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The purpose of the present study was to quantitatively determine the expression of transporters, receptors and tight junction molecules at the blood-arachnoid barrier (BAB) and blood-spinal cord barrier (BSCB) in cervical, thoracic and lumbar spines from dogs.The expression levels of 31 transporters, 3 receptors, 1 tight junction protein, and 3 marker proteins in leptomeninges and capillaries isolated from spines (3 male and 2 female dogs) were determined by quantitative Targeted Absolute Proteomics (qTAP). The units were converted from fmol/μg protein to pmol/cm (absolute abundance at the BAB and the BSCB in a 1 cm section of spine).The expression of MDR1 and BCRP were greater at the BSCB compared to the BAB (especially in the cervical cord), and the expressions at the lumbar BSCB were lower than that for the cervical BSCB. Among the organic anionic and cationic drug transporters, OAT1, OAT3, MRP1, OCT2 and MATE1/2 were detected only in the BAB, and not at the BSCB). The expression of these transporters was higher in the order: lumbar thoracic cervical BAB. The expressions of GLUT1, 4F2hc, EAAT1, 2, PEPT2, CTL1, and MCT1 at the BSCB of the cervical cord were higher than the corresponding values for the cervical BAB, and these values decreased in going down the spinal cord.These results provide a better understanding of the molecular mechanisms underlying the concentration gradients of drugs and endogenous substances in the cerebrospinal fluid and parenchyma of the spinal cord.

ZusammenfassungPharyngo-ösophageale Perforationen [PEP] sind eine sehr seltene (0,02 -1,52%) 1 jedoch lebensgefährliche Komplikation (Mortalität 16 – 50%) 2 nach anteriorer zervikaler Dekompression und Fusion. Die therapeutischen Optionen unterliegen weiterhin einer wissenschaftlichen Diskussion. Wir berichten über den Fall eines 67-jährigen Patienten mit septischem Krankheitsbild bei Aortenklappenendokarditis und Mitralklappenendokarditis sowie Nachweis einer floriden Spondylodiszitis HWK 3/4 mit knöcherner Destruktion der Endplatten sowie prävertebralem Abszess von HWK 2 – 5. Nach operativer Versorgung mittels Débridement und ACDF mit ventraler Plattenosteosynthese zeigte sich im frühen postoperativen Verlauf eine progrediente Schluckstörung mit Mediastinits, bei Arrosion und Defekt der Pharynxhinterwand. Nach Entfernung der ventralen Plattenosteosynthese konnte unter konservativer Therapie mit regelmäßigen endoskopischen Kontrollen und medikamentöser antibiotischer und antimykotischer Therapie ein Verschluss des Defektes der Pharynxhinterwand sowie ein oraler Kostaufbau erzielt werden.

The purpose of this study was to elucidate the absolute abundance of transporters, enzymes, receptors, and tight junction and marker proteins at human blood-arachnoid barrier (BAB) and compare with those of dogs and pigs. Protein expression levels in plasma membrane fractions of brain leptomeninges were determined by quantitative targeted absolute proteomics. To realistically compare the absolute abundance of target molecules at the BAB among humans, dogs, and pigs, the unit was converted from fmol/μg-protein to pmol/cm

International audience; Rho-associated kinase (ROCK) activation was shown to contribute to microvascular closure, retinal hypoxia, and to retinal pigment epithelium (RPE) barrier disruption in a rat model of diabetic retinopathy. Fasudil, a clinically approved ROCK inhibitor, improved retinal perfusion and reduced edema in this model, indicating that ROCK inhibition could be a promising new therapeutic approach for the treatment of diabetic retinopathy. However, due to its short intravitreal half-life, fasudil is not suitable for long-term treatment. In this study, we evaluated a very potent ROCK1/2 inhibitor (BIRKI) in a depot formulation administered as a single intravitreal injection providing a slow release for at least four weeks. Following BIRKI intravitreal injection in old Goto-Kakizaki (GK) type 2 diabetic rats, we observed a significant reduction in ROCK1 activity in the retinal pigment epithelium/choroid complex after 8 days and relocation of ROCK1 to the cytoplasm and nucleus in retinal pigment epithelium cells after 28 days. The chronic ROCK inhibition by the BIRKI depot formulation restored retinal pigment epithelial cell morphology and distribution, favored retinal capillaries dilation, and reduced hypoxia and inner blood barrier leakage observed in the diabetic retina. No functional or morphological negative effects were observed, indicating suitable tolerability of BIRKI after intravitreous injection. In conclusion, our data suggest that sustained ROCK inhibition, provided by BIRKI slow-release formulation, could be a valuable treatment option for diabetic retinopathy, especially with regard to the improvement of retinal vascular infusion and protection of the outer retinal barrier.

Inflammation is implicated in the etiology of diverse retinopathies including uveitis, age-related macular degeneration or diabetic retinopathy. Tumor necrosis factor alpha (TNF-α) is a well-known proinflammatory cytokine that is described as a biomarker for inflammation in diverse retinopathies and therefore emerged as an interesting target to treat inflammation in the eye by neutralizing anti-TNF-α antibodies.Recently, we have demonstrated that Adeno-associated virus (AAV)-mediated expression of human TNF-α in the murine eye induces retinal inflammation including vasculitis and fibrosis, thereby mimicking human disease-relevant pathologies. In a proof-of-mechanism study, we now tested whether AAV-TNF-α induced pathologies can be reversed by neutralizing TNF-α antibody treatment.Strikingly, a single intravitreal injection of the TNF-α antibody golimumab reduced AAV-TNF-α-induced retinal inflammation and retinal thickening. Furthermore, AAV-TNF-α-mediated impaired retinal function was partially rescued by golimumab as revealed by electroretinography recordings. Finally, to study TNF-α-induced vasculitis in human in vitro cell culture assays, we established a monocyte-to-endothelium adhesion co-culture system. Indeed, also in vitro TNF-α induced monocyte adhesion to human retinal endothelial cells, which was prevented by golimumab.Overall, our study describes valuable in vitro and in vivo approaches to study the function of TNF-α in retinal inflammation and demonstrated a preclinical proof-of-mechanism treatment with golimumab.The AAV-based model expressing human TNF-α allows us to investigate TNF-α-driven pathologies supporting research in mechanisms of retinal inflammation.

Purpose Retinopathies display complex pathologies, including vasculopathies, inflammation, and fibrosis, leading ultimately to visual impairment. However, animal models accurately reflecting these pathologies are lacking. In this study, we evaluate the suitability of using Adeno-associated virus (AAV)-mediated long-term expression of cytokines to establish retinal pathology in the murine retina. Methods We administered recombinant, Muller-glia targeted AAV-ShH10 into the mouse vitreous to induce retinal expression of either human vascular endothelial growth factor (VEGF)-A165, tumor necrosis factor alpha (TNF-α), or interleukin-6 (IL-6) and evaluated consequent effects by optical coherence tomography, fluorescein angiography, and histology. Results Intravitreal injection of AAVs resulted in rapid and stable expression of the transgenes within 1 to 6 weeks. Akin to the role of VEGF-A in wet age-related macular degeneration, expression of VEGF-A led to several vasculopathies in mice, including neovascularization and vascular leakage. In contrast, the expression of the proinflammatory cytokines TNF-α or IL-6 induced retinal inflammation, as indicated by microglial activation. Furthermore, the expression of TNF-α, but not of IL-6, induced immune cell infiltration into the vitreous as well as vasculitis, and subsequently induced the development of fibrosis and epiretinal membranes. Conclusions In summary, the long-term expression of human VEGF-A165, TNF-α, or IL-6 in the mouse eye induced specific pathologies within 6 weeks that mimic different aspects of human retinopathies. Translational relevance AAV-mediated expression of human genes in mice is an attractive approach to provide valuable insights into the underlying molecular mechanisms causing retinopathies and is easily adaptable to other genes and preclinical species supporting drug discovery for retinal diseases.

Transporters at the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) play a pivotal role as gatekeepers for efflux or uptake of endogenous and exogenous molecules. The protein expression of a number of them has already been determined in the brains of rodents, nonhuman primates, and humans using quantitative targeted absolute proteomics (QTAP). The dog is an important animal model for drug discovery and development, especially for safety evaluations. The purpose of the present study was to clarify the relevance of the transporter protein expression for drug distribution in the dog brain and CSF. We used QTAP to examine the protein expression of 17 selected transporters and receptors at the dog BBB and BCSFB. For the first time, we directly linked the expression of two efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), to regional brain and CSF distribution using specific substrates. Two cocktails, each containing one P-gp substrate (quinidine or apafant) and one BCRP substrate (dantrolene or daidzein) were infused intravenously prior to collection of the brain. Transporter expression varied only slightly between the capillaries of different brain regions and did not result in region-specific distribution of the investigated substrates. There were, however, distinct differences between brain capillaries and choroid plexus. Largest differences were observed for BCRP and P-gp: both were highly expressed in brain capillaries, but no BCRP and only low amounts of P-gp were detected in the choroid plexus. K

N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) is an established cellular model underlying learning and memory, and involves intracellular signaling mediated by the second messenger cyclic guanosine monophosphate (cGMP). As phosphodiesterase (PDE)9A selectively hydrolyses cGMP in areas of the brain related to cognition, PDE9A inhibitors may improve cognitive function by enhancing NMDA receptor-dependent LTP. This study aimed to pharmacologically characterize BI 409306, a novel PDE9A inhibitor, using in vitro assays and in vivo determination of cGMP levels in the brain. Further, the effects of BI 409306 on synaptic plasticity evaluated by LTP in ex vivo hippocampal slices and on cognitive performance in rodents were also investigated. In vitro assays demonstrated that BI 409306 is a potent and selective inhibitor of human and rat PDE9A with mean concentrations at half-maximal inhibition (IC50) of 65 and 168 nM. BI 409306 increased cGMP levels in rat prefrontal cortex and cerebrospinal fluid and attenuated a reduction in mouse striatum cGMP induced by the NMDA-receptor antagonist MK-801. In ex vivo rat brain slices, BI 409306 enhanced LTP induced by both weak and strong tetanic stimulation. Treatment of mice with BI 409306 reversed MK-801-induced working memory deficits in a T-maze spontaneous-alternation task and improved long-term memory in an object recognition task. These findings suggest that BI 409306 is a potent and selective inhibitor of PDE9A. BI 409306 shows target engagement by increasing cGMP levels in brain, facilitates synaptic plasticity as demonstrated by enhancement of hippocampal LTP, and improves episodic and working memory function in rodents. SIGNIFICANCE STATEMENT: This preclinical study demonstrates that BI 409306 is a potent and selective PDE9A inhibitor in rodents. Treatment with BI 409306 increased brain cGMP levels, promoted long-term potentiation, and improved episodic and working memory performance in rodents. These findings support a role for PDE9A in synaptic plasticity and cognition. The potential benefits of BI 409306 are currently being investigated in clinical trials.

Half-life extension strategies to reduce the intravitreal dosing frequency of biomolecules for the treatment of retinal neovascular diseases are attracting increasing interest. This study investigated ocular and systemic pharmacokinetics of the trivalent nanobody BI-X (with affinity to VEGF, Ang-2 and human albumin) in cynomolgus monkeys after intravitreal injection. BI-X concentrations were measured in serial samples of plasma, vitreous humor, aqueous humor and retina. Ocular pharmacokinetics of BI-X exhibited two phases. Initially up to 2-4 weeks after dosing, BI-X concentrations in vitreal, aqueous humor and retina declined with half-lives of around 3 days, which is comparable to macromolecules with a similar molecular weight. Thereafter, only vitreal concentrations were measurable, with a terminal half-life of 13.2 days, which is considerably longer than expected based on the BI-X molecular weight or hydrodynamic radius. It is hypothesized that binding of BI-X to low levels of intraocular albumin resulted in this half-life extension. BI-X was detectable in plasma up to 10 weeks post-dosing. Plasma pharmacokinetics of BI-X exhibited a similar biphasic disposition profile to the vitreous body, with a terminal half-life of 11.8 days, thus reflecting input kinetics from the eye. In conclusion, an important half-life extension principle based on vitreal albumin binding could be confirmed in a primate model, and the data obtained can potentially be translated to humans taking into account the differing vitreal albumin concentrations.

Linagliptin is a selective, competitive dipeptidyl peptidase-4 (DPP-4) inhibitor, recently approved in the USA, Japan and Europe for the treatment of type 2 diabetes. It has non-linear pharmacokinetics and, unlike other DPP-4 inhibitors, a largely non-renal excretion route. It was hypothesised that P-glycoprotein (P-gp)-mediated intestinal transport could influence linagliptin bioavailability, and might contribute to its elimination. Two studies evaluated the role of P-gp-mediated transport in the bioavailability and intestinal secretion of linagliptin in rats. In the bioavailability study, male Wistar rats received single oral doses of linagliptin, 1 or 15 mg/kg, plus either the P-gp inhibitor, zosuquidar trihydrochloride, or vehicle. For the intestinal secretion study, rats underwent bile duct cannulation, and urine, faeces, and bile were collected. At the end of the study, gut content was sampled. Inhibition of intestinal P-gp increased the bioavailability of orally administered linagliptin, indicating that this transport system plays a role in limiting the uptake of linagliptin from the intestine. This effect was dependent on linagliptin dose, and could play a role in its non-linear pharmacokinetics after oral dosing. Systemically available linagliptin was mainly excreted unchanged via bile (49% of i.v. dose), but some (12%) was also excreted directly into the gut independently of biliary excretion. Thus, direct excretion of linagliptin into the gut may be an alternative excretion route in the presence of liver and renal impairment. The primarily non-renal route of excretion is likely to be of benefit to patients with type 2 diabetes, who have a high prevalence of renal insufficiency.

Kurzfassung In modernen Fahrwerkssystemen sind, beispielsweise als Traggelenke, vielfach sphärische, vorgespannte Stahl-Polymergleitlager eingesetzt. Die mehrachsige Lastbeaufschlagung in Verbindung mit der sphärischen Wirkgeometrie führt während des Betriebs im Inneren des Gelenkes zu einer Verschleißverteilung auf der Gleitfläche. Entsprechend stellt sich die Beanspruchung im Inneren des Gelenkes erst in Wechselwirkung mit dieser Verschleiß- bzw. mit einer möglichen irreversiblen Verformungsverteilung ein und verändert sich insbesondere über die Betriebsdauer des Gelenkes. Dieser Beitrag stellt einen Ansatz innerhalb einer Gelenkverschleißsimulation vor, der die Rückkopplung zwischen der Verschleiß- und Pressungsverteilung berücksichtigt. Besondere Herausforderung ist dabei, eine Modellierung zu finden, die sich auf der einen Seite für verschiedene Gelenke einfach mit den üblicherweise vorhandenen Gelenkdaten (CAD, FEM) parametrieren lässt und auf der anderen Seite Rechenzeiten benötigt, welche die Simulation von mehrstündigen Beaufschlagungen zulässt. Die Vorstellung exemplarischer Simulationsergebnisse sowie eine ausblickende Betrachtung möglicher Erweiterungen, beispielsweise durch die Implementierung von plastischen Verformungen in ähnlicher Weise, runden den Beitrag ab.

Purpose Postoperative intravaginal brachytherapy for endometrial carcinoma is usually performed with 192 Ir high-dose rate (HDR) afterloading. A potential alternative is treatment with a broadband 50kV X-ray point source, the advantage being its low energy and the consequential steep dose gradient. The aim of this study was to create and evaluate a homogeneous cylindrical energy deposition around a newly designed vaginal applicator. Methods and Materials To create constant isodose layers along the cylindrical plastic vaginal applicator, the source (INTRABEAM system) was moved in steps of 17–19.5 mm outward from the tip of the applicator. Irradiation for a predetermined time was performed at each position. The axial shift was established by a stepping mechanism that was mounted on a table support. The total dose/dose distribution was determined using film dosimetry (Gafchromic EBT) in a "solid water" phantom. The films were evaluated with Mathematica 5.2 and OmniPro-I'mRT 1.6. The results (dose D0/D5/D10 in 0/5/10 mm tissue depth) were compared with an 192 Ir HDR afterloading plan for multiple sampling points around the applicator. Results Three different dose distributions with lengths of 3.9–7.3 cm were created. The irradiation time based on the delivery of 5/7 Gy to a 5 mm tissue depth was 19/26 min to 27/38 min. D0/D5/D10 was 150%/100%/67% for electronic brachytherapy and 140%/100%/74% for the afterloading technique. The deviation for repeated measurements in the phantom was Conclusions It is possible to create a homogeneous cylindrical dose distribution, similar to 192 Ir HDR afterloading, through the superimposition of multiple spherical dose distributions by stepping a kilovolt point source.

A new chemical class of potent DPP-4 inhibitors structurally derived from the xanthine scaffold for the treatment of type 2 diabetes has been discovered and evaluated. Systematic structural variations have led to 1 (BI 1356), a highly potent, selective, long-acting, and orally active DPP-4 inhibitor that shows considerable blood glucose lowering in different animal species. 1 is currently undergoing clinical phase IIb trials and holds the potential for once-daily treatment of type 2 diabetics.

Background Pyogenic infections of the lumbar spine are a rare but critical pathology, yet with considerably high mortality rates. In cases indicating surgical therapy, the implantation of titanium cages or autologous bone grafts represent today's gold standard. Although non-metallic implants such as poly-ether-ether-ketone (PEEK) have proven to be advantageous in diverse degenerative conditions, their saftey and practicability in lumbar spine infection remains questionable. Moreover, the efficacy of a single-step radical debridement of the infected disc space with subsequent fusion from a strictly posterior approach continues to be an issue of debate. We therefore sought to evaluate the feasibility, clinical and radiological outcome of a single-step TLIF procedure using oblique PEEK cages in the surgical management of patients with lumbar pyogenic spondylodiscitis. Methods From January 2009 through December 2013, all patients meeting the indication for surgical treatment of lumbar pyogenic spondylodiscitis were included. Patients demonstrating intact cortical bone on preoperative CT received a single-step radical debridement of the infected intervertebral disc space, posterior screw-and-rod instrumentation and implantation of an oblique PEEK cage using the TLIF technique. Oral antibiotics were continued for 12 weeks postoperatively and clinical and radiological results recorded throughout a minimum 1-year clinical follow-up. Results A total of 104 patients were admitted to receive surgical therapy for lumbar pyogenic spondylodiscitis. Within this patient population, 18 patients met the diagnostic criteria to receive the implantation of an oblique PEEK cage. Pathogens were detected in 13 cases with Staph. aureus being the predominant causative organism. All patients were discharged to recover in their domestic environment. Throughout the first year of clinical and radiological follow-up and beyond, none of the 18 patients demonstrated any signs of residual neurologic deficits or recurrent infection. Furthermore, two-plane conventional X-rays showed no significant implant subsidence or failure at any of the given time-points in up to 5 years postoperatively. Conclusions In patients meeting the criteria for surgical treatment of lumbar pyogenic spondylodiscitis, the implantation of PEEK cages using a single-step TLIF approach is a safe and feasible procedure. Based on our experience, the concern of a recurrent infection when implanting non-metallic cages may be refuted in carefully selected patients.

Typical antipsychotic drugs with a high extrapyramidal motor side-effects liability markedly increase extracellular dopamine in the caudate-putamen, while atypical antipsychotic drugs with a low incidence of extrapyramidal motor side-effects have less pronounced stimulating actions on striatal dopamine. Therefore, it has been suggested that the extrapyramidal motor side-effects liability of antipsychotic drugs (APD) is correlated with their ability to increase extracellular dopamine in the caudate-putamen. The globus pallidus (GP) is another basal ganglia structure probably mediating extrapyramidal motor side-effects of typical antipsychotic drugs. Therefore, the present study sought to determine whether extracellular dopamine in the globus pallidus might be a further indicator to differentiate neurochemical actions of typical and atypical antipsychotic drugs. Using in vivo microdialysis we compared effects on pallidal dopamine induced by typical and atypical antipsychotic drugs in rats. Experiment I demonstrated that systemic administration of haloperidol (1 mg/kg; i.p.) and clozapine (20 mg/kg; i.p.) induced a significant pallidal dopamine release to about 160 and 180% of baseline, respectively. Experiment II revealed that reverse microdialysis of raclopride and clozapine using a cumulative dosing regimen did not stimulate extracellular dopamine in the globus pallidus if low (1microM) or intermediate (10 and 100 microM) concentrations were used. Only at a high concentration (1,000 microM), raclopride and clozapine induced a significant pallidal dopamine release to about 130 and 300% of baseline values, respectively. Thus, effects of typical and atypical antipsychotic drugs on pallidal dopamine were similar and thus, may not be related to their differential extrapyramidal motor side-effects liability. Furthermore, the finding that reverse microdialysis of raclopride over a wide range of concentrations did not stimulate pallidal dopamine concentrations tentatively suggests that pallidal dopamine release under basal conditions is not regulated by D2 autoreceptors.

The globus pallidus (GP) receives a prominent dopamine (DA) input by collaterals from the nigrostriatal pathway. However, the neurochemical regulation of extracellular DA in the GP is unknown at present. In the present study we investigated whether activation of ionotropic glutamate receptors by reverse microdialysis of NMDA and AMPA elicits changes in extracellular DA in the GP. Results show that reverse microdialysis of 70 microM AMPA and of 1 mM NMDA produced a significant and reversible increase of dialysate DA levels to about 130% and 160%, respectively. These data provide evidence that ionotropic glutamate receptors in the GP exert excitatory actions on DA release which might be brought about by actions on presynaptic ionotropic glutamate receptors in the GP or involve stimulation of midbrain DA neurons through trans-synaptic pathways.

W e describe a system capable of measuring spatially resolved re- ¯ ectance spectra from 380 to 950 nm anduorescence excitation emission m atrices from 330 to 500 nm excitation and 380 to 700 nm emission in vivo. System performance was compared to that of a standard scanning spectrouorimeter. This ``FastEEM`` system was used to interrogate human normal and neoplastic oral cavity mu- cosa in vivo. Measurem ents were m ade through a ® ber-optic probe and require 4 min total m easurement time. W e present a method based on autocorrelation vectors to identify excitation and em ission wavelengths where the spectra of normal and pathologic tissues dif- fer most. The FastEEM system provides a tool with which to study the relative diagnostic ability of changes in absorption, scattering, anduorescence properties of tissue. Index Headings: Fluorescence; Reectance; EEM; Tissue; Head and neck; Neoplasia.

Linagliptin (BI 1356) is a novel dipeptidyl peptidase-4 (DPP-4) inhibitor in clinical development for the treatment of type 2 diabetes. It exhibits non-linear pharmacokinetics and shows concentration-dependent plasma protein binding to its target, DPP-4. The aim of this study was to investigate the impact of saturable binding of linagliptin to plasma and tissue DPP-4 by comparing the pharmacokinetics of linagliptin in wildtype and DPP-4 deficient Fischer rats using non-compartmental and model-based data analysis. The non-compartmental analysis revealed a significantly reduced AUC in DPP-4 deficient rats compared with wildtype rats when single intravenous doses ⩽1 mg/kg were administered, but the exposure was similar in both strains at higher doses. The terminal half-lives were significantly shorter in DPP-4 deficient rats compared with wildtype rats. For doses ⩽1 mg/kg, DPP-4 deficient rats exhibited linear pharmacokinetics, whereas the pharmacokinetics of wildtype rats was non-linear. In the model-based analysis these differences could be accounted for by assuming concentration-dependent protein binding in the central and one peripheral compartment in wildtype rats. In the model, disposition parameters for unbound linagliptin were assumed to be identical in both rat strains. Simulations with different doses of linagliptin and different concentrations of binding sites further illustrated that the interdependence of linagliptin and DPP-4 in plasma and in the periphery has a major influence on the disposition of linagliptin in wildtype rats. In conclusion, the study showed that the concentration-dependent binding of linagliptin to its target DPP-4 has a major impact on the plasma pharmacokinetics of linagliptin. Copyright © 2009 John Wiley & Sons, Ltd.

BI 1356 (INN: linagliptin) is an inhibitor of dipeptidyl peptidase-4 (DPP-4). This study investigated whether saturable binding of BI 1356 to its target DPP-4 occurs in tissues and whether drug accumulation occurs at these sites in vivo. In order to test these hypotheses, the tissue distribution of BI 1356 was determined in wild-type and DPP-4 deficient rats at different dose levels by means of whole body autoradiography and measurement of tissue radioactivity concentrations after single i.v. dosing of [(14)C]-radio labeled BI 1356. The accumulation behavior of drug-related radioactivity in tissues was further explored in an oral repeat dose study. Tissue levels of [(14)C]BI 1356 related radioactivity were markedly lower in all investigated tissues of the DPP-4 deficient rats and the difference of the dose-dependent increase of radioactivity tissue levels between both rat strains indicates that tissue distribution at low doses of BI 1356 is dominated by binding of BI 1356 to DPP-4 in tissues. As the binding to DPP-4 is strong but reversible, the tissue binding results in a long terminal half-life in several tissues including plasma. The binding capacity to DPP-4 is, however, limited. In the rat, saturation of DPP-4 binding is suggested at an intravenous dose above 0.01-0.1 mg/kg [(14)C]BI 1356. As the DPP-4 binding capacity is saturated already at low doses, accumulation of BI 1356 in tissues is unlikely, despite the long persistence of low amounts in the body.

A microwave-induced plasma (MIP) source produced through a surface-wave propagation process (surfatron) and a furnace atomization non-thermal excitation spectrometry source have been used to determine N and O in gaseous and aqueous samples, respectively, through the formation and emission of NH and OH radicals. Water is removed through liquid nitrogen cold trapping and hydrogen is added to the He plasma gas. The spectral characteristics of the two sources have been compared.

The present study examined the dopaminergic innervation of the rat globus pallidus by in vivo microdialysis and immunohistochemistry in more detail. Using tyrosine hydroxylase immunohistochemistry, two classes of dopaminergic fibers were distinguished morphologically in the globus pallidus. Unilateral infusion of 6-hydroxydopamine into the substantia nigra produced a loss of dopaminergic fiber density in the globus pallidus which was correlated with the nigral extent of the lesion. These findings are in line with the notion that a degenerative loss of nigral dopaminergic cell bodies might also affect the dopamine input of extrastriatal structures such as the globus pallidus. Using in vivo microdialysis, we tested whether dopamine measured in the globus pallidus is of neuronal origin. Perfusion of tetrodotoxin induced a strong and transient decrease of pallidal dopamine. The tetrodotoxin-sensitivity of pallidal dopamine demonstrates the functional significance of the nigropallidal dopaminergic innervation.

There is no satisfactory mechanism to detect premalignant lesions in the upper aero-digestive tract. Fluorescence spectroscopy has potential to bridge the gap between clinical examination and invasive biopsy; however, optimal excitation wavelengths have not yet been determined. The goals of this study were to determine optimal excitation‐emission wavelength combinations to discriminate normal and precancerous/cancerous tissue, and estimate the performance of algorithms based on fluorescence. Fluorescence excitation‐emission matrices (EEM) were measured in vivo from 62 sites in nine normal volunteers and 11 patients with a known or suspected premalignant or malignant oral cavity lesion. Using these data as a training set, algorithms were developed based on combinations of emission spectra at various excitation wavelengths to determine which excitation wavelengths contained the most diagnostic information. A second validation set of fluorescence EEM was measured in vivo from 281 sites in 56 normal volunteers and three patients with a known or suspected premalignant or malignant oral cavity lesion. Algorithms developed in the training set were applied without change to data from the validation set to obtain an unbiased estimate of algorithm performance. Optimal excitation wavelengths for detection of oral neoplasia were 350, 380 and 400 nm. Using only a single emission wavelength of 472 nm, and 350 and 400 nm excitation, algorithm performance in the training set was 90% sensitivity and 88% specificity and in the validation set was 100% sensitivity, 98% specificity. These results suggest that fluorescence spectroscopy can provide a simple, objective tool to improve in vivo identification of oral cavity neoplasia.

Brain microdialysis has been used to examine the in vivo effects of potassium and calcium on dopamine release in the dorsal globus pallidus (GP) of rats. Furthermore, the effects of food presentation and consumption on dopamine release in the GP were investigated. Basal dopamine levels in the GP were below the detection limit, therefore nomifensine (30 microM) was added to the perfused artificial cerebrospinal fluid (aCSF). A prominent increase of dopamine release to 370% was observed after perfusion with elevated potassium (100 mM), while perfusion with calcium-free aCSF produced a significant decrease of dopamine efflux to 36% of control levels. Furthermore, presentation and consumption of food resulted in a rapid increase of extracellular dopamine to 130%. The present experiments demonstrate that in the GP extracellular dopamine can be measured by in vivo brain microdialysis. The data suggest that the dopamine release in the GP can be stimulated by a depolarising agent and involves a partially calcium-dependent release mechanism. The data further suggest that dopamine in basal ganglia structures downstream the striatum as the GP is involved in signalling of important stimuli in the environment, e.g. food.

Fluorescence and reflectance spectroscopy are promising techniques for early diagnosis of neoplasia. Consistent differences have been found in spectra of normal and neoplastic oral mucosa. However, the optimal wavelengths to discriminate between normal, and neoplastic tissue in vivo have not been identified, nor has the diagnostic ability of reflectance and fluorescence spectroscopy been compared. To answer this question, the authors have designed and tested a system to collect autofluorescence emission spectra at 18 different excitation wavelengths (330 to 500 nm). These spectra are assembled into excitation emission matrices (EEM). Reflectance spectra are measured from 300-900 nm. In vivo fluorescence EEMs and reflectance spectra of normal and neoplastic oral mucosa were obtained. The system consists of: an Xe arc lamp, monochromator and filter wheel, which provides excitation; a fiber-optic probe, which directs light to the tissue and collects fluorescence from one location and diffusely reflected light from three locations; and a filter wheel, imaging spectrograph and CCD, which detects the reflectance and fluorescence. The probe consists of 46 optical fibers in two concentric bundles.

Distributed programming and object-oriented programming are two popular programming paradigms. The former is driven by advances in networking technology whereas the latter provides vigorous software principles needed in developing complex software systems. While more and more distributed object-oriented software has appeared, not much work exists on the testing of these systems in an integrated manner. Instead, the distributed and object features have been tested separately. In this paper, we propose an integrated framework known as DOOT for incremental testing of distributed object-oriented software systems. It combines various testing techniques to provide comprehensive test coverage at four levels — class testing, intra-cluster testing, inter-cluster testing and system testing. Each level uses a specific fault model, test strategy and test case generation that build on the previous test level to reduce the overall test effort. They are designed to handle the distinct requirements of the two paradigms at each level. Moreover, a reduction algorithm for testing the inherited class is also included in the framework. The approach is illustrated using a real life example of a conferencing system.

Background Afatinib (BIBW 2992) is an oral, irreversible ErbB Family Blocker. It was found to be mainly excreted via faeces and is highly protein bound. To define the impact of different degrees of hepatic impairment (HI) on the pharmacokinetics (PK) and safety of afatinib, a dedicated Phase I trial was conducted. Table: 468P 50 mg afatinib, mild HI (N = 8) 50 mg afatinib, HV matched to mild HI (N = 8) 50 mg afatinib, moderate HI (N = 8) 50 mg afatinib, HV matched to moderate HI (N = 8) Parameter Unit gMean gCV [%] gMean gCV [%] gMean gCV [%] gMean gCV [%] AUC0-∞ [ng*h/mL] 886 53.7 956 22.7 934 31.0 985 32.3 Cmax [ng/mL] 33.7 51.7 30.7 33.7 39.5 40.1 31.1 46.0 AUC0-tz [ng*h/mL] 842 50.8 930 22.5 904 31.4 956 33.3 Methods This single-dose, open-label, dose-escalation study in a matched group design included male or female subjects with HI (mild: Child Pugh A, score 5 or 6; moderate: Child Pugh B, score 7 to 9) and healthy volunteers (HV) matched to the HI subjects based on age, weight, sex and creatinine clearance. Dose escalation of afatinib from 30 to 50 mg was performed depending on tolerability and PK. Plasma and urine sampling was performed for up to 240 hours (h) and 72 h after afatinib administration, respectively. Primary endpoints were comparison of AUC0-∞ and Cmax of afatinib between subjects with HI and matched healthy subjects. For all other parameters, descriptive statistics were calculated. Additionally, blood samples were drawn to determine in vitro plasma protein binding of afatinib. Results Thirty-five subjects entered the trial: 3 subjects with mild HI received single-dose 30 mg afatinib and 32 subjects received single-dose 50 mg afatinib. Tolerability of afatinib was good, with 6/35 subjects showing AEs, which were related to afatinib in 3/35 subjects. Main PK parameters are given in Table 1. After a single dose of 50 mg, PK parameters were similar between patients with HI and their matched HV controls. Conclusions Mild and moderate HI had no influence on the PK and tolerability of a single dose of 50 mg afatinib. Table 1: Comparison of main PK parameters after single-dose administration of 50 mg afatinib. Disclosure D. Schnell: Employee of SocraMetrics / Boehringer Ingelheim. S. Buschke: Employee of Boehringer Ingelheim. H. Fuchs: Employee of Boehringer Ingelheim. R. Goldner: Employee of Boehringer Ingelheim. M. Uttenreuther-Fischer: Employee of Boehringer Ingelheim. P. Stopfer: Employee of Boehringer Ingelheim. S. Wind: Employee of Boehringer Ingelheim. A. Halabi: Employee of CRS Clinical Research Services / Boehringer Ingelheim. R. Koenen: Employee of Boehringer Ingelheim.

Background: Uremic cardiomyopathy contributes substantially to mortality in chronic kidney disease (CKD) patients. Glucagon-like peptide-1 (GLP-1) may improve cardiac function, but is mainly degraded by dipeptidyl peptidase-4 (DPP-4). Methodology/Principal Findings: In a rat model of chronic renal failure, 5/6-nephrectomized [5/6N] rats were treated orally with DPP-4 inhibitors (linagliptin, sitagliptin, alogliptin) or placebo once daily for 4 days from 8 weeks after surgery, to identify the most appropriate treatment for cardiac dysfunction associated with CKD. Linagliptin showed no significant change in blood level AUC(0-infinity) in 5/6N rats, but sitagliptin and alogliptin had significantly higher AUC(0-infinity) values; 41% and 28% (p=0.0001 and p=0.0324), respectively. No correlation of markers of renal tubular and glomerular function with AUC was observed for linagliptin, which required no dose adjustment in uremic rats. Linagliptin 7 mu mol/kg caused a 2-fold increase in GLP-1 (AUC 201.0 ng/l*h) in 5/6N rats compared with sham-treated rats (AUC 108.6 ng/l*h) (p=0.01). The mRNA levels of heart tissue fibrosis markers were all significantly increased in 5/6N vs control rats and reduced/normalized by linagliptin. Conclusions/Significance: DPP-4 inhibition increases plasma GLP-1 levels, particularly in uremia, and reduces expression of cardiac mRNA levels of matrix proteins and B-type natriuretic peptides (BNP). Linagliptin may offer a unique approach for treating uremic cardiomyopathy in CKD patients, with no need for dose-adjustment.

A method for the determination of phosphorus by the molecular non-thermal excitation spectrometry (MONES) of PO and HPO has been developed. The phosphorus was introduced into the furnace atomic non-thermal excitation spectrometry (FANES) source as a solution of sodium dihydrogen phosphate. The measurements were carried out at the bands that gave the maximum signals, for PO at 246.4 nm and for HPO at 507 nm. The electrical, thermal and chemical conditions were optimised and are discussed. In addition, the influence of La3+ as a chemical modifier on the MONES of PO and HPO was investigated. The best values for the detection limit were 700 pg of P (MONES of PO) and 3400 pg of P (MONES of HPO). By comparison with the best values obtained with commercial electrothermal atomisation atomic absorption spectrometry instruments, these values are better by factors of 8 and 1.6, respectively. However, the determination of P by FANES at 213.5/213.6 nm is the best method for investigations of phosphorus using the FANES source, in the presence of La3+ as a chemical modifier.

Afatinib, an oral irreversible ErbB family blocker, undergoes minimal metabolism by non-enzyme-catalysed adduct formation with proteins or nucleophilic small molecules and is predominantly non-renally excreted via the entero-hepatic system. This trial assessed whether mild or moderate hepatic impairment influences the pharmacokinetics of afatinib. This was an open-label single-dose study. Pharmacokinetic parameters after afatinib 50 mg were investigated in subjects with mild (n = 8) or moderate (n = 8) hepatic impairment (Child-Pugh A and B) and healthy controls (n = 16) matched for age, weight and gender. Plasma and urine samples for pharmacokinetic assessment were collected before and up to 10 days after dosing. Additional blood samples were drawn to determine ex vivo plasma protein binding of afatinib. Primary endpoints were comparisons of afatinib C max and AUC0–∞ between subjects with hepatic impairment and healthy matched controls. Study progression was based on drug-related toxicity (CTCAE v. 3.0) and C max of afatinib. Afatinib pharmacokinetic profiles and plasma protein binding were similar in subjects with impaired liver function and healthy controls. Compared with matched controls, the afatinib-adjusted geometric mean ratio for AUC0–∞ was 92.6 % (90 % CI 68.0–126.3 %) and C max was 109.5 % (90 % CI 82.7–144.9 %) for subjects with mild hepatic impairment, and 94.9 % (90 % CI 72.3–124.5 %) and 126.9 % (90 % CI 86.0–187.2 %), respectively, for subjects with moderate hepatic impairment. For all parameters, the 90 % CI included 100 %. Afatinib was generally well tolerated with no serious adverse events reported. Mild to moderate hepatic impairment had no clinically relevant effect on the pharmacokinetics of a single 50 mg dose of afatinib, implying that adjustments to the starting dose of afatinib are not considered necessary in this patient population.

A method of determining phosphorus by furnace atomic non-thermal excitation spectrometry (FANES) has been developed. The phosphorus is introduced into the FANES source as a solution of sodium dihydrogen phosphate. The measurements are carried out at 213.5/213.6 and 253.3/253.5 nm, the transition at 213.5/213.6 nm being the more sensitive one. The thermal and chemical conditions were optimised, the best chemical modifier being La3+ ions as they stabilise phosphate ions as LaPO4. Using the optimum amount of lanthanum (2 µg) the pre-treatment temperatures could be increased considerably (300 to 800 °C at normal pressure and 350 to 400 °C at low pressure). The best detection limit obtained is 90 pg of phosphorus, which is an improvement by a factor of 60 in comparison with the best values obtained with commercially available electrothermal atomisation atomic absorption spectrometric (ETAAS) instruments. Examples for real analyses with associated matrix interferences are given.

The application of two methods of trace analysis of microsamples, electrothermal atomisation atomic absorption spectrometry (ETA-AAS) and furnace atomisation non-thermal excitation spectrometry (FANES) to rare earth elements (REE) was studied. The evaporation and atomisation could be improved in both methods by the introduction of a tungsten platform into the graphite tubes to prevent the formation of REE carbides. Depending on the boiling-points of the REE, the absolute detection limits for both methods are in the picogram range. The thermal conditions for evaporation and atomisation were optimised. It was found that in ETA-AAS only the use of maximum heating conditions is successful. In FANES determinations on pure solutions the heating conditions can be reduced with the use of a vacuum, but in the presence of inorganic matrices it is again advantageous to use maximum heating. A new variation of FANES, furnace ionisation non-thermal excitation spectrometry (FINES), was developed for easily ionisable elements and was applied successfully to the determination of Eu, Sc and Sm. The limits of determination of REE in other REE are 1–100 p.p.m., depending on the conditions used, which were mainly influenced by the volatility of the elements.

A method for the determination of the halides F– and Cl– by non-thermal excitation of MgF and MgCl molecules in a glow discharge established in a hot graphite tube (2000 °C, 2 kPa) has been developed. The MgX molecules are formed by simultaneous evaporation of Mg- and F-containing species (Mg being the additive and F the sample). The detection limits are 0.5 ng of fluoride and 0.24 ng of chloride, respectively. The method is called ETE-MONES (molecular non-thermal excitation spectrometry with electrothermal evaporation) by analogy with the FANES technique (furnace atomisation non-thermal excitation spectrometry).

The determination of trace amounts of 99Tc was performed using electrothermal atomisation atomic absorption spectrometry (ETA-AAS) and furnace atomisation non-thermal excitation spectrometry (FANES). The thermal and chemical conditions were optimised. Tube-wall atomisation and matrix modification with 0.05 M Ni(NO3)2 in 0.1 M HNO3[forming Ni(TcO4)2] gave the best analytical results. The detection limits for 99Tc were 85 and 90 pg by ETA-AAS and FANES, respectively.