Coadministration of fluconazole to boost subtherapeutic sirolimus concentrations: A case report.

Individual sirolimus whole blood concentrations are highly variable, critically influenced by the concomitant use of cytochrome P450 (CYP) 3A inducers or inhibitors, and also modulated by food. Therapeutic drug monitoring is therefore recommended, especially at treatment start or in circumstances that can influence sirolimus exposure. In this case report, we highlight the challenge of achieving therapeutic sirolimus concentrations and present pragmatic solutions with regimen adaptions, pharmacokinetic enhancement (use of a drug­drug interaction), concentration monitoring, and subsequent modeling of population pharmacokinetics to support treatment decisions. In a 69-year-old female patient with allogeneic hematopoietic stem cell transplantation, sirolimus concentrations were stable until she developed cerebral toxoplasmosis with tonic­clonic seizures. During treatment of this acute infection, sirolimus concentrations dropped to subtherapeutic levels and remained largely unaffected by dose increases. [Correction added on 4 May 2024, after first online publication: The word "tacrolimus concentrations" has been changed to "sirolimus concentrations" in the preceding sentence.] Only the simultaneous administration of the CYP3A4 inhibitor fluconazole and a shortening of the sirolimus dosing intervals to a (non-approved) twice-daily administration led to successful control of the concentrations, which ultimately even made a dose reduction possible. This intervention resulted in an increase of sirolimus mean trough concentration to 5.85 ng/mL, i.e., into the desired target range. Additionally, a higher ratio of sirolimus trough levels/daily dose from 26.9 to 109 ng/mL/mg/kg/day was achieved with the initiation of fluconazole. Thus, this case report describes the use of clinical pharmacological concepts and pharmacokinetic modeling to optimize treatment strategies in an individual patient. This strategy could be generalized to other CYP inhibitors and other treatment regimens.

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.

RNA is a pro-apoptotic target of cisplatin in cancer cell lines and C. elegans.

Cisplatin not only targets DNA but also RNA. However, it is largely unknown whether platinated RNA (Pt-RNA) causes apoptosis and thus contributes to the cytotoxic effects of cisplatin. Consequently, cellular RNA was isolated from HepG2 and LS180 cells, exposed to cisplatin, and the resulting Pt-RNA (20 ng Pt/µg RNA) was transfected into these cancer cell lines or used to treat an apoptosis reporter Caenorhabditis elegans (C. elegans) strain (MD701, expressing CED-1::GFP). Cellular and molecular effects of Pt-RNA were evaluated by luminogenic caspase 3/7 assays, PCR array analysis, and fluorescence microscopy-based quantification of apoptosis in C. elegans gonads. Assuming RNA cross-linking (pseudo double-stranded RNA), the contribution of the Toll-like receptor 3 (TLR3, a sensor of double-stranded RNA) to apoptosis induction in cancer cell lines was investigated by pharmacological TLR3 inhibition and overexpression. In contrast to controls, Pt-RNA significantly enhanced apoptosis in C. elegans (2-fold) and in the cancer cell lines (2-fold to 4-fold). TLR3 overexpression significantly enhanced the pro-apoptotic effects of Pt-RNA in HepG2 cells. TLR3 inhibition reduced the pro-apoptotic effects of Pt-RNA and cisplatin, but not of paclitaxel (off-target control). Gene expression analysis showed that Pt-RNA (but not RNA) significantly enhanced the mRNA levels of nuclear factor kappa B subunit 2 and interleukin-8 in HepG2 cells, suggesting that Pt-RNA is a damage-associated molecular pattern that additionally causes pro-inflammatory responses. Together, this data suggests that not only DNA but also cellular RNA is a functionally relevant target of cisplatin, leading to pro-apoptotic and immunogenic effects.

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.

Evaluation of Hydroxychloroquine as a Perpetrator on Cytochrome P450 (CYP) 3A and CYP2D6 Activity with Microdosed Probe Drugs in Healthy Volunteers.

BACKGROUND AND OBJECTIVE: Although polypharmacy is a particular challenge in daily rheumatological practice, clinical research on the effects of hydroxychloroquine (HCQ), a commonly used drug for patients with rheumatic diseases, is sparse on cytochrome P450 (CYP)-mediated metabolism. We have shown that pre-treatment with pantoprazole does not alter HCQ absorption in healthy volunteers. In this paper, we report the effects of a single 400 mg dose of HCQ on specific CYP3A and CYP2D6 substrates in healthy volunteers. METHODS: In the trial, participants were randomized into two groups (HCQ plus a 9-day course of pantoprazole, or HCQ only). As a secondary endpoint, the effects of a single oral dose of HCQ on the exposure of the oral microdosed CYP3A probe drug midazolam (30 µg) and the oral microdosed CYP2D6 probe drug yohimbine (50 µg) were studied in 23 healthy volunteers (EudraCT no. 2020-001470-30, registered 31 March 2020). RESULTS: The exposure of the probe drugs after intake of HCQ compared with baseline values was quantified by the partial area under the plasma concentration-time curve 0-6 h after administration (AUC0-6 h) for yohimbine and the partial AUC2-4 h for midazolam. Under HCQ, yohimbine AUC0-6 h was unchanged, independent of CYP2D6 genotypes and pantoprazole exposure. Midazolam AUC2-4 h was 25% higher on the day of HCQ administration than at baseline (p = 0.0007). This significant increase was driven by the pantoprazole subgroup, which showed a 46% elevation of midazolam AUC2-4 h as compared with baseline (p < 0.0001). The ratio of midazolam to 1-OH-midazolam partial AUC2-4 h significantly increased from 3.03 ± 1.59 (baseline) to 3.60 ± 1.56 (HCQ) in the pantoprazole group (p = 0.0026). CONCLUSION: In conclusion, we observed an increased midazolam exposure most likely related to pantoprazole.

Extracellular Vesicles as Surrogates for Drug Metabolism and Clearance: Promise vs. Reality.

Drug-metabolizing enzymes (DMEs) and transporters play a major role in drug efficacy and safety. They are regulated at multiple levels and by multiple factors. Estimating their expression and activity could contribute to predicting drug pharmacokinetics and their regulation by drugs or pathophysiological situations. Determining the expression of these proteins in the liver, intestine, and kidney requires the collection of biopsy specimens. Instead, the isolation of extracellular vesicles (EVs), which are nanovesicles released by most cells and present in biological fluids, could deliver this information in a less invasive way. In this article, we review the use of EVs as surrogates for the expression and activity of DMEs, uptake, and efflux transporters. Preliminary evidence has been provided for a correlation between the expression of some enzymes and transporters in EVs and the tissue of origin. In some cases, data obtained in EVs reflect the induction of phase I-DMEs in the tissues. Further studies are required to elucidate to what extent the regulation of other DMEs and transporters in the tissues reflects in the EV cargo. If an association between tissues and their EVs is firmly established, EVs may represent a significant advancement toward precision therapy based on the biotransformation and excretion capacity of each individual.

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.

Evaluation of the drug-drug interaction potential of the novel hepatitis B and D virus entry inhibitor bulevirtide at OATP1B in healthy volunteers.

Introduction: Bulevirtide is a first-in-class antiviral drug to treat chronic hepatitis B/D. We investigated the drug-drug interaction potential and pharmacokinetics of high-dose subcutaneous bulevirtide (5 mg twice daily) with organic anion transporting polypeptide 1B1 (OATP1B1) and cytochrome P450 (CYP) 3A4. Methods: This was a single-center, open-label, fixed-sequence drug-drug interaction trial in 19 healthy volunteers. Before and at bulevirtide steady state, participants ingested a single 40 mg dose of pravastatin. A midazolam microdose was applied to quantify CYP3A4 activity. Results: At bulevirtide steady state, pravastatin area under the concentration-time curve (AUC0-∞) increased 1.32-fold (90% CI 1.08-1.61). The 5 mg bulevirtide twice-daily treatment resulted in a mean AUC0-12 of 1210 h*ng/ml (95% CI 1040-1408) and remained essentially unchanged under the influence of pravastatin. CYP3A4 activity did not change to a clinically relevant extent. As expected, total bile acids increased substantially (35-fold) compared to baseline during bulevirtide treatment. All study medication was well tolerated. Discussion: The study demonstrated that high-dose bulevirtide inhibited OATP1B-mediated hepatic uptake of the marker substrate pravastatin but the extent is considered clinically not relevant. Changes in CYP3A4 activity were also not clinically relevant. In conclusion, this study suggests that OATP1B substrate drugs as well as CYP3A4 substrates may safely be used without dose adjustment in patients treated with bulevirtide. However, in patients using high statin doses and where concomitant factors potentially further increase statin exposure, caution may be required when using bulevirtide.

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.

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.

Does the circulating ketoconazole metabolite N-deacetyl ketoconazole contribute to the drug-drug interaction potential of the parent compound?

Ketoconazole is a strong inhibitor of cytochrome P450 3A4 (CYP3A4) and of P-glycoprotein (P-gp) and is often used as an index inhibitor especially for CYP3A4-mediated drug metabolism. A preliminary physiologically based pharmacokinetic (PBPK) model for drug-drug interactions indicated possible involvement of a metabolite to the perpetrator potential of ketoconazole. Still unknown for humans, in rodents, N-deacetyl ketoconazole (DAK) has been identified as the major ketoconazole metabolite. We therefore investigated in vitro, whether DAK also inhibits the human CYPs and drug transporters targeted by ketoconazole and quantified DAK in human plasma from healthy volunteers after receiving a single oral dose of 400 mg ketoconazole. Our data demonstrated that DAK also inhibits CYP3A4 (2.4-fold less potent than ketoconazole), CYP2D6 (13-fold more potent than ketoconazole), CYP2C19 (equally potent), P-gp (3.4-fold less potent than ketoconazole), breast cancer resistance protein (more potent than ketoconazole) and organic anion transporting polypeptide 1B1 and 1B3 (7.8-fold and 2.6-fold less potent than ketoconazole). After a single oral dose of 400 mg ketoconazole, maximum concentrations of DAK in human plasma were only 3.1 ‰ of the parent compound. However, assuming that DAK also highly accumulates in the human liver as demonstrated for rodents, inhibition of the proteins investigated could also be conceivable in vivo. In conclusion, DAK inhibits several CYPs and drug transporters, which might contribute to the perpetrator potential of ketoconazole.

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.

Regulation of PXR Function by Coactivator and Corepressor Proteins: Ligand Binding Is Just the Beginning.

The pregnane X receptor (PXR, NR1I2) is a nuclear receptor which exerts its regulatory function by heterodimerization with the retinoid-X-receptor α (RXRα, NR2B1) and binding to the promoter and enhancer regions of diverse target genes. PXR is involved in the regulation of drug metabolism and excretion, metabolic and immunological functions and cancer pathogenesis. PXR activity is strongly regulated by the association with coactivator and corepressor proteins. Coactivator proteins exhibit histone acetyltransferase or histone methyltransferase activity or associate with proteins having one of these activities, thus promoting chromatin decondensation and activation of the gene expression. On the contrary, corepressor proteins promote histone deacetylation and therefore favor chromatin condensation and repression of the gene expression. Several studies pointed to clear cell- and ligand-specific differences in the activation of PXR. In this article, we will review the critical role of coactivator and corepressor proteins as molecular determinants of the specificity of PXR-mediated effects. As already known for other nuclear receptors, understanding the complex mechanism of PXR activation in each cell type and under particular physiological and pathophysiological conditions may lead to the development of selective modulators with therapeutic potential.

Investigating the Central Nervous System Disposition of Actinomycin D: Implementation and Evaluation of Cerebral Microdialysis and Brain Tissue Measurements Supported by UPLC-MS/MS Quantification.

Actinomycin D is a potent cytotoxic drug against pediatric (and other) tumors that is thought to barely cross the blood-brain barrier. To evaluate its potential applicability for the treatment of patients with central nervous system (CNS) tumors, we established a cerebral microdialysis model in freely moving mice and investigated its CNS disposition by quantifying actinomycin D in cerebral microdialysate, brain tissue homogenate, and plasma. For this purpose, we developed and validated an ultraperformance liquid chromatography-tandem mass spectrometry assay suitable for ultra-sensitive quantification of actinomycin D in the pertinent biological matrices in micro-samples of only 20 µL, with a lower limit of quantification of 0.05 ng/mL. In parallel, we confirmed actinomycin D as a substrate of P-glycoprotein (P-gp) in in vitro experiments. Two hours after intravenous administration of 0.5 mg/kg, actinomycin D reached total brain tissue concentrations of 4.1 ± 0.7 ng/g corresponding to a brain-to-plasma ratio of 0.18 ± 0.03, while it was not detectable in intracerebral microdialysate. This tissue concentration exceeds the concentrations of actinomycin D that have been shown to be effective in in vitro experiments. Elimination of the drug from brain tissue was substantially slower than from plasma, as shown in a brain-to-plasma ratio of approximately 0.53 after 22 h. Because actinomycin D reached potentially effective concentrations in brain tissue in our experiments, the drug should be further investigated as a therapeutic agent in potentially susceptible CNS malignancies, such as ependymoma.

IFITM proteins promote SARS-CoV-2 infection and are targets for virus inhibition in vitro.

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) can restrict viral pathogens, but pro- and anti-viral activities have been reported for coronaviruses. Here, we show that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. However, endogenous IFITM expression supports efficient infection of SARS-CoV-2 in human lung cells. Our results indicate that the SARS-CoV-2 Spike protein interacts with IFITMs and hijacks them for efficient viral infection. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. IFITM-derived peptides and targeting antibodies inhibit SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are cofactors for efficient SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and are potential targets for therapeutic approaches.

In vitro evidence suggesting that the toll-like receptor 7 and 8 agonist resiquimod (R-848) unlikely affects drug levels of co-administered compounds.

Resiquimod (R-848) is an immune response modifier activating toll-like receptor 7 and 8. Its potential to cause pharmacokinetic interactions with concurrently administered drugs is unknown. To study the time course of the effect of resiquimod in LS180 cells as a model for intestinal tissue, luciferase-based reporter gene assays and reverse transcription polymerase chain reaction were used to investigate whether resiquimod affects the activities of nuclear factor kappa B (NF-ĸB), pregnane x receptor (PXR) or the transcription of selected central genes for drug disposition (cytochrome P-450 isozyme 3A4 (CYP3A4), CYP1A1, UDP-glucuronosyltransferase 1A1 (UGT1A1), ATP-binding cassette transporters ABCC2, ABCB1). Its impact on the activities of organic anion transporting polypeptides 1 or 3 (OATP1B1/3), breast cancer resistance protein (BCRP), P-glycoprotein (P-gp) or CYP3A4 was evaluated using fluorescence- or luminescence-based activity assays. Resiquimod irrelevantly increased NF-ĸB activity after 2 h (1 µM: 1.07-fold, P = 0.0188; 10 µM: 1.09-fold, P = 0.0142), and diminished it after 24 h (1 µM: 0.64-fold, P < 0.0001; 10 µM: 0.68-fold, P < 0.0001) and 30 h (10 µM: 0.68-fold, P = 0.0003). Concurrently, PXR activity after 24 h was marginally increased by 10 µM (1.05-fold, P = 0.0019). Resiquimod did not alter mRNA expression levels, activities of uptake or efflux transporters, or CYP3A4 activity. Given the marginal effects on NF-ĸB, PXR, expression levels of selected PXR target genes, and activities of important drug transporters and CYP3A4 in vitro, resiquimod is not expected to cause major pharmacokinetic drug-drug interactions in vivo.

Low risk of the TMPRSS2 inhibitor camostat mesylate and its metabolite GBPA to act as perpetrators of drug-drug interactions.

Camostat mesylate, a potent inhibitor of the human transmembrane protease, serine 2 (TMPRSS2), is currently under investigation for its effectiveness in COVID-19 patients. For its safe application, the risks of camostat mesylate to induce pharmacokinetic drug-drug interactions with co-administered drugs should be known. We therefore tested in vitro the potential inhibition of important efflux (P-glycoprotein (P-gp, ABCB1), breast cancer resistance protein (BCRP, ABCG2)), and uptake transporters (organic anion transporting polypeptides OATP1B1, OATP1B3, OATP2B1) by camostat mesylate and its active metabolite 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA). Transporter inhibition was evaluated using fluorescent probe substrates in transporter over-expressing cell lines and compared to the respective parental cell lines. Moreover, possible mRNA induction of pharmacokinetically relevant genes regulated by the nuclear pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR) was analysed in LS180 cells by quantitative real-time PCR. The results of our study for the first time demonstrated that camostat mesylate and GBPA do not relevantly inhibit P-gp, BCRP, OATP1B1 or OATP1B3. Only OATP2B1 was profoundly inhibited by GBPA with an IC50 of 11 µM. Induction experiments in LS180 cells excluded induction of PXR-regulated genes such as cytochrome P450 3A4 (CYP3A4) and ABCB1 and AhR-regulated genes such as CYP1A1 and CYP1A2 by camostat mesylate and GBPA. Together with the summary of product characteristics of camostat mesylate indicating no inhibition of CYP1A2, 2C9, 2C19, 2D6, and 3A4 in vitro, our data suggest a low potential of camostat mesylate to act as a perpetrator in pharmacokinetic drug-drug interactions. Only inhibition of OATP2B1 by GBPA warrants further investigation.

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.

Interaction of Hydroxychloroquine with Pharmacokinetically Important Drug Transporters.

(1) Background: Hydroxychloroquine is used to treat malaria and autoimmune diseases, and its potential use against COVID-19 is currently under investigation. Thus far, information on interactions of hydroxychloroquine with drug transporters mediating drug-drug interactions is limited. We assessed the inhibition of important efflux (P-glycoprotein (P-gp), breast cancer resistance protein (BCRP)) and uptake transporters (organic anion transporting polypeptide (OATP)-1B1, OATP1B3, OATP2B1) by hydroxychloroquine, tested its P-gp and BCRP substrate characteristics, and evaluated the induction of pharmacokinetically relevant genes regulated by the nuclear pregnane X (PXR) (CYP3A4, ABCB1) and aryl hydrocarbon receptor (AhR) (CYP1A1, CYP1A2). (2) Methods: Transporter inhibition was evaluated in transporter over-expressing cell lines using fluorescent probe substrates. P-gp and BCRP substrate characteristics were assessed by comparing growth inhibition of over-expressing and parental cell lines. Possible mRNA induction was analysed in LS180 cells by quantitative real-time PCR. (3) Results: Hydroxychloroquine did not inhibit BCRP or the OATPs tested but inhibited P-gp at concentrations exceeding 10 µM. P-gp overexpressing cells were 5.2-fold more resistant to hydroxychloroquine than control cells stressing its substrate characteristics. Hydroxychloroquine did not induce genes regulated by PXR or AhR. (4) Conclusions: This is the first evidence that hydroxychloroquine's interaction potential with drug transporters is low, albeit bioavailability of simultaneously orally administered P-gp substrates might be increased by hydroxychloroquine.

Potential Risk of Food-Drug Interactions: Citrus Polymethoxyflavones and Flavanones as Inhibitors of the Organic Anion Transporting Polypeptides (OATP) 1B1, 1B3, and 2B1.

BACKGROUND AND OBJECTIVES: Citrus flavonoids are not only components of daily nutrition, they are also promoted as dietary supplements and are important ingredients in traditional medicines. Interactions of flavonoids with synthetic drugs represent an often neglected issue. We therefore investigated in vitro whether the polymethoxyflavones nobiletin, sinensetin, and tangeretin and the flavonoid rutinosides didymin, hesperidin, and narirutin can inhibit human organic anion transporting polypeptides (OATP) 1B1, 1B3, and 2B1, which are important transporters mediating drug-drug and food-drug interactions. METHODS: Inhibition was investigated by quantifying the decreased uptake of the fluorescent OATP1B1 and OATP1B3 substrate 8-fluorescein-cAMP in HEK293 cells overexpressing OATP1B1 or OATP1B3 and of the fluorescent OATP2B1 substrate 4',5'-dibromofluorescein in HEK293 cells overexpressing OATP2B1. RESULTS: We demonstrate that all flavonoids investigated inhibit OATP2B1 in the lower micromolar range (IC50 between 1.6 and 14.2 µM), but only the polymethoxyflavones also inhibit OATP1B1 and 1B3 (IC50 between 2.1 and 21 µM). CONCLUSIONS: All flavonoids investigated might contribute to the intestinal OATP2B1-based interactions with drugs observed with citrus juices or fruits. In contrast, the concentration of the polymethoxyflavones after consumption of citrus juices or fruits is most likely too low to reach relevant systemic concentrations and thus to inhibit hepatic OATP1B1 and OATP1B3, but there might be a risk when they are consumed as medicines or as dietary supplements.