Discovery of Novel 5-Lipoxygenase-Activating Protein (FLAP) Inhibitors by Exploiting a Multistep Virtual Screening Protocol.

Leukotrienes (LTs) are proinflammatory mediators derived from arachidonic acid (AA), which play significant roles in inflammatory diseases. The 5-lipoxygenase-activating protein (FLAP) is an integral membrane protein, which is essential for the initial step in LT biosynthesis. The aim of this study was to discover novel and chemically diverse FLAP inhibitors for treatment of inflammatory diseases requiring anti-LT therapy. Both ligand- and structure-based approaches were applied to explain the activities of known FLAP inhibitors in relation to their predicted binding modes. We gained valuable insights into the binding modes of the inhibitors by molecular modeling and generated a multistep virtual screening (VS) workflow in which 6.2 million compounds were virtually screened, and the molecular hypotheses were validated by testing VS-hit compounds biologically. The most potent hit compounds showed significant inhibition of FLAP-dependent cellular LT biosynthesis with IC50 values in the range from 0.13 to 0.87 µM. Collectively, this study provided novel bioactive chemotypes with potential for further development as effective anti-inflammatory drugs.

Development and optimization of an ultra-fast microextraction followed by HPLC-UV of tetracycline residues in milk products.

The present study developed a new ultra-fast microextraction (within 8 min) with acetonitrile followed by gradient reversed-phase HPLC-UV method to determinate six tetracyclines simultaneously in various milk products with HPLC analysis time of 9 min. Chromatographic separations were achieved with a phenomenex™ Synergi 4 µm Fusion-RP (150 × 4.60 mm) column at 220 nm, using acetonitrile/KH2PO4 buffer (4 mmol·L-1, pH 2.5) as mobile phase. The HPLC method showed excellent peak resolutions (3.3-8.8) and peak symmetry (0.99-1.11) inspite of short analysis time. The extraction rates from milk products showed consistently very good values over all tetracyclines (milk 80.58 ± 5.39 %, yoghurt 82.61 ± 5.63 %, cream cheese 80.13 ± 6.32 %, buttermilk 81.07 ± 6.49 %, kefir 79.69 ± 6.51 %, skyr 78.12 ± 5.22 %, quark 65.37 ± 4.72 %). The optimized method was found to be specific, reproducible, robust. This study combines for the first time a fast, cheap quantification of six tetracyclines via HPLC-UV with a reliable microextraction applicable to various milk products using only standard laboratory equipment.

Stereoselective plasma protein binding of amlodipine.

The binding of the (R)- and (S)-enantiomers of amlodipine to bovine serum albumin (BSA), human serum albumin (HSA), alpha(1)-acid glycoprotein (AGP), and human plasma (HP) was studied by equilibrium dialysis over the concentration range of 75-200 microM at a protein concentration of 150 microM. Unbound drug concentrations were determined by enantioselective capillary electrophoresis using 50 mM phosphate buffer, pH 2.5, containing 18 mM alpha-cyclodextrin as background electrolyte. Saturation of the protein binding sites was not observed over the concentration range tested. Upon application of racemic amlodipine besylate, (S)-amlodipine was bound to a higher extend by HSA and HP compared with (R)-amlodipine, whereas the opposite binding of the enantiomers was observed for BSA and AGP. Scatchard analysis was used to illustrate the different binding affinities of amlodipine besylate enantiomers to BSA, HSA and AGP.

Adverse Drug Events in Patients with Chronic Kidney Disease Associated with Multiple Drug Interactions and Polypharmacy.

BACKGROUND AND PURPOSE: Chronic kidney disease (CKD) is associated with adverse drug events due to medication errors and the risks of polypharmacy. The aim of this study was to investigate whether multiple pharmacodynamic interactions are a significant problem in CKD patients to improve medication safety. METHODS: The discharge medication of 200 elderly patients with stage 3, 4 and 5/5D CKD was analysed in a retrospective observational study with respect to kidney-related medication errors and multiple pharmacodynamic interactions. The clinical relevance of the most common and hazardous multiple interactions was assessed by evaluating adverse events at the primary or the subsequent hospital stay. RESULTS: Findings showed that 29.5% of the study cohort were at risk of QTc-interval prolongation in association with their medication combinations and half of them exhibited QTc-interval prolongation. The QTc interval was extended among all patients receiving a combination of two or more drugs with 'known' risk of Torsades de pointes. Amiodarone, citalopram and ciprofloxacin turned out to be the most hazardous drugs in this context. Eight percent of the patient population received a regimen of 4-6 potassium-enhancing drugs during their hospital stay, which was not de-escalated in 75.0% in the ambulatory setting. Despite close monitoring in the clinical setting, 37.5% of these patients developed hyperkalaemic episodes during their primary stay and 66.7% during rehospitalization. Of the study cohort, 8.5% received a combination of three drugs with antithrombotic or antiplatelet effects. Of these, 64.7% developed haemorrhagic events with two of them proving fatal. CONCLUSION: Multiple pharmacodynamic interactions related to QTc prolongation, hyperkalaemia and haemorrhage are frequently associated with a negative outcome in older adults with CKD and often require recurrent medical treatment or rehospitalization.

NO donors. Part 18: Bioactive metabolites of GTN and PETN--synthesis and vasorelaxant properties.

The vasodilators glyceryl trinitrate (GTN) and pentaerythrityl tetranitrate (PETN) are supposed to be degraded in vivo to the lower nitrates PETriN, PEDN, PEMN, 1,2-GDN, 1,3-GDN, 1-GMN, and 2-GMN. We synthesized these bioactive metabolites as reference compounds for pharmacokinetic studies. The use of HPLC-methods for monitoring the stepwise reduction of PETN to lower nitrates and the syntheses of the glyceryl dinitrates proved advantageous. Furthermore, we measured the vasorelaxant properties of all metabolites by performing organ bath experiments with porcine pulmonary arteries. In general, the vasodilator potency increases with the number of nitrate moieties in the compound.

Analysis of secondary care data to evaluate the clinical relevance of the drug-drug interaction between amlodipine and simvastatin.

BACKGROUND: Pharmacokinetic analyses revealed an increase in the bioavailability of simvastatin when co-administered with amlodipine [Nishio S et al. Hypertensin research 2005; Son H et al. Drug metabolism and pharmacokinetics 2014]. This may induce an increased risk of muscle toxicity for patients who receive this combination. So far, no in vivo data on the clinical relevance of this interaction exist. The objective of the present analysis was to determine the number of patients with concomitant treatment of amlodipine and simvastatin. Subsequently, the data was analyzed for the indication of muscular discomfort. Patients with combined prescription of amlodipine and another hydroxymethylglutaryl-CoA-reductase inhibitor except simvastatin or patients receiving simvastatin without amlodipine served as control groups. METHODS: The present analysis used secondary data from the health insurance company AOK PLUS including information regarding diagnosis and drug prescriptions. RESULTS: In total, 67.081 patients corresponding to 4.93% of the analyzed collective received a combined prescription of amlodipine and simvastatin. The absolute frequency increased continuously over time. Muscular discomfort was detected in a) 6.20% of the patients receiving amlodipine and simvastatin, b) 6.60% of the patients receiving amlodipine and another hydroxymethylglutaryl-CoA- reductase inhibitor and c) 8.04% of the patients with simvastatin only. CONCLUSIONS: The present analysis shows an increasing trend of combined prescriptions of amlodipine and simvastatin. Evidence for simvastatin dose adaptation or therapy switch to another hydroxymethylglutaryl-CoA-reductase inhibitor, however, was not found. Muscular discomfort does not occur more often in patients with amlodipine and simvastatin compared to the two control groups. The results of the present analysis reveal no evidence for a clinically relevant interaction between amlodipine and simvastatin.

Heme oxygenase-1: a novel key player in the development of tolerance in response to organic nitrates.

OBJECTIVE: Nitrate tolerance is likely attributable to an increased production of reactive oxygen species (ROS) leading to an inhibition of the mitochondrial aldehyde dehydrogenase (ALDH-2), representing the nitroglycerin (GTN) and pentaerythrityl tetranitrate (PETN) bioactivating enzyme, and to impaired nitric oxide bioactivity and signaling. We tested whether differences in their capacity to induce heme oxygenase-1 (HO-1) might explain why PETN and not GTN therapy is devoid of nitrate and cross-tolerance. METHODS AND RESULTS: Wistar rats were treated with PETN or GTN (10.5 or 6.6 microg/kg/min for 4 days). In contrast to GTN, PETN did not induce nitrate tolerance or cross-tolerance as assessed by isometric tension recordings in isolated aortic rings. Vascular protein and mRNA expression of HO-1 and ferritin were increased in response to PETN but not GTN. In contrast to GTN therapy, NO signaling, ROS formation, and the activity of ALDH-2 (as assessed by an high-performance liquid chromatography-based method) were not significantly influenced by PETN. Inhibition of HO-1 expression by apigenin induced "tolerance" to PETN whereas HO-1 gene induction by hemin prevented tolerance in GTN treated rats. CONCLUSIONS: HO-1 expression and activity appear to play a key role in the development of nitrate tolerance and might represent an intrinsic antioxidative mechanism of therapeutic interest.

In Vitro Studies on Physiological and Chemical Stability of New LE404-Derivatives with Extended Half-Life.

Dibenzoazecines are a class of potential neuroleptics with high affinity to dopamine and serotonin receptors. The efficacy and high therapeutic range has already been demonstrated in vivo with the lead structure 7-methyl-5,6,7,8,9,14-hexahydrodibenzo[d,g]azecin-3-ol (LE404: ) and selected derivatives. There is a variety of new synthesized structurally different dibenzoazecine derivatives with the aim to improve pharmacokinetic parameters, all of which contain the lead structure LE404: . For a multitude of these substances is still a lack of information, inclusive of stability, physicochemical parameters, pharmacokinetics and metabolism. Therefore, the present study investigated the stability properties of 17 new azecine derivatives, including esterase cleavage, stability in simulated gastrointestinal fluid, stability at different pH-values and determination of octanol/water-partition coefficients. These findings, in correlation to the properties and efficacy of the already in vivo tested substances, will be useful for safety and efficacy in further in vivo tests.

Synthesis and Characterization of new Azecine-Derivatives as Potential Neuroleptics.

Dibenzo- and benzindolo-azecines represent a class of potential neuroleptics. To characterize the effectiveness at the dopamine and 5-HT2A-receptor representative structures were synthesized and tested by radio ligand binding studies, in vivo and in vitro studies.Neuroleptic potency and the risk of side effects of the prodrug 7-methyl-5,6,7,8,9,14-hexahydrodibenzo[d,g]azecin-3-yl isobutyrate, an ester derivative of the most promising azecine 7-methyl-5,6,7,8,9,14-hexahydrodibenzo[d,g]azecin-3-ol (LE404), was tested in vivo concerning conditioned avoidance response inhibition, locomotor activity and triggering of catalepsy vs. haloperidol as a reference. Also ester hydrolysis was examined using porcine liver esterase to thereby obtain an indication of the stability of the prodrug in vivo. An HPLC method was developed for purity control and determination of octanol/water-distribution coefficients.It has been shown that the tested substances in their efficacy are comparable to haloperidol and risperidone, but the therapeutic index in most cases is larger. Esterification as a prodrug principle leads to significantly prolonged effectiveness.

Impact of pharmacokinetics on the toxicity and efficacy of clofarabine in patients with relapsed or refractory acute myeloid leukemia.

Common side effects of clofarabine (CFB) are liver toxicity, particularly a transient elevation of transaminases and skin toxicity. We studied the correlation of pharmacokinetic (PK) parameters with these toxicities and the efficacy of CFB in patients with relapsed or refractory acute myeloid leukemia. Clofarabine PK parameters showed large inter-individual variability. A higher CFB area under the curve was significantly associated with higher transaminase levels (p = .011 for aspartate aminotransferase (AST), adjusted for age, sex, cumulated CFB dosage, baseline AST, and glomerular filtration rate (GFR)). No significant association could be found between maximum concentration and the liver toxicity parameters. The occurrence of skin toxicity and the response to re-induction chemotherapy evaluated at day 15 were also not associated with PK. In conclusion, a higher individual CFB exposure is associated with increased liver toxicity reflected by elevated liver enzymes, without having an impact on anti-leukemic efficacy.

NO-donors, part X [1] : investigations on the stability of pentaerythrityl tetranitrate (PETN) by HPLC-chemoluminescence-N-detection (CLND) versus UV-detection in HPLC.

HPLC in combination with chemoluminescence-N-detection (CLND) is very useful for the analysis of pentaerythrityl tetranitrate (PETN) and its possible biological and chemical degradation products pentaerythrityl trinitrate (PETriN), pentaerythrityl dinitrate (PEDiN) and pentaerythrityl mononitrate (PEMonoN). Quantification is more convenient and sensitivity of this method is about four times higher compared to UV-detection. The present study demonstrates that PETN is a chemically more stable compound in vitro than expected. No degradation was observed in aqueous buffers (37 degrees C, pH 5.6, 7.4), human plasma, and simulated intestinal or gastric fluid. On the other hand, the addition of increasing amounts of thioles (cysteine, thioglycolic acid) induced an increasing degradation of PETN.

Simultaneous determination of clofarabine and cytarabine in human plasma by LC-MS/MS.

Combination of cytostatic agents is a basic principle in the treatment of cancer. For the treatment of acute myeloid leukemia (AML), purine analogs, like clofarabine and cytarabine act synergistically. Little is known, however, on their interaction in vivo. We developed a method for the simultaneous determination of clofarabine and cytarabine in human plasma. The substances were extracted from plasma samples by protein precipitation with acetonitrile. Cladribine was the internal standard (IS). The analytes were separated on Synergi HydroRP column (150mm×2.0mm, 4µm) and a triple-quadrupole mass spectrometry with an electrospray ionisation (ESI) source was applied for detection. The mobile phase consisted of acetonitrile, ammonium acetate 2mM and 0.5% formic acid in a gradient mode at a flow rate of 0.5ml/min. The injection volume was 10µl and the total run time was 6.0min. Retention times were 2.46min for clofarabine, 0.97min for cytarabine and 2.43min for the IS. Calibration ranges were 8-1000ng/ml for clofarabine and 20-2500ng/ml for cytarabine. The intra-day and inter-day precision was less than 15% and the relative standard deviation was all within ±15%. This new method allows a rapid and simple determination of both clofarabine and cytarabine in human plasma. It was applied to a pharmacokinetic investigation within a hematological trial in adult patients with AML.

A synopsis on different homologous series of fomocaine derivatives. In vitro interactions with the cytochrome P450 system, toxicity, and local anaesthetic effects in rats--Part 1.

Fomocaine (CAS 56583-43-6) is a basic ether-type local anaesthetic used in dermatological practice for surface anaesthesia. For many years, modifications of the fomocaine molecule have been pursued, e.g. to improve its physicochemical properties and also in view of possible new (systemic) applications, e.g. in the treatment of migraine or as antiarrhythmic. The present paper provides a survey of the investigations undertaken with all the different series of fomocaine derivatives synthesized so far with respect to their in vitro interaction capacity at the cytochrome P450 system, in vivo toxicity (LD50; paresis of the N. ischiadicus) and local anaesthetic effects (conduction anaesthesia at the N. ischiadicus; surface anaesthesia of the cornea) in rats. The main objective of this systematic comparison of the effects of all these substances was to assess possible basic structure-activity relationships.

[Conjugation reactions of ciprofibrate with human and laboratory animals]. / Konjugationsreaktionen von Ciprofibrat bei Menschen und Versuchstieren.

An open problem of the lipid lowering agent ciprofibrate (rac-2-[4-(2,2-dichlorocyclopropyl)-phenoxy]-2-methylpropanoic acid, CAS 52214-84-3) is its metabolism concerning the conjugation with amino acids and glucuronic acid. It could be solved by syntheses of the needed reference compounds--unknown up to now--and administration of ciprofibrate to volunteers and rats. Unexpectedly the conjugation compounds with amino acids are stable in vitro and in metabolism. There was no evidence for any conjugation reaction with amino acids by investigating samples of urine and faeces. On the contrary the urine of humans contains 90-97% of beta-O-acylglucuronide, whereas rat urine shows only 10% of the calculated amount.