Novel 3-aryl indoles as progesterone receptor antagonists for uterine fibroids.

We report the synthesis and characterization of novel 3-aryl indoles as potent and efficacious progesterone receptor (PR) antagonists with potential for the treatment of uterine fibroids. These compounds demonstrated excellent selectivity over other steroid nuclear hormone receptors such as the mineralocorticoid receptor (MR). They were prepared from 2-bromo-6-nitro indole in four to six steps using a Suzuki cross-coupling as the key step. Compound 8f was orally active in the complement 3 model of progesterone antagonism in the rat uterus and demonstrated partial antagonism in the McPhail model of progesterone activity.

Longitudinal in vivo analysis of the region-specific efficacy of parathyroid hormone in a rat cortical defect model.

PTH has been shown to enhance fracture repair; however, exactly when and where PTH acts in this process remains to be elucidated. Therefore, we conducted a longitudinal, region-specific analysis of bone regeneration in mature, osteopenic rats using a cortical defect model. Six-month-old rats were ovariectomized, and allowed to lose bone for 2 months, before being subjected to bilateral 2-mm circular defects in their femoral diaphyses. They were then treated for 5 wk with hPTH1-38 at doses of 0, 3, 10, or 30 microg/kg . d and scanned weekly by in vivo quantitative computed tomography. Quantitative computed tomography analyses showed temporal, dose-dependent increases in mineralization in the defects, intramedullary (IM) spaces, and whole diaphyses at the defect sites. Histomorphometry confirmed PTH stimulation of primarily woven bone in the defects and IM spaces, but not the periosteum. After necropsy, biomechanical testing identified an increase in strength at the highest PTH dose. Serum procollagen type I N-terminal propeptide concentration showed a transient increase due to drilling, but procollagen type I N-terminal propeptide also increased with PTH treatment, whereas tartrate-resistant acid phosphatase unexpectedly decreased. Analyses of lumber vertebra confirmed systemic efficacy of PTH at a nonfracture site. In summary, PTH dose dependently induced new bone formation within defects, at endocortical surfaces, and in IM spaces, resulting in faster and greater bone healing, as well as efficacy at other skeletal sites. The effects of PTH were kinetic, region specific, and most apparent at high doses that may not be entirely clinically relevant; therefore, clinical studies are necessary to clarify the therapeutic utility of PTH in bone healing.

Development of a highly sensitive, high-throughput, mass spectrometry-based assay for rat procollagen type-I N-terminal propeptide (PINP) to measure bone formation activity.

Type-I procollagen aminoterminal propeptide (PINP) is a useful biomarker for bone formation activity that is used to monitor treatment of bone disorders including osteoporosis. Studies with human patients under long-term therapy for osteoporosis by daily injection of parathyroid hormone (PTH) demonstrated that the circulating level of PINP at 3 months of treatment, measured by radioimmunoassay, was a good predictor for bone mineral density (BMD) at 18 months. It is important to have PINP assays for other species to elucidate processes of bone formation and for the development of new therapeutic options that can enhance bone formation activity. Currently, only a human PINP radioimmunoassay is commercially available for clinical use, which may not be cross reactive with PINP from other species. For example, rat PINP has little amino acid sequence homology to human PINP. Therefore, we developed a new, highly sensitive, high-throughput mass spectrometry-based assay for PINP from rat plasma or serum that does not rely on antibody reagents. Circulating levels of PINP showed age-dependent changes in rats. Prednisolone treatment, which is known to retard bone formation activity, led to a significant decrease in PINP, whereas PTH treatment dose-dependently increased PINP. The throughput of the assay parallels that of most antibody-based assays so that it can handle a large number of samples that are generated from preclinical animal studies. PINP in rats may serve as a biomarker for bone formation activity, and this assay could be instrumental in studying bone physiology in rat experimental models.

Structure-activity relationships of SERMs optimized for uterine antagonism and ovarian safety.

Structure-activity relationship studies are described, which led to the discovery of novel selective estrogen receptor modulators (SERMs) for the potential treatment of uterine fibroids. The SAR studies focused on limiting brain exposure and were guided by computational properties. Compounds with limited impact on the HPO axis were selected using serum estrogen levels as a biomarker for ovarian stimulation.

DNA microarray data integration by ortholog gene analysis reveals potential molecular mechanisms of estrogen-dependent growth of human uterine fibroids.

BACKGROUND: Uterine fibroids or leiomyoma are a common benign smooth muscle tumor. The tumor growth is well known to be estrogen-dependent. However, the molecular mechanisms of its estrogen-dependency is not well understood. METHODS: Differentially expressed genes in human uterine fibroids were either retrieved from published papers or from our own statistical analysis of downloaded array data. Probes for the same genes on different Affymetrix chips were mapped based on probe comparison information provided by Affymetrix. Genes identified by two or three array studies were submitted for ortholog analysis. Human and rat ortholog genes were identified by using ortholog gene databases, HomoloGene and TOGA and were confirmed by synteny analysis with MultiContigView tool in the Ensembl genome browser. RESULTS: By integrated analysis of three recently published DNA microarray studies with human tissue, thirty-eight genes were found to be differentially expressed in the same direction in fibroid compared to adjacent uterine myometrium by at least two research groups. Among these genes, twelve with rat orthologs were identified as estrogen-regulated from our array study investigating uterine expression in ovariectomized rats treated with estrogen. Functional and pathway analyses of the twelve genes suggested multiple molecular mechanisms for estrogen-dependent cell survival and tumor growth. Firstly, estrogen increased expression of the anti-apoptotic PCP4 gene and suppressed the expression of growth inhibitory receptors PTGER3 and TGFBR2. Secondly, estrogen may antagonize PPARgamma signaling, thought to inhibit fibroid growth and survival, at two points in the PPAR pathway: 1) through increased ANXA1 gene expression which can inhibit phospholipase A2 activity and in turn decrease arachidonic acid synthesis, and 2) by decreasing L-PGDS expression which would reduce synthesis of PGJ2, an endogenous ligand for PPARgamma. Lastly, estrogen affects retinoic acid (RA) synthesis and mobilization by regulating expression of CRABP2 and ALDH1A1. RA has been shown to play a significant role in the development of uterine fibroids in an animal model. CONCLUSION: Integrated analysis of multiple array datasets revealed twelve human and rat ortholog genes that were differentially expressed in human uterine fibroids and transcriptionally responsive to estrogen in the rat uterus. Functional and pathway analysis of these genes suggest multiple potential molecular mechanisms for the poorly understood estrogen-dependent growth of uterine fibroids. Fully understanding the exact molecular interactions among these gene products requires further study to validate their roles in uterine fibroids. This work provides new avenues of study which could influence the future direction of therapeutic intervention for the disease.

Regulation of the human ADAMTS-4 promoter by transcription factors and cytokines.

ADAMTS-4 (aggrecanase-1) is a metalloprotease that plays a role in aggrecan degradation in the cartilage extracellular matrix. In order to understand the regulation of ADAMTS-4 gene expression we have cloned and characterized a functional 4.5kb human ADAMTS-4 promoter. Sequence analysis of the promoter revealed the presence of putative binding sites for nuclear factor of activated T cells (NFAT) and Runx family of transcription factors that are known to regulate chondrocyte maturation and differentiation. Using promoter-reporter assays and mRNA analysis we have analyzed the role of chondrocyte-expressed transcription factors NFATp and Runx2 and have shown that ADAMTS-4 is a potential downstream target of these two factors. Our results suggest that inhibition of the expression/function of NFATp and/or Runx2 may enable us to modulate aggrecan degradation in normal physiology and/or in degenerative joint diseases. The ADAMTS-4 promoter would serve as a valuable mechanistic tool to better understand the regulation of ADAMTS-4 expression by signaling pathways that modulate cartilage matrix breakdown.

A selective estrogen receptor modulator for the treatment of hot flushes.

A selective estrogen receptor modulator (SERM) for the potential treatment of hot flushes is described. (R)-(+)-7,9-difluoro-5-[4-(2-piperidin-1-ylethoxy)phenyl]-5H-6-oxachrysen-2-ol, LSN2120310, potently binds ERalpha and ERbeta and is an antagonist in MCF-7 breast adenocarcinoma and Ishikawa uterine cancer cell lines. The compound is a potent estrogen antagonist in the rat uterus. In ovariectomized rats, the compound lowers cholesterol, maintains bone mineral density, and is efficacious in a morphine dependent rat model of hot flush efficacy.

A selective estrogen receptor modulator designed for the treatment of uterine leiomyoma with unique tissue specificity for uterus and ovaries in rats.

The design of a novel selective estrogen receptor modulator (SERM) for the potential treatment of uterine leiomyoma is described. 16 (LY2066948-HCl) binds with high affinity to estrogen receptors alpha and beta (ERalpha and ERbeta, respectively) and is a potent uterine antagonist with minimal effects on the ovaries as determined by serum biomarkers and histologic evaluation.

A new selective estrogen receptor modulator with potent uterine antagonist activity, agonist activity in bone, and minimal ovarian stimulation.

The use of selective estrogen receptor modulators for the treatment of estrogen-dependent diseases in premenopausal women has been hindered by undesirable ovarian stimulation and associated risks of ovarian cysts. We have identified a selective estrogen receptor modulator compound (LY2066948) that is a strong estrogen antagonist in the uterus yet has minimal effects on the ovaries of rats. LY2066948 binds with high affinity to both estrogen receptors and has potent estrogen antagonist activity in human uterine and breast cancer cells. Oral administration of LY2066948 to immature rats blocked uterine weight gain induced by ethynyl estradiol with an ED50 of 0.07 mg/kg. Studies in mature rats demonstrated that LY2066948 decreases uterine weight by 51% after 35 d treatment, confirming potent uterine antagonist activity over several estrous cycles. This strong uterine response contrasted with the minimal effects on the ovaries: serum estradiol levels remained within the normal range, whereas histologic evaluation showed granulosa cell hyperplasia in few of the rats. Bone studies demonstrated that LY2066948 prevented ovariectomy-induced bone loss and treatment of ovary-intact rats caused no bone loss, confirming estrogen receptor agonist skeletal effects. Collectively, these data show that LY2066948 exhibits a tissue-specific profile consistent with strong antagonist activity in the uterus, agonist activity in bone, and minimal effects in the ovaries.

Benzothiophene and naphthalene derived constrained SERMs.

For selective estrogen receptor modulators (SERMs), the orientation of the basic side chain relative to the SERM core has a significant impact on function. The synthesis and biological evaluation of two series of SERMs are disclosed, where the ligand side chain is constrained to adopt a defined orientation. Compounds where the side chain is forced into the plane of the SERM core have a different profile compared to those compounds where the side chain is pseudo-orthogonal, particularly with regard to antagonism of estradiol action on an Ishikawa uterine cell line.

Parathyroid hormone bone anabolic action requires Cbfa1/Runx2-dependent signaling.

The Cbfa1/Runx2 (referred to herein as Cbfa1) transcription factor has been shown to be essential for osteoblast differentiation and bone formation during embryogenesis. PTH given intermittently is a proven bone anabolic agent. Here, we investigated whether PTH regulates the expression and/or activity of Cbfa1 in osteoblastic cells and in a rat metatarsal organ culture assay. PTH was found to regulate Cbfa1 mRNA in the rat osteosarcoma cell line UMR106 in a concentration-dependent manner. The effect of PTH was mimicked by forskolin, an activator of adenylate cyclase leading to the protein kinase A pathway. PTH administered intermittently for 5 d in vivo was found to stimulate Cbfa1 protein in the rat proximal tibiae metaphysis. To demonstrate PTH regulation of Cbfa1 activity, a construct containing six tandem Cbfa1 binding elements fused to luciferase was shown to be rapidly stimulated in response to PTH. This stimulation preceded the effects on mRNA regulation and resulted from a protein kinase A-mediated increase in Cbfa1 activity. Finally, using a neonate rat metatarsal organ culture system, we demonstrated dose-dependent anabolic responsiveness to PTH and to Cbfa1 overexpression from an adenoviral construct. We further showed that Cbfa1 antisense oligonucleotides that blocked adenoviral Cbfa1-induced anabolic effects in this organ culture model also abolished the PTH-mediated anabolic increase. These findings suggest a requirement for Cbfa1 in mediating the anabolic effects of PTH. Thus, regulation of Cbfa1 expression or activity is an important mechanism by which PTH controls osteoblast function.