Discovery and Clinical Evaluation of MK-8150, A Novel Nitric Oxide Donor With a Unique Mechanism of Nitric Oxide Release.

BACKGROUND: Nitric oxide donors are widely used to treat cardiovascular disease, but their major limitation is the development of tolerance, a multifactorial process to which the in vivo release of nitric oxide is thought to contribute. Here we describe the preclinical and clinical results of a translational drug development effort to create a next-generation nitric oxide donor with improved pharmacokinetic properties and a unique mechanism of nitric oxide release through CYP3A4 metabolism that was designed to circumvent the development of tolerance. METHODS AND RESULTS: Single- and multiple-dose studies in telemetered dogs showed that MK-8150 induced robust blood-pressure lowering that was sustained over 14 days. The molecule was safe and well tolerated in humans, and single doses reduced systolic blood pressure by 5 to 20 mm Hg in hypertensive patients. Multiple-dose studies in hypertensive patients showed that the blood-pressure-lowering effect diminished after 10 days, and 28-day studies showed that the hemodynamic effects were completely lost by day 28, even when the dose of MK-8150 was increased during the dosing period. CONCLUSIONS: The novel nitric oxide donor MK-8150 induced significant blood-pressure lowering in dogs and humans for up to 14 days. However, despite a unique mechanism of nitric oxide release mediated by CYP3A4 metabolism, tolerance developed over 28 days, suggesting that tolerance to nitric oxide donors is multifactorial and cannot be overcome solely through altered in vivo release of nitric oxide. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifiers: NCT01590810 and NCT01656408.

Early Clinical Development Planning via Biomarkers, Clinical Endpoints, and Simulation: A Case Study to Optimize for Phase 3 Dose Selection.

BACKGROUND: This study investigated a framework that leverages the relationship between biomarkers and a target clinical endpoint to optimize an early development plan. METHODS: Different biomarker designs were assessed for proof of concept (PoC) and dose finding (DF) to improve phase 2b (Ph2b) design as well as phase 3 (Ph3) dose choice. A case study using a Bayesian trivariate normal distribution model for 2 biomarkers and a clinically relevant endpoint was utilized with simulation to assess performance characteristics. RESULTS: We found the following: (1) at typical sample sizes for early development trials, biomarkers appear useful for PoC but not for clinical endpoint DF; and (2) even with large amounts of prior information and near perfect correlation between biomarkers and clinical endpoints, Ph2b variability is only overcome by increased Ph2b sample sizes to improve Ph3 dose choice. CONCLUSIONS: For highly variable clinical endpoints, the fastest path should be to demonstrate PoC by biomarkers and then go directly to Ph2b to measure the target clinical endpoint.

Sex disparity in colonic adenomagenesis involves promotion by male hormones, not protection by female hormones.

It recently has been recognized that men develop colonic adenomas and carcinomas at an earlier age and at a higher rate than women. In the Apc(Pirc/+) (Pirc) rat model of early colonic cancer, this sex susceptibility was recapitulated, with male Pirc rats developing twice as many adenomas as females. Analysis of large datasets revealed that the Apc(Min/+) mouse also shows enhanced male susceptibility to adenomagenesis, but only in the colon. In addition, WT mice treated with injections of the carcinogen azoxymethane (AOM) showed increased numbers of colonic adenomas in males. The mechanism underlying these observations was investigated by manipulation of hormonal status. The preponderance of colonic adenomas in the Pirc rat model allowed a statistically significant investigation in vivo of the mechanism of sex hormone action on the development of colonic adenomas. Females depleted of endogenous hormones by ovariectomy did not exhibit a change in prevalence of adenomas, nor was any effect observed with replacement of one or a combination of female hormones. In contrast, depletion of male hormones by orchidectomy (castration) markedly protected the Pirc rat from adenoma development, whereas supplementation with testosterone reversed that effect. These observations were recapitulated in the AOM mouse model. Androgen receptor was undetectable in the colon or adenomas, making it likely that testosterone acts indirectly on the tumor lineage. Our findings suggest that indirect tumor-promoting effects of testosterone likely explain the disparity between the sexes in the development of colonic adenomas.

Identification of coregulators influenced by estrogen receptor subtype specific binding of the ER antagonists 4-hydroxytamoxifen and fulvestrant.

The aim of the present study was to investigate modulation of the interaction of ERα and ERß with coregulators in the ligand dependent responses induced by the ER antagonistic compounds 4OHT and fulvestrant. Comparison with the modulation index (MI) profiles for the ER agonist estradiol (E2) will elucidate whether differences in the (ant)agonist dependent interaction of ERα and ERß with coregulators expressed in MI profiles contribute to the differences in (ant)agonist responses. To this end, the selected ER antagonistic compounds were first characterized for intrinsic relative potency and efficacy towards ERα and ERß using ER selective U2OS reporter gene assays, and subsequently tested for ligand dependent modulation of the interaction of ERα and ERß with coregulators using the MARCoNI assay. Results obtained indicate a preference of 4OHT to antagonize ERß and find fulvestrant to be less ER specific. MARCoNI assay responses reveal that ERα and ERß mediated interaction with coregulators expressed in MI profiles are similar for 4OHT and fulvestrant and generally opposite to the MI profile of the ER agonist E2. Hierarchical clustering based on the MI profiles appeared able to clearly discriminate the two compounds with ER antagonistic properties from the ER agonist E2. Taken together the data reveal that modulation of the interaction of ERs with coregulators discriminates ER agonists from antagonists but does not discriminate between the less specific ER antagonist fulvestrant and the preferential ERß antagonistic compound 4OHT. It is concluded that differences in modulation of the interaction of ERα and ERß with coregulators contribute to the differences in ligand dependent responses induced by ER agonists and ER antagonists but the importance of the subtle differences in modulation of the interaction of ERs with coregulators between the ER antagonistic compounds 4OHT and fulvestrant for the ultimate biological effect remains to be established.

Cell proliferation and modulation of interaction of estrogen receptors with coregulators induced by ERα and ERß agonists.

The aim of the present study was to investigate modulation of the interaction of the ERα and ERß with coregulators in the ligand responses induced by estrogenic compounds. To this end, selective ERα and ERß agonists were characterized for intrinsic relative potency reflected by EC50 and maximal efficacy towards ERα and ERß mediated response in ER selective reporter gene assays, and subsequently tested for induction of cell proliferation in T47D-ERß cells with variable ERα/ERß ratio, and finally for ligand dependent modulation of the interaction of ERα and ERß with coregulators using the MARCoNI assay, with 154 unique nuclear receptor coregulator peptides derived from 66 different coregulators. Results obtained reveal an important influence of the ERα/ERß ratio and receptor selectivity of the compounds tested on induction of cell proliferation. ERα agonists activate cell proliferation whereas ERß suppresses ERα mediated cell proliferation. The responses in the MARCoNI assay reveal that upon ERα or ERß activation by a specific agonist, the modulation of the interaction of the ERs with coregulators is very similar indicating only a limited number of differences upon ERα or ERß activation by a specific ligand. Differences in the modulation of the interaction of the ERs with coregulators between the different agonists were more pronounced. Based on ligand dependent differences in the modulation of the interaction of the ERs with coregulators, the MARCoNI assay was shown to be able to classify the ER agonists discriminating between different agonists for the same receptor, a characteristic not defined by the ER selective reporter gene or proliferation assays. It is concluded that the ultimate effect of the model compounds on proliferation of estrogen responsive cells depends on the intrinsic relative potency of the agonist towards ERα and ERß and the cellular ERα/ERß ratio whereas differences in the modulation of the interaction of the ERα and ERß with coregulators contribute to the ligand dependent responses induced by estrogenic compounds.

Oestrogens promote tumorigenesis in a mouse model for colitis-associated cancer.

BACKGROUND: Hormone replacement therapy increases the risk of developing ulcerative colitis in postmenopausal women. Chronic intestinal inflammation predisposes to colon cancer development, but effects of female hormones on colitis-associated cancer development have not been examined. AIM: To investigate the role of female hormones in the dextran sodium sulfate (DSS)-azoxymethane (AOM) mouse model for colitis-associated cancer. DESIGN: We performed ovariectomies, or sham operations, on mice, and supplemented these animals with indicated hormones. Additionally, we used oestrogen receptor α or ß (Erα or Erß) mutant mice. To study colitis or colitis-associated cancer, we used DSS only, or DSS and AOM, respectively. RESULTS: Ovariectomy protects female mice against colitis-associated tumour development. Hormone replacement in ovariectomised mice with either oestradiol (E2), medroxyprogesterone acetate or a combination of both suggests that oestrogens are the ovary-derived factor that promotes tumour development in the context of inflammatory damage. E2-treated animals showed increased clinical symptoms and Il-6 production upon DSS-induced colitis and enhanced epithelial proliferation. Treatment with E2 markedly increased the numbers of polyps in ovariectomised mice and also strongly promoted tumour progression with all E2-treated animals developing at least one invasive adenocarcinoma, whereas, placebo-treated animals developed adenomas only. Using Er mutant mice, we find that the protumorigenic effect of oestrogen depends on both Erα and Erß. CONCLUSIONS: Our results suggest that oestrogens promote inflammation-associated cancer development by impairing the mucosal response to inflammatory damage.

Correction: Intestinal Tumorigenesis Is Not Affected by Progesterone Signaling in Rodent Models.

[This corrects the article DOI: 10.1371/journal.pone.0022620.].

Intestinal tumorigenesis is not affected by progesterone signaling in rodent models.

Clinical data suggest that progestins have chemopreventive properties in the development of colorectal cancer. We set out to examine a potential protective effect of progestins and progesterone signaling on colon cancer development. In normal and neoplastic intestinal tissue, we found that the progesterone receptor (PR) is not expressed. Expression was confined to sporadic mesenchymal cells. To analyze the influence of systemic progesterone receptor signaling, we crossed mice that lacked the progesterone receptor (PRKO) to the Apc(Min/+) mouse, a model for spontaneous intestinal polyposis. PRKO-Apc(Min/+) mice exhibited no change in polyp number, size or localization compared to Apc(Min/+). To examine effects of progestins on the intestinal epithelium that are independent of the PR, we treated mice with MPA. We found no effects of either progesterone or MPA on gross intestinal morphology or epithelial proliferation. Also, in rats treated with MPA, injection with the carcinogen azoxymethane did not result in a difference in the number or size of aberrant crypt foci, a surrogate end-point for adenoma development. We conclude that expression of the progesterone receptor is limited to cells in the intestinal mesenchyme. We did not observe any effect of progesterone receptor signaling or of progestin treatment in rodent models of intestinal tumorigenesis.

Olfactomedin-4 regulation by estrogen in the human endometrium requires epidermal growth factor signaling.

Olfactomedin-4 (OLFM-4) is an extracellular matrix protein that is highly expressed in human endometrium. We have examined the regulation and function of OLFM-4 in normal endometrium and in cases of endometriosis and endometrial cancer. OLFM-4 expression levels are highest in proliferative-phase endometrium, and 17ß-estradiol up-regulates OLFM-4 mRNA in endometrial explant cultures. Using the luciferase reporter under control of the OLFM-4 promoter, it was shown that both 17ß-estradiol and OH-tamoxifen induce luciferase activity, and epidermal growth factor receptor-1 is required for this estrogenic response. In turn, EGF activates the OLFM-4 promoter, and estrogen receptor-α is needed for the complete EGF response. The cellular functions of OLFM-4 were examined by its expression in OLFM-4-negative HEK-293 cells, which resulted in decreased vimentin expression and cell adherence as well as increased apoptosis resistance. In cases of endometriosis and endometrial cancer, OLFM-4 expression correlated with the presence of epidermal growth factor receptor-1 and estrogen receptor-α (or estrogen signaling). An increase of OLFM-4 mRNA was observed in the endometrium of endometriosis patients. No change in OLFM-4 expression levels were observed in patients with endometrial cancer relative with controls. In conclusion, cross-talk between estrogen and EGF signaling regulates OLFM-4 expression. The role of OLFM-4 in endometrial tissue remodeling before the secretory phase and during the predisposition and early events in endometriosis can be postulated but requires additional investigation.

Evaluation of a 99mTc-labeled AnnexinA5 variant for non-invasive SPECT imaging of cell death in liver, spleen and prostate.

PURPOSE: We investigate radio-labeling and pharmacokinetics of a new AnnexinA5 variant (HYNIC-cys-AnxA5) and then assess its utility for the non-invasive detection of cell death in liver, spleen and prostate. METHODS: AnnexinA5 binds to phosphatidylserine expressed on the surface of apoptotic and necrotic cells. Contrary to other AnnexinA5 variants, the new cys-AnxA5 allows for site-specific conjugation of a hydrazinonicotinamide-maleimide moiety and subsequent radio-labeling with (99m)Tc at a position not involved in the AnxA5-phosphatidylserine interaction. Distribution of (99m)Tc-HYNIC-cys-AnxA5 was studied in rats, both invasively and via SPECT/CT. Cycloheximide was used to induce cell death in liver and spleen, whereas apoptosis in the prostate was induced by castration. RESULTS: HYNIC-cys-AnxA5 was efficiently and reproducibly labeled with (99m)Tc. Blood clearance of radioactivity after iv-injection was adequately described by a two-compartment model, the renal cortex representing the main site of accumulation. Cycloheximide treatment resulted in increased accumulation of intravenous-injected (99m)Tc-HYNIC-cys-AnxA5 in liver and spleen over controls, which correlated well with TUNEL staining for cell death in corresponding tissue sections. However, the increase in TUNEL-positive prostate epithelial cells observed following castration was not paralleled by greater (99m)Tc-HYNIC-cys-AnxA5 accumulation. CONCLUSION: (99m)Tc-HYNIC-cys-AnxA5 appears a suitable tracer for assessment of cell death in liver and spleen, but not prostate.

The estrogenic component of tibolone reduces adiposity in female aromatase knockout mice.

OBJECTIVE: To explore the effects of tibolone on adiposity in the absence of aromatase and determine which of the hormonal properties of tibolone are exerting these effects. METHODS: In this study, vehicle; tibolone; estrogenic (ethinyl estradiol [EE]), progestogenic (ORG2058), or androgenic (dihydrotestosterone) compounds; or a combination of ORG2058 + EE was administered to 6-month-old ovariectomized aromatase knockout (ArKO) mice for a period of 6 weeks. RESULTS: In response to tibolone or EE-alone treatments, omental adipose tissue and infrarenal adipose tissue weights were significantly reduced (P = 0.004 and P = 0.01; P = 0.009 and P = 0.014, respectively) compared with those in ovariectomized and vehicle-treated ArKO mice. In contrast, adipose tissue weight tended to increase after ORG2058-alone treatment. Furthermore, EE in the presence of ORG2058 (ORG2058 + EE group) results in little effect on adiposity when compared with that in ovariectomized and vehicle-treated ArKO mice, showing that ORG2058 can negate the effect of EE. Dihydrotestosterone treatment did not have an impact on adipose tissue mass. Adipocyte volume and numbers followed the same treatment trends. CONCLUSIONS: In summary, our study in the ArKO mouse has confirmed the efficacy of tibolone as a hormone therapy to reduce adipose tissue accumulation after menopause and also shows that aromatization of tibolone is not required to elicit these estrogenic effects.

Oestrogen-mediated cardioprotection following ischaemia and reperfusion is mimicked by an oestrogen receptor (ER)alpha agonist and unaffected by an ER beta antagonist.

Oestrogen protects the heart from ischaemic injury. The current study aims to characterise two novel oestrogen receptor (ER) ligands, an ER alpha agonist ERA-45 and an ER beta antagonist ERB-88, and then use them to investigate the roles of ER alpha and ER beta in mediating the cardioprotection by E from ischaemia-reperfusion injury in the rat. The ER ligands were characterised by gene transactivation assay using ER-transfected Chinese hamster ovary (CHO) cells and in bioavailability studies in vivo. Female rats (n=48) were ovariectomised and implanted with 17beta-oestradiol (17 beta E(2)) releasing or placebo pellets. ERA-45, ERB-88 or vehicle was administered for 5 days prior to ischaemia-reperfusion studies. Necrosis, neutrophil infiltration (myeloperoxidase activity) and oxidant stress production (electron paramagnetic resonance) from the area-at-risk were measured to assess reperfusion injury. The ER alpha agonist ERA-45 showed more than 35-fold selectivity for ER alpha compared with ER beta gene transactivation. In vitro, the ER beta antagonist ERB-88 inhibited transactivation by 17 beta E(2) via ER beta with 46-fold selectivity relative to inhibition via ER alpha. In vivo, 17 beta E(2) significantly reduced neutrophil infiltration, oxidant stress and necrosis following ischaemia and reperfusion. Cardioprotection by 17 beta E(2) was not inhibited by ERB-88 but was completely reproduced by ERA-45. In conclusion, protection of the rat heart after ischaemia-reperfusion by 17 beta E(2) is achieved through the reduction of cardiomyocyte death, neutrophil infiltration and oxygen-free radical availability.The results of this study indicate that these effects are primarily mediated via activation of ER alpha.

Bone loss dynamics result in trabecular alignment in aging and ovariectomized rats.

Because of the destructive nature of techniques used to measure bone morphometry, studies of architectural changes and bone loss have utilized cross-sectional study designs, with all its inherent limitations in nuances. Here, the results of a longitudinal study using in vivo micro-CT are presented elucidating the dynamics of bone loss and architectural adaptation in rat models of aging and postmenopausal bone loss. Using 3-D methodology, we observed the changes in bone architecture in the proximal tibia of normally aging and ovariectomized rats for 54 weeks. Spatial patterns in bone resorption were observed that were similar for both groups. Remaining trabeculae increased in thickness or were remodeled into new trabecular structures, especially in the ovariectomized animals. The combination of bone loss and bone formation resulted in alignment of trabeculae across the growth plate. Cortical modeling that was associated with growth continued after cessation of longitudinal growth in the ovariectomized animals, resulting in shape changes of the proximal tibia. The organized nature of the changes in bone architecture that occurred after ovariectomy and the high similarity with the changes observed in the normally aging animals, suggest that estrogen depletion resulted in an acceleration of a normal bone adaptation process. The observed aligning of trabeculae suggests regulation through mechanical loading.

Effects of tibolone on bone quality in ovariectomized monkeys.

OBJECTIVE: The purpose of this report is to examine the effects of two doses of tibolone on bone quality (bone biomarkers, bone density, and bone strength) in ovariectomized cynomolgus monkeys fed high-fat diets. DESIGN: Ovariectomized cynomolgus monkeys were randomized into one of five treatment groups: placebo-treated control, tibolone (0.2 mg/kg/day), tibolone (0.05 mg/kg/day), conjugated equine estrogens (Premarin, 0.042 mg/kg/day), and conjugated equine estrogens plus medroxyprogesterone acetate (0.042 and 0.167 mg/kg/day, respectively). Bone quality was assessed by determining bone strength and density in vertebrae and femora collected after 24 months of treatment. RESULTS: Monkeys treated for 24 months with tibolone had increased bone mineral density in the distal femur and improved biomechanical properties in the midshaft femur compared with placebo-treated ovariectomized monkeys, as did monkeys treated with conjugated equine estrogens with or without medroxyprogesterone acetate. No treatment effects were seen in lumbar vertebra bone density or strength. There was no significant difference between tibolone and estrogen on biomechanical properties of the femur. CONCLUSION: These data show that tibolone is comparable to conjugated equine estrogens with or without medroxyprogesterone acetate in decreasing bone turnover and increasing bone strength in ovariectomized monkeys.