Lack of CYP3A4 protein induction despite mRNA induction in primary hepatocytes exposed to rifabutin as a possible explanation for its low interaction risk in vivo.

Rifampicin is a strong inducer of cytochrome P450 (CYP3A4) and P-glycoprotein (P-gp/ABCB1), leading to profound drug-drug interactions. In contrast, the chemically related rifabutin does not show such pronounced induction properties in vivo. The aim of our study was to conduct a comprehensive analysis of the different induction potentials of rifampicin and rifabutin in primary human hepatocytes and to analyze the mechanism of potential differences. Therefore, we evaluated CYP3A4/ABCB1 mRNA expression (polymerase chain reaction), CYP3A4/P-gp protein expression (immunoaffinity-liquid chromatography-mass spectrometry, IA-LC-MS/MS), CYP3A4 activity (testosterone hydroxylation), and considered intracellular drug uptake after treatment with increasing rifamycin concentrations (0.01-10 µM). Furthermore, rifamycin effects on the protein levels of CYP2C8, CYP2C9, and CYP2C19 were analyzed (IA-LC-MS/MS). Mechanistic analysis included the evaluation of possible suicide CYP3A4 inhibition (IC50 shift assay) and drug impact on translational efficiency (cell-free luminescence assays). Rifabutin accumulated 6- to 15-fold higher in hepatocytes than rifampicin, but induced CYP3A4 mRNA comparably to rifampicin (e. g. rifampicin 61-fold vs. rifabutin 44-fold, 72 h). While rifampicin for example enhanced protein (10 µM: 21-fold) and activity levels considerably (53-fold), rifabutin only slightly increased CYP3A4 protein expression (10 µM: 3.3-fold) or activity (11-fold) compared to rifampicin after 72 h. Both rifamycins similarly influenced expression of other eliminating proteins. A potential CYP3A4 suicide inhibition by a specific rifabutin metabolite or disruption of ribosome function were excluded experimentally. In conclusion, the lack of protein enhancement, could explain rifabutin's weaker induction-related drug-drug interaction risk in vivo.

Rifabutin but not rifampicin can partly out-balance P-glycoprotein induction by concurrent P-glycoprotein inhibition through high affinity binding to the inhibitory site.

Physiology-based pharmacokinetic modeling suggests that rifabutin can out-balance P-glycoprotein (P-gp) induction by concurrent P-gp inhibition. However, clinical or experimental evidence for this Janus-faced rifabutin effect is missing. Consequently, LS180 cells were exposed to a moderately (2 µM) and strongly (10 µM) P-gp-inducing concentration of rifampicin or rifabutin for 6 days. Cellular accumulation of the fluorescent P-gp substrate rhodamine 123 was evaluated using flow cytometry, either without (induction only) or with adding rifamycin drug to the cells during the rhodamine 123 efflux phase (induction + potential inhibition). Rhodamine 123 accumulation was decreased similarly by both drugs after 6-day exposure (2 µM: 55% residual fluorescence compared to non-induced cells, P < 0.01; 10 µM: 30% residual fluorescence compared to non-induced cells, P < 0.001), indicating P-gp induction. Rhodamine 123 influx transporters mRNA expressions were not affected, excluding off-target effects. Acute re-exposure to rifabutin, however, considerably re-increased rhodamine 123 accumulation (2 µM induction: re-increase by 55%, P < 0.01; 10 µM induction: 49% re-increase, P < 0.001), suggesting P-gp inhibition. In contrast, rifampicin only had weak effects (2 µM induction: no re-increase; 10 µM induction: 16% re-increase; P < 0.05). Molecular docking analysis eventually revealed that rifabutin has a higher binding affinity to the inhibitor binding site of P-gp than rifampicin (ΔG (kcal/mol) = -11.5 vs -5.3). Together, this study demonstrates that rifabutin can at least partly mask P-gp induction by P-gp inhibition, mediated by high affinity binding to the inhibitory site of P-gp.

In vitro evaluation of the reductive carbonyl idarubicin metabolism to evaluate inhibitors of the formation of cardiotoxic idarubicinol via carbonyl and aldo-keto reductases.

The most important dose-limiting factor of the anthracycline idarubicin is the high risk of cardiotoxicity, in which the secondary alcohol metabolite idarubicinol plays an important role. It is not yet clear which enzymes are most important for the formation of idarubicinol and which inhibitors might be suitable to suppress this metabolic step and thus would be promising concomitant drugs to reduce idarubicin-associated cardiotoxicity. We, therefore, established and validated a mass spectrometry method for intracellular quantification of idarubicin and idarubicinol and investigated idarubicinol formation in different cell lines and its inhibition by known inhibitors of the aldo-keto reductases AKR1A1, AKR1B1, and AKR1C3 and the carbonyl reductases CBR1/3. The enzyme expression pattern differed among the cell lines with dominant expression of CBR1/3 in HEK293 and MCF-7 and very high expression of AKR1C3 in HepG2 cells. In HEK293 and MCF-7 cells, menadione was the most potent inhibitor (IC50 = 1.6 and 9.8 µM), while in HepG2 cells, ranirestat was most potent (IC50 = 0.4 µM), suggesting that ranirestat is not a selective AKR1B1 inhibitor, but also an AKR1C3 inhibitor. Over-expression of AKR1C3 verified the importance of AKR1C3 for idarubicinol formation and showed that ranirestat is also a potent inhibitor of this enzyme. Taken together, our study underlines the importance of AKR1C3 and CBR1 for the reduction of idarubicin and identifies potent inhibitors of metabolic formation of the cardiotoxic idarubicinol, which should now be tested in vivo to evaluate whether such combinations can increase the cardiac safety of idarubicin therapies while preserving its efficacy.

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.

Influence of a Short Course of Ritonavir Used as Booster in Antiviral Therapies Against SARS-CoV-2 on the Exposure of Atorvastatin and Rosuvastatin.

PURPOSE: Early antiviral treatment with nirmatrelvir/ritonavir is recommended for SARS-CoV-2-infected patients at high risk for severe courses. Such patients are usually chronically ill and susceptible to adverse drug interactions caused by ritonavir. We investigated the interactions of short-term low-dose ritonavir therapy with atorvastatin and rosuvastatin, two statins commonly used in this population. METHOD: We assessed exposure changes (area under the concentration-time curve (AUC∞) and maximum concentration (Cmax)) of a single dose of 10 mg atorvastatin and 10 mg rosuvastatin before and on the fifth day of ritonavir treatment (2 × 100 mg/day) in healthy volunteers and developed a semi-mechanistic pharmacokinetic model to estimate dose adjustment of atorvastatin during ritonavir treatment. RESULTS: By the fifth day of ritonavir treatment, the AUC∞ of atorvastatin increased 4.76-fold and Cmax 3.78-fold, and concurrently, the concentration of atorvastatin metabolites decreased to values below the lower limit of quantification. Pharmacokinetic modelling indicated that a stepwise reduction in atorvastatin dose during ritonavir treatment with a stepwise increase up to 4 days after ritonavir discontinuation can keep atorvastatin exposure within safe and effective margins. Rosuvastatin pharmacokinetics were only mildly modified; ritonavir significantly increased the Cmax 1.94-fold, while AUC∞ was unchanged. CONCLUSION: Atorvastatin doses should likely be adjusted during nirmatrelvir/ritonavir treatment. For patients on a 20-mg dose, we recommend half of the original dose. In patients taking 40 mg or more, a quarter of the dose should be taken until 2 days after discontinuation of nirmatrelvir/ritonavir. Patients receiving rosuvastatin do not need to change their treatment regimen. TRIAL REGISTRATION: EudraCT number: 2021-006634-39. DRKS00027838.

Comprehensive in vitro analysis evaluating the variable drug-drug interaction risk of rifampicin compared to rifabutin.

Compared to rifampicin (600 mg/day), standard doses of rifabutin (300 mg/day) have a lower risk of drug-drug interactions due to induction of cytochrome P450 3A4 (CYP3A4) or P-glycoprotein (Pgp/ABCB1) mediated by the pregnane X receptor (PXR). However, clinical comparisons with equal rifamycin doses or in vitro experiments respecting actual intracellular concentrations are lacking. Thus, the genuine pharmacological differences and the potential molecular mechanisms of the discordant perpetrator effects are unknown. Consequently, the cellular uptake kinetics (mass spectrometry), PXR activation (luciferase reporter gene assays), and impact on CYP3A4 and Pgp/ABCB1 expression and activity (polymerase chain reaction, enzymatic assays, flow cytometry) were evaluated in LS180 cells after treatment with different rifampicin or rifabutin concentrations for variable exposure times and eventually normalized to actual intracellular concentrations. In addition, inhibitory effects on CYP3A4 and Pgp activities were investigated. While rifampicin is poorly taken up by LS180 cells, it strongly activates PXR and leads to enhanced expression and activity of CYP3A4 and Pgp. In contrast, rifabutin is a significantly less potent and less efficient PXR activator and gene inducer, despite sixfold to eightfold higher intracellular accumulation. Finally, rifabutin is a potent inhibitor of Pgp (IC50 = 0.3 µM) compared to rifampicin (IC50 = 12.9 µM). Together, rifampicin and rifabutin significantly differ by their effects on the regulation and function of CYP3A4 and Pgp, even when controlled for intracellular concentrations. Rifabutin's concurrent Pgp inhibitory action might partly compensate the inducing effects, explaining its weaker clinical perpetrator characteristics.

Correction: Bay et al. Functional Characterization of the Solute Carrier LAT-1 (SLC7A5/SLC3A2) in Human Brain Capillary Endothelial Cells with Rapid UPLC-MS/MS Quantification of Intracellular Isotopically Labelled L-Leucine. Int. J. Mol. Sci. 2022, 23, 3637.

The authors would like to make the following corrections to the publication [...].

A Combination of the Immunotherapeutic Drug Anti-Programmed Death 1 with Lenalidomide Enhances Specific T Cell Immune Responses against Acute Myeloid Leukemia Cells.

Immune checkpoint inhibitors can block inhibitory molecules on the surface of T cells, switching them from an exhausted to an active state. One of these inhibitory immune checkpoints, programmed cell death protein 1 (PD-1) is expressed on T cell subpopulations in acute myeloid leukemia (AML). PD-1 expression has been shown to increase with AML progression following allo-haematopoeitic stem cell transplantation, and therapy with hypomethylating agents. We have previously shown that anti-PD-1 can enhance the response of leukemia-associated antigen (LAA)-specific T cells against AML cells as well as leukemic stem and leukemic progenitor cells (LSC/LPCs) ex vivo. In concurrence, blocking of PD-1 with antibodies such as nivolumab has been shown to enhance response rates post-chemotherapy and stem cell transplant. The immune modulating drug lenalidomide has been shown to promote anti-tumour immunity including anti-inflammatory, anti-proliferative, pro-apoptotic and anti-angiogenicity. The effects of lenalidomide are distinct from chemotherapy, hypomethylating agents or kinase inhibitors, making lenalidomide an attractive agent for use in AML and in combination with existing active agents. To determine whether anti-PD-1 (nivolumab) and lenalidomide alone or in combination could enhance LAA-specific T cell immune responses, we used colony-forming immune and ELISpot assays. Combinations of immunotherapeutic approaches are believed to increase antigen-specific immune responses against leukemic cells including LPC/LSCs. In this study we used a combination of LAA-peptides with the immune checkpoint inhibitor anti-PD-1 and lenalidomide to enhance the killing of LSC/LPCs ex vivo. Our data offer a novel insight into how we could improve AML patient responses to treatment in future clinical studies.

[Interprofessional corporation between registered community nurses and general practitioner: A qualitative study]. / Interprofessionelle Zusammenarbeit zwischen diplomierten Pflegefachpersonen der ambulanten Pflege und Hausärzt_innen.

Interprofessional corporation between registered community nurses and general practitioner: A qualitative study Abstract. Background: The quality and effectiveness of the work of certified nurses and physicians in the inpatient setting is significantly influenced by interprofessional collaboration (IPC). There is a lack of knowledge about IPC in outpatient primary care at home. Aim: In highlighting the differences between inpatient and outpatient settings, the challenges of IPC for the outpatient setting should become clear. Method: The data for this study was collected in interprofessional group discussions. The data were methodically extracted by knowledge mapping and structuring content analysis according to Kuckartz (2018). The results were compared with the meta-matrix for interprofessional collaboration in inpatient settings by Schärli et al. (2017). Results: The qualitative analysis showed that the outpatient IPC of qualified nurses and general practitioners is challenged by structural conditions, which are given in the inpatient area by the form of organization. Lack of agreements and coordination of processes complicate IPC in outpatient health care, as there is no recognized common management structure. Conclusions: In order to ensure that IPC in the outpatient setting is not dependent on the individual commitment of individuals, nurses and GPs must jointly define criteria for their IPC, define processes and communication channels, and develop a common approach and team spirit.

Participative development and evaluation of a communication skills-training program for oncologists-patient perspectives on training content and teaching methods.

BACKGROUND: Using the 6-step approach to curriculum development for medical education, we developed a communication skills training (CST) curriculum for oncology and evaluated this curriculum from the perspective of cancer patients. METHODS: We conducted a qualitative interview study with cancer patients, collecting data using semi-structured face-to-face or telephone interviews with a short standardized survey. We fully transcribed the audiotaped interviews and conducted the content analysis using MAXQDA 2020. We analyzed the quantitative sociodemographic data descriptively. RESULTS: A total of 22 cancer patients participated, having a mean age of 60.6 (SD, 13.2) years and being predominantly female (55%). The patients believed that the CST curriculum addressed important aspects of patient-centered communication in cancer care. They emphasized the importance of physicians acquiring communication skills to establish a trusting relationship between doctor and patient, show empathy, inform patients, and involve them in treatment decisions. The patients had some doubts concerning the usefulness of strict protocols or checklists (e.g., they feared that protocol adherence might disturb the conversation flow). DISCUSSION: Although it was a challenge for some participants to take the perspective of a trainer and comment on the CST content and teaching methods, the patients provided a valuable perspective that can help overcome blind spots in CST concepts.

How to avoid misinterpretation of dual reporter gene assay data affected by cell damage.

The activity of nuclear receptors (e.g., pregnane x receptor, PXR) can be assessed by luminescence-based dual reporter gene assays. Under most conditions, receptor-activated firefly luminescence is normalized to Renilla luminescence, which is triggered by a constitutively active promoter. Simultaneous damage to the cells can however disrupt these signals and thus impair the interpretation of the data. Consequently, this study addressed three important aspects: First, idealized models were described, each highlighting crucial characteristics and important pitfalls of dual PXR reporter gene assays used to evaluate PXR activation or inhibition. Second, these models were supported by experimental data obtained with a strong PXR activator (rifampicin) with low cytotoxicity, a PXR activator with high cytotoxicity (dovitinib), a proposed PXR inhibitor that reportedly has no toxic effects (triptolide), and a cytotoxic control (oxaliplatin). Data were evaluated for relative PXR activity data, individual firefly or Renilla luminescence, and anti-proliferative effects of the compounds (assessed by crystal violet staining). Finally, a step-by-step guide is proposed to avoid misleading set-up of the assay or misinterpretation of the data obtained. Key considerations here include (1) omission of drug concentrations beyond 10-20% proliferation inhibition; (2) observation of Renilla luminescence, because this tends to indicate 'false PXR activation' when it inexplicably decreases; (3) parallel decrease of relative PXR activity and proliferation below baseline levels in conjunction with a sharp decrease in Renilla luminescence indicates 'false PXR antagonism'; (4) non-sigmoidal relationships suggest the absence of concentration dependency.

Functional Characterization of the Solute Carrier LAT-1 (SLC7A5/SLC2A3) in Human Brain Capillary Endothelial Cells with Rapid UPLC-MS/MS Quantification of Intracellular Isotopically Labelled L-Leucine.

The solute carrier L-type amino acid transporter 1 (LAT-1/SLC7A5) is a viable target for drug delivery to the central nervous system (CNS) and tumors due to its high abundance at the blood-brain barrier and in tumor tissue. LAT-1 is only localized on the cell surface as a heterodimer with CD98, which is not required for transporter function. To support future CNS drug-delivery development based on LAT-1 targeting, we established an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for stable isotopically labeled leucine ([13C6, 15N]-L-leucine), with a dynamic range of 0.1-1000 ng/mL that can be applied for the functional testing of LAT-1 activity when combined with specific inhibitors and, consequently, the LAT-1 inhibition capacity of new compounds. The assay was established in a 96-well format, facilitating high-throughput experiments, and, hence, can support the screening for novel inhibitors. Applicable recommendations of the US Food and Drug Administration and European Medicines Agency for bioanalytical method validation were followed to validate the assay. The assay was applied to investigate the IC50 of two well-known LAT-1 inhibitors on hCMEC/D3 cells: the highly specific LAT-1 inhibitor JPH203, which was also used to demonstrate LAT-1 specific uptake, and the general system L inhibitor BCH. In addition, the [13C6, 15N]-L-leucine uptake was determined on two human brain capillary endothelial cell lines (NKIM-6 and hCMEC/D3), which were characterized for their expressional differences of LAT-1 at the protein and mRNA level and the surface amount of CD98. The IC50 values of the inhibitors were in concordance with previously reported values. Furthermore, the [13C6, 15N]-L-leucine uptake was significantly higher in hCMEC/D3 cells compared to NKIM-6 cells, which correlated with higher expression of LAT-1 and a higher surface amount of CD98. Therefore, the UPLC-MS/MS quantification of ([13C6, 15N]-L-leucine is a feasible strategy for the functional characterization of LAT-1 activity in cells or tissue.

Metabolic Effect of Blocking Sodium-Taurocholate Co-Transporting Polypeptide in Hypercholesterolemic Humans with a Twelve-Week Course of Bulevirtide-An Exploratory Phase I Clinical Trial.

Bile acids (BA) play an important role in cholesterol metabolism and possess further beneficial metabolic effects as signalling molecules. Blocking the hepatocellular uptake of BA via sodium-taurocholate co-transporting polypeptide (NTCP) with the first-in-class drug bulevirtide, we expected to observe a decrease in plasma LDL cholesterol. In this exploratory phase I clinical trial, volunteers with LDL cholesterol > 130 mg/dL but without overt atherosclerotic disease were included. Thirteen participants received bulevirtide 5 mg/d subcutaneously for 12 weeks. The primary aim was to estimate the change in LDL cholesterol after 12 weeks. Secondary endpoints included changes in total cholesterol, HDL cholesterol, lipoprotein(a), inflammatory biomarkers, and glucose after 12 weeks. In addition, cardiac magnetic resonance imaging (CMR) was performed at four time points. BA were measured as biomarkers of the inhibition of hepatocellular uptake. After 12 weeks, LDL cholesterol decreased not statistically significantly by 19.6 mg/dL [−41.8; 2.85] (Hodges−Lehmann estimator with 95% confidence interval). HDL cholesterol showed a significant increase by 5.5 mg/dL [1.00; 10.50]. Lipoprotein(a) decreased by 1.87 mg/dL [−7.65; 0]. Inflammatory biomarkers, glucose, and cardiac function were unchanged. Pre-dose total BA increased nearly five-fold (from 2026 nmol/L ± 2158 (mean ± SD) at baseline to 9922 nmol/L ± 7357 after 12 weeks of treatment). Bulevirtide was generally well tolerated, with most adverse events being administration site reactions. The exploratory nature of the trial with a limited number of participants allows the estimation of potential effects, which are crucial for future pharmacological research on bile acid metabolism in humans.

A nuclear factor kappa B reporter cell line used to evaluate ex vivo the net inflammatory effect of plasma samples from patients with rheumatoid arthritis, psoriasis, or COVID-19.

BACKGROUND: The overall clinical outcome of inflammatory conditions is the result of the balance between pro-inflammatory and anti-inflammatory mediators. Because nuclear factor kappa B (NF-ĸB) is at the bottom of many inflammatory conditions, methods to evaluate the net effect of inflammation modulators on this master regulator have been conceptualized for years. METHODS: Using an ex vivo NF-ĸB reporter cell line-based assay, plasma samples of patients with rheumatoid arthritis (n = 27), psoriasis (n = 15), or severe coronavirus disease-19 (COVID-19) (n = 21) were investigated for NF-ĸB activation compared to plasma samples from 9 healthy volunteers. RESULTS: When separated by C-reactive protein (CRP) threshold levels, samples of patients exhibiting increased CRP levels (≥5 mg/l) activated NF-ĸB more efficiently than samples from patients with levels below 5 mg/l (P = 0.0001) or healthy controls (P = 0.04). Overall, there was a moderate association of CRP levels with NF-ĸB activation (Spearman r = 0.66; p < 0.0001). Plasma from COVID-19 patients activated NF-ĸB more efficiently (mean 2.4-fold compared to untreated reporter cells) than samples from any other condition (healthy controls, 1.8-fold, P = 0.0025; rheumatoid arthritis, 1.7-fold, P < 0.0001; psoriasis, 1.7-fold, P < 0.0001). In contrast, effects of rheumatoid arthritis, psoriasis, or healthy volunteer samples did not differ. CONCLUSION: This study shows that a NF-ĸB reporter cell line can be used to evaluate the net inflammatory effect of clinical plasma samples. Patients with chronic but stable rheumatoid arthritis or psoriasis do not exhibit increased plasma levels of NF-ĸB-activating compounds as opposed to COVID-19 patients with high inflammatory burden.

Approaching sites of action of drugs in clinical pharmacology: New analytical options and their challenges.

Clinical pharmacology is an important discipline for drug development aiming to define pharmacokinetics (PK), pharmacodynamics (PD) and optimum exposure to drugs, i.e. the concentration-response relationship and its modulators. For this purpose, information on drug concentrations at the anatomical, cellular and molecular sites of action is particularly valuable. In pharmacological assays, the limited accessibility of target cells in readily available samples (i.e. blood) often hampers mass spectrometry-based monitoring of the absolute quantity of a compound and the determination of its molecular action at the cellular level. Recently, new sample collection methods have been developed for the specific capture of rare circulating cells, especially for the diagnosis of circulating tumour cells. In parallel, new advances and developments in mass spectrometric instrumentation now allow analyses to be scaled down to the cellular level. Together, these developments may permit the monitoring of minute drug quantities and show their effect at the cellular level. In turn, such PK/PD associations on a cellular level would not only enrich our pharmacological knowledge of a given compound but also expand the basis for PK/PD simulations. In this review, we describe novel concepts supporting clinical pharmacology at the anatomical, cellular and molecular sites of action, and highlight the new challenges in mass spectrometry-based monitoring. Moreover, we present methods to tackle these challenges and define future needs.

Reporter cell assay-based functional quantification of TNF-α-antagonists in serum - a proof-of-principle study for adalimumab.

Quantification of therapeutic antibodies is commonly based on physico-chemical assays such as enzyme-linked immunoabsorption assays (ELISA) and lately on mass spectrometry. However, the functional integrity of evaluated immunoglobulins is yet not assessed. Consequently, a commercially available reporter cell line was used to quantify the functional concentration of the anti-tumor necrosis factor alpha (TNF-α) antibody adalimumab present in serum of a healthy beagle dog treated with 3 mg intravenous adalimumab (Humira®). HEK-Blue™-hTLR3 cells express a secreted alkaline phosphatase under the control of a nuclear factor kappa B (NF-κB) response element. Its enzymatic activity can be recorded using colorimetry, which reports activity of extracellular NF-κB stimuli such as TNF-α. Using an adalimumab concentration-response calibration curve, the functional concentration of serum adalimumab was estimated to be 4.9 ± 1.4 µg/ml, which was in excellent agreement with ELISA results (4.8 µg/ml). The obtained data suggest that this simple, easy-to-handle reporter cell assay can be used for the functional quantification of adalimumab present in samples from in vitro or pre-clinical in vivo experiments. Moreover, this assay could be used in vitro to compare the pharmacodynamics of adalimumab biosimilars or different anti-TNF-α compounds, respectively.

Functional role of miR-148a in oropharyngeal cancer: influence on pregnane X receptor and P-glycoprotein expression.

MicroRNAs are short noncoding RNAs of about 19-25 nucleotides that usually target the 3' untranslated regions of mRNAs thus mediating post-transcriptional regulation of gene expression. Previous data indicate a role for miR-148a in the regulation of the pregnane X receptor (PXR/NR1I2), a nuclear receptor that regulates the expression of drug transporters like P-glycoprotein (P-gp/ABCB1). Our study investigated the effect of miR-148a on the post-transcriptional regulation of PXR and its target gene ABCB1 in oropharyngeal cancer cell lines (OPSCC). miR-148a was over-expressed and knocked-down in three OPSCC cell lines (HNO41, HNO206, and HNO413) by transfection with miR-148a mimic and miR-148a antagomir, respectively. Expression of miR-148a, NR1I2, and ABCB1 mRNA was quantified via real-time qPCR, protein expression of PXR was assessed by immunoblotting. Transfection of miR-148a mimic led to increased miR-148a levels in all cell lines and transfection of miR-148a antagomir reduced miR-148a expression in HNO206 and HNO413. Whereas these changes had no significant effect on PXR mRNA expression, protein expression was reduced in HNO41 by transfection with miR-148a and increased in HNO413 by transfection with miR-148a antagomir. Transfection of miR-148a downregulated ABCB1 mRNA in all cell lines, whereas antagonizing miR-148a had no significant effect. Our data demonstrate a modulation of PXR/NR1I2 and ABCB1 expression in OPSCC by miR-148a, however the effect was not uniform in all cell lines and depended on the range of expression of miR-148 and the genotype of rs1054190 SNP in NR1I2 3'UTR. Thus, our findings argue against an unequivocal association between miR-148a and PXR levels in OPSCC.

Perpetrator effects of ciclosporin (P-glycoprotein inhibitor) and its combination with fluconazole (CYP3A inhibitor) on the pharmacokinetics of rivaroxaban in healthy volunteers.

AIMS: Rivaroxaban exposure is considerably increased by drugs that are combined P-glycoprotein (P-gp) and strong cytochrome P450 (CYP) 3A inhibitors (e.g. ketoconazole). The aim of the present study was to investigate the effects of the potent P-gp inhibitor ciclosporin and its combination with the moderate CYP3A inhibitor fluconazole on rivaroxaban pharmacokinetics and on CYP3A activity. METHODS: Twelve healthy volunteers received 20 mg rivaroxaban orally alone, in combination with ciclosporin (dose-individualized oral regimen), and in combination with ciclosporin and fluconazole (400 mg day-1 orally). CYP3A4 activity was estimated using a midazolam microdose. Pharmacokinetics was analysed using noncompartmental and compartmental methods. RESULTS: Compared to baseline, ciclosporin increased rivaroxaban average exposure by 47% (90% confidence interval 28-68%), maximum concentration by 104% (70-146%), and decreased CYP3A4 activity by 34% (25-42%). Ciclosporin combined with fluconazole increased rivaroxaban average exposure by 86% (58-119%) and maximum concentration by 115% (83-153%), which was considerably stronger than observed in historical controls receiving rivaroxaban with fluconazole alone, and decreased CYP3A4 activity by 79% (76-82%). CONCLUSION: Patients treated with rivaroxaban in combination with single modulators of multiple elimination pathways or multiple modulators of single elimination pathways (CYP3A, P-gp) require particular care.

The phytoestrogens daidzein and equol inhibit the drug transporter BCRP/ABCG2 in breast cancer cells: potential chemosensitizing effect.

PURPOSE: The soy isoflavone genistein has been described to up-regulate breast cancer resistance protein (BCRP) and, thus, enhance chemoresistance in breast cancer cells. The aim of this work was to assess the effect of long- and short-term incubation with daidzein, the second most abundant soy isoflavone and its metabolite equol on the expression and activity of P-glycoprotein, multidrug resistance-associated proteins 1 and 2 (MRP1 and MRP2) and BCRP in breast cancer cells. METHODS: MCF-7 and MDA-MB-231 cells were treated with phytoestrogen concentrations within the range achieved in individuals with a high isoflavone intake. Transporter expression was evaluated at protein and mRNA level through western blot and qRT-PCR, respectively. Transporter activity was determined using doxorubicin, mitoxantrone and carboxy-dichlorofluorescein as substrates. RESULTS: Daidzein (5 µM) up-regulated MRP2- and down-regulated MRP1 protein expressions in MCF-7 and MDA-MB-231 cells, respectively. Both effects were ER-dependent, as determined using the antagonist ICI 182,780. The decrease in MRP1 mRNA in MDA-MB-231 cells indicates a transcriptional mechanism. On the contrary, MRP2 induction in MCF-7 cells takes place post-transcriptionally. Whereas changes in the transporter expression had a minor effect on the transporter activity, acute incubation with daidzein, R-equol and S-equol led to a strong inhibition of BCRP activity and an increase in the IC50 of BCRP substrates. CONCLUSIONS: In contrast to previous reports for genistein, daidzein and equol do not provoke a major up-regulation of the transporter expression but instead an inhibition of BCRP activity and sensitization to BCRP substrates.

Herb⁻Drug Interaction Potential of Anti-Borreliae Effective Extracts from Uncaria tomentosa (Samento) and Otoba parvifolia (Banderol) Assessed In Vitro.

Samento (extract from Uncaria tomentosa) and Banderol (extract from Otoba parvifolia) have been demonstrated to have anti-inflammatory and antimicrobial properties, e.g., against different morphological forms of Borrelia burgdorferi. However, there is hardly any data on the pharmacological safety of these two herbal medicines. This in vitro study aimed at scrutinizing their possible characteristics as perpetrators in pharmacokinetic herbal⁻drug interactions. Inhibition of cytochrome P450 enzymes (CYPs) was quantified by commercial kits and inhibition of drug transporters by use of fluorescent probe substrates. Induction was quantified by real-time RT-PCR and activation of pregnane x receptor (PXR) and aryl hydrocarbon receptor (AhR) by reporter gene assays. Organic anion transporting polypeptide 1B1 (OATP1B1) (IC50 = 0.49 ± 0.28%) and OATP1B3 (IC50 = 0.65 ± 0.29%) were potently inhibited by Banderol, but only weakly by Samento. CYP3A4 was inhibited about 40% at a Samento concentration of 1%. Samento significantly induced mRNA expression of CYP2J2, UGT1A3, UGT1A9, ABCB1, and SLCO1B1 and strongly activated PXR, but hardly AhR. In conclusion, the perpetrator profiles of Samento and Banderol for herb⁻drug interactions completely differ. Clinical studies are strongly recommended to clarify whether the effects observed in vitro are of clinical relevance.