Drug concentration response relationships in normal volunteers after oral administration of losartan, an angiotensin II receptor antagonist.

The aim of this study was to investigate the relationships between plasma concentrations of losartan, an orally active angiotensin II inhibitor, its active metabolite EXP3174, and angiotensin II blockade. Six healthy subjects received single oral doses of 40, 80, or 120 mg losartan and placebo at 1-week intervals in a crossover study. Angiotensin II blockade was assessed by the blood pressure response to exogenous angiotensin II before and after losartan administration. EXP3174 reached higher plasma concentrations and was eliminated more slowly than its parent compound; its levels paralleled the profile of angiotensin II blockade closer than losartan. Inhibition of the pressure response was dose dependent. The Hill-shaped relationship between response and EXP3174 concentration (or time-integrated variables) approached a plateau with 80 mg. The dose-dependent increase in plasma renin and angiotensin II exhibited a considerable individual scatter. We conclude that losartan produces a dose-dependent, effective angiotensin II blockade that is largely determined by the active metabolite EXP3174.

Suprachoroidal electrotransfer: a nonviral gene delivery method to transfect the choroid and the retina without detaching the retina.

Photoreceptors and retinal pigment epithelial cells (RPE) targeting remains challenging in ocular gene therapy. Viral gene transfer, the only method having reached clinical evaluation, still raises safety concerns when administered via subretinal injections. We have developed a novel transfection method in the adult rat, called suprachoroidal electrotransfer (ET), combining the administration of nonviral plasmid DNA into the suprachoroidal space with the application of an electrical field. Optimization of injection, electrical parameters and external electrodes geometry using a reporter plasmid, resulted in a large area of transfected tissues. Not only choroidal cells but also RPE, and potentially photoreceptors, were efficiently transduced for at least a month when using a cytomegalovirus (CMV) promoter. No ocular complications were recorded by angiographic, electroretinographic, and histological analyses, demonstrating that under selected conditions the procedure is devoid of side effects on the retina or the vasculature integrity. Moreover, a significant inhibition of laser induced-choroidal neovascularization (CNV) was achieved 15 days after transfection of a soluble vascular endothelial growth factor receptor-1 (sFlt-1)-encoding plasmid. This is the first nonviral gene transfer technique that is efficient for RPE targeting without inducing retinal detachment. This novel minimally invasive nonviral gene therapy method may open new prospects for human retinal therapies.

Extended access cocaine self-administration differentially activates dorsal raphe and amygdala corticotropin-releasing factor systems in rats.

Cocaine-induced neuroadaptation of stress-related circuitry and increased access to cocaine each putatively contribute to the transition from cocaine use to cocaine dependence. The present study tested the hypothesis that rats receiving extended versus brief daily access to cocaine would exhibit regional differences in levels of the stress-regulatory neuropeptide corticotropin-releasing factor (CRF). A secondary goal was to explore how CRF levels change in relation to the time since cocaine self-administration. Male Wistar rats acquired operant self-administration of cocaine and were assigned to receive daily long access (6 hours/day, LgA, n = 20) or short access (1 hour/day, ShA, n = 18) to intravenous cocaine self-administration (fixed ratio 1, ∼0.50 mg/kg/infusion). After at least 3 weeks, tissue CRF immunoreactivity was measured at one of three timepoints: pre-session, post-session or 3 hours post-session. LgA, but not ShA, rats showed increased total session and first-hour cocaine intake. CRF immunoreactivity increased within the dorsal raphe (DR) and basolateral, but not central, nucleus of the amygdala (BLA, CeA) of ShA rats from pre-session to 3 hours post-session. In LgA rats, CRF immunoreactivity increased from pre-session to 3 hours post-session within the CeA and DR but tended to decrease in the BLA. LgA rats showed higher CRF levels than ShA rats in the DR and, pre-session, in the BLA. Thus, voluntary cocaine intake engages stress-regulatory CRF systems of the DR and amygdala. Increased availability of cocaine promotes greater tissue CRF levels in these extrahypothalamic brain regions, changes associated here with a model of cocaine dependence.

The JNK inhibitor XG-102 protects from ischemic damage with delayed intravenous administration also in the presence of recombinant tissue plasminogen activator.

BACKGROUND: XG-102 (formerly D-JNKI1), a TAT-coupled dextrogyre peptide which selectively inhibits the c-Jun N-terminal kinase, is a powerful neuroprotectant in mouse models of middle cerebral artery occlusion (MCAo) with delayed intracerebroventricular injection. We aimed to determine whether this neuroprotection could also be achieved by intravenous injection of XG-102, which is a more feasible approach for future use in stroke patients. We also tested the compatibility of the compound with recombinant tissue plasminogen activator (rtPA), commonly used for intravenous thrombolysis and known to enhance excitotoxicity. METHODS: Male ICR-CD1 mice were subjected to a 30-min-suture MCAo. XG-102 was injected intravenously in a single dose, 6 h after ischemia. Hippocampal slice cultures were subjected to oxygen (5%) and glucose (1 mM) deprivation for 30 min. rtPA was added after ischemia and before XG-102 administration, both in vitro and in vivo. RESULTS: The lowest intravenous dose achieving neuroprotection was 0.0003 mg/kg, which reduced the infarct volume after 48 h from 62 +/- 19 mm(3) (n = 18) for the vehicle-treated group to 18 +/- 9 mm(3) (n = 5, p < 0.01). The behavioral outcome was also significantly improved at two doses. Addition of rtPA after ischemia enhanced the ischemic damage both in vitro and in vivo, but XG-102 was still able to induce a significant neuroprotection. CONCLUSIONS: A single intravenous administration of XG-102 several hours after ischemia induces a powerful neuroprotection. XG-102 protects from ischemic damage in the presence of rtPA. The feasibility of systemic administration of this promising compound and its compatibility with rtPA are important steps for its development as a drug candidate in ischemic stroke.

Oxidation of methane at the CH4/H2O-(CO2) transition zone in the external part of the Central Alps, Switzerland: Evidence from stable isotope investigations

With the aim of understanding the mechanisms that control the metamorphic transition from the CH4- to the H2O-(CO2)-dominated fluid zone in the Helvetic domain of the Central Alps of Switzerland, fluid inclusions in quartz, illite ``crystallinity'' index, vitrinite reflectance, and the stable isotope compositions of vein and whole rock minerals and fluids trapped in quartz were investigated along four cross-sections. Increasing temperature during prograde metamorphism led to the formation of dry gas by hydrocarbon cracking in the CH4-zone. Fluid immiscibility in the H2O-CH4-(CO2)-NaCl system resulted in cogenetic, CH4- and H2O-dominated fluid inclusions. In the CH4-zone, fluids were trapped at temperatures <= 270 +/- 5 degrees C. The end of the CH4-zone is markedby a sudden increase of CO2 content in the gas phase of fluid inclusions. At temperatures > 270 +/- 5 degrees C, in the H2O-zone, the total amount of volatiles within the fluid decreased below 1 mol% with no immiscibility. This resulted m total homogenization temperatures of H2O-(CO2-CH4)-NaCl inclusions below 180 degrees C. Hydrogen isotope compositions of methane in fluid inclusion have delta D values of less than -100 parts per thousand in the CH4-zone, typical for an origin through cracking of higher hydrocarbons, but where the methane has not equilibrated with the pore water. delta D values of fluid inclusion water are around -40 parts per thousand., in isotopic equilibrium with phyllosilicates of the whole rocks. Within the CH4 to H2O(CO2) transition zone, delta D(H2O) values in fluid inclusions decrease to -130 parts per thousand interpreted to reflect the contribution of deuterium depleted water from methane oxidation. In the H2O-zone, delta D(H2O) values increase again towards an average of -30 parts per thousand which is again consistent with isotopic equilibrium with host-rock phyllosilicates. delta C-13 values of methane in fluid inclusions from the CH4-zone are around -27 parts per thousand in isotopic equilibrium with calcite in veins and whole rocks. The delta C-13(CH4) values decrease to less than -35 parts per thousand at the transition to the H2O-zone and are no longer in equilibrium with the carbonates in the whole rocks. delta C-13 values of CO, are variable but too low to be in equilibrium with the wall rock fluids, compatible with a contribution of CO2 from closed system oxidation of methane. Differences in isotopic composition between host-rock and Alpine fissure carbonate are generally small, suggesting that the amount of CO2 produced by oxidation of methane was small compared to the C-budget in the rocks and local pore fluids were buffered by the wall rocks during precipitation of calcite within the fissures. (c) 2006 Elsevier B.V. All rights reserved.

Profiling of steroid metabolites after transdermal and oral administration of testosterone by ultra-high pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometry.

The screening of testosterone (T) misuse for doping control is based on the urinary steroid profile, including T, its precursors and metabolites. Modifications of individual levels and ratio between those metabolites are indicators of T misuse. In the context of screening analysis, the most discriminant criterion known to date is based on the T glucuronide (TG) to epitestosterone glucuronide (EG) ratio (TG/EG). Following the World Anti-Doping Agency (WADA) recommendations, there is suspicion of T misuse when the ratio reaches 4 or beyond. While this marker remains very sensitive and specific, it suffers from large inter-individual variability, with important influence of enzyme polymorphisms. Moreover, use of low dose or topical administration forms makes the screening of endogenous steroids difficult while the detection window no longer suits the doping habit. As reference limits are estimated on the basis of population studies, which encompass inter-individual and inter-ethnic variability, new strategies including individual threshold monitoring and alternative biomarkers were proposed to detect T misuse. The purpose of this study was to evaluate the potential of ultra-high pressure liquid chromatography (UHPLC) coupled with a new generation high resolution quadrupole time-of-flight mass spectrometer (QTOF-MS) to investigate the steroid metabolism after transdermal and oral T administration. An approach was developed to quantify 12 targeted urinary steroids as direct glucuro- and sulfo-conjugated metabolites, allowing the conservation of the phase II metabolism information, reflecting genetic and environmental influences. The UHPLC-QTOF-MS(E) platform was applied to clinical study samples from 19 healthy male volunteers, having different genotypes for the UGT2B17 enzyme responsible for the glucuroconjugation of T. Based on reference population ranges, none of the traditional markers of T misuse could detect doping after topical administration of T, while the detection window was short after oral TU ingestion. The detection ability of the 12 targeted steroids was thus evaluated by using individual thresholds following both transdermal and oral administration. Other relevant biomarkers and minor metabolites were studied for complementary information to the steroid profile, including sulfoconjugated analytes and hydroxy forms of glucuroconjugated metabolites. While sulfoconjugated steroids may provide helpful screening information for individuals with homozygotous UGT2B17 deletion, hydroxy-glucuroconjugated analytes could enhance the detection window of oral T undecanoate (TU) doping.

Preclinical evaluation of the immunogenicity of C-type HIV-1-based DNA and NYVAC vaccines in the Balb/C mouse model.

As part of a European initiative (EuroVacc), we report the design, construction, and immunogenicity of two HIV-1 vaccine candidates based on a clade C virus strain (CN54) representing the current major epidemic in Asia and parts of Africa. Open reading frames encoding an artificial 160-kDa GagPolNef (GPN) polyprotein and the external glycoprotein gp120 were fully RNA and codon optimized. A DNA vaccine (DNA-GPN and DNA-gp120, referred to as DNA-C), and a replication-deficient vaccinia virus encoding both reading frames (NYVAC-C), were assessed regarding immunogenicity in Balb/C mice. The intramuscular administration of both plasmid DNA constructs, followed by two booster DNA immunizations, induced substantial T-cell responses against both antigens as well as Env-specific antibodies. Whereas low doses of NYVAC-C failed to induce specific CTL or antibodies, high doses generated cellular as well as humoral immune responses, but these did not reach the levels seen following DNA vaccination. The most potent immune responses were detectable using prime:boost protocols, regardless of whether DNA-C or NYVAC-C was used as the priming or boosting agent. These preclinical findings revealed the immunogenic response triggered by DNA-C and its enhancement by combining it with NYVAC-C, thus complementing the macaque preclinical and human phase I clinical studies of EuroVacc.

A comparison of Bayesian and frequentist approaches to incorporating external information for the prediction of prostate cancer risk.

We present the most comprehensive comparison to date of the predictive benefit of genetics in addition to currently used clinical variables, using genotype data for 33 single-nucleotide polymorphisms (SNPs) in 1,547 Caucasian men from the placebo arm of the REduction by DUtasteride of prostate Cancer Events (REDUCE®) trial. Moreover, we conducted a detailed comparison of three techniques for incorporating genetics into clinical risk prediction. The first method was a standard logistic regression model, which included separate terms for the clinical covariates and for each of the genetic markers. This approach ignores a substantial amount of external information concerning effect sizes for these Genome Wide Association Study (GWAS)-replicated SNPs. The second and third methods investigated two possible approaches to incorporating meta-analysed external SNP effect estimates - one via a weighted PCa 'risk' score based solely on the meta analysis estimates, and the other incorporating both the current and prior data via informative priors in a Bayesian logistic regression model. All methods demonstrated a slight improvement in predictive performance upon incorporation of genetics. The two methods that incorporated external information showed the greatest receiver-operating-characteristic AUCs increase from 0.61 to 0.64. The value of our methods comparison is likely to lie in observations of performance similarities, rather than difference, between three approaches of very different resource requirements. The two methods that included external information performed best, but only marginally despite substantial differences in complexity.

Rhythmic growth explained by coincidence between internal and external cues.

Most organisms use circadian oscillators to coordinate physiological and developmental processes such as growth with predictable daily environmental changes like sunrise and sunset. The importance of such coordination is highlighted by studies showing that circadian dysfunction causes reduced fitness in bacteria and plants, as well as sleep and psychological disorders in humans. Plant cell growth requires energy and water-factors that oscillate owing to diurnal environmental changes. Indeed, two important factors controlling stem growth are the internal circadian oscillator and external light levels. However, most circadian studies have been performed in constant conditions, precluding mechanistic study of interactions between the clock and diurnal variation in the environment. Studies of stem elongation in diurnal conditions have revealed complex growth patterns, but no mechanism has been described. Here we show that the growth phase of Arabidopsis seedlings in diurnal light conditions is shifted 8-12 h relative to plants in continuous light, and we describe a mechanism underlying this environmental response. We find that the clock regulates transcript levels of two basic helix-loop-helix genes, phytochrome-interacting factor 4 (PIF4) and PIF5, whereas light regulates their protein abundance. These genes function as positive growth regulators; the coincidence of high transcript levels (by the clock) and protein accumulation (in the dark) allows them to promote plant growth at the end of the night. Thus, these two genes integrate clock and light signalling, and their coordinated regulation explains the observed diurnal growth rhythms. This interaction may serve as a paradigm for understanding how endogenous and environmental signals cooperate to control other processes.

Arterial wall dosimetry for non-Hodgkin lymphoma patients treated with radioimmunotherapy.

Tumors in non-Hodgkin lymphoma (NHL) patients are often proximal to the major blood vessels in the abdomen or neck. In external-beam radiotherapy, these tumors present a challenge because imaging resolution prevents the beam from being targeted to the tumor lesion without also irradiating the artery wall. This problem has led to potentially life-threatening delayed toxicity. Because radioimmunotherapy has resulted in long-term survival of NHL patients, we investigated whether the absorbed dose (AD) to the artery wall in radioimmunotherapy of NHL is of potential concern for delayed toxicity. SPECT resolution is not sufficient to enable dosimetric analysis of anatomic features of the thickness of the aortic wall. Therefore, we present a model of aortic wall toxicity based on data from 4 patients treated with (131)I-tositumomab. METHODS: Four NHL patients with periaortic tumors were administered pretherapeutic (131)I-tositumomab. Abdominal SPECT and whole-body planar images were obtained at 48, 72, and 144 h after tracer administration. Blood-pool activity concentrations were obtained from regions of interest drawn on the heart on the planar images. Tumor and blood activity concentrations, scaled to therapeutic administered activities-both standard and myeloablative-were input into a geometry and tracking model (GEANT, version 4) of the aorta. The simulated energy deposited in the arterial walls was collected and fitted, and the AD and biologic effective dose values to the aortic wall and tumors were obtained for standard therapeutic and hypothetical myeloablative administered activities. RESULTS: Arterial wall ADs from standard therapy were lower (0.6-3.7 Gy) than those typical from external-beam therapy, as were the tumor ADs (1.4-10.5 Gy). The ratios of tumor AD to arterial wall AD were greater for radioimmunotherapy by a factor of 1.9-4.0. For myeloablative therapy, artery wall ADs were in general less than those typical for external-beam therapy (9.4-11.4 Gy for 3 of 4 patients) but comparable for 1 patient (32.6 Gy). CONCLUSION: Blood vessel radiation dose can be estimated using the software package 3D-RD combined with GEANT modeling. The dosimetry analysis suggested that arterial wall toxicity is highly unlikely in standard dose radioimmunotherapy but should be considered a potential concern and limiting factor in myeloablative therapy.