Identification of R146225 as a novel, orally active inhibitor of interleukin-5 biosynthesis.

Interleukin (IL)-5 regulates the growth, differentiation, and activation of eosinophils. When activated, eosinophils release an array of proinflammatory and cytotoxic products and act as prominent effector cells in the process of allergic inflammation. Depriving eosinophils of IL-5 may therefore represent a viable approach to treat allergic disorders. This study describes the identification of R146225, a novel six-substituted azauracil derivative, as a potent, orally active inhibitor of IL-5 biosynthesis, capable of reducing pulmonary eosinophilia in mice. In vitro, R146225 inhibited IL-5 protein formation by activated human whole blood (IC(50) = 34 nM), human peripheral blood mononuclear cells (IC(50) = 24 nM), and murine spleen cells (IC(50) = 6 nM). In contrast, the compound enhanced generation of interferon-gamma and had little or no inhibitory effect on the production of IL-2 and IL-4. Reverse transcription-polymerase chain reaction analysis of stimulated whole blood cells indicated R146225's ability to down-regulate IL-5 mRNA expression. In vivo p.o. administration of R146225 (2.5 mg/kg) to mice before an i.v. anti-CD3 antibody challenge reduced IL-5 but enhanced interferon-gamma serum levels, without affecting IL-2 and IL-4 production. Analogous to the in vitro results, R146225 suppressed splenic IL-5 mRNA expression, while message levels of the other cytokines remained unchanged. Moreover, p.o. dosing of R146225 (0.6-2.5 mg/kg) dose dependently reduced the pulmonary accumulation of eosinophils induced in mice by an intranasal instillation of Cryptococcus neoformans. Based on these data, R146225 may be useful in the therapy of eosinophil-driven allergic conditions.

R115866 inhibits all-trans-retinoic acid metabolism and exerts retinoidal effects in rodents.

All-trans-retinoic acid (RA) regulates epithelial differentiation and growth through activation of specific nuclear RA receptors (RARs). Because high-rate metabolism largely impairs the biological efficacy of RA, we have sought for compounds capable of inhibiting the metabolic breakdown of the retinoid. This study identifies R115866 as a novel inhibitor of the cytochrome P450 (CYP)-mediated metabolism of RA. In vitro, nanomolar concentrations of R115866 inhibited the conversion of RA by CYP26, a RA-inducible RA metabolizing enzyme. In vivo, oral administration of R115866 (2.5 mg/kg) to rats induced marked and transient increases of endogenous RA levels in plasma, skin, fat, kidney, and testis. Consistent with its ability to enhance endogenous RA content in tissues, R115866 was found to exert retinoidal activities. Like RA, the title compound: 1) inhibited vaginal keratinization in estrogen-stimulated rats; 2) induced epidermal hyperplasia in mouse ear skin; 3) transformed mouse tail epidermis from a para- to an orthokeratotic skin type; and 4) up-regulated the CYP26 mRNA expression in rat liver. Furthermore, we found that the keratinization-suppressive and CYP26-inducing activities of R115866 could be reversed by concomitant administration of the RAR antagonist, AGN193109. Our data characterize R115866 as a potent, orally active inhibitor of RA metabolism, capable of enhancing RA levels and displaying retinoidal actions. These activities are reversed by RAR antagonism, supporting the idea that the actions of R115866 result from increased availability of endogenous RA and improved RAR triggering.

Liarozole fumarate inhibits the metabolism of 4-keto-all-trans-retinoic acid.

The metabolism of 4-keto-all-trans-retinoic-acid (4-keto-RA), a biologically active oxygenated metabolite of all-trans-retinoic (RA), has been examined. In vitro, incubation of [14C]4-keto-RA with hamster liver microsomes in the presence of NADPH produced two major radioactive metabolites which were more polar than the parent compound. Following isolation, appropriate derivatization and analysis by GC-MS, these compounds were tentatively identified as 2-hydroxy- and 3-hydroxy-4-ketoretinoic acid. Formation of both hydroxy-keto derivatives was suppressed by the imidazole-containing P450 inhibitor liarozole fumarate (IC50, 1.3 microM). In vitro, an i.v. injection of 4-keto-RA (20 micrograms) into rats was followed by rapid disappearance of the retinoid from plasma with a half-life of 7 min. Pretreatment with liarozole fumarate (40 mg/kg, -60 min) reduced the elimination rate of 4-keto-RA: it prolonged the plasma half-life of the retinoid to 12 min, without affecting its distribution volume. These results indicate the important role of the P450 enzyme system in the metabolism of 4-keto-RA both in vitro and in vivo. The inhibitory effect of liarozole fumarate on this metabolic process may contribute to the reported retinoid-mimetic activity of this drug.

Liarozole, an inhibitor of retinoic acid metabolism, exerts retinoid-mimetic effects in vivo.

Liarozole is an imidazole-containing compound that inhibits the cytochrome P-450-dependent metabolism of all-trans-retinoic acid (RA). In vitro, liarozole (IC50, 2.2 microM) suppressed the P-450-mediated conversion of RA to more polar metabolites by hamster liver microsomes. In vivo, it enhanced the plasma level of RA from mostly undetectable values (less than 0.5 ng/ml) in control rats to 1.4 +/- 0.1 and 2.9 +/- 0.1 ng/ml in animals treated p.o. with 5 and 20 mg/kg of liarozole, respectively. Moreover, liarozole possessed antikeratinizing activity: when dosed subchronically (5-20 mg/kg, once daily for 3 days) to ovariectomized rats, the compound reversed the vaginal keratinization induced in these animals by estrogenic stimulation. Dose response experiments indicated that the antikeratinizating effect of liarozole was as potent as that of RA. One-dimensional electrophoresis and immunoblotting of extracted vaginal epithelia showed that liarozole shared with RA the ability to inhibit the synthesis of high molecular weight (57-60 kDa) keratin proteins, and to enhance the expression of the 45 to 47 kDa keratin polypeptides. Furthermore, we found that antikeratinizing doses of liarozole doubled the RA concentration in the vagina of ovariectomized rats: the mean amount of RA extracted from 200 mg of vaginal tissue was increased from 1.1 +/- 0.1 ng in vehicle-treated animals to 2.2 +/- 0.2 and 2.6 +/- 0.2 ng after treatment with 5 and 20 mg/kg of liarozole, respectively. These findings indicate that liarozole, an inhibitor of RA metabolism and RA produce similar morphologic and biochemical effects on the differentiation process of rat vaginal epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Suggested mechanism for the formation of 15-hydroxyeicosatrienoic acid by rat epidermal microsomes.

We have previously demonstrated that rat epidermal microsomes NADPH-dependently convert 15(S)-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) into 15-hydroxy-5,8,11-eicosatrienoic acid (15-HETrE). The present study examines the mechanism of this reductive conversion. Rat epidermal microsomes were incubated with [1-14C]15-HPETE in the presence and absence of NADPH. Major reaction products were purified by high performance liquid chromatography (HPLC) and analyzed by gas chromatography-mass spectrometry (GC-MS), UV spectroscopy and/or cochromatography with standard products. In the presence of NADPH, 15-HPETE was transformed to 13-hydroxy-14,15-epoxy-5,8,11-eicosatrienoic acid (13-HEpETrE), 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), 15-keto-5,8,11-eicosatrienoic acid (15-KETrE) and 15-hydroxy-5,8,11-eicosatrienoic acid (15-HETrE). In the absence of NADPH, the microsomes reacted with 15-HPETE to form 13-HEpETrE, 15-keto-5,8,11,13-eicosatetraenoic acid (15-KETE) and 15-HETE. Furthermore, when supplemented with NADPH, epidermal microsomes converted 15-KETE to 15-KETrE, which was subsequently reduced to 15-HETrE. These data suggest that rat epidermal microsomes are capable of metabolizing 15-HPETE to 15-HETrE via the following reaction steps: conversion of HPETE to KETE, NADPH-dependent double bond saturation in KETE to KETrE and keto-reduction of the latter compound to HETrE.

NADPH-dependent formation of 15- and 12-hydroxyeicosatrienoic acid from arachidonic acid by rat epidermal microsomes.

Rat epidermal microsomes were incubated with [1-14C]-arachidonic acid for 30 min at 37 degrees C in the absence and presence of NADPH. The arachidonate metabolites that eluted in the "monohydroxy acid fraction" on reverse-phase high performance liquid chromatography (HPLC) were methylated, purified by straight-phase HPLC and analyzed by chromatography with standard compounds, UV spectroscopy and/or gas chromatography-mass spectrometry (GC-MS). In the absence of NADPH, epidermal microsomes converted arachidonic acid to two major products identified as 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15(S)-HETE) and 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE). In the presence of NADPH, the microsomal reaction produced, besides 15(S)- and 12(S)-HETE, two less polar metabolites which were characterized as 15-hydroxy-5,8,11,-eicosatrienoic acid (15-HETrE) and 12-hydroxy-5,8,14-eicosatrienoic acid (12-HETrE). Stereochemical analysis by chiral-phase HPLC showed that the biosynthesized 12-HETrE consisted of a mixture of optical isomers in a S/R ratio of 65:35. Formation of 15- and 12-HETrE was blocked by the mixed cyclooxygenase-lipoxygenase inhibitors quercetin and phenidone but was not affected by the cyclooxygenase inhibitor indomethacin or the cytochrome P-450 monooxygenase inhibitor metyrapone. These data indicate that rat epidermal microsomes, supplemented with NADPH, are capable of metabolizing arachidonic acid to 15- and 12-HETrE. The production of these compounds may be initiated by lipoxygenase-mediated hydroperoxidation of arachidonic acid.

Inhibition of lymphocyte proliferation by monoclonal antibody directed against the T3 antigen on human T cells.

Peripheral blood mononuclear cells from 40% of normal donors are mitogenically unresponsive to UCHT1, a monoclonal antibody reactive to the T3 surface molecule on human T lymphocytes. Cell preparations from non-UCHT1 responders were used to examine whether and how interaction of UCHT1 with the T3 molecule affects T-cell functionality. It was found that UCHT1 profoundly (greater than 85%) suppressed lymphocyte proliferation induced by plant mitogens (phytohemagglutinin (PHA) and concanavalin A (Con A], recall antigen (candidin), and allogeneic non-T cells. The antibody abrogated both the production of interleukin 2 (IL-2) by and the expression of IL-2-specific receptors on T lymphocytes stimulated by PHA or allogeneic non-T cells. UCHT1 was maximally suppressive when added to cells within 2 hr (PHA stimulation) or 1 day (allogeneic non-T cell activation) after the initiation of the culture period. The inhibiting activity of UCHT1 could be related to its ability to modulate T3 molecules from the T-cell surface: both actions displayed the same antibody concentration dependence and had a comparable time dependence. Moreover, after modulation, unresponsive lymphocytes regained responsiveness to PHA in parallel with reexpression of surface T3 molecules. These findings are consistent with the idea that the human T3 molecule functions as an essential signal transducer during the early phases of T-cell activation.