Kazunori Imada

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.

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.

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.

Distinct regulation of plasma LDL cholesterol by eicosapentaenoic acid and docosahexaenoic acid in high fat diet-fed hamsters: Participation of cholesterol ester transfer protein and LDL receptor

Despite established anti-atherogenic action, previous reports have shown that fish oils or n-3 poly-unsaturated fatty acid (PUFA) increase plasma LDL-C in animals and humans. However, which component of n-3 PUFAs and what mechanisms contribute to this increase are unclear. We investigated the effects of the major components of n-3 PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on plasma LDL-C in high fat diet-fed hamsters. While LDL-C increased significantly with n-3 PUFA oil and DHA, EPA had no effect on LDL-C. Interestingly, a positive correlation was found between plasma cholesterol ester transfer protein (CETP) activity and LDL-C. Only DHA increased plasma CETP activity and significantly decreased LDL receptor expression in the liver. Our data suggest that DHA, not EPA, is a major factor in the LDL-C increasing effect of n-3 PUFA oil. These differential effects on LDL-C may arise from differences in plasma CETP activity and LDL receptor expression.

Palmitic Acid Induces Osteoblastic Differentiation in Vascular Smooth Muscle Cells through ACSL3 and NF-κB, Novel Targets of Eicosapentaenoic Acid

Free fatty acids (FFAs), elevated in metabolic syndrome and diabetes, play a crucial role in the development of atherosclerotic cardiovascular disease, and eicosapentaenoic acid (EPA) counteracts many aspects of FFA-induced vascular pathology. Although vascular calcification is invariably associated with atherosclerosis, the mechanisms involved are not completely elucidated. In this study, we tested the hypothesis that EPA prevents the osteoblastic differentiation and mineralization of vascular smooth muscle cells (VSMC) induced by palmitic acid (PA), the most abundant long-chain saturated fatty acid in plasma. PA increased and EPA abolished the expression of the genes for bone-related proteins, including bone morphogenetic protein (BMP)-2, Msx2 and osteopontin in human aortic smooth muscle cells (HASMC). Among the long-chain acyl-CoA synthetase (ACSL) subfamily, ACSL3 expression was predominant in HASMC, and PA robustly increased and EPA efficiently inhibited ACSL3 expression. Importantly, PA-induced osteoblastic differentiation was mediated, at least in part, by ACSL3 activation because acyl-CoA synthetase (ACS) inhibitor or siRNA targeted to ACSL3 completely prevented the PA induction of both BMP-2 and Msx2. Conversely, adenovirus-mediated ACSL3 overexpression enhanced PA-induced BMP-2 and Msx2 expression. In addition, EPA, ACSL3 siRNA and ACS inhibitor attenuated calcium deposition and caspase activation induced by PA. Notably, PA induced activation of NF-κB, and NF-κB inhibitor prevented PA-induction of osteoblastic gene expression and calcium deposition. Immunohistochemistry revealed the prominent expression of ACSL3 in VSMC and macrophages in human non-calcifying and calcifying atherosclerotic plaques from the carotid arteries. These results identify ACSL3 and NF-κB as mediators of PA-induced osteoblastic differentiation and calcium deposition in VSMC and suggest that EPA prevents vascular calcification by inhibiting such a new molecular pathway elicited by PA.

Eicosapentaenoic acid inhibits interleukin-6 production in interleukin-1β-stimulated C6 glioma cells through peroxisome proliferator-activated receptor-gamma

Epidemiological studies suggest that intake of omega-3 polyunsaturated fatty acids improves neurological disorders such as Alzheimers disease which exhibit inflammatory pathology. We therefore investigated the anti-inflammatory effects of eicosapentaenoic acid (EPA) on interleukin (IL)-1beta-stimulated C6 glioma cells. In the present study, EPA inhibited pro-inflammatory cytokine IL-6 production, a characteristic of certain neurodegenerative disorders, in IL-1beta-stimulated C6 glioma cells in dose-dependent fashion. EPA down-regulated the expression of IL-6 at mRNA level, indicating that the effect of EPA occurs at the transcriptional level. In addition, peroxisome proliferator-activated receptor (PPAR) gamma antagonists abolished the inhibitory effect of EPA on IL-1beta-induced IL-6 production, whereas PPARalpha antagonist did not block the inhibitory effect of EPA. EPA might thus contribute to the regulation of pro-inflammatory cytokine production in astrocytes through interaction with PPARgamma. Among the PPARgamma ligands tested in this study, ciglitazone, a synthetic agonist of PPARgamma, effectively inhibited IL-6 production, but while neither rosiglitazone nor 15-deoxy-Delta(12,14)-prostaglandin J2 did. These findings indicate that the coordination of PPAR gamma ligands is important in inhibiting the production of IL-6 in C6 glioma cells.

Dienogest, a selective progestin, reduces plasma estradiol level through induction of apoptosis of granulosa cells in the ovarian dominant follicle without follicle-stimulating hormone suppression in monkeys

Dienogest is a selective progestin that has been shown to arrest ovarian follicular development in women, without affecting gonadotropin secretion. As luteal progesterone or exogeneous progestins are known to suppress ovarian folliculogenesis via the inhibition of gonadotropin secretion, this action of dienogest on ovaries seems to be unique. To examine the underlying mechanism of the antifolliculogenic effect of dienogest, female cynomolgus monkeys were treated with a single oral dose of 0.1 mg/kg dienogest on day 7 of the menstrual cycle. Plasma FSH, estradiol (E2), and progesterone levels were measured up to 15 days after dosing. In an additional experiment, ovaries were excised 24 h after dosing for histological examinations. As a result, plasma E2 level declined within 24 h after dosing, while dienogest did not decreased FSH level prior to E2 decline. After decline of E2 level, the low level of E2 was sustained for more than 11 days. It is considered that a single oral dose of dienogest induced atresia of the dominant follicle. In the histological examination, two out of three animals showed decline in E2 level. The ovarian dominant follicles from these animals showed apoptotic changes in granulosa cells with scattered aromatase expression within 24 h after dosing. These results indicate that the induction of atresia of the ovarian dominant follicle by direct action would be a possible mechanism of dienogest to inhibit plasma E2 level.

Dienogest inhibits nerve growth factor expression induced by tumor necrosis factor-α or interleukin-1β

OBJECTIVE Dienogest (DNG), a selective P receptor (PR) agonist, is used to treat endometriosis. To investigate whether DNG affects nerve growth factor (NGF) expression, we stimulated human endometrial epithelial cells (hEECs) with inflammatory cytokines. DESIGN Prospective basic research study using immortalized hEEC lines. SETTING Development Research, Mochida Pharmaceutical Co., Ltd., Japan. PATIENT(S) None. INTERVENTION(S) Not applicable. MAIN OUTCOME MEASURE(S) In immortalized hEECs, NGF production induced by tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β) was evaluated in the presence or absence of the synthetic progestin DNG or endogenous P. The NGF messenger RNA (mRNA) and protein were measured using real-time reverse transcriptase-polymerase chain reaction (PCR) and ELISA, respectively. The NGF bioactivity in the culture medium was measured by assaying neurite outgrowth of PC-12 cells. RESULT(S) Tumor necrosis factor-α and IL-1β induced NGF mRNA and protein and increased NGF bioactivity in the culture medium. These activities were inhibited by DNG in a hEEC line that stably expresses PR. In contrast, in an hEEC line that constitutively expresses faint levels of PR, no inhibitory effect of DNG on NGF mRNA was detected. The NGF mRNA was also inhibited in hEEC lines that express only PR-A or only PR-B. CONCLUSION(S) Nerve growth factor is one of the key mediators that generates the pain associated with endometriosis. Dienogest inhibits NGF expression through PR-A and PR-B in hEEC, which may contribute to the pharmacological mechanisms of how DNG relieves pain in endometriosis.

Eicosapentaenoic acid suppresses palmitate-induced cytokine production by modulating long-chain acyl-CoA synthetase 1 expression in human THP-1 macrophages

BACKGROUND Chronic inflammation caused by macrophages may be associated with progression of arteriosclerosis or obesity, both risk factors for cardiovascular events. In the Japan EPA Lipid Intervention Study (JELIS), eicosapentaenoic acid (EPA), an n-3 polyunsaturated fatty acid, was found to reduce the incidence of cardiovascular events. METHODS The effect of EPA on the expression of inflammatory factors induced by palmitate, a saturated fatty acid, was investigated using human THP-1 macrophages. RESULTS Palmitate induced expression of inflammatory cytokines and activated NF-κB, similar to lipopolysaccharide (LPS). EPA strongly suppressed palmitate-induced up-regulation of inflammatory factors while slightly suppressing LPS-induced factors. Both palmitate and LPS up-regulated expression of long-chain acyl-CoA synthetase (ACSL) 1, while EPA preferentially suppressed palmitate-induced ACSL1 expression. Although an acyl-CoA synthetase inhibitor and ACSL1 siRNA both suppressed palmitate-induced tumor necrosis factor (TNF)-α expression, the former had no effect on LPS-induced TNF-α expression. Palmitate may therefore stimulate cytokine production through a different mechanism than LPS mediated through Toll-like receptor 4, at least partly, and ACSL1 may play an important role in this mechanism. Finally, palmitate induced expression of sterol regulatory element-binding protein-1a and ACSL1, while EPA suppressed the expression of these genes. CONCLUSION The suppressive effects of EPA on palmitate-induced cytokine production may be mediated by the suppression of ACSL1 expression, at least partly. This anti-inflammatory effect of EPA may contribute to suppression of chronic inflammation caused by macrophages in atherosclerotic plaques.

Suppression of hepatic fat accumulation by highly purified eicosapentaenoic acid prevents the progression of D-galactosamine-induced hepatitis in mice fed with a high-fat/high-sucrose diet

The pathogenesis of non-alcoholic fatty liver disease (NAFLD) remains largely unknown. Here, we assessed the importance of hepatic fat accumulation on the progression of hepatitis. BALB/cA mice were fed with a standard diet (STD) or a high-fat and high-sucrose diet (HFHSD) for 14 days followed by intraperitoneal injection of d-galactosamine (DGalN) or vehicle. After 20-21 h, plasma and liver tissue were collected and analyzed. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in plasma were increased significantly in HFHSD-fed mice treated with DGalN compared to STD-fed mice treated with DGalN. This exacerbation by the HFHSD was also observed in the plasma soluble tumor necrosis factor receptor (sTNFR) levels, and hepatic levels of reactive oxygen species (ROS) and the fibrogenic gene expression, such as tissue inhibitor of matrix metalloproteinase 1 (TIMP-1), connective tissue growth factor (CTGF) and osteopontin (OPN) in HFHSD-fed mice treated with DGalN. The triglyceride contents of the liver were significantly increased by the HFHSD. When eicosapentaenoic acid (EPA), a suppressor of sterol regulatory element binding protein 1 (SREBP-1), was administered to HFHSD-fed mice, the sensitivity of DGalN, as a result of plasma ALT and AST levels, was suppressed accompanied by reduced plasma sTNFR2 level and hepatic levels of triglyceride, ROS, and fibrogenic parameters, and by increased plasma adiponectin levels. These data suggest that the progression of steatotic liver injury closely depends on the accumulation of fat in the liver and is prevented by EPA through the suppression of the fatty liver change.