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In an attempt to seek increased understanding of compound attributes that influence successful drug pipeline progression, GlaxoSmithKline's portfolio of oral candidates was compared with reference sets of marketed oral drugs. The approach differs from other attrition studies by explicitly focusing on choosing 'the right compound' by applying relevant, experimentally derived properties. The analysis led to four proposed compound quality categories, created by combining specific criteria for three measures: dose, solubility and the property forecast index, a composite measure of lipophilicity using chromatographically determined LogD and aromaticity. The 'three properties' provide benchmarked guidelines for project teams to use when seeking and selecting clinical candidates, because they reflect the property distribution of marketed oral drugs., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The aim of the present investigation was to compare the onset of action and intrinsic activity of the long-acting β(2)-agonist GW597901 with the fast- and short-acting salbutamol as model compounds using an isolated human lung reperfusion model. Twelve resected human lung lobes were challenged with methacholine (MCh) and subsequently nebulised with either GW597901 or salbutamol. Prostaglandin E(2) (PGE(2)) concentrations in the perfusion fluid were compared with the dose of MCh that was required to induce a bronchoconstriction. After successful MCh provocation, nebulisation of GW597901 and salbutamol fully reversed any observed bronchoconstriction. The bronchodilating effect was more pronounced for GW597901. Salbutamol revealed an immediate onset of action while the effect of GW597901 was observed with an approximate delay of 6 min. Higher doses of MCh were required for a successful bronchial challenge in the presence of elevated PGE(2) levels (r=0.8171, p ≤ 0.05). For the first time, an isolated perfused human lung model has been established for comparing the onset of action and potency of a short- and long-acting β(2)-agonist. We therefore conclude that it is an alternative for determination of drug effect characteristics and suitable for supplementing or predicting clinical data., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: The aim of the present investigation was to compare the pulmonary absorption of the novel long-acting β(2)-agonist GW597901 with salbutamol and to determine the influence of an induced bronchoconstriction on the pharmacokinetics of the compounds using a human lung reperfusion model., Methods: In an initial study with six lung perfusions the pharmacokinetic properties of the β(2)-agonists were determined. We then investigated the influence of an induced bronchoconstriction on the pulmonary absorption in six lung lobes for each drug. Therefore, methacholine (MCh) challenge agent was nebulised prior to administration of the β(2)-agonists., Results: As expected, the extent of pulmonary absorption of salbutamol into the perfusate was more pronounced than for the more lipophilic GW597901. Although the observed differences were not statistically significant they were further supported by analysis of tissue concentrations. In contrast, we observed a statistically significant influence of the bronchoprovocation with MCh on the pulmonary absorption of both β(2)-agonists, but this effect was not limited to a successfully induced bronchoconstriction. A prominent decline of salbutamol distribution into perfusion fluid was also observed when the organic cation transporter substrate carnitine was nebulised prior to the bronchodilator., Conclusions: Nebulised methacholine had a significant influence on the pharmacokinetics of bronchodilators. Since we observed this effect independently of a successfully induced bronchoconstriction and also after nebulisation of carnitine we suggest a significant delay of pulmonary absorption of inhaled salbutamol and GW597901 due to competition for a cation/carnitine drug transporter, most likely OCTN2., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The pulmonary and hepatic expression and catalytic activities of phase I and II drug-metabolizing enzymes were compared using human lung and liver tissue, and lung parenchymal cells (LPCs) and cryopreserved hepatocytes. Cytochrome P450 gene expression was generally lower in lung than in liver and CYP3A4 expression in lung was negligible. Esterase gene expression was similar in lung and liver. Expression of all sulfotransferase isoforms in lung was similar to or higher than that in liver. Lung tissue expressed low levels of UGT. However, the expression of UGT2A1 in lung was higher than that in liver. There was a range of catalytic activities in LPCs, including cytochrome P450, esterase, and sulfation pathways. Phase I activities were generally less than 10% of those determined in hepatocytes. Rates of ester hydrolysis and sulfation in LPCs were similar to those in hepatocytes. When measurable, glucuronidation in LPCs was present at very low levels, reflecting the gene expression data. The metabolism of salbutamol, formoterol, and budesonide was also investigated. Production of salbutamol-4-O-sulfate and budesonide oleate was observed in LPCs from at least two of three donor preparations studied. Formoterol sulfate and low levels of formoterol glucuronide were detected in one of three donors. In general, drug-metabolizing capability of LPCs is low compared with liver, although some evidence for substantial sulfation and deesterification capacity was observed. Therefore, these data support the use of this cell-based system for the investigation of key routes of xenobiotic metabolism in human lung parenchyma.

The metabolism of the structurally related 5HT3 antagonists ondansetron, alosetron and GR87442 in the rat, dog and human was determined in hepatocytes, liver microsomes and human recombinant microsomes. The profiles of phase I metabolites were similar in human hepatocytes and microsomes. The metabolites of all three compounds produced in rat, dog and human microsomes and hepatocytes were similar to those seen in vivo, with the major routes of metabolism being N-dealkylation and/or hydroxylation. There was more extensive metabolic processing in hepatocytes than in microsomes; however, sequential metabolism was less extensive in vitro compared with in vivo. The pharmacokinetics of the three 5HT3 antagonists investigated were dominated by CYP3A4 (and/or 2C9) compared with CYP1A2 in man, possibly determined by enzyme capacity rather than relative enzyme affinity. These data support the use of rat, dog and human hepatocytes for the prediction of in vivo metabolites of ondansetron, alosetron and GR87442.

The in vitro clearances of the 5HT3 antagonists, ondansetron, alosetron and GR87442 were investigated. Intrinsic clearances using either metabolite formation or substrate depletion methods were equivalent (R2 = 0.95). Hepatocytes from preclinical species were superior to microsomes for the prediction of hepatic clearance (CL(H)), whereas the predictions from human microsomes and hepatocytes were similar. Using a non-restrictive model, seven of the nine CL(H) predictions using hepatocytes were within 2-fold of the in vivo CL(H) values. If the unbound fraction was included, the clearance of the compounds was generally under-predicted by both in vitro models. However, for the most metabolically stable compound, GR87442, the non-restrictive model over-predicted CLp. This and the possibility of extrahepatic metabolism indicate that the restrictive model is more appropriate for prediction of CL(H). The rank order of metabolic stability correlated with that in vivo. All three compounds were more metabolically stable in human than in the preclinical animal species examined.

Reported predictions of human in vivo hepatic clearance from in vitro data have used a variety of values for the scaling factors human microsomal protein (MPPGL) and hepatocellularity (HPGL) per gram of liver, generally with no consideration of the extent of their inter-individual variability. We have collated and analysed data from a number of sources, to provide weighted meangeo values of human MPPGL and HPGL of 32 mg g-1 (95% Confidence Interval (CI); 29-34 mg.g-1) and 99x10(6) cells.g-1 (95% CI; 74-131 mg.g-1), respectively. Although inter-individual variability in values of MPPGL and HPGL was statistically significant, gender, smoking or alcohol consumption could not be detected as significant covariates by multiple linear regression. However, there was a weak but statistically significant inverse relationship between age and both MPPGL and HPGL. These findings indicate the importance of considering differences between study populations when forecasting in vivo pharmacokinetic behaviour. Typical clinical pharmacology studies, particularly in early drug development, use young, fit, healthy male subjects of around 30 years of age. In contrast, the average age of patients for many diseases is about 60 years of age. The relationship between age and MPPGL observed in this study estimates values of 40 mg.g-1 for a 30 year old individual and 31 mg.g-1 for a 60 year old individual. Investigators may wish to consider the reported covariates in the selection of scaling factors appropriate for the population in which estimates of clearance are being predicted. Further studies are required to clarify the influence of age (especially in paediatric subjects), donor source and ethnicity on values of MPPGL and HPGL. In the meantime, we recommend that the estimates (and their variances) from the current meta-analysis be used when predicting in vivo kinetic parameters from in vitro data.

Profound technological advances in the drug discovery process have led to the identification of an increasingly large number of promising compounds at the hits-to-leads stage. Higher-throughput pharmacokinetic screens have therefore been developed to enhance the tractability of selected leads and minimize the risk of failure in the later stages of drug development.

Rational drug discovery requires an early appraisal of all factors impacting on the likely success of a drug candidate in the subsequent preclinical, clinical and commercial phases of drug development. The study of absorption, distribution, metabolism, excretion and pharmacokinetics (ADME/PK) has developed into a relatively mature discipline in drug discovery through the application of well-established in vitro and in vivo methodologies. The availability of improved analytical and automation technologies has dramatically increased our ability to dissect out the fundamentals of ADME/PK through the development of increasingly powerful in silico methods. This is fuelling a shift away from the traditional, empirical nature of ADME/PK towards a more rational, in cerebro approach to drug design.

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.

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.

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.

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.

The profound technological advances that are now occurring early in the drug discovery process have enabled lead identification groups to deliver very large numbers of promising compounds to the project teams responsible for lead optimization and candidate selection. This success has applied significant pressure to the 'traditional' selection processes performed during the preclinical optimization stages of a new medicine, where compounds with the optimal balance of potency, selectivity, safety and pharmacokinetics, are identified for progression using an iterative synthesis and testing process. Thus, the need exists for higher-throughput methods of determining pharmacokinetic parameters to enable rational decisions to be made on large numbers of compounds. Protocols detailing the administration of mixtures of compounds, cassette dosing, to single animals have been used successfully to increase throughput, and, at the same time address ethical considerations by reducing animal usage. Typically, cassettes of up to ten compounds have been administered in one dose via the intravenous or oral routes. The samples produced have then been analyzed by mass spectrometry.

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.

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.

The liver performs a wide range of physiologically important functions, including the synthesis and secretion of albumin, fibrinogen, and other plasma proteins; the synthesis of cholesterol and bile acids, and the metabolism of drugs, steroids, and amino acids. The liver has a central role in energy metabolism as the major store of glycogen, as the site of gluconeogenesis, and in the synthesis of fatty acids and triglycerides. The liver is, therefore, a vital organ, but it is difficult to study specific liver functions in vivo owing to interfering influences from other organs, e g., the kidney, gut, and lungs, which metabolize drugs and the muscle involvement in glucose homeostasis. An isolated liver preparation seems necessary, and the isolated human hepatocyte appears to be a suitable experimental model for the study of liver-specific functions.

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.

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.