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Background.Bezafibrate, a fabric acid analogue, has well-established lipid- and fibrinogen-lowering properties. Some data exist pointing towards a blood pressure-lowering effect of bezafibrate. Thus the aim of this study was to examine the acute effect of bezafibrate on blood pressure and renal haemodynamics in hypertensive and normotensive rats. [ABSTRACT FROM PUBLISHER]

Elastic recoil, neointima formation and vessel narrowing after balloon angioplasty or stent implantation were compared in 17 non-atherosclerotic New Zealand White rabbits. The implantation of a balloon-expandable Palmaz-Schatz stent was performed in one iliac artery and a balloon angioplasty alone was performed in the contralateral artery (n=34 arteries). Quantitative histomorphometry was performed by a computer-assisted analysis 1 h and 4, 10 and 24 weeks after the initial procedure. The histological appearance of the neointima was similar to that of human restenosis. The amount of the neointima was increased within sten ted vessels as compared to balloon angioplasty alone (1·0±0·1 vs 0·4±0·1 mm at 4 weeks, <0·001). However, the neointimal lumen narrowing was smaller in the stented vessels due to persistent increase in vessel perimeter as compared to balloon angio plasty alone (16·5±0·9 vs 34·7±16·5% lumen narrowing at 4 weeks, <0·05). In conclusion, stent implantation enhances neointima formation as compared to angioplasty in non-atherosclerotic rabbits. The prevention of elastic recoil after stent implantation, however, reduces the neointimal lumen narrowing. This study supports clinical observations demonstrating lower restenosis rates after stent implantation compared to standard balloon angioplasty. [ABSTRACT FROM PUBLISHER]

Eliciting information from patients is fundamental to medical professionals' capacity to deliver good healthcare outcomes in Emergency Departments (EDs). There are different kinds of utterances that "do questioning", and health professionals can variously attend to the medical agenda and the interpersonal aspects of their interactions with those attending the ED in the way that they construct these utterances. We investigate a corpus of ED interactions to determine the prevalence and range of utterances produced by doctors and directed at patients that "do questioning." We developed a questioning utterance typology, informed by previous research on the formulation of such utterances and extended according to observations of our data. We subsequently manually coded 4,355 questioning utterances and report the variety of forms that such utterances can take, considering how these are distributed across doctors at different levels of seniority. We found that doctors at different seniority levels favored similar questioning utterance types and the most frequently used appeared to restrict the contributions of patients. We conclude that our extended typology of questioning utterances has value for understanding the ways in which doctors may encourage patients to provide more extensive responses.

We developed novel zwitterionic near infrared (NIR) fluorescent agents (ABZWCY-HPβCD and AAZWCY-HPβCD), which exhibit favorable hydrophilicity, low plasma protein binding, high stability and non-toxicity. These attractive characteristics ensure that they are excreted rapidly, without any skin accumulation or metabolism in vivo . More importantly, zwitterionic HPβCD based agents can be efficiently filtrated by the glomerulus and completely excreted through the kidneys into urine without reabsorption or secretion in the kidney proximal tubule. Relying on these novel zwitterionic NIR agents and a transcutaneous device, we demonstrate a rapid, robust and biocompatible approach for assessing kidney function in rat models of both healthy rats and those with kidney disease, without the need for time-consuming blood/urine sample preparation. Our work provides a promising tool for in vivo real-time non-invasive kidney function assessment in preclinical applications.

Transcutaneous measurement of the glomerular filtration rate ( t GFR) is now frequently used in animal studies. t GFR allows consecutive measurements on the same animal, including multiple measurements on a daily basis, because no blood sampling is required. Here we derive and validate a novel kinetic model for the description of transcutaneously measured FITC-Sinistrin excretion kinetics. In contrast to standard 1- to 3-compartment models, our model covers the complete kinetic, including injection and distribution of the tracer in the plasma compartment. Because the model describes the complete progression of the measurement, it allows further refinement by correcting for baseline shifts observed occasionally during measurement. Possible reasons for shifts in the background signal include photo bleaching of the skin, autofluorescence, changes of physiological state of the animals during the measurements, or effects arising from the attachment of the measurement device. Using the new 3-compartment kinetic model with modulated baseline ( t GFR 3cp.b.m ), t GFR measurements in rats can reach comparable precision as those from GFR measurements assessed using a gold standard technique based on constant infusion of a tracer. Moreover, the variability of simultaneous (parallel) measurements, as well as repeated t GFR measurements in the same animals, showed higher precision when t GFR 3cp.b.m was compared with the 1-compartment t GFR 1cp model., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Evaluation of renal function is crucial for a number of clinical situations. Here, we reported a novel exogenous fluorescent marker (FITC-HPβCD) to real-time assess renal function by using a transcutaneous fluorescent detection technique. FITC-HPβCD was designed based on the principle of renal clearance of designed drugs. It displays favorable fluorescent properties, high hydrophilicity, low plasma protein binding, and high stability in porcine liver esterase as well as in plasma and nontoxicity. More importantly, FITC-HPβCD can be efficiently and rapidly filtered by glomerulus and completely excreted into urine without proximal tubular reabsorption or secretion in rat models. Additionally, the marker was well-tolerated, with nearly 100% urinary recovery of the given doses, and no metabolism were found. Relying on this novel kidney function marker and transcutaneous devices, we demonstrate a rapid, robust, and convenient approach for real-time assessing renal function without the need of time-consuming blood and urine sample preparation. Our work provides a promising tool for noninvasive real-time monitoring of renal function in vivo.

Background: Some International Medical Graduates (IMGs) need to develop language and communication skills for patient-centred care but have limited opportunities to do so., Aim: To develop an evidence-based, language and communication skills web resource for IMG doctors and supervisors, focussing on culturally challenging patient interviews., Methods: Forty-eight IMGs participated in four practice OSCEs. We video-recorded the interactions and applied discourse analytic methods to investigate salient language and communication features., Results: The findings from the OSCE workshops showed that many participants demonstrated aspects of patient-centred interviewing but were hindered by limited interactional competence to elicit information and negotiate behaviours as well as a limited repertoire of English grammar, vocabulary, and phonological phrasing for effective interaction. These findings guided the choice of content and pedagogy for the development of the web-based resource Doctors Speak Up., Conclusion: Evaluation and uptake of the Doctors Speak Up website confirm the demand for a resource combining targeted communication skills and language instruction. Over 19 500 users visited the website between March 2012 and November 2013.

Aim: To examine the feedback given by nurse educators and clinicians on the quality of communication skills of nurses in interactions with simulated patients., Background: The quality of communication in interactions between nurses and patients has a major influence on patient outcomes. To support the development of effective nursing communication in clinical practice, a good understanding of what constitutes effective communication is helpful., Design: An exploratory design was used involving individual interviews, focus groups and written notes from participants and field notes from researchers to investigate perspectives on nurse-patient communication., Methods: Focus groups and individual interviews were held between August 2010-September 2011 with a purposive sample of 15 nurse educators and clinicians who observed videos of interactions between nurses and simulated patients. These participants were asked to give oral feedback on the quality and content of these interactions. Verbatim transcriptions were undertaken of all data collected. All written notes and field notes were also transcribed. Thematic analysis of the data was undertaken., Findings: Four major themes related to nurse-patient communication were derived from the educators' and clinicians' feedback: approach to patients and patient care, manner towards patients, techniques used for interacting with patients and generic aspects of communication., Conclusion: This study has added to previous research by contributing grounded evidence from a group of nurse educators and clinicians on the aspects of communication that are relevant for effective nurse-patient interactions in clinical practice., (© 2013 John Wiley & Sons Ltd.)

Glomerular filtration rate (GFR) is considered the best parameter for the assessment of renal function, being usually determined on the basis of urine or plasma clearance of exogenous renal markers. The common methodology is invasive, time consuming and cumbersome, with multiple blood and/or urine sampling and following laboratory assays required. The method detailed here allows to transcutaneously determine the renal function in awake animals, in a non-invasive and efficient manner by using an electronic device which detects the fluorescence emitted through the skin from the renal marker FITC-Sinistrin. A crucial target has been to improve the fixation of the device, which is dependent on the skin structure. For validation, the technique has been compared with the classical clearance method, and its robustness has been demonstrated in healthy and diseased murine models. Moreover, the method allows sequential measurements in the same individual. Thus progression and recovery of renal failure can be followed. Therefore, its future application in humans would allow an accurate and appropriate prediction and monitoring of patients with established kidney disease over time. Furthermore, it will be possible to observe those patients under other pathological conditions with associated risk of developing renal problems.

Measuring renal function in laboratory animals using blood and/or urine sampling is not only labor-intensive but puts also a strain on the animal. Several approaches for fluorescence based transcutaneous measurement of the glomerular filtration rate (GFR) in laboratory animals have been developed. They allow the measurement of GFR based on the elimination kinetics of fluorescent exogenous markers. None of the studies dealt with the reproducibility of the measurements in the same animals. Therefore, the reproducibility of a transcutaneous GFR assessment method was investigated using the fluorescent renal marker FITC-Sinistrin in conscious mice in the present study. We performed two transcutaneous GFR measurements within three days in five groups of mice (Balb/c, C57BL/6, SV129, NMRI at 3-4 months of age, and a group of 24 months old C57BL/6). Data were evaluated regarding day-to-day reproducibility as well as intra- and inter-strain variability of GFR and the impact of age on these parameters. No significant differences between the two subsequent GFR measurements were detected. Fastest elimination for FITC-Sinistrin was detected in Balb/c with significant differences to C57BL/6 and SV129 mice. GFR decreased significantly with age in C57BL/6 mice. Evaluation of GFR in cohorts of young and old C57BL/6 mice from the same supplier showed high consistency of GFR values between groups. Our study shows that the investigated technique is a highly reproducible and reliable method for repeated GFR measurements in conscious mice. This gentle method is easily used even in old mice and can be used to monitor the age-related decline in GFR.

Determination of glomerular filtration rate (GFR) in conscious mice is cumbersome for the experimenter and stressful for the animals. Here we report on a simple new technique allowing the transcutaneous measurement of GFR in conscious mice. This approach extends our previously developed technique for rats to mice. The technique relies on a miniaturized device equipped with an internal memory that permits the transcutaneous measurement of the elimination kinetics of the fluorescent renal marker FITC-sinistrin. This device is described and validated compared with FITC-sinistrin plasma clearance in healthy, unilaterally nephrectomized and pcy mice. In summary, we describe a technique allowing the measurement of renal function in freely moving mice independent of blood or urine sampling as well as of laboratory assays.

Constant infusion clearance techniques using exogenous renal markers are considered the gold standard for assessing the glomerular filtration rate. Here we describe a constant infusion clearance method in rats allowing the real-time monitoring of steady-state conditions using an automated closed-loop approach based on the transcutaneous measurement of the renal marker FITC-sinistrin. In order to optimize parameters to reach steady-state conditions as fast as possible, a Matlab-based simulation tool was established. Based on this, a real-time feedback-regulated approach for constant infusion clearance monitoring was developed. This was validated by determining hourly FITC-sinistrin plasma concentrations and the glomerular filtration rate in healthy and unilaterally nephrectomized rats. The transcutaneously assessed FITC-sinistrin fluorescence signal was found to reflect the plasma concentration. Our method allows the precise determination of the onset of steady-state marker concentration. Moreover, the steady state can be monitored and controlled in real time for several hours. This procedure is simple to perform since no urine samples and only one blood sample are required. Thus, we developed a real-time feedback-based system for optimal regulation and monitoring of a constant infusion clearance technique.

Determination of the urinary or plasma clearance of exogenous renal markers, such as inulin or iohexol, is considered to be the gold standard for glomerular filtration rate (GFR) measurement. Here, we describe a technique allowing determination of renal function based on transcutaneously measured elimination kinetics of fluorescein isothiocyanate (FITC)-sinistrin, the FITC-labeled active pharmaceutical ingredient of a commercially available marker of GFR. A low cost device transcutaneously excites FITC-sinistrin at 480  nm and detects the emitted light through the skin at 520  nm. A radio-frequency transmission allows remote monitoring and real-time analysis of FITC-sinistrin excretion as a marker of renal function. Due to miniaturization, the whole device fits on the back of freely moving rats, and requires neither blood sampling nor laboratory assays. As proof of principle, comparative measurements of transcutaneous and plasma elimination kinetics of FITC-sinistrin were compared in freely moving healthy rats, rats showing reduced kidney function due to unilateral nephrectomy and PKD/Mhm rats with cystic kidney disease. Results show highly comparable elimination half-lives and GFR values in all animal groups. Bland-Altman analysis of enzymatically compared with transcutaneously measured GFR found a mean difference (bias) of 0.01 and a -0.30 to 0.33 ml/min per 100 g body weight with 95% limit of agreement. Thus, with this device, renal function can be reliably measured in freely moving rats eliminating the need for and influence of anesthesia on renal function.

Background: Inulin/sinistrin (I/S) clearance is a gold standard for an accurate assessment of glomerular filtration rate (GFR). Here we describe and validate an approach for a transcutaneous determination of GFR by using fluorescein-isothiocyanate-labelled sinistrin (FITC-S) in rats., Methods: Using a small animal imager, fluorescence is measured over the depilated ear of a rat after the injection of FITC-S. The decay curve of fluorescence is used for the calculation of half-life and GFR. The thus obtained transcutaneous data were validated by simultaneously performed enzymatic and fluorometric measurements in plasma of both FITC-S and sinistrin., Results: The results of enzymatic sinistrin determination versus transcutaneous half-life of FITC-S or plasma fluorescence correlated well with each other (R(2) > 0.90). Furthermore, Bland-Altman analyses proved a good degree of agreement of the three methods used. The measurements performed in healthy animals as well as different models of renal failure demonstrate its appropriateness in a wide range of renal function., Conclusions: The transcutaneous method described offers a precise assessment of GFR in small animals. As neither blood and/or urine sampling nor time-consuming lab work is required, GFR can be determined immediately after the clearance procedure is finished. This method, therefore, simplifies and fastens GFR determinations in small lab animals compared to conventional bolus clearance techniques based on blood sampling. A low-cost device for the measurement of transcutaneous fluorescence intensity over time is under construction.

Considerable progress in improved control of disturbed glucose metabolism can be expected by continuous glucose monitoring. The aim of the study was to evaluate in male Sprague-Dawley rats tissue response to implantation of a new amperometric glucose-oxidase-based glucose sensor (NTS) compared to a commercially available sensor system CGMS of MiniMed. Both sensors were tested under working conditions over a period of 3 days. Using NTS, glucose in interstitial fluid reflected glucose in arterial blood even in rapidly changing hyper- and hypoglycaemia whereas the CGMS did not detect the experimentally induced glucose changes adequately. Gene expression profiling was performed using Affymetrix chips. Acute phase response to injury by sensor application for a short time is indicated by down regulation of the increase in mRNA of proteases e.g. metallothionein-1alpha and matrix metalloprotease-3 at day 3. Improvement of anabolic situation is suggested by decrease in mRNA of insulin-like growth factor binding protein whereas increase of heme oxygenase and hypoxia-inducible factor may be related to defense mechanisms. Changes of mRNA together with slight fibrous capsule formation suggest good histocompatibility. Comparability of the patterns of changed mRNA in tissue surrounding SCGM with and without operating voltage as shown in dendrogram indicates no contribution of hydrogen peroxide to worsening biocompatibility. Beside established histological investigations of foreign body reaction weeks or months after implantation, gene expression profiling provides additional information to biocompatibility already early after implantation.

Glucose monitoring is of importance for success of complex therapeutic interventions in diabetic patients. Its impact on treatment and glycemic control is demonstrated in large clinical trials. Up to eight blood glucose measurements per day are recommended. Notwithstanding, a substantial number of diabetic patients cannot or will not monitor their blood glucose appropriately. Considerable progress in control of disturbed metabolism in diabetic patients can be expected by continuous glucose monitoring. The aim of the study was to evaluate the performance of a new amperometric glucose oxidase-based glucose sensor in vitro and in vivo after subcutaneous implantation into rats. For in vitro testing current output of sensors was measured by exposure to increasing and decreasing glucose concentrations up to 472 mg dL(-1) over a time period of 7 days. After subcutaneous implantation of sensors into interscapular region of male rats glucose in interstitial fluid was evaluated and compared to glucose in arterial blood up to 7 days. Hyper- and hypoglycaemia were induced by intravenous application of glucose and insulin, respectively. Current of each implanted sensor was converted into glucose concentration using the first blood glucose measurement only. A change of current with glucose of 0.35 nA mg(-1)dL(-1) indicates high sensitivity of the sensor in vitro. The response time (90% of steady state) was calculated by approximately 60s. Test strips for blood glucose measurement as reference for sensor readings was found as an appropriate and rapidly available method in rats by comparison with established hexokinase method in an automated lab analyzer with limits of agreement of +32.8 and -25.7 mg dL(-1) in Bland-Altman analysis. In normo- and hypoglycaemic range sensor readings in interstitial fluid correlated well with blood glucose measurements whereas hyperglycaemia was not reflected by the sensor completely when blood glucose was changing rapidly. The data given characterize a sensor with high sensitivity, long term stability and short response time. A single calibration of the sensor is required only in measurement periods up to 7 days. The findings demonstrate that the sensor is a highly promising candidate for assessment in humans.

There is an evident and growing medical need for an accurate determination of kidney function for a broad spectrum of indications. The glomerular filtration rate (GFR) is the most accepted indicator of renal function. Due to difficulties in performing the test, GFR is currently determined rarely in clinical practice. A procedure for such GFR determination has to be safe, accurate and easy to handle. By using the new compound fluorescein isothiocyanate-sinistrin (FS) these requirements are met. The pharmacological profile and tolerability of FS, selected from among various newly synthesized, labelled compounds intended for use as GFR markers, was characterized in male Sprague-Dawley rats following i.v. application. Using the newly described fluorometric method, FS can be determined much more easily in serum and urine than with the established enzymatic method. After i.v. dosing, FS concentrations in serum declined rapidly in various experimental groups to a comparable extent (t (1/2), mean+/-SD: 22.4+/-8.3 to 26.2+/-5.4 min). Its increase after unilateral nephrectomy reflects the loss of filtration capacity. Comparable concentration-time curves of FS in serum measured fluorometrically and enzymatically suggest no relevant alteration of pharmacokinetic behaviour by the labelling. This notion is supported by the high urinary excretion rate and absence of biliary excretion. The higher sensitivity of the fluorometric method suggests a dose of FS of 100 mg in humans compared with 5 g of sinistrin or inulin. FS was well tolerated after single and multiple applications. On the basis of these results, the kinetics of FS are comparable with the gold standard inulin or sinistrin, but FS is superior in handling. Providing the data can be transferred from rat to human, determination of GFR using the new method should result in an improvement of acceptance by both physicians and patients.

There is an obvious and growing medical need for an accurate and easy to handle determination of glomerular filtration rate (GFR) for a broad spectrum of indications. Newly synthesized fluorescein-isothiocyanate (FITC)-sinistrin (FS) with various degrees of labeling was selected by its physicochemical properties and good tolerability out of a number of dye-labeled compounds intended for use as GFR markers for characterization of its pharmacological profile. With respect to solubility FS is more convenient in handling compared to FITC-inulin (FI). Up to 100 mg ml(-1) of FS can be dissolved in aqueous solvents at room temperature, whereas FI can only be solubilized after warming up to 55 degrees C. This reveals a considerable advantage of FS over FI in preparation of galenical formulations for intended i.v. application. A fluorometric method was established to determine FS concentration in blood serum with a comparable accuracy to the established enzymatic method for polyfructosanes. Similar concentration time curves in blood serum of FS measured fluorometrically and enzymatically suggest no relevant change of pharmacokinetic behavior by dye labeling. This notion is supported by the rapid renal and missing of biliary excretion. On the basis of these results, FS is superior in handling to the available GFR markers and makes it highly interesting as a novel diagnostic drug.

There is an obvious and growing medical need for an accurate determination of kidney function in the diagnosis and management of renal diseases. The glomerular filtration rate (GFR) is the accepted gold standard measurement of kidney function. Several approaches to estimate the GFR are available, but most of them are inconvenient and, therefore, of limited acceptance. A new method of quantification with fluorescein-isothiocyanate (FITC) sinistrin (FS), a novel GFR marker, has been evaluated. The method is based on the fluorescence label of FS and can be performed with a standard fluorometer. To control the interference of protein with the fluorescence signal, a calibration function was developed. The accuracy of the fluorometric method established is comparable to the so-called "gold standard" of enzymatic determination of polyfructosan. Moreover, FS is easy to handle and requires low-cost instruments. Our results demonstrate the potential of the direct fluorometric analysis of the new FITC-labelled marker of being a precise, simple, rapid and cost-effective method for diagnosing disturbed kidney function and monitoring its treatment efficacy. The dramatic decrease in analytical effort will result in a significantly higher acceptability of GFR determination.

Background: The continuous monitoring of glucose allows for tighter control of the glucose concentration and thus may prevent hyper- and hypoglycemia as well as long-term complications of diabetes. While most current systems depend on the transport of fluid to a glucose sensor outside the body, we investigate the possibility of implanting a reagent-based sensor directly into the skin. In this manuscript, the biocompatibility of an electrochemical sensor for continuous glucose monitoring was assessed in vitro and in vivo., Methods: Cytotoxicity was investigated in vitro using agar diffusion testing. In vivo biocompatibility was assessed by means of histomorphological examination of the surrounding tissue 10 days after sensor implantation in rats., Results: The grade of cytotoxicity of the individual sensor components in vitro was between none and mild based on agar diffusion testing. The complete sensor also showed no cytotoxic effects when coated with the co-polymer MPC (2-methacryloyloxyethyl phosphorylcholine, Lipidure CM 5206, NOF Corp., Tokyo, Japan) and when assessed under working conditions, i.e., when a bias voltage was applied to the sensor. Additionally, the hydrogen peroxide-which is inherently generated by the enzymatic glucose detection process using glucose oxidase (GOD)-is likely to have been sufficiently decomposed under these working conditions. Finally, no toxic leachable substances were found during the cytotoxicity testing of sensors and its extracts in vitro. In the in vivo experiments, the strongest foreign body reaction (FBR) was found near the GOD-electrode using a sensor without MPC coating and without a porous membrane. Covering the sensor with MPC, a porous membrane, or both led to a gradual decrease of the FBR down to the level of the negative control., Conclusions: The electrochemical, reagent-based sensor with MPC coating and/or a porous membrane is suitable for continuous monitoring of glucose from a biocompatibility standpoint.

K-111 has been characterized as a potent peroxisome proliferator-activated receptor (PPAR)alpha activator. Antidiabetic potency and amelioration of disturbed lipid metabolism were demonstrated in rodents, which were accompanied by elevations of peroxisomal enzymes and liver weight. To examine the possible therapeutic application of K-111 we have now assessed its efficacy in non-human primates with high transferability to humans. For this purpose obese, hypertriglyceridaemic, hyperinsulinaemic prediabetic rhesus monkeys were dosed sequentially with 0, 1, 3 and 10mg/kg per day orally over a period of 4 weeks each. In addition, the effect of K-111 on the peroxisome compartment was analyzed in cynomolgus monkeys using liver samples obtained following a 13-week oral toxicity study. In prediabetic monkeys, the reduction of hyperinsulinaemia and improvement of insulin-stimulated glucose uptake rate indicated amelioration of insulin resistance. These effects were nearly maximal at a dose of 3mg/kg per day, while triglycerides and body weight were lowered significantly in a dose-dependent manner. This reduction of body weight contrasts sharply with the adipogenic response observed with thiazolidinediones, another family of insulin-sensitizing agents. In young cynomolgus monkeys at a dosage of 5mg/kg per day and more, K-111 induced an up to three-fold increase in lipid beta-oxidation enzymes with an 1.5- to 2-fold increase in peroxisome volume density. This moderate increase in peroxisomal activity by K-111 in monkeys is consistent with its role as an PPARalpha activator and corresponds to the observations with fibrates in other low responder mammalian species. The increase in beta-oxidation may explain, at least in part, the lipid modulating effect as well as the antidiabetic potency of K-111. This pharmacological profile makes K-111 a highly promising drug candidate for clinical applications in the treatment of type 2 diabetes, dyslipidaemia, obesity and the metabolic syndrome.

K-111, formerly BM 17.0744, (2,2-dichloro-12-(4-chlorophenyl)-dodecanoic acid) is a new insulin-sensitizer with peroxisome proliferator-activated receptor (PPAR) alpha activity but without PPAR gamma activity. We determined the efficacy of K-111 in non-human primates in increasing insulin-stimulated glucose uptake and improving metabolic syndrome, assessing the general health-related effects. Six adult male obese normoglycemic prediabetic and insulin-resistant rhesus monkeys were studied on vehicle and following K-111 treatment (four-week chronic dosing each of 3 doses: 1, 3, and 10 mg/kg/d) with assessment of changes in substrate, hormone, and blood pressure measurements and alterations in insulin sensitivity using the euglycemic, hyperinsulinemic clamp technique. K-111 led to significantly decreased body weight and improved hyperinsulinemia, insulin sensitivity, hypertriglyceridemia, and HDL-cholesterol levels without adipogenesis or significant effects on fasting glucose, 24-hour urine glucose excretion, systolic or diastolic blood pressure, plasma fibrinogen, total cholesterol, or chemistry and hematology profile. These benefits are similar to the health-improving effects of calorie restriction, providing preliminary evidence that K-111 has excellent potential as a calorie-restriction mimetic agent. These results indicate the necessity of future study of K-111 for metabolic syndrome in humans, and suggest potential in reducing the risks of diabetes and cardiovascular disease.

BM 17.0744, a new anti-diabetic and lipid-lowering agent, leads also to strong hepatomegaly and carnitine acetyl transferase (CAT) increase in the liver of rats, a phenomenon known from fibrates. For information on the relevance of changes in liver of rats to other species, we investigated the effects of BM 17.0744 on lipids and selected marker enzymes related to beta-oxidation in rats, dogs and guinea-pigs, so-called high and low responders to peroxisome proliferators. To examine selectivity other enzymes were also determined, e.g. esterase, urate oxidase (UOX) and cytochrome c oxidase (CYT.C.OX.). Lowering of triglycerides and cholesterol in blood serum and/or liver was observed in pharmacological dose range in the three species tested. In dogs and guinea-pigs, liver and kidney weights were unaffected even in dogs in medium and high dose groups with high systemic exposure and severe toxicity. In male Sprague-Dawley rats treatment with 1.5, 3, 6 and 12.5 mg/kg per day BM 17.0744 selectively elevated the activities of CAT and acyl-CoA oxidase (AOX) by < or =200 and 20-fold, respectively. Administration of BM 17.0744 to Beagle dogs (1.5, 4, 12 mg/kg per day) and guinea-pigs (3 and 12 mg/kg per day) enhanced the activities of CAT and AOX dose-dependently by a factor of two to three only. Immunoblotting revealed a drug-specific enhancement of the amount of beta-oxidation enzymes in rats, which is in accord with the rapid and coordinated transcriptional activation shown in Northern dot blot analysis. Nuclear run-on assays demonstrated a real transcriptional activation. BM 17.0744 activates peroxisome proliferator-activated receptor alpha (PPARalpha), which could be shown by transactivation assays. The stimulation of PPARalpha by BM 17.0744 was stronger than that of the known ligands WY 14.643 and ETYA. Activation of PPARgamma can be excluded. Taken collectively, the data demonstrate an enhancement of the beta-oxidation system by BM 17.0744 paralleled by lipid-lowering in all species investigated. The activation of the nuclear factor PPARalpha may explain the changes in liver and the metabolic effects on the molecular level. The lack of an increase in liver and kidney weights and the relatively moderate enhancement of activities of beta-oxidation-related enzymes in dogs and guinea-pigs indicate that the excessive response observed in rats is not applicable to other, predominantly non-rodent, species. On the basis of these data and the experience with fibrates a specific risk for humans is not expected.

BM 17.0744 (2,2-dichloro-12-(p-chlorophenyl)-dodecanoic acid) is a substance from a group of omega-substituted alkyl carboxylic acids with the general formula, ring-spacer-carboxylic acid. With BM 17.0744-a compound structurally unrelated to thiazolidinediones--antihyperglycemic and antihyperinsulinemic potency has been demonstrated in various animal models of type II diabetes. The antidiabetic effect is independent of the genetic background of the disease, gender, and animal species. The 24-hour blood glucose profile was dose- and time-dependently improved in ob/ob mice after a single and fourth oral administration of 0.3, 1, and 3 mg/kg/d. A dose-dependent reduction of hyperglycemia (10%, 15%, 28%, and 66%) was found in db/db mice after the fifth oral administration of 3, 10, 30, and 100 mg/kg/d. Hyperinsulinemia was reduced dose-dependently in yellow KK mice by 1%, 24%, 34%, and 66% after the fifth oral administration of 0.3, 1, 3, and 10 mg/kg/d. Overall glucose metabolism was predominantly higher in euglycemic-hyperinsulinemic clamp studies in obese fa/fa rats pretreated for 14 days with 10 mg/kg/d BM 17.0744. The data in diabetic and insulin-resistant animals suggest an improvement of insulin action that is supported by enhancement of insulin effects in vitro. There is no evidence of a risk for hypoglycemia in diabetic and metabolically healthy animals. Triglyceride (TG) and cholesterol were reduced in the serum of metabolically healthy rats, as well as serum lipids in db/db mice, which suggests this effect is independent of amelioration of the diabetic status. Lipid-lowering effects in diabetic and healthy animals show an additional property of BM 17.0744. Because of its antidiabetic and lipid-lowering potency, the substance is of great interest in treating the metabolic syndrome. Lipid decreases in rats are associated with a dose-dependent increase in carnitine acetyltransferase activity in the liver to about 100-fold (12.5 mg/kg/d). This together with hepatomegaly in small rodents may indicate peroxisomal proliferation, a phenomenon considered species-specific. Its relevance for humans is well documented for other classes of compounds including fibrates. Specific side effects of insulin sensitizers of the thiazolidinedione type, such as an increase in body weight and heart weight, could not be observed after 4-week oral application of BM 17.0744 in rats. In general, BM 17.0744 was well tolerated in the pharmacological dose range in all species tested.

Oxidized low density lipoprotein (oxLDL) induces apoptosis in human macrophages (Mphi), a significant feature in atherogenesis. We found that induction of apoptosis in Mphi by oxLDL, C2-ceramide, tumor necrosis factor alpha (TNF-alpha), and hydrogen peroxide (H2O2) was associated with enhanced expression of manganese superoxide dismutase (MnSOD) and p53. Treatment of cells with p53 or MnSOD antisense oligonucleotides prior to stimulation with oxLDL, C2-ceramide, TNF-alpha, or H2O2 caused an inhibition of the expression of the respective protein together with a marked reduction of apoptosis. Exposure to N-acetylcysteine before treatment with oxLDL, C2-ceramide, TNF-alpha, or H2O2 reversed a decrease in cellular glutathione concentrations as well as the enhanced production of p53 and MnSOD mRNA and protein. In apoptotic macrophages of human atherosclerotic plaques, colocalization of MnSOD and p53 immunoreactivity was found. These results indicate that in oxLDL-induced apoptosis, a concomitant induction of p53 and MnSOD is critical, and suggest that it is at least in part due to an enhancement of the sphingomyelin/ceramide pathway.

We investigated the effect of alterations of blood cholesterol levels on macrophages (mphi) in the myocardium of New Zealand White (NZW) rabbits. Three groups of NZW rabbits were used: controls, rabbits fed a 0.5% cholesterol-enriched diet (CH-D) for 96 days, and rabbits fed a 0.5% CH-D for 96 days followed by normal chow for 4 months. Immunohistochemical analysis by mAbs directed against mphi (RAM-11) and Mn superoxide dismutase (MnSOD) were quantified by computer-assisted morphometry. Using cultured human and rabbit mphi, a cross-reaction of the human MnSOD mAbs was found as well as the predominant localization of MnSOD-immunoreactivity (IR) in mitochondria. In group 1, only a very few RAM-11-immunoreactive (ir) mphi occurred in the interstitial space of the myocardium. In group II blood cholesterol levels significantly increased in parallel with the numbers of mphi, which often contained lipid droplets (foam cells). Although blood cholesterol concentrations regressed about 10-fold in group III, mphi in the myocardium were found to be reduced only about 20%. Most mphi were also MnSOD-ir. In atherosclerotic coronary arteries RAM-11-IR was located in mphi and also extracellularly, whereas MnSOD-IR was found only in mphi. Drastically induced MnSOD in the mitochondria of mphi is suggested as an indicator of increased oxidative stress caused by in vitro conditions or by phagocytosis of low-density lipoprotein in vivo. Elevation of the cholesterol level leads to a long-term increase and its regression results in a delayed reduction of such mphi, which seem to play a key role in the atherogenesis of the coronary arteries as well.

The objective of the study was to analyze the intracellular antioxidative response of macrophages (Mphi) exposed to increased levels of low density lipoprotein (LDL). We studied manganese superoxide dismutase (MnSOD) and, in part, GSH in cultured human and rabbit Mphi, and in atheromatous arterial tissue of humans and heritable hyperlipidemic (HHL) rabbits. Incubation of human Mphi with oxidized-LDL (ox-LDL) resulted in an induction of MnSOD mRNA production as shown by RT-PCR. MnSOD immunoreactivity (IR) was found to be located in the mitochondria of Mphi. In HHL rabbits, MnSOD activity and GSH concentration were significantly increased in atherosclerotic intima compared to the media of the aorta, but significantly decreased (P<0.01) in larger plaques compared with smaller ones, resulting in a significant inverse correlation of MnSOD activity (r=-0.67, P<0.001) and GSH concentration (r=-0.57, P<0.01) with plaque size. Immunohistology of the atherosclerotic intima revealed MnSOD-IR in Mac-1 (CD 11b/CD 18)-immunoreactive (ir) Mphi of human arteries and, similarly, in RAM-11-ir Mphi of rabbit ones. The relation of MnSOD-ir Mphi decreased with plaque advancement, which is consistent with biochemical findings. Most MnSOD-ir Mphi in atherosclerotic plaques revealed TUNEL-positive nuclei, indicating DNA strand breaks, and p53-IR. We conclude that mitochondrial antioxidants such as MnSOD are induced in Mphi in vitro and in atherosclerotic arteries as a reply to increased mitochondrial oxidation. As normal consequences of an increased oxidative stress due to the exposure to ox-LDL nuclear DNA strand breaks occur, which are suggested to be a signal to increase p53 protein levels. Reactive oxygen species-mediated mitochondrial-dependent pathways are suggested as major contributing pathomechanisms to nuclear damage, which eventually may result in apoptosis. A common response to increased oxidative stress due to modified LDL is presumed in rabbit and human atherosclerotic plaques.