Synthesis and biological evaluation of novel orally available 1-phenyl-6-aminouracils containing dimethyldihydrobenzofuranol structure for the treatment of allergic skin diseases.

We have designed and efficiently synthesized novel 1-phenyl-6-aminouracils by replacing the chroman moiety in CX-659S, a nonsteroidal dermatologic candidate, with dimethyldihydrobenzofuranol to cancel CX-659S asymmetric center. Medicinal chemistry effort culminated in the discovery of 13d bearing a 3-methyl group at the 1-phenyl group as a promising compound. Compound 13d, having good in vitro ADME profile and moderate oral bioavailability in mice, showed potent anti-inflammatory activity against hapten-induced contact hypersensitivity reaction in mice following topical and oral administration. The effects of 13d were equipotent to that of tacrolimus or prednisolone. In addition, compound 13d, having potent hydroxyl radical-scavenging activity, showed more potent suppressive effect on substance P-induced pruritus in mice than oxatomide.

Bis(L-cysteinato)zincate(lI) as a coordination compound that induces metallothionein gene transcription without inducing cell-stress-related gene transcription.

Zinc is an essential micronutrient, deficiency of which results in growth retardation, immunodeficiency, and neurological diseases such as dysgeusia. Several zinc coordination compounds are used for zinc supplementation; however, supplemented zinc ions have no specificity and interact with various groups of molecules. Here, we found that, from a library of 30 zinc coordination compounds, bis(L-cysteinato)zincate(II), designated Z01, functioned as a metallothionein (MT) inducer. Z01 induced MT expression mediated by the transcription factor MTF-1, without inducing cell-stress-related heme oxygenase-1 gene expression at specific concentration. The zinc ion was necessary for the MT induction. (65)Zn incorporation following treatment with (65)Zn-labeled Z01 suggested that Z01 did not act as zinc ionophore despite its hydrophilicity. Electrophoretic mobility shift assays revealed that Z01 facilitates MTF-1-MRE complex formation, and, by inference, transfer of zinc from Z01 to MTF-1. Phosphorylated ERK levels were increased by ZnSO(4) treatment but not by Z01. Although our data do not definitely prove that Z01 is an MTF-1-specific activator, our observations suggest that zinc coordination compounds can regulate zinc distribution and act as zinc donors for specific molecules.

Chromium (VI) inhibits mouse metallothionein-I gene transcription by modifying the transcription potential of the co-activator p300.

The production of the heavy metal-binding proteins, the metallothioneins (MTs), is induced by heavy metals such as Zn, Cd, and Hg. MTs maintain Zn homeostasis and attenuate heavy metal-induced cytotoxicity by sequestering these metals and lowering their intracellular concentrations. Previously, we had reported that Zn induced the formation of a co-activator complex containing metal response element-binding transcription factor-1 (MTF-1) and the histone acetyltransferase (HAT), p300, which plays an essential role in the activation of MT-1 transcription. In addition, we had shown that Cr(VI) inhibits Zn-induced MT-1 transcription by preventing the Zn-dependent formation of the MTF-1-p300 complex. In the current study, we have shown that the inhibition by Cr(VI) was partially overcome by the overexpression of p300 or MTF-1 in an MT-I promoter-driven luciferase reporter assay system and have used real-time RT-PCR to determine MT-I mRNA levels. It has been reported that Cr(VI) inhibits CYP1A1 transcription by crosslinking histone deacetylase (HDAC) to the promoter. The crosslink inhibits the recruitment of p300 to the MT-1 promoter and blocks HAT-dependent transactivation by p300. However, our results demonstrate that trichostatin A, an HDAC inhibitor, could not block the inhibitory effects of Cr(VI) on MT-1 transcription and that there were no significant differences in the in vitro inhibitory effects of Cr(VI), Cr(III), and Zn on p300 HAT activity. This suggests that the inhibitory effects of Cr(VI) on MT-I transcription may be due to its effects on the HAT-independent transactivation ability rather than the HAT-dependent, HDAC release-related transactivation ability of p300.

The zinc-sensing transcription factor MTF-1 mediates zinc-induced epigenetic changes in chromatin of the mouse metallothionein-I promoter.

Metallothionein (MT) is a small, cysteine-rich protein active in zinc homeostasis, cadmium detoxification, and protection against reactive oxygen species. Mouse MT-I gene transcription is regulated by metal response element-binding transcription factor-1 (MTF-1), which is recruited to the promoter by zinc. We examined alterations in the chromatin structure of the MT-I promoter associated with enhanced transcriptional activation. MTF-1 proved essential for zinc-induced epigenetic changes in the MT-I promoter. Chromatin immunoprecipitation assays demonstrated that zinc treatment rapidly decreased Lys4-trimethylated and Lys9-acetylated histone H3 in the promoter and decreased total histone H3 but not histone H3.3. Micrococcal nuclease sensitivity of the MT-I promoter was increased by zinc. Thus, the chromatin structure in the promoter may be locally disrupted by zinc-induced nucleosome removal. Without MTF-1 these changes were not observed, and an MTF-1 deletion mutant recruited to the MT-I promoter by zinc that did not recruit the coactivator p300 or activate MT-I transcription did not affect histone H3 in the MT-I promoter in response to zinc. Interleukin-6, which induces MT-I transcription independently of MTF-1, did not reduce histone H3 levels in the promoter. Rapid disruption of nucleosome structure at the MT-I promoter is mediated by zinc-responsive recruitment of an active MTF-1-coactivator complex.

Involvement of carboxylesterase 1 and 2 in the hydrolysis of mycophenolate mofetil.

Mycophenolate mofetil (MMF) is the ester prodrug of the immunosuppressant agent mycophenolic acid (MPA) and is rapidly activated by esterases after oral administration. However, the role of isoenzymes in MMF hydrolysis remains unclear. Although human plasma, erythrocytes, and whole blood contain MMF hydrolytic activities, the mean half-lives of MMF in vitro were 15.1, 1.58, and 3.20 h, respectively. Thus, blood esterases seemed to contribute little to the rapid MMF disappearance in vivo. In vitro analyses showed that human intestinal microsomes exposed to 5 and 10 µM MMF exhibited hydrolytic activities of 2.38 and 4.62 nmol/(min · mg protein), respectively. Human liver microsomes exhibited hydrolytic activities of 14.0 and 26.1 nmol/(min · mg protein), respectively, approximately 6-fold higher than those observed for intestinal microsomes. MMF hydrolytic activities in human liver cytosols were 1.40 and 3.04 nmol/(min · mg protein), respectively. Because hepatic cytosols generally contain 5-fold more protein than microsomes, MMF hydrolysis in human liver cytosols corresponded to approximately 50% of that observed in microsomes. Fractions obtained by 9000g centrifugation of supernatants from COS-1 cells expressing human carboxylesterase (CES) 1 or 2 exhibited MMF hydrolytic activity, with CES1-containing fractions showing higher catalytic efficiency than CES2-containing fractions. The CES inhibitor bis-p-nitrophenylphosphate inhibited MMF hydrolysis in human liver microsomes and cytosols with IC(50) values of 0.51 and 0.36 µM, respectively. In conclusion, both intestinal and hepatic CESs and in particular CES1 may be involved in MMF hydrolysis and play important roles in MMF bioactivation. Hepatic CES1 activity levels may help explain the between-subject variability observed for MMF usage.

Ethanol-induced expression of glutamate-cysteine ligase catalytic subunit gene is mediated by NF-kappaB.

Glutamate-cysteine ligase is a rate-limiting enzyme in the de novo synthesis of glutathione, a known scavenger of electrophiles and reactive oxygen species. Glutamate-cysteine ligase catalytic subunit (GCLC) is regulated transcriptionally by nuclear factor erythroid 2-related factor 2 (Nrf2). It has been reported that ethanol induces human GCLC production via Nrf2-mediated transactivation of the antioxidant-responsive element (ARE). Here, the luciferase reporter assay revealed the presence of an ethanol-responsive element in the human GCLC promoter; it spanned bases -1432 to -832 in hepatocytes and HepG2 cells transfected with cytochrome P450 2E1 (CYP2E1). The region lacked an ARE but had a putative nuclear factor-kappaB (NF-kappaB) element. NF-kappaB DNA-binding activity was activated in response to ethanol treatment. CYP2E1 expression was required for GCLC promoter-driven gene expression and the activation of NF-kappaB. Thus ethanol-induced GCLC transcription is mediated by not only Nrf2 but also NF-kappaB.

Chromium(VI) inhibits mouse metallothionein-I gene transcription by preventing the zinc-dependent formation of an MTF-1-p300 complex.

Mouse MT-I (metallothionein-I) transcription is regulated by MTF-1 (metal-response-element-binding transcription factor-1) which is recruited to the promoter in response to zinc. Cr(VI) [chromium(VI)] pretreatment blocks zinc-activation of the endogenous MT-I gene and attenuates zinc-activation of MT-I-promoter-driven luciferase reporter genes in transient transfection assays. Chromatin immunoprecipitation assays revealed that Cr(VI) only modestly reduces recruitment of MTF-1 to the MT-I promoter in response to zinc, but drastically reduces the recruitment of RNA polymerase II. These results suggest that Cr(VI) inhibits the ability of MTF-1 to transactivate this gene in response to zinc. Zinc has recently been shown to induce the formation of a co-activator complex containing MTF-1 and the histone acetyltransferase p300 which plays an essential role in the activation of MT-I transcription. In the present study, co-immunoprecipitation assays demonstrated that Cr(VI) pretreatment blocks the zinc-induced formation of this co-activator complex. Thus Cr(VI) inhibits mouse MT-I gene expression in response to zinc by interfering with the ability of MTF-1 to form a co-activator complex containing p300 and recruiting RNA polymerase II to the promoter.

The in vitro metabolism of a pyrethroid insecticide, permethrin, and its hydrolysis products in rats.

The in vitro metabolism of permethrin and its hydrolysis products in rats was investigated. Cis- and trans-permethrin were mainly hydrolyzed by liver microsomes, and also by small-intestinal microsomes of rats. trans-Permethrin was much more effectively hydrolyzed than the cis-isomer. When NADPH was added to the incubation mixture of the liver microsomes, three metabolites, 3-phenoxybenzyl alcohol (PBAlc), 3-phenoxybenzaldehyde (PBAld) and 3-phenoxybenzoic acid (PBAcid), were formed. However, only PBAlc was formed by rat liver microsomes in the absence of cofactors. The microsomal activities of rat liver and small intestine were inhibited by bis-p-nitrophenyl phosphate, an inhibitor of carboxylesterase (CES). ES-3 and ES-10, isoforms of the CES 1 family, exhibited significant hydrolytic activities toward trans-permethrin. When PBAlc was incubated with rat liver microsomes in the presence of NADPH, PBAld and PBAcid were formed. The NADPH-linked oxidizing activity was inhibited by SKF 525-A. Rat recombinant cytochrome P450, CYP 2C6 and 3A1, exhibited significant oxidase activities with NADPH. When PBAld was incubated with the microsomes in the presence of NADPH, PBAcid was formed. CYP 1A2, 2B1, 2C6, 2D1 and 3A1 exhibited significant oxidase activities in this reaction. Thus, permethrin was hydrolyzed by CES, and PBAlc formed was oxidized to PBAld and PBAcid by the cytochrome P450 system in rats.

Involvement of human blood arylesterases and liver microsomal carboxylesterases in nafamostat hydrolysis.

Metabolism of nafamostat, a clinically used serine protease inhibitor, was investigated with human blood and liver enzyme sources. All the enzyme sources examined (whole blood, erythrocytes, plasma and liver microsomes) showed nafamostat hydrolytic activity. V(max) and K(m) values for the nafamostat hydrolysis in erythrocytes were 278 nmol/min/mL blood fraction and 628 microM; those in plasma were 160 nmol/min/mL blood fraction and 8890 microM, respectively. Human liver microsomes exhibited a V(max) value of 26.9 nmol/min/mg protein and a K(m) value of 1790 microM. Hydrolytic activity of the erythrocytes and plasma was inhibited by 5, 5'-dithiobis(2-nitrobenzoic acid), an arylesterase inhibitor, in a concentration-dependent manner. In contrast, little or no suppression of these activities was seen with phenylmethylsulfonyl fluoride (PMSF), diisopropyl fluorophosphate (DFP), bis(p-nitrophenyl)phosphate (BNPP), BW284C51 and ethopropazine. The liver microsomal activity was markedly inhibited by PMSF, DFP and BNPP, indicating that carboxylesterase was involved in the nafamostat hydrolysis. Human carboxylesterase 2 expressed in COS-1 cells was capable of hydrolyzing nafamostat at 10 and 100 microM, whereas recombinant carboxylesterase 1 showed significant activity only at a higher substrate concentration (100 microM). The nafamostat hydrolysis in 18 human liver microsomes correlated with aspirin hydrolytic activity specific for carboxylesterase 2 (r=0.815, p<0.01) but not with imidapril hydrolysis catalyzed by carboxylesterase 1 (r=0.156, p=0.54). These results suggest that human arylesterases and carboxylesterase 2 may be predominantly responsible for the metabolism of nafamostat in the blood and liver, respectively.

Role of metal-responsive transcription factor-1 (MTF-1) in EGF-dependent DNA synthesis in primary hepatocytes.

Metal-responsive transcription factor-1 (MTF-1), which is involved in sensing heavy metal load, induces the transcription of several protective genes. The mouse Mtf-1 gene is essential, and Mtf-1(-/-) embryos die from liver degeneration. We showed that DNA synthesis induced in hepatocytes by epidermal growth factor (EGF) was delayed by inhibition of MTF-1. To inhibit MTF-1 activity, MTFDeltaC, a C-terminal deletion mutant of MTF-1, was expressed by infection with the virus Ad5MTFDeltaC. Lactate dehydrogenase (LDH) release and/or caspase-3/7 activation was not observed under our experimental conditions. The inhibitory effect of MTFDeltaC on EGF-dependent DNA synthesis in hepatocytes was not eliminated by zinc addition. EGF-dependent extracellular signal-related kinase (ERK) phosphorylation, an essential reaction for EGF-dependent DNA synthesis, was decreased in MTF-1-inhibited hepatocytes. Moreover, decrease of ERK phosphorylation was observed by using siRNA in MTF-1-downregulated hepatocytes. These results indicate that MTF-1 is particularly important for proper hepatocyte proliferation. This is the first report to suggest the function of MTF-1 in the ERK pathway.

C-terminal deletion mutant of MRE-binding transcription factor-1 inhibits MRE-driven gene expression.

Heavy metal-induced transcriptional activation of the genes coding for metallothionein (MT) is mediated by a cis-acting DNA element, the metal-responsive element (MRE). MRE-binding transcription factor-1 (MTF-1) is a highly conserved heavy metal-induced transcriptional activator. MTF-1 also activates transcription in response to oxidative stress and regulates the expression of several cytoprotective factor genes, including MT, gamma-glutamylcysteine synthetase, and Cu/Zn-superoxide dismutase. It is thus thought that MTF-1 plays a role in cellular stress response. The physiological role of MTF-1 remains unclear because of the lack of MTF-1-specific activators and/or inhibitors. To obtain an MTF-1-specific inhibitor, we constructed an MTFDeltaC (amino acids 1-317), a C-terminal deletion mutant of MTF-1. MTFDeltaC could bind MRE and competed with MTF-1 for MTF-MRE complex formation. Transient expression of MTFDeltaC in HepG2 cells reduced MRE-driven gene expression, demonstrating that MTFDeltaC is dominant to MTF-1. HepG2 cells stably expressing MTFDeltaC showed increased susceptibility to the cytotoxic effects of tert-butyl hydroperoxide (tBH). Furthermore, we constructed Ad5MTFDeltaC, a recombinant adenovirus that expresses MTFDeltaC. Infection with the virus induced MTFDeltaC expression and increased susceptibility to the cytotoxic effects of tBH. These results indicate that MTF-1 participates in controlling the cellular redox state.

Structure and activity relationships of novel uracil derivatives as topical anti-inflammatory agents.

In order to create novel, topical anti-inflammatory compounds exhibiting more potent activities than lead compound CX-659S (1), we designed and synthesized various derivatives of 1 focusing on the uracil N(1)- and N(3)-substituents, and evaluated their anti-inflammatory activities via inhibition of the picryl chloride-induced contact hypersensitivity reaction (CHR) in mice. In the course of our structure and activity relationship study, we found that compounds 6k, 6q, and 6r inhibited by approximately 50% the CHR, at 0.1 mg/ear. These activities were essentially equipotent with that of Tacrolimus, a strong immunosuppressant.

Inhibitory activity of CX-659S, a novel diaminouracil derivative, against the rebound phenomenon following withdrawal of corticosteroid therapy for chronic contact hypersensitivity responses.

BACKGROUND: CX-659S, a newly discovered anti-inflammatory compound, exerts inhibitory effects on chronic contact hypersensitivity responses (CHRs) induced by repeated application with picryl chloride (PC), which is known to mimic many, if not all, events occurring within lesional skin of patients with atopic dermatitis (AD). CX-659S suppresses the expression of mRNA for interleukin (IL)-4 and IL-10 but not that for IFN-gamma, and inhibits serum IgE production in a chronic CHR model. Although topical corticosteroids have been widely utilized in steroid-responsive dermatoses such as AD, their chronic use may be associated with significant side effects. In addition, a rebound phenomenon often occurs after discontinuation of prolonged use of topical corticosteroids, with enhanced production of IgE and Th2 cell cytokines. The purpose of this study was to assess whether CX- 659S inhibits the rebound phenomenon after discontinuation of chronic treatment with prednisolone in a chronic CHR model in mice. METHODS: The efficacy of CX-659S as a sequential therapeutic agent after discontinuation of chronic treatment with prednisolone was tested on PC-treated ears of BALB/c mice with chronic CHR. Effects were quantified by measurements of ear thickness, serum IgE and cytokine mRNA expression. RESULTS: The rebound phenomenon was confirmed after discontinuation of chronic treatment with prednisolone in chronic CHR in mice, i.e. by evidence of flare thickening of the ear, enhanced expression of mRNA for IL-4 and IL-10 and increased serum IgE. Sequentially applied CX-659S suppressed these rebound phenomena with a good cosmetic result. CONCLUSIONS: CX-659S is the first promising compound with inhibitory activity on the rebound phenomenon following withdrawal of corticosteroid therapy without immunosuppression.

The combined effect of topical CX-659S, a novel diaminouracil derivative, with topical corticosteroid on the three types of allergic responses in mice or guinea pigs.

CX-659S ((S)-6-amino-5-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamido)-3-methyl-1-phenyl-2,4(1H,3H)-pyrimidinedione), a newly discovered anti-inflammatory compound, exerts inhibitory effects against picryl chloride-, oxazolone-, and dinitrochlorobenzene-induced acute contact hypersensitivity responses (CHRs) characterized by Th1-type reactions. Furthermore, this compound suppressed chronic CHRs characterized by Th2-type reactions, which is well known to mimic many, if not all, events occurring within the lesional skin of patients with atopic dermatitis (AD). The present study was conducted to determine the combined effect of topical CX-659S with topical corticosteroid on immediate type (ITR), late type (LTR), and delayed type hypersensitivity (DTHR) allergic reactions that are involved in AD. An ineffective dose of CX-659S (0.03 mg/ear) combined with betamethasone valerate (BV) significantly potentiated inhibitory activity of BV alone (0.1 micro g/ear and 0.3Shizuokag/ear) on both the ITR and the LTR in mice with the ovalbumin (OVA)-induced biphasic cutaneous reaction. Furthermore, the combined effect of CX-659S with BV was also observed on dinitrochlorobenzene (DNCB)-induced DTHR in guinea pigs. These results indicate that CX-659S has a combined effect with corticosteroids on every ITR, LTR, and DTHR. Proper treatment with corticosteroids for a safe and effective treatment of AD is needed. Thus, the combination therapy of topical CX-659S with topical corticosteroid would be one of the potential approaches for devising a proper treatment with corticosteroids.

Inhibitory activities of novel pyrimidine derivatives on the contact hypersensitivity reaction.

In order to obtain novel topically applied anti-inflammatory compounds containing an inexpensive anti-oxidative moiety without chirality, we synthesized compound 2c derivatives having a di-tert-butylphenol moiety, and evaluated by topical administration their anti-inflammatory potentials on picryl chloride-(PC) induced contact hypersensitivity reaction (CHR) in mice. In the course of our structure-activity relationship (SAR) studies on the pyrimidine or the anti-oxidative moiety and the linker between them, the most potent compounds (10, 11) were obtained by the insertion of a C2 unit in compound 2c. The potencies of these compounds were 2-fold greater than that of 1. Compounds 10 and 11 were considered to be useful lead compounds having inexpensive anti-oxidative moieties without chirality.