Alan P. Watt

In Vitro and In Vivo Inhibition of Inositol Monophosphatase by the Bisphosphonate L‐690,330

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.

Chiral Base-Mediated Asymmetric Synthesis of Tricarbonyl(.eta.6-arene)chromium Complexes

Enantiomerically enriched tricarbonyl(η 6 -arene)chromium complexes can be obtained in up to 84% ee from the reaction of certain monosubstituted complexes with Me 3 SiCl, mediated by a chiral lithium amide base

2-Aryl Indole NK1 receptor antagonists: optimisation of indole substitution

The synthesis and biological evaluation of a series of 2-aryl indoles with high affinity for the human neurokinin-1 (hNK1) receptor are reported, concentrating on optimisation of the indole substitution.

An Increased Throughput Method for the Determination of Partition Coefficients

AbstractPurpose. To present an increased throughput automated shake-flask method for the direct determination of the partition coefficients of solutes between octan-1-ol and buffer. Method. The traditional shake-flask method has been transferred onto 96-well plate technology and a robotic liquid handler has been used for sample preparation. A custom programmed Gilson auto- sampler samples the organic and aqueous phases directly from the plate, circumventing the need for any manual separation. Analyses are performed by reverse phase high performance liquid chromatography (RP-HPLC). Generic fast gradient RP-HPLC conditions are used to eliminate chromatographic method development time and reduce analysis time. Results. A full validation of the automated method is presented for a range of compounds with log D values between −2 and 4. Conclusions. The advantages and limitations of this direct measurement method are discussed. The use of this methodology provides a means to rapidly assess log D values for large compound arrays.

Approaches to higher-throughput pharmacokinetics (HTPK) in drug discovery.

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.

A study of the synthesis and racemisation of a chiral lithiated tricarbonyl(η6-anisole)chromium complex

Abstract The non-racemic metallated complex derived from tricarbonyl(η 6 -anisole)chromium 1 , formed using the homochiral lithium amide base 2 , undergoes rapid racemisation by intermolecular proton transfer if the neutral chromium complex is present, this problem being minimised if the deprotonation is conducted in the presence of LiCl.

L-708,474: the C5-cyclohexyl analogue of L-365,260, a selective high affinity ligand for the CCKB/gastrin receptor

Abstract The C5-cyclohexyl analogue of the cholecystokinin type-B (CCKB) receptor antagonist L-365,260 has been prepared. This derivative has significantly higher CCKB affinity and markedly improved CCKB/CCKA receptor selectivity (6,500 v. 87-fold) than the parent compound. It is one of the most potent and selective CCKB ligands reported to date.

POTENT, SELECTIVE, WATER-SOLUBLE BENZODIAZEPINE-BASED CCKB RECEPTOR ANTAGONISTS THAT CONTAIN LIPOPHILIC CARBOXYLATE SURROGATES

Abstract Acylsulphonamide analogues of the meta-tolylurea L-708,474 have been synthesised and evaluated as CCKB receptor antagonists. Such derivatives retain very high affinity and subtype selectivity for the CCKB receptor, and have good aqueous solubility. The ortho-tolyl acylsulphonamide L-736,309 is orally bioavailable and brain penetrant in rat.

Measurement of lithium-induced changes in mouse inositol(1)phosphate levels in vivo

Abstract: An anion‐exchange HPLC mass assay was used to characterize Swiss‐Webster mouse brain and peripheral tissue inositol(1)phosphate [Ins(1)P] levels. Ins(1)P was identified in all tissues studied but Ins(4)P could be identified only in brain, and then only as a part of a peak containing an additional, unidentified component. As a result, it was not possible to quantify Ins(4)P levels. Following a single subcutaneous dose of lithium (10 mmol/kg), brain Ins(1)P levels were maximally elevated after 6 h (corresponding to peak brain lithium concentrations) and were increased to levels 35‐ and 20‐fold higher than in saline‐treated animals in cholinergic agonist (pilocarpine)‐stimulated and unstimulated animals, respectively. The ED50 for the lithium‐induced accumulation of brain Ins(1)P 6 h after administration was 4–6 mmol/kg. The pilocarpine stimulation of lithium‐induced brain Ins(1)P accumulation had an ED50 of 22 mg/kg, with maximal accumulation occurring 120 min after pilocarpine administration. Atropine reduced Ins(1)P levels, in both the absence and the presence of lithium, by 40%, indicating that cholinergic systems contribute a large (40%) component of basal brain phosphatidylinositol (PI) cycle activity. In peripheral tissues, there were lithium‐induced accumulations of Ins(1)P in kidney, heart, and liver (but not testes) but these were less than that seen in the brain, suggesting that under basal (and pilocarpine‐stimulated) conditions, the brain has a higher turnover of the PI cycle than the various peripheral tissues studied. These data support the hypothesis that lithium exerts its effects in vivo via modulation of the PI cycle. In addition, the susceptibility of brain rather than peripheral tissue to the pharmacological effects of lithium may be a consequence of higher PI cycle turnover in brain.

Utility of Long-Term Cultured Human Hepatocytes as an in Vitro Model for Cytochrome P450 Induction

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.