Elimination of the antimicrobial action of the organoarsenical cancer therapeutic, 4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid, before finished product sterility testing.

OBJECTIVES: Arsenical compounds have been used therapeutically for over 2000 years finding particular relevance as antimicrobials. After being replaced by more selective and consequently less toxic antibiotics in the last century, arsenicals have recently made a resurgence as anticancer drugs (specifically arsenic trioxide and its derivatives). Arsenical parenteral formulations require post-manufacture sterility testing; however, their intrinsic antimicrobial activity must be neutralised before testing to eliminate the possibility of false (no-growth) test results. METHODS: A range of thiol-containing compounds was screened to establish a suitable deactivation agent for the novel organoarsenical compound, 4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid (GSAO). Dimercatopropanol (DMP) was found to successful deactivate GSAO and was validated according to pharmacopoeial sterility test guidelines (specifically the method suitability test/sterility validation test). KEY FINDINGS: DMP is an effective way of deactivating GSAO before sterility testing and can be used for pharmacopoeial sterility tests. Our results affirm previous research highlighting the sensitivity of Staphylococcus aureus to arsenical compounds CONCLUSIONS: A method of deactivating the arsenical drug GSAO before the post-manufacture sterility test was established and validated. DMP is a commonly used chelator/deactivation agent so this work may have implications for other inorganic therapeutic agents.

Improved pharmaceutical stability of a boronphenylalanine mannitol formulation for boron neutron capture therapy.

Boron neutron capture therapy (BNCT) is a radiotherapy based cancer treatment requiring the availability of a low energy thermal neutron beam and a boron containing drug. These requirements limit BNCT availability with the latter pharmaceutical issue related to the extremely short shelf-life and clinical acceptability of the current fructose based L-boronphenylalanine (BPA) formulation. Resolution of the formulation issues would remove this factor and therefore the stability of an alternative mannitol BPA formulation has been investigated. A mannitol BPA solution formulation was prepared and either lyophilised or stored as a solution at varying temperatures. After suitable periods the formulation was analysed by HPLC for BPA and degradation products. Lyophilised and solution mannitol BPA formulations exhibited a temperature and time dependent loss of BPA with concomitant increases in degradation products. Autoclaving the solution induced and accelerated degradation. A solution or lyophilised mannitol BPA formulation has a shelf-life of between 1 and 4 years respectively, a marked improvement over the current fructose formulation. Due to temperature dependent degradation the formulation cannot be terminally sterilised by autoclaving. The enhanced stability of the mannitol formulation removes the requirement for extemporaneous aseptic preparation of BPA just prior to treatment and eliminates one of the issues complicating the delivery of BNCT.

Boron phenylalanine and related impurities: HPLC analysis, stability profile and degradation pathways.

Boron phenylalanine is one of the lead drug candidates in the field of Boron Neutron Capture Therapy. Its inherent low toxicity allows large doses to be administered, but this makes it important to identify, rationalise and quantify impurities. Here we report a chromatographic assay method, the conditions under which the parent compound is unstable, and the suggested degradation mechanisms.

Integrative analysis of the Caenorhabditis elegans genome by the modENCODE project.

We systematically generated large-scale data sets to improve genome annotation for the nematode Caenorhabditis elegans, a key model organism. These data sets include transcriptome profiling across a developmental time course, genome-wide identification of transcription factor-binding sites, and maps of chromatin organization. From this, we created more complete and accurate gene models, including alternative splice forms and candidate noncoding RNAs. We constructed hierarchical networks of transcription factor-binding and microRNA interactions and discovered chromosomal locations bound by an unusually large number of transcription factors. Different patterns of chromatin composition and histone modification were revealed between chromosome arms and centers, with similarly prominent differences between autosomes and the X chromosome. Integrating data types, we built statistical models relating chromatin, transcription factor binding, and gene expression. Overall, our analyses ascribed putative functions to most of the conserved genome.

The C. elegans dosage compensation complex propagates dynamically and independently of X chromosome sequence.

BACKGROUND: The C. elegans dosage compensation complex (DCC) associates with both X chromosomes of XX animals to reduce X-linked transcript levels. Five DCC members are homologous to subunits of the evolutionarily conserved condensin complex, and two noncondensin subunits are required for DCC recruitment to X. RESULTS: We investigated the molecular mechanism of DCC recruitment and spreading along X by examining gene expression and the binding patterns of DCC subunits in different stages of development, and in strains harboring X;autosome (X;A) fusions. We show that DCC binding is dynamically specified according to gene activity during development and that the mechanism of DCC spreading is independent of X chromosome DNA sequence. Accordingly, in X;A fusion strains, DCC binding propagates from X-linked recruitment sites onto autosomal promoters as a function of distance. Quantitative analysis of spreading suggests that the condensin-like subunits spread from recruitment sites to promoters more readily than subunits involved in initial X targeting. CONCLUSIONS: A highly conserved chromatin complex is appropriated to accomplish domain-scale transcriptional regulation during C. elegans development. Unlike X recognition, which is specified partly by DNA sequence, spreading is sequence independent and coupled to transcriptional activity. Similarities to the X recognition and spreading strategies used by the Drosophila DCC suggest mechanisms fundamental to chromosome-scale gene regulation.