P450-mediated O-demethylated metabolite is responsible for rat hepatobiliary toxicity of pyridyltriazine-containing PI3K inhibitors.

The dysregulation of phosphatidylinositol 3-kinase (PI3K)-dependent pathways is implicated in several human cancers making it an attractive target for small molecule PI3K inhibitors. A series of potent pyridyltriazine-containing inhibitors of class Ia PI3Ks were synthesized and a subset of compounds was evaluated in exploratory repeat-dose rat toxicology studies. Daily oral dosing of compound 1: in Sprague Dawley rats for four consecutive days was associated with hepatobiliary toxicity that included biliary epithelial hyperplasia and hypertrophy, periductular edema, biliary stasis, and acute peribiliary inflammatory infiltrates. These histological changes were associated with clinical pathology changes that included increased serum liver enzymes, total bile acids, and bilirubin. The predominant clearance pathway of 1: was shown in vitro and in a bile-duct cannulated rat (14)C-ADME study to be P450-mediated oxidative metabolism. An O-demethylated pyridine metabolite, M3: , was identified as a candidate proximal metabolite that caused the hepatotoxicity. Co-administration of the pan-P450 inhibitor 1-aminobenzotriazole with 1: to rats significantly reduced the formation of M3: and prevented liver toxicity, whereas direct administration of M3: reproduced the toxicity. Structural changes were introduced to 1: to make the methoxypyridine ring less susceptible to P450 oxidation (compound 2: ), and addition of a methyl group to the benzylic carbon (compound 3: ) improved the pharmacokinetic profile. These changes culminated in the successful design of a clinical candidate 3: (AMG 511) that was devoid of liver toxicity in a 14-day rat toxicity study. Herein, we describe how a metabolism-based structure-activity relationship analysis allowed for the successful identification of a PI3K inhibitor devoid of off-target toxicity.

Reproductive toxicity of denosumab in cynomolgus monkeys.

Denosumab is a monoclonal antibody that inhibits bone resorption by targeting RANKL, an essential mediator of osteoclast formation, function, and survival. Reproductive toxicity of denosumab was assessed in cynomolgus monkeys in an embryofetal development study (dosing GD20-50) and a pre-postnatal toxicity study (dosing GD20-parturition). In the embryofetal toxicity study, denosumab did not elicit maternal toxicity, fetal harm or teratogenicity. In the pre-postnatal toxicity study, there were increased stillbirths, and one maternal death due to dystocia. There was no effect on maternal mammary gland histomorphology, lactation, or fetal growth. In infants exposed in utero, there was increased postnatal mortality, decreased body weight gain, and decreased growth/development. Denosumab-related effects in infants were present in bones and lymph nodes. There was full recovery at 6 months of age from most bone-related changes observed earlier postpartum. The effects observed in mothers and infants were consistent with the pharmacological action of denosumab.

RANK induces epithelial-mesenchymal transition and stemness in human mammary epithelial cells and promotes tumorigenesis and metastasis.

Paracrine signaling through receptor activator of NF-κB (RANK) pathway mediates the expansion of mammary epithelia that occurs during pregnancy, and activation of RANK pathway promotes mammary tumorigenesis in mice. In this study we extend these previous data to human cells and show that the RANK pathway promotes the development of mammary stem cells and breast cancer. Overexpression of RANK (FL-RANK) in a panel of tumoral and normal human mammary cells induces the expression of breast cancer stem and basal/stem cell markers. High levels of RANK in untransformed MCF10A cells induce changes associated with both stemness and transformation, including mammary gland reconstitution, epithelial-mesenchymal transition (EMT), increased migration, and anchorage-independent growth. In addition, spheroids of RANK overexpressing MCF10A cells display disrupted acinar formation, impair growth arrest and polarization, and luminal filling. RANK overexpression in tumor cells with nonfunctional BRCA1 enhances invasiveness in acinar cultures and increases tumorigenesis and metastasis in immunodeficient mice. High levels of RANK were found in human primary breast adenocarcinomas that lack expression of the hormone receptors, estrogen and progesterone, and in tumors with high pathologic grade and proliferation index; high RANK/RANKL expression was significantly associated with metastatic tumors. Together, our findings show that RANK promotes tumor initiation, progression, and metastasis in human mammary epithelial cells by increasing the population of CD44(+)CD24(-) cells, inducing stemness and EMT. These results suggest that RANK expression in primary breast cancer associates with poor prognosis.

RANK ligand mediates progestin-induced mammary epithelial proliferation and carcinogenesis.

RANK ligand (RANKL), a TNF-related molecule, is essential for osteoclast formation, function and survival through interaction with its receptor RANK. Mammary glands of RANK- and RANKL-deficient mice develop normally during sexual maturation, but fail to form lobuloalveolar structures during pregnancy because of defective proliferation and increased apoptosis of mammary epithelium. It has been shown that RANKL is responsible for the major proliferative response of mouse mammary epithelium to progesterone during mammary lactational morphogenesis, and in mouse models, manipulated to induce activation of the RANK/RANKL pathway in the absence of strict hormonal control, inappropriate mammary proliferation is observed. However, there is no evidence so far of a functional contribution of RANKL to tumorigenesis. Here we show that RANK and RANKL are expressed within normal, pre-malignant and neoplastic mammary epithelium, and using complementary gain-of-function (mouse mammary tumour virus (MMTV)-RANK transgenic mice) and loss-of function (pharmacological inhibition of RANKL) approaches, define a direct contribution of this pathway in mammary tumorigenesis. Accelerated pre-neoplasias and increased mammary tumour formation were observed in MMTV-RANK transgenic mice after multiparity or treatment with carcinogen and hormone (progesterone). Reciprocally, selective pharmacological inhibition of RANKL attenuated mammary tumour development not only in hormone- and carcinogen-treated MMTV-RANK and wild-type mice, but also in the MMTV-neu transgenic spontaneous tumour model. The reduction in tumorigenesis upon RANKL inhibition was preceded by a reduction in pre-neoplasias as well as rapid and sustained reductions in hormone- and carcinogen-induced mammary epithelial proliferation and cyclin D1 levels. Collectively, our results indicate that RANKL inhibition is acting directly on hormone-induced mammary epithelium at early stages in tumorigenesis, and the permissive contribution of progesterone to increased mammary cancer incidence is due to RANKL-dependent proliferative changes in the mammary epithelium. The current study highlights a potential role for RANKL inhibition in the management of proliferative breast disease.

An antibody against the colony-stimulating factor 1 receptor depletes the resident subset of monocytes and tissue- and tumor-associated macrophages but does not inhibit inflammation.

The development of the mononuclear phagocyte system requires macrophage colony-stimulating factor (CSF-1) signaling through the CSF-1 receptor (CSF1R, CD115). We examined the effect of an antibody against CSF1R on macrophage homeostasis and function using the MacGreen transgenic mouse (csf1r-enhanced green fluorescent protein) as a reporter. The administration of a novel CSF1R blocking antibody selectively reduced the CD115(+)Gr-1(neg) monocyte precursor of resident tissue macrophages. CD115(+)Gr-1(+) inflammatory monocytes were correspondingly increased, supporting the view that monocytes are a developmental series. Within tissue, the antibody almost completely depleted resident macrophage populations in the peritoneum, gastrointestinal tract, liver, kidney, and skin, but not in the lung or female reproductive organs. CSF1R blockade reduced the numbers of tumor-associated macrophages in syngeneic tumor models, suggesting that these cells are resident type macrophages. Conversely, it had no effect on inflammatory monocyte recruitment in models, including lipopolysaccharide-induced lung inflammation, wound healing, peritonitis, and severe acute graft-versus-host disease. Depletion of resident tissue macrophages from bone marrow transplantation recipients actually resulted in accelerated pathology and exaggerated donor T-cell activation. The data indicate that CSF1R signaling is required only for the maturation and replacement of resident-type monocytes and tissue macrophages, and is not required for monocyte production or inflammatory function.