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Evidence theory by Dempster-Shafer for determination of hormone receptor status in breast cancer samples was introduced in our previous paper. One major topic pointed out here is the link between pieces of evidence found from different origins. In this paper the challenge of selecting appropriate ways of fusing evidence, depending on the type and quality of data involved is addressed. A parameterized family of evidence combination rules, covering the full range of potential needs, from emphasizing discrepancies in the measurements to aspiring accordance, is covered. The consequences for real patient samples are shown by modeling different decision strategies.

Estrogen and progesterone receptors being present or not represents one of the most important biomarkers for therapy selection in breast cancer patients. Conventional measurement by immunohistochemistry (IHC) involves errors, and numerous attempts have been made to increase precision by additional information from gene expression. This raises the question of how to fuse information, in particular, if there is disagreement. It is the primary domain of Dempster–Shafer decision theory (DST) to deal with contradicting evidence on the same item (here: receptor status), obtained through different techniques. DST is widely used in technical settings, such as self-driving cars and aviation, and is also promising to deliver significant advantages in medicine. Using data from breast cancer patients already presented in previous work, we focus on comparing DST with classical statistics in this work, to pave the way for its application in medicine. First, we explain how DST not only considers probabilities (a single number per sample), but also incorporates uncertainty in a concept of ‘evidence’ (two numbers per sample). This allows for very powerful displays of patient data in so-called ternary plots, a novel and crucial advantage for medical interpretation. Results are obtained according to conventional statistics (ODDS) and, in parallel, according to DST. Agreement and differences are evaluated, and the particular merits of DST discussed. The presented application demonstrates how decision theory introduces new levels of confidence in diagnoses derived from medical data.

Aims Animal studies suggest that inhibition of dipeptidyl peptidase 4 (DPP‐IV) may improve heart function and survival after myocardial infarction by increasing cardiac myocytes’ regenerative capacity. Parenterally administered dutogliptin may provide continuous strong DPP‐IV inhibition to translate these results into humans. This trial investigated the safety and tolerability, as well as pharmacokinetics and pharmacodynamics, of parenterally administered dutogliptin after single and repeated doses. Methods In an open‐label trial, volunteers received dutogliptin at increasing doses of 30–120 mg subcutaneously or 30 mg intravenously in the single‐dose cohorts. Subjects in the multiple‐dose cohort received 60, 90 or 120 mg dutogliptin subcutaneously once daily on 7 consecutive days. Results Forty healthy males were included in the trial. No related serious adverse events occurred. Mild local injection site reactions with no requirement for intervention comprised 147 of 153 (96%) related adverse events. Subcutaneous bioavailability was approximately 100%. Multiple injections at daily intervals did not lead to the accumulation of the study drug. The accumulation ratios based on AUC0‐24h range from 0.90 to 1.03, supporting this argument. All subjects receiving ≥60 mg dutogliptin yielded a maximum DPP‐IV inhibition >90%. The duration of DPP‐IV inhibition over time increased in a dose‐dependent manner and was highest in the 120‐mg multiple‐dosing cohort with a maximum AUEC0‐24h of 342 h % (standard deviation: 73), translating into 86% DPP‐IV inhibition 24 hours after dosing. Conclusion Parenteral injection of dutogliptin was safe and subcutaneous bioavailability is excellent. DPP‐IV inhibition increased dose dependently to >86% over 24 hours after multiple doses of 120 mg dutogliptin.

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.

The interactions between peptide(p)-bound major histocompatibility complexes (MHCs) and T cell receptors (TCRs) play an important role during the immune response. Here, we report on a molecular dynamics (MD) simulation study of free and TCR-bound pMHC-I proteins of the LC13-HLA-B* 44:05-pEEYLQAFTY complex. We performed long-term MD simulations of 4 μs each for both free and bound pMHC-TCR systems, employing 10 independent 400 ns replicas for each state. We observed a reduced dynamic flexibility in the central residues of the peptide and the MHC α1-helix upon TCR ligation, indicating a restricted conformational space in the MHC binding groove. Moreover, our data suggest a minimum of 50 to 100 ns simulation time of individual runs and 5 to 10 independent replicas to permit reliable conclusions for biomolecules of comparable complexity and similar descriptors.

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...

Ciprofloxacin is a commonly prescribed fluoroquinolone antibiotic which is cleared by active tubular secretion and intestinal excretion. Ciprofloxacin is a known substrate of the ATP-binding cassette (ABC) transporters breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 4 (MRP4). In this work, we used positron emission tomography (PET) imaging to investigate the influence of BCRP, MRP4, MRP2 and P-glycoprotein (P-gp) on the excretion of [18F]ciprofloxacin in mice. Dynamic 90-min PET scans were performed after intravenous injection of [18F]ciprofloxacin in wild-type mice without and with pre-treatment with the broad-spectrum MRP inhibitor MK571. Moreover, [18F]ciprofloxacin PET scans were performed in Abcc4(-/-), Abcc2(-/-), Abcc4(-/-)Abcg2(-/-) and Abcb1a/b(-/-)Abcg2(-/-) mice. In addition to non-compartmental pharmacokinetic (PK) analysis, a novel three-compartment PK model was developed for a detailed assessment of the renal disposition of [18F]ciprofloxacin. In MK571 pre-treated mice, a significant increase in the blood exposure to [18F]ciprofloxacin was observed along with a significant reduction in the renal and intestinal clearances. PK modelling revealed a significant reduction in renal radioactivity uptake (CL1) and in the rate constants for transfer of radioactivity from the corticomedullary renal region into blood (k2) and urine (k3), respectively, after MK571 administration. No changes in the renal clearance or in the estimated kidney PK model parameters were observed in any of the studied knockout models, while a significant reduction in the intestinal clearance was observed in Abcc2(-/-) and Abcc4(-/-)Abcg2(-/-) mice. Our data failed to reveal a role of any of the studied ABC transporters in the tubular secretion of ciprofloxacin. This may indicate that ciprofloxacin is handled in the kidneys by more than one transporter family, most likely with a great degree of mutual functional redundancy. Our study highlights the potential of PET imaging for an assessment of transporter-mediated renal excretion of radiolabelled drugs.

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.

Positron emission tomography (PET) imaging with radiolabeled drugs holds great promise to assess the influence of membrane transporters on hepatobiliary clearance of drugs. To exploit the full potential of PET, quantitative pharmacokinetic models are required. In this study, we evaluated the suitability of different compartment models to describe the hepatic disposition of [11C]erlotinib as a small-molecule model drug which undergoes transporter-mediated hepatobiliary excretion. We analyzed two different, previously published data sets in healthy volunteers, in which a baseline [11C]erlotinib PET scan was followed by a second PET scan either after oral intake of unlabeled erlotinib (300 mg) or after intravenous infusion of the prototypical organic anion-transporting polypeptide inhibitor rifampicin (600 mg). We assessed a three-compartment (3C) and a four-compartment (4C) model, in which either a sampled arterial blood input function or a mathematically derived dual input function (DIF), which takes the contribution of the portal vein to the liver blood supply into account, was used. Both models provided acceptable fits of the observed PET data in the liver and extrahepatic bile duct and gall bladder. Changes in model outcome parameters between scans were consistent with the involvement of basolateral hepatocyte uptake and canalicular efflux transporters in the hepatobiliary clearance of [11C]erlotinib. Our results demonstrated that inclusion of a DIF did not lead to substantial improvements in model fits. The models developed in this work represent a step forward in applying PET as a tool to assess the impact of hepatic transporters on drug disposition and their involvement in drug-drug interactions. Electronic supplementary material The online version of this article (10.1208/s12248-019-0323-0) contains supplementary material, which is available to authorized users.

Our previous immunoprecipitation analysis of nicotinic acetylcholine receptors (nAChRs) in the mouse superior cervical ganglion (SCG) revealed that approximately 55%, 24%, and 21% of receptors are comprised of α3β4, α3β4α5, and α3β4β2 subunits, respectively. Moreover, mice lacking β4 subunits do not express α5‐containing receptors but still express a small number of α3β2 receptors. Here, we investigated how synaptic transmission is affected in the SCG of α5β4‐KO and α5β2‐KO mice. Using an ex vivo SCG preparation, we stimulated the preganglionic cervical sympathetic trunk and measured compound action potentials (CAPs) in the postganglionic internal carotid nerve. We found that CAP amplitude was unaffected in α5β4‐KO and α5β2‐KO ganglia, whereas the stimulation threshold for eliciting CAPs was significantly higher in α5β4‐KO ganglia. Moreover, intracellular recordings in SCG neurons revealed no difference in EPSP amplitude. We also found that the ganglionic blocking agent hexamethonium was the most potent in α5β4‐KO ganglia (IC 50: 22.1 μmol/L), followed by α5β2‐KO (IC 50: 126.7 μmol/L) and WT ganglia (IC 50: 389.2 μmol/L). Based on these data, we estimated an IC 50 of 568.6 μmol/L for a receptor population consisting solely of α3β4α5 receptors; and we estimated that α3β4α5 receptors comprise 72% of nAChRs expressed in the mouse SCG. Similarly, by measuring the effects of hexamethonium on ACh‐induced currents in cultured SCG neurons, we found that α3β4α5 receptors comprise 63% of nAChRs. Thus, in contrast to our results obtained using immunoprecipitation, these data indicate that the majority of receptors at the cell surface of SCG neurons consist of α3β4α5.

P-glycoprotein (ABCB1) is expressed at the blood-retina barrier (BRB), where it may control distribution of drugs from blood to the retina and thereby influence drug efficacy and toxicity. Methods: We performed PET scans with the ABCB1 substrate (R)-11C-verapamil on 5 healthy male volunteers without and with concurrent infusion of the ABCB1 inhibitor tariquidar. We estimated the rate constants for radiotracer transfer across the BRB (K1, k2) and total retinal distribution volume VTResults: During ABCB1 inhibition, retinal VT and influx rate constant K1 were significantly, by 1.4 ± 0.5-fold and 1.5 ± 0.3-fold, increased compared with baseline. Retinal efflux rate constant k2 was significantly decreased by 2.8 ± 1.0-fold. Conclusion: We found a significant increase in (R)-11C-verapamil distribution to the retina during ABCB1 inhibition, which provides first in vivo evidence for ABCB1 transport activity at the human BRB. The increase in retinal distribution was approximately 2.5-fold less pronounced than previously reported for the blood-brain barrier.

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 (11)C-metoclopramide in humans to elucidate the impact of ABCB1 function on its brain kinetics. Methods: Ten healthy male subjects underwent 2 consecutive (11)C-metoclopramide PET scans without and with ABCB1 inhibition using cyclosporine A (CsA). Pharmacokinetic modeling was performed to estimate the total volume of distribution (V(T)) and the influx (K(1)) and efflux (k(2)) rate constants between plasma and selected brain regions. Furthermore, (11)C-metoclopramide washout from the brain was estimated by determining the elimination slope (k(E,brain)) of the brain time–activity curves. Results: In baseline scans, (11)C-metoclopramide showed appreciable brain distribution (V(T) = 2.11 ± 0.33 mL/cm(3)). During CsA infusion, whole-brain gray matter V(T) and K(1) were increased by 29% ± 17% and 9% ± 12%, respectively. K(2) was decreased by 15% ± 5%, consistent with a decrease in k(E,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.

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), [

The purpose of this study was to establish physiologically based pharmacokinetic models to predict in humans the brain concentration–time profiles and P-glycoprotein (Pgp)–mediated brain drug–drug interactions between the model Pgp substrate ( R )-[ 11 C]verapamil (VPM), the model dual Pgp/breast cancer resistance protein (BCRP) substrate [ 11 C]tariquidar (TQD), and the Pgp inhibitor tariquidar. The model predictions were validated with results from positron emission tomography studies in humans. Using these physiologically based pharmacokinetic models, the differences between predicted and observed areas under the concentration–time curves (AUC) of VPM and TQD in the brain were within a 1.2-fold and 2.5-fold range, respectively. Also, brain AUC increases of VPM and TQD after Pgp inhibitor administration were predicted with 2.5-fold accuracy when in vitro inhibition constant or half-maximum inhibitory concentration values of tariquidar were used. The predicted rank order of the magnitude of AUC increases reflected the results of the clinical positron emission tomography studies. Our results suggest that the established models can predict brain exposure from the respective blood concentration–time profiles and rank the magnitude of the Pgp-mediated brain drug–drug interaction potential for both Pgp and Pgp/BCRP substrates in humans.

P-glycoprotein (ABCB1) is expressed at the blood-retina barrier (BRB), where it may control distribution of drugs from blood to the retina and thereby influence drug efficacy and toxicity.

The aim of this work is to find semi-rigid domains within large proteins as reference structures for fitting molecular dynamics trajectories. We propose an algorithm, multistage consensus clustering, MCC, based on minimum variation of distances between pairs of Cα-atoms as target function. The whole dataset (trajectory) is split into sub-segments. For a given sub-segment, spatial clustering is repeatedly started from different random seeds, and we adopt the specific spatial clustering with minimum target function: the process described so far is stage 1 of MCC. Then, in stage 2, the results of spatial clustering are consolidated, to arrive at domains stable over the whole dataset. We found that MCC is robust regarding the choice of parameters and yields relevant information on functional domains of the major histocompatibility complex (MHC) studied in this paper: the α-helices and β-floor of the protein (MHC) proved to be most flexible and did not contribute to clusters of significant size. Three alleles of the MHC, each in complex with ABCD3 peptide and LC13 T-cell receptor (TCR), yielded different patterns of motion. Those alleles causing immunological allo-reactions showed distinct correlations of motion between parts of the peptide, the binding cleft and the complementary determining regions (CDR)-loops of the TCR. Multistage consensus clustering reflected functional differences between MHC alleles and yields a methodological basis to increase sensitivity of functional analyses of bio-molecules. Due to the generality of approach, MCC is prone to lend itself as a potent tool also for the analysis of other kinds of big data.

Molecular dynamics simulations have to be sufficiently long to draw reliable conclusions. However, no method exists to prove that a simulation has converged. We suggest the method of “lagged RMSD-analysis” as a tool to judge if an MD simulation has not yet run long enough. The analysis is based on RMSD values between pairs of configurations separated by variable time intervals Δt. Unless RMSD(Δt) has reached a stationary shape, the simulation has not yet converged.

Using positron emission tomography (PET) imaging we assessed, in vivo, the interaction between a microdose of (R)-[(11)C]verapamil (a P-glycoprotein (Pgp) substrate) and escalating doses of the Pgp inhibitor tariquidar (3, 4, 6, and 8 mg/kg) at the blood-brain barrier (BBB) in healthy human subjects. We compared the dose-response relationship of tariquidar in humans with data obtained in rats using a similar methodology. Tariquidar was equipotent in humans and rats in its effect of increasing (R)-[(11)C]verapamil brain uptake (expressed as whole-brain volume of distribution (V(T))), with very similar half-maximum-effect concentrations. Both in humans and in rats, brain V(T) approached plateau levels at plasma tariquidar concentrations >1,000 ng/ml. However, Pgp inhibition in humans led to only a 2.7-fold increase in brain V(T) relative to baseline scans (before administration of tariquidar) as compared with 11.0-fold in rats. The results of this translational study add to the accumulating evidence that there are marked species-dependent differences in Pgp expression and functionality at the BBB.

Abscesses are often treated with antibiotics in addition to incision or when incision is unfeasible, but accurate information about antibiotic abscess penetration in humans is missing. This study aimed at evaluating the penetration of moxifloxacin into human abscesses. After administration of a single dose of 400 mg moxifloxacin, drug concentrations were measured in 10 differently located abscesses at incision, and in plasma over 8 h. At incision performed 0.9-4.8 h after administration, moxifloxacin concentrations in abscesses ranged from ≤0.01 to 9.2 mg/l (1.9 ± 3.4 mg/l), indicating pronounced drug accumulation in some abscesses. The degree of abscess penetration could not be explained by covariates like the ratio of surface area to volume or pH of abscesses, or by moxifloxacin plasma concentrations. Concluding, moxifloxacin was detectable in most abscesses and may be a useful antibiotic for this indication. However, antibiotic abscess penetration was highly variable and unpredictable, suggesting surgical abscess incision whenever possible.

Overactivity of the multidrug efflux transporter P-glycoprotein (P-gp) at the blood-brain barrier (BBB) is believed to play an important role in resistance to central nervous system drug treatment. (R)-[(11)C]verapamil (VPM) PET can be used to measure the function of P-gp at the BBB, but low brain uptake of VPM hampers the mapping of regional differences in cerebral P-gp function and expression. The aim of this study was to evaluate the dose-response relationship of two potent P-gp inhibitors and to investigate if increased brain uptake of VPM mediated by P-gp inhibition can be used to assess regional differences in P-gp activity.Two groups of Sprague-Dawley rats (n = 12) underwent single VPM PET scans at 120 min after administration of different doses of the P-gp inhibitors tariquidar and elacridar. In an additional six rats, paired VPM PET scans were performed before and after administration of 3 mg/kg tariquidar.Inhibitor administration resulted in an up to 11-fold increase in VPM brain distribution volumes (DV) with half-maximum effective dose (ED(50)) values of 3.0 +/- 0.2 and 1.2 +/- 0.1 mg/kg for tariquidar and elacridar, respectively. In paired PET scans, 3 mg/kg tariquidar resulted in regionally different enhancement of brain activity distribution, with lowest DV in cerebellum and highest DV in thalamus.Our data show that tariquidar and elacridar are able to increase VPM brain distribution in rat brain up to 11-fold over baseline at maximum effective doses, with elacridar being about three times more potent than tariquidar. Regional differences in tariquidar-induced modulation of VPM brain uptake point to regional differences in cerebral P-gp function and expression in rat brain.

We evaluated the usefulness of small animal brain positron emission tomography (PET) imaging with the amyloid-beta (A beta) probe 2-(1-{6-[(2-[(18)F]fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)malonitrile ([(18)F]FDDNP) and with 2-deoxy-2-[F-18]fluoro-D-glucose (FDG) for detection and quantification of pathological changes occurring in a transgenic mouse model of Alzheimer's disease (Tg2576 mice).[(18)F]FDDNP (n = 6) and FDG-PET scans (n = 3) were recorded in Tg2576 mice (age 13-15 months) and age-matched wild-type litter mates. Brain volumes of interest were defined by co-registration of PET images with a 3D MOBY digital mouse phantom. Regional [(18)F]FDDNP retention in mouse brain was quantified in terms of the relative distribution volume (DVR) using Logan's graphical analysis with cerebellum as a reference region.Except for a lower maximum brain uptake of radioactivity in transgenic animals, the regional brain kinetics as well as DVR values of [(18)F]FDDNP appeared to be similar in both groups of animals. Also for FDG, regional radioactivity retention was almost identical in the brains of transgenic and control animals.We could not detect regionally increased [(18)F]FDDNP binding and regionally decreased FDG binding in the brains of Tg2576 transgenic versus wild-type mice. However, small group differences in signal might have been masked by inter-animal variability. In addition, technical limitations of the applied method (partial volume effect, spatial resolution) for measurements in such small organs as mouse brain have to be taken into consideration.

Purpose The aim of this study was to assess the influence of age on the functional activity of the multidrug efflux transporter P-glycoprotein (P-gp) at the human blood-brain barrier. Methods Seven young (mean age: 27±4 years) and six elderly (mean age: 69±9 years) healthy volunteers underwent dynamic (R)-[ 11 C]verapamil (VPM) positron emission tomography (PET) scans and arterial blood sampling. Parametric distribution volume (DV) images were generated using Logan linearisation, and age groups were compared with statistical parametric mapping (SPM). Brain regions that SPM analysis had shown to be most affected by age were analysed by a region of interest (ROI)-based approach using a maximum probability brain atlas, before and after partial volume correction (PVC). Results SPM analysis revealed significant clusters of DV increases in cerebellum, temporal and frontal lobe of elderly compared to younger subjects. In the ROI-based analysis, elderly subjects showed significant DV increases in amygdala (+30%), insula (+26%) and cerebellum (+25%) before PVC, and in insula (+33%) after PVC. Conclusions Increased VPM DV values in the brains of elderly subjects suggest a decrease in cerebral P-gp function with increasing age.

Purpose and Methods: Regional overexpression of the multidrug transporter P-glycoprotein (P-gp) in epileptic brain tissue may lower target site concentrations of antiepileptic drugs and thus contribute to pharmacoresistance in epilepsy. We used the P-gp substrate R-[(11)C]verapamil and positron emission tomography (PET) to test for differences in P-gp activity between epileptogenic and nonepileptogenic brain regions of patients with drug-resistant unilateral temporal lobe epilepsy (n = 7). We compared R-[(11)C]verapamil kinetics in homologous brain volumes of interest (VOIs) located ipsilateral and contralateral to the seizure focus. Results: Among different VOIs, radioactivity was highest in the choroid plexus. The hippocampal VOI could not be used for data analysis because it was contaminated by spill-in of radioactivity from the adjacent choroid plexus. In several other temporal lobe regions that are known to be involved in seizure generation and propagation ipsilateral influx rate constants K(1) and efflux rate constants k(2) of R-[(11)C]verapamil were descriptively increased as compared to the contralateral side. Parameter asymmetries were most prominent in parahippocampal and ambient gyrus (K(1), range: -3.8% to +22.3%; k(2), range: -2.3% to +43.9%), amygdala (K(1), range: -20.6% to +31.3%; k(2), range: -18.0% to +38.9%), medial anterior temporal lobe (K(1), range: -8.3% to +14.5%; k(2), range: -14.5% to +31.0%) and lateral anterior temporal lobe (K(1), range: -20.7% to +16.8%; k(2), range: -24.4% to +22.6%). In contrast to temporal lobe VOIs, asymmetries were minimal in a region presumably not involved in epileptogenesis located outside the temporal lobe (superior parietal gyrus, K(1), range: -3.7% to +4.5%; k(2), range: -4.2% to +5.8%). In 5 of 7 patients, ipsilateral efflux (k(2)) increases were more pronounced than ipsilateral influx (K(1)) increases, which resulted in ipsilateral reductions (10%-26%) of R-[(11)C]verapamil distribution volumes (DV). However, for none of the examined brain regions, any of the differences in K(1), k(2) and DV between the epileptogenic and the nonepileptogenic hemisphere reached statistical significance (p > 0.05, Wilcoxon matched pairs test). Conclusions: Even though we failed to detect statistically significant differences in R-[(11)C]verapamil model parameters between epileptogenic and nonepileptogenic brain regions, it cannot be excluded from our pilot data in a small sample size of patients that regionally enhanced P-gp activity might contribute to drug resistance in some patients with temporal lobe epilepsy.

Molecular dynamics was used to simulate large molecules of the immune system (major histocompatibility complex class I, presented epitope, T-cell receptor, and a CD8 coreceptor.) To characterize the relative orientation and movements of domains local coordinate systems (based on principal component analysis) were generated and directional cosines and Euler angles were computed. As a most interesting result, we found that the presence of the coreceptor seems to influence the dynamics within the protein complex, in particular the relative movements of the twoα-helices, Gα1and Gα2.

MHCα-helices form the antigen-binding cleft and are of particular interest for immunological reactions. To monitor these helices in molecular dynamics simulations, we applied a parsimonious fragment-fitting method to trace the axes of theα-helices. Each resulting axis was fitted by polynomials in a least-squares sense and the curvature integral was computed. To find the appropriate polynomial degree, the method was tested on two artificially modelled helices, one performing a bending movement and another a hinge movement. We found that second-order polynomials retrieve predefined parameters of helical motion with minimal relative error. From MD simulations we selected those parts ofα-helices that were stable and also close to the TCR/MHC interface. We monitored the curvature integral, generated a ruled surface between the two MHCα-helices, and computed interhelical area and surface torsion, as they changed over time. We found that MHCα-helices undergo rapid but small changes in conformation. The curvature integral of helices proved to be a sensitive measure, which was closely related to changes in shape over time as confirmed by RMSD analysis. We speculate that small changes in the conformation of individual MHCα-helices are part of the intrinsic dynamics induced by engagement with the TCR.

We propose a Gibbs point field model for the pattern of coronary capillaries in transverse histologic sections from human hearts, based on the physiology of oxygen supply from capillaries to tissue. To specify the potential energy function of the Gibbs point field, we draw on an analogy between the equation of steady-state oxygen diffusion from an array of parallel capillaries to the surrounding tissue and Poisson’s equation for the electrostatic potential of a two-dimensional distribution of identical point charges. The influence of factors other than diffusion is treated as a thermal disturbance. On this basis, we arrive at the well-known two-dimensional one-component plasma, a system of identical point charges exhibiting a weak (logarithmic) repulsive interaction that is completely characterized by a single dimensionless parameter. By variation of this parameter, the model is able to reproduce many characteristics of real capillary patterns. r 2006 Elsevier B.V. All rights reserved.

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)-[(11)C]verapamil in five healthy volunteers during continuous intravenous tariquidar infusion. Total distribution volume (VT) of (R)-[(11)C]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.

The present study was performed to evaluate the ability of fosfomycin, a broad-spectrum antibiotic, to penetrate into abscess fluid. Twelve patients scheduled for surgical or computer tomography-guided abscess drainage received a single intravenous dose of 8 g of fosfomycin. The fosfomycin concentrations in plasma over time and in pus upon drainage were determined. A pharmacokinetic model was developed to estimate the concentration-time profile of fosfomycin in pus. Individual fosfomycin concentrations in abscess fluid at drainage varied substantially, ranging from below the limit of detection up to 168 mg/liter. The fosfomycin concentrations in pus of the study population correlated neither with plasma levels nor with the individual ratios of abscess surface area to volume. This finding was attributed to highly variable abscess permeability. The average concentration in pus was calculated to be 182 ± 64 mg/liter at steady state, exceeding the MIC 50/90 s of several bacterial species which are commonly involved in abscess formation, such as streptococci, staphylococci, and Escherichia coli . Hereby, the exceptionally long mean half-life of fosfomycin of 32 ± 39 h in abscess fluid may favor its antimicrobial effect because fosfomycin exerts time-dependent killing. After an initial loading dose of 10 to 12 g, fosfomycin should be administered at doses of 8 g three times per day to reach sufficient concentrations in abscess fluid and plasma. Applying this dosing regimen, fosfomycin levels in abscess fluid are expected to be effective after multiple doses in most patients.

Background and Objective Single nucleotide polymorphisms in the human multidrug-resistance gene ABCB1 have been reported to be associated with altered expression and function of P-glycoprotein, an efflux transporter, expressed at the blood-brain barrier. To test whether certain ABCB1 haplotypes contribute to interindividual differences in central nervous system drug distribution, brain distribution of a model P-glycoprotein substrate, the calcium channel inhibitor verapamil, was measured by positron emission tomography (PET) in 2 groups of healthy volunteers. Methods Ten homozygous carriers (cases) of the TTT haplotype (3435T, 1236T, and 2677T) and 10 controls homozygous for the wild-type CGC haplotype (3435C, 2677G, and 1236C) were administered a mean intravenous bolus of 412 ± 114 MBq carbon 11-labeled verapamil containing less than 15 nmol of unlabeled verapamil. PET imaging of brain tissue and venous blood sampling were performed for 1 hour after dosing. Results As a measure of brain penetration, the ratio of PET area under the time-radioactivity curve (AUC) to plasma AUC was calculated from time-radioactivity curves, with a mean ratio of 1.1 ± 0.3 (SD) (95% confidence interval, 0.9–1.3) for cases and 1.1 ± 0.2 (95% confidence interval, 0.9–1.2) for controls, respectively (P = .96). Mean brain AUC values were 31.2 ± 3.9 and 35.7 ± 5.7 for the TTT and CGC haplotype, respectively (P = .11). Plasma AUCs were not significantly different. Conclusion No difference in the brain distribution of [11C]verapamil could be detected in healthy volunteers differing in ABCB1 haplotypes. Clinical Pharmacology & Therapeutics (2005) 78, 182–190; doi: 10.1016/j.clpt.2005.04.011

Regional blood flows in the heart muscle are remarkably heterogeneous. It is very likely that the most important factor for this heterogeneity is the metabolic need of the tissue rather than flow dispersion by the branching network of the coronary vasculature. To model the contribution of tissue needs to the observed flow heterogeneities we use arterial trees generated on the computer by constrained constructive optimization. This method allows to prescribe terminal flows as independent boundary conditions, rather than obtaining these flows by the dispersive effects of the tree structure. We study two specific cases: equal terminal flows (model 1) and terminal flows set proportional to the volumes of Voronoi polyhedra used as a model for blood supply regions of terminal segments (model 2). Model 1 predicts, depending on the number Nterm of end-points, fractal dimensions D of perfusion heterogeneities in the range 1.20 to 1.40 and positively correlated nearest-neighbor regional flows, in good agreement with experimental data of the normal heart. Although model 2 yields reasonable terminal flows well approximated by a lognormal distribution, it fails to predict D and nearest-neighbor correlation coefficients r1 of regional flows under normal physiologic conditions: model 2 gives D = 1.69 ± 0.02 and r1 = −0.18 ± 0.03 (n = 5), independent of Nterm and consistent with experimental data observed under coronary stenosis and under the reduction of coronary perfusion pressure. In conclusion, flow heterogeneity can be modeled by terminal positions compatible with an existing tree structure without resorting to the flow-dispersive effects of a specific branching tree model to assign terminal flows.

Topological and metric properties of Voronoi poly- hedra ~VP! generated by the distal end points of terminal seg- ments in arterial tree models grown by the method of con- strained constructive optimization ~CCO! are analyzed with the aim to characterize the spatial distribution of their supply sites relative to randomly distributed points as a reference model. The distributions of the number N f of Voronoi cell faces, cell volume V, surface area S, area A of individual cell faces, and asphericity parameter a of the CCO models are all significantly different from the ones of random points, whereas the distribu- tions of V, S, and a are also significantly different among CCO models optimized for minimum intravascular volume and minimum segment length (p,0.0001). The distributions of N f , V, and S of the CCO models are reasonably well approxi- mated by two-parameter gamma distributions. We study scaling of intravascular blood volume and arterial cross-sectional area with the volume of supplied tissue, the latter being represented by the VP of the respective terminal segments. We observe scaling exponents from 1.2060.007 to 1.0860.005 for intra- vascular blood volume and 0.7760.01 for arterial cross- sectional area. Setting terminal flows proportional to the asso- ciated VP volumes during tree construction yields a relative dispersion of terminal flows of 37% and a coefficient of skew- ness of 1.12. © 2003 Biomedical Engineering Society. @DOI: 10.1114/1.1566444#

Objectives: 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. Methods: 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. Results: 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. Conclusions: 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.