-
Andrew Pike,
Susan M Cook, Alan P Watt,
Paul Scott-Stevens,
Thomas W Rosahl,
Ruth M McKernan,
Luanda J Pym,
Alec Guiblin,
Christopher Moyes,
Bindi Sohal,
John R Atack
[show abstract]
[hide abstract]
ABSTRACT: The in vivo occupancy of brain benzodiazepine binding sites by compounds A and B was measured using a [(3)H]Ro 15-1788 binding assay and related to plasma and brain drug concentrations. The plasma concentration associated with 50% occupancy was higher for compound A than compound B (73 and 3.7 nM, respectively), however, there was little difference in the brain concentrations required (73 and 63 nM). Both compounds showed a non-linear relationship between plasma and brain concentrations such that above brain concentrations of approximately 100 nM increasing plasma concentrations did not result in a concomitant increase in brain concentrations. This is consistent with brain concentrations being dependent on a saturable compartment which was postulated to be the benzodiazepine binding site-containing GABA(A) receptors. This hypothesis was tested in alpha1H101R mice, in which the alpha1 subunit of the GABA(A) receptor is rendered insensitive to benzodiazepine binding resulting in an approximate 50% reduction in the total benzodiazepine-containing GABA(A) receptor population. It was shown that the Occ(50) brain concentrations in the alpha1H101R animals was lower (17 nM) than in wild type mice (63 nM), as was the plateau concentration in the brain (105 and 195 nM, respectively). These data suggest measured concentrations of compounds A and B in brain tissue are dependent on receptor expression with a minimal contribution from unbound and non-specifically bound compound.
Biopharmaceutics & Drug Disposition 10/2007; 28(6):275-82. · 2.07 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cytochrome P450 (P450) induction may have considerable implications for drug therapy. Therefore, understanding the induction potential of a new chemical entity at an early stage in discovery is crucial to reduce the risk of failure in the clinic and help the identification of noninducing chemical structures. Availability of human viable tissue often limits evaluation of induction potential in human hepatocytes. A solution is to increase the time period during which the hepatocytes remain viable. In this study we have investigated the induction of several P450 isozymes in long-term cultured hepatocytes compared with short-term cultured hepatocytes from the same individuals. Short- and long-term cultured primary hepatocytes isolated from each individual were cultured in a 96-well format and treated for 24 h with a range of prototypical P450 inducers and Merck Research Laboratories compounds. CYP3A4, 1A1, 1A2, 2B6, and 2C9 mRNA levels were measured using quantitative real-time reverse transcriptase-polymerase chain reaction (TaqMan) from the same cultured hepatocyte wells. CYP3A4, 1A1, 1A2, 2B6, and 2C9 were shown to be inducible in long-term cultured hepatocytes. The -fold induction varied between donors, and between short- and long-term cultured hepatocytes from the same donor. However, this variability can be controlled by normalizing data from each hepatocyte preparation to a positive control. The use of long-term cultured hepatocytes on 96-well plates has proven to be sensitive, robust, and convenient for assessing P450 induction potential of new compound entities during the drug discovery process.
Drug Metabolism and Disposition 03/2007; 35(2):215-20. · 3.73 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We have previously described the synthesis of bis-phosphonate-containing inhibitors of inositol monophosphatase. In the present study, a more detailed examination of the in vitro and in vivo properties of one of these compounds, L-690,330, is described. L-690,330 is a competitive inhibitor of inositol monophosphatase with a K1, depending on the source of IMPase, of between 0.2 and 2 μM. Although ∼1,000-fold more potent in vitro than lithium, in muscarinic m1 receptor-transfected Chinese hamster ovary cells prelabelled with [3H]inositol, L-690,330 only produced 40% of the accumulation of [3H]inositol monophosphates achieved by lithium at the same concentration (10 mM), suggesting that the ability of L-690,330 to cross the cell membrane is limited. Nevertheless, under conditions of cholinergic stimulation (100 mg/kg of pilocarpine s.c.), high doses of L-690,330 were able to increase brain inositol(1)phosphate levels in vivo to three- to fourfold control levels. This effect was dose dependent (ED50= 0.3 mmol/kg s.c.) and was maximal after 1 h. In peripheral tissues, the effects of L-690,330 on inositol(1)phosphate levels mimicked those of lithium both qualitatively and quantitatively. However, in the brain, the effects of L-690,330 were much less than seen with lithium, consistent with the blood-brain barrier restricting access of the polar L-690,330 into the CNS, thereby further limiting entry of compound into cells in the brain. In the future, it may be possible to develop prodrugs of this compound, which circumvent many of the cell permeability problems inherent in bisphosphonate compounds.
Journal of Neurochemistry 10/2006; 60(2):652 - 658. · 4.06 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Idiosyncratic adverse drug reactions (ADRs) are one of the most common causes of pharmaceutical withdrawals and labeling changes. Most ADRs are caused by drugs that form reactive species that can bind covalently to macromolecules such as proteins. The current methodology for the measurement of covalent binding relies on the use of radiolabeled material that requires an investment in time and resources not typically expended until later in the discovery process. Efforts are also made to identify reactive intermediates by the use of chemical trapping agents, such as reduced glutathione and cyanide, to form stable adducts that are characterized by liquid chromatography-tandem mass spectrometry and/or nuclear magnetic resonance spectroscopy. Here, we describe a high-throughput assay for the measurement of reactive intermediate formation. The method involves incubation of cold compound with liver microsomes in the presence of [14C]potassium cyanide. Hard electrophilic species would react with the trapping agent, resulting in the formation of a radiolabeled conjugate. Unreacted trapping agent is removed using solid-phase extraction, and the amount of radiolabeled conjugate present is determined by liquid scintillation counting. This newly developed screen has proved to be specific, sensitive, robust, and a powerful tool for assessing bioactivation potential.
Analytical Biochemistry 05/2006; 351(2):266-72. · 3.00 Impact Factor
-
10/2005;
-
[show abstract]
[hide abstract]
ABSTRACT: It is generally accepted that there is neither a well-defined nor a consistent link between the formation of drug-protein adducts and organ toxicity. Because the potential does exist, however, for these processes to be causally related, the general strategy at Merck Research Laboratories has been to minimize reactive metabolite formation to the extent possible by appropriate structural modification during the lead optimization stage. This requires a flexible approach to defining bioactivation issues in a variety of metabolism vectors and typically involves the initial use of small molecule trapping agents to define the potential for bioactivation. At some point, however, there is a requirement to synthesize a radiolabeled tracer and to undertake covalent binding studies in vitro, usually in liver microsomal (and sometimes hepatocyte) preparations from preclinical species and human, and also in vivo, typically in the rat. This paper serves to provide one pragmatic approach to addressing the issue of bioactivation from an industry viewpoint based on protocols adopted by Merck Research Laboratories. The availability of a dedicated Labeled Compound Synthesis group, coupled to a close working relationship between Drug Metabolism and Medicinal Chemistry, represents a framework within which this perspective becomes viable; the overall aim is to bring safer drugs to patients.
Chemical Research in Toxicology 02/2004; 17(1):3-16. · 3.78 Impact Factor
-
Su-Er W Huskey,
Brian J Dean,
Ray Bakhtiar,
Rosa I Sanchez,
F David Tattersall,
Wayne Rycroft,
Richard Hargreaves, Alan P Watt,
Gary G Chicchi,
Carolann Keohane,
Donald F Hora,
Shuet-Hing L Chiu
[show abstract]
[hide abstract]
ABSTRACT: The pharmacokinetics, metabolism, and brain penetration of the neurokinin 1 (NK1) receptor antagonist (substance P receptor antagonist), aprepitant (MK-0869), were examined in ferrets. This species exhibits human-type NK1receptor pharmacology and is of proven value in the identification of clinically useful drugs for the treatment of chemotherapy-induced nausea and vomiting in humans. After a single p.o. dose of aprepitant at 1 or 2 mg/kg, plasma levels of the compound were between approximately 200 and 270 ng/ml, 24 h after dosing. In the brain cortex, concentrations of aprepitant reached between approximately 80 and 150 ng/g of tissue 24 h after dosing. The predominant radioactive component present in the plasma and the brain of ferrets at 24 or 48 h after a single oral dose of [14C]aprepitant at 3 mg/kg was the parent compound itself. The slow plasma clearance of aprepitant ( approximately 1.5 ml/min/kg) and its abundance in ferret brain were in accord with its efficacy in blocking the retching and vomiting at 24 and 48 h postdose when ferrets were challenged with the emetic anticancer drug, cisplatin. When aprepitant and some of its metabolites were assessed for their in vitro binding affinity to the human NK1receptor, aprepitant demonstrated the highest affinity. Collectively, these data suggested that aprepitant, rather than its metabolites, was responsible, primarily, for the antiemetic activity of this compound in the male ferret.
Drug Metabolism and Disposition 07/2003; 31(6):785-91. · 3.73 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Recent developments in the technologies and approaches to identify metabolites in a drug discovery environment are reviewed. Samples may be generated using either in vitro systems--typically, but not exclusively, liver subcellular fractions, such as microsomes, or whole cells, such as hepatocytes. Alternatively, metabolites are generated in vivo using excreta obtained following dosing in preclinical species. Recombinant drug metabolizing enzymes or microorganisms may offer alternate vectors. New techniques, such as the use of solid-phase microextraction, have found application in the isolation of metabolites from biological matrices. However, this is still dominated by the use of preparative chromatography, which has advanced through the use of mass-directed detection. Detection and structural elucidation by mass spectrometry have improved markedly with increases in sensitivity, allowing lower abundance metabolites to be detected, and increases in selectivity, with the use of high-resolution time-of-flight and quadrupole-time-of-flight instruments. Finally, higher field strength magnets coupled with novel probe designs and increased use of liquid chromatographic hyphenation techniques continue to drive the capabilities of nuclear magnetic resonance spectroscopy as the definitive structural elucidation tool.
Current opinion in drug discovery & development 02/2003; 6(1):57-65. · 4.20 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The enantiomeric separation of a series of 2-arylindoles, developed as 5HT(2A) receptor antagonists for the treatment of schizophrenia, was investigated. Evaluation of a number of chiral stationary phases (CSPs) suggested that Chiralcel OD-H and Chiralpak AD were the most versatile for these compounds, and were employed for more detailed studies. A degree of complementarity between the CSPs was observed, such that Chiralcel OD-H was more effective for piperidine-containing molecules and Chiralpak AD for piperazine- and morpholine-containing molecules. The presence of a basic secondary amine was detrimental chromatographically, but resolution was improved substantially by employing diethylamine (DEA) in the mobile phase. All separations were either enthalpy-controlled or showed no temperature dependence. Differential temperature effects between series highlighted the possibility of multiple binding modes on these CSPs. Based on this study, it is possible to make a more rational selection of chromatographic conditions for future novel analogues.
Journal of Biochemical and Biophysical Methods 01/2003; 54(1-3):275-86. · 2.33 Impact Factor
-
Cornelis E C A Hop,
Yanfeng Wang,
Sanjeev Kumar,
Maria Victoria Silva Elipe,
Conrad E Raab,
Dennis C Dean,
Grace K Poon,
Carol-Ann Keohane,
John Strauss,
Shuet-Hing L Chiu,
Neil Curtis,
Jason Elliott,
Ute Gerhard,
Karen Locker,
Denise Morrison,
Russell Mortishire-Smith,
Steven Thomas, Alan P Watt,
David C Evans
[show abstract]
[hide abstract]
ABSTRACT: [3R,5R,6S]-3-(2-cyclopropyloxy-5-trifluoromethoxyphenyl)-6-phenyl-1-oxa-7-azaspiro[4.5]decane is a substance P (Neurokinin 1 receptor) antagonist. Substance P antagonists are proven in concept to have excellent potential for the treatment of major depression, and they allow superior and sustained protection from acute and delayed chemotherapy-induced emesis. The metabolism of this compound was investigated in rat hepatocytes, and circulating rat plasma metabolites were identified following oral and intravenous dosing. The turnover in rat hepatocytes within 4 h was about 30%, and the major metabolites were identified as two nitrones and a lactam associated with the piperidine ring. Although these metabolites were also observed in rat plasma, the major circulating metabolite was a keto acid following oxidative de-amination of the piperidine ring. Liquid chromatography/tandem mass spectrometry and nuclear magnetic resonance were used to confirm the structure of the latter metabolite. A mechanism leading to the formation of the keto acid metabolite has been suggested, and most intermediates were observed in rat plasma.
Drug Metabolism and Disposition 09/2002; 30(8):937-43. · 3.73 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A four-channel multiplexed electrospray inlet system (MUX) coupled to a triple quadrupole mass spectrometer was investigated as a higher throughput approach to quantitative analysis. Four discrete samples may be simultaneously analyzed by virtue of a rotating sampler with a concomitant 4-fold increase in analytical throughput. Although absolute sensitivity was reduced using the MUX interface compared with analysis using traditional single electrospray interface, reproducibility of response was shown to be comparable. Source robustness was established for the analysis of both aqueous drug standards and drugs in biological media, and linearity of response for a test compound, diazepam, was demonstrated over 2 orders of magnitude. Analyte-dependent response differences were exhibited between the four channels of the interface, and this led to the overall conclusion that samples to be compared quantitatively must be analyzed through the same sprayer. In addition, each channel must be independently calibrated to afford true quantification. Should a deuterated internal standard be employed, however, quantitative comparisons can be made across channels. An HPLC pumping system providing individual back-pressure regulation to each channel was shown to provide adequate chromatography even in the event of a channel blockage. Furthermore, following multiple injections of biological samples onto the MUX interface, an eluent flow diversion was integrated into the first part of each analytical run. This served to prevent source fouling, and thus, no detrimental effects to response reproducibility or sensitivity were observed.
Analytical Chemistry 05/2002; 74(8):1896-902. · 5.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: This report presents a highly automated procedure for the determination of drug concentrations in plasma samples. The method is generic, in that it has been applied without adaptation to many different drug candidate molecules, but is also flexible, in that variations in the nature and number of samples to be analyzed can be readily accommodated. The method includes preparation of dilutions of analyte stock solutions, spiking these into control plasma to generate analytical standards, and preparation of samples suitable for analysis by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) by precipitation of plasma proteins with acetonitrile, centrifugation, and dilution of the supernatants with HPLC buffer. All of these steps, apart from centrifugation, are performed without manual intervention on an automated liquid-handling workstation using 96-well plates. Analysis is by HPLC/MS/MS, using a generic HPLC gradient. Commercially available software was used for optimization of parameters for analysis by HPLC/MS/MS, integration of chromatographic peaks, and quantification of drug concentrations. The use of this methodology in our laboratory has greatly facilitated the analysis of small sample sets for a large number of analytes, a situation regularly encountered in an early drug discovery environment.
Rapid Communications in Mass Spectrometry 02/2002; 16(11):1065-71. · 2.79 Impact Factor
-
Monique B. van Niel,
Ian Collins,
Margaret S. Beer,
Howard B. Broughton,
Susan K. F. Cheng,
Simon C. Goodacre,
Anne Heald,
Karen L. Locker,
Angus M. MacLeod,
Denise Morrison, [......],
Desmond O'Connor,
Andrew Pike,
Michael Rowley,
Michael G. N. Russell,
Balbinder Sohal,
Josephine A. Stanton,
Steven Thomas,
Hugh Verrier, Alan P. Watt,
José L. Castro
[show abstract]
[hide abstract]
ABSTRACT: It has previously been reported that a 3-(3-(piperazin-1-yl)propyl)indole series of 5-HT1D receptor ligands have pharmacokinetic advantages over the corresponding 3-(3-(piperidin-1-yl)propyl)indole series and that the reduced pKa of the piperazines compared to the piperidines may be one possible explanation for these differences. To investigate this proposal we have developed versatile synthetic strategies for the incorporation of fluorine into these ligands, producing novel series of 4-fluoropiperidines, 3-fluoro-4-aminopiperidines, and both piperazine and piperidine derivatives with one or two fluorines in the propyl linker. Ligands were identified which maintained high affinity and selectivity for the 5-HT1D receptor and showed agonist efficacy in vitro. The incorporation of fluorine was found to significantly reduce the pKa of the compounds, and this reduction of basicity was shown to have a dramatic, beneficial influence on oral absorption, although the effect on oral bioavailability could not always be accurately predicted.
05/1999;
-
[show abstract]
[hide abstract]
ABSTRACT: With pressure on pharmaceutical companies to reduce time-to-market and improve the success rate of new drug candidates, higher-throughput pharmacokinetic (HTPK) support has become an integral part of many drug discovery programmes. This report details the amalgamation of robotics, new sample preparation technologies and highly sensitive and selective mass spectrometric detection systems to deliver the promise of HTPK. A historical perspective on automated bioanalysis with the current approaches and future prospects for the discipline are described.
Drug Discovery Today.
