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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.

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.

Recombinant human erythropoietin (rhEPO) is used to treat anemia in chronic renal insufficiency. Erythropoietin (EPO) immunogenicity can lead to EPO-resistant anemia. Conjugating proteins with polyethylene glycol (PEG) can prolong elimination half-life and diminish protein immunogenicity. We investigated the efficacy of new erythropoietic agents, synthesized by single (Ro 50-3821) and multiple (MIX) integrations of PEG and succinimidyl butanoic acid with rhEPO, in rats with chronic renal insufficiency. Sprague-Dawley rats with surgically induced renal insufficiency received Ro 50-3821 or MIX subcutaneously (s.c.) over 4-12 weeks compared to rhEPO and NaCl. Hemoglobin and antibody levels served as primary efficacy and safety variables. Dosing intervals and dose-response characteristics were investigated. Ro 50-3821 (2.5 microg/kg once weekly) increased hemoglobin levels by 7 g/dl after 4 weeks compared to 1 g/dl in NaCl controls (P0.05). MIX (2.5 microg/kg once weekly) and rhEPO (0.25 microg/kg three times weekly) increased hemoglobin levels by 3 g/dl. Ro 50-3821 administered for 12 weeks (0.75 microg/kg once weekly) increased hemoglobin levels (from 13 to 19 g/dl) more effectively than rhEPO (0.75 microg/kg once weekly, decline from 13 to 11 g/dl, P0.05). No antibodies against Ro 50-3821 were detected after 12 weeks of treatment. Antibodies against rhEPO were seen in 69% of animals (P0.00001). Ro 50-3821 increased hemoglobin levels with once weekly s.c. dosing. Multiple pegylated EPO is less effective. In rats, rhEPO failed to increase hemoglobin levels with once weekly long-term dosing. Antibody formation following rhEPO may explain this finding. Therefore, Ro 50-3821 may provide important clinical advantages compared to unpegylated EPO. It can be administered in longer dosing intervals and has a lower risk of unfavorable immunological responses.

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.

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.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.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.The electrochemical, reagent-based sensor with MPC coating and/or a porous membrane is suitable for continuous monitoring of glucose from a biocompatibility standpoint.

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.

Determining true glomerular filtration rate (GFR) using an exogenous marker is time-consuming and cumbersome. Therefore, creatinine-based estimates of GFR are used. Recent papers using new population-specific/local parameters in their prediction equations, standardizing creatinine determination or adding other endogenous surrogate markers of GFR, like cystatin C, could demonstrate an improvement of bias inherent in the results of the prediction equations. Precision, however, is still poor. Currently, we have to accept a precision (as defined in the so-called Bland-Altman plot) of +/-20% in adults and +/-30-40% in children. This problem of poor precision/uncertainty is especially bothering in the higher, near normal GFR range. Caution should be exercised when applying prediction equations in individuals in need of an accurate GFR determination. In that case, a real clearance procedure has to be performed. In the long run, the true clearance procedure should be simplified using new exogenous GFR markers and developing new devices, allowing GFR measurements to be performed, for example, transcutaneously. Such a procedure would be more acceptable for both patients and physicians.

In screening experiments certain ω-substituted alkyl carboxylic acids, were found to produce an increase in insulin-stimulated 14C-acetate incorporation into triglycerides, which may indicate an improvement in the action of insulin. Antidiabetic and lipid-lowering properties in genetically diabetic ob/ob mice demonstrated the in vivo relevance of the insulin-potentiating effects seen in vitro. The chemical structures of the ω-substituted alkyl carboxylic acids with insulin-potentiating effects correspond to the general formula ring-spacer-COOH. A close structure-activity relationship was observed. The most potent compound in ob/ob mice was 3e, which normalized blood glucose as well as hyperinsulinaemia and lowered serum triglycerides and cholesterol by 52% and 37%, respectively. On the basis of these results, ω-substituted alkyl carboxylic acids are interesting as a new class of oral antidiabetic agents with insulin-sensitizing and lipid-lowering activity.

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.

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.

The effects of bezafibrate administered at 10 and 50 mg/kg/day for 7 days to male Sprague-Dawley (SD) and Lewis rats were investigated in order to determine the interrelation between the changes in serum and hepatic lipid contents and activities of selected peroxisomal, microsomal and mitochondrial enzymes in the two rat strains. In both strains, bezafibrate effectively reduced serum and hepatic lipids, increased the liver weight, induced a proliferation of peroxisomes, and selectively elevated the activities of carnitine acetyltransferase and of the enzymes of the peroxisomal beta-oxidation system. Moreover, immunoblotting revealed that the drug specifically enhanced the concentration of only those peroxisomal enzymes involved in fatty acid beta-oxidation. The data obtained demonstrate that although the responses initiated by bezafibrate are qualitatively similar in both strains, they differ in their magnitude in a dose-dependent manner, with the Lewis strain exhibiting a more pronounced response than the SD rats. These results show that dose-dependent strain differences as well as the generally known species differences should be taken into account in pharmacological and toxicological evaluations of fibrates in rodents. Furthermore, generalization and extrapolation from rodent studies should be treated with great caution.

In vivo experiments have shown that the magnitude of peroxisomal proliferation induced by xenobiotics varies markedly in different mammalian species. Hepatocytes from rats and guinea pigs In vivo also exhibit species dependent differences in the response to hypolipidaemic drugs. In order to investigate the mechanisms underlying this phenomenon and to rule out the influence of different culture conditions we established a technique for the co-cultivation of primary hepatocytes of rat and guinea pig. Hepatocytes from rats and guinea pigs were isolated by a modified collagenase perfusion technique. Cells were either seeded as homogeneous or mixed populations on standard culture dishes. For their species specific distinction the cells were fixed, stained with the alkaline DAB method for cytochemical demonstration of catalase and embedded in Epon 812. In contrast to rat hepatocytes those of guinea pigs possess not only a peroxisomal but also a cytosolic catalase resulting in a darker cytoplasmic label. Whereas in guinea pig hepatocytes peroxisomes are smaller, forming clusters, they are larger and more sparse in rat hepatocytes. These characteristics are sufficient for the distinction of the hepatocytes of rat and guinea pig in coculture permitting the separate study of their peroxisomes by automatic image analysis. The cocultivation of primary hepatocytes from different mammalian species provides a novel approach for the investigation of interspecies differences in drug metabolism and toxicity studies.

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.

The use of the colorimetric Jaffé method for the measurement of creatinine in mouse and rat plasma has been criticized as prior studies have shown a dramatic overestimation. We compared a colorimetric picric acid, an enzymatic, and a high-performance liquid chromatography (HPLC) method to assess their appropriateness for routine measurements of creatinine in plasma of healthy and diseased mice (n=61) and rats (n=56). For the colorimetric Jaffé method a pronounced overestimation is confirmed. Additionally the method showed interference with hemoglobin already in a very low, non-visible concentration range in rat plasma. The enzymatic measurement demonstrated a hemoglobin interference in mice, only when hemolysis was visible. The comparison between HPLC and the enzymatic measurement gave a good agreement between both methods in both species. Therefore the enzymatic method fulfills the requirements for a routine screening test for plasma creatinine in healthy as well as diseased mice and rats Kiover a broad concentration range.

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 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.

K-111 has been characterized as a potent peroxisome proliferator-activated receptor (PPAR)α 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 10 mg/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 3 mg/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 5 mg/kg per day and more, K-111 induced an up to three-fold increase in lipid β-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 PPARα activator and corresponds to the observations with fibrates in other low responder mammalian species. The increase in β-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.

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) byor =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.

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 (P0.01) in larger plaques compared with smaller ones, resulting in a significant inverse correlation of MnSOD activity (r=-0.67, P0.001) and GSH concentration (r=-0.57, P0.01) 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.

The effect of hypercholesterolemia on manganese superoxide dismutase (MnSOD)-containing macrophages was investigated in male New Zealand white rabbits. Macrophages from control animals, which were marked with the RAM-11 antibody, demonstrated co-localization with MnSOD immunoreactivity, e.g. in the peri- and paravascular space within the myocardium, but not in the bone marrow. In rabbits fed a 0.5% cholesterol-enriched diet for 42 days, a significant increase (P < 0.01) of MnSOD-immunoreactive macrophages within the myocardium was found concomitant to the drastic elevation of serum cholesterol level. In the bone marrow, MnSOD immunoreactivity did not change after cholesterol feeding. Thus in cholesterol-fed rabbits, the increase of MnSOD-containing macrophages seems to parallel that of lipoproteins. MnSOD is considered as being protective against the cytotoxic effects of those superoxide anions, possibly generated in macrophages, which are involved in the metabolism of modified lipoproteins.

In rat hepatocyte cultures daltroban reduced 14C-acetate incorporation stronger into cholesterol (CH) esters than into free CH. Further data suggest that the reduction of cellular sterols by daltroban is independent from its TXA2 receptor antagonistic activity and caused by reduced capacity of ACAT depending CH esterification. In rabbits fed CH-enriched diet treatment with daltroban led to an inhibition of platelet aggregation and to a significant reduction of progression of atherosclerosis. Both reduced CH esterification and TXA2 receptor antagonism may contribute to the diminution of progression of atherosclerosis by daltroban.

Summary These studies have examined aortic atherogenesis in cholesterol-fed rabbits and have correlated the effects of daltroban to the pathomechanism of the vessel wall lesions. After feeding a 0.5% cholesterol-enriched diet for 96 d atherosclerotic alterations were seen, which exhibited a proximo-distal pattern, to which the branching of the aorta contributed considerably. Depending on their localization and size a varying cellular constitution of the plaques was obvious. Large plaques, which were mainly seen in the aortic arch and the proximal descending thoracic aorta, consisted of numerous proliferating cells, masses of fibrillar ground substance, clusters of foam cells, and rarely contained cholesterol crystals and necroses. Emerging plaques mainly found in distal thoracic and abdominal aorta imposed as fatty streaks. Daltroban treatment, used in a clinically relevant dosis of 10 mg/kg b.wt. per day, reduced extension and protrusional area of plaques to about 40%, which was evaluated using a newly developed computerized morphometric method, in association with significant reductions in free cholesterol content within the aorta. The results suggest that daltroban inhibits the progression of atherosclerosis in cholesterolfed rabbits. This effect may be related to its antagonistic interaction with the thromboxane A2 receptor and also to an inhibition of the cholesterol metabolism.

The effects of mevinolin, a 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor and bezafibrate, a modulator of lipoprotein metabolism, were measured on BM 15.766-induced 7-dehydrocholesterol (7-DHC) accumulation in liver and serum of rats. BM 15.766, an inhibitor of delta 7 sterol reductase, leads to an accumulation of 7-DHC, which can be used as a measure of cholesterol (CH) synthesis de novo. The investigations were carried out to evaluate the usefulness of this new non-isotopic in vivo method for testing compounds that affect directly and indirectly the CH-biosynthetic pathway. Mevinolin showed a dose-dependent reduction of BM 15.766-induced 7-DHC accumulation after a single oral dose. The dose range for reduction of 7-DHC in the liver of rats was comparable with that for serum CH-lowering in humans. Bezafibrate reduced the BM 15.766-induced 7-DHC accumulation in liver in a dose- and time-dependent manner. These findings agree with the reported reduced activity of HMG-CoA reductase and support the view, that bezafibrate reduces CH biosynthesis by modulation of lipoprotein metabolism. The 7-DHC levels in serum do not reflect those in the liver and cannot be used as a measure of CH biosynthesis. The investigations show that BM 15.766-induced 7-DHC accumulation in liver of rats is an appropriate measure for CH de novo synthesis and can be used for testing compounds that interfere directly and indirectly with the CH-biosynthetic pathway. In contrast to previously described methods, no radiolabelled precursors are necessary.(ABSTRACT TRUNCATED AT 250 WORDS)

beta,beta,beta',beta'-Tetrasubstituted, long-chain dioic acids of the general formula HOOC-C(XY)-C(R2)-Q-C-(R2)-C(XY)-COOH have been synthesized and evaluated as hypotriglyceridemic-hypocholesterolemic agents in rats and as antidiabetogenic agents in ob/ob diabetic mice. The free carboxyl function of analogues of the series was mandatory for their hypolipidemic-antidiabetogenic effect while nonhydrolyzable diesters were inactive. Other structure-activity relationships were determined as a function of the overall chain length (C12-C22), alpha,alpha'-substitutions (X, Y = H, F, Cl, Br, OH, CN), beta, beta'-substitutions (R = CH3, C6H5), and core substitutions [Q = (CH2)10, (CH2)4CH = CH(CH2)4, 1,4-C6H10[(CH2)3]2, 1,4-C6H4[(CH2)3]2, 1,4-C6H4(CH = CHCH2)2, CH2(OCH2CH2)3OCH2)]. The most effective hypolipidemic-antidiabetogenic members of the series were alpha,alpha'-nonsubstituted, beta,beta'-methyl-substituted analogues of 14-18-carbon chains having either a saturated aliphatic core or a 1,4-bis(propenyl)benzene core in the cis/trans configuration. The hypotriglyceridemic rather than the hypocholesterolemic capacity of members of the series was found to correlate with their respective capacities as liver peroxisomal proliferators in rats.

The effect of the piperazine derivative BM 15.766 (4-(2-[1-(4-chlorocinnamyl)piperazin-4-yl]ethyl]-benzoic acid) on the biosynthesis of sterols was investigated in adult rat hepatocytes in primary monolayer culture. The substance led to a dose-dependent reduction of cholesterol in the serum of various species of animals such as rat, dog and marmoset. BM 15.766 showed a dose-dependent action on the incorporation of 14C-acetate in neutral, nonsaponifiable lipids. The inhibition of the overall incorporation was 10-12% (10(-5) M). No inhibition was observed in the hepatocytes over the entire dose range of 10(-8) M to 2 X 10(-5) M, while the release of the neutral lipids from the hepatocytes into the culture medium was reduced by up to 40%. The biosynthesis of cholesterol could be reduced by more than 90%. Simultaneously, 7-dehydrocholesterol levels rose in the cells and, to a less marked extent, in the medium. This can be interpreted as an indication that 7-dehydrocholesterol is incorporated into the cell membrane, which results in a lower release of 7-dehydrocholesterol into the medium in comparison with controls. The site of attack is the inhibition of the delta 5.7-sterol delta 7-reductase. The formation of desmosterol and cholestatrienol as well as other 7-dehydrocholesterol precursors could also be demonstrated. After longer incubation, there was an additional accumulation of squalene and lanosterol with simultaneous reduction of 7-dehydrocholesterol by BM 15.766, whereas the total 14C-acetate incorporation in neutral lipids was increased.

Durch Abanderung unserer fruheren Synthesevorschrift fur die Dithiapropellane werden die Oxathiapropellane 1–5 in praparativem Masstab hergestellt. Die Konfigurationen der aus 2–5 erhaltenen Monochloroxathiapropellandioxide 11–16 werden aufgeklart. Die Uberfuhrung von 11–16 in die entsprechenden Oxapropellene 17–19 ist von der Ringgrose des Carbocyclus abhangig. Das 3-Oxa[3.3.2]propell-9-en (17) entsteht nur in ca. 1 proz. Ausb. und ist damit das Oxapropellen mit dem kleinsten Carbocyclus, das nach diesem Syntheseweg hergestellt werden kann. Condensed Ring Systems, XII. On the Influence of the Carbocycle on the Reaction Course of the Ramberg-Backlund Reaction of Oxathiapropellanes By modification of our previous specifications for the syntheses of dithiapropellanes, oxathiapropellanes 1–5 are synthesized on a preparative scale. The configurations of monochloro-oxathiapropellane dioxides 11–16 obtained from 2–5 are elucidated. The transformation of 11–16 into the analogous oxapropellenes 17–19 is dependent on the ring size of the carbocycles. 3-Oxa[3.3.2]propell-9-ene (17) is obtained in about 1% yield only, and thus represents the oxapropellene with the smallest carbocycle preparable by this synthetic route.

Marked proliferation of rat hepatic peroxisomes is observed after treatment with a new potent hypolipidemic drug BM 15766, as well as after bezafibrate. Whereas the relative specific activity of the peroxisomal fatty acid beta-oxidation system is not affected by BM 15766 it is significantly increased after bezafibrate. This is also confirmed by immunoblot analysis of individual beta-oxidation enzymes in highly purified peroxisome fractions. These observations suggest that peroxisome proliferation and the induction of the fatty acid beta-oxidation are regulated separately.

Rat liver cells derived from male and female animals in primary monolayer cultures were investigated for suitability as a test system for xenobiotics affecting the cholesterol pathway. An appropriate mode of extraction and separation of newly formed cholesterol and precursors is described. This system can be widely applied. Rat liver cells from females in oestrus cycle had a higher synthesis rate of cholesterol than those from males. The disadvantages related to the cycle phases make male rats more appropriate donor animals for the test system developed. The altered in vitro cholesterol synthesis is relevant to that in vivo. The extraction of newly synthesized cholesterol and its precursors by means of columns packed with large-pore kieselgur is precise and time saving. The modified separation by thin-layer chromatography on silica gel layers impregnated with silver nitrate enables direct separation from the extract and is sufficient to recognize cholesterol and its precursors. The method in this form is suitable for processing a large number of specimens e.g. for screening.

Treatment of rats by β,β'-methyl-substituted hexadecanedioic acid (MEDICA 16) resulted in a dose- and time-dependent increase in liver peroxisomal enoyl-CoA hydratase and cyanide-insensitive palmitoyl-CoA oxidation with a concomitant increase in the volume density of peroxisomes as determined by morphometry. The induced peroxisomal proliferation was sustained as long as treatment was maintained and was accompanied by an increase in liver weight. Incubation of cultured rat hepatocytes in the presence of MEDICA 16 added to the culture medium resulted in a dose-dependent increase in peroxisomal β-oxidation activities with a concomitant elevation of the volume density of peroxisomes. The induction of peroxisomal proliferation by MEDICA 16 in culture could be prevented in the presence of carnitine palmitoyltransferase inhibitors added to the culture medium, e.g. 2-bromopalmitate, 2-tetradecylglycidic acid or 2-[5-(4-chlorophenyl)-pentyl]oxirane-2-carboxylate. The induction of liver peroxisomes by MEDICA 16 conforms to the previously denned requirement for an amphipathic carboxylate in initiating peroxisomal proliferation. The prevention of peroxisomal proliferation by carnitine acyltransferase inhibitors may implicate the involvement of this acyltransferase in the induction of peroxisomal proliferation by xenobiotic or native amphipathic carboxylates.