John Dawson

GW274150 and GW273629 are potent and highly selective inhibitors of inducible nitric oxide synthase in vitro and in vivo

1 GW274150 ([2‐[(1‐iminoethyl) amino]ethyl]‐L‐homocysteine) and GW273629 (3‐[[2‐[(1‐iminoethyl)amino]ethyl]sulphonyl]‐L‐alanine) are potent, time‐dependent, highly selective inhibitors of human inducible nitric oxide synthase (iNOS) vs endothelial NOS (eNOS) (>100‐fold) or neuronal NOS (nNOS) (>80‐fold). GW274150 and GW273629 are arginine competitive, NADPH‐dependent inhibitors of human iNOS with steady state Kd values of <40 and <90 nM, respectively. 2 GW274150 and GW273629 inhibited intracellular iNOS in J774 cells in a time‐dependent manner, reaching IC50 values of 0.2±0.04 and 1.3±0.16 μM, respectively. They were also acutely selective in intact rat tissues: GW274150 was >260‐fold and 219‐fold selective for iNOS against eNOS and nNOS, respectively, while GW273629 was >150‐fold and 365‐fold selective for iNOS against eNOS and nNOS, respectively. 3 The pharmacokinetic profile of GW274150 was biphasic in healthy rats and mice with a terminal half‐life of ∼6 h. That of GW273629 was also biphasic in rats, producing a terminal half‐life of ∼3 h. In mice however, elimination of GW273629 appeared monophasic and more rapid (∼10 min). Both compounds show a high oral bioavailability (>90%) in rats and mice. 4 GW274150 was effective in inhibiting LPS‐induced plasma NOx levels in mice with an ED50 of 3.2±0.7 mg kg−1 after 14 h intraperitoneally (i.p.) and 3.8±1.5 mg kg−1 after 14 h when administered orally. GW273629 showed shorter‐lived effects on plasma NOx and an ED50 of 9±2 mg kg−1 after 2 h when administered i.p. 5 The effects of GW274150 and GW273629 in vivo were consistent with high selectivity for iNOS, as these inhibitors were of low potency against nNOS in the rat cerebellum and did not cause significant effects on blood pressure in instrumented mice.

Pyrazolopyridines as a novel structural class of potent and selective PDE4 inhibitors.

Optimisation of a high-throughput screening hit resulted in the discovery of 4-(substituted amino)-1-alkyl-pyrazolo[3,4-b]pyridine-5-carboxamides as potent and selective inhibitors of Phosphodiesterase 4 (PDE4). Herein, we describe early SAR studies around this novel template highlighting preferred substituents and rationalization of SAR through X-ray crystal structures of analogues bound to the PDE4 active site. Pyrazolopyridine 20a was found to be a potent and selective PDE4 inhibitor which also inhibits LPS induced TNF-alpha production from isolated human peripheral blood mononuclear cells and has an encouraging rat PK profile suitable for oral dosing.

Quinolines as a novel structural class of potent and selective PDE4 inhibitors. Optimisation for inhaled administration.

Crystallography-driven optimisation of a lead derived from similarity searching of the GSK compound collection resulted in the discovery of a series of quinoline derivatives that were highly potent and selective inhibitors of PDE4 with a good pharmacokinetic profile in the rat. Quinolines 43 and 48 have potential as oral medicines for the treatment of COPD.

Uptake of acetaminophen (paracetamol) by isolated rat liver cells

The characteristics of the uptake of acetaminophen (N-acetyl-p-aminophenol or paracetamol, APAP) in incubations of isolated rat liver cells were consistent with diffusion of the drug being the predominant mechanism of APAP influx in these cells at concentrations above 0.5 mM. At lower substrate concentrations (below 0.5 mM) a saturable component was apparent. Both uptake processes could have a role in the control of the metabolism of APAP, because, at low concentrations, there was no intracellular accumulation of unconjugated drug, all the APAP entering the cell being converted to sulphate and glucuronide. After addition of drug, there was a lag phase of approximately 5 min before APAP-glucuronide and APAP-sulphate appeared in the incubation medium; during this time both conjugates accumulated inside the cells. These results have implications for our understanding of the mechanisms of APAP transport, and indicate how these processes may affect the drugs overall metabolism.

y+ LAT-1 mediates transport of the potent and selective iNOS inhibitor, GW274150, in control J774 macrophages

Summary.This study has characterised the transport mechanism(s) for the novel and selective inhibitor of inducible nitric oxide synthase (iNOS), GW274150, in murine macrophage J774 cells. Transport of GW274150 was saturable (Km = 0.24 ± 0.01 mM and Vmax of 8.5 ± 0.12 pmol·µg protein−1 min−1), pH-insensitive and largely Na+-independent. Transport was also susceptible to trans-stimulation and was significantly inhibited by a 10-fold excess of L-arginine, L-lysine, L-leucine, L-methionine, L-glutamine and 6-diazo-5-oxo-L-norleucine but not by other amino acids or by N-ethylmaleimide. More importantly, the inhibitions caused by the neutral amino acids were critically dependent on Na+. These results strongly implicate system y+L in the transport of GW274150. Northern blot analysis confirmed this by revealing the presence of transcripts for y+LAT-1 but not y+LAT-2. Thus, taken together, our data show for the first time that J774 macrophages express y+LAT-1 transporters and that these carriers mediate transport of GW2741500 at least in these cells.

Measurement of glucuronidation by isolated rat liver cells using [14C]fructose

We have developed a simple and sensitive method for the study of the relative rates of glucuronidation of compounds, in isolated liver cells, based on the incorporation of 14C from fructose into glucuronide conjugates. Liver cells from fasted rats are used to minimize any reduction of the specific activity by glycogenolysis. Although rates of glucuronidation are lower in isolated liver cells from fasted rats than in those from fed rats, because of a reduction in the concentration of UDP-glucuronic acid, it is possible to compare the rates of glucuronidation of different compounds. Radiolabelled glucuronides are separated from [14C]fructose and [14C]glucose, produced by the liver cells, by normal-phase HPLC on a polar amino-cyano column. The specific activity of the glucuronide was found to be approximately 50% of that of the [14C]fructose. Absolute amounts of glucuronide can be determined by measuring the specific activity of the [14C]glucose, also produced by liver cells from fructose, which reflects that of the glucose-6-phosphate and hence the UDP-glucuronic acid used for glucuronidation, although for the measurement of relative rates this would not be necessary. We have used this method to examine the kinetics of the glucuronidation of N-acetyl-p-aminophenol (acetaminophen), 4-nitrophenol and 1-naphthol in isolated rat liver cells. The method should be applicable to the study of the rates of glucuronidation of a range of aglycones and, unlike other methods, does not require glucuronide standards or radiolabelled aglycone.

Quantitative studies of sulphate conjugation by isolated rat liver cells using [35S]sulphate

We have developed a simple, rapid and sensitive method for the study of sulphate conjugation in isolated liver cells based on the incorporation of 35S from [35S]sulphate. Excess [35S]sulphate is removed by a barium precipitation procedure, leaving [35S]sulphate conjugates in solution. We have used this method to examine the kinetics of sulphation of N-acetyl-p-aminophenol (acetaminophen), 4-nitrophenol and 1-naphthol in isolated rat liver cells. The efficiency of recovery of the sulphate conjugates was greater than 86%. The method is applicable to the quantitative study of sulphate conjugation of any substrate which forms a sulphate conjugate that is soluble in the presence of barium, without the need for standards or radiolabelled sulphate acceptors.

7-aminoalkyl-2-amino-1,2,3,4-tetrahydro-2-naphthoic acids as inhibitors of tryptophan uptake

Abstract A series of non-alkylating DL 7-amino-substituted 2-amino-1,2,3,4-tetrahydro-2-naphthoic acids (6–c, 8–b Table) have been prepared and shown to be potent inhibitors (Ki-μM) of tryptophan uptake in WiDr cells.

Heteroalicyclic carboxamidines as inhibitors of inducible nitric oxide synthase; the identification of (2R)-2-pyrrolidinecarboxamidine as a potent and selective haem-co-ordinating inhibitor.

Heteroalicyclic carboxamidines were synthesised and evaluated as inhibitors of nitric oxide synthases. (2R)-2-Pyrrolidinecarboxamidine, in particular, was shown to be a highly potent in vitro (IC(50)=0.12 μM) and selective iNOS inhibitor (>100-fold vs both eNOS and nNOS), with probable binding to the key anchoring glutamate residue and co-ordination to the haem iron.