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Green and sensitive spectrofluorometric methods have been developed and validated for the determination of timolol maleate (TML)/hydrochlorothiazide (HCT) and amiloride hydrochloride (AMH)/hydrochlorothiazide in tablets. The proposed spectrofluorometric procedures were found to be linear in the range of 4–12, 5–35 and 0.025–0.2 mg L−1 for HCT, TML and AMH, resp. The excitation and emission wavelengths for HCT, TML and AMH at room temperature were 270 and 375, 295 and 435, 330 and 415 nm, resp. The methods were validated with respect to ICH guidelines. The AMH showed higher sensitivity with lower values of LOD and LOQ values compared to HCT and TML. The proposed methods were applied to two pharmaceutical formulations; the method for HCT and AMH has proven as reliable assaying method, whereas the method for TML, when combined with HCT, is applicable to screening semi-quantitative analyses.

Chagas disease is a chronic infection caused by the parasite Trypanosoma cruzi (T. cruzi). The development of the parasite in the human heart muscle or digestive tract causes severe damage, resulting in organ failure. The current treatments, benznidazole and nifurtimox, have a limited efficacy, show significant side effects, and increasing resistance. Post-translational modifications (PTMs) play a crucial role in the life cycle of protozoan parasites. Bromodomains (BDs) are readers of lysine acetylation, one of the PTMs found on histone tails. The function of BDs remains uncharacterised in T. cruzi. The life cycle of the parasite is complex and is thought to rely heavily on epigenetic control and therefore our main interest is to validate the T. cruzi BDs as therapeutic targets. Optimisation of expression and purification protocols of the protein BD-containing factors (BDFs) in E. coli (Chapter 1) was followed by the development of a waterLOGSY- based assay to screen acetyl-lysine mimic containing compounds (Figure 0.1). The binding of the hits was further characterised by 1H NMR ligand-observed protein titration to obtain KD. Excitingly, the human BD and kinase ligand, BI-2536 (8), was shown to bind to TcBDF3 with a KD value between 25 and 55 μM. The binding was confirmed to be 14.6±3.0 μM (n=3) using ITC. BI-2536 (8) is to date the highest affinity ligand identified and was chosen as a lead structure for further development (Chapter 2). Based on this initial work, eight functional probes were designed and synthesised to enable the development of higher-throughput assays for T. cruzi BDs (Chapter 3). The 19F NMR reporter molecules, photo-activatable diazirines, and fluorescent probes were assessed against TcBDF3 to establish novel assays (Chapter 4). Firstly, the fluorinated probes were used as 19F NMR reporter molecules to observe their binding and displacement to and from TcBDF3. Secondly, a protein LCMS assay was established to characterise binding and subsequent cross-linking of the diazirine probes. Finally, fluorescent probes were validated by fluorescence polarisation. Satisfactorily, one probe binds to TcBDF3, with a KD value of 3 μM. Displacement of the fluorescent probe by the parent compound BI-2536 (8) results in a dose-dependent response, however, a full curve and an IC50 value could not be obtained due to assay interference at high ligand concentrations. Further validation is required, using higher affinity ligands to show that the fluorescence polarisation displacement can generate IC50 values. With GSK collaborators, 556 diazirines of the GSK PhaBits library were screened against TcBDF3 and 5-1, identifying one and six new binding fragments, respectively. Additionally, a DNA-encoded library of ca. 1.9 trillion encodings were screened against TcBDF2 and 5- 1, giving 82 and 134 hit clusters, respectively, to be investigated for binding. The low hit rates for both screening campaigns highlights that the T. cruzi BDs are challenging targets. In this work we report the first production of soluble T. cruzi BD proteins and their study using NMR assays such as waterLOGSY and NMR titration. Critically, this initial work led to the development of functional probes to provide a tool box to study TcBDF3 at higher throughput with diverse and orthogonal assays. The novel biophysical assays established here can be used to screen efficiently for binding compounds and opens up the possibility of rapid SAR. Identified ligands will enable validation of the bromodomains of T. cruzi as targets for Chagas disease for the first time. This will have a wide range of implications in the use of epigenetics targets for treatments of parasites more broadly.

Serotonin (5-HT) is a hormone and neurotransmitter that modulates neural activity as well as a wide range of other physiological processes including cardiovascular function, bowel motility, and platelet aggregation. 5-HT synthesis is catalyzed by tryptophan hydroxylase (TPH) which exists as two distinct isoforms; TPH1 and TPH2, which are responsible for peripheral and central 5-HT, respectively. Due to the implication of 5-HT in a number of pathologies, including depression, anxiety, autism, sexual dysfunction, irritable bowel syndrome, inflammatory bowel disease, and carcinoid syndrome, there has been a growing interest in finding modulators of these enzymes in recent years. We thus performed high-throughput screening (HTS) using a fluorescence-based thermal shift assay (DSF) to search the Prestwick Chemical Library containing 1,280 compounds, mostly FDA-approved drugs, for TPH1 binders. We here report the identification of omeprazole, a proton pump inhibitor, as an inhibitor of TPH1 and TPH2 with low micromolar potency and high selectivity over the other aromatic amino acid hydroxylases. The S-enantiomer of omeprazole, esomeprazole, has recently also been described as an inhibitor of monoamine oxidase-A (MAO-A), the main enzyme responsible for 5-HT degradation, albeit with lower potency compared to the effect on TPH1 and TPH2. In order to investigate the net effect of simultaneous inhibition of TPH and MAO-A in vivo, we administered high-dose (100 mg/kg) omeprazole to CD-1 mice for 4 days, after which the animals were subjected to the tail suspension test. Finally, central (whole brain) and peripheral (serum) 5-HT content was measured using liquid chromatography-mass spectrometry (LC-MS). Omeprazole treatment significantly increased 5-HT concentrations, both in brain and in serum, and reduced the time spent immobile in the tail suspension test relative to vehicle control. Thus, the MAO-A inhibition afforded by high-dose omeprazole appears to overcome the opposing effect on 5-HT produced by inhibition of TPH1 and TPH2. Further modification of proton pump inhibitor scaffolds may yield more selective modulators of 5-HT metabolism.

Annual Report for Utah Agricultural College.

Background: Linezolid is the first synthetic compound of a new group of antimicrobials, the oxazolidinones, which inhibit protein synthesis. It shows a broad spectrum of activity against Gram positive organisms. With respect to its pharmacokinetics, linezolid shows a relatively high volume of distribution and good penetration into inflammatory fluids, bone, fat and muscle. Methods: A reversed-phase isocratic high-performance liquid chromatographic method for linezolid analysis in piglet pulmonary tissue is described. Tissue samples and controls were prepared in 1×TBE (1 M Tris, 0.9 M boric acid, 0.01 M EDTA). The mobile phase consisted of 20% ultrafiltered water and 80% of (A) 15 mM potassium monohydrogen phosphate buffer (pH=5) with (B) acetonitrile (80%/20%; v/v). Samples were homogenized and precipitated with HClO4 3% (1/1, v/v). The injection volume was 100 μL. Ofloxacin was used as an internal standard. Results: The assay was linear over a linezolid concentration range: 1.6–100 μg/mL. The method provided good validation data (n=15): inaccuracy (3.6%), intra and inter-day variability (4.2% and 5.2%, respectively), recovery (91.8%), limit of detection (0.8 μg/mL) and quantitation (1.6 μg/mL) and acceptable stability within 24 h in the auto-sampler. Conclusions: The method offers a fast and simple approach to determine linezolid in pulmonary tissue which could be of use in pharmacokinetic studies. Clin Chem Lab Med 2010;48:391–8.