Photodynamic therapy with mTHPC and polyethylene glycol-derived mTHPC: a comparative study on human tumour xenografts.

The photosensitizing properties of m-tetrahydroxyphenylchlorin (mTHPC) and polyethylene glycol-derivatized mTHPC (pegylated mTHPC) were compared in nude mice bearing human malignant mesothelioma, squamous cell carcinoma and adenocarcinoma xenografts. Laser light (20 J/cm2) at 652 nm was delivered to the tumour (surface irradiance) and to an equal-sized area of the hind leg of the animals after i.p. administration of 0.1 mg/kg body weight mTHPC and an equimolar dose of pegylated mTHPC, respectively. The extent of tumour necrosis and normal tissue injury was assessed by histology. Both mTHPC and pegylated mTHPC catalyse photosensitized necrosis in mesothelioma xenografts at drug-light intervals of 1-4 days. The onset of action of pegylated mTHPC seemed slower but significantly exceeds that of mTHPC by days 3 and 4 with the greatest difference being noted at day 4. Pegylated mTHPC also induced significantly larger photonecrosis than mTHPC in squamous cell xenografts but not in adenocarcinoma at day 4, where mTHPC showed greatest activity. The degree of necrosis induced by pegylated mTHPC was the same for all three xenografts. mTHPC led to necrosis of skin and underlying muscle at a drug-light interval of 1 day but minor histological changes only at drug-light intervals from 2-4 days. In contrast, pegylated mTHPC did not result in histologically detectable changes in normal tissues under the same treatment conditions at any drug-light interval assessed. In this study, pegylated mTHPC had advantages as a photosensitizer compared to mTHPC. Tissue concentrations of mTHPC and pegylated mTHPC were measured by high-performance liquid chromatography in non-irradiated animals 4 days after administration. There was no significant difference in tumour uptake between the two sensitizers in mesothelioma, adenocarcinoma and squamous cell carcinoma xenografts. Tissue concentration measurements were of limited use for predicting photosensitization in this model.

Methionine enkephalin: immunomodulator in normal volunteers, cancer and AIDS patients

Clinical studies of the immunological effects of methionine enkephalin in normal volunteers, cancer, and AIDS patients are summarized. The major immunology changes seen were increases in T cell subsets, natural killer activity, as well as mitogen blastogenesis. Clinically, the cancer and ARC patients did not develop infections.

Photodynamic therapy : a pathway for enhanced delivery of liposomal doxorubicin to tumors

Abstract Part I : Background : Isolated lung perfusion (ILP) was designed for the treatment of loco-regional malignancies of the lung. In contrast to intravenous (IV) drug application, ILP allows for a selective administration of cytostatic agents such as doxorubicin to the lung while sparing non-affected tissues. However, the clinical results with ILP were disappointing. Doxorubicinbased ILP on sarcoma rodent lungs suggested high overall doxorubicin concentrations within the perfused lung but a poor penetration of the cytostatic agent into tumors. The same holds true for liposomal-encapsulated macromolecular doxorubicin (LiporubicinTM) In specific conditions, low-dose photodynamic therapy (PDT) can enhance the distribution of macromolecules across the endothelial bamer in solid tumors. It was recently postulated that tumor neovessels were more responsive to PDT than the normal vasculature. We therefore hypothesized that Visudyne®-mediated PDT could selectively increase liposomal doxorubicin (LiporubicinTM) uptake in sarcoma tumors to rodent lungs during intravenous (IV) drug administration and isolated lung perfusion (ILP). Material and Methods : A sarcoma tumor was generated in the left lung of Fisher rats by subpleural injection of a sarcoma cell ,suspension via thoracotomy. Ten days later, LiporubicinTM is administered IV or by single pass antegrade ILP, with or without Visudyne® -mediated low-dose PDT pre-treatment of the sarcoma bearing lung. The drug concentration and distribution were assessed separately in tumors and lung tissues by high pressure liquid chromatography (HPLC) and fluorescence microscopy (FNI~, respectively. Results : PDT pretreatment before IV LiporubicinTM administration resulted in a significantly higher tumor drug uptake and tumor to lung drug ratio compared to IV drug injection alone without affecting the blood flow and drug distribution in the lung. PDT pre-treatment before LiporubicinTM-based ILP also resulted in a higher tumor drug uptake and a higher tumor to lung drug ratio compared to ILP alone, however, these differences were not significant due to a heterogeneous blood flow drug distribution during ILP which was further accentuated by PDT. Conclusions : Low-dose Visudyne®-mediated PDT pre-treatment has the potential to selectively enhance liposomal encapsulated doxorubicin uptake in tumors but not in normal lung tissue after IV drug application in a rat model of sarcoma tumors to the lung which opens new perspectives for the treatment of superficially spreading chemoresistant tumors of the chest cavity such as mesothelioma or malignant effusion. However, the impact of PDT on macromolecular drug uptake during ILP is limited since its therapeutic advantage is circumvented by ILP-induced heterogeneicity of blood flow and drug distribution Abstract Part II Background : Photodynamic therapy (PDT) with Visudyne® acts by direct cellular phototoxicity and/or by an indirect vascular-mediated effect. Here, we demonstrate that the vessel integrity interruption by PDT can promote the extravasation of a macromolecular agent in normal tissue. To obtain extravasation in normal tissue PDT conditions were one order of magnitude more intensive than the ones in tissue containing neovessels reported in the literature. Material and Methods : Fluorescein isothiocyanate dextran (FITC-D, 2000kDa), a macromolecular agent, was intravenously injected 10 minutes before (LKO group, n=14) or 2 hours (LK2 group, n=16) after Visudyne® mediated PDT in nude mice bearing a dorsal skin fold chamber. Control animals had no PDT (CTRL group, n=8). The extravasation of FITC-D from blood vessels in striated muscle tissue was observed in both groups in real-time for up to 2500 seconds after injection. We also monitored PDT-induced leukocyte rolling in-vivo and assessed, by histology, the corresponding inflammatory reaction score in the dorsal skin fold chambers. Results : In all animals, at the applied PDT conditions, FITC-D extravasation was significantly enhanced in the PDT treated areas as compared to the surrounding non-treated areas (p<0.0001). There was no FITC-D leakage in the control animals. Animals from the LKO group had significantly less FITC-D extravasation than those from the LK2 group (p = 0.0002). In the LKO group FITC-D leakage correlated significantly with the inflammation (p < 0.001). Conclusions: At the selected conditions, Visudyne-mediated PDT promotes vascular leakage and FITC-D extravasation into the interstitial space of normal tissue. The intensity of vascular leakage depends on the time interval between PDT and FITC-D injection. This concept could be used to locally modulate the delivery of macromolecules in vivo. Résumé : La perfusion cytostatique isolée du poumon permet une administration sélective des agents cytostatiques sans implication de la circulation systémique avec une forte accumulation au niveau du poumon mais une faible pénétration dans les tumeurs. La thérapie photodynamique (PDT) qui consiste en l'application d'un sensibilisateur activé par lumière laser non- thermique d'une longueur d'onde définie permet dans certaines conditions, une augmentation de la pénétration des agents cytostatiques macromoléculaires à travers la barrière endothéliale tumorale. Nous avons exploré cet avantage thérapeutique de la PDT dans un modèle expérimental afin d'augmenter d'une manière sélective la pénétration tumorale de la doxorubicin pegylée, liposomal- encapsulée macromoléculaire (Liporubicin). Une tumeur sarcomateuse a été générée au niveau du poumon de rongeur suivie d'administration de Liporubicin, soit par voie intraveineuse soit par perfusion isolée du poumon (ILP). Une partie des animaux ont reçus un prétraitement de la tumeur et du poumon sous jacent par PDT avec Visudyne comme photosensibilisateur. Les résultats ont démontrés que la PDT permet, sous certaines conditions, une augmentation sélective de Liporubicin dans les tumeurs mais pas dans le parenchyme pulmonaire sous jacent. Après administration intraveineuse de Liporubicin et prétraitement par PDT, l'accumulation dans les tumeurs était significative par rapport au poumon, et aux tumeurs sans PDT. Le même phénomène est observé après ILP du poumon. Cependant, les différences avec ou sans PDT n'étaient pas significatives lié à und distribution hétérogène de Liporubicin dans le poumon perfusé après ILP. Dans une deuxième partie de l'expérimentation, nous avons exploré la microscopie intra-vitale pour déterminer l'extravasion des substances macromoléculaires (FITS) à travers la barrière endothéliale avec ou sans Visudyne-PDT au niveau des chambres dorsales des souris nues. Les résultats montrent qu'après PDT, l'extravasion de FITS a été augmentée de manière significative par rapport au tissu non traité. L'intensité de l'extravasion de FITS dépendait également de l'intervalle entre PDT et injection de FITS. En conclusion, les expérimentations montrent que la PDT est capable, sous certaines conditions, d'augmenter de manière significative l'extravasion des macromolécules à travers la barrière endothéliale et leur accumulation dans des tumeurs mais pas dans le parenchyme pulmonaire. Ces résultats permettent une nouvelle perspective de traitement pour des tumeurs superficielles intrathoraciques chimio-résistent comme l'épanchement pleural malin ou le mésothéliome pleural.

Effects of hyperglycemia on glucose metabolism before and after oral glucose ingestion in normal men.

The plasma glucose excursion may influence the metabolic responses after oral glucose ingestion. Although previous studies addressed the effects of hyperglycemia in conditions of hyperinsulinemia, it has not been evaluated whether the route of glucose administration (oral vs. intravenous) plays a role. Our aim was to determine the effects of moderately controlled hyperglycemia on glucose metabolism before and after oral glucose ingestion. Eight normal men underwent two oral glucose clamps at 6 and 10 mmol/l plasma glucose. Glucose turnover and cycling rates were measured by infusion of [2H7]glucose. The oral glucose load was labeled by D-[6,6-2H2]glucose to monitor exogenous glucose appearance, and respiratory exchanges were measured by indirect calorimetry. Sixty percent of the oral glucose load appeared in the systemic circulation during both the 6 and 10 mmol/l plasma glucose tests, although less endogenous glucose appeared during the 10 mmol/l tests before glucose ingestion (P &lt; 0.05). This inhibitory effect of hyperglycemia was not detectable after oral glucose ingestion, although glucose utilization was increased (+28%, P &lt; 0.05) due to increased nonoxidative glucose disposal [10 vs. 6 mmol/l: +20%, not significant (NS) before oral glucose ingestion; +40%, P &lt; 0.05 after oral glucose ingestion]. Glucose cycling rates were increased by hyperglycemia (+13% before oral glucose ingestion, P &lt; 0.001; +31% after oral glucose ingestion, P &lt; 0.05) and oral glucose ingestion during both the 6 (+10%, P &lt; 0.05) and 10 mmol/l (+26%, P &lt; 0.005) tests. A moderate hyperglycemia inhibits endogenous glucose production and contributes to glucose tolerance by enhancing nonoxidative glucose disposal. Hyperglycemia and oral glucose ingestion both stimulate glucose cycling.

Quantification of clenbuterol at trace level in human urine by ultra-high pressure liquid chromatography-tandem mass spectrometry.

Clenbuterol is a β2 agonist agent with anabolic properties given by the increase in the muscular mass in parallel to the decrease of the body fat. For this reason, the use of clenbuterol is forbidden by the World Anti-Doping Agency (WADA) in the practice of sport. This compound is of particular interest for anti-doping authorities and WADA-accredited laboratories due to the recent reporting of risk of unintentional doping following the eating of meat contaminated with traces of clenbuterol in some countries. In this work, the development and the validation of an ultra-high pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method for the quantification of clenbuterol in human urine is described. The analyte was extracted from urine samples by liquid-liquid extraction (LLE) in basic conditions using tert butyl-methyl ether (TBME) and analyzed by UHPLC-MS/MS with a linear gradient of acetonitrile in 9min only. The simple and rapid method presented here was validated in compliance with authority guidelines and showed a limit of quantification at 5pg/mL and a linearity range from 5pg/mL to 300pg/mL. Good trueness (85.8-105%), repeatability (5.7-10.6% RSD) and intermediate precision (5.9-14.9% RSD) results were obtained. The method was then applied to real samples from eighteen volunteers collecting urines after single oral doses administration (1, 5 and 10μg) of clenbuterol-enriched yogurts.

Rapid high-performance liquid chromatographic determination with fluorescence detection of furosemide in human body fluids and its confirmation by gas chromatography-mass spectrometry.

Furosemide (FD: Lasix) is a loop diuretic which strongly increases both urine flow and electrolyte urinary excretion. Healthy volunteers were administered 40 mg orally (dissolved in water) and concentrations of FD were determined in serum and urine for up to 6 h for eight subjects, who absorbed water at a rate of 400 ml/h. Quantification was performed by HPLC with fluorescence detection (excitation at 233 nm, emission at 389 nm) with a limit of detection of 5 ng/ml for a 300-microliters sample. The elution of FD was completed within 4 min using a gradient of acetonitrile concentration rising from 30 to 50% in 0.08 M phosphoric acid. The delay to the peak serum concentration ranged from 60 to 120 min. FD was still easily measurable in the sera from all subjects 6 h after administration. In urine, the excretion rates reached their maximum between 1 and 3 h. The total amount of FD excreted in the urine averaged 11.2 mg (range 7.6-14.0 mg), with a mean urine volume of 3024 ml (range 2620-3596 ml). Moreover, the urine density was lower than 1.010 (recommended as an upper limit in doping analysis to screen diuretics) only for 2 h. An additional volunteer was administered 40 mg of FD and his urine was collected over a longer period. FD was still detectable 48 h after intake. Gas chromatography-mass spectrometry with different types of ionization was used to confirm the occurrence of FD after permethylation of the extract. Negative-ion chemical ionization, with ammonia as reactant gas, was found to be the most sensitive method of detection.

Effectiveness of three different walking prescription durations on total physical activity in normal- and overweight women.

OBJECTIVE: While there is a dose-response relationship between physical activity (PA) and health benefit, little is known about the effectiveness of different PA prescriptions on total daily PA. AIM: To test, under real-life conditions and using an objective, non-invasive measurement technique (accelerometry), the effect of prescribing additional physical activity (walking only) of different durations (30, 60 and 90 min/day) on compliance (to the activity prescribed) and compensation (to total daily PA). Participants in each group were prescribed 5 sessions of walking per week over 4 weeks. METHODS: 55 normal-weight and overweight women (mean BMI 25 ± 5 kg/m(2), height 165 ± 1 cm, weight 68 ± 2 kg and mean age 27 ± 1 years) were randomly assigned to 3 prescription groups: 30, 60 or 90 min/day PA. RESULTS: Walking duration resulted in an almost linear increase in the number of steps per day during the prescription period from an average of about 10,000 steps per day for the 30-min prescription to about 14,000 for the 90-min prescription. Compliance was excellent for the 30-min prescription but decreased significantly with 60-min and 90-min prescriptions. In parallel, degree of compensation subsequent to exercise increased progressively as length of prescription increased. CONCLUSION: A 30-min prescription of extra walking 5 times per week was well tolerated. However, in order to increase total PA further, much more than 60 min of walking may need to be prescribed in the majority of individuals. While total exercise 'volume' increased with prescriptions longer than 30 min, compliance to the prescription decreased and greater compensation was evident. © 2014 S. Karger GmbH, Freiburg.

Extensor carpi ulnaris (ECU) subsheath: Normal MRI appearance and findings in athletic injuries : 40

Purpose: First, to report ECU subsheath's normal MRI appearance and the findings in athletic injuries. Second, to determine the best MRI sequence for diagnosis. Methods and materials: Sixteen patients (13 males, 3 females, mean age 30.3 years) with ECU subsheath's athletic injuries sustained between January 2003 and June 2009 were retrospectively reviewed. Wrist MRI studies were performed on 1.5-T units and consisted of at least transverse T1 and STIR sequences in pronation, and FS Gd T1 in pronation and supination. Two radiologists assessed the following items, in consensus: injury type (A to C according to Inoue), ECU tendon stability, and associated lesions (ulnar head oedema, extensor retinaculum injury, ECU tendinosis and tenosynovitis). Then, each reader independently rated the sequences' diagnostic value: 0 = questionable, 1 = suggestive, 2 = certain. Follow-up studies were present in 8 patients. ECU subsheath's normal visibility (medial, central and lateral parts) was retrospectively evaluated in 30 consecutive control MRI studies. Results: FS Gd T1 sequences in supination (1.63) and pronation (1.59) were the most valuable for diagnosis, compared to STIR (1.22) and T1 (1). The study group included 9 type A, 1 type B and 6 type C injuries. There were trends towards diminution in pouches' size, signal intensity and enhancement in follow-up studies, along with tendon stabilization within the ulnar groove. In control studies, ECU subsheath's visibility in medial, central and lateral parts were noted in 66.7-80%, 63.3-80% and 30-50% respectively. Conclusion: ECU subsheath's athletic injuries are visible on 1.5-T MRI studies. FS Gd T1 sequences in supination and pronation are the most valuable.

Long-term fluctuation of relative afferent pupillary defect in subjects with normal visual function.

PURPOSE: To determine whether the relative afferent pupillary defect (RAPD) remains constant over time in normal subjects. METHODS: Seventeen normal subjects were tested with infrared pupillography and automated perimetry in four sessions over 3 years. The changes in RAPD and visual field asymmetry between testing sessions were compared. RESULTS: The range of RAPD was 0.0 to 0.3 log unit, and the difference in the mean deviation between the eyes on automated static perimetry was 0 to 3 dB. Eight subjects repeatedly had an RAPD in the same eye. There was no correlation between the RAPD and the visual field asymmetry at the same visit. Changes in the magnitude of the RAPD between any two sessions were typically small (median, 0.08 log unit; 25th percentile, 0.04 log unit; 75th percentile, 0.15 log unit). CONCLUSIONS: Some normal subjects may show a persistent but small RAPD in the absence of detectable pathologic disease. Therefore, an isolated RAPD in the range of 0.3 log unit that is not associated with any other significant historical or clinical finding should probably be considered benign.

Pareto or log-normal? A recursive-truncation approach to the distribution of (all) cities

Traditionally, it is assumed that the population size of cities in a country follows a Pareto distribution. This assumption is typically supported by nding evidence of Zipf's Law. Recent studies question this nding, highlighting that, while the Pareto distribution may t reasonably well when the data is truncated at the upper tail, i.e. for the largest cities of a country, the log-normal distribution may apply when all cities are considered. Moreover, conclusions may be sensitive to the choice of a particular truncation threshold, a yet overlooked issue in the literature. In this paper, then, we reassess the city size distribution in relation to its sensitivity to the choice of truncation point. In particular, we look at US Census data and apply a recursive-truncation approach to estimate Zipf's Law and a non-parametric alternative test where we consider each possible truncation point of the distribution of all cities. Results con rm the sensitivity of results to the truncation point. Moreover, repeating the analysis over simulated data con rms the di culty of distinguishing a Pareto tail from the tail of a log-normal and, in turn, identifying the city size distribution as a false or a weak Pareto law.

Multiplex ultra-performance liquid chromatography-tandem mass spectrometry method for simultaneous quantification in human plasma of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole, voriconazole-N-oxide, anidulafungin, and caspofungin.

Therapeutic drug monitoring (TDM) may contribute to optimizing the efficacy and safety of antifungal therapy because of the large variability in drug pharmacokinetics. Rapid, sensitive, and selective laboratory methods are needed for efficient TDM. Quantification of several antifungals in a single analytical run may best fulfill these requirements. We therefore developed a multiplex ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method requiring 100 μl of plasma for simultaneous quantification within 7 min of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole, voriconazole-N-oxide, caspofungin, and anidulafungin. Protein precipitation with acetonitrile was used in a single extraction procedure for eight analytes. After reverse-phase chromatographic separation, antifungals were quantified by electrospray ionization-triple-quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. Deuterated isotopic compounds of azole antifungals were used as internal standards. The method was validated based on FDA recommendations, including assessment of extraction yields, matrix effect variability (<9.2%), and analytical recovery (80.1 to 107%). The method is sensitive (lower limits of azole quantification, 0.01 to 0.1 μg/ml; those of echinocandin quantification, 0.06 to 0.1 μg/ml), accurate (intra- and interassay biases of -9.9 to +5% and -4.0 to +8.8%, respectively), and precise (intra- and interassay coefficients of variation of 1.2 to 11.1% and 1.2 to 8.9%, respectively) over clinical concentration ranges (upper limits of quantification, 5 to 50 μg/ml). Thus, we developed a simple, rapid, and robust multiplex UPLC-MS/MS assay for simultaneous quantification of plasma concentrations of six antifungals and two metabolites. This offers, by optimized and cost-effective lab resource utilization, an efficient tool for daily routine TDM aimed at maximizing the real-time efficacy and safety of different recommended single-drug antifungal regimens and combination salvage therapies, as well as a tool for clinical research.

Normal kinetics of intestinal glucose absorption in the absence of GLUT2: evidence for a transport pathway requiring glucose phosphorylation and transfer into the endoplasmic reticulum.

Glucose is absorbed through the intestine by a transepithelial transport system initiated at the apical membrane by the cotransporter SGLT-1; intracellular glucose is then assumed to diffuse across the basolateral membrane through GLUT2. Here, we evaluated the impact of GLUT2 gene inactivation on this transepithelial transport process. We report that the kinetics of transepithelial glucose transport, as assessed in oral glucose tolerance tests, was identical in the presence or absence of GLUT2; that the transport was transcellular because it could be inhibited by the SGLT-1 inhibitor phlorizin, and that it could not be explained by overexpression of another known glucose transporter. By using an isolated intestine perfusion system, we demonstrated that the rate of transepithelial transport was similar in control and GLUT2(-/-) intestine and that it was increased to the same extent by cAMP in both situations. However, in the absence, but not in the presence, of GLUT2, the transport was inhibited dose-dependently by the glucose-6-phosphate translocase inhibitor S4048. Furthermore, whereas transport of [(14)C]glucose proceeded with the same kinetics in control and GLUT2(-/-) intestine, [(14)C]3-O-methylglucose was transported in intestine of control but not of mutant mice. Together our data demonstrate the existence of a transepithelial glucose transport system in GLUT2(-/-) intestine that requires glucose phosphorylation and transfer of glucose-6-phosphate into the endoplasmic reticulum. Glucose may then be released out of the cells by a membrane traffic-based pathway similar to the one we previously described in GLUT2-null hepatocytes.