4-Oxypiperidine Ethers as Multiple Targeting Ligands at Histamine H3 Receptors and Cholinesterases.

This study examines the properties of a novel series of 4-oxypiperidines designed and synthesized as histamine H3R antagonists/inverse agonists based on the structural modification of two lead compounds, viz., ADS003 and ADS009. The products are intended to maintain a high affinity for H3R while simultaneously inhibiting AChE or/and BuChE enzymes. Selected compounds were subjected to hH3R radioligand displacement and gpH3R functional assays. Some of the compounds showed nanomolar affinity. The most promising compound in the naphthalene series was ADS031, which contained a benzyl moiety at position 1 of the piperidine ring and displayed 12.5 nM affinity at the hH3R and the highest inhibitory activity against AChE (IC50 = 1.537 µM). Eight compounds showed over 60% eqBuChE inhibition and hence were qualified for the determination of the IC50 value at eqBuChE; their values ranged from 0.559 to 2.655 µM. Therapy based on a multitarget-directed ligand combining H3R antagonism with additional AChE/BuChE inhibitory properties might improve cognitive functions in multifactorial Alzheimer's disease.

Guanidines: Synthesis of Novel Histamine H3R Antagonists with Additional Breast Anticancer Activity and Cholinesterases Inhibitory Effect.

This study examines the properties of novel guanidines, designed and synthesized as histamine H3R antagonists/inverse agonists with additional pharmacological targets. We evaluated their potential against two targets viz., inhibition of MDA-MB-231, and MCF-7 breast cancer cells viability and inhibition of AChE/BuChE. ADS10310 showed micromolar cytotoxicity against breast cancer cells, combined with nanomolar affinity at hH3R, and may represent a promising target for the development of an alternative method of cancer therapy. Some of the newly synthesized compounds showed moderate inhibition of BuChE in the single-digit micromolar concentration ranges. H3R antagonist with additional AChE/BuChE inhibitory effect might improve cognitive functions in Alzheimer's disease. For ADS10310, several in vitro ADME-Tox parameters were evaluated and indicated that it is a metabolically stable compound with weak hepatotoxic activity and can be accepted for further studies.

First- and Second-Line Targeted Systemic Therapy in Hepatocellular Carcinoma-An Update on Patient Selection and Response Evaluation.

Advanced hepatocellular carcinoma (HCC) with vascular invasion and/or extrahepatic spread and preserved liver function, according to stage C of the Barcelona Clinic Liver Cancer (BCLC) classification, has a dismal prognosis. The multi-targeted tyrosine-kinase receptor inhibitor (TKI) sorafenib is the only proven active substance in systemic HCC therapy for first-line treatment. In this review, we summarize current aspects in patient selection and management of side effects, and provide an update on response evaluation during first-line sorafenib therapy. Since second-line treatment options have been improved with the successful completion of the RESORCE trial, demonstrating a survival benefit for second-line treatment with the TKI regorafenib, response monitoring during first-line therapy will be critical to deliver optimal systemic therapy in HCC. To this regard, specific side effects, in particular worsening of arterial hypertension and diarrhea, might suggest treatment response during first-line sorafenib therapy; however, clear predictive clinical markers, as well as laboratory test or serum markers, are not established. Assessment of radiologic response according to the modified Response Evaluation Criteria in Solid Tumors (mRECIST) is helpful to identify patients who do not benefit from sorafenib treatment.

Time-resolved cell culture assay analyser (TReCCA Analyser) for the analysis of on-line data: data integration--sensor correction--time-resolved IC50 determination.

Time-resolved cell culture assays circumvent the need to set arbitrary end-points and reveal the dynamics of quality controlled experiments. However, they lead to the generation of large data sets, which can represent a complexity barrier to their use. We therefore developed the Time-Resolved Cell Culture Assay (TReCCA) Analyser program to perform standard cell assay analyses efficiently and make sophisticated in-depth analyses easily available. The functions of the program include data normalising and averaging, as well as smoothing and slope calculation, pin-pointing exact change time points. A time-resolved IC50/EC50 calculation provides a better understanding of drug toxicity over time and a more accurate drug to drug comparison. Finally the logarithmic sensor recalibration function, for sensors with an exponential calibration curve, homogenises the sensor output and enables the detection of low-scale changes. To illustrate the capabilities of the TReCCA Analyser, we performed on-line monitoring of dissolved oxygen in the culture media of the breast cancer cell line MCF-7 treated with different concentrations of the anti-cancer drug Cisplatin. The TReCCA Analyser is freely available at www.uni-heidelberg.de/fakultaeten/biowissenschaften/ipmb/biologie/woelfl/Research.html. By introducing the program, we hope to encourage more systematic use of time-resolved assays and lead researchers to fully exploit their data.

Complications and risk factors in 2731 diagnostic mini-laparoscopies in patients with liver disease.

BACKGROUND/AIMS: Mini-laparoscopy (ML) allows macroscopic assessment and biopsy under direct vision and therefore is a valuable technique in the diagnosis of liver disease. Herein we report procedure-related complications and risk factors. METHODS: A total of 2731 consecutive patients underwent diagnostic ML at two university hospitals (June 1996-December 2007). ML was performed using standard technique with a 1.9mm optical instrument. Coagulation of the liver biopsy site was performed with APC. The following variables were analysed as risk factors for complications: platelet count (<50/nL), international normalized ratio (INR) (>1.5), Cirrhosis, signs of portal hypertension, prior abdominal surgery. RESULTS: Major complications occurred in 1.0% (n=27) of patients and these were, delayed bleeding from the liver biopsy site or abdominal wall (in 0.7% of patients) and intestinal perforation (in 0.3% of patients). Two patients died after severe haemorrhage (mortality 0.07%); the other patients recovered without sequelae. Bleeding risk was increased in patients with low platelets (OR=6.1), increased INR (OR=8.9), cirrhosis (OR=1.9) and portal hypertension (OR=2.1). Logistic regression showed a significant correlation only for the concurrence of low platelets and increased INR (P = 0.001; OR=14.1); bootstrap analysis identified INR >1.5 as significant predictor (P = 0.0002). Prior abdominal surgery did not carry a significant risk for intestinal perforation (OR=1.1; P = 0.142), unless abdominal adhesions were present (OR=9.5; P = 0.0002). None of the patients required surgery for intestinal perforation. CONCLUSION: Mini-laparoscopy is a diagnostic technique with a low complication rate. However, in patients with increased INR, low platelets or after extensive abdominal surgery, complications may occur in up to 5%.

Effectiveness and safety of minilaparoscopy-guided spleen biopsy: a retrospective series of 57 cases.

BACKGROUND: Minilaparoscopy is an accepted method for liver biopsy. We report our experience with minilaparoscopy for splenic biopsy. METHODS: We reviewed the records of all minilaparoscopy procedures performed from 1996 to 2004 at the University of Mainz Medical Center and from 2005 to mid-2011 at the University of Hamburg Medical Center to identify patients who underwent a minilaparoscopy-guided splenic biopsy. All procedures were performed using the previously described method (2.75-mm trocar, 2.3-mm Veress needle, 1.9-mm laparoscope) with the patient under conscious sedation (midazolam/meperidine/propofol). Splenic biopsies were performed using a second trocar with an 18-G Tru-Cut needle. Argon plasma coagulation (APC) and/or fibrin glue (FG) were used to control postbiopsy bleeding. RESULTS: Fifty-seven patients underwent minilaparoscopy-guided biopsy of the spleen (27 females, 30 males; median age = 41 years, range = 16-76). A specimen suitable for histopathologic evaluation was obtained in all patients. Grouped by preprocedure indication, a definitive diagnosis was obtained in 70% (7/10) of patients who had splenic mass lesions in prior imaging (3 B-NHL, 2 hemangioma, 1 tuberculosis, 1 sarcoidosis; p < 0.01) compared to 29% (10/34) in the group with unexplained fever or suspected lymphoma (3 tuberculosis, 2 B-NHL, 1 hepatosplenic T-cell lymphoma, 1 sarcoidosis, 1 Still's disease, 1 EBV, 1 Q-fever) and 0/13 with unexplained splenomegaly. Focal lesions noted at laparoscopy yielded to a histologic diagnosis in 38% (11/29) of 42 patients compared to 21% (6/28) without laparoscopic abnormality (p = 0.25). Bleeding from the biopsy site was noted in 96.5% (55/57) and was classified as brisk in 9. Control of hemorrhage was achieved in all patients (APC: 47, FG: 1, APC/FG: 7). There was no postprocedure bleeding or other complications. CONCLUSION: Splenic biopsy guided by minilaparoscopy can be performed safely. Postprocedure bleeding is readily controlled with APC with or without fibrin glue. The highest diagnostic yield is in patients with focal splenic lesions.

Beta-amyloid (Abeta40, Abeta42) binding to modified LDL accelerates macrophage foam cell formation.

Apart from its role as a risk factor in arteriosclerosis, plasma cholesterol is increasingly recognized to play a major role in the pathogenesis of Alzheimer's disease (AD). Moreover, alterations of intracellular cholesterol metabolism in neuronal and vascular cells are of considerable importance for the understanding of AD. Cellular cholesterol accumulation enhances the deposition of insoluble beta-amyloid peptides, which is considered a hallmark in the pathogenesis of AD. In order to test the hypothesis, whether exogenous beta-amyloid peptides (Abeta42, Abeta40) might contribute to cellular cholesterol accumulation by opsonization of lipoproteins, we compared the binding and uptake of native LDL, enzymatically modified LDL (E-LDL), copper oxidized LDL (Ox-LDL) and HDL as control, preincubated either in the absence or presence of Abeta42 or Abeta40, by human monocytes or monocyte-derived macrophages. Incubation of monocytes and macrophages with Abeta-lipoprotein-complexes lead to increased cellular free and esterified cholesterol when compared to non-opsonized lipoproteins, except for HDL. Furthermore, the cellular uptake of these complexes regulated Abeta-receptors such as FPRL-1 or LRP/CD91. In summary, our results suggest that Abeta42 and Abeta40 act as potent opsonins for LDL, E-LDL and Ox-LDL and enhance cellular cholesterol accumulation as well as Abeta-deposition in vessel wall macrophages.

E-LDL and Ox-LDL differentially regulate ceramide and cholesterol raft microdomains in human Macrophages.

Atherosclerosis is characterized by the generation of lipid-loaded macrophage-derived foam cells. To study the effect of different types of atherogenic lipoproteins, human macrophages were loaded with enzymatically degraded low density lipoprotein (E-LDL) or oxidized LDL (Ox-LDL). Cellular cholesterol content was increased by E-LDL, whereas Ox-LDL increased the ceramide content. Cell surface expression analysis by flow cytometry and confocal microscopy revealed that Ox-LDL increased ceramide and lactosylceramide expression compared to E-LDL loading and induced ceramide rafts, whereas loading with E-LDL induced cholesterol-rich microdomains. Formation of different rafts may have consequences for raft associated signaling in cholesterol homeostasis and apoptosis in human macrophages.

Ezetimib influences the expression of raft-associated antigens in human monocytes.

Aminopeptidase N (CD13) was recently identified as a molecular target of the cholesterol absorption inhibitor Ezetimib. Regarding that CD13 is expressed in lipid rafts of monocytic cells, we have investigated whether Ezetimib influences raft function in these cells. Expression of raft-associated antigens (CD11b, CD13, CD14, CD16, CD36, and CD64) was followed by flow cytometry and/or immunoblot in human monocyte-derived macrophages in response to in vitro administration of Ezetimib. Cellular redistribution of CD13 was assessed by confocal imaging. Ezetimib significantly decreased the surface expression of CD13, CD16, CD64, and CD36; furthermore, it induced a shift of CD13 from plasma membrane to intracellular vesicles, and thus it quite likely modulated monocytic raft-assembly.

Evaluation of a high-content screening fluorescence-based assay analyzing the pharmacological modulation of lipid homeostasis in human macrophages.

BACKGROUND: For understanding cholesterol and phospholipid efflux pathways there is a need for cellular fluorescence-based high-content screens (HCS) to investigated the cholesterol and phospholipid content in human macrophages. METHODS: Making use of fluorescence imaging based on HCS we have developed a tool to evaluate new agents that can act as inducers of cholesterol efflux. The fluorescence assay is based on the different staining patterns of cholesterol-loaded (E-LDL) and deloaded (HDL3) differentiated monocytes by the saturated, fluorescent lipid probe (1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine)-tetramethyl-rhodamin. RESULTS: Morphologic examination and statistical evaluation of the staining pattern such as gray value, threshold area, shape factor and the spot size distribution provides evidence for a significant pattern change when cholesterol enriched and cholesterol depleted differentiated monocytes were imaged.