Expression of a skin cholesterol sulfotransferase,St2b2, is a trigger of epidermal cell differentiation

1.?St2b2, a mouse cytosolic sulfotransferase, predominantly catalyses epidermal cholesterol sulfation. St2b2 was found in the basement layer by immunohistochemical analysis of normal mouse skin. The highest expression level was detected in epidermis from 3-day-old mice and then decreased before maturation. There was a good correlation between expression levels of skin St2b2 and a differentiation marker, involucrin.

2.?To understand the role of St2b2 in epidermal cell differentiation, recombinant St2b2 was expressed in primary epidermal cells. The expression of St2b2 enhanced the involucrin expression with an increase of cholesterol sulfate. Furthermore, by down-regulation of the St2b2 gene expression, involucrin was decreased in dorsal skin of 1–3-day-old mice by 67% of the control.

3.?These results strongly suggest a possibility that St2b2 expression plays a trigger of epidermal cell differentiation by controlling cholesterol sulfate level in the cells.     

2-Furoyl-LIGRL-NH2, a potent agonist for proteinase-activated receptor-2, as a gastric mucosal cytoprotective agent in mice

1. Proteinase-activated receptor-2 (PAR(2)), expressed in capsaicin-sensitive sensory neurons, plays a protective role in gastric mucosa. The present study evaluated gastric mucosal cytoprotective effect of 2-furoyl-LIGRL-NH(2), a novel highly potent PAR(2) agonist, in ddY mice and in wild-type and PAR(2)-knockout mice of C57BL/6 background. 2. Gastric mucosal injury was created by oral administration of HCl/ethanol solution in the mice. The native PAR(2)-activating peptide SLIGRL-NH(2), administered intraperitoneally (i.p.) at 0.3-1 micromol kg(-1) in combination with amastatin, an aminopeptidase inhibitor, but not alone, revealed gastric mucosal protection in ddY mice, which was abolished by ablation of capsaicin-sensitive sensory neurons. 3. I.p. administration of 2-furoyl-LIGRL-NH(2) at 0.1 micromol kg(-1), without combined treatment with amastatin, exhibited gastric mucosal cytoprotective activity in ddY mice, the potency being much greater than SLIGRL-NH(2) in combination with amastatin. This effect was also inhibited by capsaicin pretreatment. 4. Oral administration of 2-furoyl-LIGRL-NH(2) at 0.003-0.03 micromol kg(-1) also protected against gastric mucosal lesion in a capsaicin-reversible manner in ddY mice. 5. I.p. 2-furoyl-LIGRL-NH(2) at 0.1-0.3 micromol kg(-1) caused prompt salivation in anesthetized mice, whereas its oral administration at 0.003-1 micromol kg(-1) was incapable of eliciting salivation. 6. In wild-type, but not PAR(2)-knockout, mice of C57BL/6 background, i.p. administration of 2-furoyl-LIGRL-NH(2) caused gastric mucosal protection. 7. Thus, 2-furoyl-LIGRL-NH(2) is considered a potent and orally available gastric mucosal protective agent. Our data also substantiate a role for PAR(2) in gastric mucosal protection and the selective nature of 2-furoyl-LIGRL-NH(2).     

Expression of a skin cholesterol sulfotransferase, St2b2, is a trigger of epidermal cell differentiation

1. St2b2, a mouse cytosolic sulfotransferase, predominantly catalyses epidermal cholesterol sulfation. St2b2 was found in the basement layer by immunohistochemical analysis of normal mouse skin. The highest expression level was detected in epidermis from 3-day-old mice and then decreased before maturation. There was a good correlation between expression levels of skin St2b2 and a differentiation marker, involucrin. 2. To understand the role of St2b2 in epidermal cell differentiation, recombinant St2b2 was expressed in primary epidermal cells. The expression of St2b2 enhanced the involucrin expression with an increase of cholesterol sulfate. Furthermore, by down-regulation of the St2b2 gene expression, involucrin was decreased in dorsal skin of 1-3-day-old mice by 67% of the control. 3. These results strongly suggest a possibility that St2b2 expression plays a trigger of epidermal cell differentiation by controlling cholesterol sulfate level in the cells.     

EZH2抑制剂GSK126对Neuro-2a细胞增殖分化的影响及其可能机制

<正>神经母细胞瘤(neuroblastoma,NB)是常见的儿童恶性肿瘤,在手术、放化疗及介入等多种手段综合治疗下,总体疗效仍然极差。近年来研究发现,果蝇zeste基因增强子同源物2(enhancer of zeste homolog 2,EZH2)能抑制相关抑癌基因,明显增加神经母细胞瘤发生、发展的风险~([1])。GSK126是一种新型的选择性EZH2抑制剂~([2-3]),其在实体瘤方面的药效探究目前相对较少。本文将对GSK126在神经母细胞瘤     

COX-2抑制剂戊地昔布的合成

目的:合成COX-2抑制剂戊地昔布.方法:以苯乙酸为起始原料,经酰氯化、傅克酰化生成二苯乙酮后,与盐酸羟胺反应生成肟,再环合,磺化,氨化得戊地昔布.结果:得到的产品经IR,1H-NMR,MS和元素分析,证明是戊地昔布.结论:本合成路线简化了操作步骤,缩短反应时间,提高了收率.     

Design, synthesis, and antiviral activity of 2'-deoxy-2'-fluoro-2'-C-methylcytidine, a potent inhibitor of hepatitis C virus replication

The pyrimidine nucleoside beta-d-2'-deoxy-2'-fluoro-2'-C-methylcytidine (1) was designed as a hepatitis C virus RNA-dependent RNA polymerase (HCV RdRp) inhibitor. The title compound was obtained by a DAST fluorination of N(4)-benzoyl-1-(2-methyl-3,5-di-O-benzoyl-beta-d-arabinofuranosyl]cytosine to provide N(4)-benzoyl-1-[2-fluoro-2-methyl-3,5-di-O-benzoyl-beta-d-ribofuranosyl]cytosine. The protected 2'-C-methylcytidine was obtained as a byproduct from the DAST fluorination and allowed for the preparation of two biologically active compounds from a common precursor. Compound 1 and 2'-C-methylcytidine were assayed in a subgenomic HCV replicon assay system and found to be potent and selective inhibitors of HCV replication. Compound 1 shows increased inhibitory activity in the HCV replicon assay compared to 2'-C-methylcytidine and low cellular toxicity.     

Characterization of etoricoxib,a novel,selective COX-2 inhibitor

Etoricoxib is a potent selective COX-2 inhibitor in man. Ex vivo whole-blood assays assessed COX-2 inhibition after oral administration of etoricoxib in single (5-500 mg) and multiple (25-150 mg) once-daily doses to healthy human subjects. A separate study examined ex vivo gastric mucosal PGE2 synthesis after etoricoxib (120 mg qd), naproxen (500 mg bid), or placebo for 5 days. The effect of etoricoxib 120 mg qd on the COX-1-mediated antiplatelet effects of low-dose aspirin (ASA) was also assessed. The mean (time)-weighted average inhibition (WAI) of lipopolysaccharide (LPS)-stimulated PGE2 (COX-2 assay) vcrsus placebo was dose related after single (range: 3.1%-99.1%) and multiple doses (range: 52.5%-96.7%). PGE2 remained significantly inhibited 24 hours postdose at steady state. Inhibition of LPS-stimulated PGE2 showed a strong relationship with etoricoxib plasma concentrations; ex vivo, IC50 was almost identical to in vitro. Multiple dosing of etoricoxib (up to 150 mg qd) showed no important effects on serum TXB2, bleeding time, or platelet aggregation (COX-1-mediated effects). The nonselective nonsteroidal anti-inflammatory (NSAID) naproxen significantly inhibited (approximately 78%) ex vivo prostaglandin synthesis in gastric mucosa; etoricoxib had no effect. Etoricoxib did not interfere with the antiplatelet effects of low-dose ASA, as assessed by serum TXB2 and platelet aggregation. Etoricoxib was generally well tolerated, even at doses above the clinical dose range. Based on these results, etoricoxib is a potent selective inhibitor of COX-2 after single and multiple dosing regimens and does not inhibit prostaglandin synthesis in the gastric mucosa, even at doses above the clinical dose range of 60 to 120 mg.     

2-Methoxyestradiol,a promising anticancer agent

Estrogens occurring naturally in the body are metabolized to catecholestrogens (2- and 4-hydroxyestradiol) by the cytochrome P450 enzymes. 2-Hydroxy catecholestrogens are further metabolized by catechol-O-methyltransferase to 2-methoxyestradiol, which is known to be protective against tumor formation. 2-Methoxyestradiol exhibits potent apoptotic activity against rapidly growing tumor cells. It also possesses antiangiogenic activity through a direct apoptotic effect on endothelial cells. Other molecular mechanisms, including microtubule stabilization by inhibition of the colchicine-binding site, have been reported. The exact mechanism of action of 2-methoxyestradiol is still unclear, but it has been shown to be effective in preventing tumor growth in a variety of cell lines. 2-Methoxyestradiol also possesses cardioprotective activity by inhibiting vascular smooth muscle cell growth in arteries. It has a lower binding affinity for estrogen receptor alpha compared with that of estradiol, and its affinity for estrogen receptor beta is even lower than that of estrogen receptor alpha, thus it has minimal estrogenic activity. 2-Methoxyestradiol is distinct because of its inability to engage estrogen receptors as an agonist, and its unique antiproliferative and apoptotic activities are mediated independently of estrogen receptors alpha and beta. A phase I clinical trial of 2-methoxyestradiol 200, 400, 600, 800, and 1,000 mg/day in 15 patients with breast cancer showed significant reduction in bone pain and analgesic intake in some patients, with no significant adverse effects. Another phase I study of 2-methoxyestradiol 200-1,000 mg/day in combination with docetaxel 35 mg/m2/week for 4-6 weeks performed in 15 patients with advanced refractory metastatic breast cancer showed no serious drug-related adverse effects. A phase II randomized, double-blind trial of 2-methoxyestradiol 400 and 1,200 mg/day in 33 patients with hormone-refractory prostate cancer showed that it was well tolerated and showed prostate specific antigen stabilizations and declines. We have started a phase I clinical trial to explore dosages greater than 1,000 mg/day.     

Pharmacokinetic assessment of a five-probe cocktail for CYPs 1A2, 2C9, 2C19, 2D6 and 3A

AIMS

To assess the pharmacokinetics (PK) of selective substrates of CYP1A2 (caffeine), CYP2C9 (S-warfarin), CYP2C19 (omeprazole), CYP2D6 (metoprolol) and CYP3A (midazolam) when administered orally and concurrently as a cocktail relative to the drugs administered alone.

METHODS

This was an open-label, single-dose, randomized, six-treatment six-period six-sequence William''s design study with a wash-out of 7 or 14 days. Thirty healthy male subjects received 100 mg caffeine, 100 mg metoprolol, 0.03 mg kg⁻¹ midazolam, 20 mg omeprazole and 10 mg warfarin individually and in combination (cocktail). Poor metabolizers of CYP2C9, 2C19 and 2D6 were excluded. Plasma samples were obtained up to 48 h for caffeine, metoprolol and omeprazole, 12 h for midazolam, 312 h for warfarin and the cocktail. Three different validated liquid chromatography tandem mass spectrometry methods were used. Noncompartmental PK parameters were calculated. Log-transformed C_max, AUC_last and AUC for each analyte were analysed with a linear mixed effects model with fixed term for treatment, sequence and period, and random term for subject within sequence. Point estimates (90% CI) for treatment ratios (individual/cocktail) were computed for each analyte C_max, AUC_last and AUC.

RESULTS

There was no PK interaction between the probe drugs when administered in combination as a cocktail, relative to the probes administered alone, as the 90% CI of the PK parameters was within the prespecified bioequivalence limits of 0.80, 1.25.

CONCLUSION

The lack of interaction between probes indicates that this cocktail could be used to evaluate the potential for multiple drug–drug interactions in vivo.     

Pharmacokinetics of T-2 tetraol, a urinary metabolite of the trichothecene mycotoxin, T-2 toxin, in dog

1. The urinary metabolites of T-2 toxin were identified and analysed quantitatively after i.v. administration to dogs. 2. A new routine assay for T-2 tetraol was developed and a pharmacokinetic study was carried out on this final hydrolytic metabolite of T-2 toxin. T-2 tetraol was excreted in urine for 2-3 days. Its 'sigma minus' plot demonstrated a significantly longer apparent half-life than its precursors (T-2 toxin and HT-2 toxin). This fact was explained by extraplasma binding causing prolongation of the metabolism and excretion of T-2 toxin metabolites. 3. The urinary metabolites of T-2 toxin were: HT-2 toxin, T-2 triol and T-2 tetraol. The metabolites were excreted in free and conjugated forms. In two dogs T-2 toxin was found in the urine in an amount which accounts for 3.2 and 16% of the administered dose respectively. The cumulative amount of the identified metabolites and toxins formed in the urine ranged from 9.7 to 17.3% in four dogs and 44.7% in one dog.     

Pharmacokinetics of T-2 tetraol,a urinary metabolite of the trichothecene mycotoxin,T-2 toxin,in dog

1. The urinary metabolites of T-2 toxin were identified and analysed quantitatively after i.v. administration to dogs.

2. A new routine assay for T-2 tetraol was developed and a pharmacokinetic study was carried out on this final hydrolytic metabolite of T-2 toxin. T-2 tetraol was excreted in urine for 2–3 days. Its ‘sigma minus' plot demonstrated a significantly longer apparent half-life than its precursors (T-2 toxin and HT-2 toxin). This fact was explained by extraplasma binding causing prolongation of the metabolism and excretion of T-2 toxin metabolites.

3. The urinary metabolites of T-2 toxin were: HT-2 toxin, T-2 triol and T-2 tetraol. The metabolites were excreted in free and conjugated forms. In two dogs T-2 toxin was found in the urine in an amount which accounts for 3.2 and 16% of the administered dose respectively. The cumulative amount of the identified metabolites and toxins formed in the urine ranged from 9.7 to 17.3% in four dogs and 44.7% in one dog.     

pD2-, pA2- and pD2'-values of a series of compounds in a histaminic and a cholinergic system.

(1) Affinity and intrinsic activity values of 75 compounds for a histaminergic and a cholinergic system are presented. The quantitative correlations between the affinity values of 35 compounds and some physicochemical constants (Van der Waals volume, lipophilicity, number of hydrogen atoms on the protonated amine) are discussed. (2) Absence of systematic differences between pD2 and pA2 of partial agonists supports the assumption that these values are equivalent expressions of the same affinity. (3) The "mimetic moiety" in a number of the antihistaminic test compounds hardly contributes to their affinity. The affinity mainly depends on an interaction tendency with additional receptor areas. (4) The correlation between pA2 and pD2' of the whole series of compounds in the histaminergic system is artificial. The method only allows determination of both values if their ratio lies between certain limits. (5) The correlation between pA2 and pD2' for 16 closely related compounds in the guinea pig ileum and for nearly all compounds in the rat intestine has to be explained by an influence of the structural differences on drug transference and/or the less specific binding forces. (6) The metactoid receptors in the two systems are different structures. (7) Possible molecular modifications to maximize the separation of antihistaminic form cholinergic affinity are suggested.     

2-Arachidonoylglycerol, a candidate of endothelium-derived hyperpolarizing factor

We investigated whether 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, is involved in acetylcholine- and calcium ionophore A23187-induced relaxations in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME) and indomethacin, which is considered to be mediated by endothelium-derived hyperpolarizing factor (EDHF). In rabbit mesenteric arterial rings pre-constricted with noradrenaline, 2-arachidonoylglycerol caused concentration-dependent relaxation. The 2-arachidonoylglycerol-induced relaxations were not affected by endothelium removal. N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-caroxamide (SR141716A) and 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morholinyl-1H-pyrazole-3-carboxamide (AM281), cannabinoid CB(1) receptor antagonists, significantly attenuated 2-arachidonoylglycerol-induced relaxation and the acetylcholine-induced relaxation only slightly, but not the calcium ionophore A23187-induced relaxation. On the other hand, charybdotoxin plus apamin, K(+) channel blockers, significantly attenuated acetylcholine and calcium ionohore A23187-induced relaxations but not 2-arachidonoylglycerol-induced relaxations. These results suggest that 2-arachidonoylglycerol can cause relaxations via cannabinoid CB(1) receptors, but is not involved in EDHF-mediated relaxations.     

Synthesis of a 2'-deoxyguanosine adduct of cis-2-butene-1,4-dial, a reactive metabolite of furan

Furan is a liver and kidney toxicant and a hepatocarcinogen in rodents. Its reactive metabolite, cis-2-butene-1,4-dial, reacts with nucleosides to form adducts in vitro. The reaction with 2'-deoxyguanosine generates 3-(2'-deoxy-beta-D-erythropentafuranosyl)-3,5,6,7-tetrahydro-6-hydroxy-7-(ethane-2"-al)-9H-imidazo[1,2-alpha]purine-9-one as the major reaction product. A synthetic approach to this adduct is presented in this report. The key step in this synthesis is the preparation of 2'-deoxy-3',5'-O-bis(tert-butyldimethylsilyl)-1-(1,2,5,6-tetrahydroxyhexan-3-yl)guanosine. Treatment of this intermediate with sodium periodate gave three reaction products: a one-substituted adduct, 2'-deoxy-3',5'-O-bis(tert-butyldimethylsilyl)-1-(2,5-dihydroxy-tetrahydrofuran-3-yl)guanosine; a 1,N(2)-cyclic adduct, 3-[2'-deoxy-3',5'-O-bis(tert-butyldimethylsilyl)-beta-D-erythropentafuranosyl]-6-hydroxy-8-formyl-5,6,7,8-tetrahydropyrimidino[1,2-alpha]purin-10(3H)-one; and the 1,N(2)-bicyclic adduct, 3-[2'-deoxy-3',5'-O-bis(tert-butyldimethylsilyl)-beta-D-erythropentafuranosyl]-3,5,6,7-tetrahydro-6-hydroxy-7-(ethane-2"-al)-9H-imidazo[1,2-alpha]purine-9-one. The one-substituted and 1,N(2)-cyclic reaction products were unstable and rearranged over time to yield the 1,N(2)-bicyclic 2'-deoxyguanosine adducts. The desired reaction product was obtained as a mixture of four diastereomers by removing the tert-butyldimethylsilyl groups with hydrogen fluoride. This synthetic approach to the cis-2-butene-1,4-dial-derived dGuo adducts confirms our previous structural characterization of the in vitro cis-2-butene-1,4-dial-dGuo reaction product. These studies demonstrate that the observed 1,N(2) bicyclic structure is the thermodynamically stable isomer, supporting our previous observations that this adduct is the major product formed in vitro. Finally, these studies provide the necessary groundwork for the preparation of oligonucleotides with site specifically incorporated cis-2-butene-1,4-dial-derived adducts.     

Zuclopenthixol, a combined dopamine D1/D2 antagonist, versus haloperidol, a dopamine D2 antagonist, in tardive dyskinesia.

Animal data suggest that a D₁ antagonistic component in neuroleptic drugs counteracts development of dopamine supersensitivity and of tolerance to cataleptic effect. This has led to the hypothesis that neuroleptics with D₁ antagonistic activity should cause a better suppression of tardive dyskinesia (TD) and less rebound aggravation after withdrawal than pure D₂ antagonists. In this study the effect of zuclopenthixol (mixed D₁/D₂ antagonist) and haloperidol (D₂ antagonist) was evaluated in chronic psychotic patients with TD. Fifteen patients completed a randomized crossover study with blind evaluation of TD and parkinsonism. The test medications, haloperidol and zuclopenthixol, caused a significant suppression of TD and a significant increase of parkinsonism. No significant differences between haloperidol and zuclopenthixol were observed. No TD aggravation was seen. The lack of differences between the mixed D₁/D₂ antagonist and a D₂ antagonist suggest that tolerance and DA supersensitivity play no or a minor role for development of TD.     

2'-Fluoro-2',3'-dideoxyarabinosyladenine: a metabolically stable analogue of the antiretroviral agent 2',3'-dideoxyadenosine

In this report, we have compared the uptake, metabolism, and relevant enzymology of a novel anti-acquired immunodeficiency syndrome drug, 2'-fluoro-2',3'-dideoxyarabinosyladenine (2'-F-dd-ara-A) with the corresponding properties of its parent compound 2',3'-dideoxyadenosine (2',3'-ddAdo) in three human T cell lines, MOLT-4, ATH8, and CEM. In previous communications, we have reported that the primary route of metabolism of 2',3'-ddAdo in human T lymphoblasts is catabolic, i.e., deamination to 2',3'-dideoxyinosine (2',3'-ddlno). At this point, the metabolic pathway diverges, to result in either cleavage and inactivation of 2',3'-ddlno by purine nucleoside phosphorylase or in 5'-phosphorylation by a phosphotransferase, a reaction that generates 2',3'-inosine monophosphate and ultimately the putative active metabolite 2',3'-dideoxy-ATP. Studies with kinase-deficient mutant CEM lines indicate, however, that 2'-F-dd-ara-A favors a more direct anabolic route toward formation of 2'-fluoro-dideoxynucleotides, catalyzed initially by 2'-deoxycytidine kinase. In MOLT-4 cells, amounts of 2'-fluoro-dideoxyarabinosyladenine di- and triphosphate formed were approximately 20-fold and 5-fold greater than the respective accumulation of 2',3'-dideoxy-ADP and 2',3'-dideoxy-ATP over the same time of exposure. This metabolic profile was supported by enzymological studies, which revealed that 2'-F-dd-ara-A is deaminated 10 times less rapidly than ddAdo and that the resulting deaminated product is resistant to hydrolysis by purine nucleoside phosphorylase. Under similar conditions, ddAdo was rapidly degraded through cleavage of its deamination product ddlno. Like ddAdo, 2'-F-dd-ara-A was found to be transported by passive diffusion and does not enter cells via the purine nucleoside transport carrier system. However, the rate of entry of 2'-F-dd-ara-A was about half that of ddAdo (9.7 pmol/10(6) cells/min for 2'-F-dd-ara-A versus 18.4 pmol/10(6) cells/min for ddAdo). This investigation, therefore, demonstrates that, under the conditions studied, 2'-F-dd-ara-A and its deamination product 2'-fluoro-2',3'-dideoxyarabinosylhypoxanthine have metabolic properties that differ significantly from those of their parent compounds ddAdo and ddlno. These properties, combined with the previously reported resistance of the fluorinated nucleosides to acid degradation, make these compounds interesting candidates for further study as orally administered agents for the inhibition of human immunodeficiency virus replication in patients with acquired immunodeficiency syndrome.     

抗肿瘤新生血管形成作用的靶点Bcl-2蛋白

肿瘤的生长、转移依赖新生血管形成 ,以新生血管形成各环节为靶标的化合物在过去 10年中成为研发新型抗肿瘤药物的热点。本文从Bcl 2蛋白家族的生物学作用、与肿瘤新生血管形成的关系及抑制Bcl 2蛋白后的药理学作用等方面综述了Bcl 2作为抗肿瘤新生血管形成的新靶点肿瘤的生长、转移依赖新生血管形成 ,以新生血管形成各环节为靶标的化合物在过去 10年中成为研发新型抗肿瘤药物的热点。本文从Bcl 2蛋白家族的生物学作用、与肿瘤新生血管形成的关系及抑制Bcl 2蛋白后的药理学作用等方面综述了Bcl 2作为抗肿瘤新生血管形成的新靶点