associated with hypercalcemia, at diagnosis or relapse, with a group of controls matched for histological and prognostic factors. Sixty-two and 118 comparable patients treated between 2000 and 2016 were included in hypercalcemia and control cohorts, respectively. Hypercalcemia was found mainly at diagnosis (71%) in higher-risk patients (prognosis scores ≥ 3, 76%) and those with diffuse large B cell lymphoma (67.7%), stage III/IV disease (91.9%), and elevated LDH (90.3%). Two-year progression-free survival (PFS) was shorter in the hypercalcemia than control cohort [30.1% (95% confidence interval (95% CI) 18.3-41.9) vs 63.9% (95% CI 5.1-72.7), p < 0.001]. Two-year overall survival (OS) was 40.6% (95% CI 28.1-53.1) and 77.7% (95% CI 70.1-85.3) in the hypercalcemia and control cohorts, respectively (p < 0.001). Hypercalcemia was independently associated with poor PFS [HR = 2.5 (95% CI 1.4-3.5)] and OS [HR = 4.7 (95% CI 2.8-7.8)] in multivariate analysis. Among the 40 patients who received autologous stem cell transplantation (ASCT), hypercalcemia was still associated with shorter OS [2-year OS: 65% (95% CI 40.1-89.9) vs 88.0 (95% CI 75.3-100), p = 0.04]. Hypercalcemia may be associated with chemo-resistance, given its impact on PFS and OS. Hence, these data suggest that alternate strategies for lymphoma patients with hypercalcemia should be developed.

Assessment of the glomerular filtration rate (GFR) in kidney transplant recipients using Bayesian estimation of the iohexol clearance.

Background Plasma iohexol clearance (CLiohexol) is a reference technique for glomerular filtration rate (GFR) determination. In routine practice, CLiohexol is calculated using one of several formulas, which have never been evaluated in kidney transplant recipients. We aimed to model iohexol pharmacokinetics in this population, evaluate the predictive performance of three simplified formulas and evaluate whether a Bayesian algorithm improves CLiohexol estimation. Methods After administration of iohexol, six blood samples were drawn from 151 patients at various time points. The dataset was split into two groups, one to develop the population pharmacokinetic (POPPK) model (n = 103) and the other (n = 48) to estimate the predictive performances of the various GFR estimation methods. GFR reference values (GFRref) in the validation dataset were obtained by non-compartmental pharmacokinetic (PK) analysis. Predictive performances of each method were evaluated in terms of bias (ME), imprecision (root mean square error [RMSE]) and number of predictions out of the ±10% or 15% error interval around the GFRref. Results A two-compartment model best fitted the data. The Bayesian estimator with samples drawn at 30, 120 and 270 min allowed accurate prediction of GFRref (ME = 0.47%, RMSE = 3.42%), as did the Brøchner-Mortensen (BM) formula (ME = - 0.0425%, RMSE = 3.40%). With both methods, none of the CL estimates were outside the ±15% interval and only 2.4% were outside the ±10% for the BM formula (and none for the Bayesian estimator). In patients with GFR ≤30 mL/min/1.73 m2, the BM formula performed very well, while the Bayesian method could not be evaluated in depth due to too small a number of patients with adequate sampling times. Conclusions GFR can be estimated with acceptable accuracy in kidney transplant patients using the BM formula, but also using a Bayesian algorithm.

Glomerular Hyper- and Hypofiltration During Acute Circulatory Failure: Iohexol-Based Gold-Standard Descriptive Study.

OBJECTIVE: To assess glomerular filtration rate in the early phase of acute circulatory failure by measuring iohexol plasma clearance. DESIGN: Interventional prospective multicentric study. SETTING: Three French ICUs in tertiary teaching hospitals. PATIENTS: Patients with acute circulatory failure within 12 hours after ICU admission. INTERVENTIONS: IV administration of a nontoxic 5-mL dose of iohexol. Collection of nine arterial blood samples over 24 hours for iohexol plasma concentration measurements. Iohexol clearance calculation with a population pharmacokinetic model. Iohexol clearance was an estimation of the mean glomerular filtration rate over 24 hours. MEASUREMENTS AND MAIN RESULTS: Among 99 included patients, we could calculate iohexol clearance for 85. The median iohexol clearance was 31 mL/min (interquartile range, 16-44). According to iohexol clearance, 41 patients (48%) had severe hypofiltration (clearance, < 30 mL/min), 29 (34%) had moderate hypofiltration, and 10 (12%) had mild hypofiltration (clearance, 30-60 and 60-90 mL/min, respectively). Four patients (5%) had normal glomerular filtration rate, and only one (1%) showed hyperfiltration (clearance, > 130 mL/min). Urinary creatinine clearance underestimated renal impairment in one patient out of two; the bias of iohexol clearance toward 24-hour urinary creatinine clearance over the same period was -18.1 mL/min (limits of agreement, -73.5 to 37.4). CONCLUSIONS: We demonstrated the feasibility of iohexol clearance measurement in unstable critically ill patients. Normal kidney function is exceptional during the early phase of acute circulatory failure. Glomerular filtration rate estimation by urinary creatinine clearance frequently fails to detect renal impairment. Hyperfiltration is very infrequent.

Ischemia/reperfusion-associated tubular cells injury in renal transplantation: Can metabolomics inform about mechanisms and help identify new therapeutic targets?

Tubular cells are central targets of ischemia-reperfusion (I/R) injury in kidney transplantation. Inflammation and metabolic disturbances occurring within these cells are deleterious by themselves but also favor secondary events, such as activation of immune response. It is critical to have an in depth understanding of the mechanisms governing tubular cells response to I/R if one wants to define pertinent biomarkers or to elaborate targeted therapeutic interventions. As oxidative damage was shown to be central in the patho-physiological mechanisms, the impact of I/R on proximal tubular cells metabolism has been widely studied, contrary to its effects on expression and activity of membrane transporters of the proximal tubular cells. Yet, temporal modulation of transporters over ischemia and reperfusion periods appears to play a central role, not only in the induction of cells injury but also in graft function recovery. Metabolomics in cell models or diverse biofluids has the potential to provide large pictures of biochemical consequences of I/R. Metabolomic studies conducted in experimental models of I/R or in transplanted patients indeed retrieved metabolites belonging to the pathways known to be particularly affected. Interestingly, they also revealed that metabolic disturbances and transporters activities are in very close mutual interplay. As well as helping to select diagnostic biomarkers, such analyses could also contribute to identify new pharmacological targets and to set up innovative nephroprotective strategies for the future. Even if various therapeutic approaches have been evaluated for a long time to prevent or treat I/R injuries, metabolomics has helped identifying new ones, those related to membrane transporters seeming to be of particular interest. However, considering the very complex and multifactorial effects of I/R in the context of kidney transplantation, all tracks must be followed if one wants to prevent or limit its deleterious consequences.

Endogenous metabolites that are substrates of organic anion transporter's (OATs) predict methotrexate clearance.

Variable pharmacokinetics of high-dose-methotrexate (MTX) is responsible for severe toxicities. Unpredictable overexposure still occurs during some courses despite having controlled the main factors known to play a role in its elimination. The aim of our study was to evaluate whether the urine metabolomic profile measured at the time of MTX administration is predictive of the drug's clearance and/or of treatment-related toxicity. We analyzed the urine content of endogenous metabolites before MTX administration in a cohort of adult patients treated for lymphoid malignancies. Individual MTX clearance (MTXCL) was estimated from population pharmacokinetic analyses of therapeutic drug monitoring data. We determined the urine metabolite content by gas chromatography-mass spectrometry (GC-MS) and applied Partial Least Square (PLS) analysis to assess the relationship between the urine metabolome and MTXCL. External validation was applied to evaluate the performances of the PLS model. We used orthogonal partial least squares discriminant analysis (OPLS-DA) to distinguish patients with normal or delayed elimination, and patients with or without toxicity. Sixty-two patients were studied. We obtained a very good prediction of individual MTX clearance using a set of 28 metabolites present in patient urine at baseline. The mean prediction error and precision were -0.36% and 21.4%, respectively, for patients not included in the model. The model included a set of endogenous organic anions, of which the tubular secretion depends on organic anion transporter (OAT) function. Our analyses did not allow us to discriminate between patients with or without delayed elimination or those who did or did not experience toxicity. Urinary metabolomics can be informative about an individual's ability to clear MTX. More broadly, it paves the way for the development of a biomarker of tubular secretion, easily measurable from endogenous substances.

A Time-Dependent Model Describes Methotrexate Elimination and Supports Dynamic Modification of MRP2/ABCC2 Activity.

BACKGROUND: Multidrug resistance protein-2 encoded by the ABCC2 gene (MRP2/ABCC2), an efflux transporter expressed at the proximal renal tubule, is rate-limiting for urine excretion of coproporphyrin (UCP) isomers I and III, translating in high UCP [I/(I + III)] ratio in MRP2-deficient patients presenting with the Dubin-Johnson Syndrome. MRP2 is also a major contributor to methotrexate (MTX) clearance. As MTX is both a substrate and an inhibitor of MRP2, time course of the concentrations of MTX in blood could induce functional modification of MRP2 over time, which in turn can modify its own elimination rate. METHODS: A 3-parameter time-dependent MTX population pharmacokinetic (PK) model based on a power function accounting for nonlinearity in its clearance was developed using Pmetrics in a first cohort of 41 patients (76 PK profiles) and compared with a previously published 2-compartment model developed with NONMEM and a 3-compartment model developed with ITSIM. In a second cohort (62 patients and 62 PK profiles), the association between the UCP [I/(I + III)] ratio at 3 periods [before MTX administration (P1), at the end of infusion (P2), and at hospital discharge (P3)] and the time-dependent PK parameters of MTX was investigated. Effects of genetic polymorphisms and of coadministered drugs were also studied. RESULTS: The model developed tightly fitted the data in both cohorts. A significant inverse correlation was found between log (k1) (ie, the rate constant explaining MTX concentration decrease) and the difference in UCP [I/(I + III)] ratio between P3 and P2 (DP3) (ß ± SD = -0.025 ± 0.008, P = 0.00443). CONCLUSIONS: Self-inhibition of the MRP2-dependent secretion of MTX is a plausible explanation for the time-dependent PKs of this drug. Additional studies specifically designed to evaluate this hypothesis are required.

Pharmacogenetics-based personalized therapy: Levels of evidence and recommendations from the French Network of Pharmacogenetics (RNPGx).

More than 50 laboratories offer pharmacogenetic testing in France. These tests are restricted to a limited number of indications: prevention of serious adverse drug reactions; choice of most appropriate therapeutic option; dose adjustment for a specific drug. A very small proportion of these tests are mentioned in drug information labeling and the data provided (if any) are generally insufficient to ascertain whether a test is required and if it is useful. This article discusses the rationale for evaluating the performance and clinical usefulness of pharmacogenetics and provides, on behalf of the French national network of pharmacogenetics (RNPGx), three levels of recommendation for testing: essential, advisable, and possibly helpful.

Iohexol clearance in unstable critically ill patients: a tool to assess glomerular filtration rate.

BACKGROUND: Acute kidney injury (AKI) is associated with significant morbidity and mortality, particularly in unstable critically ill patients. In this context, serum creatinine concentration is an imperfect tool for estimating glomerular filtration rate (GFR), an index of renal function. The objective of this pilot study was to evaluate the feasibility of measuring iohexol clearance for GFR assessment in critically ill patients with acute circulatory failure at intensive care unit (ICU) admission. METHODS: ICU patients were prospectively included within 12 h of acute circulatory failure; a non-toxic dose of iohexol (5 mL) was infused intravenously and iohexol plasma concentration decrease was measured over 24 h. Urinary iohexol concentration was measured in urine samples collected four times, every 6 h for 24 h. The Kidney Disease Improving Global Outcome score, measuring AKI, was calculated each day. RESULTS: Among 18 patients with acute circulatory failure, AKI developed in 15; 14 showed decreased serum creatinine concentration during the first 24 h even though 10 presented AKI. The absolute variation in serum creatinine concentration was correlated with fluid balance over 24 h. Median [min; max] plasma clearance of iohexol was 39.4 mL/min [6.1; 154.0] and iohexol urinary clearance 32.8 mL/min [0.8-170.4]. The correlation between plasma and urinary clearance was ρ=0.97, p<0.0001. CONCLUSIONS: GFR may be estimated by plasma iohexol clearance in unstable critically ill patients. This method is reliable, correlates very well with urinary iohexol clearance and does not depend on input/output fluid balance and fluid infusion, as compared with serum creatinine concentration.

Safety study of Ciprofloxacin in newborn mice.

Ciprofloxacin, a broad-spectrum antimicrobial agent belonging to the fluoroquinolone family, is prescribed off-label in infants less than one year of age. Ciprofloxacin is included in the European Medicines Agency priority list of off-patent medicinal products requiring evaluation in neonates. This evaluation is undergoing within the TINN (Treat Infections in Neonates) FP7 EU project. As part of the TINN project, the present preclinical study was designed to assess the potential adverse effects of Ciprofloxacin on neurodevelopment, liver and joints in mice. Newborn mice received subcutaneous Ciprofloxacin at 10, 30 and 100 mg/kg/day from 2 to 12 postnatal days. Peak plasma levels of Ciprofloxacin were in the range of levels measured in human neonates. We examined vital functions in vivo, including cardiorespiratory parameters and temperature, psychomotor development, exploratory behavior, arthro-, nephro- and hepato-toxic effects. We found no effect of Ciprofloxacin at 10 and 30 mg/kg/day. In contrast, administration at 100 mg/kg/day delayed weight gain, impaired cardiorespiratory and psychomotor development, caused inflammatory infiltrates in the connective tissues surrounding the knee joint, and moderately increased extramedullary hematopoiesis. The present study pleads for careful watching of cardiorespiratory and motor development in neonates treated with Ciprofloxacin, in addition to the standard surveillance of arthrotoxicity.

Population pharmacokinetics of ciprofloxacin in neonates and young infants less than three months of age.

Ciprofloxacin is used in neonates with suspected or documented Gram-negative serious infections. Currently, its use is off-label partly because of lack of pharmacokinetic studies. Within the FP7 EU project TINN (Treat Infection in NeoNates), our aim was to evaluate the population pharmacokinetics of ciprofloxacin in neonates and young infants <3 months of age and define the appropriate dose in order to optimize ciprofloxacin treatment in this vulnerable population. Blood samples were collected from neonates treated with ciprofloxacin and concentrations were quantified by high-pressure liquid chromatography-mass spectrometry. Population pharmacokinetic analysis was performed using NONMEM software. The data from 60 newborn infants (postmenstrual age [PMA] range, 24.9 to 47.9 weeks) were available for population pharmacokinetic analysis. A two-compartment model with first-order elimination showed the best fit with the data. A covariate analysis identified that gestational age, postnatal age, current weight, serum creatinine concentration, and use of inotropes had a significant impact on ciprofloxacin pharmacokinetics. Monte Carlo simulation demonstrated that 90% of hypothetical newborns with a PMA of <34 weeks treated with 7.5 mg/kg twice daily and 84% of newborns with a PMA ≥34 weeks and young infants receiving 12.5 mg/kg twice daily would reach the AUC/MIC target of 125, using the standard EUCAST MIC susceptibility breakpoint of 0.5 mg/liter. The associated risks of overdose for the proposed dosing regimen were <8%. The population pharmacokinetics of ciprofloxacin was evaluated in neonates and young infants <3 months old, and a dosing regimen was established based on simulation.

Urinary coproporphyrin I/(I + III) ratio as a surrogate for MRP2 or other transporter activities involved in methotrexate clearance.

AIMS: The urinary coproporphyrin I/(I + III) ratio may be a surrogate for MRP2 activity. We conducted a prospective study in patients receiving methotrexate (MTX) to examine the relationship between this ratio and the pharmacokinetics of a MRP2 substrate. METHODS: Three urine samples were collected from 81 patients for UCP I/(I + III) ratio determination: one before (P1), one at the end of MTX infusion (P2), and one on the day of hospital discharge (P3). Three polymorphisms of ABCC2 were analysed and their relationships with basal UCP I/(I + III) ratio values assessed. All associated drugs were recorded and a drug interaction score (DIS) was assigned. Population pharmacokinetic analysis was conducted to assess whether MTX clearance (MTXCL) was associated with the basal UCP I/(I + III) ratio, its variation during MTX infusion, the DIS or other common covariates. RESULTS: The basal UCP I/(I + III) ratio was not associated with ABCC2 polymorphisms and did not differ according to the DIS. Significant changes in the ratio were observed over time, with an increase between P1 and P2 and a decrease at P3 (P < 0.001). No association was found between basal UCP I/(I + III) ratio and MTXCL. The final model indicates that MTXCL was dependent on the change in the ratio between P1 and P3, DIS and creatinine clearance. CONCLUSION: The basal UCP I/(I + III) ratio is not predictive of MTXCL. However, it is sensitive to the presence of MTX, so it is plausible that it reflects a function modified in response to the drug.

Population pharmacokinetics of rituximab with or without plasmapheresis in kidney patients with antibody-mediated disease.

AIMS: Both rituximab and plasmapheresis can be associated in the treatment of immune-mediated kidney diseases. The real impact of plasmapheresis on rituximab pharmacokinetics is unknown. The aim of this study was to compare rituximab pharmacokinetics between patients requiring plasmapheresis and others without plasmapheresis. METHODS: The study included 20 patients receiving one or several infusions of rituximab. In 10 patients, plasmapheresis sessions were also performed (between two and six sessions per patient). Rituximab concentrations were measured in blood samples in all patients and in discarded plasma obtained by plasmapheresis using an enzyme-linked immunosorbent assay method. Data were analysed according to a population pharmacokinetic approach. RESULTS: The mean percentage of rituximab removed during the first plasmapheresis session ranged between 47 and 54% when plasmapheresis was performed between 24 and 72 h after rituximab infusion. Rituximab pharmacokinetics was adequately described by a two-compartment model with first-order elimination. Plasmapheresis had a significant impact on rituximab pharmacokinetics, with an increase of rituximab clearance by a factor of 261 (95% confidence interval 146-376), i.e. from 6.64 to 1733 ml h(-1) . Plasmapheresis performed 24 h after rituximab infusion decreased the rituximab area under the curve by 26%. CONCLUSIONS: Plasmapheresis removed an important amount of rituximab when performed less than 3 days after infusion. The removal of rituximab led to a significant decrease of the area under the curve. This pharmacokinetic observation should be taken into account for rituximab dosing, e.g. an additional third rituximab infusion may be recommended when three plasmapheresis sessions are performed after the first rituximab infusion.

Substrate binding and lipid-mediated allostery in the human organic anion transporter 1 at the atomic-scale.

The Organic Anion Transporter 1 is a membrane transporter known for its central role in drug elimination by the kidney. hOAT1 is an antiporter translocating substrate in exchange for a-ketoglutarate. The understanding of hOAT1 structure and function remains limited due to the absence of resolved structure of hOAT1. Benefiting from conserved structural and functional patterns shared with other Major Facilitator Superfamily transporters, the present study intended to investigate fragments of hOAT1 transport function and modulation of its activity in order to make a step forward the understanding of its transport cycle. µs-long molecular dynamics simulation of hOAT1 were carried out suggesting two plausible binding sites for a typical substrate, adefovir, in line with experimental observations. The well-known B-like motif binding site was observed in line with previous studies. However, we here propose a new inner binding cavity which is expected to be involved in substrate translocation event. Binding modes of hOAT1 co-substrate α-ketoglutarate were also investigated suggesting that it may bind to highly conserved intracellular motifs. We here hypothesise that α-ketoglutarate may disrupt the pseudo-symmetrical intracellular charge-relay system which in turn may participate to the destabilisation of OF conformation. Investigations regarding allosteric communications along hOAT1 also suggest that substrate binding event might modulate the dynamics of intracellular charge relay system, assisted by surrounding lipids as active partners. We here proposed a structural rationalisation of transport impairments observed for two single nucleotide polymorphisms, p.Arg50His and p.Arg454Gln suggesting that the present model may be used to transport dysfunctions arising from hOAT1 mutations.

Current diagnostic and clinical issues of screening for dihydropyrimidine dehydrogenase deficiency.

Fluoropyrimidine drugs (FP) are the backbone of many chemotherapy protocols for treating solid tumours. The rate-limiting step of fluoropyrimidine catabolism is dihydropyrimidine dehydrogenase (DPD), and deficiency in DPD activity can result in severe and even fatal toxicity. In this review, we survey the evidence-based pharmacogenetics and therapeutic recommendations regarding DPYD (the gene encoding DPD) genotyping and DPD phenotyping to prevent toxicity and optimize dosing adaptation before FP administration. The French experience of mandatory DPD-deficiency screening prior to initiating FP is discussed.

A case of severe toxicity during coadministration of vincristine and piperacillin: are drug transporters involved in vincristine hypersensitivity and drug-drug interactions?

Neurotoxicity is frequent with vincristine treatment, but severe autonomic neuropathy is rare. A decreased activity of drug transporters in the presence of an interacting drug may favor such events by increasing systemic or tissue exposure to the drug. We encountered severe autonomic neuropathy and cholestasis in a child receiving vincristine, after the introduction of piperacillin-tazobactam. A causality assessment of the adverse reaction identified the antibiotic as the most probable cause of the observation. The patient was heterozygous for several common polymorphisms of ABCC2 (multidrug-related protein-2), CYP3A5, and ABCB1 (multidrug-related protein-1, P-glycoprotein), but their role in the toxicity cannot be ascertained.

Insights into the structure and function of the human organic anion transporter 1 in lipid bilayer membranes.

The human SLC22A6/OAT1 plays an important role in the elimination of a broad range of endogenous substances and xenobiotics thus attracting attention from the pharmacological community. Furthermore, OAT1 is also involved in key physiological events such as the remote inter-organ communication. Despite its significance, the knowledge about hOAT1 structure and the transport mechanism at the atomic level remains fragmented owing to the lack of resolved structures. By means of protein-threading modeling refined by µs-scaled Molecular Dynamics simulations, the present study provides the first robust model of hOAT1 in outward-facing conformation. Taking advantage of the AlphaFold 2 predicted structure of hOAT1 in inward-facing conformation, we here provide the essential structural and functional features comparing both states. The intracellular motifs conserved among Major Facilitator Superfamily members create a so-called "charge-relay system" that works as molecular switches modulating the conformation. The principal element of the event points at interactions of charged residues that appear crucial for the transporter dynamics and function. Moreover, hOAT1 model was embedded in different lipid bilayer membranes highlighting the crucial structural dependence on lipid-protein interactions. MD simulations supported the pivotal role of phosphatidylethanolamine components to the protein conformation stability. The present model is made available to decipher the impact of any observed polymorphism and mutation on drug transport as well as to understand substrate binding modes.