Bioavailability of repeated oral administration of MDL 100,240, a dual inhibitor of angiotensin-converting enzyme and neutral endopeptidase in healthy volunteers.

BACKGROUND: MDL 100,240 (pyrido[2,1-a] [2]benzazepine-4-carboxylic acid,7-[[2-(acetylthio)-1-oxo-3-phenylpropyl]amino]-1,2,3,4,6,7,8, 12b-octahydro-6-oxo, [4S-[4alpha,7alpha(R(*)),12bbeta]]-) is a molecule possessing an inhibiting ability on both angiotensin converting enzyme (ACE) and neutral endopeptidase, the enzyme responsible for atrial natriuretic peptide (ANP) degradation. Such a dual mechanism of action presents a potential clinical interest for the treatment of hypertension and congestive heart failure. OBJECTIVES: To evaluate the bioavailability of MDL 100,240 and its accumulation over repeated oral administration, using ACE inhibition as a surrogate for plasma drug level and determining its profile after oral and i.v. administration. METHODS: First, in an open, one-period, single-dose study, the ACE inhibition profile was characterised following a 12.5 mg MDL 100,240 i.v. infusion. Second, in a three-group, parallel, randomised, double-blind study, each group of four subjects received q.d., over 8 days, 2.5, 10 or 20 mg of MDL 100,240 orally. The ACE inhibition profile was determined on day 1 and day 8. Trough plasma ACE was measured on days 2, 3 and 4. The recovery of ACE activity was monitored up to 72 h after the last dose of MDL 100,240. RESULTS: ACE inhibition profile was similar on day 1 and day 8, and trough inhibition remained unchanged after the 8 days of treatment with 10 mg or 20 mg. Following repeated 2.5-mg ingestion, trough inhibition increased from 33% to 44% after the eighth dose. The oral bioavailability of MDL 100,240 was estimated at 85%, not statistically different from 100%. The accumulation ratio at steady state was estimated at 112%. Expressing the accumulation ratio in terms of half-life, a t(1/2) of 0.31 days or 7. 5 h was estimated. CONCLUSION: MDL 100,240 (oral solution) has a good bioavailability, as estimated by ACE inhibition, and no drug accumulation seems to occur over 8 days with the 10-mg and 20-mg doses, but a slight rise in the trough level is observed with the 2. 5-mg dose.

Stimulation of thermogenesis in men after combined glucose-long-chain triglyceride infusion.

The effect of combined long-chain triglyceride infusion (Intralipid 20%) with graded doses of insulin/glucose on energy expenditure was examined in 17 healthy young male volunteers by using the euglycemic insulin clamp technique in combination with indirect calorimetry. Intralipid was infused for 90 min at a constant rate of 0.23 g/min; plasma free fatty acids increased from base-line values of 380 +/- 8 mumol/l to steady state levels of 650 +/- 12 mumol/l. After 90 min the Intralipid was continued and insulin was infused at three rates (0.5, 2, and 4 mU/kg . min) to achieve steady state hyperinsulinemic plateaus of 63 +/- 4, 167 +/- 10, and 410 +/- 15 microU/ml. Plasma glucose concentration was maintained constant at basal euglycemic levels (insulin clamp technique) by infusing glucose at 0.24, 0.48, and 0.59 g/min, respectively. Glucose storage during the insulin clamp (ie, glucose uptake minus glucose oxidation) was 0.13, 0.33, and 0.40 g/min for each group and exogenous lipid storage was 0.17, 0.18, and 0.19 g/min, respectively. The net increment in energy expenditure was 0.15, 0.24, and 0.26 kcal/min, respectively, which represents 8.5% of the energy content of the total amount of glucose and lipid stored. The experimentally determined value (approximately 9%) for the cost of storing both glucose and lipid was found to be significantly greater than predicted by stoichiometric calculations. However, the experimental value for the combined infusion was less than that observed for glucose storage alone (12%). This finding provides support for the use of combined glucose/fat infusions in parenteral nutrition as it is used more economically than when glucose is infused alone.

Effects of SCH 34826, an orally active inhibitor of atrial natriuretic peptide degradation, in healthy volunteers.

Atrial natriuretic peptide is cleared from plasma by clearance receptors and by enzymatic degradation by way of a neutral metalloendopeptidase. Inhibition of neutral metalloendopeptidase activity appears to provide an interesting approach to interfere with metabolism of atrial natriuretic peptide to enhance the renal and haemodynamic effects of endogenous atrial natriuretic peptide. In this study, the effects of SCH 34826, a new orally active neutral metalloendopeptidase inhibitor, have been evaluated in a single-blind, placebo-controlled study involving eight healthy volunteers who had maintained a high sodium intake for 5 days. SCH 34826 had no effect on blood pressure or heart rate in these normotensive subjects. SCH 34826 promoted significant increases in excretion of urinary sodium, phosphate, and calcium. The cumulative 5-hour urinary sodium excretion was 15.7 +/- 7.3 mmol for the placebo and 22.9 +/- 5, 26.7 +/- 6 (p less than 0.05), and 30.9 +/- 6.8 mmol (p less than 0.01) for the 400, 800, and 1600 mg SCH 34826 doses, respectively. During the same time interval, the cumulative urinary phosphate excretion increased by 0.3 +/- 0.4 mmol after placebo and by 1.5 +/- 0.3 (p less than 0.01), 1.95 +/- 0.3 (p less than 0.01), and 2.4 +/- 0.4 mmol (p less than 0.001) after 400, 800, and 1600 mg SCH 34826, respectively. There was no change in diuresis or excretion of urinary potassium and uric acid. The natriuretic response to SCH 34826 occurred in the absence of any change in plasma atrial natriuretic peptide levels but was associated with a dose-dependent elevation of urinary atrial natriuretic peptide and cyclic guanosine monophosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Régulation du canal à sodium épithélial par le sodim extracellulaire et développement d'un microsystème intégré pour l'étude électrophysiologique sur ovocytes de "Xenopus laevis"

Résumé : La première partie de ce travail de thèse est consacrée au canal à sodium épithélial (ENaC), l'élément clé du transport transépithélial de Na+ dans le néphron distal, le colon et les voies aériennes. Ce canal est impliqué dans certaines formes génétiques d'hypo- et d'hypertension (PHA I, syndrome de Liddle), mais aussi, indirectement, dans la mucoviscidose. La réabsorption transépithéliale de Na+ est principalement régulée par des hormones (aldostérone, vasopressine), mais aussi directement par le Na+, via deux phénomènes distincts, la « feedback inhibition » et la « self-inhibition » (SI). Ce second phénomène est dépendant de la concentration de Na+ extracellulaire, et montre une cinétique rapide (constante de temps d'environ 3 s). Son rôle physiologique serait d'assurer l'homogénéité de la réabsorption de Na+ et d'empêcher que celle-ci soit excessive lorsque les concentrations de Na+ sont élevées. Différents éléments appuient l'hypothèse de la présence d'un site de détection de la concentration du Na+ extracellulaire sur ENaC, gouvernant la SI. L'objectif de ce premier projet est de démontrer l'existence du site de détection impliqué dans la SI et de déterminer ses propriétés physiologiques et sa localisation. Nous avons montré que les caractéristiques de la SI (en termes de sélectivité et affinité ionique) sont différentes des propriétés de conduction du canal. Ainsi, nos résultats confirment l'hypothèse de l'existence d'un site de détection du Na+ (responsable de la transmission de l'information au mécanisme de contrôle de l'ouverture du canal), différent du site de conduction. Par ailleurs, ce site présente une affinité basse et indépendante du voltage pour le Na+ et le Li+ extracellulaires. Le site semble donc être localisé dans le domaine extracellulaire, plutôt que transmembranaire, de la protéine. L'étape suivante consiste alors à localiser précisément le site sur le canal. Des études précédentes, ainsi que des résultats préliminaires récemment obtenus, mettent en avant le rôle dans la self-inhibition du premiers tiers des boucles extracellulaires des sous-unités α et γ du canal. Le second projet tire son origine des limitations de la méthode classique pour l'étude des canaux ioniques, après expression dans les ovocytes de Xenopus laevis, par la méthode du voltage-clamp à deux électrodes, en particulier les limitations dues à la lenteur des échanges de solutions. En outre, cette méthode souffre de nombreux désavantages (manipulations délicates et peu rapides, grands volumes de solution requis). Plusieurs systèmes améliorés ont été élaborés, mais aucun ne corrige tous les désavantages de la méthode classique Ainsi, l'objectif ici est le développement d'un système, pour l'étude électrophysiologique sur ovocytes, présentant les caractéristiques suivantes : manipulation des cellules facilitée et réduite, volumes de solution de perfusion faibles et vitesse rapide d'échange de la perfusion. Un microsystème intégré sur une puce a été élaboré. Ces capacités de mesure ont été testées en utilisant des ovocytes exprimant ENaC. Des résultats similaires (courbes IV, courbes dose-réponse au benzamil) à ceux obtenus avec le système traditionnel ont été enregistrés avec le microsystème. Le temps d'échange de solution a été estimé à ~20 ms et des temps effectifs de changement ont été déterminés comme étant 8 fois plus court avec le nouveau système comparé au classique. Finalement, la SI a été étudiée et il apparaît que sa cinétique est 3 fois plus rapide que ce qui a été estimé précédemment avec le système traditionnel et son amplitude de 10 à 20 % plus importante. Le nouveau microsystème intégré apparaît donc comme adapté à la mesure électrophysiologique sur ovocytes de Xenopus, et possèdent des caractéristiques appropriées à l'étude de phénomènes à cinétique rapide, mais aussi à des applications de type « high throughput screening ». Summary : The first part of the thesis is related to the Epithelial Sodium Channel (ENaC), which is a key component of the transepithelial Na+ transport in the distal nephron, colon and airways. This channel is involved in hypo- and hypertensive syndrome (PHA I, Liddle syndrome), but also indirectly in cystic fibrosis. The transepithelial reabsorption of Na+ is mainly regulated by hormones (aldosterone, vasopressin), but also directly by Na+ itself, via two distinct phenomena, feedback inhibition and self-inhibition. This latter phenomenon is dependant on the extracellular Na+ concentration and has rapid kinetics (time constant of about 3 s). Its physiological role would be to prevent excessive Na+ reabsorption and ensure this reabsorption is homogenous. Several pieces of evidence enable to propose the hypothesis of an extracellular Na+ sensing site on ENaC, governing self-inhibition. The aim of this first project is to demonstrate the existence of the sensing site involved in self-inhibition and to determine its physiological properties and localization. We show self-inhibition characteristics (ionic selectivity and affinity) are different from the conducting properties of the channel. Our results support thus the hypothesis that the Na+ sensing site (responsible of the transmission of the information about the extracellular Na+ concentration to the channel gating mechanism), is different from the channel conduction site. Furthermore, the site has a low and voltage-insensitive affinity for extracellular Na+ or Li+. This site appears to be located in the extracellular domain rather than in the transmembrane part of the channel protein. The next step is then to precisely localize the site on the channel. Some previous studies and preliminary results we recently obtained highlight the role of the first third of the extracellular loop of the α and γ subunits of the channel in self-inhibition. The second project originates in the limitation of the classical two-electrode voltageclamp system classically used to study ion channels expressed in Xenopus /aevis oocytes, in particular limitations related to the slow solution exchange time. In addition, this technique undergoes several drawbacks (delicate manipulations, time consumption volumes). Several improved systems have been built up, but none corrected all these detriments. The aim of this second study is thus to develop a system for electrophysiological study on oocytes featuring an easy and reduced cell handling, small necessary perfusion volumes and fast fluidic exchange. This last feature establishes the link with the first project, as it should enable to improve the kinetics analysis of self-inhibition. A PDMS chip-based microsystem has been elaborated. Its electrophysiological measurement abilities have been tested using oocytes expressing ENaC. Similar measurements (IV curves of benzamil-sensitive currents, benzamil dose-response curves) have been obtained with this system, compared to the traditional one. The solution exchange time has been estimated at N20 ms and effective exchange times (on inward currents) have been determined as 8 times faster with the novel system compared to the classical one. Finally, self-inhibition has been studied and it appears its kinetics is 3 times faster and its amplitude 10 to 20 % higher than what has been previously estimated with the traditional system. The novel integrated microsystem appears therefore to be convenient for electrophysiological measurement on Xenopus oocytes, and displays features suitable for the study of fast kinetics phenomenon, but also high throughput screening applications. Résumé destiné large public : Le corps humain est composé d'organes, eux-mêmes constitués d'un très grand nombre de cellules. Chaque cellule possède une paroi appelée membrane cellulaire qui sépare l'intérieur de cette cellule (milieu intracellulaire) du liquide (milieu extracellulaire) dans lequel elle baigne. Le maintien de la composition stable de ce milieu extracellulaire est essentiel pour la survie des cellules et donc de l'organisme. Le sodium est un des composants majeurs du milieu extracellulaire, sa quantité dans celui-ci doit être particulièrement contrôlée. Le sodium joue en effet un rôle important : il conditionne le volume de ce liquide extracellulaire, donc, par la même, du sang. Ainsi, une grande quantité de sodium présente dans ce milieu va de paire avec une augmentation du volume sanguin, ce qui conduit l'organisme à souffrir d'hypertension. On se rend donc compte qu'il est très important de contrôler la quantité de sodium présente dans les différents liquides de l'organisme. Les apports de sodium dans l'organisme se font par l'alimentation, mais la quantité de sodium présente dans le liquide extracellulaire est contrôlée de manière très précise par le rein. Au niveau de cet organe, on appelle urine primaire le liquide résultant de la filtration du sang. Elle contient de nombreuses substances, des petites molécules, dont l'organisme a besoin (sodium, glucose...), qui sont ensuite récupérées dans l'organe. A la sortie du rein, l'urine finale ne contient plus que l'excédent de ces substances, ainsi que des déchets à éliminer. La récupération du sodium est plus ou moins importante, en fonction des ajustements à apporter à la quantité présente dans le liquide extracellulaire. Elle a lieu grâce à la présence de protéines, dans les membranes des cellules du rein, capables de le transporter et de le faire transiter de l'urine primaire vers le liquide extracellulaire, qui assurera ensuite sa distribution dans l'ensemble de l'organisme. Parmi ces protéines « transporteurs de sodium », nous nous intéressons à une protéine en particulier, appelée ENaC. Il a été montré qu'elle jouait un rôle important dans cette récupération de sodium, elle est en effet impliquée dans des maladies génétiques conduisant à l'hypo- ou à l'hypertension. De précédents travaux ont montré que lorsque le sodium est présent en faible quantité dans l'urine primaire, cette protéine permet d'en récupérer une très grande partie. A l'inverse, lorsque cette quantité de sodium dans l'urine primaire est importante, sa récupération par le biais d'ENaC est réduite. On parle alors d'autorégulation : la protéine elle-même est capable d'adapter son activité de transport en fonction des conditions. Ce phénomène d'autorégulation constitue a priori un mécanisme préventif visant à éviter une trop grande récupération de sodium, limitant ainsi les risques d'hypertension. La première partie de ce travail de thèse a ainsi consisté à clarifier le mécanisme d'autorégulation de la protéine ENaC. Ce phénomène se caractérise en particulier par sa grande vitesse, ce qui le rend difficile à étudier par les méthodes traditionnelles. Nous avons donc, dans une deuxième partie, développé un nouveau système permettant de mieux décrire et analyser cette « autorégulation » d'ENaC. Ce second projet a été mené en collaboration avec l'équipe de Martin Gijs de l'EPFL.

Preclinical vaccine study of Plasmodium vivax circumsporozoite protein derived-synthetic polypeptides formulated in montanide ISA 720 and montanide ISA 51 adjuvants.

Plasmodium vivax circumsporozoite (CS) protein is a leading malaria vaccine candidate previously assessed in animals and humans. Here, combinations of three synthetic polypeptides corresponding to amino (N), central repeat (R), and carboxyl (C) regions of the CS protein formulated in Montanide ISA 720 or Montanide ISA 51 adjuvants were assessed for immunogenicity in rodents and primates. BALB/c mice and Aotus monkeys were divided into test and control groups and were immunized three times with doses of 50 and 100 μg of vaccine or placebo. Antigen-specific antimalarial antibodies were determined by enzyme-linked immunosorbent assay, immunofluorescent antibody test, and IFN-γ responses by enzyme-linked immunosorbent spot (ELIspot). Both vaccine formulations were highly immunogenic in both species. Mice developed better antibody responses against C and R polypeptides, whereas the N polypeptide was more immunogenic in monkeys. Anti-peptide antibodies remained detectable for several months and recognized native proteins on sporozoites. Differences between Montanide ISA 720 and Montanide ISA 51 formulations were not significant.

Changes in sleeping and basal energy expenditure and substrate oxidation induced by short term thyroxin administration in man.

The present study was designed to explore the thermogenic effect of thyroid hormone administration and the resulting changes in nitrogen homeostasis. Normal male volunteers (n = 7) received thyroxin during 6 weeks. The first 3-week period served to suppress endogenous thyroid secretion (180 micrograms T4/day). This dose was doubled for the next 3 weeks. Sleeping energy expenditure (respiratory chamber) and BMR (hood) were measured by indirect calorimetry, under standardized conditions. Sleeping heart rate was continuously recorded and urine was collected during this 12-hour period to assess nitrogen excretion. The changes in energy expenditure, heart rate and nitrogen balance were then related to the excess thyroxin administered. After 3 weeks of treatment, serum TSH level fell to 0.15 mU/L, indicating an almost complete inhibition of the pituitary-thyroid axis. During this phase of treatment there was an increase in sleeping EE and sleeping heart rate, which increased further by doubling the T4 dose (delta EE: +8.5 +/- 2.3%, delta heart rate +16.1 +/- 2.2%). The T4 dose, which is currently used as a substitutive dose, lead to a borderline hyperthyroid state, with an increase in EE and heart rate. Exogenous T4 administration provoked a significant increase in urinary nitrogen excretion averaging 40%. It is concluded that T4 provokes an important stimulation of EE, which is mostly mediated by an excess protein oxidation.

Bolus injections of synthetic atrial natriuretic peptide in patients with chronic renal failure or nephrotic syndrome.

The diuretic and natriuretic responses to exogenous synthetic atrial natriuretic peptide (ANP) were evaluated in patients with chronic renal failure (CRF) or nephrotic syndrome (NS). Patients were studied after an oral water load (8 ml/kg in CRF and 20 ml/kg in NS patients). A short intravenous bolus of either a placebo or ANP was administered when urine output was stable. In each group of patients, three doses of ANP were injected at 24 h intervals, i.e., 1.0, 1.5, and 2.0 micrograms/kg in the CRF and 1.0, 1.5, and 3.0 micrograms/kg in the NS group. Blood pressure and heart rate were monitored throughout the study and urinary volume and electrolyte excretion were measured every 20 min up to 3 h after the bolus. An acute and transient fall in blood pressure was observed immediately after the ANP injection. It was more pronounced in CRF than in NS patients. In CRF patients, ANP caused only a slight increase in urinary volume (13.5-44% over baseline) but a significant increase in urinary sodium excretion (45-114% over baseline). In NS patients, significant increases in both urine volume (60-105%) and sodium excretion (149-248%) were also found. In these latter patients, the renal response to ANP appeared to be better preserved. The hemodynamic and renal changes induced by ANP occurred mainly during the first 20 min following the ANP administration, when the peak plasma ANP levels were obtained. However, no clear dose-response effect could be evidenced in either group with the three doses of ANP chosen in this study.

Determination of four sequential stages during microautophagy in vitro.

Microautophagy is the transfer of cytosolic components into the lysosome by direct invagination of the lysosomal membrane and subsequent budding of vesicles into the lysosomal lumen. This process is topologically equivalent to membrane invagination during multivesicular body formation and to the budding of enveloped viruses. Vacuoles are lysosomal compartments of yeasts. Vacuolar membrane invagination can be reconstituted in vitro with purified yeast vacuoles, serving as a model system for budding of vesicles into the lumen of an organelle. Using this in vitro system, we defined different reaction states. We identified inhibitors of microautophagy in vitro and used them as tools for kinetic analysis. This allowed us to characterize four biochemically distinguishable steps of the reaction. We propose that these correspond to sequential stages of vacuole invagination and vesicle scission. Formation of vacuolar invaginations was slow and temperature-dependent, whereas the final scission of the vesicle from a preformed invagination was fast and proceeded even on ice. Our observations suggest that the formation of invaginations rather than the scission of vesicles is the rate-limiting step of the overall reaction.

Canakinumab (ACZ885) vs triamcinolone acetonide for treatment of acute flares and prevention of recurrent flares in gouty arthritis patients refractory to or contraindicated to nsaids and/or colchicine.

Purpose: Current treatments for arthritis flares in gout (gouty arthritis) are not effective in all patients and may be contraindicated in many due to underlying comorbidities. Urate crystals activate the NALP 3 inflammasome which stimulate production of IL-1β, driving inflammatory processes. Targeted IL-1β blockade may be an alternative treatment for gouty arthritis. Canakinumab (ACZ885) is a fully human monoclonal anti- IL-1β antibody with a long half-life (28 days). Method: This was an 8-weeks, dose-ranging, multicenter, blinded, double-dummy, active-controlled trial of patients ≥18 to ≤80 y with an acute gouty arthritis flare, refractory to or contraindicated to NSAIDs and/or colchicine. Patients were randomized to 1 subcutanous (sc) dose of canakinumab (10, 25, 50, 90, or 150 mg) or 1 intra muscular (im) dose of triamcinolone acetonide (TA) [40 mg]. The primary variable was assessed 72 h post-dose, measured on a 0-100 mm VAS pain scale. Secondary variables included pain intensity 24 and 48 h post dose, time to 50% reduction in pain intensity, and time to recurrence of gout flares up to 8 weeks post dose. Results: 200 patients were enrolled (canakinumab n=143, TA n=57) and 191 completed the study. A statistically significant dose response was observed at 72 h. The 150 mg dose reached superior pain relief compared to TA starting from 24h: estimated mean difference in pain intensity on 0-100 mm VAS was -11.5 at 24 h, -18.2 at 48 h, and -19.2 at 72 h (all p<0.05). Canakinumab 150 mg provided a rapid onset of pain relief: median time to 50% reduction in pain was reached at 1 day with canakinumab 150 mg vs 2 days for the TA group (p=0.0006). The probability of recurrent gout flares was 3.7% with canakinumab 150 mg vs. 45.4% with TA 8 weeks post treatment, a relative risk reduction of 94% (p=0.006). Serious AEs occurred in 2 patients receiving canakinumab (appendicitis and carotid artery stenosis) and 1 receiving TA (cerebrovascular disorder). Investigator's reported these events as not study drug related. There were no discontinuations due to AEs. Conclusion: Canakinumab 150 mg provided faster onset and superior pain relief compared to TA for acute flares in gouty arthritis patients refractory to or contraindicated to standard treatments. The 150 mg dose of canakinumab prevented recurrence of gout flares with a relative risk reduction compared to TA of 94% at 8 weeks post-dose, and was well tolerated.

Ätiologie und Management der Gicht [The etiology and management of gout].

Gout is an inflammatory arthritis caused by monosodium urate (MSU) crystal deposits in and around the joint. The formation of urinary calculi can also occur in gout, but are less common than arthritis. Gout usually presents with recurrent episodes of joint inflammation, which over time lead to tophus formation and joint destruction. In the last decade, significant advances have been made regarding not only the epidemiology and genetics of gout and hyperuricemia but also the mechanisms of inflammation and treatment of gout. In addition, knowledge concerning the key role of interleukin 1 (IL-1) has provided new therapeutic perspectives. However, the current management of gout is often suboptimal, with many Patienten either not receiving adequate treatment or being unable to tolerate existing treatments. New therapeutic agents provide interesting new options for Patienten with difficult-to-treat gouty arthritis.The English full-text version of this is available at SpringerLink (under "Supplemental").

Effect of age and gender on citalopram and desmethylcitalopram steady-state plasma concentrations in adults and elderly depressed patients.

The effect of aging on steady-state plasma concentrations of citalopram (CIT) and desmethylcitalopram (DCIT) was investigated in 128 depressive patients treated with 10-80 mg/day CIT. They were separated into three groups, with age up to 64 years (mean age+/-S.D.: 47+/-12 years; n=48), between 65 and 79 years (72+/-1 years; n=57), and from 80 years or older (84+/-1 years; n=23). Body mass index (BMI), renal and hepatic functions were similar in the three groups. A large interindividual variability of plasma levels of CIT (16-fold) and DCIT (12-fold) was measured for a given dose. The mean plasma levels of CIT corrected for a 20 mg daily dose were 55% higher in the very elderly (>=80 years) patients (65+/-30 ng/ml; p<0.001) and 38% higher in the elderly (65-79 years) patients (58+/-24 ng/ml; p<0.001) when compared to the adult patients (42+/-17 ng/ml). DCIT mean plasma level was 38% higher (p<0.05) in the group of very elderly patients (22+/-10 ng/ml) when compared to the adult patients (16+/-9 ng/ml). As a consequence, the mean plasma concentration of CIT+DCIT was 48% higher in the very elderly patients (86+/-36 ng/ml; p<0.001) and 33% higher in the elderly patients (77+/-28 ng/ml; p<0.001) when compared to the adult patients (58+/-21 ng/ml). Age correlated significantly with CIT (r=0.43, p<0.001), DCIT (r=0.28, p<0.01), and CIT+DCIT plasma levels (r=0.44, p<0.001), and thus accounts for 18% of the variability of CIT plasma levels, with no influence of gender. The recommended dose reduction of CIT in elderly patients seems therefore justified.

Asymmetric cancer cell division regulated by AKT.

Human tumors often contain slowly proliferating cancer cells that resist treatment, but we do not know precisely how these cells arise. We show that rapidly proliferating cancer cells can divide asymmetrically to produce slowly proliferating "G0-like" progeny that are enriched following chemotherapy in breast cancer patients. Asymmetric cancer cell division results from asymmetric suppression of AKT/PKB kinase signaling in one daughter cell during telophase of mitosis. Moreover, inhibition of AKT signaling with small-molecule drugs can induce asymmetric cancer cell division and the production of slow proliferators. Cancer cells therefore appear to continuously flux between symmetric and asymmetric division depending on the precise state of their AKT signaling network. This model may have significant implications for understanding how tumors grow, evade treatment, and recur.

An effector-reduced anti-β-amyloid (Aβ) antibody with unique aβ binding properties promotes neuroprotection and glial engulfment of Aβ.

Passive immunization against β-amyloid (Aβ) has become an increasingly desirable strategy as a therapeutic treatment for Alzheimer's disease (AD). However, traditional passive immunization approaches carry the risk of Fcγ receptor-mediated overactivation of microglial cells, which may contribute to an inappropriate proinflammatory response leading to vasogenic edema and cerebral microhemorrhage. Here, we describe the generation of a humanized anti-Aβ monoclonal antibody of an IgG4 isotype, known as MABT5102A (MABT). An IgG4 subclass was selected to reduce the risk of Fcγ receptor-mediated overactivation of microglia. MABT bound with high affinity to multiple forms of Aβ, protected against Aβ1-42 oligomer-induced cytotoxicity, and increased uptake of neurotoxic Aβ oligomers by microglia. Furthermore, MABT-mediated amyloid plaque removal was demonstrated using in vivo live imaging in hAPP((V717I))/PS1 transgenic mice. When compared with a human IgG1 wild-type subclass, containing the same antigen-binding variable domains and with equal binding to Aβ, MABT showed reduced activation of stress-activated p38MAPK (p38 mitogen-activated protein kinase) in microglia and induced less release of the proinflammatory cytokine TNFα. We propose that a humanized IgG4 anti-Aβ antibody that takes advantage of a unique Aβ binding profile, while also possessing reduced effector function, may provide a safer therapeutic alternative for passive immunotherapy for AD. Data from a phase I clinical trial testing MABT is consistent with this hypothesis, showing no signs of vasogenic edema, even in ApoE4 carriers.

Effects of exercise on the isoelectric patterns of erythropoietin.

OBJECTIVES: Recombinant erythropoietin has a strong impact on aerobic power and is therefore one of the most potent doping agents in endurance sports. The anti-doping control of this synthetic hormone relies on the detection, in the urine, of its isoelectric pattern, which differs from that of the corresponding natural hormone, the latter being typically more acidic than the former. However, a small number of natural urinary patterns, referred to as "atypical patterns," are less acidic than the dominant form. Based on anecdotal evidence, the occurrence of such patterns seems to be related to particular strenuous exercises. This study aimed to demonstrate this relation using a strenuous exercise protocol. DESIGN: Seven athletes took part in a training protocol including a series of supramaximal short-duration exercises. Urine and blood samples were collected throughout the protocols. SETTINGS: World Cycling Center, Aigle, Switzerland, and research laboratories. PARTICIPANTS: Seven top-level athletes (cyclists) were involved in this study. MAIN OUTCOME MEASURES: Erythropoietin (EPO) isoelectric patterns were obtained by submitting blood and urine samples to isoelectric focusing. Additional protein dosages were performed. RESULTS: Supramaximal short-duration exercises induced the transformation of typical urinary natural EPO patterns into atypical ones. None of the obtained atypical patterns fulfilled the 3 criteria mandatory for reporting an adverse analytical finding. Serum EPO patterns were not affected by the exercises that caused the transformation of urinary patterns. CONCLUSION: An exercise-induced transient renal dysfunction is proposed as a hypothetic explanation for these observations that rely on parallel investigations of proteinuria in the same samples.