An integrated pharmacokinetic and pharmacodynamic study of a new drug of abuse, methylone, a synthetic cathinone sold as 'bath salts'

Material and methods. Methylone was administered to male Sprague-Dawley rats intravenously (10 mg/kg) and orally (15 and 30 mg/kg). Plasma concentrations and metabolites were characterized by LC/MS and LC-MS/MS fragmentation patterns. Locomotor activity was monitored for 180-240 min. Results. Oral administration of methylone induced a dose-dependent increase in locomotor activity in rats. The plasma concentrations after i.v. administration were described by a two-compartment model with distribution and terminal elimination phases of α = 1.95 h− 1 and β = 0.72 h− 1. For oral administration, peak methylone concentrations were achieved between 0.5 and 1 h and fitted to a flip-flop model. Absolute bioavailability was about 80% and the percentage of methylone protein binding was of 30%. A relationship between methylone brain levels and free plasma concentration yielded a ratio of 1.42 ± 0.06, indicating access to the central nervous system. We have identified four Phase I metabolites after oral administration. The major metabolic routes are N-demethylation, aliphatic hydroxylation and O-methylation of a demethylenate intermediate. Discussion. Pharmacokinetic and pharmacodynamic analysis of methylone showed a correlation between plasma concentrations and enhancement of the locomotor activity. A contribution of metabolites in the activity of methylone after oral administration is suggested. Present results will be helpful to understand the time course of the effects of this drug of abuse in humans.

An integrated pharmacokinetic and pharmacodynamic study of a new drug of abuse, methylone, a synthetic cathinone sold as 'bath salts'

Material and methods. Methylone was administered to male Sprague-Dawley rats intravenously (10 mg/kg) and orally (15 and 30 mg/kg). Plasma concentrations and metabolites were characterized by LC/MS and LC-MS/MS fragmentation patterns. Locomotor activity was monitored for 180-240 min. Results. Oral administration of methylone induced a dose-dependent increase in locomotor activity in rats. The plasma concentrations after i.v. administration were described by a two-compartment model with distribution and terminal elimination phases of α = 1.95 h− 1 and β = 0.72 h− 1. For oral administration, peak methylone concentrations were achieved between 0.5 and 1 h and fitted to a flip-flop model. Absolute bioavailability was about 80% and the percentage of methylone protein binding was of 30%. A relationship between methylone brain levels and free plasma concentration yielded a ratio of 1.42 ± 0.06, indicating access to the central nervous system. We have identified four Phase I metabolites after oral administration. The major metabolic routes are N-demethylation, aliphatic hydroxylation and O-methylation of a demethylenate intermediate. Discussion. Pharmacokinetic and pharmacodynamic analysis of methylone showed a correlation between plasma concentrations and enhancement of the locomotor activity. A contribution of metabolites in the activity of methylone after oral administration is suggested. Present results will be helpful to understand the time course of the effects of this drug of abuse in humans.

Power performance with gated clocks of a pipelined Cordic Core

This paper presents the evaluation in power consumption of gated clocks pipelined circuits with different register configurations in Virtex-based FPGA devices. Power impact of a gated clock circuitry aimed at reducing flip-flops output rate at the bit level is studied. Power performance is also given for pipeline stages based on the implementation of a double edge-triggered flip-flop. Using a pipelined Cordic Core circuit as an example, this study did not find evidence in power benefits either when gated clock at the bit-level or double-edge triggered flip-flops used when synthesized with FPGA logic resources.

Proton flux induced by free fatty acids across phospholipid bilayers: New evidences based on short-circuit measurements in planar lipid membranes

Free fatty acids (FFA) are important mediators of proton transport across membranes. However, information concerning the influence of the Structural features of both FFA and the membrane environment on the proton translocation mechanisms across phospholipid membranes is relatively scant. The effects of FFA chain length, unsaturation and membrane composition on proton transport have been addressed in this study by means of electrical measurements in planar lipid bilayers. Proton conductance (G(H)(+)) was calculated from open-circuit voltage and short-circuit current density measurements. We found that cis-unsaturated FFA caused a more pronounced effect on proton transport as compared to Saturated and trans-unsaturated FFA. Cholesterol and cardiolipin decreased membrane leak conductance. Cardiolipin also decreased proton conductance. These effects indicate a dual modulation of protein-independent proton transport by FFA: through a flip-flop mechanism and by modifying a proton diffusional pathway. Moreover the membrane phospholipid composition was shown to importantly affect both processes. (C) 2009 Elsevier Inc. All rights reserved.

A multisampling time-domain CMOS imager with synchronous readout circuit

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A multisampling time-domain CMOS imager with synchronous readout circuit

A novel multisampling time-domain architecture for CMOS imagers with synchronous readout and wide dynamic range is proposed. The architecture was implemented in a prototype of imager with 32x32 pixel array fabricated in AMS CMOS 0.35νm and was characterized for sensitivity and color response. The pixel is composed of an n+/psub photodiode, a comparator and a D flip-flop having 16% fill-factor and 30νmx26νm dimensions. The multisampling architecture requires only a 1 bit per pixel memory instead of 8 bits which is typical for time-domain active pixel architectures. The advantage is that the number of transistors in the pixel is low, saving area and providing higher fill-factor. The maximum frame rate is analyzed as a function of number of bits and array size. The analysis shows that it is possible to achieve high frame rates and operation in video mode with 10 bits. Also, we present analysis for the impact of comparator offset voltage in the fixed pattern noise. Copyright 2007 ACM.

Glukose-konjugierte Hemmstoffe als Strategie der gezielten Hemmung des DNA-Reparaturproteins O 6 -Methylguanin-DNA-Methyltransferase in Tumorzellen und der Einfluss von ATP-binding cassette Transportern

Das DNA-Reparaturprotein O6-Methylguanin-DNA-Methyltransferase [MGMT] ist der Hauptresistenzfaktor gegenüber der zytotoxischen Wirkung von SN1-alkylierenden Zytostatika in der Tumortherapie. Die Verwendung der MGMT-Hemmstoffe O6-Benzylguanin [O6BG] und O6-(4-Bromothenyl)guanin [O6BTG] führte zu einer Sensibilisierung des Normalgewebes, was eine Dosis-Reduktion der Zytostatika erforderlich machte und die erhoffte Therapieverbesserung verhinderte. Aus diesem Grund ist eine Strategie der selektiven Hemmung des MGMT-Proteins (Targeting-Strategie) erforderlich, um die systemische Toxizität in der Kombinationsbehandlung zu reduzieren. In dieser Arbeit wurde die Anwendbarkeit der Glukose-Konjugation als Targeting-Strategie untersucht, da Tumorzellen einen erhöhten Glukoseverbrauch aufweisen und demzufolge Glukosetransporter überexprimieren. Die Glukose-Konjugate O6BG-Glu und O6BTG-Glu inhibierten MGMT in Tumorzellen und sensibilisierten die Zellen gegenüber den alkylierenden Agenzien Temozolomid [TMZ] und Lomustin [CCNU]. Des Weiteren inaktivierten die Glukose-Konjugate die MGMT-Aktivität im Tumor eines Xenograft-Mausmodells und reduzierten das Tumorwachstum nach einer TMZ-Behandlung im gleichen Ausmass wie die Inhibitoren O6BG und O6BTG. Trotzdem war auch mit den Glukose-Konjugaten keine Steigerung der Zytostatika-Dosis im Mausmodell möglich. Die Untersuchungen der Aufnahme von O6BG-Glu und O6BTG-Glu wiederlegten eine Involvierung der Glukosetransporter. Der Einsatz von spezifischen Glukosetransporter-Inhibitoren und Kompetitions-Experimenten führte zu keiner Verminderung der MGMT-Hemmung oder Aufnahme vom radioaktiven H3-O6BTG-Glu in die Zelle. Dies legt nahe, dass die Glukose-Konjugate über einen unspezifischen Mechanismus (aktiv) in die Zellen gelangen. Der Grund für eine mögliche unselektive Aufnahme könnte im hydrophoben Alkyllinker, der für die Konjugation des Glukosemoleküls verwendet wurde, begründet sein. Dies führt zur Generierung von amphipathischen Konjugaten, die eine initiale Bindung an die Plasmamembran aufweisen und eine Aufnahme über den Flip-Flop-Mechanismus (transbilayer transport) wahrscheinlich machen. Die amphipathische Molekülstruktur der Glukose-Konjugate führte zu einer Partikelbildung in wässrigen Lösungen, die eine Reduktion der Menge an aktiven Monomeren von O6BG-Glu und O6BTG-Glu bewirken, die zur Hemmung von MGMT zur Verfügung stehen. Der zweite Teil der Arbeit befasste sich mit der Rolle von ABC-Transportern hinsichtlich einer Targeting-Strategie von MGMT-Hemmstoffen. Obwohl eine hohe Expression dieser ABC-Transporter in Tumoren zur Resistenzentwicklung gegenüber Zytostatika führt, wurde ihr Einfluss auf MGMT-Hemmstoffe oder einer MGMT-Targeting-Strategie niemals untersucht. In dieser Arbeit wurde zum ersten Mal ein aktiver Efflux von MGMT-Hemmstoffen durch ABC-Transporter nachgewiesen. Die Inhibition von ABC-Transportern bewirkte eine schnellere Inaktivierung von MGMT durch die Glukose-Konjugate. Des Weiteren zeigten Kompetitions-Experimente mit den MGMT-Hemmstoffen eine verminderte Efflux-Rate von Fluoreszenzfarbstoffen, die spezifisch von ABC-Transportern exportiert werden. ABC-Transporter reduzieren die wirksame Konzentration des Hemmstoffes in der Zelle und beeinträchtigen somit die Effektivität der MGMT-Inhibition. Eine simultane Hemmung der ABC-Transporter P-glycoprotein (P-gp), multi resistance protein 1 (MRP1) and breast cancer resistance protein (BCRP) erhöhte die Effektivität der MGMT-Hemmstoffe (O6BG, O6BTG, O6BG-Glu, O6BTG-Glu) und verstärkte auf diese Weise die TMZ-induzierte Toxizität in Tumorzelllinien. Die Involvierung von ABC-Transportern in der intrazellulären Speicherung von MGMT-Hemmstoffen ist wahrscheinlich die Ursache für die beobachteten Unterschiede in der Sensibilisierung verschiedener Tumorzelllinien gegenüber Zytostatika durch das Glukose-Konjugat O6BG-Glu. Eine Strategie, den Einfluss von ABC-Transportern zu reduzieren und zukünftliche MGMT-Targeting-Strategien effizienter umzusetzen, ist die Verwendung von O6BTG als Ausgangssubstanz. Die höhere Inhibitionsfähigkeit der Bromthiophenmoleküle vermindert die erforderliche intrazelluläre Konzentration für eine vollständige MGMT-Hemmung und reduziert auf diese Weise den Einfluss von ABC-Transportern.

pH-dependent distribution of chlorin e6 derivatives across phospholipid bilayers probed by NMR spectroscopy

The pH-dependent membrane adsorption and distribution of three chlorin derivatives, chlorin e6 (CE), rhodin G7 (RG), and monoaspartyl-chlorin e6 (MACE), in the physiological pH range (pH 6-8) were probed by NMR spectroscopy. Unilamellar vesicles consisting of dioleoyl-phosphatidyl-choline (DOPC) were used as membrane models. The chlorin derivatives were characterized with respect to their aggregation behavior, the pK(a) values of individual carboxylate groups, the extent of membrane adsorption, and their flip-flop rates across the bilayer membrane for pH 6-8. External membrane adsorption was found to be lower for RG than for CE and MACE. Both electrostatic interactions and the extent of aggregation seemed to be the main determinants of membrane adsorption. Rate constants for chlorin transfer across the membrane were found to correlate strongly with the pH of the surrounding medium, in particular, for CE and RG. In acidic solution, CE and RG transfer across the membrane was strongly accelerated, and in basic solution, all compounds were retained, mostly in the outer monolayer. In contrast, MACE flip-flop across the membrane remained very low even at pH 6. The protonation of ionizable groups is suggested to be a major determinant of chlorin transfer rates across the bilayer. pK(a) values of CE and RG were found to be between 6 and 8, and two of the carboxylate groups in MACE had pK(a) values below 6. For CE and RG, the kinetic profiles at acidic pH indicated that the initial fast membrane distribution was followed by secondary steps that are discussed in this article.

Time-dependent interactions of the two porphyrinic compounds chlorin e6 and mono-L-aspartyl-chlorin e6 with phospholipid vesicles probed by NMR spectroscopy

The distribution processes of chlorin e6 (CE) and monoaspartyl-chlorin e6 (MACE) between the outer and inner phospholipid monolayers of 1,2-dioleoyl-phosphatidylcholine (DOPC) vesicles were monitored by 1H NMR spectroscopy through analysis of chemical shifts and line widths of the DOPC vesicle resonances. Chlorin adsorption to the outer vesicle monolayer induced changes in the DOPC 1H NMR spectrum. Most pronounced was a split of the N-methyl choline resonance, allowing for separate analysis of inner and outer vesicle layers. Transbilayer distribution of the chlorin compounds was indicated by time-dependent characteristic spectral changes of the DOPC resonances. Kinetic parameters for the flip-flop processes, that is, half-lives and rate constants, were obtained from the experimental data points. In comparison to CE, MACE transbilayer movement was significantly reduced, with MACE remaining more or less attached to the outer membrane layer. The distribution coefficients for CE and MACE between the vesicular and aqueous phase were determined. Both CE and MACE exhibited a high affinity for the vesicular phase. For CE, a positive correlation was found between transfer rate and increasing molar ratio CE/DOPC. Enhanced membrane rigidity induced by increasing amounts of cholesterol into the model membrane was accompanied by a decrease of CE flip-flop rates across the membrane. The present study shows that the movement of porphyrins across membranes can efficiently be investigated by 1H NMR spectroscopy and that small changes in porphyrin structure can have large effects on membrane kinetics.

Mechanism of oligonucleotide release from cationic liposomes.

We propose a mechanism for oligonucleotide (ODN) release from cationic lipid complexes in cells that accounts for various observations on cationic lipid-nucleic acid-cell interactions. Fluorescent confocal microscopy of cells treated with rhodamine-labeled cationic liposome/ fluorescein-labeled ODN (F-ODN) complexes show the F-ODN separates from the lipid after internalization and enters the nucleus leaving the fluorescent lipid in cytoplasmic structures. ODN displacement from the complex was studied by fluorescent resonance energy transfer. Anionic liposome compositions (e.g., phosphatidylserine) that mimic the cytoplasmic facing monolayer of the cell membrane released ODN from the complex at about a 1:1 (-/+) charge ratio. Release was independent of ionic strength and pH. Physical separation of the F-ODN from monovalent and multivalent cationic lipids was confirmed by gel electrophoresis. Fluid but not solid phase anionic liposomes are required, whereas the physical state of the cationic lipids does not effect the release. Water soluble molecules with a high negative linear charge density, dextran sulfate, or heparin also release ODN. However, ATP, spermidine, spermine, tRNA, DNA, polyglutamic acid, polylysine, bovine serum albumin, or histone did not release ODN, even at 100-fold charge excess (-/+). Based upon these results, we propose that the complex, after internalization by endocytosis, induces flip-flop of anionic lipids from the cytoplasmic facing monolayer. Anionic lipids laterally diffuse into the complex and form a charged neutralized ion-pair with the cationic lipids. This leads to displacement of the ODN from the cationic lipid and its release into the cytoplasm.

Intracellular pH in adipocytes: effects of free fatty acid diffusion across the plasma membrane, lipolytic agonists, and insulin.

The main function of white adipose tissue is to store nutrient energy in the form of triglycerides. The mechanism by which free fatty acids (FFA) move into and out of the adipocyte has not been resolved. We show here that changes in intracellular pH (pH1) in adipocytes correlate with the movement of FFA across cellular membranes as predicted by the Kamp and Hamilton model of passive diffusion of FFA. Exposure of fat cells to lipolytic agents or external FFA results is a rapid intracellular acidification that is reversed by metabolism of the FFA or its removal by albumin. In contrast, insulin causes an alkalinization of the cell, consistent with its main function to promote esterification. Inhibition of Na+/H+ exchange in adipocytes does not prevent the changes in pHi caused by FFA, lipolytic agents, or insulin. A fatty acid dimer, which diffuses into the cell but is not metabolized, causes an irreversible acidification. Taken together, the data suggest that changes in pHi occur in adipocytes in response to the passive diffusion of un-ionized FFA (flip-flop) into and out of the cell and in response to their metabolism and production within the cell. These changes in pHi may, in turn, modulate hormonal signaling and metabolism with significant impact on cell function.

Toxicokinetics of domoic acid in a nonhuman primate model (Macaca fascicularis)

Thesis (Master's)--University of Washington, 2016-06

A bang-bang PLL employing dynamic gain control for low jitter and fast lock times

Bang-bang phase detector based PLLs are simple to design, suffer no systematic phase error, and can run at the highest speed a process can make a working flip-flop. For these reasons designers are employing them in the design of very high speed Clock Data Recovery (CDR) architectures. The major drawback of this class of PLL is the inherent jitter due to quantized phase and frequency corrections. Reducing loop gain can proportionally improve jitter performance, but also reduces locking time and pull-in range. This paper presents a novel PLL design that dynamically scales its gain in order to achieve fast lock times while improving fitter performance in lock. Under certain circumstances the design also demonstrates improved capture range. This paper also analyses the behaviour of a bang-bang type PLL when far from lock, and demonstrates that the pull-in range is proportional to the square root of the PLL loop gain.

Spin gaps and spin-flip energies in density-functional theory

Energy gaps are crucial aspects of the electronic structure of finite and extended systems. Whereas much is known about how to define and calculate charge gaps in density-functional theory (DFT), and about the relation between these gaps and derivative discontinuities of the exchange-correlation functional, much less is known about spin gaps. In this paper we give density-functional definitions of spin-conserving gaps, spin-flip gaps and the spin stiffness in terms of many-body energies and in terms of single-particle (Kohn-Sham) energies. Our definitions are as analogous as possible to those commonly made in the charge case, but important differences between spin and charge gaps emerge already on the single-particle level because unlike the fundamental charge gap spin gaps involve excited-state energies. Kohn-Sham and many-body spin gaps are predicted to differ, and the difference is related to derivative discontinuities that are similar to, but distinct from, those usually considered in the case of charge gaps. Both ensemble DFT and time-dependent DFT (TDDFT) can be used to calculate these spin discontinuities from a suitable functional. We illustrate our findings by evaluating our definitions for the Lithium atom, for which we calculate spin gaps and spin discontinuities by making use of near-exact Kohn-Sham eigenvalues and, independently, from the single-pole approximation to TDDFT. The many-body corrections to the Kohn-Sham spin gaps are found to be negative, i.e., single-particle calculations tend to overestimate spin gaps while they underestimate charge gaps.

Spin-polarized current and shot noise in the presence of spin flip in a quantum dot via nonequilibrium Green's functions

Using nonequilibrium Green's functions we calculate the spin-polarized current and shot noise in a ferromagnet-quantum-dot-ferromagnet system. Both parallel (P) and antiparallel (AP) magnetic configurations are considered. Coulomb interaction and coherent spin flip (similar to a transverse magnetic field) are taken into account within the dot. We find that the interplay between Coulomb interaction and spin accumulation in the dot can result in a bias-dependent current polarization p. In particular, p can be suppressed in the P alignment and enhanced in the AP case depending on the bias voltage. The coherent spin flip can also result in a switch of the current polarization from the emitter to the collector lead. Interestingly, for a particular set of parameters it is possible to have a polarized current in the collector and an unpolarized current in the emitter lead. We also found a suppression of the Fano factor to values well below 0.5.