Extracellular Vesicles as Surrogates for the Regulation of the Drug Transporters ABCC2 (MRP2) and ABCG2 (BCRP).

Drug efflux transporters of the ATP-binding-cassette superfamily play a major role in the availability and concentration of drugs at their site of action. ABCC2 (MRP2) and ABCG2 (BCRP) are among the most important drug transporters that determine the pharmacokinetics of many drugs and whose overexpression is associated with cancer chemoresistance. ABCC2 and ABCG2 expression is frequently altered during treatment, thus influencing efficacy and toxicity. Currently, there are no routine approaches available to closely monitor transporter expression. Here, we developed and validated a UPLC-MS/MS method to quantify ABCC2 and ABCG2 in extracellular vesicles (EVs) from cell culture and plasma. In this way, an association between ABCC2 protein levels and transporter activity in HepG2 cells treated with rifampicin and hypericin and their derived EVs was observed. Although ABCG2 was detected in MCF7 cell-derived EVs, the transporter levels in the vesicles did not reflect the expression in the cells. An analysis of plasma EVs from healthy volunteers confirmed, for the first time at the protein level, the presence of both transporters in more than half of the samples. Our findings support the potential of analyzing ABC transporters, and especially ABCC2, in EVs to estimate the transporter expression in HepG2 cells.

Low Exposure to Direct Oral Anticoagulants Is Associated with Ischemic Stroke and Its Severity.

BACKGROUND AND PURPOSE: In acute stroke patients, plasma concentrations of direct oral anticoagulants (DOAC) at hospital admission only poorly mirror DOAC exposure or the coagulation status at the time of the event. Here, we evaluated whether DOAC exposure and DOAC plasma concentration at the time of transient ischemic attacks (TIA) and ischemic strokes correlate with their likelihood of occurrence. METHODS: Prospectively, consecutive DOAC patients with acute ischemic stroke or TIA were included. Admission DOAC plasma concentrations were measured by ultraperformance liquid chromatography- tandem mass spectrometry. Individual DOAC exposure (area under the curve) and DOAC concentrations at event onset were derived from population pharmacokinetic analyses. RESULTS: DOAC exposure was successfully modeled in 211 patients (ischemic stroke 74.4%, TIA 25.6%). Compared to published values, 63.0% had relatively lower DOAC exposure and they more often received lower DOAC doses than recommended (odds ratio [OR], 2.125; 95% confidence interval [CI], 1.039 to 4.560; P=0.044). These patients more likely suffered ischemic strokes than TIA (OR, 2.411; 95% CI, 1.254 to 4.638; P=0.008) and their strokes were more severe (slope, 3.161; 95% CI, 0.741 to 5.58; P=0.011). Low relative DOAC concentrations at event onset were likewise associated with ischemic strokes (OR, 4.123; 95% CI, 1.834 to 9.268; P=0.001), but not to stroke severity (P=0.272). DOAC exposure had a higher explanatory value for stroke severity than concentrations at event. CONCLUSIONS: Low DOAC exposure is strongly associated to ischemic stroke and its severity. By monitoring DOAC plasma concentrations, patients prone to ischemic stroke might be identified.

Differential Effect of a Continental Breakfast on Tacrolimus Formulations With Different Release Characteristics.

Food reduces tacrolimus bioavailability after immediate-release tacrolimus (IR-Tac) and after a new prolonged-release tacrolimus formulation (PR-Tac), when using a high-fat breakfast, but the effects of a continental breakfast on PR-Tac are unknown. In an open-label, 4-phase, randomized, 2-sequence, crossover pharmacokinetic trial, 36 healthy volunteers (18 females) received single 5-mg tacrolimus doses as PR-Tac and as IR-Tac fasted or with a standardized continental breakfast. Tacrolimus pharmacokinetics were analyzed using noncompartmental methods and mixed-model analysis of variance. The continental breakfast significantly decreased average tacrolimus exposure (area under the plasma concentration-time curve) with both preparations (IR-Tac, 67%; 90% confidence interval [CI], 59%-75%; P < .01; and PR-Tac, 79%; 90%CI, 70%-89%; P < .01) with a nonsignificant difference between both preparations (P = .10). The maximum concentration (Cmax ) and the time to maximum concentration (tmax ) were significantly affected only after IR-Tac (Cmax IR-Tac, 39%; 90%CI, 34%-45%; P < .01; and PR-Tac, 87%; 90%CI, 76%-101%; P = .11; tmax IR-Tac, 212%, 90%CI, 179%-252%; P < .01; and PR-Tac, 101%; 90%CI, 86%-120%; P = .89), which was significantly different between both preparations (P < .01). Considering switching from IR-Tac to PR-Tac, predicted dose requirements differed according to the timing of drug intake in relation to food. In conclusion, a continental breakfast decreased average tacrolimus exposure of both preparations to a similar extent. Cmax and tmax were affected only after IR-Tac. The effect of a standardized continental breakfast on PR-Tac was considerably smaller than previously reported effects of a high-fat breakfast on PR-Tac.

Unexpected excessive apixaban exposure: case report of a patient with polymorphisms of multiple apixaban elimination pathways.

BACKGROUND: Apixaban effectively lowers the risk of ischemic stroke and systemic embolism in patients with non-valvular atrial fibrillation. Systemic exposure to a given apixaban dose depends on multiple clearance pathways. Though routine quantification of direct oral anticoagulants (DOACs) in neurological emergency situations has not been widely established, suspected associations of DOAC peak concentrations with bleeding events and DOAC trough concentrations with efficacy and safety suggest that such information might support clinical decision making. CASE PRESENTATION: We describe the case of a 75 year-old woman with atrial fibrillation maintained on apixaban who was admitted due to suspected acute stroke. Clinical work-up did not confirm ischemic or hemorrhagic stroke but routine quantification of apixaban revealed an excessively high apixaban plasma concentration (~ 3 h after the last drug intake: 1100 ng/ml (expected range: 91-321 ng/ml); ~ 12 h after drug intake: 900 ng/ml (expected range: 41-230 ng/ml)) and a substantially prolonged elimination half-life (~ 31 h). The corresponding apixaban concentration-to-dose ratio was 9900 (ng/ml)/(mg/kg/d) and 8100 (ng/ml)/(mg/kg/d), respectively (expected range: 249-463 (ng/ml)/(mg/kg/d)). Renal function was only moderately impaired (creatinine 1.36 mg/dl (0.5-1.1 mg/dl), creatinine clearance 40 ml/min). Genotype analyses revealed that the patient was a CYP3A5*3/*3 non-expressor, a heterozygous carrier of the ABCG2 c.421C/A alleles, and a homozygous carrier of ABCB1 c.2677 T/T and ABCB1 c.3435 T/T. In the absence of known drug interactions explaining apixaban clearance impairment, excessive apixaban concentrations were most probably caused by moderate renal impairment combined with multiple functional polymorphisms of apixaban clearance pathways. CONCLUSIONS: This case suggests that concurrent genetic polymorphisms can impair multiple apixaban elimination pathways and thus substantially increase its exposure.

Prolonged-Release Tacrolimus Is Less Susceptible to Interaction With the Strong CYP3A Inhibitor Voriconazole in Healthy Volunteers.

The nature and extent of drug-drug interactions between oral drugs is affected by numerous modulators. The effect of the formulation (prolonged release (PR) vs. immediate release (IR)) of a victim drug during treatment with a CYP3A (cytochrome P450 enzyme 3A4) inhibitor is unknown but expected to be smaller with PR. We studied PR and IR tacrolimus during treatment with the strong CYP3A inhibitor voriconazole in 18 healthy volunteers in a pharmacokinetic, four-phase, crossover trial. The exposure increase was significantly smaller after PR tacrolimus than after IR tacrolimus (AUC (area under the curve) 2.62-fold vs. 6.02-fold, P < 0.001; Cmax (maximum concentration) 2.02-fold vs. 2.7-fold, P = 0.026) and less variable (AUC increase 1.6 to 4.8-fold vs. 1.8 to 19-fold). CYP3A5 genotype, voriconazole exposure, and CYP3A4 phenotype (determined with a midazolam microdose) were not related to the relative change in tacrolimus exposure. Thus, when considering drug-drug interactions with CYP3A inhibitors, the formulation of orally administered victim drugs should also be considered.

Rivaroxaban and macitentan can be coadministered without dose adjustment but the combination of rivaroxaban and St John's wort should be avoided.

AIMS: We assessed the potential mutual interaction of oral macitentan (cytochrome P450 (CYP) 3A4 substrate) at steady-state with single-dose oral rivaroxaban (CYP3A4 and P-glycoprotein substrate) and evaluated the effect of the CYP3A and P-glycoprotein inducer St John's wort (SJW) on the pharmacokinetics of these drugs in healthy volunteers. METHODS: Twelve healthy volunteers completed this open-label, monocentre, two-period, one-sequence phase I clinical trial. The pharmacokinetics of macitentan (10 mg) was assessed on study days 3 (single dose), 15 (steady-state), 16 (impact of rivaroxaban) and 29 (after induction by oral SJW), and of rivaroxaban on days 2 (single dose), 16 (impact of macitentan at steady-state) and 29 (after induction by SJW). Concurrently, we quantified changes of CYP3A activity using oral microdoses of midazolam (30 µg). RESULTS: Rivaroxaban and macitentan did not significantly change the pharmacokinetics of each other. After induction with SJW, CYP3A activity increased by 272% and geometric mean ratios of macitentan AUC decreased by 48% and of Cmax by 45%. Concurrently, also geometric mean ratios of rivaroxaban AUC and Cmax decreased by 25%. CONCLUSIONS: There is no evidence for a relevant pharmacokinetic interaction between macitentan and rivaroxaban suggesting that these two drugs can be combined without dose adjustment. SJW strongly increased CYP3A activity and substantially reduced rivaroxaban and macitentan exposure while estimated net endothelin antagonism only decreased by 20%, which is considered clinically irrelevant. The combination of SJW with rivaroxaban should be avoided.

Dried-Blood-Spot Technique to Monitor Direct Oral Anticoagulants: Clinical Validation of a UPLC-MS/MS-Based Assay.

Plasma concentrations of direct oral anticoagulants (DOACs) vary largely between individuals, and they correlate well with desired and adverse outcomes. Although regular concentration monitoring of DOACs is not recommended, information on DOAC exposure could be useful in situations when multiple DOAC-clearance pathways are impaired or nonadherence is suspected. Self-sampling techniques, like the use of dried-blood spots (DBSs), would be particularly useful because they enable the collection of information in ambulatory patients at relevant points in time of the dosing interval (e.g., trough). We developed and validated a DBS-based assay to quantify all currently marketed DOACs (apixaban, dabigatran, edoxaban, and rivaroxaban) in a single ultraperformance-liquid-chromatography-tandem-mass-spectrometry assay. It fulfilled all validation standards within a hematocrit range of 0.33-0.65 and was linear over the calibration ranges of 2.5-750 ng/mL (apixaban and rivaroxaban), 4.4-750 ng/mL (dabigatran), and 9.3-750 ng/mL (edoxaban). Only minor ion suppression (matrix effect ≤13%) was present, inter- and intra-assay precision was ≤13%, and inter- and intra-assay accuracies ranged between 88 and 110%. All DOACs were stable in DBSs up to 52 days at room temperature, if the DBSs were protected from light and humidity. The correlation between (whole blood) DBS and plasma concentrations was assessed in 33 patients under regular DOAC therapy. Deming-regression coefficients between simultaneously collected capillary DBSs and plasma samples were used to predict plasma concentrations from DBSs. Bland-Altman plots revealed a strong agreement between predicted and observed plasma concentrations, thus confirming the suitability of DBSs for DOAC monitoring as an important step toward the important aim of self-sampling at home.

Simultaneous quantification of direct oral anticoagulants currently used in anticoagulation therapy.

Direct oral anticoagulants (DOACs) are among the most effective options to prevent serious thromboembolic events in patients with atrial fibrillation. Coagulation assays are used to assess DOAC activity, but lack the possibility to quantify drugs with concurrent pharmacodynamic effect. We developed a selective multi-drug assay to analyze apixaban, betrixaban, dabigatran, edoxaban, edoxaban M4, and rivaroxaban with ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC/MS/MS) in plasma fulfilling all requirements of the FDA und EMA guidelines for bioanalytical method validation. Plasma samples were extracted using solid phase extraction in a 96-well micro volume format. Chromatographic separation was performed on a Waters BEH Phenyl 1.7µm column coupled to tandem mass spectrometry. Extraction recoveries exceeded 80 %. Concentrations of 1-1000 ng/ml can be precisely quantified (correlation coefficient of >0.99) using 100 µL plasma volume. Intra-day and inter-day accuracies ranged between 91.0 % and 116 %. Precisions at low and high concentrations were below 13.3 %. The method was applied within a clinical drug trial and eight short pharmacokinetic profiles of patients under DOAC therapy were analyzed. The assay allows for highly sensitive and selective simultaneous quantification of DOACs in patient plasma samples.

Pharmacokinetics and Clinical Outcomes of Generic Tacrolimus (Hexal) Versus Branded Tacrolimus in De Novo Kidney Transplant Patients: A Multicenter, Randomized Trial.

BACKGROUND: Scrupulous comparison of the pharmacokinetic and clinical characteristics of generic tacrolimus formulations versus the reference drug (Prograf) is essential. The pharmacokinetics of the Tacrolimus Hexal (TacHexal) formulation is similar to Prograf in stable renal transplant patients, but data in de novo patients are lacking. METHODS: De novo kidney transplant patients were randomized to generic tacrolimus (TacHexal) or Prograf in a 6-month open-label study. RESULTS: The primary end point, the dose-normalized area under the curve0-12h at month 1 posttransplant, was similar with TacHexal or Prograf; back-transformed geometric means of adjusted log-transformed values (analysis of variance) were 18.99 ng·h·L (TacHexal) and 20.48 ng·h·L (Prograf) (ratio, 1.08; 90% confidence interval, 0.84-1.38; P = 0.605). The dose-normalized peak concentration geometric means at month 1 was also comparable between treatments (ratio, 1.16; 90% confidence interval, 0.88-1.54; P = 0.377). There were no relevant differences in other pharmacokinetic parameters at month 1 or in area under the curve0-4h and trough concentration when measured at months 3 and 6. The adjusted change in mean estimated glomerular filtration rate from baseline to month 6 (Nankivell) was noninferior for TacHexal versus Prograf using observed values (47.7 vs 38.6 mL/min per 1.73 m, P < 0.001) and was superior based on observed values (P = 0.044) but not using last observation-carried forward method. Rates of biopsy-proven acute rejection (5.7% vs 7.9%), adverse events, and serious adverse events were similar with TacHexal or Prograf. CONCLUSION: Tacrolimus pharmacokinetics is similar with TacHexal and Prograf early after kidney transplantation. Efficacy and safety in this limited data set were comparable, with at least equivalent graft function under TacHexal.

Plasma Drug Concentrations in Patients with Pulmonary Arterial Hypertension on Combination Treatment.

BACKGROUND: Combination therapy with the phosphodiesterase type 5 inhibitors (PDE-5i) sildenafil or tadalafil and the endothelin receptor antagonists (ERA) bosentan, ambrisentan, or macitentan may cause mutual pharmacokinetic interactions in patients with pulmonary arterial hypertension (PAH). OBJECTIVE: The objective of this study was to analyze plasma drug concentrations in PAH patients receiving different combination treatments. METHODS: PAH patients receiving a stable combination treatment with ERA and PDE-5i with targeted dosage for at least 1 month were routinely assessed, including clinical parameters and plasma drug concentrations. Concentrations were normalized considering dose and time from last medication intake and presented as multiples of the expected mean (MoM) of the respective monotherapies. RESULTS: A total of 125 PAH patients (84 female, 41 male, 57% idiopathic/heritable) were included. Sildenafil and tadalafil concentrations were lowest in combination with bosentan (MoM 0.44 ± 0.42, 95% confidence interval [CI] 0.30-0.57, and MoM 0.89 ± 0.53, 95% CI 0.50-1.28, respectively) compared to the combination with ambrisentan (MoM 1.3 ± 0.97, 95% CI 0.86-1.73, and MoM 1.67 ± 0.63, 95% CI 1.40-1.94, respectively) and macitentan (MoM 1.16 ± 0.87, 95% CI 0.86-1.46, and MoM 1.59 ± 0.99, 95% CI 0.80-2.38, respectively). The combination of sildenafil and bosentan led to more than twice the expected bosentan concentrations in 53.8%. Patients switching from sildenafil-bosentan to macitentan showed a significant increase in sildenafil concentrations (p < 0.001). CONCLUSIONS: Only the combination with macitentan or ambrisentan led to targeted mean PDE-5i plasma concentrations and should therefore be preferred to combination with bosentan. Sildenafil-bosentan showed the strongest interaction, with low sildenafil and high bosentan concentrations. The study was not powered to analyze whether lower PDE-5i concentrations cause unsatisfying clinical response. However, plasma concentrations within a targeted range are desirable and may become of increasing importance.

Impact of de novo donor-specific HLA antibodies detected by Luminex solid-phase assay after transplantation in a group of 88 consecutive living-donor renal transplantations.

De novo donor-specific HLA antibodies (DSA) after renal transplantation are known to be correlated with poor graft outcome and the development of acute and chronic rejection. Currently, data for the influence of de novo DSA in patient cohorts including only living-donor renal transplantations (LDRT) are limited. A consecutive cohort of 88 LDRT was tested for the occurrence of de novo DSA by utilizing the highly sensitive Luminex solid-phase assay for antibody detection. Data were analyzed for risk factors for de novo DSA development and correlated with acute rejection (AR) and graft function. Patients with de novo DSA [31 (35%)] showed a trend for inferior graft function [mean creatinine change (mg/dL/year) after the first year: 0.15 DSA (+) vs. 0.02 DSA (-) (P = 0.10)] and a higher rate of AR episodes, especially in case of de novo DSA of both class I and II [6 (55%), (P = 0.05)]. Antibody-mediated rejection (AMR) appeared in five patients and was significantly correlated with de novo DSA (P = 0.05). Monitoring for de novo DSA after LDRT may help to identify patients at risk of declining renal function. Especially patients with simultaneous presence of de novo DSA class I and class II are at a high risk to suffer AR episodes.

Effector T-cell infiltration positively impacts survival of glioblastoma patients and is impaired by tumor-derived TGF-ß.

PURPOSE: In glioma-in contrast to various other cancers-the impact of T-lymphocytes on clinical outcome is not clear. We investigated the clinical relevance and regulation of T-cell infiltration in glioma. EXPERIMENTAL DESIGN: T-cell subpopulations from entire sections of 93 WHO°II-IV gliomas were computationally identified using markers CD3, CD8, and Foxp3; survival analysis was then done on primary glioblastomas (pGBM). Endothelial cells expressing cellular adhesion molecules (CAM) were similarly computationally quantified from the same glioma tissues. Influence of prominent cytokines (as measured by ELISA from 53 WHO°II-IV glioma lysates) on CAM-expression in GBM-isolated endothelial cells was determined using flow cytometry. The functional relevance of the cytokine-mediated CAM regulation was tested in a transmigration assay using GBM-derived endothelial cells and autologous T-cells. RESULTS: Infiltration of all T-cell subsets increased in high-grade tumors. Most strikingly, within pGBM, elevated numbers of intratumoral effector T cells (T(eff), cytotoxic and helper) significantly correlated with a better survival; regulatory T cells were infrequently present and not associated with GBM patient outcome. Interestingly, increased infiltration of T(eff) cells was related to the expression of ICAM-1 on the vessel surface. Transmigration of autologous T cells in vitro was markedly reduced in the presence of CAM-blocking antibodies. We found that TGF-ß molecules impeded transmigration and downregulated CAM-expression on GBM-isolated endothelial cells; blocking TGF-ß receptor signaling increased transmigration. CONCLUSIONS: This study provides comprehensive and novel insights into occurrence and regulation of T-cell infiltration in glioma. Specifically, targeting TGF-ß1 and TGF-ß2 might improve intratumoral T-cell infiltration and thus enhance effectiveness of immunotherapeutic approaches.