Klaus Gebhardt

Endophenazines A-D, New Phenazine Antibiotics from the Arthropod Associated Endosymbiont Streptomyces anulatus

Four new members of the phenazine family, endophenazines A-D, and the already known phenazine-1-carboxylic acid (tubermycin B) were detected in the culture broth of various endosymbiotic Streptomyces anulatus strains by chemical screening in a combination of TLC-staining reagents and HPLC-diode array analysis. The endosymbiotic strains were isolated from four different arthropod hosts at various sites. The new phenazine compounds showed antimicrobial activities against Gram-positive bacteria and some filamentous fungi, and herbicidal activity against Lemna minor (duckweed).

In Vitro and In Vivo Properties of BAL30376, a β-Lactam and Dual β-Lactamase Inhibitor Combination with Enhanced Activity against Gram-Negative Bacilli That Express Multiple β-Lactamases

ABSTRACT BAL30376 is a triple combination comprising a siderophore monobactam, BAL19764; a novel bridged monobactam, BAL29880, which specifically inhibits class C β-lactamases; and clavulanic acid, which inhibits many class A and some class D β-lactamases. The MIC90 was ≤4 μg/ml (expressed as the concentration of BAL19764) for most species of the Enterobacteriaceae family, including strains that produced metallo-β-lactamases and were resistant to all of the other β-lactams tested. The MIC90 for Stenotrophomonas maltophilia was 2 μg/ml, for multidrug-resistant (MDR) Pseudomonas aeruginosa it was 8 μg/ml, and for MDR Acinetobacter and Burkholderia spp. it was 16 μg/ml. The presence of the class C β-lactamase inhibitor BAL29880 contributed significantly to the activity of BAL30376 against strains of Citrobacter freundii, Enterobacter species, Serratia marcescens, and P. aeruginosa. The presence of clavulanic acid contributed significantly to the activity against many strains of Escherichia coli and Klebsiella pneumoniae that produced class A extended-spectrum β-lactamases. The activity of BAL30376 against strains with metallo-β-lactamases was largely attributable to the intrinsic stability of the monobactam BAL19764 toward these enzymes. Considering its three components, BAL30376 was unexpectedly refractory toward the development of stable resistance.

Combined effects of the siderophore monosulfactam BAL30072 and carbapenems on multidrug-resistant Gram-negative bacilli

OBJECTIVES Carbapenem resistance in Gram-negative bacteria, mediated by restricted net influx and carbapenem-hydrolysing β-lactamases, is a growing problem. The monosulfactam antibiotic BAL30072 is stable to most carbapenemases, suggesting that it could be complementary to carbapenems. We have investigated the antimicrobial activity of BAL30072 combined with imipenem, meropenem and doripenem. METHODS The in vitro activities of the combinations were evaluated using broth microdilution susceptibility and agar disc diffusion tests, broth dilution chequerboard titration and time-kill studies, using strains of Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter with carbapenem MICs ≥ 2 mg/L. RESULTS The combinations were effective against 70%-80% of the isolates tested in the presence of 1 mg/L of each antibiotic, whereas the carbapenems were ineffective and BAL30072 alone was effective against 20%-40% of the strains. Synergistic effects were observed with many Enterobacteriaceae and P. aeruginosa, but were less common among the Acinetobacter, although additive effects, where the activity of one partner compensated for lack of activity of the other, were common. None of the combinations exhibited an antagonistic effect in all tests, in contrast to other β-lactams where negative interactions were frequently observed. Animal models of septicaemia demonstrated that the synergy observed in vitro with BAL30072 and meropenem can translate into greater in vivo efficacy. CONCLUSIONS BAL30072/carbapenem combinations were effective against a broader range of multidrug-resistant Gram-negative bacteria than either of the single agents. Additive and synergistic effects were observed in Enterobacteriaceae and P. aeruginosa, and this enhanced activity was frequently associated with suppression of resistance development. The in vitro activity translated into improved in vivo efficacy.

Biosynthetic Capacities of Actinomysetes. No.27. Endophenazines A-D, New Phenazine Antibiotics from the Athropod Associated Endosymbiont Streptomyces anulatus. II. Structure Elucidation.

A detailed screening of the secondary metabolite pattern produced by different athropod associated strains of the species Streptomyces anulatus resulted in the isolation and structure elucidation of the endophenazines A-D (2, 4-6). The structures were assigned by spectroscopic methods and chemical transformations. 4 represents a chromophoric system based on a phenazin-7-one, 5 and 6 are new 5, 10-dihydrophenazine derivatives.

Phenalinolactones A–D, terpenoglycoside antibiotics from Streptomyces sp. Tü 6071

Four new terpenoglycoside antibiotics, phenalinolactones A–D were isolated from Streptomyces sp. Tü 6071. The structures were elucidated on the basis of detailed NMR and MS analyses. Phenalinolactones combine a diterpenoid tricycle, a 2,3,6-trideoxysugar, a pyrrole-carboxylic acid and an uncommonly oxidized unsaturated γ-lactone in a unique manner. Phenalinolactones show an inhibitory activity against Gram-positive bacteria.

Investigation of the species-dependent in vitro metabolism of BAL30630 by stable isotope labeling and isotope exchange experiments analyzed by capillary liquid chromatography coupled to mass spectrometry.

The in vitro metabolic profile of BAL30630, an antifungal piperazine propanol derivative, which inhibits the 1,3-beta-D-glucansynthase, was investigated by incubation with microsomes of several species and with rat hepatocytes. For the spotting of the metabolites, mixtures of BAL30630 with a stable isotope (deuterium) labeled analogue were incubated. The metabolic pattern comprises several oxidized metabolites. Based on isotope exchange experiments, their structures could be assigned to epoxide- and hydroxylated metabolites. In hepatocyte incubations, several glucuronides formed from these oxidized metabolites could be observed. From the analysis of the metabolic pattern in microsomes, products of carbamate hydrolysis were characterized. This hydrolysis was highly species dependent. In activated incubations and in rat hepatocytes, those metabolites were further oxidized. In incubations without NADPH activation, the resulting hydrolytic metabolites could be enriched without the subsequent oxidation. Final structural elucidation of the metabolites was performed using accurate mass determination and isotope exchange experiments, in which incubations were analyzed by deuterium exchange and capillary HPLC-QTof-MS and MS/MS. The use of non-radioactive, stabile isotope labeled drug analogues in combination with isotope exchange studies was essential in particular for a defined assignment of the functional groups in the structures of the investigated metabolites.

Experiments for a systematic comparison between stable-isotope-(deuterium) labeling and radio-(14C) labeling for the elucidation of the in vitro metabolic pattern of pharmaceutical drugs

A systematic comparison between two labeling approaches for the investigation of the in vitro metabolic pattern of pharmaceutical drugs was performed by examining the use of (i) radiolabeled drugs analyzed with LC-MS-offline radiodetection and (ii) stable-isotope labeled drugs, used in a defined mixture with the unlabeled drug and analyzed by LC-MS with recognition of the specific isotopic pattern. (14)C was used for the radioisotope-approach and deuterium for the stable-isotope approach. Olanzapine, diclofenac and ketoconazole were chosen as model drugs, as they are commercially available in their non-, radio- and stable-isotope labeled forms. For all three model drugs, liver microsome- and hepatocyte-incubations (both from rat) were performed with various concentrations and incubation times for both, the radio- and the stable-isotope approaches. The metabolic pattern, including structure elucidation of all detected metabolites, was performed independently for all individual compounds and incubations. Subsequently, the metabolic patterns of the radio-, and the stable-isotope approaches were compared. In conclusion, all metabolites found with the radioisotope approach could also be found with the stable-isotope approach. Although the stable-isotope approach does not provide a quantitative result, it can be considered to be a highly suited analytical alternative for early in vitro metabolism investigations, especially when radiolabeled drug analogues are not yet available and quantitative results are not yet necessary.

Biaryl-Bridged Lipopeptides from Streptomyces sp. TÜ 6075

HPLC-DAD screening for new metabolites [1] from Streptomyces sp. TU 6075 revealed two series of compounds composed of five colorless and seven yellow fractions. For structure elucidation, ES-FTICR-MS, chiral GC-MS, Edman sequencing, and NMR spectroscopy were used. The isolated compounds, termed arylomycins, represent the first example of lipopeptides containing biaryl-bridged amino acids. The majority of the arylomycins exhibit antimicrobial activity against Gram-positive bacteria.

Abstract 1347: BAL101553: An optimized prodrug of the microtubule destabilizer BAL27862 with superior antitumor activity

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: [BAL27862][1] is a novel small molecule, inducing apoptosis in cancer cells through microtubule destabilization. A series of amino acid-derived [BAL27862][1] prodrugs was evaluated for solubility and in vivo conversion into drug. The Lys-prodrug BAL101553 was further compared with [BAL27862][1] in animal models of human cancer. Methods: Kinetic solubility of compounds was determined by diluting DMSO stock solutions with aqueous buffer. Pharmacokinetics and prodrug conversion were evaluated in mice. In vivo efficacy was analyzed in colon carcinoma SW480 and patient-derived mammary MaCa4151 xenografts. MTD dosing was used in all cases, adapted for each mouse strain. Results: All amino acid-derived prodrugs showed significantly increased aqueous solubility compared to [BAL27862][1], most pronounced at pH 3. At pH 5 and 6.5, the dibasic Lys-prodrug BAL101553 proved to be the most soluble compound (>200 μM). In vivo conversion rates differed significantly between the prodrugs. Highest exposure to the parent drug was obtained with the Lys, Ala and Gly derivatives, whereas the exposure achieved with other amino acid prodrugs (e.g. Phe, Asn, Ser, Trp) was more than twofold lower. Combining high solubility, good conversion and oral bioavailability, BAL101553 was selected for further evaluation. In tumor models, [BAL27862][1] was rapidly distributed into SW480 tumors after i.v. administration of drug or BAL101553. Strikingly, [BAL27862][1] was retained in tumor ∼1.5 times longer after BAL101553 (T1/2: 8.3h) vs. [BAL27862][1] (T1/2: 5.4h) administration. In all models, a higher MTD was reached with the prodrug, related to ∼60% prodrug conversion in vivo. In MaCa4151 xenografts, twice-weekly i.v. administration of BAL101553 (17 mg/kg [BAL27862][1] equivalents [BE]) elicited superior antitumor effects (final T/C=30%; p<0.05 vs. controls) as compared to [BAL27862][1] (10 mg/kg; T/C=66%). Once-weekly dosing of BAL101553 (14 mg/kg BE) and [BAL27862][1] (8 mg/kg) in the SW480 model resulted in final T/Cs of 34% and 49% (both p<0.001), resp. Strikingly, fractionation of the same total weekly dose did not significantly affect outcome for either BAL101553 (3x per day once-weekly: T/C=40%; 3x per week: T/C=26%; both p<0.001) or [BAL27862][1] (3x per day once-weekly: T/C=54%; 3x per week: T/C=54%; p=0.001 and p=0.002, resp.), suggesting antitumor response is related to AUC. Again, indications of superior antitumor responses were observed with all prodrug schedules in this model. Conclusions: BAL101553 has been identified as a highly soluble prodrug of [BAL27862][1], which can be administered p.o. or i.v. in the absence of solubilizing excipients known to be associated with adverse side-effects. Its administration facilitates higher tumor exposure to the active agent, with more profound responses in some tumor models. These data, together with a flexible dosing potential, support profiling of BAL101553 in cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1347. doi:10.1158/1538-7445.AM2011-1347 [1]: /lookup/external-ref?link_type=GENPEPT&access_num=BAL27862&atom=%2Fcanres%2F71%2F8_Supplement%2F1347.atom

Abstract C233: BAL27862: A unique microtubule‐targeted agent with a potential for the treatment of human brain tumors

Background: BAL27862 is a synthetic small molecule, potently inducing apoptosis in cancer cells through tubulin depolymerization characterized by a unique microtubule phenotype. BAL27862 has demonstrated a broad in vitro anti‐proliferative activity against a range of human tumor histotypes (low nM IC50s). Significant antitumor responses occur in animal models of human cancer after oral or intravenous (i.v.) administration, including tumors refractory to conventional treatments. The purpose of this study was to investigate the tissue distribution of BAL27862 in tumor‐bearing mice. Tumor and brain penetration, together with anti‐proliferative activity in glioblastoma (GBM) cell lines, is described. Materials and Methods: Anti‐proliferative activity and induction of tumor cell death in vitro were analyzed using the YO‐PRO® assay (48h incubation). CD1 nu/nu female mice were implanted with human colon carcinoma SW480 cells. For pharmacokinetic studies, tumor‐bearing (approx. 150 mm 3 ) mice were administered i.v. with either 2 mg/kg 14 C‐BAL27862 (n=6, single dose) or 8 mg/kg cold BAL27862 (n=33, once‐weekly during 4 weeks). Animals were culled at serial time points. Radioactivity was measured in slices, with Bio‐Imaging Analyzer read‐outs of the exposed phosphor imaging plates quantified with Aida software. Cold BAL27862 was determined in plasma, brain and tumors using a specific LC‐MS/MS method after sample homogenization and protein precipitation. Results: BAL27862 elicited a potent anti‐proliferative activity in 6 GBM cell lines (IC50 range: 10–20nM), which was independent of PTEN status. Strikingly, at optimal concentrations, a dramatic loss of cell viability was observed (% cell death at 50nM BAL27862: >15% in 5 lines, >30% in 2 lines), demonstrating that BAL27862 potently drives GBM cells into a cell death program..In mice, after i.v. administration of 14 C‐BAL27862, radioactivity was distributed to all organs: notably brain and tumor. A delayed peak was observed in slowly perfused organs such as skin, tumor and fat‐tissue. There was no tissue‐specific retention of radioactivity, as halflives were comparable between tissues and blood. 48h after administration, radioactivity was almost undetectable in most tissues. Following administration of 8 mg/kg i.v. cold BAL27862 to mice, the excellent brain and tumor penetration was confirmed. Specifically, similar levels of BAL27862 were found in brain and plasma (Cmax of 6.86 µg/g and 7.34 µg/mL, respectively). The ratio tumor/plasma was also around 1. There was no brain or tumor accumulation over time, as concentrations paralleled those in plasma. Conclusions: BAL27862 is efficiently distributed to tissues and tumor in a mouse model of human cancer. Significant brain penetration, coupled with cytotoxic activity in GBM tumor cell lines, would support further evaluation of BAL27862 for the treatment of human brain cancers. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C233.