Binding of Metallocenes to Short Oligonucleotides

Cancer is one of the most severe and widespread diseases and an ideal treatment has not yet been found. In the last decades, cisplatinum was commonly applied in cancer therapy with very good results. However, serious side effects and resistant tumors necessitated the development of new antineoplastic agents, such as metallocenes dihalides. These are metal-based compounds exhibiting two cyclopentadienyl ligands and a cis-dihalide motif. They resemble the cis-chloro configuration of cisplatinum, which propounds a similar mode of action. Metallocenes comprising one of the transition metals titanium, molybdenum, vanadium, niobium, and zirconium as the metal center have been shown to be effective against several cancer cell lines. Evidence for the accumulation of metallocenes in the nucleus implied that DNA is one of the major targets. Although several studies reported adduct formation of metallocenes with nuclear DNA, as yet substantial information about the general binding pattern and the binding to higher-order structures is lacking. Mass spectrometry can fill this gap as it constitutes a powerful technique to investigate the formation of organometallic adducts. Presented data demonstrate that the two agents titanocene dichloride and molybdenocene dichloride bind to single-stranded DNA and RNA. Distinct fragment ions formed upon collision-induced dissociation help to unravel preferential binding sites within the oligonucleotides. Moreover, adducts with duplexes and quadruplexes shed light on the molecular mechanism of action.

Tandem Mass Spectrometric Analysis of Metallocene Adducts with Short Oligonucleotides

Metallocene dichlorides constitute a remarkable class of antineoplastic agents that are highly effective against several cancer cell lines. They were shown to accumulate in the DNA-rich region, which suggests DNA as the primary target. These compounds exhibit two cyclopentadienyl ligands and two labile halide ligands, resulting in a bent sandwich structure. The cis-dihalide motif is structurally related to the cis-chloro configuration of cisplatin and similar modes of action can thus be assumed. Cisplatin binds to two neighboring guanine nucleobases in DNA and consequently, distorts the double-helix, thereby inhibiting DNA replication and transcription. Platinum is classified as a soft Lewis acid and binds preferentially to the nitrogen atoms within the nucleobases. The metallocene dichlorides investigated in this study comprise the metal centers Ti, V, Nb, Mo, Hf, and W, which are classified as hard or intermediate Lewis acids, and thus, favor binding to the phosphate oxygen. Although several studies reported adduct formation of metallocene dichlorides with nucleic acids, substantial information about the adduct composition, the binding pattern, and the nucleobase selectivity has not been provided yet. ESI-MS analyses gave evidence for the formation of metallocene adducts (M = Ti, V, Mo, and W) with single-stranded DNA homologues at pH 7. No adducts were formed with Nb and Hf at neutral pH, albeit adducts with Nb were observed at a low pH. MS2 data revealed considerable differences of the adduct compositions. The product ion spectra of DNA adducts with hard Lewis acids (Ti, V) gave evidence for the loss of metallocene ligands and only moderate backbone fragmentation was observed. By contrast, adducts with intermediate Lewis acids (Mo, W) retained the hydroxy ligands. Preliminary results are in good agreement with the Pearson concept and DFT calculations. Since the metallodrugs were not lost upon CID, the nucleobase selectivity, stoichiometry, and binding patterns can be elucidated by means of tandem mass spectrometry.

Role of electrostatic interactions for ligand recognition and specificity of peptide transporters.

BACKGROUND Peptide transporters are membrane proteins that mediate the cellular uptake of di- and tripeptides, and of peptidomimetic drugs such as β-lactam antibiotics, antiviral drugs and antineoplastic agents. In spite of their high physiological and pharmaceutical importance, the molecular recognition by these transporters of the amino acid side chains of short peptides and thus the mechanisms for substrate binding and specificity are far from being understood. RESULTS The X-ray crystal structure of the peptide transporter YePEPT from the bacterium Yersinia enterocolitica together with functional studies have unveiled the molecular bases for recognition, binding and specificity of dipeptides with a charged amino acid residue at the N-terminal position. In wild-type YePEPT, the significant specificity for the dipeptides Asp-Ala and Glu-Ala is defined by electrostatic interaction between the in the structure identified positively charged Lys314 and the negatively charged amino acid side chain of these dipeptides. Mutagenesis of Lys314 into the negatively charged residue Glu allowed tuning of the substrate specificity of YePEPT for the positively charged dipeptide Lys-Ala. Importantly, molecular insights acquired from the prokaryotic peptide transporter YePEPT combined with mutagenesis and functional uptake studies with human PEPT1 expressed in Xenopus oocytes also allowed tuning of human PEPT1's substrate specificity, thus improving our understanding of substrate recognition and specificity of this physiologically and pharmaceutically important peptide transporter. CONCLUSION This study provides the molecular bases for recognition, binding and specificity of peptide transporters for dipeptides with a charged amino acid residue at the N-terminal position.

Regulation of human (adrenal) androgen biosynthesis-New insights from novel throughput technology studies

Androgens are precursors for sex steroids and are predominantly produced in the human gonads and the adrenal cortex. They are important for intrauterine and postnatal sexual development and human reproduction. Although human androgen biosynthesis has been extensively studied in the past, exact mechanisms underlying the regulation of androgen production in health and disease remain vague. Here, the knowledge on human androgen biosynthesis and regulation is reviewed with a special focus on human adrenal androgen production and the hyperandrogenic disorder of polycystic ovary syndrome (PCOS). Since human androgen regulation is highly specific without a good animal model, most studies are performed on patients harboring inborn errors of androgen biosynthesis, on human biomaterials and human (tumor) cell models. In the past, most studies used a candidate gene approach while newer studies use high throughput technologies to identify novel regulators of androgen biosynthesis. Using genome wide association studies on cohorts of patients, novel PCOS candidate genes have been recently described. Variant 2 of the DENND1A gene was found overexpressed in PCOS theca cells and confirmed to enhance androgen production. Transcriptome profiling of dissected adrenal zones established a role for BMP4 in androgen synthesis. Similarly, transcriptome analysis of human adrenal NCI-H295 cells identified novel regulators of androgen production. Kinase p38α (MAPK14) was found to phosphorylate CYP17 for enhanced 17,20 lyase activity and RARB and ANGPTL1 were detected in novel networks regulating androgens. The discovery of novel players for androgen biosynthesis is of clinical significance as it provides targets for diagnostic and therapeutic use.

Current state of evidence on 'off-label' therapeutic options for systemic lupus erythematosus, including biological immunosuppressive agents, in Germany, Austria and Switzerland--a consensus report

Systemic lupus erythematosus (SLE) can be a severe and potentially life-threatening disease that often represents a therapeutic challenge because of its heterogeneous organ manifestations. Only glucocorticoids, chloroquine and hydroxychloroquine, azathioprine, cyclophosphamide and very recently belimumab have been approved for SLE therapy in Germany, Austria and Switzerland. Dependence on glucocorticoids and resistance to the approved therapeutic agents, as well as substantial toxicity, are frequent. Therefore, treatment considerations will include 'off-label' use of medication approved for other indications. In this consensus approach, an effort has been undertaken to delineate the limits of the current evidence on therapeutic options for SLE organ disease, and to agree on common practice. This has been based on the best available evidence obtained by a rigorous literature review and the authors' own experience with available drugs derived under very similar health care conditions. Preparation of this consensus document included an initial meeting to agree upon the core agenda, a systematic literature review with subsequent formulation of a consensus and determination of the evidence level followed by collecting the level of agreement from the panel members. In addition to overarching principles, the panel have focused on the treatment of major SLE organ manifestations (lupus nephritis, arthritis, lung disease, neuropsychiatric and haematological manifestations, antiphospholipid syndrome and serositis). This consensus report is intended to support clinicians involved in the care of patients with difficult courses of SLE not responding to standard therapies by providing up-to-date information on the best available evidence.

Use of ampicillin-sulbactam for treatment of experimental meningitis caused by a beta-lactamase-producing strain of Escherichia coli K-1

We evaluated the pharmacokinetics and therapeutic efficacy of ampicillin combined with sulbactam in a rabbit model of meningitis due to a beta-lactamase-producing strain of Escherichia coli K-1. Ceftriaxone was used as a comparison drug. The MIC and MBC were 32 and greater than 64 micrograms/ml (ampicillin), greater than 256 and greater than 256 micrograms/ml (sulbactam), 2.0 and 4.0 micrograms/ml (ampicillin-sulbactam [2:1 ratio, ampicillin concentration]) and 0.125 and 0.25 micrograms/ml (ceftriaxone). All antibiotics were given by intravenous bolus injection in a number of dosing regimens. Ampicillin and sulbactam achieved high concentrations in cerebrospinal fluid (CSF) with higher dose regimens, but only moderate bactericidal activity compared with that of ceftriaxone was obtained. CSF bacterial titers were reduced by 0.6 +/- 0.3 log10 CFU/ml/h with the highest ampicillin-sulbactam dose used (500 and 500 mg/kg of body weight, two doses). This was similar to the bactericidal activity achieved by low-dose ceftriaxone (10 mg/kg), while a higher ceftriaxone dose (100 mg/kg) produced a significant increase in bactericidal activity (1.1 +/- 0.4 log10 CFU/ml/h). It appears that ampicillin-sulbactam, despite favorable CSF pharmacokinetics in animals with meningitis, may be of limited value in the treatment of difficult-to-treat beta-lactamase-producing bacteria, against which the combination shows only moderate in vitro activity.

Cladophialophora bantiana: a rare cause of fungal brain abscess. Clinical aspects and new therapeutic options

Black molds or dematiaceous fungi are rare etiologic agents of intracerebral abscesses and such infections carry a high mortality of up to 70% despite combined surgical and antifungal therapy. While the growing use of immunosuppressive therapies and organ transplantation have caused an increase in the incidence of rare fungal cerebral infections, occurrence in immunocompetent hosts is also possible. We describe a 60-year-old female patient with a cerebral abscess caused by Cladophialophora bantiana. The case illustrates the clinical and radiological similarities between glioblastomas and brain abscesses and emphasizes the need to perform histological and microbiological studies prior to the initiation of any form of therapy. Long-term survival from cerebral black mold abscesses has been reported only when complete surgical resection was possible. The recommended antifungal treatment involves the use of amphotericin B combined with a triazole and, if possible, flucytosine. Highly-active new generation triazole antifungal compounds (voriconazole or posaconazole) are likely to offer improved survival rates for patients with rare mold infections. In particular, posaconazole could be a new therapeutic option given its better tolerance, lower toxicity and fewer drug-drug interactions. We discuss clinical, microbiological and practical pharmacological aspects and review current and evolving treatment options.

Clinical usefulness of therapeutic concentration monitoring for imatinib dosage individualization: results from a randomized controlled trial.

PURPOSE This study assessed whether a cycle of "routine" therapeutic drug monitoring (TDM) for imatinib dosage individualization, targeting an imatinib trough plasma concentration (C min) of 1,000 ng/ml (tolerance: 750-1,500 ng/ml), could improve clinical outcomes in chronic myelogenous leukemia (CML) patients, compared with TDM use only in case of problems ("rescue" TDM). METHODS Imatinib concentration monitoring evaluation was a multicenter randomized controlled trial including adult patients in chronic or accelerated phase CML receiving imatinib since less than 5 years. Patients were allocated 1:1 to "routine TDM" or "rescue TDM." The primary endpoint was a combined outcome (failure- and toxicity-free survival with continuation on imatinib) over 1-year follow-up, analyzed in intention-to-treat (ISRCTN31181395). RESULTS Among 56 patients (55 evaluable), 14/27 (52 %) receiving "routine TDM" remained event-free versus 16/28 (57 %) "rescue TDM" controls (P = 0.69). In the "routine TDM" arm, dosage recommendations were correctly adopted in 14 patients (median C min: 895 ng/ml), who had fewer unfavorable events (28 %) than the 13 not receiving the advised dosage (77 %; P = 0.03; median C min: 648 ng/ml). CONCLUSIONS This first target concentration intervention trial could not formally demonstrate a benefit of "routine TDM" because of small patient number and surprisingly limited prescriber's adherence to dosage recommendations. Favorable outcomes were, however, found in patients actually elected for target dosing. This study thus shows first prospective indication for TDM being a useful tool to guide drug dosage and shift decisions. The study design and analysis provide an interesting paradigm for future randomized TDM trials on targeted anticancer agents.

Current developments in the use of stem cell for therapeutic neovascularisation: is the future therapy "cell-free"?

The plasticity and self-regenerative properties of stem cells have opened new avenues in regenerative medicine. Greater understanding of the biology of stem cells is followed by growing expectations of a rapid translation into alternative therapeutic options. Recent preclinical studies and clinical trials employing stem and progenitor cells from different sources have shown encouraging results. However, their underlying mechanisms are still poorly understood, the potential adverse effects and the discrepancy in efficacy remain to be further investigated. Their essential role in vessel regeneration has made endothelial progenitor cells (EPC) a suitable candidate for therapeutic applications aiming at tissue revascularisation. Recent evidence suggests that EPC contribute to neovascularisation not only by direct participation in tissue homeostasis but mainly via paracrine mechanisms. In future, novel therapeutic strategies could be based on EPC paracrine factors or synthetic factors, and replace cell transplantation.

EAACI IG Biologicals task force paper on the use of biologic agents in allergic disorders.

Biologic agents (also termed biologicals or biologics) are therapeutics that are synthesized by living organisms and directed against a specific determinant, for example, a cytokine or receptor. In inflammatory and autoimmune diseases, biologicals have revolutionized the treatment of several immune-mediated disorders. Biologicals have also been tested in allergic disorders. These include agents targeting IgE; T helper 2 (Th2)-type and Th2-promoting cytokines, including interleukin-4 (IL-4), IL-5, IL-9, IL-13, IL-31, and thymic stromal lymphopoietin (TSLP); pro-inflammatory cytokines, such as IL-1β, IL-12, IL-17A, IL-17F, IL-23, and tumor necrosis factor (TNF); chemokine receptor CCR4; and lymphocyte surface and adhesion molecules, including CD2, CD11a, CD20, CD25, CD52, and OX40 ligand. In this task force paper of the Interest Group on Biologicals of the European Academy of Allergy and Clinical Immunology, we review biologicals that are currently available or tested for the use in various allergic and urticarial pathologies, by providing an overview on their state of development, area of use, adverse events, and future research directions.

Directional DBS improves therapeutic window: from model prediction to human use

Deep brain stimulation of different targets has been shown to drastically improve symptoms of a variety of neurological conditions. However, the occurrence of disabling side effects may limit the ability to deliver adequate amounts of current necessary to reach the maximal benefit. Computed models have suggested that reduction in electrode size and the ability to provide directional stimulation could increase the efficacy of such therapies. This has never been demonstrated in humans. In the present study, we assess the effect of directional stimulation compared to omnidirectional stimulation.

Drug-induced torsades de pointes in an underserved urban population. Methadone: is there therapeutic equipoise?

BACKGROUND Although it has been well established that methadone use can result in prolonged QTc/torsades de pointes (TdP) and has been labeled as one of the main drugs that cause TdP, it is still prescribed indiscriminately, and several cases of methadone-associated TdP have been seen in our community. METHODS Our objective was to determine the associated factors for prolonged QTc and the development of torsades de pointes (TdP) in our underserved patient population. We found 12,550 ECGs with prolonged QTc between 2002 and 2013. Medical records were reviewed in order to identify precipitating factors for prolonged QTc and to detect incidence of TdP. RESULTS We identified 2735 patients with prolonged QTc who met the inclusion criteria. Of these, 89 (3%) experienced TdP. There was a greater prevalence of HIV infection in the TdP group (11.2 vs. 3.7%, p < 0.001). Furosemide, hydrochlorothiazide, selective serotonin reuptake inhibitors (SSRIs), amiodarone, ciprofloxacin, methadone, haloperidol, and azithromycin were the drugs most often associated with prolonged QTc (31, 8.2, 7.6, 7.1, 3.9, 3.4 and 3.3%, respectively). However, the agents most commonly associated with TdP were furosemide (39.3%), methadone (27%), SSRIs (19.1%), amiodarone (18%), and dofetilide (9%). The medications with statistical significance in the multivariate analysis for TdP development in descending order were as follows: ranolazine (odds ratios [OR] = 53.61, 95% confidence interval [CI] 5.4-524, p < 0.001), dofetilide (OR = 25, CI 6.47-103.16, p < 0.001), voriconazole (OR = 21.40, CI 3.24-124.25, p < 0.001), verapamil (OR = 10.98, CI 2.62-44.96, p < 0.001), sotalol (OR = 12.72, 1.95-82.81, p = 0.008), methadone (OR = 9.89, CI 4.05-24.15, p < 0.001), and SSRI (OR = 2.26, CI 1.10-5.96, p < 0.001). This multivariate analysis revealed that amiodarone and HIV infection were not implicated in TdP. CONCLUSION Methadone was by far the leading medication implicated in the development of TdP and an independent predictor in both univariate and multivariate analyses despite the fact that it was not the most common QT-prolonging medication in our population.