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M K Bayliss

Publications and source records attributed to M K Bayliss.

14 recordsLinked to original sources
PubMed2000

Parallel ultra-high flow rate liquid chromatography with mass spectrometric detection using a multiplex electrospray source for direct, sensitive determination of pharmaceuticals in plasma at extremely high throughput.

Recent years have seen increasing usage of large particle size stationary phases and ultra-high flow rate liquid chromatography/mass spectrometry (LC/MS) for rapid determination of pharmaceuticals in plasma without prior sample preparation. This lack of sample preparation prior to analysis, together with the extremely high throughput of the chromatography, makes the technique extremely attractive to the bioanalyst. Further, the introduction of multiple sprayer interfaces to mass spectrometers provides the potential for even higher throughput. In this paper, we present parallel ultra-high flow rate liquid chromatography using four columns in parallel and a four-way multiple sprayer interface to the mass spectrometer. We have applied this on both the narrow-bore and capillary scale. This technique enables the quantification of drugs from four plasma samples simultaneously, at nanogram per millilitre concentrations, from small aliquots of plasma without sample preparation and with throughputs of up to 120 samples per hour.

Calibration↗
PubMed2000

A commentary on the use of hepatocytes in drug metabolism studies during drug discovery and development.

Isolated hepatocytes and liver slices, in short-term suspension or longer-term culture, offer the prospect of providing qualitative metabolic information and quantitative pharmacokinetic parameters from key animal species and man at early stages of the drug discovery-development continuum. The propensity for changes in the fidelity of drug metabolism after removal of hepatocytes from the organ has long been recognized. The many and varied approaches which have been undertaken in an attempt to compensate for physiological shortcomings of in vitro hepatocyte systems are reviewed. In this respect, short-term suspension culture may provide a baseline against which to measure the success of extended culture methods, but it should be remembered that even freshly isolated hepatocyte preparations have deficiencies and liabilities that may affect the nature of information gathered. This article discusses the current advances and shortcomings of hepatocyte suspensions and cultures, along with liver slice technology, at both quantitative and qualitative levels.

Animals↗
PubMed1999

Microsomal prediction of in vivo clearance of CYP2C9 substrates in humans.

AIMS: To assess the utility of human hepatic microsomes for predicting in vivo intrinsic clearance (CLint ) via the use of four cytochrome P450 2C9 substrates: phenytoin, tolbutamide (S)-ibuprofen (two pathways) and diclofenac, and to examine the role of exogenous albumin within the microsomal incubation. METHODS: V max, Km and CLint (defined as V max/Km ratio) were estimated under initial rate conditions for five pathways of metabolism in a bank of 15 human hepatic microsomal samples and were scaled to in vivo units using the microsomal protein index. Non-metabolic related binding in microsomes was measured for phenytoin and tolbutamide in the presence and absence of albumin. RESULTS: Microsomal CLint values differed by over two orders of magnitude, with the means ranging from 0.18 (phenytoin) to 40.70 (diclofenac) microl min-1 mg-1 microsomal protein. When these data were scaled and compared with published in vivo studies a similar rank order was obtained, however, the actual CLint tended to be underpredicted. While the in vivo unbound Km for phenytoin, 1-5 micron is substantially lower than the value determined in microsomes based on total concentrations (56 micron), correction for the in vitro binding reduces this value to 20 micron and 6 micron in the absence and presence of albumin, respectively. Similar trends were seen with tolbutamide Km. CONCLUSIONS: An appreciation of the utility of in vitro prediction can be best achieved when the range of CLint values predicted from the individual hepatic microsomal samples are compared with the range of individual in vivo CLint values reported in the literature. The degree of underprediction is less evident using the range than the mean data and no consistent advantage in adding albumin to the incubation media is apparent.

Aryl Hydrocarbon Hydroxylases↗
PubMed1999

Utility of hepatocytes to model species differences in the metabolism of loxtidine and to predict pharmacokinetic parameters in rat, dog and man.

1. The metabolism of loxtidine (1-methyl-5-[3-[3-[(1-piperidinyl) methyl] phenoxy] propyl] amino-1H-1,2,4-triazole-3-methanol) was studied in freshly isolated rat, dog and human hepatocytes. Metabolism in vitro was comparable with previously available in vivo data in all three species with the marked species differences observed in vivo being reproduced in the hepatocyte model. 2. The major route for the metabolism of loxtidine by rat hepatocytes was N-dealkylation to form the propionic acid and hydroxymethyl triazole metabolites. A minor metabolic route was the oxidation of loxtidine to a carboxylic acid metabolite. The major route of metabolism for loxtidine in dog hepatocytes was glucuronidation with oxidation to the carboxylic acid metabolite being of minor importance. Incubation of loxtidine with human hepatocytes resulted in the drug remaining largely unchanged but with the carboxylic acid metabolite being produced in minor amounts. 3. In vitro studies were undertaken with rat, dog and human hepatocytes to determine the Michaelis-Menten parameters Vmax and Km for the sum of all the metabolic pathways. These kinetic parameters were used to calculate the intrinsic clearance of loxtidine. Using appropriate scaling factors, the predicted in vivo hepatic clearance was then calculated. The predicted intrinsic clearances were 51.4 +/- 12.4, 8.0 +/- 0.8 and 1.0 +/- 0.6 ml/min/kg for rat, dog and human hepatocytes respectively. These data were then used to calculate hepatic clearances of 24.5, 3.1 and 0.2 ml/min/kg for rat, dog and man respectively. 4. In vivo hepatic and intrinsic clearances for loxtidine were determined in rat, dog and human volunteers. The hepatic clearances of loxtidine were 26.6, 6.6 and 0.4 ml/min/kg in rat, dog and man respectively and intrinsic clearances were 58.5, 18.6 and 2.0 ml/min/kg in rat, dog and man respectively. 5. The present studies demonstrate that the hepatocyte model may be a valuable in vitro tool for predicting both qualitative and quantitative aspects of the metabolism of a drug in animals and man at an early stage of the drug development process.

Adult↗
PubMed1998

The characterisation of the major metabolite of salmeterol in the dog.

Salmeterol xinafoate is the first of a new class of long acting, selective beta2-adrenoceptor agonists introduced for the treatment of asthma. The major metabolite of salmeterol in the dog has been identified as the 3-catechol sulphate of the benzoic acid derivative. This metabolite was isolated from dog bile and was shown to have very similar physiochemical properties to a major endogenous component of bile, the bile acids, creating a complex analytical challenge. Initial experiments, involving hydrolysis with the enzyme sulphatase, suggested that the metabolite was a sulphate conjugate. However, complete identification of the metabolite was complicated in part due to the loss, by metabolism, of deuterium atoms added to the compound, specifically as a marker for mass spectrometry. Subsequently, a synthesis of salmeterol was completed with deuterium labels in different positions. This material was used as a substrate for dog liver slices, a simpler matrix than dog bile, which provided the basis for the metabolite's identification. The metabolite was characterised by the use of spectroscopic techniques, in particular LC/MS, LC/MS/MS and NMR.

Albuterol↗
PubMed1996

The aliphatic oxidation of salmeterol to alpha-hydroxysalmeterol in human liver microsomes is catalyzed by CYP3A.

Salmeterol xinafoate (Serevent) is a long-acting beta2-adrenoceptor agonist, used in the treatment of asthma, that has bronchodilator and anti-inflammatory action. Salmeterol is extensively metabolized by aliphatic oxidation in humans, with the major metabolite being alpha-hydroxysalmeterol. The aim of this investigation was to identify the specific cytochrome P450 (P450) isoform or isoforms involved in the formation of alpha-hydroxysalmeterol in human liver microsomes. [14C]Salmeterol was incubated with a pooled sample (N = 19) of human liver microsomes in the absence or presence of selective chemical inhibitors of the major human P450 isoforms. One microM ketoconazole, a selective inhibitor of CYP3A, substantially inhibited the metabolism of salmeterol to alpha-hydroxysalmeterol. Disulfiram caused a small but consistent decrease in the amount of alpha-hydroxysalmeterol formed, possibly reflecting less than total selectivity for CYP2E1 under the conditions used. Other selective inhibitors had no significant effect on the metabolism of salmeterol. The rates of formation of alpha-hydroxysalmeterol in 10 individual liver microsomal samples showed an approximately 10-fold variation and were found to be highly correlated (r2 = 0.94; p < 0.001) with rates of metabolism of midazolam to 1'-hydroxymidazolam, a marker of CYP3A activity, in the same microsomal samples. No significant correlation was evident for the metabolism of salmeterol with levels of total P450 or other markers of human P450 activities in the same microsomal samples, thus indicating that the formation of alpha-hydroxysalmeterol is catalyzed predominantly by CYP3A. Insect cell microsomes that coexpressed human CYP3A and NADPH-P450 reductase were able to metabolize [14C]salmeterol to alpha-hydroxysalmeterol, thus confirming the role of CYP3A in catalyzing this reaction. The therapeutic dose of salmeterol is very low, so it is unlikely that any clinically relevant interactions will be observed as a consequence of the coadministration of salmeterol and other pharmaceutical agents that are metabolized by CYP3A.

Adrenergic beta-Agonists↗
PubMed1994

Evaluation of pyrazole and ethanol induced S9 fraction in bacterial mutagenicity testing.

A major constitutive enzyme in the liver of the uninduced rat is cytochrome P450-2E1. This isozyme has been shown to metabolize a number of carcinogens, including low molecular weight nitrosamines and a number of compounds normally regarded as non-mutagenic in the Ames test, e.g. aniline, urethane and benzene. Using the standard induction procedures [Aroclor 1254 or a combination of phenobarbitone (PB) and beta-naphthoflavone (beta-NF)] the level of CYP2E1 in rat liver is actually suppressed and it has been suggested that this may account for the negative findings with these compounds in the Ames test. S9 fractions were prepared from rats pre-treated with pyrazole or ethanol (inducers of CYP2E1) and then used in the Ames test (or pre-incubation modification) with urethane, acetaminophen, aniline, benzene, procarbazine and N-nitrosopyrrolidine. Both pyrazole and ethanol induced S9 were superior to PB/beta-NF-S9 and uninduced-S9 for the activation of N-nitrosopyrrolidine, a known CYP2E1 substrate. However, there was no evidence of mutagenic activity with urethane, aniline, benzene, procarbazine or acetaminophen. As these compounds have demonstrated genotoxicity in vivo, additional important metabolic pathways must be required which are not present in rat liver S9 fraction.

Animals↗
PubMed1994

7-Ethoxycoumarin O-deethylase kinetics in isolated rat, dog and human hepatocyte suspensions.

1. A comparative study of the kinetics of the O-deethylation of 7-ethoxycoumarin has been carried out in freshly isolated rat, dog and human hepatocytes. 2. Biphasic kinetics were observed for all three species with apparent Km and Vmax values of 11.5, 2.2 and 3.9 microM and 0.30, 0.21, 0.007 nmol/min/10(6) cells from rat, dog and man, respectively, for the high affinity-low capacity component, and 560, 40, 470 microM and 1.52, 0.74, 0.057 nmol/min/10(6) cells, respectively, for the low affinity-high capacity component. 3. These observed kinetic parameters in hepatocytes from rat and man were similar to published values for microsomes for the same two species. 4. Values for intrinsic clearance of 7-ethoxycoumarin for the three species calculated from the Km and Vmax data were 152, 631 and 6 ml/min/kg for rat, dog and human hepatocytes, respectively. These intrinsic clearance values predict that 7-ethoxycoumarin would be subject to a high hepatic clearance in rat and dog, and low hepatic clearance in man. These values are supported by published data on rat which show that 7-ethoxycoumarin is subject to high clearance.

7-Alkoxycoumarin O-Dealkylase↗