St. John's wort extract with a high hyperforin content does not induce P-glycoprotein activity at the human blood-brain barrier.

St. John's wort (SJW) extract, a herbal medicine with antidepressant effects, is a potent inducer of intestinal and/or hepatic cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp), which can cause clinically relevant drug interactions. It is currently not known whether SJW can also induce P-gp activity at the human blood-brain barrier (BBB), which may potentially lead to decreased brain exposure and efficacy of certain central nervous system (CNS)-targeted P-gp substrate drugs. In this study, we used a combination of positron emission tomography (PET) imaging and cocktail phenotyping to gain a comprehensive picture on the effect of SJW on central and peripheral P-gp and CYP activities. Before and after treatment of healthy volunteers (n = 10) with SJW extract with a high hyperforin content (3-6%) for 12-19 days (1800 mg/day), the activity of P-gp at the BBB was assessed by means of PET imaging with the P-gp substrate [11C]metoclopramide and the activity of peripheral P-gp and CYPs was assessed by administering a low-dose phenotyping cocktail (caffeine, omeprazole, dextromethorphan, and midazolam or fexofenadine). SJW significantly increased peripheral P-gp, CYP3A, and CYP2C19 activity. Conversely, no significant changes in the peripheral metabolism, brain distribution, and P-gp-mediated efflux of [11C]metoclopramide across the BBB were observed following the treatment with SJW extract. Our data suggest that SJW does not lead to significant P-gp induction at the human BBB despite its ability to induce peripheral P-gp and CYPs. Simultaneous intake of SJW with CNS-targeted P-gp substrate drugs is not expected to lead to P-gp-mediated drug interactions at the BBB.

Pilot Pharmacokinetic Study in Healthy Adults Using Intravascular Microdialysis Catheters Modified for Use in Paediatric Patients to Assess Vancomycin Blood Levels.

BACKGROUND AND OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Meropenem Population Pharmacokinetics and Simulations in Plasma, Cerebrospinal Fluid, and Brain Tissue.

Meropenem is a broad spectrum carbapenem used for the treatment of cerebral infections. There is a need for data describing meropenem pharmacokinetics (PK) in the brain tissue to optimize therapy in these infections. Here, we present a meropenem PK model in the central nervous system and simulate dosing regimens. This was a population PK analysis of a previously published prospective study of patients admitted to the neurointesive care unit between 2016 and 2019 who received 2 g of meropenem intravenously every 8 h. Meropenem concentration was determined in blood, cerebrospinal fluid (CSF), and brain microdialysate. Meropenem was described by a six-compartment model: two compartments in the blood, two in the CSF, and two in the brain tissue. Creatinine clearance and brain glucose were included as covariates. The median elimination rate constant was 1.26 h-1, the central plasma volume was 5.38 L, and the transfer rate constants from the blood to the CSF and from the blood to the brain were 0.001 h-1 and 0.02 h-1, respectively. In the first 24 h, meropenem 2 g, administered every 8 h via intermittent and extended infusions achieved good target attainment in the CSF and brain, but continuous infusion (CI) was better at steady-state. Administering a 3 g loading dose (LD) followed by 8 g CI was beneficial for early target attainment. In conclusion, a meropenem PK model was developed using blood, CSF, and brain microdialysate samples. An 8 g CI may be needed for good target attainment in the CSF and brain. Giving a LD prior to the CI improved the probability of early target attainment.

Pharmacokinetics of metronomic temozolomide in cerebrospinal fluid of children with malignant central nervous system tumors.

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.

In the Model Cell Lines of Moderately and Poorly Differentiated Endometrial Carcinoma, Estrogens Can Be Formed via the Sulfatase Pathway.

Endometrial cancer (EC) is the most common gynecological malignancy in resource-abundant countries. The majority of EC cases are estrogen dependent but the mechanisms of estrogen biosynthesis and oxidative metabolism and estrogen action are not completely understood. Here, we evaluated formation of estrogens in models of moderately and poorly differentiated EC: RL95-2 and KLE cells, respectively. Results revealed high expression of estrone-sulfate (E1-S) transporters (SLCO1A2, SLCO1B3, SLCO1C1, SLCO3A1, SLC10A6, SLC22A9), and increased E1-S uptake in KLE vs RL95-2 cells. In RL95-2 cells, higher levels of sulfatase and better metabolism of E1-S to E1 were confirmed compared to KLE cells. In KLE cells, disturbed balance in expression of HSD17B genes led to enhanced activation of E1 to E2, compared to RL95-2 cells. Additionally, increased CYP1B1 expression and down-regulation of genes encoding phase II metabolic enzymes: COMT, NQO1, NQO2, and GSTP1 suggested decreased detoxification of carcinogenic metabolites in KLE cells. Results indicate that in model cell lines of moderately and poorly differentiated EC, estrogens can be formed via the sulfatase pathway.

Meropenem concentrations in brain tissue of neurointensive care patients exceed CSF levels.

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 < 0.001), but only moderately with plasma concentrations (R = 0.51, P < 0.001). Bactericidal thresholds were achieved in both plasma and brain tissue for MIC values ≤16 mg/L. In CSF, bactericidal effects were only reached for MIC values ≤1 mg/L. 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.

Altered Profile of E1-S Transporters in Endometrial Cancer: Lower Protein Levels of ABCG2 and OSTß and Up-Regulation of SLCO1B3 Expression.

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)αß. 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 (OSTß) 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.

Actaea racemosa L. extract inhibits steroid sulfation in human breast cancer cells: Effects on androgen formation.

BACKGROUND: Actaea racemosa L., also known as black cohosh, is a popular herb commonly used for the treatment of menopausal symptoms. Because of its purported estrogenic activity, black cohosh root extract (BCE) may trigger breast cancer growth. STUDY DESIGN/METHODS: The potential effects of standardized BCE and its main constituent actein on cellular growth rates and steroid hormone metabolism were investigated in estrogen receptor alpha positive (ERα+) MCF-7 and -negative (ERα-) MDA-MB-231 human breast cancer cells. Cell numbers were determined following incubation of both cell lines with the steroid hormone precursors dehydroepiandrosterone (DHEA) and estrone (E1) for 48 h, in the presence and absence of BCE or actein. Using a validated liquid chromatography-high resolution mass spectrometry assay, cell culture supernatants were simultaneously analyzed for the ten main steroids of the estrogen pathway. RESULTS: Inhibition of MCF-7 and MDA-MB-231 cell growth (up to 36.9%) was observed following treatment with BCE (1-25 µg/ml) or actein (1-50 µM). Incubation of MCF-7, but not of MDA-MB-231 cells, with DHEA and BCE caused a 20.9% reduction in DHEA-3-O-sulfate (DHEA-S) formation, leading to a concomitant increase in the androgens 4-androstene-3,17-dione (AD) and testosterone (T). Actein was shown to exert an even stronger inhibitory effect on DHEA-S formation in MCF-7 cells (up to 89.6%) and consequently resulted in 12- to 15-fold higher androgen levels compared with BCE. The formation of 17ß-estradiol (E2) and its glucuronidated and sulfated metabolites was not affected by BCE or actein after incubation with the estrogen precursor estrone (E1) in either cell line. CONCLUSIONS: The results of the present study demonstrated that actein and BCE do not promote breast cancer cell growth or influence estrogen levels. However, androgen formation was strongly stimulated by BCE and actein, which may contribute to their ameliorating effects on menopausal symptoms in women. Future studies monitoring the levels of AD and T upon BCE supplementation of patients are warranted to verify an association between BCE and endogenous androgen metabolism.

Metabolite profiling of natural substances in human: in vitro study from fecal bacteria to colon carcinoma cells (Caco-2).

Flavonoids, including anthocyanins, are polyphenolic compounds present in fruits, vegetables and dietary supplements. They can be absorbed from the intestine to the bloodstream or pass into the large intestine. Various bacterial species and enzymes are present along the entire intestine. The aim of the present work was to investigate the intestinal metabolism of selected dietary polyphenol and polyphenol glycosides (quercetin, cyanidin-3-O-glucoside, cyanidin-3-O-galactoside, and delphinidin-3-O-galactoside) by human fecal bacteria. Moreover, the metabolism of metabolites formed from these compounds in human colon carcinoma cells (Caco-2) was also point of the interest. Test compounds were added to fresh human stool in broth or to Caco-2 cells in medium and then incubated for 6 or 20 h at 37°C. After incubation, samples were prepared for LC/MS determination. Main metabolic pathways were deglycosylation, hydrogenation, methylation, hydroxylation, and decomposition. 2,4,5-trihydroxybenzaldehyde, as a metabolite of cyanidin glycosides, was detected after incubation for the first time. Metabolites formed by fecal bacteria were further glucuronidated or methylated by intestinal enzymes. This metabolite profiling of natural compounds has helped to better understand the complex metabolism in the human intestine and this work also has shown the connection of metabolism of natural substances by intestinal bacteria followed by metabolism in intestinal cells.

Metabolism of Estrogens: Turnover Differs Between Platinum-Sensitive and -Resistant High-Grade Serous Ovarian Cancer Cells.

High-grade serous ovarian cancer (HGSOC) is currently treated with cytoreductive surgery and platinum-based chemotherapy. The majority of patients show a primary response; however, many rapidly develop drug resistance. Antiestrogens have been studied as low toxic treatment options for HGSOC, with higher response rates in platinum-sensitive cases. Mechanisms for this difference in response remain unknown. Therefore, the present study investigated the impact of platinum resistance on steroid metabolism in six established HGSOC cell lines sensitive and resistant against carboplatin using a high-resolution mass spectrometry assay to simultaneously quantify the ten main steroids of the estrogenic metabolic pathway. An up to 60-fold higher formation of steroid hormones and their sulfated or glucuronidated metabolites was observed in carboplatin-sensitive cells, which was reversible by treatment with interleukin-6 (IL-6). Conversely, treatment of carboplatin-resistant cells expressing high levels of endogenous IL-6 with the monoclonal anti-IL-6R antibody tocilizumab changed their status to "platinum-sensitive", exhibiting a decreased IC50 value for carboplatin, decreased growth, and significantly higher estrogen metabolism. Analysis of these metabolic differences could help to detect platinum resistance in HGSOC patients earlier, thereby allowing more efficient interventions.

Impact of thrombocytes, on bacterial growth and antimicrobial activity of selected antibiotics.

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.

Resveratrol and other dietary polyphenols are inhibitors of estrogen metabolism in human breast cancer cells.

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'.

Inhibition of ABCB1 and ABCG2 at the Mouse Blood-Brain Barrier with Marketed Drugs To Improve Brain Delivery of the Model ABCB1/ABCG2 Substrate [11C]erlotinib.

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.

Impact of erythrocytes on bacterial growth and antimicrobial activity of selected antibiotics.

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.

A Proof-of-Concept Study to Inhibit ABCG2- and ABCB1-Mediated Efflux Transport at the Human Blood-Brain Barrier.

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.

Effect of Rifampicin on the Distribution of [11C]Erlotinib to the Liver, a Translational PET Study in Humans and in Mice.

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-dose rifampicin (100 mg/kg). Rifampicin caused a moderate reduction in the liver distribution of [11C]erlotinib in humans, while a more pronounced effect of rifampicin was observed in mice, in which rifampicin plasma concentrations were higher than in humans. In vitro uptake experiments in an OATP2B1-overexpressing cell line indicated that rifampicin inhibited OATP2B1 transport of [11C]erlotinib in a concentration-dependent manner with a half-maximum inhibitory concentration of 72.0 ± 1.4 µM. Our results suggest that rifampicin-inhibitable uptake transporter(s) contributed to the liver distribution of [11C]erlotinib in humans and mice and that [11C]erlotinib PET in combination with rifampicin may be used to measure the activity of this/these uptake transporter(s) in vivo. Furthermore, our data suggest that a standard clinical dose of rifampicin may exert in vivo a moderate inhibitory effect on hepatic OATP2B1.

Resveratrol Inhibits Key Steps of Steroid Metabolism in a Human Estrogen-Receptor Positive Breast Cancer Model: Impact on Cellular Proliferation.

The role of resveratrol (RES) in preventing breast cancer is controversial, as low concentrations may stimulate the proliferation of estrogen-receptor alpha positive (ERα+) breast cancer cells. As metabolism is the key factor in altering cellular estrogens, thereby influencing breast tumor growth, we investigated the effects of RES on the formation of estrogen metabolites, namely 4-androstene-3,17-dione (AD), dehydroepiandrosterone (DHEA), dehydroepiandrosterone-3-O-sulfate (DHEA-S), estrone (E1), estrone-3-sulfate (E1-S), 17ß-estradiol (E2), 17ß-estradiol-3-O-(ß-D-glucuronide) (E2-G), 17ß-estradiol-3-O-sulfate (E2-S), 16α-hydroxy-17ß-estradiol (estriol, E3), and testosterone (T) in ERα- MDA-MB-231 and ERα+ MCF-7 cells. Incubation of both of the cell lines with the hormone precursors DHEA and E1 revealed that sulfation and glucuronidation were preferred metabolic pathways for DHEA, E1 and E2 in MCF-7 cells, compared with in MDA-MB-231 cells, as the Vmax values were significantly higher (DHEA-S: 2873.0 ± 327.4 fmol/106 cells/h, E1-S: 30.4 ± 2.5 fmol/106 cells/h, E2-S: 24.7 ± 4.9 fmol/106 cells/h, E2-G: 7.29 ± 1.36 fmol/106 cells/h). RES therefore significantly inhibited DHEA-S, E1-S, E2-S and E2-G formation in MCF-7, but not in MDA-MB-231 cells (Kis: E2-S, 0.73 ± 0.07 µM < E1-S, 0.94 ± 0.03 µM < E2-G, 7.92 ± 0.24 µM < DHEA-S, 13.2 ± 0.2 µM). Suppression of these metabolites subsequently revealed twofold higher levels of active E2, concomitant with an almost twofold increase in MCF-7 cell proliferation, which was the most pronounced upon the addition of 5 µM RES. As the content of RES in food is relatively low, an increased risk of breast cancer progression in women is likely to only be observed following the continuous consumption of high-dose RES supplements. Further long-term human studies simultaneously monitoring free estrogens and their conjugates are therefore highly warranted to evaluate the efficacy and safety of RES supplementation, particularly in patients diagnosed with ERα+ breast cancer.

Influence of OATPs on Hepatic Disposition of Erlotinib Measured With Positron Emission Tomography.

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.

The Impacts of Genistein and Daidzein on Estrogen Conjugations in Human Breast Cancer Cells: A Targeted Metabolomics Approach.

The beneficial effect of dietary soy food intake, especially for women diagnosed with breast cancer, is controversial, as in vitro data has shown that the soy isoflavones genistein and daidzein may even stimulate the proliferation of estrogen-receptor alpha positive (ERα+) breast cancer cells at low concentrations. As genistein and daidzein are known to inhibit key enzymes in the steroid metabolism pathway, and thus may influence levels of active estrogens, we investigated the impacts of genistein and daidzein on the formation of estrogen metabolites, namely 17ß-estradiol (E2), 17ß-estradiol-3-(ß-D-glucuronide) (E2-G), 17ß-estradiol-3-sulfate (E2-S) and estrone-3-sulfate (E1-S) in estrogen-dependent ERα+ MCF-7 cells. We found that both isoflavones were potent inhibitors of E1 and E2 sulfation (85-95% inhibition at 10 µM), but impeded E2 glucuronidation to a lesser extent (55-60% inhibition at 10 µM). The stronger inhibition of E1 and E2 sulfation compared with E2 glucuronidation was more evident for genistein, as indicated by significantly lower inhibition constants for genistein [Kis: E2-S (0.32 µM) < E1-S (0.76 µM) < E2-G (6.01 µM)] when compared with those for daidzein [Kis: E2-S (0.48 µM) < E1-S (1.64 µM) < E2-G (7.31 µM)]. Concomitant with the suppression of E1 and E2 conjugation, we observed a minor but statistically significant increase in E2 concentration of approximately 20%. As the content of genistein and daidzein in soy food is relatively low, an increased risk of breast cancer development and progression in women may only be observed following consumption of high-dose isoflavone supplements. Further long-term human studies monitoring free estrogens and their conjugates are therefore highly warranted to evaluate the potential side effects of high-dose genistein and daidzein, especially in patients diagnosed with ERα+ breast cancer.

Human Bile Reduces Antimicrobial Activity of Selected Antibiotics against Enterococcus faecalis and Escherichia coli In Vitro.

It has been known from previous studies that body fluids, such as cerebrospinal fluid, lung surfactant, and urine, have a strong impact on the bacterial killing of many anti-infective agents. However, the influence of human bile on the antimicrobial activity of antibiotics is widely unknown. Human bile was obtained and pooled from 11 patients undergoing cholecystectomy. After sterilization of the bile fluid by gamma irradiation, its effect on bacterial killing was investigated for linezolid (LZD) and tigecycline (TGC) against Enterococcus faecalis ATCC 29212. Further, ciprofloxacin (CIP), meropenem (MEM), and TGC were tested against Escherichia coli ATCC 25922. Time-kill curves were performed in pooled human bile and Mueller-Hinton broth (MHB) over 24 h. Bacterial counts (in CFU per milliliter after 24 h) of bile growth controls were approximately equal to MHB growth controls for E. coli and approximately 2-fold greater for E. faecalis, indicating a promotion of bacterial growth by bile for the latter strain. Bile reduced the antimicrobial activity of CIP, MEM, and TGC against E. coli as well as the activity of LZD and TGC against E. faecalis This effect was strongest for TGC against the two strains. Degradation of TGC in bile was identified as the most likely explanation. These findings may have important implications for the treatment of bacterial infections of the gallbladder and biliary tract and should be explored in more detail.