Shizuka Mita

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.

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.

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.

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.

Krüppel-like factor 4 mediates anti-proliferative effects of progesterone with G0/G1 arrest in human endometrial epithelial cells

Activation of the progesterone receptor (PR) inhibits cell proliferation in various reproductive tissues. However, the molecular mechanisms underlying the regulation of cell proliferation by PR remain poorly understood. It is well established that Krüppel-like factor 4 (KLF4), a family of zinc finger-containing transcription factors, induces cell cycle arrest in epithelial cells. In this study, we investigated whether KLF4 served as a target of PR activation during cell proliferation using human endometrial epithelial cells. PR agonists, progesterone and dienogest, were found to produce a lasting increase in the expression of KLF4 mRNA, followed by a decrease in cyclin D1 mRNA, and inhibit cell proliferation with G0/G1 arrest. KLF4 knockdown using KLF4 small interferingRNA abrogated the inhibition of cell proliferation by PR agonists. In addition, forced expression of KLF4 inhibited cyclin D1 promoter transactivation. These results suggest that PR agonists induce KLF4 expression and then inhibit cyclin D1 expression, and consequently inhibit cell proliferation in human endometrial epithelial cells. In terms of human reproductive tissue, KLF4 may be a factor concerning cell cycle, directly responsive to PR activation.

Dienogest, a synthetic progestin, down-regulates expression of CYP19A1 and inflammatory and neuroangiogenesis factors through progesterone receptor isoforms A and B in endometriotic cells.

Dienogest (DNG) is a selective progesterone receptor (PR) agonist and oral administration of DNG is used for the treatment of endometriosis. DNG is considered to act on PR to down-regulate pathophysiological factors associated with endometriosis. PR exists as two major isoforms, PR-A and PR-B, and their physiological functions are mostly distinct. It was suggested that PR isoform expression patterns are altered in endometriosis, but it is unknown whether the pharmacological effects of DNG are exerted through PR-A, PR-B or both. In the present study, we investigated the pharmacological effects of DNG through these PR isoforms on the expression of CYP19A1 which encodes aromatase and inflammatory and neuroangiogenesis factors associated with the pain and progression of endometriosis. We used immortalized human endometriotic epithelial cell lines that specifically express PR-A or PR-B in a spheroid cell culture system, and treated them with DNG. We evaluated messenger RNA (mRNA) expression of CYP19A1, prostaglandin (PG)E2 synthase (cyclooxygenase (COX)-2 and microsomal PGE2 synthase (mPGES)-1), inflammatory cytokines (interleukin (IL)-6, IL-8, and monocyte chemoattractant protein (MCP)-1) and neuroangiogenesis factors (vascular endothelial growth factor (VEGF) and nerve growth factor (NGF)) using real-time polymerase chain reaction. In addition, PGE2 production was measured by enzyme immunoassay. We found that DNG down-regulated mRNA expression of CYP19A1, COX-2, mPGES-1, IL-6, IL-8, MCP-1, NGF and VEGF, and PGE2 production in human endometriotic epithelial cell lines that specifically express either PR-A or PR-B. These results demonstrate that DNG activates both PR-A and PR-B and down-regulates the expression of pathophysiological factors associated with pain and progression of endometriosis. Our results suggest that DNG exerts therapeutic efficacy against the pain and progression of endometriosis regardless of PR isoform expression patterns.

Dienogest improves human leucocyte antigen-DR underexpression and reduces tumour necrosis factor-α production in peritoneal fluid cells from women with endometriosis

OBJECTIVE To determine the immunological effect of dienogest (DNG), an oral anti-endometriosis drug, on peritoneal fluid (PF) macrophages collected from women with endometriosis. Although it has been suggested that DNG has direct effects on endometriotic cells, including decreased cell proliferation and decreased anti-inflammatory cytokine production, the effects of DNG on PF cells are unclear. STUDY DESIGN The effects of DNG on PF cells from 34 women with endometriosis and 22 women without endometriosis (controls) were investigated. Expression of human leucocyte antigen (HLA)-DR in PF macrophages, obtained from the peritoneal cavity during laparoscopic surgery, was determined by flow cytometry. HLA-DR expression was measured again after PF cells had been cultured for 72 h in a humidified atmosphere at 37 °C in 5% CO₂-95% air with or without DNG. After 72 h of incubation, the concentration of pro-inflammatory tumour necrosis factor (TNF)-α in the media was measured by enzyme-linked immunosorbent assay. RESULTS HLA-DR expression was lower in PF macrophages from women with endometriosis compared with controls. However, after DNG treatment, HLA-DR expression in PF macrophages from women with endometriosis was increased to the same level as in controls. The TNF-α concentration in the media was decreased by DNG. CONCLUSIONS DNG can restore the antigen-presenting ability of PF macrophages by increased HLA-DR expression, and may have an anti-inflammatory effect on PF macrophages in women with endometriosis.

Dienogest suppresses the activation of primordial follicles and preserves the primordial follicle stockpile for fertility in mice

The aim of the present study was to characterize the effect of long-term usage of dienogest, a fourth-generation progestin that possesses progestogen and anti-androgen activities, on the stockpile of oocytes and fertility after administration. Female ICR mice (100 days old) were divided into a dienogest group and a control group. The mice received 16 consecutive subcutaneous injections of 5 mg dienogest dissolved in corn oil or corn oil as a vehicle control every 4 days. The mice treated with dienogest had more total offspring and larger litter sizes after the final administration than the mice treated with the vehicle control. Greater numbers of primordial follicles were detected at both 4 and 80 days after the final administration. No significant differences were found in serum anti-Müllerian hormone concentrations at 4 and 80 days after the final dienogest administration. The ratio of primary to primordial follicles was decreased in 3-day-old newborn ovaries cultured for 4 days with dienogest (10-7, 10-6 and 10-5 mol/l) compared with ovaries cultured without dienogest. The results of the present study indicate that dienogest suppresses the activation of primordial follicles during its administration and preserves the primordial follicle stockpile and subsequent fertility in mice.

Endometriotic cyst fluid induces reactive oxygen species (ROS) in human immortalized epithelial cells derived from ovarian endometrioma

ABSTRACT Objectives: Endometriotic cyst fluid (ECF) contains a large amount of reactive oxygen species (ROS), and endometriotic cysts are exposed to strong oxidative stress, which may cause malignant transformation. In this study, ROS production by ECF was clinically analysed. Methods: Human immortalized epithelial cells derived from ovarian endometrioma (EMosis-CC/TERT 1) were treated with ECF. In addition, ROS production in EMosis-CC/TERT 1 was measured, and its clinical significance was analysed. Results: A total of 38 ECF samples were obtained from patients diagnosed with endometriotic cysts. In EMosis-CC/TERT1, significantly higher levels of ROS were induced by ECF than by the vehicle control and ferric nitrilotriacetate. There were no significant differences in ROS production by laterality and preoperative serum CA125 values. There were several patients whose cyst sizes were approximately 5 cm and had relatively high ROS production. Production of ROS by ECF was relatively higher in patients older than 40 years of age than in those younger than 40. Discussion: Our study revealed that ROS are highly produced by ECF in EMosis-CC/TERT1 cells; therefore, exposure to ECF induced strong oxidative stress. Development of a therapeutic strategy to reduce ROS production might be useful for preventing malignant transformation of endometriotic cysts.

Dienogest inhibits C-C motif chemokine ligand 20 expression in human endometriotic epithelial cells

OBJECTIVE C-C motif chemokine ligand 20 is thought to contribute to the development of endometriosis by recruiting Th17 lymphocytes into endometriotic foci. The present study investigated the effects of dienogest, a progesterone receptor agonist used to treat endometriosis, on C-C motif chemokine ligand 20 expression by endometriotic cells. STUDY DESIGN Effects of dienogest on mRNA expression and protein secretion of C-C motif chemokine ligand 20 induced by interleukin 1β were assessed in three immortalized endometriotic epithelial cell lines, parental cells (EMosis-CC/TERT1), and stably expressing human progesterone receptor isoform A (EMosis-CC/TERT1/PRA+) or isoform B (EMosis-CC/TERT1/PRA-/PRB+). RESULTS Dienogest markedly inhibited interleukin 1β-stimulated C-C motif chemokine ligand 20 mRNA expression and protein secretion in EMosis-CC/TERT1/PRA-/PRB+, which was abrogated by the progesterone receptor antagonist RU486. In EMosis-CC/TERT1/PRA+, dienogest slightly inhibited C-C motif chemokine ligand 20 mRNA and protein. In EMosis-CC/TERT1, dienogest slightly inhibited C-C motif chemokine ligand 20 mRNA, but had no effect on C-C motif chemokine ligand 20 protein. CONCLUSION Dienogest inhibited interleukin 1β-induced up-regulation of C-C motif chemokine ligand 20 in endometriotic epithelial cells, mainly mediated by progesterone receptor B.