Kenneth E.J. Dickinson

Evaluation of 170 Xenobiotics as Transactivators of Human Pregnane X Receptor (hPXR) and Correlation to Known CYP3A4 Drug Interactions

The human transcription factor pregnane X receptor (hPXR) is a key regulator of enzyme expression, especially cytochrome P450 3A4 (CYP3A4). Due to the prominence of CYP3A4 in the elimination of many drugs, the development of high throughput in vitro models to predict the effect of drugs on CYP3A4 expression have increased. To better interpret and predict potential drug-drug interactions due to CYP3A4 enzyme induction, we evaluated 170 xenobiotics in a hPXR transactivation assay and compared these results to known clinical drug-drug interactions. Of the 170 xenobiotics tested, 54% of them demonstrated some level of hPXR transactivation. By taking into consideration cell culture conditions (solubility, cytotoxicity, appropriate drug concentration in media), as well as in vivo pharmacokinetics (therapeutic plasma C(max), distribution, route of administration, dosing regimen, liver exposure, potential to inhibit CYP3A4), the risk potential of CYP3A4 enzyme induction for most compounds reduced dramatically. By employing this overall interpretation strategy, the final percentage of compounds predicted to significantly induce CYP3A4 reduced to 5%, all of which are known to cause drug-drug interactions. Also, this is the first report that identifies several potent compounds that have the ability to transactivate hPXR that previously have not been identified, such as terbinafine, diclofenac, sildenafil, glimepiride, montelukast, and ticlopidine.

Molecular characterization, pharmacological properties and chromosomal localization of the human GALR2 galanin receptor

The neuropeptide galanin mediates a diverse spectrum of biological activities by interacting with specific G protein-coupled receptors. We have used homology genomic library screening and polymerase chain reaction (PCR) techniques to isolate both genomic and cDNA clones encoding the human homolog of the recently cloned rat GALR2 galanin receptor. By fluorescence in situ hybridization, the gene encoding human GALR2 (GALNR2) has been localized to chromosome 17q25.3. The two coding exons of the human GALNR2 gene, interrupted by an intron positioned at the end of transmembrane domain III, encode a 387 amino acid G protein-coupled receptor with 87% overall amino acid identity with rat GALR2. In HEK-293 cells stably expressing human GALR2, binding of [125I]porcine galanin is saturable and can be displaced by galanin, amino-terminal galanin fragments and chimeric galanin peptides but not by carboxy-terminal galanin fragments. In HEK-293 cells, human GALR2 couples both to Galphaq/11 to stimulate phospholipase C and increase intracellular calcium levels and to Galphai/o to inhibit forskolin-stimulated intracellular cAMP accumulation. A wide tissue distribution is observed by reverse transcriptase (RT)-PCR analysis, with human GALR2 mRNA being detected in many areas of the human central nervous system as well as in peripheral tissues.

The endothelin receptor antagonist, BQ-123, inhibits angiotensin II-induced contractions in rabbit aorta

The purpose of this study was to examine the specificity of the cyclic pentapeptide ET(A) receptor antagonist BQ-123. BQ-123 competitively antagonized endothelin-1-induced contractions in rabbit aorta, increases in inositol phosphates in cultured rat vascular smooth muscle A10 cells, and binding of [125I]endothelin-1 to the cloned ETA receptor cDNA expressed in Cos 7 cells. In contrast, BQ-123 was a weak antagonist of [125I]endothelin-3 binding to rat cerebellar membranes and to membranes from Cos 7 cells transfected with the cloned ETB receptor cDNA. BQ-123 shifted concentration-response curves in isolated rabbit aorta elicited by angiotensin II, but did not bind to angiotensin II receptors nor affect angiotensin II-induced increases in inositol phosphates. BQ-123 also did not affect contractions induced by KCl or norepinephrine. These data suggest that endothelin may play a role in angiotensin II-induced contractions of rabbit aorta.

Correlation of High-Throughput Pregnane X Receptor (PXR) Transactivation and Binding Assays

Pregnane X receptor (PXR) transactivation and binding assays have been developed into high-throughput assays, which are robust and reproducible (Z′ > 0.5). For most compounds, there was a good correlation between the results of the transactivation and binding assays. EC50 values of compounds in the transactivation assay correlated reasonably well with their IC50 values in the binding assay. However, there were discrepancies with some compounds showing high binding affinity in the binding assay translated into low transactivation. The most likely cause for these discrepancies was an agonist-dependent relationship between binding affinity and transactivation response. In general, compounds that bound to human PXR and transactivated PXR tended to be large hydrophobic molecules.

BMS-180560, an insurmountable inhibitor of angiotensin II-stimulated responses: comparison with losartan and EXP3174

1 This study compares the activity of BMS‐180560 (2‐butyl‐4‐chloro‐1‐[[1‐[2‐(2H‐tetrazol‐5‐yl)phenyl]‐1H‐indol‐4‐yl]methyl]‐1H‐imidazole‐5‐carboxylic acid), an insurmountable angiotensin II (AII) receptor antagonist, with that of losartan and EXP3174 in functional and biochemical models of AII‐receptor activation. 2 BMS‐180560 selectively inhibited [125I]‐Sar1Ile8AII ([125I]SI‐AII) binding to rat aortic smooth muscle (RASM) cell and rat adrenal cortical AT1 receptors (Ki = 7.6 ± 1.2 and 18.4 ± 3.9 nm respectively) compared to adrenal cortical AT2 receptors (Ki = 37.6 ± 1.3 μm). The Ki values of BMS‐180560 and EXP3174, but not losartan, varied as a function of the BSA concentration used in the assays, indicating that the diacid drugs bound to albumin. 3 BMS‐180560 (3–300 nm) increased the KD of SI‐AII for RASM cell AT1 receptors. Only at high concentrations of BMS‐180560 (300 nm) were Bmax values decreased. 4 BMS‐180560 inhibited AII‐stimulated contraction of rabbit aorta with a calculated KB = 0.068 ± 0.048 nm and decreased maximal AII‐stimulated contraction at 1 nm BMS‐180560 by 75%. In the presence of 0.1% BSA, a higher KB value (5.2 ± 0.92 nm) was obtained. Losartan behaved as a competitive antagonist with a KB = 2.6 ± 0.13 nm. Contraction stimulated by endothelin‐1, noradrenaline, KC1, or the TXA2 receptor agonist U‐46619 were unaffected by BMS‐180560 (1 nm). 5 All stimulated the acidification rates of RASM cells as measured by a Cytosensor microphysiometer with an EC50 of 18 nm. Losartan (30 nm) shifted the AII concentration‐effect curves in a competitive manner whereas BMS‐180560 (0.01 and 0.1 nm) decreased the maximum responses by 60 and 75% respectively. Inhibition by losartan and BMS‐180560 could be reversed following washout although recovery took longer for BMS‐180560. 6 In [3H]‐myoinositol‐labelled RASM cells, losartan (30 and 200 nm), shifted the EC50 for AII‐stimulated [3H]‐inositol monophosphate formation to higher values, with no change in the maximal response. By contrast, EXP3174 (0.1 to 1 nm) decreased the maximal response in a concentration‐dependent manner (17–55%). BMS‐180560 (3 and 10 nm) increased the EC50 for AII and decreased the maximum response by 30 and 80% respectively. The inhibition by EXP3174 and BMS‐180560 could be reversed by inclusion of losartan (200 nm) indicating that the inhibition was not irreversible. 7 In conclusion, BMS‐180560 is a potent, specific, predominantly competitive, reversible AII receptor antagonist, which displays insurmountable receptor antagonism. At concentrations of BMS‐180560 which have no effect on receptor number, BMS‐180560 produced insurmountable antagonism of AII‐stimulated second messenger formation, extracellular acidification, and smooth muscle contraction.

Use of Cryopreserved Transiently Transfected Cells in High-Throughput Pregnane X Receptor Transactivation Assay

Cryopreserved, transiently transfected HepG2 cells were compared to freshly transfected HepG2 cells for use in a pregnane X receptor (PXR) transactivation assay. Assay performance was similar for both cell preparations; however, cryopreserved cells demonstrated less interassay variation. Validation with drugs of different PXR activation potencies and efficacies demonstrated an excellent correlation (r 2 > 0.95) between cryopreserved and fresh cells. Cryopreservation did not change the effect of known CYP3A4 inducers that have poor cell permeability, indicating that cryopreservation had little effect on membrane permeability. In addition, cryopreserved HepG2 cells did not exhibit enhanced susceptibility to cytotoxic compounds compared to transiently transfected control cells. The use of cryopreserved cells enables this assay to run with enhanced efficiency.

BMS-187257, a potent, selective, and novel heterocyclic β3 adrenergic receptor agonist

Abstract Novel heterocyclic β 3 adrenergic receptor agonists 2 were prepared and evaluated for their ability to bind to human β 1 , β 2 , and β 3 adrenergic receptors. Stimulatory effects on the β 3 adrenergic receptor were also measured. BMS-187257 ( 4b ) was found to be a potent and selective β 3 agonist.

Carboxyl-promoted enhancement of selectivity for the β3 adrenergic receptor. Negative charge of the sulfonic acid BMS-187413 introduces-β3 binding selectivity

Abstract Carboxyl and other negatively charged groups were found to be most effective at producing human β3 adrenergic receptor binding selectivity in 1 (BRL 37344) and related compounds. The sulfonic acid analog 7 (BMS-187413) is a novel and potent β3 adrenergic agonist that binds selectively, and thus has an in vitro profile that compares favorably with that of BRL 37344.

VASCULAR A10 CELL MEMBRANES CONTAIN AN ENDOTHELIN METABOLIZING NEUTRAL ENDOPEPTIDASE

We have investigated the possible presence of endothelin-metabolizing neutral endopeptidase (NEP, EC 3.4.24.11) on A10 cell membranes using [125I]-ET-1 binding and direct measurements of NEP. NEP activity of A10 cell membranes has been compared to that of solubilized rat kidney brush border membranes (KNEP). Specific [125I]-ET-1 (50 pM) binding (defined with 100 nM ET-1) to A10 cell membranes was increased in a concentration dependent manner by the selective NEP inhibitors thiorphan, phosphoramidon, and SQ 28,603 [(+/-)-N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-beta-alanine] with EC50 values of 9.4, 28.4, and 5.7 nM respectively. At equilibrium (150 min), 70% more specific binding was apparent in the presence of these inhibitors. Phosphoramidon (2 microM) did not alter Bmax values, but it decreased the apparent KD for [125I] ET-1 from 63 (+/- 3) to 27 (+/- 2) pM. Thiorphan, phosphoramidon, and SQ 28,603 inhibited A10 cell NEP activity with IC50 values of 5.3, 36.5, and 6.0 nM respectively, which was similar to values obtained with KNEP (3.6, 22.6, and 3.5 nM). ET-1 inhibited A10 cell NEP, and KNEP with IC50 values of 30 and 21.3 microM respectively. The order of inhibitory potencies: ET-3 greater than ET-1 = ET-2 greater than or equal to sarafotoxin-6b was similar for both systems. These data suggest A10 cell membranes contain a NEP which has similar characteristics to NEP 24.11, and which actively metabolizes [125I]-ET-1.

Beta 3 agonists. Part 1: evolution from inception to BMS-194449.

Screening of the BMS collection identified 4-hydroxy-3-methylsulfonanilidoethanolamines as full beta 3 agonists. Substitution of the ethanolamine nitrogen with a benzyl group bearing a para hydrogen bond acceptor promoted beta(3) selectivity. SAR elucidation established that highly selective beta(3) agonists were generated upon substitution of C(alpha) with either benzyl to form (R)-1,2-diarylethylamines or with aryl to generate 1,1-diarylmethylamines. This latter subset yielded a clinical candidate, BMS-194449 (35).(1)