BMS-229724 is a tight-binding inhibitor of cytosolic phospholipase A2 that acts at the lipid/water interface and possesses anti-inflammatory activity in skin inflammation models.

Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. BMS-229724 (4-[4-[2-[2-[bis(4-chlorophenyl)methoxy]ethyl-sulfonyl]ethoxy]phenyl]-1,1,1-trifluoro-2-butanone) was found to be a selective inhibitor of cPLA2 (IC50 = 2.8 microM) in that it did not inhibit secreted phospholipase A2 in vitro, nor phospholipase C and phospholipase D in cells. The compound was active in inhibiting arachidonate and eicosanoid production in U937 cells, neutrophils, platelets, monocytes, and mast cells. With a synthetic covesicle substrate system, the dose-dependent inhibition could be defined by kinetic equations describing competitive inhibition at the lipid/water interface. The apparent equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface (K(I)*(app)) was determined to be 1. 10(-5) mol% versus an apparent dissociation constant for the arachidonate-containing phospholipid of 0.35 mol%. The unit of concentration in the interface is mole fraction (or mol%), which is related to the surface concentration of substrate, rather than bulk concentration that has units of molarity. Thus, BMS-229724 represents a novel inhibitor of cPLA2, which partitions into the phospholipid bilayer and competes with phospholipid substrate for the active site. This potent inhibition of the enzyme translated into anti-inflammatory activity when applied topically (5%, w/v) to a phorbol ester-induced chronic inflammation model in mouse ears, inhibiting edema and neutrophil infiltration, as well as prostaglandin and leukotriene levels in the skin. In hairless guinea pigs, BMS-229724 was active orally (10 mg/kg) in a UVB-induced skin erythema model in hairless guinea pigs.

Assessment of P450 induction in the marmoset monkey using targeted anti-peptide antibodies.

The identity and expression of hepatic P450 enzymes in marmosets was investigated using a panel of anti-peptide antibodies originally targeted against human P450 enzymes. In immunoblotting, of 12 antibodies examined, 10 bound specifically to bands in marmoset liver microsomal fraction corresponding to P450 enzymes. It is proposed that these represent marmoset CYP1A1, CYP1A2, CYP2A, CYP2B, CYP2C forms (CYP2C-1 and CYP2C-2), CYP2D19, CYP3A21 and another CYP3A form (CYP3A-m). The antibodies, together with an anti-marmoset CYP2E1 antibody, were used to investigate the expression of 10 P450 enzymes in marmosets treated with P450-inducing chemicals. Treatment with phenobarbitone caused CYP2B, CYP2C-2 and CYP3A21 levels to increase, rifampicin caused increases in CYP2B and CYP2C-1 and a decrease in CYP3A21 levels, whereas dioxin caused CYP1A1 and CYP1A2 levels to increase and CYP2E1 levels to decrease. Clofibric acid did not induce any P450. P450 enzyme activities were assessed using 8 different substrates and increases were found after treatment with phenobarbitone, rifampicin, and dioxin. However, due to species differences in substrate selectivity, it proved difficult to ascribe these changes to individual P450 enzymes. Thus, the use of anti-peptide antibodies provides a more informative way of assessing the levels of specific P450 enzymes than enzyme activity measurements.

Reduced hepatic expression of CYP7A1 and CYP2C13 in rats with spontaneous hyperlipidaemia.

A strain of hyperlipidaemic Sprague-Dawley (HSD) rat was compared with normal Sprague-Dawley (SD) rats for expression of cholesterol 7alpha-hydroxylase activity (CYP7A1) and other cytochrome P450 (P450) enzymes in liver. Hepatic microsomal CYP7A1 activity in male HSD rats was 2-3-fold lower than in male SD rats with CYP7A1 apoprotein levels being similarly reduced. CYP7A1 expression was subject to diurnal variation in HSD rats as found in SD rats. Treatment of HSD rats with cholestyramine caused an increase in hepatic microsomal cholesterol 7alpha-hydroxylase activity of 3.3-fold compared with a 3.5-fold increase in SD rats with similar changes in apoprotein levels. These results indicate that the lower activity in HSD rats is not due to a defect in the catalytic activity of the enzyme, regulation affecting diurnal variation or regulation through bile acid feedback inhibition. No difference between hepatic microsomal methoxyresorufin-O-demethylase, benzoxyresorufin-O-debenzylase or chlorzoxazone 6-hydroxylase activities in SD and HSD rats was found, nor was there any difference in the levels of CYP1A2, CYP2D1, CYP2E1, CYP3A1, CYP3A2 or NADPH cytochrome P450 reductase determined by immunoblotting using specific anti-peptide antibodies. However, unlike in male SD rats, CYP2C13 was absent in male HSD rats and this was associated with a two-fold reduction in testosterone 6beta-hydroxylase activity. In conclusion, while HSD rats do not have a general reduction in P450 levels, they do lack CYP2C13 and have lowered cholesterol 7alpha-hydroxylase activity, as a result of a reduced level of expression of the enzyme.

Expression and distribution of cholesterol 7 alpha-hydroxylase in rat liver.

The hydroxylation of cholesterol by cholesterol 7 alpha-hydroxylase (CYP7) to 7 alpha-hydroxycholesterol is the rate-limiting step in the production of bile acids. An anti-peptide antibody targeted to the C-terminus of CYP7 was produced by immunising rabbits with the synthetic peptide Tyr-Lys-Leu-Lys-His. The antibody bound to a single band of 54 kDa from rat hepatic microsomal fractions. The intensity of the band was subject to a diurnal variation and showed a significant increase (P < 0.01) in apoprotein at night. Treatment of rats with cholestyramine increased CYP7 apoprotein in the morning (P < 0.005) and at night (P < 0.005), but diurnal variation was maintained. CYP7 catalytic activity, measured using a specific gas chromatography/mass spectrometry assay, showed similar changes in the pattern of diurnal variation and induction. The distribution of CYP7 in rat liver tissue sections was investigated by immunocytochemistry. In sections from rats treated with cholestyramine, there was an even distribution of immunoreactivity, except in the proximal perivenous hepatocytes where immunoreactivity was slightly more intense. A similar distribution was found in sections from untreated rat liver, except immunoreactivity was overall slightly less intense. This study shows that the C-terminus of CYP7 is a useful epitope for the targeting of anti-peptide antibodies.