Inhibition of the PI3K but not the MEK/ERK pathway sensitizes human glioma cells to alkylating drugs.

BACKGROUND: Intrinsic chemoresistance of glioblastoma (GBM) is frequently owed to activation of the PI3K and MEK/ERK pathways. These signaling cascades are tightly interconnected however the quantitative contribution of both to intrinsic resistance is still not clear. Here, we aimed at determining the activation status of these pathways in human GBM biopsies and cells and investigating the quantitative impact of both pathways to chemoresistance. METHODS: Receptor tyrosine kinase (RTK) pathways in temozolomide (TMZ) treatment naive or TMZ resistant human GBM biopsies and GBM cells were investigated by proteome profiling and immunoblotting of a subset of proteins. Resistance to drugs and RTK pathway inhibitors was assessed by MTT assays. Apoptotic rates were determined by Annexin V staining and DNA damage with comet assays and immunoblotting. RESULTS: We analyzed activation of RTK pathways by proteome profiling of tumor samples of patients which were diagnosed a secondary GBM and underwent surgery and patients which underwent a second surgery after TMZ treatment due to recurrence of the tumor. We observed substantial activation of the PI3K and MEK/ERK pathways in both groups. However, AKT and CREB phosphorylation was reduced in biopsies of resistant tumors while ERK phosphorylation remained unchanged. Subsequent proteome profiling revealed that multiple RTKs and downstream targets are also activated in three GBM cell lines. We then systematically describe a mechanism of resistance of GBM cell lines and human primary GBM cells to the alkylating drugs TMZ and cisplatin. No specific inhibitor of the upstream RTKs sensitized cells to drug treatment. In contrast, we were able to restore sensitivity to TMZ and cisplatin by inhibiting PI3K in all cell lines and in human primary GBM cells. Interestingly, an opposite effect was observed when we inhibited the MEK/ERK signaling cascade with two different inhibitors. CONCLUSIONS: Temozolomide treatment naive and TMZ resistant GBM biopsies show a distinct activation pattern of the MEK/ERK and PI3K signaling cascades indicating a role of these pathways in resistance development. Both pathways are also activated in GBM cell lines, however, only the PI3K pathway seems to play a crucial role in resistance to alkylating agents and might serve as drug target for chemosensitization.

Thioredoxin Confers Intrinsic Resistance to Cytostatic Drugs in Human Glioma Cells.

Thioredoxin (Trx) overexpression is known to be a cause of chemotherapy resistance in various tumor entities. However, Trx effects on resistance are complex and depend strictly on tissue type. In the present study, we analyzed the impact of the Trx system on intrinsic chemoresistance of human glioblastoma multiforme (GBM) cells to cytostatic drugs. Resistance of GBM cell lines and primary cells to drugs and signaling inhibitors was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Impact of Trx inhibition on apoptosis was investigated by proteome profiling of a subset of proteins and annexin V apoptosis assays. Trx-interacting protein (TXNIP) was overexpressed by transfection and protein expression was determined by immunoblotting. Pharmacological inhibition of Trx by 1-methyl-2-imidazolyl-disulfide (PX-12) reduced viability of three GBM cell lines, induced expression of active caspase-3, and reduced phosphorylation of AKT-kinase and expression of ß-catenin. Sensitivity to cisplatin could be restored by both PX-12 and recombinant expression of the upstream Trx inhibitor TXNIP, respectively. In addition, PX-12 also sensitized primary human GBM cells to temozolomide. Combined inhibition of Trx and the phosphatidylinositide 3-kinase (PI3K) pathway resulted in massive cell death. We conclude that the Trx system and the PI3K pathway act as a sequential cascade and could potentially present a new drug target.

Is sunitinib a Narrow Therapeutic Index Drug? - A systematic review and in vitro toxicology-analysis of Sunitinib vs. Imatinib in cells from different tissues.

Narrow Therapeutic Index Drugs (NTIDs) are characterized by a small range between therapeutic and toxicological effect. Missing international harmonized definition for NTIDs the EMA does not even have a definition of NTIDs in contrast to the U.S. FDA, Health Canada, and the Japanese NIHS. Sunitinib, a tyrosine kinase inhibitor (TKI), indicated for the treatment of certain cancer types, will be running off-patent soon. Falling into the category of NTID would have a major impact on regulatory requirements for generic applications. Our analyses of metadata revealed numerous arguments in favor of a NTID designation. We used in vitro experiments to also give initial experimental answers. Five cell types of different tissue origin were examined for determination of IC50-values in cell viability assays. For comparison, the first-in-class TKI Imatinib was used as reference non-NTID drug. In addition, apoptotic proteins were investigated with respect to their expression and phosphorylation status. These in vitro experiments showed systematically higher toxicity of Sunitinib compared to Imatinib and a different expression and phosphorylation pattern of apoptotic proteins. In vitro data can only give preliminary results and further experiments with clinical blood samples and tumor biopsies are needed to finally clarify NTID status of Sunitinib.

Clinical pharmacology and regulatory consequences of GnRH analogues in prostate cancer.

INTRODUCTION: GnRH (gonadotropin-releasing hormone) analogues are long-term known to be safe and effective in the clinical management of hormone-dependent advanced prostate cancer. However, their unusual mechanism of action of de-sensitizing pituitary receptors makes generic market entry challenging. In addition, safety aspects like initial flare-up, breakthrough escape, and miniflares render planning and organization of clinical registration trials a complex project. REGULATORY REQUIREMENTS THERAPEUTIC EQUIVALENCE: Regulatory requirements are high as these medicines are compared to bilateral surgical castration with a 100% success rate. GnRH analogues will be used probably even wider in the near future due to demographic development and extension of indications. However, they are challenged by their antagonistic counterparts, which are avoiding flare-up phenomena. The following article deals with regulatory requirements of GnRH analogues in regard to their clinical characteristics.

Investigations on the in vitro and in vivo metabolic fate of the new synthetic opioid desmethylmoramide using HPLC-HRMS/MS for toxicological screening purposes.

New synthetic opioids are an increasing challenge for clinical and forensic toxicologists that developed over the recent years. Desmethylmoramide (DMM), a structural analogue of methadone, is one of the most recent appearances on the drug market. This study investigated its metabolic fate in rat and pooled human liver S9 fraction (pHLS9) to allow the identification of suitable urinary screening targets beyond the parent compound. The analysis of rat urine after the administration of DMM revealed five metabolites, which were the result of pyrrolidine ring or morpholine ring hydroxylation and combinations of them. Additionally, an N',N-bisdesalkyl metabolite was formed. Incubations of DMM using pHLS9 revealed a pyrrolidine hydroxy metabolite, as well as an N-oxide. No Phase II metabolites were detected in either rat urine or incubations using pHLS9. The metabolism of DMM did in part comply with that of its archetype dextromoramide (DXM). Although morpholine ring hydroxylation and N-oxidation were described for DXM and detected for DMM, phenyl ring hydroxylation was not found for DMM but described for DXM. An analysis of 24 h pooled rat urine samples after DMM administration identified the hydroxy and dihydroxy metabolite as the most abundant excretion products, and they may, thus, serve as screening targets, as the parent compound was barely detectable.

Characterization and Metabolism of Drug Products Containing the Cocaine-Like New Psychoactive Substances Indatraline and Troparil.

With a rising demand of cocaine over the last years, it is likely that unregulated new psychoactive substances with similar effects such as indatraline ((1R,3S)-3-(3,4-dichlorophenyl)-N-methyl-2,3-dihydro-1H-inden-1-amine) and troparil (Methyl (1R,2S,3S,5S)-8-methyl-3-phenyl-8-azabicyclo[3.2.1]octane-2-carboxylate) become popular as well. Both substances share a similar pharmacological profile as cocaine, while their potency is higher, and their duration of action is longer. This study investigated their metabolic fate in rat urine and incubations using pooled human liver S9 fraction (pHLS9). Indatraline formed two phase I and four phase II metabolites, with aromatic hydroxylation and glucuronidation being the main metabolic steps. All metabolites were detected in rat urine, while the parent compound was not detectable. Although low in abundance, indatraline metabolites were well identifiable due to their specific isotopic patterns caused by chlorine. Troparil formed four phase I and three phase II metabolites, with demethylation being the main metabolic step. Hydroxylation of the tropane ring, the phenyl ring, and combinations of these steps, as well as glucuronidation, were found. Phase I metabolites were detectable in rat urine and pHLS9, while phase II metabolites were only detectable in rat urine.

Analytical toxicology of the semi-synthetic cannabinoid hexahydrocannabinol studied in human samples, pooled human liver S9 fraction, rat samples and drug products using HPLC-HRMS-MS.

Hexahydrocannabinol (HHC) is an emerging semi-synthetic cannabinoid, which is obtained from cyclization of cannabidiol and subsequent hydrogenation. As a potentially legal alternative of ∆9-tetrahydrocannabinol (∆9-THC), it is increasingly seized in the USA and Europe. The aims of this study were to investigate the metabolism of HHC in pooled human liver S9 fraction (pHLS9), rat and human samples. Additionally, a locally obtained low-THC cannabis product was investigated, which was advertised with an elevated concentration of HHC. Overall, HHC formed an 11-hydroxy (HO) metabolite, as well as a carboxy metabolite. While only the parent compound was detected in rat urine and feces, the hydroxy metabolite was additionally detected in pHLS9 and human plasma. The carboxy metabolite was only detectable in human plasma. The metabolism corresponded well to that of ∆9-THC, although glucuronidation and the formation of an 8-HO metabolite were not observed. Detectability of HHC and its carboxy metabolite in rat urine was investigated using gas chromatography-mass spectrometry, but neither the parent compound nor the metabolite were detectable. The investigated low-THC cannabis product appeared to be an actual cannabis product since, in addition to HHC, cannabinol, cannabidiol and ∆9-THC were detected after qualitative analysis. Estimation of its content revealed not only 30.6% of HHC but also 4% of ∆9-THC.

Methadone-mediated sensitization of glioblastoma cells is drug and cell line dependent.

PURPOSE: D,L-methadone (MET), an analgesic drug used for pain treatment and opiate addiction, has achieved attention from oncologists and social media as possible chemoensitizing agent in cancer therapy, notably brain cancer (glioblastoma multiforme, GBM). MET has been reported to enhance doxorubicin-induced cytotoxicity in GBM cells via activation of the µ-opioid receptor (MOR). Here, we extended this work and quantified the toxic effect of MET in comparison to other opioids alone and in combination with doxorubicin and the clinically more relevant alkylating drug temozolomide (TMZ), using a set of GBM cell lines and primary GBM cells. METHODS: MOR expression in GBM cells was investigated by immunofluorescence and immunoblotting. Resistance to drugs alone and in combination with anticancer drugs was assessed by MTT assays. Concentration effect curves were fitted by nonlinear regression analysis and IC50 values were calculated. Apoptosis and necrosis rates were determined by annexin V/propidium iodide (PI)-flow cytometry. RESULTS: MET alone was cytotoxic in all GBM cell lines and primary GBM cells at high micromolar concentrations (IC50 ~ 60-130 µM), observed both in the metabolic MTT assay and by quantifying apoptosis and necrosis, while morphine and oxycodone were not cytotoxic in this concentration range. Naloxone was not able to block MET-induced cytotoxicity, indicating that cell death-inducing effects of MET are not MOR-dependent. We recorded doxorubicin and TMZ concentration- response curves in combination with fixed MET concentrations. MET enhanced doxorubicin-induced cytotoxicity in only one cell line, and in primary cells it was observed only in a particular MET concentration range. In all assays, MET was not effective in sensitizing cells to TMZ. In two cell lines, MET even decreased the cell's sensitivity to TMZ. CONCLUSION: MET was found to be cytotoxic in GBM cells in vitro only at high, clinically not relevant concentrations, where it was effective in inducing apoptosis and necrosis. Sensitizing effects were only observed in combination with doxorubicin, but not with TMZ, and are dependent on cell line and the applied drug concentration. Therefore, our findings do not support the use of MET in the treatment of GBM in combination with TMZ, as no sensitizing effect of MET was observed.

Studies on the In Vitro and In Vivo Metabolic Fate of the New Psychoactive Substance N-Ethyl-N-Propyltryptamine for Analytical Purposes.

Prerequisites for the reliable identification of substances in terms of forensic and clinical toxicology or doping control include knowledge about their metabolism and their excretion patterns in urine. N-Ethyl-N-propyltryptamine (N-ethyl-N-[2-(1H-indol-3-yl)ethyl]propan-1-amine, EPT) is an N,N-dialkylated tryptamine derivative, sold as new psychoactive substance, and supposed to act as a partial agonist at the 5-HT2A receptor. The aims of the presented study were to elucidate in vitro metabolites of EPT after incubations with pooled human liver S9 fraction (pS9) and in vivo metabolites excreted into rat urine. Finally, suitable analytical target compounds should be identified. Analysis of pS9 incubations using liquid chromatography-high-resolution tandem mass spectrometry revealed EPT metabolites formed after N-dealkylation as well as alkyl and aryl hydroxylation and formation of a hydroxy sulfate. Investigations using rat urine after oral dosing showed that the metabolic pathways of EPT shifted from in vitro hydroxylation of the alkyl amine group to an increased in vivo hydroxylation of the indole ring with several N-dealkyl metabolites. A glucuronic acid conjugate after hydroxylation of the indole ring was additionally found in vivo. The parent compound could not be detected in the rat urine samples. Therefore, analytical methods using mass spectrometry should include hydroxy-EPT and two hydroxy-EPT glucuronide isomers for reliable identification.

Flubromazolam-Derived Designer Benzodiazepines: Toxicokinetics and Analytical Toxicology of Clobromazolam and Bromazolam.

Flubromazolam is widely known as highly potent designer benzodiazepine (DBZD). Recently, the two flubromazolam-derived new psychoactive substances (NPS) clobromazolam and bromazolam appeared on the drugs of abuse market. Since no information concerning their toxicokinetics in humans is available, the aims of the current study were to elucidate their metabolic profile and to identify the isozymes involved in their phase I and phase II metabolism. In vitro incubations with pooled human liver S9 fraction were performed and analyzed by liquid chromatography coupled to orbitrap-based high-resolution tandem mass spectrometry (LC-HRMS-MS). Biosamples after the ingestion of bromazolam allowed the identification of metabolites in human plasma and urine as well as the determination of bromazolam plasma concentrations by LC-HRMS-MS using the standard addition method. In total, eight clobromazolam metabolites were identified in vitro as well as eight bromazolam metabolites in vitro and in vivo. Predominant metabolic steps were hydroxylation, glucuronidation and combinations thereof. Alpha-hydroxy bromazolam glucuronide and bromazolam N-glucuronide are recommended as screening targets in urine. Bromazolam and its alpha-hydroxy metabolite are recommended if conjugate cleavage is part of the sample preparation procedure. The bromazolam plasma concentrations were determined to be 6 and 29 µg/L, respectively. Several cytochrome P450 (CYP) and uridine 5'-diphospho-glucuronosyltransferase (UGT) isozymes were shown to catalyze their metabolic transformations. CYP3A4 was involved in the formation of all phase I metabolites of both NPS, while UGT1A4 and UGT2B10 catalyzed their N-glucuronidation. Several UGT isoforms catalyzed the glucuronidation of the hydroxy metabolites. In conclusion, the determined bromazolam plasma concentrations in the low micrograms per liter range underlined the need for sensitive analytical methods and the importance of suitable urine screening procedures including DBZD metabolites as targets. Such an analytical strategy should be also applicable for clobromazolam.

NF546 [4,4'-(carbonylbis(imino-3,1-phenylene-carbonylimino-3,1-(4-methyl-phenylene)-carbonylimino))-bis(1,3-xylene-alpha,alpha'-diphosphonic acid) tetrasodium salt] is a non-nucleotide P2Y11 agonist and stimulates release of interleukin-8 from human monocyte-derived dendritic cells.

The G protein-coupled P2Y(11) receptor is involved in immune system modulation. In-depth physiological evaluation is hampered, however, by a lack of selective and potent ligands. By screening a library of sulfonic and phosphonic acid derivatives at P2Y(11) receptors recombinantly expressed in human 1321N1 astrocytoma cells (calcium and cAMP assays), the selective non-nucleotide P2Y(11) agonist NF546 [4,4'-(carbonylbis(imino-3,1-phenylene-carbonylimino-3,1-(4-methyl-phenylene)carbonylimino))-bis(1,3-xylene-alpha,alpha'-diphosphonic acid) tetrasodium salt] was identified. NF546 had a pEC(50) of 6.27 and is relatively selective for P2Y(11) over P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(12), P2X(1), P2X(2), and P2X(2)-X(3). Adenosine-5'-O-(3-thio)triphosphate (ATPgammaS), a nonhydrolyzable analog of the physiological P2Y(11) agonist ATP, and NF546 use a common binding site as suggested by molecular modeling studies and their competitive behavior toward the nanomolar potency antagonist NF340 [4,4'-(carbonylbis(imino-3,1-(4-methyl-phenylene)carbonylimino))bis(naphthalene-2,6-disulfonic acid) tetrasodium salt] in Schild analysis. The pA(2) of NF340 was 8.02 against ATPgammaS and 8.04 against NF546 (calcium assays). NF546 was further tested for P2Y(11)-mediated effects in monocyte-derived dendritic cells. Similarly to ATPgammaS, NF546 led to thrombospondin-1 secretion and inhibition of lipopolysaccharide-stimulated interleukin-12 release, whereas NF340 inhibited these effects. Further, for the first time, it was shown that ATPgammaS or NF546 stimulation promotes interleukin 8 (IL-8) release from dendritic cells, which could be inhibited by NF340. In conclusion, we have described the first selective, non-nucleotide agonist NF546 for P2Y(11) receptors in both recombinant and physiological expression systems and could show a P2Y(11)-stimulated IL-8 release, further supporting the immunomodulatory role of P2Y(11) receptors.

The metabolic fate of two new psychoactive substances - 2-aminoindane and N-methyl-2-aminoindane - studied in vitro and in vivo to support drug testing.

The aim of this study was to characterize the in vitro and in vivo metabolism of 2-aminoindane (2,3-dihydro-1H-inden-2-amine, 2-AI), and N-methyl-2-aminoindane (N-methyl-2,3-dihydro-1H-inden-2-amine, NM-2-AI) after incubations using pooled human liver microsomes (pHLMs), pooled human liver S9 fraction (pS9), and rat urine after oral administration. After analysis using liquid chromatography coupled to high-resolution mass spectrometry, pHLM incubations revealed that 2-AI was left unmetabolized, while NM-2-AI formed a hydroxylamine and diastereomers of a metabolite formed after hydroxylation in beta position. Incubations using pS9 led to the formation of an acetyl conjugation in the case of 2-AI and merely a hydroxylamine for NM-2-AI. Investigations on rat urine showed that 2-AI was hydroxylated also forming diasteromers as described for NM-2-AI or acetylated similar to incubations using pS9. All hydroxylated metabolites of NM-2-AI except the hydroxylamine were found in rat urine as additional sulfates. Assuming similar patterns in humans, urine screening procedures might be focused on the parent compounds but should also include their metabolites. An activity screening using human recombinant N-acetyl transferase (NAT) isoforms 1 and 2 revealed that 2-AI was acetylated exclusively by NAT2, which is polymorphically expressed.

Liquid Chromatography-High-Resolution Mass Spectrometry-Based In Vitro Toxicometabolomics of the Synthetic Cathinones 4-MPD and 4-MEAP in Pooled Human Liver Microsomes.

Synthetic cathinones belong to the most often seized new psychoactive substances on an international level. This study investigated the toxicometabolomics, particularly the in vitro metabolism of 2-(methylamino)-1-(4-methylphenyl)-1-pentanone (4-MPD) and 2-(ethylamino)-1-(4-methylphenyl)-1-pentanone (4-MEAP) in pooled human liver microsomes (pHLM) using untargeted metabolomics techniques. Incubations were performed with the substrates in concentrations ranging from 0, 12.5, and 25 µM. Analysis was done by means of high-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS/MS) in full scan only and the obtained data was evaluated using XCMS Online and MetaboAnalyst. Significant features were putatively identified using a separate parallel reaction monitoring method. Statistical analysis was performed using Kruskal-Wallis test for prefiltering significant features and subsequent hierarchical clustering, as well as principal component analysis (PCA). Hierarchical clustering or PCA showed a distinct clustering of all concentrations with most of the features z-scores rising with the concentration of the investigated substances. Identification of significant features left many of them unidentified but revealed metabolites of both 4-MPD and 4-MEAP. Both substances formed carboxylic acids, were hydroxylated at the alkyl chain, and formed metabolites after combined hydroxylation and reduction of the cathinone oxo group. 4-MPD additionally formed a dihydroxy metabolite and a hydroxylamine. 4-MEAP was additionally found reduced at the cathinone oxo group, N-dealkylated, and formed an oxo metabolite. These findings are the first to describe the metabolic pathways of 4-MPD and to extend our knowledge about the metabolism of 4-MEAP. Findings, particularly the MS data of the metabolites, are essential for setting up metabolite-based toxicological (urine) screening procedures.

Toxicokinetics and Analytical Toxicology of Flualprazolam: Metabolic Fate, Isozyme Mapping, Human Plasma Concentration and Main Urinary Excretion Products.

An increasing number of benzodiazepine-type compounds are appearing on the new psychoactive substances market. 8-Chloro-6-(2-fluorophenyl)-1-methyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine (well known as flualprazolam) represents a potent 'designer benzodiazepine' that has been associated with sedation, loss of consciousness, memory loss and disinhibition. The aims of the present study were to tentatively identify flualprazolam metabolites using in vitro incubations with pooled human liver S9 fraction or HepaRG cells by means of liquid-chromatography-high resolution tandem mass spectrometry. Isozymes involved in phase I and II biotransformation were identified in vitro. Results were then confirmed using human biosamples of an 18-year old male who was admitted to the emergency department after suspected flualprazolam ingestion. Furthermore, the plasma concentration was determined using the standard addition method. Seven flualprazolam metabolites were tentatively identified. Several cytochrome P450 and UDP-glucuronosyltransferase isozymes, amongst them CYP3A4 and UGT1A4, were shown to be involved in flualprazolam biotransformation reactions, and an influence of polymorphisms as well as drug-drug or drug-food interactions cannot be excluded. Alpha-hydroxy flualprazolam glucuronide, 4-hydroxy flualprazolam glucuronide and the parent glucuronide were identified as most abundant signals in urine, far more abundant than the parent compound flualprazolam. These metabolites are thus recommended as urine-screening targets. If conjugate cleavage was performed during sample preparation, the corresponding phase I metabolites should be added as targets. Both hydroxy metabolites can also be recommended for blood screening. The flualprazolam plasma concentration determined in the intoxication case was as low as 8 µg/L underlining the need of analytical methods with sufficient sensitivity for blood-screening purposes.

Acquired cisplatin resistance in the head-neck cancer cell line Cal27 is associated with decreased DKK1 expression and can partially be reversed by overexpression of DKK1.

Head and neck cancers are treated by a combination of surgery, radiotherapy and/or chemotherapy. The clinical success of cisplatin-based chemotherapy, mostly in combination with 5-FU or a taxane, is however limited by multifactorial intrinsic or acquired resistance. So far, known genes involved in cisplatin resistance do not sufficiently allow the prediction of cancer chemosensitivity. Thus, the purpose of this study was to search for further genes involved in cisplatin resistance by differential gene expression analysis of the parental tongue cancer cell line Cal27 and its 10-fold more resistant sub-cell line Cal27cis, which was obtained by treating Cal27 with increasing concentrations of cisplatin. As found by the suppression subtractive hybridization, expression of DKK1, an inhibitor of canonical WNT signaling, was decreased in Cal27cis. Microarray analysis, qPCR and ELISA confirmed the approximately 2-fold difference in expression. Cisplatin treatment and serum starvation increased by 2-fold the secretion of DKK1 in Cal27 and Cal27cis, thus rendering DKK1-levels significantly different in both cell lines under basal and stress conditions. Recombinant overexpression of DKK1 in Cal27 and Cal27cis resulted in clonal cell lines, which were both 2.2- to 3-fold more sensitive toward cisplatin in cell viability (MTT) and in proliferation (BrdU) assays. In conclusion, acquired (10-fold) resistance of Cal27 against cisplatin is associated with decreased DKK1 expression and could partially be reversed by DKK1 overexpression, thus suggesting DKK1 and the WNT signaling pathway as a marker and target for cisplatin chemosensitivity.

Functional screening of G protein-coupled receptors by measuring intracellular calcium with a fluorescence microplate reader.

Ligand binding studies reveal information about affinity to G protein-coupled receptors (GPCRs) rather than functional properties. Increase in intracellular Ca(2+) appears to represent a universal second messenger signal for a majority of recombinant GPCRs. Here, we exploit Ca(2+) signaling as a fast and sensitive functional screening method for a number of GPCRs coupled to different G proteins. Ca(2+) fluorescence measurements are performed using Oregon Green 488 BAPTA-1/AM and a microplate reader equipped with an injector. Buffer alone or test compounds dissolved in buffer are injected into a cell suspension, and fluorescence intensity is recorded for 30 s. Each of the GPCRs tested--G(q)-coupled P2Y(2), G(s)-coupled dopamine D1 and D5, G(i)-coupled dopamine D2L, and G(q/11)-coupled muscarinic acetylcholine M1--yielded a significant rise in intracellular free [Ca(2+)] on agonist stimulation. Agonist stimulation was dose dependent, as shown for ATP or UTP stimulation of P2Y(2) receptors (EC(50) = 1 microM), SKF38393 stimulation of hD1 and hD5 (EC(50) = 18.1 nM and 2.7 nM), and quinpirole at hD2L (EC(50) = 6.5 nM). SCH23390 (at hD1 and hD5) and spiperone, haloperidol, and clozapine (at hD2L) competitively antagonized the Ca(2+) response. Furthermore, the Ca(2+) assay served to screen suramin analogs for antagonistic activity at P2Y(2) receptors. Screening at dopamine receptors revealed LE300, a new lead for a dopamine receptor antagonist. Advantages of the assay include fast and simple 96- or 384-well plate format (high-throughput screening), use of a visible light-excitable fluorescent dye, applicability to a majority of GPCRs, and simultaneous analysis of distinct Ca(2+) fluxes.

Pharmacological characterization of the benz[d]indolo[2,3-g]azecine LE300, a novel type of a nanomolar dopamine receptor antagonist.

LE300 (7-methyl-6,7,8,9,14,15-hexahydro-5 H-benz[d]indolo[2,3-g]azecine), a previously reported subnanomolar antagonist at rat striatal dopamine D1 receptors, and three of its azecine-N-substituted congeners combining structural elements of serotonin and dopamine were comprehensively characterised (binding and function) at recombinant human dopamine receptors. Radioligand competition experiments at D1 and D2L receptors were performed by using [(3)H]SCH23390 and [(3)H]spiperone, respectively. Functional assays included measurements of cAMP, intracellular [Ca(2+)], and [(35)S]GTPgammaS-binding. LE300 was the most potent compound with a 10- to 20-fold selectivity for D1 over D2L receptors as measured in equilibrium binding experiments [competition radioligand binding: K(i)(D1)=1.9 nM, K(i)(D2L)=44.7 nM; [(35)S]GTPgammaS-binding: K(i)(D1)=1.8 nM, K(i)(D2L)=21.5 nM]. In functional (non-equilibrium) experiments, LE300 did not reveal a D1 over D2L selectivity but retained nanomolar K(i) values at human dopamine receptors (measurement of cAMP: K(i)(D1)=25.9 nM, K(i)(D2L)=5.2 nM; measurement of intracellular [Ca(2+)]: K(i)(D1)=60.4 nM, K(i)(D2L)=19.0 nM). LE300 is currently under investigation for usefulness as positrone emission tomography ligand. In conclusion, LE300 is a novel type of a nanomolar dopamine receptor antagonist combining structural core elements of dopamine and serotonin, and may become useful as positrone emission tomography ligand.

Systemic therapy of Cushing's syndrome.

Cushing's disease (CD) in a stricter sense derives from pathologic adrenocorticotropic hormone (ACTH) secretion usually triggered by micro- or macroadenoma of the pituitary gland. It is, thus, a form of secondary hypercortisolism. In contrast, Cushing's syndrome (CS) describes the complexity of clinical consequences triggered by excessive cortisol blood levels over extended periods of time irrespective of their origin. CS is a rare disease according to the European orphan regulation affecting not more than 5/10,000 persons in Europe. CD most commonly affects adults aged 20-50 years with a marked female preponderance (1:5 ratio of male vs. female). Patient presentation and clinical symptoms substantially vary depending on duration and plasma levels of cortisol. In 80% of cases CS is ACTH-dependent and in 20% of cases it is ACTH-independent, respectively. Endogenous CS usually is a result of a pituitary tumor. Clinical manifestation of CS, apart from corticotropin-releasing hormone (CRH-), ACTH-, and cortisol-producing (malign and benign) tumors may also be by exogenous glucocorticoid intake. Diagnosis of hypercortisolism (irrespective of its origin) comprises the following: Complete blood count including serum electrolytes, blood sugar etc., urinary free cortisol (UFC) from 24 h-urine sampling and circadian profile of plasma cortisol, plasma ACTH, dehydroepiandrosterone, testosterone itself, and urine steroid profile, Low-Dose-Dexamethasone-Test, High-Dose-Dexamethasone-Test, after endocrine diagnostic tests: magnetic resonance imaging (MRI), ultra-sound, computer tomography (CT) and other localization diagnostics. First-line therapy is trans-sphenoidal surgery (TSS) of the pituitary adenoma (in case of ACTH-producing tumors). In patients not amenable for surgery radiotherapy remains an option. Pharmacological therapy applies when these two options are not amenable or refused. In cases when pharmacological therapy becomes necessary, Pasireotide should be used in first-line in CD. CS patients are at an overall 4-fold higher mortality rate than age- and gender-matched subjects in the general population. The following article describes the most prominent substances used for clinical management of CS and gives a systematic overview of safety profiles, pharmacokinetic (PK)-parameters, and regulatory framework.

Clinical pharmacology of tyrosine kinase inhibitors becoming generic drugs: the regulatory perspective.

Over the last decades, billions have been spent and huge efforts have been taken in basic and clinical cancer research [CA Cancer J Clin63:11-30]. About a decade ago, the arms race between drugs and cancer cells reached a new level by introduction of tyrosine kinase inhibitors (TKI) into pharmacological anti-cancer therapy. According to their molecular mechanism of action, TKI in contrast to so-called "classic" or "conventional" cytostatics belong to the group of targeted cancer medicines, characterized by accurately fitting with biological structures (i.e. active centers of kinases). Numerous (partly orphan) indications are covered by this new class of substances. Approximately ten years after the first substances of this class of medicines were authorized, patent protection will end within the next years. The following article covers clinical meaning and regulatory status of anti-cancer TKI and gives an outlook to what is expected from the introduction of generic anti-cancer TKI.

Targeting adipose tissue.

Two different types of adipose tissues can be found in humans enabling them to respond to starvation and cold: white adipose tissue (WAT) is generally known and stores excess energy in the form of triacylglycerol (TG), insulates against cold, and serves as a mechanical cushion. Brown adipose tissue (BAT) helps newborns to cope with cold. BAT has the capacity to uncouple the mitochondrial respiratory chain, thereby generating heat rather than adenosine triphosphate (ATP). The previously widely held view was that BAT disappears rapidly after birth and is no longer present in adult humans. Using positron emission tomography (PET), however, it was recently shown that metabolically active BAT occurs in defined regions and scattered in WAT of the adult and possibly has an influence on whole-body energy homeostasis. In obese individuals adipose tissue is at the center of metabolic syndrome. Targeting of WAT by thiazolidinediones (TZDs), activators of peroxisome proliferator-activated receptor γ (PPARγ) a 'master' regulator of fat cell biology, is a current therapy for the treatment of type 2 diabetes. Since its unique capacity to increase energy consumption of the body and to dissipate surplus energy as heat, BAT offers new perspectives as a therapeutic target for the treatment of obesity and associated diseases such as type 2 diabetes and metabolic syndrome. Recent discoveries of new signaling pathways of BAT development give rise to new therapeutic possibilities in order to influence BAT content and activity.