Liesegang pattern formations are widely spread in nature. In spite of a comparably simple experimental setup under laboratory conditions, a variety of spatio-temporal structures may arise. Presumably because of easier control of the experimental conditions, Liesegang pattern formation was mainly studied in gel systems during more than a century. Here we consider pattern formation in a gas phase, where beautiful but highly complex reaction-diffusion-convection dynamics are uncovered by means of a specific laser technique. A quantitative analysis reveals that two different, apparently independent processes, both highly correlated and synchronized across the extension of the reaction cloud, act on different time scales. Each of them imprints a different structure of salt precipitation at the tube walls.
The obligate intracellular bacterium Ehrlichia ruminantium (ER) causes heartwater, a fatal tick-borne disease in livestock. In the field, ER strains present different levels of virulence, limiting vaccine efficacy, for which the molecular basis remains unknown. Moreover, there are no genetic tools currently available for ER manipulation, thus limiting the knowledge of the genes/proteins that are essential for ER pathogenesis and biology. As such, to identify proteins and/or mechanisms involved in ER virulence, we performed the first exhaustive comparative proteomic analysis between a virulent strain (ERGvir) and its high-passaged attenuated strain (ERGatt). Despite their different behaviors in vivo and in vitro, our results from 1DE-nanoLC-MS/MS showed that ERGvir and ERGatt share 80% of their proteins; this core proteome includes chaperones, proteins involved in metabolism, protein-DNA-RNA biosynthesis and processing, and bacterial effectors. Conventional 2DE revealed that 85% of the identified proteins are proteoforms, suggesting that post-translational modifications (namely glycosylation) are important in ER biology. Strain-specific proteins were also identified: while ERGatt has an increased number and overexpression of proteins involved in cell division, metabolism, transport and protein processing, ERGvir shows an overexpression of proteins and proteoforms (DIGE experiments) involved in pathogenesis such as Lpd, AnkA, VirB9 and B10, providing molecular evidence for its increased virulence in vivo and in vitro. Overall, our work reveals that ERGvir and ERGatt proteomes are streamlined to fulfill their biological function (maximum virulence for ERGvir and replicative capacity for ERGatt), and we provide both pioneering data and novel insights into the pathogenesis of this obligate intracellular bacterium.
The absolute levels of 5-hydroxymethylcytosine (hmC) and 5-methylcytosine (mC) in human brain tissues at various ages were determined. Additionally, absolute levels of 5-formylcytosine (fC) in adult individuals and cytosine modification levels in sorted neurons were quantified. These data were compared with age-related fC, hmC, and mC levels in mouse brain samples. For hmC, an initial steady increase is observed, which levels off with age to a final steady-state value of 1.2 % in human brain tissue. This level is nearly twice as high as in mouse cerebral cortex. In contrast, fC declines rapidly with age during early developmental stages, thus suggesting that while hmC is a stable epigenetic mark, fC is more likely an intermediate of active DNA demethylation during early brain development. The trends in global cytosine modification dynamics during the lifespan of an organism are conserved between humans and mice and show similar patterns in different organs.
5-formylcytosine; 5-hydroxymethylcytosine; aging; epigenetics; cerebral cortex
Epilepsy surgery is a potentially curative treatment option for pharmacoresistent patients. If non-invasive methods alone do not allow to delineate the epileptogenic brain areas the surgical candidates undergo long-term monitoring with intracranial EEG. Visual EEG analysis is then used to identify the seizure onset zone for targeted resection as a standard procedure.
Despite of its great potential to assess the epileptogenicty of brain tissue, quantitative EEG analysis has not yet found its way into routine clinical practice. To demonstrate that quantitative EEG may yield clinically highly relevant information we retrospectively investigated how post-operative seizure control is associated with four selected EEG measures evaluated in the resected brain tissue and the seizure onset zone. Importantly, the exact spatial location of the intracranial electrodes was determined by coregistration of pre-operative MRI and post-implantation CT and coregistration with post-resection MRI was used to delineate the extent of tissue resection. Using data-driven thresholding, quantitative EEG results were separated into normally contributing and salient channels.
In patients with favorable post-surgical seizure control a significantly larger fraction of salient channels in three of the four quantitative EEG measures was resected than in patients with unfavorable outcome in terms of seizure control (median over the whole peri-ictal recordings). The same statistics revealed no association with post-operative seizure control when EEG channels contributing to the seizure onset zone were studied.
We conclude that quantitative EEG measures provide clinically relevant and objective markers of target tissue, which may be used to optimize epilepsy surgery. The finding that differentiation between favorable and unfavorable outcome was better for the fraction of salient values in the resected brain tissue than in the seizure onset zone is consistent with growing evidence that spatially extended networks might be more relevant for seizure generation, evolution and termination than a single highly localized brain region (i.e. a “focus”) where seizures start.
adenosine triphosphate-binding cassette transporter P-glycoprotein
(ABCB1/Abcb1a) restricts at the blood–brain barrier (BBB) brain
distribution of many drugs. ABCB1 may be involved in drug–drug
interactions (DDIs) at the BBB, which may lead to changes in brain
distribution and central nervous system side effects of drugs. Positron
emission tomography (PET) with the ABCB1 substrates (R)-[11C]verapamil and [11C]-N-desmethyl-loperamide and the ABCB1 inhibitor tariquidar has allowed
direct comparison of ABCB1-mediated DDIs at the rodent and human BBB.
In this work we evaluated different factors which could influence
the magnitude of the interaction between tariquidar and (R)-[11C]verapamil or [11C]-N-desmethyl-loperamide at the BBB and thereby contribute to previously
observed species differences between rodents and humans. We performed in vitro transport experiments with [3H]verapamil
and [3H]-N-desmethyl-loperamide in ABCB1
and Abcb1a overexpressing cell lines. Moreover we conducted in vivo PET experiments and biodistribution studies with
(R)-[11C]verapamil and [11C]-N-desmethyl-loperamide in wild-type mice without and with
tariquidar pretreatment and in homozygous Abcb1a/1b(−/−) and heterozygous Abcb1a/1b(+/−) mice. We found no differences for in vitro transport of [3H]verapamil and [3H]-N-desmethyl-loperamide by ABCB1 and Abcb1a and its inhibition
by tariquidar. [3H]-N-Desmethyl-loperamide
was transported with a 5 to 9 times higher transport ratio than [3H]verapamil in ABCB1- and Abcb1a-transfected cells. In vivo, brain radioactivity concentrations were lower for
[11C]-N-desmethyl-loperamide than for
(R)-[11C]verapamil. Both radiotracers
showed tariquidar dose dependent increases in brain distribution with
tariquidar half-maximum inhibitory concentrations (IC50) of 1052 nM (95% confidence interval CI: 930–1189) for (R)-[11C]verapamil and 1329 nM (95% CI: 980–1801)
for [11C]-N-desmethyl-loperamide. In homozygous Abcb1a/1b(−/−) mice brain radioactivity
distribution was increased by 3.9- and 2.8-fold and in heterozygous Abcb1a/1b(+/−) mice by 1.5- and 1.1-fold,
for (R)-[11C]verapamil and [11C]-N-desmethyl-loperamide, respectively, as compared
with wild-type mice. For both radiotracers radiolabeled metabolites
were detected in plasma and brain. When brain and plasma radioactivity
concentrations were corrected for radiolabeled metabolites, brain
distribution of (R)-[11C]verapamil and
[11C]-N-desmethyl-loperamide was increased
in tariquidar (15 mg/kg) treated animals by 14.1- and 18.3-fold, respectively,
as compared with vehicle group. Isoflurane anesthesia altered [11C]-N-desmethyl-loperamide but not (R)-[11C]verapamil metabolism, and this had a
direct effect on the magnitude of the increase in brain distribution
following ABCB1 inhibition. Our data furthermore suggest that in the
absence of ABCB1 function brain distribution of [11C]-N-desmethyl-loperamide but not (R)-[11C]verapamil may depend on cerebral blood flow. In conclusion,
we have identified a number of important factors, i.e., substrate
affinity to ABCB1, brain uptake of radiolabeled metabolites, anesthesia,
and cerebral blood flow, which can directly influence the magnitude
of ABCB1-mediated DDIs at the BBB and should therefore be taken into
consideration when interpreting PET results.
barrier; drug−drug interaction; positron
emission tomography; (R)-[11C]verapamil; [11C]-N-desmethyl-loperamide; tariquidar
Capacitance probes have the potential to revolutionize bioprocess control due to their safe and robust use and ability to detect even the smallest capacitors in the form of biological cells. Several techniques have evolved to model biomass statistically, however, there are problems with model transfer between cell lines and process conditions. Errors of transferred models in the declining phase of the culture range for linear models around +100% or worse, causing unnecessary delays with test runs during bioprocess development. The goal of this work was to develop one single universal model which can be adapted by considering a potentially mechanistic factor to estimate biomass in yet untested clones and scales. The novelty of this work is a methodology to select sensitive frequencies to build a statistical model which can be shared among fermentations with an error between 9% and 38% (mean error around 20%) for the whole process, including the declining phase. A simple linear factor was found to be responsible for the transferability of biomass models between cell lines, indicating a link to their phenotype or physiology.
CHO cell culture; capacitance; fed batch; PLS; statistical model
As P-glycoprotein (Pgp) inhibition at the blood–brain barrier (BBB) after administration of a single dose of tariquidar is transient, we performed positron emission tomography (PET) scans with the Pgp substrate (R)-[11C]verapamil in five healthy volunteers during continuous intravenous tariquidar infusion. Total distribution volume (VT) of (R)-[11C]verapamil in whole-brain gray matter increased by 273±78% relative to baseline scans without tariquidar, which was higher than previously reported VT increases. During tariquidar infusion whole-brain VT was comparable to VT in the pituitary gland, a region not protected by the BBB, which suggested that we were approaching complete Pgp inhibition at the human BBB.
blood–brain barrier; P-glycoprotein; pituitary gland; positron emission tomography; (R)-[11C]verapamil; tariquidar
A multicomponent, evidence-based and interdisciplinary Patient Blood Management (PBM) program was introduced at the University Hospital Frankfurt in July 2013. The implementation strategy included practical and tactical components aimed to increase knowledge on the risks of preoperative anemia, to standardize hemotherapy, and to facilitate PBM components.
This article analyzes barriers to PBM implementation and outlines a strategy to introduce and manifest PBM. The effects in Frankfurt were measured in a before and after questionnaire study distributed among groups of physicians immediately before and 1 year after PBM implementation.
142 clinicians completed the questionnaire in July 2013 and 101 clinicians in August 2014. Absolute certainty that the treatment of preoperative anemia favorably influences morbidity and mortality rose from 25 to 37%. Transfusion behavior seems to have been affected: In 2014, 56% of clinicians stated that they clinically reassess the patient and analyze hemoglobin following each single red blood cell unit compared to only 38% stating this in 2013.
These results show that our implementation strategy was effective in changing physicians' risk perception, attitude, and knowledge on PBM principles. Our experience highlights key success factors for the implementation of a comprehensive PBM program.
Patient blood management; Anemia; Blood transfusion; Surgery – preoperative period; Medical decision-making; Implementation strategy
In the field of anesthesia for bariatric surgery, a wide variety of recommendations exist, but a general consensus on the perioperative management of such patients is missing. We outline the perioperative experiences that we gained in the first two years after introducing a bariatric program.
The perioperative approach was established together with all relevant disciplines. Pertinent topics for the anesthesiologists were; successful airway management, indications for more invasive monitoring, and the planning of the postoperative period and deposition. This retrospective analysis was approved by the local ethics committee. Data are mean [SD].
182 bariatric surgical procedures were performed (147 gastric bypass procedures (GBP; 146 (99.3%) performed laparascopically). GBP patients were 43  years old, 78% female, BMI 45  kg/m2, 73% ASA physical status of 2. 42 patients (28.6%) presented with obstructive sleep apnea syndrome. 117 GBP (79.6%) patients were intubated conventionally by direct laryngoscopy (one converted to fiber-optic intubation, one aspiration of gastric contents). 32 patients (21.8%) required an arterial line, 10 patients (6.8%) a central venous line. Induction lasted 25  min, the procedure itself 138  min. No blood products were required. Two patients (1.4%) presented with hypothermia (<35°C) at the end of their case. The emergence period lasted 17  min. Postoperatively, 32 patients (21.8%) were transferred to the ICU (one ventilated). The other patients spent 4.1 [0.7] h in the post anesthesia care unit. 15 patients (10.2%) required take backs for surgical revision (two laparotomies).
The physiology and anatomy of bariatric patients demand a tailored approach from both the anesthesiologist and the perioperative team. The interaction of a multi-disciplinary team is key to achieving good outcomes and a low rate of complications.
DRKS00005437 (date of registration 16th December 2013)
Anesthesia; Complications; Bariatric surgery; Obesity
Preoperative and hospital-acquired anaemia is common among surgical patients. It is associated with an increased risk of morbidity and mortality and a strong risk factor for allogeneic blood transfusions with their own inherent risks. Patient Blood Management (PBM) concepts aim to increase and preserve autologous erythrocyte volume and to optimise haemotherapy. They thus have great potential to benefit patients.
This prospective, multi-centre clinical trial tests the hypothesis that PBM programs are safe and effective in the care of adult surgical patients. Primary outcome is a composite endpoint of adverse events and in-hospital mortality.
This trial will determine whether the implementation of a PBM program is safe and effective in terms of clinical outcome compared to a pre-implementation cohort. This trial is registered at www.clinicaltrials.gov (NCT01820949).
Patient Blood Management; Red Blood Cell Transfusion Practice, Patient Safety; Anaemia; Clinical Outcome; Perioperative Care
To study the functional activity of the multidrug efflux transporter P-glycoprotein (Pgp) at the blood-brain barrier of patients with temporal lobe epilepsy using (R)-[11C]verapamil (VPM)-PET before and after temporal lobe surgery to assess whether postoperative changes in seizure frequency and antiepileptic drug load are associated with changes in Pgp function.
Seven patients with drug-resistant temporal lobe epilepsy underwent VPM-PET scans pre- and postsurgery. Patients were followed up for a median of 6 years (range 4–7) after surgery. Pgp immunoreactivity in surgically resected hippocampal specimens was determined with immunohistochemistry.
Optimal surgical outcome, defined as seizure freedom and withdrawal of antiepileptic drugs, was associated with higher temporal lobe Pgp function before surgery, higher Pgp-positive staining in surgically resected hippocampal specimens, and reduction in global Pgp function postoperatively, compared with nonoptimal surgery outcome.
The data from our pilot study suggest that Pgp overactivity in epilepsy is dynamic, and complete seizure control and elimination of antiepileptic medication is associated with reversal of overactivity, although these findings will require confirmation in a larger patient cohort.
The adenosine triphosphate-binding cassette transporters P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) are 2 major gatekeepers at the blood-brain barrier (BBB) which restrict brain distribution of several clinically used drugs. In this study we investigated the suitability of the radiolabeled Pgp/BCRP inhibitors 11C-tariquidar and 11C-elacridar to assess Pgp density in human brain with PET.
Healthy subjects underwent a first PET scan of 120 min duration with either 11C-tariquidar (n = 6) or 11C-elacridar (n = 5) followed by a second PET scan of 60 min duration with (R)-11C-verapamil. During scan 1 (at 60 min after radiotracer injection) unlabeled tariquidar (3 mg/kg) was intravenously administered. Data was analyzed using 1-tissue 2-rate-constant (1T2K) and 2-tissue 4-rate-constant (2T4K) compartment models using either metabolite-corrected or uncorrected arterial input functions.
Following injection of 11C-tariquidar or 11C-elacridar, brain PET signal corrected for radioactivity in vasculature was very low (~0.1 standardized uptake value) with slow washout. In response to tariquidar injection, a moderate, but statistically significant rise in brain PET signal was observed for 11C-tariquidar (+27 ± 15%, P = 0.014, paired t-test) and 11C-elacridar (+21 ± 15%, P = 0.014) without changes in plasma activity concentrations. Low levels of radiolabeled metabolites (<25%) were detected in plasma at time points up to 60 min after injection of 11C-tariquidar or 11C-elacridar. The 2T4K model provided better data fits than the 1T2K model. Model outcome parameters were similar when metabolite-corrected or uncorrected input functions were used. There was no significant correlation between distribution volumes (VT) of 11C-tariquidar or 11C-elacridar and VTs of (R)-11C-verapamil in different brain regions.
The in vivo behavior of 11C-tariquidar and 11C-elacridar was consistent with that of dual Pgp/BCRP substrates. Both tracers were unable to visualize cerebral Pgp density, which was most likely related to insufficiently high binding affinities in relation to the very low density of Pgp in human brain (~1.3 nM). Despite their inability to visualize Pgp density, 11C-tariquidar and 11C-elacridar may find use as a new class of radiotracers to study the interplay of Pgp and BCRP at the human BBB in limiting brain uptake of dual substrates.
P-glycoprotein; breast cancer resistance protein; blood-brain barrier; 11C-tariquidar; 11C-elacridar
Data processing, management and visualization are central and critical components of a state of the art high-throughput mass spectrometry (MS)-based proteomics experiment, and are often some of the most time-consuming steps, especially for labs without much bioinformatics support. The growing interest in the field of proteomics has triggered an increase in the development of new software libraries, including freely available and open-source software. From database search analysis to post-processing of the identification results, even though the objectives of these libraries and packages can vary significantly, they usually share a number of features. Common use cases include the handling of protein and peptide sequences, the parsing of results from various proteomics search engines output files, and the visualization of MS-related information (including mass spectra and chromatograms). In this review, we provide an overview of the existing software libraries, open-source frameworks and also, we give information on some of the freely available applications which make use of them. This article is part of a Special Issue entitled: Computational Proteomics in the Post-Identification Era. Guest Editors: Martin Eisenacher and Christian Stephan.
•A review of existing open-source software for computational proteomics.•Available software for each step in a typical MS experiment is described.•OpenMS, TPP, compomics, ProteoWizard, JPL, PRIDE toolsuite are covered in detail.•Different programming languages are considered (Java, Perl, C++ or Python).
AMT, Accurate Mass Tag; ATAQS, Automated and Targeted Analysis with Quantitative SRM; CV, Controlled Vocabulary; DAO, Data Access Object; EBI, European Bioinformatics Institute; emPAI, exponentially modified Protein Abundance Index; FDR, False Discovery Rate; (HUPO)-PSI, (Human Proteome Organization) — Proteomics Standards Initiative; GUI, Graphical User Interface; ICAT, Isotope-Coded Affinity Tags; ICPL, Isotope-Coded Protein Label; IPTL, Isobaric Peptide Termini Labeling; ISB, Institute for Systems Biology; iTRAQ, Isobaric Tag for Relative and Absolute Quantitation; JPL, Java Proteomic Library; LC-MS, Liquid Chromatography–Mass Spectrometry; LIMS, Laboratory Information Management System; MGF, Mascot Generic Format; MIAPE, Minimum Information About a Proteomics Experiment; MS, Mass Spectrometry; SILAC, Stable Isotope Labeling by Amino acids in Cell culture; PASSEL, PeptideAtlas SRM Experiment Library; PRIDE, PRoteomics IDEntifications (database); PSM, Peptide Spectrum Match; PTM, Post-Translational Modifications; RT, Retention Time; SRM, Selected Reaction Monitoring; TMT, Tandem Mass Tag; TOPP, The OpenMS Proteomics Pipeline; TPP, Trans-Proteomic Pipeline; Proteomics; Databases; Bioinformatics; Software libraries; Application programming interface; Open source software
Methanol (CH3OH) fluxes were quantified above a managed temperate mountain grassland in the Stubai Valley (Tyrol, Austria) during the growing seasons 2008 and 2009. Half-hourly methanol fluxes were calculated by means of the virtual disjunct eddy covariance (vDEC) method using 3-dimensional wind data from a sonic anemometer and methanol volume mixing ratios measured with a proton-transfer-reaction mass spectrometer (PTR-MS). During (undisturbed) mature and growing phases methanol fluxes exhibited a clear diurnal cycle with close-to-zero fluxes during nighttime and emissions, up to 10 nmol m−2 s−1, which followed the diurnal course of radiation and air temperature. Management events were found to represent the largest perturbations of methanol exchange at the studied grassland ecosystem: Peak emissions of 144.5 nmol m−2 s−1 were found during/after cutting of the meadow reflecting the wounding of the plant material and subsequent depletion of the leaf internal aqueous methanol pools. After the application of organic fertilizer, elevated methanol emissions of up to 26.7 nmol m−2 s−1 were observed, likely reflecting enhanced microbial activity associated with the applied manure. Simple and multiple linear regression analyses revealed air temperature and radiation as the dominant abiotic controls, jointly explaining 47 % and 70 % of the variability in half-hourly and daily methanol fluxes. In contrast to published leaf-level laboratory studies, the surface conductance and the daily change in the amount of green plant area, used as ecosystem-scale proxies for stomatal conductance and growth, respectively, were found to exert only minor biotic controls on methanol exchange.
disjunct eddy covariance; flux; methanol; volatile organic compounds; management; grassland; PTR-MS
Increased lipid availability reduces insulin-stimulated glucose disposal in skeletal muscle, which is generally explained by fatty acid–mediated inhibition of insulin signaling. It remains unclear whether lipids also impair transcapillary transport of insulin and glucose, which could become rate controlling for glucose disposal. We hypothesized that lipid-induced insulin resistance is induced by inhibiting myocellular glucose uptake and not by interfering with the delivery of insulin or glucose. We measured changes in interstitial glucose and insulin in skeletal muscle of healthy volunteers during intravenous administration of triglycerides plus heparin or glycerol during physiologic and supraphysiologic hyperinsulinemia, by combining microdialysis with oral glucose tolerance tests and euglycemic-hyperinsulinemic clamps. Lipid infusion reduced insulin-stimulated glucose disposal by ∼70% (P < 0.05) during clamps and dynamic insulin sensitivity by ∼12% (P < 0.05) during oral glucose loading. Dialysate insulin and glucose levels were unchanged or even transiently higher (P < 0.05) during lipid than during glycerol infusion, whereas regional blood flow remained unchanged. These results demonstrate that short-term elevation of free fatty acids (FFAs) induces insulin resistance, which in skeletal muscle occurs primarily at the cellular level, without impairment of local perfusion or transcapillary transport of insulin and glucose. Thus, vascular effects of FFAs are not rate controlling for muscle insulin-stimulated glucose disposal.
Breast cancer resistance protein (BCRP) is the most abundant multidrug efflux transporter at the human blood–brain barrier (BBB), restricting brain distribution of various drugs. In this study, we developed a positron emission tomography (PET) protocol to visualize Bcrp function at the murine BBB, based on the dual P-glycoprotein (P-gp)/Bcrp substrate radiotracer [11C]tariquidar in combination with the Bcrp inhibitor Ko143. To eliminate the contribution of P-gp efflux to [11C]tariquidar brain distribution, we studied mice in which P-gp was genetically knocked out (Mdr1a/b(−/−) mice) or chemically knocked out by pretreatment with cold tariquidar. We found that [11C]tariquidar brain uptake increased dose dependently after administration of escalating doses of Ko143, both in Mdr1a/b(−/−) mice and in tariquidar pretreated wild-type mice. After 15 mg/kg Ko143, the maximum increase in [11C]tariquidar brain uptake relative to baseline scans was 6.3-fold in Mdr1a/b(−/−) mice with a half-maximum effect dose of 4.98 mg/kg and 3.6-fold in tariquidar (8 mg/kg) pretreated wild-type mice, suggesting that the presented protocol is sensitive to visualize a range of different functional Bcrp activities at the murine BBB. We expect that this protocol can be translated to the clinic, because tariquidar can be safely administered to humans at doses that completely inhibit cerebral P-gp.
blood–brain barrier; breast cancer resistance protein; Ko143; P-glycoprotein; positron emission tomography; [11C]tariquidar
We investigate the behaviour of the recently proposed Quantum PageRank algorithm, in large complex networks. We find that the algorithm is able to univocally reveal the underlying topology of the network and to identify and order the most relevant nodes. Furthermore, it is capable to clearly highlight the structure of secondary hubs and to resolve the degeneracy in importance of the low lying part of the list of rankings. The quantum algorithm displays an increased stability with respect to a variation of the damping parameter, present in the Google algorithm, and a more clearly pronounced power-law behaviour in the distribution of importance, as compared to the classical algorithm. We test the performance and confirm the listed features by applying it to real world examples from the WWW. Finally, we raise and partially address whether the increased sensitivity of the quantum algorithm persists under coordinated attacks in scale-free and random networks.
The plasma and tumor dispositions of a commonly used antimelanoma agent, carboplatin, were evaluated in patients with cutaneous melanoma and compared with four different murine melanoma models (one genetically engineered mouse model, one human cell line xenograft, and two orthotopic syngeneic transplant models). The tumor pharmacokinetics of carboplatin in the a genetically engineered mouse model most closely resembled the tumor disposition in patients with melanoma.
Rodent studies are a vital step in the development of novel anticancer therapeutics and are used in pharmacokinetic (PK), toxicology, and efficacy studies. Traditionally, anticancer drug development has relied on xenograft implantation of human cancer cell lines in immunocompromised mice for efficacy screening of a candidate compound. The usefulness of xenograft models for efficacy testing, however, has been questioned, whereas genetically engineered mouse models (GEMMs) and orthotopic syngeneic transplants (OSTs) may offer some advantages for efficacy assessment. A critical factor influencing the predictability of rodent tumor models is drug PKs, but a comprehensive comparison of plasma and tumor PK parameters among xenograft models, OSTs, GEMMs, and human patients has not been performed.
In this work, we evaluated the plasma and tumor dispositions of an antimelanoma agent, carboplatin, in patients with cutaneous melanoma compared with four different murine melanoma models (one GEMM, one human cell line xenograft, and two OSTs).
Using microdialysis to sample carboplatin tumor disposition, we found that OSTs and xenografts were poor predictors of drug exposure in human tumors, whereas the GEMM model exhibited PK parameters similar to those seen in human tumors.
The tumor PKs of carboplatin in a GEMM of melanoma more closely resembles the tumor disposition in patients with melanoma than transplanted tumor models. GEMMs show promise in becoming an improved prediction model for intratumoral PKs and response in patients with solid tumors.
GEMM; Microdialysis; Carboplatin pharmacokinetics; Mouse tumor models; Melanoma models; Genetically engineered mouse models
Positron emission tomography (PET) with [11C]verapamil, either in racemic form or in form of the (R)-enantiomer, has been used to measure the functional activity of the adenosine triphosphate-binding cassette (ABC) transporter P-glycoprotein (Pgp) at the blood–brain barrier (BBB). There is some evidence in literature that verapamil inhibits two other ABC transporters expressed at the BBB, i.e. multidrug resistance protein 1 (MRP1) and breast cancer resistance protein (BCRP). However, previous data were obtained with micromolar concentrations of verapamil and do not necessarily reflect the transporter selectivity of verapamil at nanomolar concentrations, which are relevant for PET experiments. The aim of this study was to assess the selectivity of verapamil, in nanomolar concentrations, for Pgp over MRP1 and BCRP.
Concentration equilibrium transport assays were performed with [3H]verapamil (5 nM) in cell lines expressing murine or human Pgp, human MRP1, and murine Bcrp1 or human BCRP. Paired PET scans were performed with (R)-[11C]verapamil in female FVB/N (wild-type), Mrp1(−/−), Mdr1a/b(−/−), Bcrp1(−/−) and Mdr1a/b(−/−)Bcrp1(−/−) mice, before and after Pgp inhibition with 15 mg/kg tariquidar.
In vitro transport experiments exclusively showed directed transport of [3H]verapamil in Mdr1a- and MDR1-overexpressing cells which could be inhibited by tariquidar (0.5 μM). In PET scans acquired before tariquidar administration, brain-to-blood ratio (Kb,brain) of (R)-[11C]verapamil was low in wild-type (1.3 ± 0.1), Mrp1(−/−) (1.4 ± 0.1) and Bcrp1(−/−) mice (1.8 ± 0.1) and high in Mdr1a/b(−/−) (6.9 ± 0.8) and Mdr1a/b(−/−)Bcrp1(−/−) mice (7.9 ± 0.5). In PET scans after tariquidar administration, Kb,brain was significantly increased in Pgp-expressing mice (wild-type: 5.0 ± 0.3-fold, Mrp1(−/−): 3.2 ± 0.6-fold, Bcrp1(−/−): 4.3 ± 0.1-fold) but not in Pgp knockout mice (Mdr1a/b(−/−) and Mdr1a/b(−/−)Bcrp1(−/−)).
Our combined in vitro and in vivo data demonstrate that verapamil, in nanomolar concentrations, is selectively transported by Pgp and not by MRP1 and BCRP at the BBB, which supports the use of (R)-[11C]verapamil or racemic [11C]verapamil as PET tracers of cerebral Pgp function.
Positron emission tomography; (R)-[11C]verapamil; Blood–brain barrier; P-glycoprotein; Multidrug resistance protein 1; Breast cancer resistance protein
Aim of this study was to determine whether the carbon-11-labelled antiepileptic drug [11C]mephobarbital is a substrate of P-glycoprotein (Pgp) and can be used to assess Pgp function at the blood-brain barrier (BBB) with positron emission tomography (PET). We performed paired PET scans in rats, wild-type (FVB) and Mdr1a/b(−/−) mice, before and after intravenous administration of the Pgp inhibitor tariquidar (15 mg/kg). Brain-to-blood AUC0-60 ratios in rats and brain AUC0-60 values of [11C]mephobarbital in wild-type and Mdr1a/b(−/−) mice were similar in scan 1 and scan 2, respectively, suggesting that in vivo brain distribution of [11C]mephobarbital is not influenced by Pgp efflux. Absence of Pgp transport was confirmed in vitro by performing concentration equilibrium transport assay in cell lines transfected with MDR1 or Mdr1a. PET experiments in wild-type mice, with and without pretreatment with the multidrug resistance protein (MRP) inhibitor MK571 (20 mg/kg), and in Mrp1(−/−) mice suggested that [11C]mephobarbital is also not transported by MRPs at the murine BBB, which was also supported by in vitro transport experiments using human MRP1-transfected cells. Our results are surprising as phenobarbital, the N-desmethyl derivative of mephobarbital, has been shown to be a substrate of Pgp, which suggests that N-methylation abolishes Pgp affinity of barbiturates.
drug resistance; epilepsy; blood-brain barrier; P-glycoprotein; PET; [11C]mephobarbital
Background and Objective
In microdose studies, the pharmacokinetic (PK) profile of a drug in blood after administration of a dose up to 100 μg is measured with sensitive analytical techniques, such as accelerator mass spectrometry (AMS). As most drugs exert their effect in tissue rather than blood, methodology is needed for extending PK analysis to different tissue compartments. In the present study, we combined, for the first time, AMS analysis with positron emission tomography (PET) in order to determine the PK profile of the model drug verapamil in plasma and brain of humans. In order to assess PK dose-linearity of verapamil, data were acquired and compared after administration of an intravenous (iv) microdose and an iv microdose dosed concomitantly with an oral therapeutic dose.
Six healthy male volunteers received an iv microdose (0.05 mg) (period 1) and an iv microdose dosed concomitantly with an oral therapeutic dose (80 mg) of verapamil (period 2) in a randomized, cross-over, two-period study design. The iv dose was a mixture of (R/S)-[14C]verapamil and (R)-[11C]verapamil and the oral dose was unlabelled racemic verapamil. Brain distribution of radioactivity was measured with PET whereas plasma PK of (R)- and (S)-verapamil was determined with AMS. PET data were analyzed by kinetic modeling to estimate the rate constants for transfer of radioactivity across the blood-brain barrier.
Most PK parameters of (R)- and (S)-verapamil as well as parameters describing exchange of radioactivity between plasma and brain (K1=0.030±0.003 and 0.031±0.005 mL·mL−1·min−1 and k2=0.099±0.006 and 0.095±0.008 min−1 for period 1 and 2, respectively) were not statistically different between the two periods although there was a trend for non-linear kinetics for the (R)-enantiomer. On the other hand, all PK parameters (except for t1/2) differed significantly between the (R)- and (S)-enantiomers for both periods. Cmax, AUC(0-24) and AUC(0-inf) were higher and CL, V and VSS were lower for the (R)- than for the (S)-enantiomer.
Combining AMS and PET microdosing allows long term PK data along with information on drug tissue distribution to be acquired in the same subjects thus making it a promising approach to maximize data output from a single clinical study.
Measurement of microvascular perfusion with Intravoxel Incoherent Motion (IVIM) MRI is gaining interest. Yet, the physiological influences on the IVIM perfusion parameters (“pseudo-diffusion” coefficient D*, perfusion fraction f, and flow related parameter fD*) remain insufficiently characterized. In this article, we hypothesize that D* and fD*, which depend on blood speed, should vary during the cardiac cycle. We extended the IVIM model to include time dependence of D* = D*(t), and demonstrate in the healthy human brain that both parameters D* and fD* are significantly larger during systole than diastole, while the diffusion coefficient D and f do not vary significantly. The results non-invasively demonstrate the pulsatility of the brain’s microvasculature.
About one third of epilepsy patients are pharmacoresistant. Overexpression of P-glycoprotein and other multidrug transporters at the blood-brain barrier is thought to play an important role in drug-refractory epilepsy. Thus, quantification of regionally different P-glycoprotein activity in the brain in vivo is essential to identify P-glycoprotein overactivity as the relevant mechanism for drug-resistance in an individual patient.
Using the radiolabeled P-glycoprotein substrate (R)-[11C]verapamil and different doses of co-administered tariquidar, which is an inhibitor of P-glycoprotein, we evaluated whether small-animal positron emission tomography (PET) can quantify regional changes in transporter function in the rat brain at baseline and 48 h after a pilocarpine-induced status epilepticus. P-glycoprotein expression was additionally quantified by immunohistochemistry. To reveal putative seizure-induced changes in blood-brain barrier integrity, we performed gadolinium-enhanced magnetic resonance scans on a 7.0 Tesla small-animal scanner. Before P-glycoprotein modulation, brain uptake of (R)-[11C]verapamil was low in all regions investigated in control and post-status epilepticus rats. After administration of 3 mg/kg tariquidar, which inhibits P-glycoprotein only partially, we observed increased regional differentiation in brain activity uptake in post-status epilepticus versus control rats, which diminished after maximal P-glycoprotein inhibition. Regional increases in the efflux rate constant k2, but not in distribution volume VT or influx rate constant K1, correlated significantly with increases in P-glycoprotein expression measured by immunohistochemistry.
This imaging protocol proves to be suitable to detect seizure-induced regional changes in P-glycoprotein activity and is readily applicable to humans, with the aim to detect relevant mechanisms of pharmacoresistance in epilepsy in vivo.
Adenosine triphosphate-binding cassette (ABC) transporters, such as P-glycoprotein (Pgp, ABCB1), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance-associated proteins (MRPs) are expressed in high concentrations at various physiological barriers (e.g. blood-brain barrier, blood-testis barrier, blood-tumor barrier), where they impede the tissue accumulation of various drugs by active efflux transport. Changes in ABC transporter expression and function are thought to be implicated in various diseases, such as cancer, epilepsy, Alzheimer’s and Parkinson’s disease. The availability of a non-invasive imaging method which allows for measuring ABC transporter function or expression in vivo would be of great clinical use in that it could facilitate the identification of those patients that would benefit from treatment with ABC transporter modulating drugs. To date three different kinds of imaging probes have been described to measure ABC transporters in vivo: i) radiolabelled transporter substrates ii) radiolabelled transporter inhibitors and iii) radiolabelled prodrugs which are enzymatically converted into transporter substrates in the organ of interest (e.g. brain). The design of new imaging probes to visualize efflux transporters is inter alia complicated by the overlapping substrate recognition pattern of different ABC transporter types. The present article will describe currently available ABC transporter radiotracers for positron emission tomography (PET) and single-photon emission computed tomography (SPECT) and critically discuss strengths and limitations of individual probes and their potential clinical applications.
ABC transporter; blood-brain barrier; breast cancer resistance protein; multidrug resistance-associated protein; P-glycoprotein; positron emission tomography; single-photon emission computed tomography
Tariquidar, a potent, nontoxic, third-generation P-glycoprotein (P-gp) inhibitor, is a possible reversal agent for central nervous system drug resistance. In animal studies, tariquidar has been shown to increase delivery of P-gp substrates into brain by several-fold. The aim of this study was to measure P-gp function at the human blood-brain barrier (BBB) after tariquidar administration using PET and the model P-gp substrate (R)–11C-verapamil. Methods: 5 healthy volunteers underwent paired (R)–11C-verapamil PET scans and arterial blood sampling, before and at 2 h 50 min after i.v. administration of tariquidar (2 mg/kg body weight). Inhibition of P-gp on CD56+ peripheral lymphocytes of each volunteer was determined by means of the rhodamine-123 efflux assay. Tariquidar concentrations in venous plasma were quantified using liquid chromatography/mass spectrometry. Results: Tariquidar administration resulted in significant increases (Wilcoxon test for paired samples) in the distribution volume (DV, +24±15%) and influx rate constant (K1, +49±36%) of (R)–11C-verapamil across the BBB (DV=0.65±0.13 and 0.80±0.07, p=0.043, K1=0.034±0.009 and 0.049±0.009, p=0.043, before and after tariquidar, respectively). A strong correlation was observed between change in brain DV after administration of tariquidar and tariquidar exposure in plasma (r=0.90, p=0.037). The mean plasma concentration of tariquidar achieved during the second PET scan (490±166 ng/mL) corresponded to 100% inhibition of P-gp function in peripheral lymphocytes. Conclusion: Tariquidar significantly increased brain penetration of (R)–11C-verapamil-derived activity, due to increased influx. As opposed to peripheral P-gp function, central P-gp inhibition appeared to be far from complete after the administered tariquidar dose.
PET; (R)-11C-verapamil; tariquidar; P-glycoprotein; blood-brain barrier