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The legend of the supplemetary figures is given.

Inflammatory profile in A375 and M24met melanoma cells. Distribution of inflammatory proteins in A375 and M24met (a). M8 downregulates proteins involved in inflammation, as exemplified by the inflammatory proteins lysyl oxidase homolog 2, plasminogen activator inhibitor 1 (PAI-1), ADAMTS-1, pentraxin, granulins, that are upregulated in M24met (b,c). Correlation of the candidates with tumor microenvironment and IL-1beta and VEGF response (d).

Activity of vemurafenib and M8 against A375, M24met and V0, V0.1, V1, V10 (Proliferation assay).*Paulitschke et al. 2010

Comparison of protein expression in A375 and M24met by 2D-gel electrophoresis. Corresponding proteins which are assigned in the 2-D gel in Figure 2. Accession numbers are from the Uniprot database. Vol indicates the volume identified by 2D-gel electrophoresis n.d =not determined

Figure S1. Photographic comet images of (1A) undamaged and (1B) damaged nuclei stained with ethidium bromide. Table S1. Statistical analyses of data from the participants (n= 22) of the main study with the XN containing drink. Table S2 Statistical analyses of data obtained with the participants (n = 10) of a study with XN pills. Figure S2. Representative histograms of a γH2AX experiment with lymphocytes from a participant who consumed XN pills.

An overview of the applied workflow is depected in Figure S4. All used cell systems and applied techniques are depicted. In addition to that the read out of this study design is visualized

Morphologic characterization of A375 and M24met melanoma cells. (a) Microscopic analyses; the arrows indicate a round cell of A375 and a tube-like structure formed by M24met. Immunohistochemical staining of A375 (b) and M24met (c) with S100, HMB45, Nestin, and p75NTR. (d) FACS analysis of A375, M24met and solvent control with CD90, FE, CD45, CD31, CD34 and CD40.

Rabbits are frequently used for the examination of the pharmacokinetics and effectiveness of antibiotic substances. However, antibiotics vary substantially in protein binding affecting the concentration of the antimicrobially effective unbound drug. We hypothesized that the binding properties of vancomycin, meropenem and ceftriaxone might vary between human and rabbit plasma. In an in-vitro study we observed dose dependent variability in protein binding of antibiotics between species. Thus, in-vitro-pre-studies are required to guarantee for translational conditions.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus has been causing the COVID-19 pandemic since December 2019, with over 600 million infected persons worldwide and over six million deaths. We investigated the anti-viral effects of polyphenolic green tea ingredients and the synthetic resveratrol analogue 3,3′,4,4′,5,5′-hexahydroxy-trans-stilbene (HHS), a compound with antioxidant, antitumor and anti-HIV properties. In the TCID50 assay, four out of nine green tea constituents showed minor to modest cell protective effects, whereas HHS demonstrated the highest reduction (1103-fold) of the TCID50, indicating pronounced inhibition of virus replication. HHS was also a highly effective inhibitor of SARS-CoV-2 proliferation in VeroE6 cells with an IC50 value of 31.1 µM. HSS also inhibited the binding of the receptor-binding domain (RBD) of the spike protein to the human angiotensin-converting enzyme 2 (ACE2) receptor (RBD-ACE2) binding with 29% at 100 µM and with 9.2% at 50 µM indicating that the SARS-CoV-2 inhibitory effect might at least in part be attributed to the inhibition of virus binding to ACE2. Based on the chemical similarity to other polyphenols, the oral bioavailability of HHS is likely also very low, resulting in blood levels far below the inhibitory concentration of EGCG against SARS-CoV-2 observed in vitro. However, administration of HHS topically as a nose or throat spray would increase concentrations several-fold above the minimal inhibitory concentration (MIC) in the mucosa and might reduce virus load when administered soon after infection. Due to these promising tissue culture results, further preclinical and clinical studies are warranted to develop HHS as an additional treatment option for SARS-CoV-2 infection to complement vaccines, which is and will be the main pillar to combat the COVID-19 pandemic.

Exhaustive pharmacokinetic (PK) studies in paediatric patients are unavailable for most antibiotics and feasibility of PK studies is limited by challenges, such as low blood volume and venipuncture-related pain. Microdialysis (MD) represents a promising method to overcome these obstacles. The aim of this proof-of-concept study was to develop and validate modified MD catheters that can be used to obtain concentration-time profiles of antibiotics in paediatric patients.Following extensive in vitro MD experiments, a prospective open-labelled study in ten healthy adult volunteers (HVs) was conducted. Subjects received a single intravenous dose of 1000 mg vancomycin, then plasma and intravascular microdialysate were sampled over 24 h. In vivo MD probe calibration was conducted using the retrodialysis technique. Plasma protein binding was measured using ultrafiltration. Confirmation of the measurements was performed using a Bland-Altman plot, relevant PK parameters were calculated, and a pharmacometric model was established.No safety issues were encountered. The concentration-time curves of microdialysate and plasma measurements showed good alignment. The Bland-Altman plot yielded a mean bias of 0.19 mg/L and 95% limits of agreement of - 9.34 to 9.71 mg/L. A two-compartment model best described plasma PK, model-based estimates for recovery of the MD probes being in high agreement with the observed values. Quantified estimates of fraction unbound were comparable between plasma and microdialysate (p = 0.56).An innovative MD catheter that can be inserted into small intravenous lines was successfully developed and applied in HV. This proof-of-concept study is encouraging and opens the way to further experiments leading towards future use of MD in paediatric patients.

Background Inadequate antibiotic exposure in cerebral infections might have detrimental effects on clinical outcome. Commonly, antibiotic concentrations within the CSF were used to estimate cerebral target levels. However, the actual pharmacological active unbound drug concentration beyond the blood–brain barrier is unknown. Objectives To compare meropenem concentrations in blood, CSF and cerebral microdialysate of neurointensive care patients. Patients and methods In 12 patients suffering subarachnoid haemorrhage, 2000 mg of meropenem was administered every 8 h due to an extracerebral infection. Meropenem concentrations were determined in blood, CSF and cerebral microdialysate at steady state (n = 11) and following single-dose administration (n = 5). Results At steady state, the free AUC0–8 was 233.2 ± 42.7 mg·h/L in plasma, 7.8 ± 1.9 mg·h/L in CSF and 26.6 ± 14.0 mg·h/L in brain tissue. The brain tissue penetration ratio (AUCbrain/AUCplasma) was 0.11 ± 0.06, which was more than 3 times higher than in CSF (0.03 ± 0.01), resulting in an AUCCSF/AUCbrain ratio of 0.41 ± 0.16 at steady state. After single-dose administration similar proportions were achieved (AUCbrain/AUCplasma = 0.09 ± 0.08; AUCCSF/AUCplasma = 0.02 ± 0.00). Brain tissue concentrations correlated well with CSF concentrations (R = 0.74, P Conclusions Meropenem achieves sufficient bactericidal concentrations for the most common bacterial strains of cerebral infections in both plasma and brain tissue, even in non-inflamed brain tissue. CSF concentrations would highly underestimate the target site activity of meropenem beyond the blood–brain barrier.

Ponatinib is a small molecule multi‐tyrosine kinase inhibitor clinically approved for anticancer therapy. Molecular mechanisms by which cancer cells develop resistance against ponatinib are currently poorly understood. Likewise, intracellular drug dynamics, as well as potential microenvironmental factors affecting the activity of this compound are unknown. Cell/molecular biological and analytical chemistry methods were applied to investigate uptake kinetics/subcellular distribution, the role of lipid droplets (LDs) and lipoid microenvironment compartments in responsiveness of FGFR1‐driven lung cancer cells toward ponatinib. Selection of lung cancer cells for acquired ponatinib resistance resulted in elevated intracellular lipid levels. Uncovering intrinsic ponatinib fluorescence enabled dissection of drug uptake/retention kinetics in vitro as well as in mouse tissue cryosections, and revealed selective drug accumulation in LDs of cancer cells. Pharmacological LD upmodulation or downmodulation indicated that the extent of LD formation and consequent ponatinib incorporation negatively correlated with anticancer drug efficacy. Co‐culturing with adipocytes decreased ponatinib levels and fostered survival of cancer cells. Ponatinib‐selected cancer cells exhibited increased LD levels and enhanced ponatinib deposition into this organelle. Our findings demonstrate intracellular deposition of the clinically approved anticancer compound ponatinib into LDs. Furthermore, increased LD biogenesis was identified as adaptive cancer cell‐defense mechanism via direct drug scavenging. Together, this suggests that LDs represent an underestimated organelle influencing intracellular pharmacokinetics and activity of anticancer tyrosine kinase inhibitors. Targeting LD integrity might constitute a strategy to enhance the activity not only of ponatinib, but also other clinically approved, lipophilic anticancer therapeutics., What's new? Ponatinib is a small‐molecule multi‐tyrosine kinase inhibitor clinically approved for anticancer therapy. However, to date, the intracellular pharmacokinetics of this compound and the molecular mechanisms underlying resistance in cancer cells remain largely unknown. Here, the authors found that ponatinib was selectively scavenged by lipid droplets in cancer cells. Ponatinib accumulation into lipid droplets emerged as a critical determinant of intrinsic and acquired drug resistance. The findings suggest that lipid droplets represent an underestimated organelle influencing intracellular pharmacokinetics and anticancer tyrosine kinase inhibitor activity. Moreover, co‐targeting of lipogenic cancer cell phenotypes might enhance the efficacy of ponatinib and other lipophilic pharmaceuticals.

Purpose Although temozolomide is widely used in the treatment of childhood central nervous system (CNS) tumors, information on its pharmacokinetic profile in the brain or cerebrospinal fluid (CSF) is sparse. This study aimed at investigating whether measurable and clinically relevant concentrations of temozolomide are reached and maintained in CSF for continuous oral administration in pediatric patients. A population pharmacokinetic model was developed to quantify CSF penetration of temozolomide. Methods Eleven pediatric CNS tumor patients (aged 4–14 years) treated with oral temozolomide using a metronomic schedule (24–77 mg/m2/day) were included. Temozolomide concentrations in 28 plasma samples and 64 CSF samples were analyzed by high-performance liquid chromatography. Population pharmacokinetic modeling and simulations were performed using non-linear mixed effects modeling (NONMEM 7.4.2). Results Median temozolomide concentrations in plasma and CSF were 0.96 (range 0.24–5.99) µg/ml and 0.37 (0.06–1.76) µg/ml, respectively. A two-compartment model (central/plasma [1], CSF [2]) with first-order absorption, first-order elimination, and a transit compartment between CSF and plasma adequately described the data. Population mean estimates for clearance (CL) and the volume of distribution in the central compartment (Vc) were 3.29 L/h (95% confidence interval (CI) 2.58–3.95) and 10.5 L (8.17–14.32), respectively. Based on simulations, we found a median area under the concentration vs. time curve ratio (AUCCSF / AUCplasma ratio) of 37%. Conclusion Metronomic oral temozolomide penetrates into the CSF in pediatric patients, with even higher concentration levels compared to adults.

Background and Objective In microdose studies, drug pharmacokinetics is measured in humans after administration of subtherapeutic doses. While previous microdose studies focused primarily on plasma pharmacokinetics, we set out to evaluate the feasibility of microdosing for a pharmacokinetic assessment in subcutaneous tissue and epithelial lining fluid. Methods Healthy subjects received a single intravenous bolus injection of a microdose of [14C]ciprofloxacin (1.1 µg, 7 kBq) with (cohort A, n = 9) or without (cohort B, n = 9) a prior intravenous infusion of a therapeutic dose of unlabeled ciprofloxacin (400 mg). Microdialysis and bronchoalveolar lavage were applied for determination of subcutaneous and intrapulmonary drug concentrations. Microdose [14C]ciprofloxacin was quantified by accelerator mass spectrometry and therapeutic-dose ciprofloxacin by liquid chromatography–tandem mass spectrometry. Results The pharmacokinetics of therapeutic-dose ciprofloxacin (cohort A) in plasma, subcutaneous tissue, and epithelial lining fluid was in accordance with previous data. In plasma and subcutaneous tissue, the dose-adjusted area under the concentration–time curve of microdose ciprofloxacin was similar in cohorts A and B and within an 0.8-fold to 1.1-fold range of the area under the concentration–time curve of therapeutic-dose ciprofloxacin. Penetration of microdose ciprofloxacin into subcutaneous tissue was similar in cohorts A and B and comparable to that of therapeutic-dose ciprofloxacin with subcutaneous tissue-to-plasma area under the concentration–time curve ratios of 0.44, 0.44, and 0.38, respectively. Penetration of microdose ciprofloxacin into epithelial lining fluid was highly variable and failed to predict the epithelial lining fluid penetration of therapeutic-dose ciprofloxacin. Conclusions Our study confirms the feasibility of microdosing for pharmacokinetic measurements in plasma and subcutaneous tissue. Microdosing combined with microdialysis is a potentially useful tool in clinical antimicrobial drug development, but its applicability for the assessment of pulmonary pharmacokinetics with bronchoalveolar lavage requires further studies. Clinical Trial Registration ClinicalTrials.gov NCT03177720 (registered 6 June, 2017).

Introduction/Background* Endometrial cancer (EC) is the most common gynecological malignancy in the western world. EC has traditionally been divided into type I, which is estrogen dependent, and type II, where associations with estrogens were only recently uncovered. Both types of EC arise after menopause when tumor tissue depends on formation of estrogens from inactive steroid precursors. In EC, active estrogens can be formed from circulating estrone sulfate (E1-S) via sulfatase pathway by the sulfatase (STS) and reductive 17β-hydroxysteroid dehydrogenase type 1 (HSD17B1) enzymes. Methodology In our study, we aimed to investigate the role of estrogens in model cell lines of moderately (type I) and poorly (type II) differentiated EC: RL95-2 and KLE cells, respectively. We evaluated expression of genes involved in E1-S transport, estrogen biosynthesis and oxidative metabolism, and examined cellular uptake of E1-S, formation of estrogens from E1-S, and effects of estrogens on cell proliferation. Result(s)* Gene expression analysis revealed up-regulated expression of several E1-S uptake transporters: SLCO1A2 (3434-fold), SLCO1B3 (2302-fold), SLCO1C1 (381-fold), SLCO3A1 (19-fold), SLC22A9 (5-fold), SLC10A6 (5-fold), and functional studies showed increased E1-S uptake in KLE cells versus RL95-2 cells. Higher levels of STS were confirmed in RL95-2 cells, which also better metabolized E1-S to estrone (E1), compared to KLE cells. In KLE cells, disturbed balance in the expression of genes encoding reductive and oxidative HSD17B enzymes enhanced activation of E1 to estradiol (E2), compared to RL95-2 cells, and physiological E1 concentrations stimulated KLE cell proliferation. Additionally, gene expression analysis in KLE versus RL95-2 cells indicated increased CYP1B1 expression (17-fold) as responsible for formation of carcinogenic 4-hydroxycatechols, and down-regulation of genes that encode phase II metabolic enzymes: COMT (6-fold), NQO1 (13-fold), NQO2 (7-fold), and GSTP1 (2-fold). This suggested decreased detoxification of carcinogenic metabolites in KLE cells. Conclusion* Our results indicate that in cell lines of type I and type II EC, estrogens can be formed via the sulfatase pathway, and can promote proliferation of poorly differentiated EC. This supports the importance of estrogens in poorly differentiated (type II) EC. Further studies in tissue samples of type II EC are needed to confirm our findings. Funding Slovenian Research Agency, grant J3-8212 to T.L.R., Young Researcher grant to R.P.; Austrian Science Fund (FWF), grant I 3417-B31 to W.J.

Ceftaroline fosamil, a fifth-generation cephalosporin antibiotic with activity against methicillin-resistant Staphylococcus aureus (MRSA), is currently approved for the treatment of pneumonia and complicated skin and soft tissue infections. However, pharmacokinetics data on free lung tissue concentrations in critical patient populations are lacking. The aim of this study was to evaluate the pharmacokinetics of the high-dose regimen of ceftaroline in plasma and lung tissue in cardiac surgery patients during intermittent and continuous administration. Nine patients undergoing elective cardiac surgery on cardiopulmonary bypass were included in this study and randomly assigned to intermittent or continuous administration. Eighteen hundred milligrams of ceftaroline fosamil was administered intravenously as either 600 mg over 2 h every 8 h (q8h) (intermittent group) or 600 mg over 2 h (loading dose) plus 1,200 mg over 22 h (continuous group). Interstitial lung tissue concentrations were measured by in vivo microdialysis. Relevant pharmacokinetics parameters were calculated for each group. Plasma exposure levels during intermittent and continuous administration were comparable to those of previously published studies and did not differ significantly between the two groups. In vivo microdialysis demonstrated reliable and adequate penetration of ceftaroline into lung tissue during intermittent and continuous administration. The steady-state area under the concentration-time curve from 0 to 8 h (AUCss 0-8) and the ratio of AUCSS 0-8 in lung tissue and AUC in plasma (AUClung/plasma) were descriptively higher in the continuous group. Continuous administration of ceftaroline fosamil achieved a significantly higher proportion of time for which the free drug concentration remained above 4 times the minimal inhibitory concentration (MIC) during the dosing interval (% fT>4xMIC) than intermittent administration for pathogens with a MIC of 1 mg/liter. Ceftaroline showed adequate penetration into interstitial lung tissue of critically ill patients undergoing major cardiothoracic surgery, supporting its use for pneumonia caused by susceptible pathogens.

P-Glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) in the canalicular membrane of hepatocytes mediate the biliary excretion of drugs and drug metabolites. To measure hepatic ABCB1 and ABCG2 activity, we performed positron emission tomography (PET) scans with the ABCB1/ABCG2 substrate [11C]tariquidar in healthy volunteers and wild-type, Abcb1a/b(-/-), Abcg2(-/-), and Abcb1a/b(-/-)Abcg2(-/-) mice without and with coadministration of unlabeled tariquidar. PET data were analyzed with a three-compartment pharmacokinetic model. [11C]Tariquidar underwent hepatobiliary excretion in both humans and mice, and tariquidar coadministration caused a significant reduction in the rate constant for the transfer of radioactivity from the liver into bile (by -74% in humans and by -62% in wild-type mice), suggesting inhibition of canalicular efflux transporter activity. Radio-thin-layer chromatography analysis revealed that the majority of radioactivity (>87%) in the mouse liver and bile was composed of unmetabolized [11C]tariquidar. PET data in transporter knockout mice revealed that both ABCB1 and ABCG2 mediated biliary excretion of [11C]tariquidar. In vitro experiments indicated that tariquidar is not a substrate of major hepatic basolateral uptake transporters (SLCO1B1, SLCO1B3, SLCO2B1, SLC22A1, and SLC22A3). Our data suggest that [11C]tariquidar can be used to measure hepatic canalicular ABCB1/ABCG2 transport activity without a confounding effect of uptake transporters.

Polyphenols in foods and dietary supplements are commonly used for the prevention and treatment of a variety of malignancies, including breast cancer. However, daily intake by patients with breast cancer is controversial, as these compounds may stimulate cancer growth. Estrogens serve key roles in breast cancer cell proliferation; therefore, understanding the interaction between endogenous steroid hormones and natural dietary polyphenols is essential. Currently, comprehensive knowledge regarding these effects remains limited. The current review summarizes the dose-dependent in vitro and in vivo interactions of resveratrol and other dietary polyphenols with estrogen precursors, active estrogens, catechol estrogens and their respective glucuronidated, sulfated, glutathionated or O-methylated metabolites in estrogen receptor alpha negative (ERα-) and positive (ERα+) breast cancer. Which estrogen-metabolizing enzymes are affected by polyphenols is also reviewed in detail. Furthermore, the impacts of dose and therapy duration on disease development and progression in patients with breast cancer are discussed. The present article is part of a Special Issue titled 'CSR 2018'.

The abundance of OATP4A1 in colorectal cancer (CRC) might be related to tumor progression. This was studied by immunohistochemistry on paraffin-embedded samples obtained from 178 patients (43 patients with a relapse within 5 y) with early-stage CRC. Positivity for OATP4A1 in tumor cells and noncancerous mucosal cells was proved by double-immunofluorescence staining with antibodies against OATP4A1 and keratin 8, whereas antibodies against appropriate CD markers were used to identify immune cells. Automated microscopic image analysis was used to measure the percentage of OATP4A1-positive cells and OATP4A1 staining intensity in tumor, immune, and adjacent normal-looking mucosal cells separately, as well as in the mucosal and immune cells of 14 nonmalignant tissue samples. In CRC the percentage of OATP4A1-positive cells, but not staining intensity, was significantly higher in tumor and mucosal cells adjacent to the tumor compared to the mucosa of nonmalignant samples (P

P-Glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) are two efflux transporters at the blood-brain barrier (BBB), which effectively restrict brain distribution of diverse drugs, such as tyrosine kinase inhibitors. There is a crucial need for pharmacological ABCB1 and ABCG2 inhibition protocols for a more effective treatment of brain diseases. In the present study, seven marketed drugs (osimertinib, erlotinib, nilotinib, imatinib, lapatinib, pazopanib, and cyclosporine A) and one nonmarketed drug (tariquidar), with known in vitro ABCB1/ABCG2 inhibitory properties, were screened for their inhibitory potency at the BBB in vivo. Positron emission tomography (PET) using the model ABCB1/ABCG2 substrate [11C]erlotinib was performed in mice. Tested inhibitors were administered as i.v. bolus injections at 30 min before the start of the PET scan, followed by a continuous i.v. infusion for the duration of the PET scan. Five of the tested drugs increased total distribution volume of [11C]erlotinib in the brain ( VT,brain) compared to vehicle-treated animals (tariquidar, + 69%; erlotinib, + 19% and +23% for the 21.5 mg/kg and the 43 mg/kg dose, respectively; imatinib, + 22%; lapatinib, + 25%; and cyclosporine A, + 49%). For all drugs, increases in [11C]erlotinib brain distribution were lower than in Abcb1a/b(-/-)Abcg2(-/-) mice (+149%), which suggested that only partial ABCB1/ABCG2 inhibition was reached at the mouse BBB. The plasma concentrations of the tested drugs at the time of the PET scan were higher than clinically achievable plasma concentrations. Some of the tested drugs led to significant increases in blood radioactivity concentrations measured at the end of the PET scan (erlotinib, + 103% and +113% for the 21.5 mg/kg and the 43 mg/kg dose, respectively; imatinib, + 125%; and cyclosporine A, + 101%), which was most likely caused by decreased hepatobiliary excretion of radioactivity. Taken together, our data suggest that some marketed tyrosine kinase inhibitors may be repurposed to inhibit ABCB1 and ABCG2 at the BBB. From a clinical perspective, moderate increases in brain delivery despite the administration of high i.v. doses as well as peripheral drug-drug interactions due to transporter inhibition in clearance organs question the translatability of this concept.

It has been shown that protein binding, temperature, and pH influence in vitro pharmacodynamic (PD) models. The fact that corpuscular blood compounds might also have an important impact is something which has, until now, often been neglected. We investigated if the addition of human erythrocytes to standard growth media (Mueller Hinton Broth, MHBII) has an influence on bacterial growth behavior and on antibiotic efficacy. We did this by using bacterial growth assays and time kill curves (TKC) of selected strains (Escherichia coli ATCC25922, Staphylococcus aureus ATCC29213, and Pseudomonas aeruginosa ATCC27853) over 24 h. The final concentration of erythrocytes was set to match the physiological concentrations in the blood of a healthy human, i.e., 3 × 10^6 cells/μl in MHBII. Meropenem, ciprofloxacin, and tigecycline were tested with concentrations several-fold above and below the minimal inhibitory concentration (MIC). Moreover, HPLC analysis of antibiotic stability and distribution in erythrocytes was performed. Meropenem, ciprofloxacin, and tigecycline showed the greatest decline in activity against E. coli when erythrocytes were present. A mean difference in log10 bacterial killing between pure MHBII and 50%-Ery of 3.83, 1.33, and 2.42 was found for ciprofloxacin, meropenem, and tigecycline, respectively. In the case of ciprofloxacin, HPLC analysis revealed that less extracellular antibiotic is available in the presence of erythrocytes. We have demonstrated that erythrocytes do influence antimicrobial activity and that this might have an impact on the extrapolation of in vitro activity testing to in vivo efficacy in patients.

PET with avid substrates of P-glycoprotein (ABCB1) provided evidence of the role of this efflux transporter in effectively restricting the brain penetration of its substrates across the human blood–brain barrier (BBB). This may not reflect the situation for weak ABCB1 substrates including several antidepressants, antiepileptic drugs, and neuroleptics, which exert central nervous system effects despite being transported by ABCB1. We performed PET with the weak ABCB1 substrate 11C-metoclopramide in humans to elucidate the impact of ABCB1 function on its brain kinetics. Methods: Ten healthy male subjects underwent 2 consecutive 11C-metoclopramide PET scans without and with ABCB1 inhibition using cyclosporine A (CsA). Pharmacokinetic modeling was performed to estimate the total volume of distribution (VT) and the influx (K1) and efflux (k2) rate constants between plasma and selected brain regions. Furthermore, 11C-metoclopramide washout from the brain was estimated by determining the elimination slope (kE,brain) of the brain time–activity curves. Results: In baseline scans, 11C-metoclopramide showed appreciable brain distribution (VT = 2.11 ± 0.33 mL/cm3). During CsA infusion, whole-brain gray matter VT and K1 were increased by 29% ± 17% and 9% ± 12%, respectively. K2 was decreased by 15% ± 5%, consistent with a decrease in kE,brain (−32% ± 18%). The impact of CsA on outcome parameters was significant and similar across brain regions except for the pituitary gland, which is not protected by the BBB. Conclusion: Our results show for the first time that ABCB1 does not solely account for the “barrier” property of the BBB but also acts as a detoxifying system to limit the overall brain exposure to its substrates at the human blood–brain interface.

Background: Elimination of a drug during renal replacement therapy is not only dependent on flow rates, molecular size and protein binding, but is often influenced by difficult to predict drug membrane interactions. In vitro models allow for extensive profiling of drug clearance using a wide array of hemofilters and flow rates. We present a bovine blood based in vitro pharmacokinetic model for intermittent renal replacement therapy.Methods: Four different drugs were analyzed: gentamicin, doripenem, vancomicin and teicoplanin. The investigated drug was added to a bovine blood reservoir connected to a hemodialysis circuit. In total seven hemofilter models were analyzed using commonly employed flow rates. Pre-filter, post-filter and dialysate samples were drawn, plasmaseparated and analyzed using turbidimetric assays or HPLC. Protein binding of doripenem and vancomycin was measured in bovine plasma and compared to previously published values for human plasma.Results: Clearance values were heavily impacted by choice of membrane material and surface as well as by dialysis parameters such as blood flow rate. Gentamicin clearance ranged from a minimum of 90.12 ml/min in a Baxter CAHP-170 diacetate hemofilter up to a maximum of 187.90 ml/min in a Fresenius medical company Fx80 polysulfone model (blood flow rate 400 ml/min, dialysate flow rate 800 ml/min). Clearance of Gentamicin vs Vancomicin over the F80s hemofilter model using the same flow rates was 137.62 mL vs 103.25 ml/min. Doripenem clearance with the Fx80 was 141.25 ml/min.Conclusion: Clearance values corresponded very well to previously published data from clinical pharmacokinetic trials. In conjunction with in silico pharmacometric models. This model will allow precise dosing recommendations without the need of large scale clinical trials.

Endometrial cancer (EC) is associated with increased estrogen actions. Locally, estrogens can be formed from estrone-sulphate (E1-S) after cellular uptake by organic anion-transporting polypeptides (OATP) or organic anion transporters (OAT). Efflux of E1-S is enabled by ATP Binding Cassette transporters (ABC) and organic solute transporter (OST)αβ. Currently, 19 E1-S transporters are known but their roles in EC are not yet understood. Here, we analysed levels of E1-S transporters in Ishikawa (premenopausal EC), HEC-1-A (postmenopausal EC), HIEEC (control) cell lines, in EC tissue, examined metabolism of steroid precursor E1-S, studied effects of OATPs’ inhibition and gene-silencing on E1-S uptake, and assessed associations between transporters and histopathological data. Results revealed enhanced E1-S metabolism in HEC-1-A versus Ishikawa which could be explained by higher levels of OATPs in HEC-1-A versus Ishikawa, especially 6.3-fold up-regulation of OATP1B3 (SLCO1B3), as also confirmed by immunocytochemical staining and gene silencing studies, lower ABCG2 expression and higher levels of sulfatase (STS). In EC versus adjacent control tissue the highest differences were seen for ABCG2 and SLC51B (OSTβ) which were 3.0-fold and 2.1-fold down-regulated, respectively. Immunohistochemistry confirmed lower levels of these two transporters in EC versus adjacent control tissue. Further analysis of histopathological data indicated that SLCO1B3 might be important for uptake of E1-S in tumours without lymphovascular invasion where it was 15.6-fold up-regulated as compared to adjacent control tissue. Our results clearly indicate the importance of E1-S transporters in EC pathophysiology and provide a base for further studies towards development of targeted treatment.

The antiemetic and gastroprokinetic drug metoclopramide is a weak substrate of the blood-brain barrier (BBB) efflux transporter P-glycoprotein (P-gp) and displays central nervous system (CNS) side effects (i.e. extrapyramidal symptoms, tardive dyskinesia) caused by dopamine D2 receptor blockade in the basal ganglia. These side effects occur with a higher incidence in elderly people. We used positron emission tomography (PET) to assess the brain distribution of [11 C]metoclopramide in young (n = 11, 26 ± 3 years) and elderly (n = 7, 68 ± 9 years) healthy men both after administration of a microdose (9 ± 7 µg) and a microdose co-injected with a therapeutic dose of unlabeled metoclopramide (10 mg). For both doses, elderly subjects had a significantly higher total volume of distribution (VT ) of [11 C]metoclopramide in the basal ganglia than young subjects (microdose: +26%, therapeutic dose: +41%). Increases in VT (= K1 /k2 ) were caused by significant decreases in the transfer rate constant of [11 C]metoclopramide from brain into plasma (k2 , microdose: -18%, therapeutic dose: -30%), while the distributional clearance from plasma into brain (K1 ) remained unaltered. This reduction in the clearance of [11 C]metoclopramide (k2 ) from the brains of elderly subjects may be caused by an age-related decrease in the activity of P-gp at the BBB and may contribute to the higher incidence of CNS side effects of metoclopramide in the aged population. Our data suggest that an age-associated decrease in the clearance properties of the BBB may modulate the CNS effects or side effects of clinically used P-gp substrates.

Metastatic cancer cells cross endothelial barriers and travel through the blood or lymphatic fluid to pre‑metastatic niches, leading to their colonisation. 'S' stereoisomer 12S‑hydroxy‑5Z,8Z,10E,14Z‑eicosatetraenoic acid [12(S)‑HETE] is secreted by a variety of cancer cell types and has been indicated to open up these barriers. In the present study, another aspect of the endothelial unlocking mechanism was elucidated. This was achieved by investigating 12(S)‑HETE‑treated lymph endothelial cells (LECs) with regard to their expression and mutual interaction with v‑rel avian reticuloendotheliosis viral oncogene homolog A (RELA), intercellular adhesion molecule 1, SRY‑box transcription factor 18 (SOX18), prospero homeobox 1 (PROX1) and focal adhesion kinase (FAK). These key players of LEC retraction, which is a prerequisite for cancer cell transit into vasculature, were analysed using western blot analysis, reverse transcription‑quantitative PCR and transfection with small interfering (si)RNA. The silencing of a combination of these signalling and executing molecules using siRNA, or pharmacological inhibition with defactinib and Bay11‑7082, extended the mono‑culture experiments to co‑culture settings using HCT116 colon cancer cell spheroids that were placed on top of LEC monolayers to measure their retraction using the validated 'circular chemorepellent‑induced defect' assay. 12(S)‑HETE was indicated to induce the upregulation of the RELA/SOX18 feedback loop causing the subsequent phosphorylation of FAK, which fed back to RELA/SOX18. Therefore, 12(S)‑HETE was demonstrated to be associated with circuits involving RELA, SOX18 and FAK, which transduced signals causing the retraction of LECs. The FAK‑inhibitor defactinib and the NF‑κB inhibitor Bay11‑7082 attenuated LEC retraction additively, which was similar to the suppression of FAK and PROX1 (the target of SOX18) by the transfection of respective siRNAs. FAK is an effector molecule at the distal end of a pro‑metastatic signalling cascade. Therefore, targeting the endothelial‑specific activity of FAK through the pathway demonstrated herein may provide a potential therapeutic method to combat cancer dissemination via vascular routes.

Genetic variations in the organic-anion-transporting polypeptide (OATP)-encoding solute carrier of organic anions (SLCO) genes can promote cancer development and progression. The overexpression of solute carrier organic anion transporter family member 4A1 (OATP4A1), a transporter for steroid hormones, prostaglandins, and bile acids, has been previously associated with tumor recurrence and progression in colorectal cancer (CRC). Therefore, the present study aimed to investigate the association between 2 frequent single nucleotide polymorphisms (SNPs) in SLCO4A1 (rs34419428, R70Q; rs1047099G, V78I) and CRC predisposition. Following restriction fragment length polymorphism-PCR analysis in 178 patients with CRC [Union for International Cancer Control (UICC) stage I/II] and 65 healthy controls, no significant difference was observed in allele frequency and the number of heterozygous/homozygous individuals between the groups. Notably, the R70Q minor allele was identified to be associated with the V78I minor allele in the genome. Comparing of the individual genotypes of CRC patients to clinical data, including sex, UICC-stage and relapse revealed no increased risk for CRC. In addition, the OATP4A1 immunoreactivity assay in paraffin-embedded CRC and adjacent non-tumorous mucosa sections, examined using quantitative microscopy image analysis, did not reveal any association with these polymorphisms. No significant differences were observed in the expression levels, localization, and sodium fluorescein transport capacity among the OATP4A1 variants, which was studied using functional assays in Sf9-insect and A431 tumor cells overexpressing the 2 single and a double mutant OATP4A1 SNP variants. These results suggested that the 2 most frequent polymorphisms located in the first intracellular loop of OATP4A1 do not associate with CRC predisposition and tumor recurrence. They are unlikely to affect the outcome of CRC in patients.

The adenosine triphosphate-binding cassette transporters P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) are 2 efflux transporters at the blood-brain barrier (BBB) that effectively restrict brain distribution of dual ABCB1/ABCG2 substrate drugs, such as tyrosine kinase inhibitors. Pharmacologic inhibition of ABCB1/ABCG2 may improve the efficacy of dual-substrate drugs for treatment of brain tumors, but no marketed ABCB1/ABCG2 inhibitors are currently available. In the present study, we examined the potential of supratherapeutic-dose oral erlotinib to inhibit ABCB1/ABCG2 activity at the human BBB. Methods: Healthy men underwent 2 consecutive PET scans with 11C-erlotinib: a baseline scan and a second scan either with concurrent intravenous infusion of the ABCB1 inhibitor tariquidar (3.75 mg/min, n = 5) or after oral intake of single ascending doses of erlotinib (300 mg, n = 7; 650 mg, n = 8; or 1,000 mg, n = 2). Results: Although tariquidar administration had no effect on 11C-erlotinib brain distribution, oral erlotinib led, at the 650-mg dose, to significant increases in volume of distribution (23% ± 13%, P = 0.008), influx rate constant of radioactivity from plasma into brain (58% ± 26%, P = 0.008), and area under the brain time-activity curve (78% ± 17%, P = 0.008), presumably because of combined partial saturation of ABCG2 and ABCB1 activity. Inclusion of further subjects into the 1,000-mg dose group was precluded by adverse skin events (rash). Conclusion: Supratherapeutic-dose erlotinib may be used to enhance brain delivery of ABCB1/ABCG2 substrate anticancer drugs, but its clinical applicability for continuous ABCB1/ABCG2 inhibition at the BBB may be limited by safety concerns.

Organic anion-transporting polypeptides (OATPs) mediate the uptake of various drugs from blood into the liver in the basolateral membrane of hepatocytes. Positron emission tomography (PET) is a potentially powerful tool to assess the activity of hepatic OATPs in vivo, but its utility critically depends on the availability of transporter-selective probe substrates. We have shown before that among the three OATPs expressed in hepatocytes (OATP1B1, OATP1B3, and OATP2B1), [11C]erlotinib is selectively transported by OATP2B1. In contrast to OATP1B1 and OATP1B3, OATP2B1 has not been thoroughly explored yet, and no specific probe substrates are currently available. To assess if the prototypical OATP inhibitor rifampicin can inhibit liver uptake of [11C]erlotinib in vivo, we performed [11C]erlotinib PET scans in six healthy volunteers without and with intravenous pretreatment with rifampicin (600 mg). In addition, FVB mice underwent [11C]erlotinib PET scans without and with concurrent intravenous infusion of high...

Mechanisms how colorectal cancer (CRC) cells penetrate blood micro-vessel endothelia and metastasise is poorly understood. To study blood endothelial cell (BEC) barrier breaching by CRC emboli, an in vitro assay measuring BEC-free areas underneath SW620 cell spheroids, so called “circular chemorepellent induced defects” (CCIDs, appearing in consequence of endothelial retraction), was adapted and supported by Western blotting, EIA-, EROD- and luciferase reporter assays. Inhibition of ALOX12 or NF-κB in SW620 cells or BECs, respectively, caused attenuation of CCIDs. The FDA approved drugs vinpocetine [inhibiting ALOX12-dependent 12(S)-HETE synthesis], ketotifen [inhibiting NF-κB], carbamazepine and fenofibrate [inhibiting 12(S)-HETE and NF-κB] significantly attenuated CCID formation at low μM concentrations. In the 5-FU-resistant SW620-R/BEC model guanfacine, nifedipine and proadifen inhibited CCIDs stronger than in the naive SW620/BEC model. This indicated that in SW620-R cells formerly silent (yet unidentified) genes became expressed and targetable by these drugs in course of resistance acquisition. Fenofibrate, and the flavonoids hispidulin and apigenin, which are present in medicinal plants, spices, herbs and fruits, attenuated CCID formation in both, naive- and resistant models. As FDA-approved drugs and food-flavonoids inhibited established and acquired intravasative pathways and attenuated BEC barrier-breaching in vitro, this warrants testing of these compounds in CRC models in vivo.

To assess the hepatic disposition of erlotinib, we performed positron emission tomography (PET) scans with [11 C]erlotinib in healthy volunteers without and with oral pretreatment with a therapeutic erlotinib dose (300 mg). Erlotinib pretreatment significantly decreased the liver exposure to [11 C]erlotinib with a concomitant increase in blood exposure, pointing to the involvement of a carrier-mediated hepatic uptake mechanism. Using cell lines overexpressing human organic anion-transporting polypeptides (OATPs) 1B1, 1B3, or 2B1, we show that [11 C]erlotinib is selectively transported by OATP2B1. Our data suggest that at PET microdoses hepatic uptake of [11 C]erlotinib is mediated by OATP2B1, whereas at therapeutic doses OATP2B1 transport is saturated and hepatic uptake occurs mainly by passive diffusion. We propose that [11 C]erlotinib may be used as a hepatic OATP2B1 probe substrate and erlotinib as an OATP2B1 inhibitor in clinical drug-drug interaction studies, allowing the contribution of OATP2B1 to the hepatic uptake of drugs to be revealed.

Aims The efflux transporter P-glycoprotein (ABCB1) acts at the blood–brain barrier (BBB) to restrict the distribution of many different drugs from blood to the brain. Previous data suggest an age-associated decrease in the expression and function of ABCB1 at the BBB. In the present study, we investigated the influence of age on the magnitude of an ABCB1-mediated drug–drug interaction (DDI) at the BBB. Methods We performed positron emission tomography scans using the model ABCB1 substrate (R)-[11C]verapamil in five young [26 ± 1 years, (mean ± standard deviation)] and five elderly (68 ± 6 years) healthy male volunteers before and after intravenous administration of a low dose of the ABCB1 inhibitor tariquidar (3 mg kg−1). Results In baseline scans, the total distribution volume (VT) of (R)-[11C]verapamil in whole-brain grey matter was not significantly different between the elderly (VT = 0.78 ± 0.15) and young (VT = 0.79 ± 0.10) group. After partial (incomplete) ABCB1 inhibition, VT values were significantly higher (P = 0.040) in the elderly (VT = 1.08 ± 0.15) than in the young (VT = 0.80 ± 0.18) group. The percentage increase in (R)-[11C]verapamil VT following partial ABCB1 inhibition was significantly greater (P = 0.032) in elderly (+40 ± 17%) than in young (+2 ± 17%) volunteers. Tariquidar plasma concentrations were not significantly different between the young (786 ± 178 nmol l−1) and elderly (1116 ± 347 nmol l−1) group. Conclusions Our results provide the first direct evidence of an increased risk for ABCB1-mediated DDIs at the BBB in elderly persons, which may have important consequences for pharmacotherapy of the elderly.

Liquid chromatography–mass spectrometry (LC–MS) is the state of the art technique for quantification of steroid hormones. Currently used methods are typically limited by the need of pre-column derivatization to increase ionization efficiency; however, this causes hydrolysis of conjugated metabolites. Our newly established LC–HRMS method is able to simultaneously quantify conjugated and unconjugated steroids without prior derivatization using deuterated internal standards and solid-phase extraction. This assay was validated according to ICH Q2(R1) guidelines for the analysis of the 10 main steroids of the estrogenic pathway, namely 4-androstene-3,17-dione, dehydroepiandrosterone (DHEA), DHEA-3-sulfate, estrone, 17β-estradiol, estriol (16α-OH-17β-estradiol), estrone-3-sulfate, 17β-estradiol-3-(β- d -glucuronide), 17β-estradiol-3-sulfate and testosterone. Assay performance characteristics were excellent with results for accuracy (98.8–101.2%), precision (mean: 2.05%, all ≤2.80%), stability over five freeze–thaw-cycles (95.7–100.4%) and SPE accuracy (96.9–102.0%), as well as suitable lower and upper limits of quantification for cell culture experiments (LLOQ 0.005–2 ng/ml, ULOQ 3–2000 ng/ml). Furthermore, we demonstrated the functionality of our method for the monitoring of steroid levels in the human breast cancer cell line MCF-7. This sensitive assay allows for the first time detailed investigations on estrogen metabolomics in breast cancer cells and may also apply to other estrogen-dependent tumor entities.

Lymph node metastasis of breast cancer is a clinical marker of poor prognosis. Yet, there exist no therapies targeting mechanisms of intravasation into lymphatics. Herein we report on an effect of the antidyslipidemic drug fenofibrate with vasoprotective activity, which attenuates breast cancer intravasation in vitro, and describe the potential mechanisms. To measure intravasation in a 3-dimensional co-culture model MDA-MB231 and MCF-7 breast cancer spheroids were placed on immortalised lymphendothelial cell (LEC) monolayers. This provokes the formation of circular chemorepellent induced defects (CCIDs) in the LEC barrier resembling entry ports for the intravasating tumour. Furthermore, the expression of adhesion molecules ICAM-1, CD31 and FAK was investigated in LECs by western blotting as well as cell-cell adhesion and NF-κB activity by respective assays. In MDA-MB231 cells the activity of CYP1A1 was measured by EROD assay. Fenofibrate inhibited CCID formation in the MDA-MB231/LEC- and MCF-7/LEC models and the activity of NF-κB, which in turn downregulated ICAM-1 in LECs and the adhesion of cancer cells to LECs. Furthermore, CD31 and the activity of FAK were inhibited. In MDA-MB231 cells, fenofibrate attenuated CYP1A1 activity. Combinations with other FDA-approved drugs, which reportedly inhibit different ion channels, attenuated CCID formation additively or synergistically. In summary, fenofibrate inhibited NF-κB and ICAM-1, and inactivated FAK, thereby attenuating tumour intravasation in vitro. A combination with other FDA-approved drugs further improved this effect. Our new concept may lead to a novel therapy for cancer patients.

Bioavailability of nutrients is highly diverse and depends on a variety of endogenous and exogenous factors in humans. This data article reports on the plasma response of 10 human subjects (5 females, 5 males) to a single dose of a multivitamin drink within 6h (blood taken after 1, 2, 4, and 6h). Nutrients, which were considered in the assessment, were folate (Radioimmuno Assay), vitamin B12 (Radioimmuno Assay) and resveratrol and its plasma metabolites resveratrol-3-O-glucuronide (R3G), resveratrol-4′-O-glucuronide (R4G), resveratrol-3-O-sulfate (R3S) and resveratrol-3-O-4′-O-disulfate (RD, all HPLC). Biological outcome measures were malondialdehyde (MDA, HPLC) and Ferric Reducing ability potential (FRAP, Microplate reader).Mean plasma concentration increased over time significantly for folate (p

P-Glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) in the canalicular membrane of hepatocytes mediate the biliary excretion of drugs and drug metabolites. To measure hepatic ABCB1 and ABCG2 activity, we performed positron emission tomography (PET) scans with the ABCB1/ABCG2 substrate [

In vitro pharmacodynamic models are used to optimize in vivo dosing regimens in antimicrobial drug development. One limiting factor of such models is the lack of host factors such as corpuscular blood components as erythrocytes which have already been shown to impact activity of antibiotics and/or growth of the pathogen. However, the impact of thrombocytes has not previously been investigated. We set out to investigate if the addition of thrombocytes (set to physiological concentrations in blood of healthy human, i.e., 5 × 105 thrombocytes/μL standard growth media Mueller Hinton Broth, MHB) has an influence on bacterial growth and on the efficacy of antibiotics against Gram+ and Gram− bacteria. Growth assays and time-killing-curves (TKC) were performed with ATCC-strains of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa in triplicate over 24 h. The same approach was followed for 5 clinical isolates of Escherichia coli. Meropenem, ciprofloxacin, and tigecycline were tested as representatives of broad-spectrum antibiotics, and concentrations several-fold above and below the minimal inhibitory concentration (MIC) were simulated. No significant impact of thrombocytes was found on bacterial growth or antimicrobial stability for the investigated agents. Bacteria reduced thrombocyte content to different degree, indicating direct interaction of pathogens and thrombocytes. Impact on bacterial killing was observed but was not fully reproducible when thrombocytes from different donors where used. While interaction of bacteria and thrombocytes was evident in the present study, interaction between antibiotic activity and thrombocytes seems unlikely. Whether variability was caused by different thrombocyte concentrates needs further investigation.

Cannabinoids (CBs) from Cannabis sativa provide relief for tumor-associated symptoms (including nausea, anorexia, and neuropathic pain) in the palliative treatment of cancer patients. Additionally, they may decelerate tumor progression in breast cancer patients. Indeed, the psychoactive delta-9-tetrahydrocannabinol (THC), non-psychoactive cannabidiol (CBD) and other CBs inhibited disease progression in breast cancer models. The effects of CBs on signaling pathways in cancer cells are conferred via G-protein coupled CB-receptors (CB-Rs), CB1-R and CB2-R, but also via other receptors, and in a receptor-independent way. THC is a partial agonist for CB1-R and CB2-R; CBD is an inverse agonist for both. In breast cancer, CB1-R expression is moderate, but CB2-R expression is high, which is related to tumor aggressiveness. CBs block cell cycle progression and cell growth and induce cancer cell apoptosis by inhibiting constitutive active pro-oncogenic signaling pathways, such as the extracellular-signal-regulated kinase pathway. They reduce angiogenesis and tumor metastasis in animal breast cancer models. CBs are not only active against estrogen receptor-positive, but also against estrogen-resistant breast cancer cells. In human epidermal growth factor receptor 2-positive and triple-negative breast cancer cells, blocking protein kinase B- and cyclooxygenase-2 signaling via CB2-R prevents tumor progression and metastasis. Furthermore, selective estrogen receptor modulators (SERMs), including tamoxifen, bind to CB-Rs; this process may contribute to the growth inhibitory effect of SERMs in cancer cells lacking the estrogen receptor. In summary, CBs are already administered to breast cancer patients at advanced stages of the disease, but they might also be effective at earlier stages to decelerate tumor progression.