Kiyoshi Mizuguchi

Dienogest is a selective progesterone receptor agonist in transactivation analysis with potent oral endometrial activity due to its efficient pharmacokinetic profile.

Dienogest was introduced as an oral progestin. Yet its strong oral potency on endometrial activity is not clearly explained. To circumvent this situation, steroid hormone receptor profiling using transactivation assay and endometrial activity test in rabbits were carried out with determination of plasma drug concentration. Agonistic/antagonistic activity on human progesterone receptor (PR), androgen receptor (AR), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), estrogen receptor alpha (ERalpha), or estrogen receptor beta (ERbeta) were determined. Dienogest activate PR (EC50=3.4 or 10.5 nmol/l) with antagonistic activity on AR (EC50=420.6 or 775.0 nmol/l) but not agonistic nor antagonistic action on GR, MR (3000 nmol/l). Dienogest activate neither ERalpha nor ERbeta (3000 nmol/l). Progesterone activated PR with antagonistic activity on AR and on MR. Dydrogesterone showed a similar profile to progesterone. Norethisterone activated PR, AR, and ERalpha. Medroxyprogesterone acetate activated PR, AR, and GR. Danazol activated PR and AR. Collectively, dienogest has a good specificity to PR compared with the other drugs. By oral treatment, dienogest showed the strongest endometrial activity (ED50=0.0042 mg/kg) in McPhail test among other progestins (ED50 values for MPA, DYG, NES were 0.074, 1.9, >0.05 mg/kg, respectively). Dienogest showed higher plasma concentrations than those of the other progestins with higher doses. The estimated plasma concentration of dienogest at ED50 (3.66 nmol/l) was close to its EC50 value to activate PR. Thus, the stronger oral activity of dienogest could be explained simply by its in vitro potency on PR and its oral pharmacokinetic profile.

Antiobesity effect of eicosapentaenoic acid in high-fat/high-sucrose diet-induced obesity: importance of hepatic lipogenesis.

OBJECTIVE Given the pleiotropic effect of eicosapentaenoic acid (EPA), it is interesting to know whether EPA is capable of improving obesity. Here we examined the anti-obesity effect of EPA in mice with two distinct models of obesity. RESEARCH DESIGN AND METHODS Male C57BL/6J mice were fed a high-fat/high-sucrose diet (25.0% [w/w] fat, 32.5% [w/w] sucrose) (HF/HS group) or a high-fat diet (38.1% [w/w] fat, 8.5% [w/w] sucrose) (HF group) for 4–20 weeks. A total of 5% EPA was administered by partially substituting EPA for fat in the HF/HS + EPA and HF + EPA groups. RESULTS Both the HF/HS and HF groups similarly developed obesity. EPA treatment strongly suppresses body weight gain and obesity-related hyperglycemia and hyperinsulinemia in HF/HS-fed mice (HF/HS + EPA group), where hepatic triglyceride content and lipogenic enzymes are increased. There is no appreciable effect of EPA on body weight in HF-fed mice (HF + EPA group) without enhanced expression of hepatic lipogenic enzymes. Moreover, EPA is capable of reducing hepatic triglyceride secretion and changing VLDL fatty acid composition in the HF/HS group. By indirect calorimetry analysis, we also found that EPA is capable of increasing energy consumption in the HF/HS + EPA group. CONCLUSIONS This study is the first demonstration that the anti-obesity effect of EPA in HF/HS-induced obesity is associated with the suppression of hepatic lipogenesis and steatosis. Because the metabolic syndrome is often associated with hepatic lipogenesis and steatosis, the data suggest that EPA is suited for treatment of the metabolic syndrome.

fect of Eicosapentaenoic Acid in High-fat/High-sucrose Diet-induced Obesity: Importance of Hepatic Lipogenesis

OBJECTIVE Given the pleiotropic effect of eicosapentaenoic acid (EPA), it is interesting to know whether EPA is capable of improving obesity. Here we examined the anti-obesity effect of EPA in mice with two distinct models of obesity. RESEARCH DESIGN AND METHODS Male C57BL/6J mice were fed a high-fat/high-sucrose diet (25.0% [w/w] fat, 32.5% [w/w] sucrose) (HF/HS group) or a high-fat diet (38.1% [w/w] fat, 8.5% [w/w] sucrose) (HF group) for 4–20 weeks. A total of 5% EPA was administered by partially substituting EPA for fat in the HF/HS + EPA and HF + EPA groups. RESULTS Both the HF/HS and HF groups similarly developed obesity. EPA treatment strongly suppresses body weight gain and obesity-related hyperglycemia and hyperinsulinemia in HF/HS-fed mice (HF/HS + EPA group), where hepatic triglyceride content and lipogenic enzymes are increased. There is no appreciable effect of EPA on body weight in HF-fed mice (HF + EPA group) without enhanced expression of hepatic lipogenic enzymes. Moreover, EPA is capable of reducing hepatic triglyceride secretion and changing VLDL fatty acid composition in the HF/HS group. By indirect calorimetry analysis, we also found that EPA is capable of increasing energy consumption in the HF/HS + EPA group. CONCLUSIONS This study is the first demonstration that the anti-obesity effect of EPA in HF/HS-induced obesity is associated with the suppression of hepatic lipogenesis and steatosis. Because the metabolic syndrome is often associated with hepatic lipogenesis and steatosis, the data suggest that EPA is suited for treatment of the metabolic syndrome.

Highly purified eicosapentaenoic acid prevents the progression of hepatic steatosis by repressing monounsaturated fatty acid synthesis in high-fat/high-sucrose diet-fed mice.

Eicosapentaenoic acid (EPA) is a member of the family of n-3 polyunsaturated fatty acids (PUFAs) that are clinically used to treat hypertriglyceridemia. The triglyceride (TG) lowering effect is likely due to an alteration in lipid metabolism in the liver, but details have not been fully elucidated. To assess the effects of EPA on hepatic TG metabolism, mice were fed a high-fat and high-sucrose diet (HFHSD) for 2 weeks and were given highly purified EPA ethyl ester (EPA-E) daily by gavage. The HFHSD diet increased the hepatic TG content and the composition of monounsaturated fatty acids (MUFAs). EPA significantly suppressed the hepatic TG content that was increased by the HFHSD diet. EPA also altered the composition of fatty acids by lowering the MUFAs C16:1 and C18:1 and increasing n-3 PUFAs, including EPA and docosahexaenoic acid (DHA). Linear regression analysis revealed that hepatic TG content was significantly correlated with the ratios of C16:1/C16:0, C18:1/C18:0, and MUFA/n-3 PUFA, but was not correlated with the n-6/n-3 PUFA ratio. EPA also decreased the hepatic mRNA expression and nuclear protein level of sterol regulatory element binding protein-1c (SREBP-1c). This was reflected in the levels of lipogenic genes, such as acetyl-CoA carboxylase alpha (ACCalpha), fatty acid synthase, stearoyl-CoA desaturase 1 (SCD1), and glycerol-3-phosphate acyltransferase (GPAT), which are regulated by SREBP-1c. In conclusion, oral administration of EPA-E ameliorates hepatic fat accumulation by suppressing TG synthesis enzymes regulated by SREBP-1 and decreases hepatic MUFAs accumulation by SCD1.

Dienogest inhibits aromatase and cyclooxygenase-2 expression and prostaglandin E2 production in human endometriotic stromal cells in spheroid culture

OBJECTIVE To determine the effect of dienogest (DNG) on the expression of aromatase and cyclooxygenase-2 (COX-2) and the production of prostaglandin E(2) (PGE(2)) in human endometriotic stromal cells (ESCs). DESIGN Experimental study in vitro. SETTING University hospital. PATIENT(S) Seventeen patients with ovarian endometrioma. INTERVENTION(S) ESCs from chocolate cyst linings of ovaries were treated with DNG. MAIN OUTCOME MEASURE(S) Expression of aromatase and COX-2 evaluated in spheroid cultures of human ESCs by real-time quantitative polymerase chain-reaction and immunocytochemistry, production of PGE(2) quantified by enzyme-linked immunosorbent assay (ELISA), and nuclear factor kappa B (NF-κB) DNA-binding examined by ELISA and immunocytochemistry. RESULT(S) The pharmaceutical actions of DNG on the expression of aromatase and COX-2 and the production of PGE(2) were examined using spheroid cultures of human ESCs. More aromatase, COX-2, and PGE(2) were expressed in spheroid cultures than in conventional ESCs monolayers. In the spheroid cultures, DNG (10(-7) M) and progesterone (10(-7) M) inhibited the expression of aromatase, COX-2, and PGE(2). DNG also inhibited NF-κB DNA-binding activity and reduced the immunocytochemical protein expression of aromatase, COX-2, and NF-κB p50 nuclear localization. CONCLUSION(S) Because DNG inhibits aromatase and COX-2 expression as well as PGE(2) production in ESCs, these pharmacologic features might contribute to a therapeutic effect of DNG on endometriosis.

The protective effect of dietary eicosapentaenoic acid against impairment of spatial cognition learning ability in rats infused with amyloid β(1-40).

BACKGROUND Amyloid beta (Abeta) peptide (1-40) can cause cognitive impairment. EXPERIMENTAL DESIGN We investigated whether dietary preadministration of eicosapentaenoic acid (EPA) is conducive to cognition learning ability and whether it protects against the impairment of learning ability in rats infused with Abeta peptide (1-40) into the cerebral ventricle. RESULTS Dietary EPA administered to rats for 12 weeks before the infusion of Abeta into the rat brain significantly decreased the number of reference memory errors (RMEs) and working memory errors (WMEs), suggesting that chronic administration of EPA improves cognition learning ability in rats. EPA preadministered to the Abeta-infused rats significantly reduced the increase in the number of RMEs and WMEs, with concurrent proportional increases in the levels of corticohippocampal EPA and docosahexaenoic acid (DHA) and in the DHA/arachidonic acid molar ratio. Decrease in oxidative stress in these tissues was evaluated by determining the reactive oxygen species and lipid peroxide levels. cDNA microarray analysis revealed that altered genes included those that control synaptic signal transduction, cell communication, membrane-related vesicular transport functions, and enzymes and several other proteins. CONCLUSION The present study suggests that EPA, by acting as a precursor for DHA, ameliorates learning deficits associated with Alzheimers disease and that these effects are modulated by the expression of proteins involved in neuronal plasticity.

Effects of eicosapentaenoic acid on synaptic plasticity, fatty acid profile and phosphoinositide 3-kinase signaling in rat hippocampus and differentiated PC12 cells.

Placebo-controlled clinical studies suggest that intake of n-3 polyunsaturated fatty acids improves neurological disorders such as Alzheimers disease, Huntingtons disease and schizophrenia. To evaluate the impact of eicosapentaenoic acid (EPA), we orally administered highly purified ethyl EPA (EPA-E) to rats at a dose of 1.0 mg/g per day and measured long-term potentiation of the CA1 hippocampal region, a physiological correlate of synaptic plasticity that is thought to underlie learning and memory. The mean field excitatory postsynaptic potential slope of the EPA-E group was significantly greater than that of the control group in the CA1 region. Gene expression of hippocampal p85alpha, one of the regulatory subunits of phosphatidylinositol 3-kinase (PI3-kinase), was increased with EPA-E administration. Investigation of fatty acid profiles of neuronal and glia-enriched fractions demonstrated that a single administration of EPA-E significantly increased neuronal and glial EPA content and glial docosahexaenoic acid content, clearly suggesting that EPA was indeed taken up by both neurons and glial cells. In addition, we investigated the direct effects of EPA on the PI3-kinase/Akt pathway in differentiated PC12 cells. Phosphorylated-Akt expression was significantly increased in EPA-treated cells, and nerve growth factor withdrawal-induced increases in cell death and caspase-3 activity were suppressed by EPA treatment. These findings suggest that EPA protects against neurodegeneration by modulating synaptic plasticity and activating the PI3-kinase/Akt pathway, possibly by its own functional effects in neurons and glial cells and by its capacity to increase brain docosahexaenoic acid.

Mechanism of the lipid-lowering effect of ethyl all-cis-5,8,11,14,17-icosapentaenoate

The effect of highly purified ethyl all-cis-5,8,11,14,17-icosapentaenoate (EPA-E) on cholesterol metabolism in rats was examined to clarify the mechanism of its hypolipidemic action. Pretreatment with EPA-E reduced the increase in plasma radioactivity after oral administration of [14C]cholesterol. The conversion of [14C]3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to [14C]mevalonic acid was significantly inhibited in liver microsomes obtained from rats treated with EPA-E. There was an increase in free cholesterol and a marked rise in the eicosapentaenoic acid (EPA) content of phospholipids in these microsomes. EPA-E restored the suppression of biliary secretion induced by feeding a casein-rich diet to bile duct-cannulated rats. Furthermore, when serum lipoprotein (d < 1.210) from rats given EPA-E was i.v. injected into normal rats, a more rapid elimination of cholesterol was observed as compared to that in rats injected with lipoprotein from EPA-E-untreated rats. This rapid clearance was found in the lipoprotein fractions of d < 1.006 and 1.006 < d < 1.063. These findings suggest that EPA-E has an inhibitory effect on intestinal cholesterol absorption and hepatic cholesterol biosynthesis, and an enhancing effect on hepatic biliary secretion. EPA-E would also seem to cause modification of serum lipoproteins, whereby their clearance from the serum is increased.

Dienogest, a synthetic progestin, inhibits prostaglandin E2 production and aromatase expression by human endometrial epithelial cells in a spheroid culture system.

Prostaglandin E(2) (PGE(2)) is a major mediator in the pathophysiology, and pathogenesis of gynecological diseases associated with abnormal endometrial disease with proliferation and inflammation, such as endometriosis. In this study, we investigated the effect of dienogest, a selective progesterone receptor agonist, on PGE(2) production and the expression of aromatase, an estrogen synthase, in human immortalized endometrial epithelial cells. Compared with monolayer culture, the cells showed enhanced PGE(2) production and expression of the PGE(2) synthases cyclooxygenase-2 (COX-2), and microsomal prostaglandin E(2) synthase-1 (mPGES-1) in a spheroid culture system. Dienogest inhibited PGE(2) production and this effect was reversed by RU486, a progesterone receptor antagonist. Dienogest inhibited the PGE(2) synthases mRNA and protein expression, and the nuclear factor-κB activation. Moreover, the suppressive effect of dienogest on PGE(2) production was sustained 24h after the drug was withdrawn. Dienogest but not COX inhibitors inhibited aromatase expression. These results suggest that progesterone receptor activation reduces the gene expressions of COX-2, mPGES-1, and aromatase. Our findings suggest that the pharmacological mechanism of dienogest includes the direct inhibition of PGE(2) synthase and aromatase expression and may contribute to the therapeutic effect on the progression of endometriosis.

Effects of ethyl-all-cis-5,8,11,14,17-icosapentaenoate (EPA-E), pravastatin and their combination on serum lipids and intimal thickening of cuff-sheathed carotid artery in rabbits

The anti-arteriosclerotic effects of ethyl all-cis-5, 8, 11, 14, 17-icosapentaenoate (EPA-E), pravastatin and their combination in cuff-treated rabbits were investigated. EPA-E at 600 mg/kg, pravastatin at 50 mg/kg or their combination was orally administered once daily for 5 weeks, and each of the animals was sheathed with a cuff on the carotid artery 2 weeks after the beginning of drug administration. EPA-E, pravastatin and their combination significantly reduced serum total cholesterol compared to the control group. EPA-E also potently reduced serum triglyceride, while pravastatin only slightly reduced it. The combination of these two agents had the most potent effect on the level of serum triglyceride. Serum phospholipids were also reduced by these treatments in a similar fashion. At the end of treatment, diffuse intimal thickening was observed in the cuff-covered region in all animals in the control group, and the intima/media area ratio in this group was 0.293 +/- 0.038. Treatment with EPA-E alone tended to prevent the intimal thickening, and the intima/media area ratio was 0.209 +/- 0.058 (p = 0.094). This ratio was 0.287 +/- 0.048 (p = 0.902) when pravastatin was administered alone, indicating that it had no significant effect on intimal thickening. The ratio was 0.175 +/- 0.041 (p = 0.042) when both EPA-E and pravastatin were administered, indicating that this combination had a significant inhibitory effect on intimal thickening in the cuff-sheathed region. These findings suggest that combined treatment with EPA-E and pravastatin is more effective than respective monotherapies in lowering serum lipids and/or preventing an intimal thickening as events of atherogenesis.