Methionine is a glutathionc precursor which is effective in preventing liver damage after paracetamol overdosage. Paracetamol and N-acetyl-D-L-methioninc have been linked to form the paracetamol N-acetyl-D-L-methionate ester in an attempt to make a safer form of paracetamol. The disposition of paracetamol N-acetyl-D-L-methionate was studied in healthy subjects and patients with chronic renal failure. Each study was conducted as a double blind crossover clinical trial where each volunteer received Ig of paracetamol as 1) paracetamol and 2) paracetamol N-acetyl-D-L-mcthionate (2.146g). Existing methods of measurement of paracetamol and its metabolites in plasma and urine were validated and used. A method using solid phase extraction and analysis by HPLC with U.V. detection was developed for measurement of paracetamol N-acetyl-D-L-methionate in plasma. Using these methods, paracetamol N-acctyl-D-L-methionate hydrolysis was shown to be accelerated as pH and temperature increased. The rate of hydrolysis in plasma and whole blood at 37°C was rapid. Following administration of paracetamol to healthy subjects paracetamol was rapidly absorbed, distributed and extensively metabolised. This group provided control data consistent with other published reports. Following administration of paracetamol N-acetyl-D-L-methionate to healthy subjects the parent compound was rapidly hydrolysed by ubiquitous estcrases and the peak plasma paracetamol concentration was reduced and delayed. There was a corresponding delay in the appearance of paracetamol metabolites. Sulphate conjugation was significantly increased, however the relative bioavailability of paracetamol, the amount recovered in urine as glutathione derived conjugates and the elimination of paracetamol and its metabolites remained unaltered. Paracetamol absorption was essentially complete. Following administration of paracetamol to 7 non-dialysis and 5 haemodialysis patients with chronic renal failure, absorption of paracetamol was normal, however there was gross cumulation of metabolites and the late elimination phase of the parent compound was also impaired, the latter possibly occurring due to limited enterohepatic circulation of paracetamol metabolites with subsequent reabsorption of parent compound. In patients with chronic renal failure paracetamol metabolism was normal with the capacity for active tubular transport reduced to a greater extent than passive reabsorption. In the non-dialysis patients, the total 24 h urinary recovery of paracetamol was significantly reduced. Following administration of paracetamol H-acetyl-D-L-methionate to patients with chronic renal failure the appearance of paracetamol was delayed and the peak plasma paracetamol concentration reduced. All other phar-macokinetic variables obtained for paracetamol N-acetyl-D-L-methionate were, however, not significantly different from those obtained after administration of paracetamol alone to the same patients.

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The rat embryonic cardiomyoblast-derived H9c2 cell line is increasingly used for studies into cardioprotection, as these cells display similar properties to primary cardiomyocytes. Adenosine receptors are well known mediators of cardioprotection and trigger effectors such as the mitochondrial KATP channel – however, the role of the mitochondrial BKCa channel in adenosine receptor-mediated cardioprotection has not been investigated. GPCR assays provided evidence for functional expression of Gi-coupled adenosine A1 and κ-opioid receptors, Gs-coupled β2 adrenergic receptors and Gq-coupled UTP-binding P2Y purinergic receptors on H9c2 cells. Activation of the adenosine A1 receptor with CPA (N(6)-cyclopentyladenosine) provided significant protection against hypoxia-induced cell death in these cells, as did opening of a BKCa channel with NS1619. The location of this BKCa channel was confirmed to be the mitochondria by the probing of subcellular fractions with BKCa-specific antibodies. Interestingly, CPA-induced protection against hypoxia was blocked by inhibition of the BKCa channel. In a model of hypoxia/reoxygenation in H9c2 cells both CPA and NS1619 significantly reduced cell death when used as postconditioning agents, and in both cases the protection was abolished by blockade of the BKCa channel. This data suggests for the first time that, in H9c2 cells, the BKCa channel is involved in A1 receptor-mediated cytoprotection. To confirm this finding in a more physiologically relevant model – and validate the use of H9c2 cells as a model for cardioprotection –hypoxia/reoxygenation in isolated rat ventricle strips was investigated. It was discovered that blockade of the BKCa channel significantly attenuated protection afforded by hypoxia preconditioning and preconditioning triggered by activation of the adenosine A1 and A2A receptors. For the first time, this report has shown an important role for the BKCa channel in adenosine receptor-mediated cytoprotection.

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Critical care patients commonly experience sleep fragmentation, in which sleep quality is poor and distributed throughout the 24 hour cycle. This irregular sleep wake pattern is a form of circadian rhythm sleep disorder. The causes of sleep disturbances are multifactorial and contribute to patient morbidity. Conventional hypnotic treatment is often ineffective and, indeed, may cause delirium and reduced sleep quality. Administration of exogenous melatonin has been shown to re-enforce circadian rhythm disorders and improve sleep in other patient groups. An open evaluation of 5 mg oral melatonin was undertaken in a group of 12 critical care patients exhibiting sleep disturbances resistant to conventional hypnotics. Melatonin significantly increased observed sleep quantity by night 3, compared to baseline. An oral solution of melatonin was formulated to allow administration by enteral feeding tubes. It was shown to have a 1 year shelf life when refrigerated and protected from light. A randomised controlled trial was undertaken in 24 critical care patients weaning from mechanical ventilation. Melatonin 10 mg orally increased nocturnal bispectral index sleep quantity over nights 3 and 4 compared to placebo. Agreement of the other sleep measurement techniques with the bispectral index was poor. Actigraphy was not a useful measure of sleep in critical care patients and nurse observation overestimated sleep quantity. The clearance of melatonin appeared to be decreased in critical care patients compared to that in healthy subjects. Doses of 1-2 mg should be used in future critical care studies. 11 Acute administration of melatonin did not have a significant effect over placebo on rest-activity rhythms, which remained delayed, fragmented and reduced. Similar disturbances were present in plasma melatonin and cortisol rhythms, which were no longer phase locked. Melatonin therapy may prove beneficial in the treatment of sleep and circadian rhythms in critical care patients, and further larger studies should be pursued.

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The application of potentiometric dyes to isolated ventricular myocytes may provide the opportunity to record changes in drug-induced action potential (AP) morphology without the use of more difficult electrophysiological techniques.;Conditions were optimised for recording cardiac APs from isolated guinea pig ventricular myocytes stimulated at 1Hz using the ratiometric fluorescence emission of the dyes, di-4-ANEPPS and di-8-ANEPPS. Using di-8-ANEPPS, APs of steady duration were recorded for up to 28 min, when exposed to excitation light for 30 s in every 3 min, and using di-4-ANEPPS up to 24 min when exposed for 5 s in every 4 min. Using voltage-clamp protocols simultaneously with fluorescent recordings demonstrated a linear relationship between membrane potential and the fluorescence emission of both di-4-ANEPPS and di-8-ANEPPS.;Changes in action potential duration in response to increasing concentrations of cisapride were measured using a patch electrode or the emission of di-8-ANEPPS. Values for IC50 apparent for action potential prolongation were similar between the two assays. However, cells loaded with dye had an increased basal APD90 and a decreased sensitivity compared to patch electrode recordings, suggesting additional actions of the dye.;Screening a number of structurally similar dyes (di-4-ANEPPS, di-8-ANEPPS, di-12-ANEPPS, di-3-ANEPPDHQ and di-4-ANEPPDHQ) or demonstrated a variety of different pharmacological effects.;A double-blinded validation using the fluorescence emission of di-4-ANEPPS (loaded in guinea pig myocytes) was compared to results from standard proarrhythmia screening techniques: sharp electrode recordings from canine Purkinje fibres and M cells. The data suggest that guinea pig myocytes respond to drug-induced changes in AP morphology in a more similar manner to canine M cells from Purkinje fibres and show that di-4-ANEPPS can be used to monitor changes in AP duration and triangulation in isolated ventricular cells.;This method provides a higher throughput method for safety-pharmacology screens than standard microelectrode techniques, whilst still providing an indication of the effects of test compounds in native tissue.

hERG encodes the a-subunit of the rapid delayed rectifier potassium current, a crucial current for normal repolarisation of the cardiac action potential. Pharmacological block of hERG is associated with arrhythmias and sudden death. Given its physiological importance, aspects of both the gating and pharmacology of this channel were investigated.;hERG has unusual gating properties characterised by slow activation and deactivation gating. The roles of conserved S6 glycines (Gly648 and Gly657) in hERG as hinges for activation gating were studied. Glycine residues impart flexibility that is thought to be conducive for channel opening. However, mutations at positions 648 and 657 altered gating in a manner consistent with a role in protein packing rather than flexibility. Deactivation gating in hERG is slow due to interactions between the amino-terminus, the voltage sensor, and the pore that stabilise the open state. The pore mutation V659A dramatically slowed channel deactivation and reduced drug block. Replacing Val659 with larger hydrophobic residues resulted in faster deactivation kinetics, but in contrast, V659G hERG was constitutively open. It was concluded that Val659 mutations influence deactivation through hydrophobic interactions with the S4-S5 linker that couples S6 to the voltage sensor. Effects on drug binding correlated with deactivation rates, indicating that Val659 mutations have allosteric rather than direct effects on drug binding.;Tyr652 is thought to be a critical residue for high affinity drug binding. However, this study showed that the contribution of Tyr652 to drug binding varied considerably among 24 compounds tested, with the majority of low affinity blockers being relatively insensitive to the Y652A mutation. Pharmacophore models generated from the results suggest that higher affinity compounds are longer than lower affinity compounds and simultaneously interact with multiple inner cavity residues. The compact structure of low affinity, Y652A-insensitive drugs permits multiple binding modes, making the compounds less reliant on interactions with Tyr652.

No description available.