Drug safety is a barrier to the discovery and development of new androgen receptor antagonists.

BACKGROUND: Androgen receptor (AR) antagonists are part of the standard of care for prostate cancer. Despite the almost inevitable development of resistance in prostate tumors to AR antagonists, no new AR antagonists have been approved for over a decade. Treatment failure is due in part to mutations that increase activity of AR in response to lower ligand concentrations as well as to mutations that result in AR response to a broader range of ligands. The failure to discover new AR antagonists has occurred in the face of continued research; to enable progress, a clear understanding of the reasons for failure is required. METHODS: Non-clinical drug safety studies and safety pharmacology assays were performed on previously approved AR antagonists (bicalutamide, flutamide, nilutamide), next generation antagonists in clinical testing (MDV3100, BMS-641988), and a pre-clinical drug candidate (BMS-501949). In addition, non-clinical studies with AR mutant mice, and EEG recordings in rats were performed. Non-clinical findings are compared to disclosures of clinical trial results. RESULTS: As a drug class, AR antagonists cause seizure in animals by an off-target mechanism and are found in vitro to inhibit GABA-A currents. Clinical trials of candidate next generation AR antagonists identify seizure as a clinical safety risk. CONCLUSIONS: Non-clinical drug safety profiles of the AR antagonist drug class create a significant barrier to the identification of next generation AR antagonists. GABA-A inhibition is a common off-target activity of approved and next generation AR antagonists potentially explaining some side effects and safety hazards of this class of drugs.

Evaluation of cynomolgus monkey pregnane X receptor, primary hepatocyte, and in vivo pharmacokinetic changes in predicting human CYP3A4 induction.

Monkeys have been proposed as an animal model to predict the magnitude of human clinical drug-drug interactions caused by CYP3A4 enzyme induction. To evaluate whether the cynomolgus monkey can be an effective in vivo model, human CYP3A4 inducers were evaluated both in vitro and in vivo. First, a full-length pregnane X receptor (PXR) was cloned from the cynomolgus monkey, and the sequence was compared with those of rhesus monkey and human PXR. Cynomolgus and rhesus monkey PXR differed by only one amino acid (A68V), and both were highly homologous to human PXR (approximately 96%). When the transactivation profiles of 30 compounds, including known inducers of CYP3A4, were compared between cynomolgus and human PXR, a high degree of correlation with EC(50) values was observed. These results suggest that cynomolgus and human PXR respond in a similar fashion to these ligands. Second, two known human CYP3A4 inducers, rifampicin and hyperforin, were tested in monkey and human primary hepatocytes for induction of CYP3A enzymes. Both monkey and human hepatocytes responded similarly to the inducers and resulted in increased RNA and enzyme activity changes of CYP3A8 and CYP3A4, respectively. Lastly, in vivo induction of CYP3A8 by rifampicin and hyperforin was shown by significant reductions of midazolam exposure that were comparable with those in humans. These results show that the cynomolgus monkey can be a predictive in vivo animal model of PXR-mediated induction of human CYP3A4 and can provide a useful assessment of the resulting pharmacokinetic changes of affected drugs.

P2Y6 receptor potentiates pro-inflammatory responses in macrophages and exhibits differential roles in atherosclerotic lesion development.

BACKGROUND: P2Y(6), a purinergic receptor for UDP, is enriched in atherosclerotic lesions and is implicated in pro-inflammatory responses of key vascular cell types and macrophages. Evidence for its involvement in atherogenesis, however, has been lacking. Here we use cell-based studies and three murine models of atherogenesis to evaluate the impact of P2Y(6) deficiency on atherosclerosis. METHODOLOGY/PRINCIPAL FINDINGS: Cell-based studies in 1321N1 astrocytoma cells, which lack functional P2Y(6) receptors, showed that exogenous expression of P2Y(6) induces a robust, receptor- and agonist-dependent secretion of inflammatory mediators IL-8, IL-6, MCP-1 and GRO1. P2Y(6)-mediated inflammatory responses were also observed, albeit to a lesser extent, in macrophages endogenously expressing P2Y(6) and in acute peritonitis models of inflammation. To evaluate the role of P2Y(6) in atherosclerotic lesion development, we used P2Y(6)-deficient mice in three mouse models of atherosclerosis. A 43% reduction in aortic arch plaque was observed in high fat-fed LDLR knockout mice lacking P2Y(6) receptors in bone marrow-derived cells. In contrast, no effect on lesion development was observed in fat-fed whole body P2Y(6)xLDLR double knockout mice. Interestingly, in a model of enhanced vascular inflammation using angiotensin II, P2Y(6) deficiency enhanced formation of aneurysms and exhibited a trend towards increased atherosclerosis in the aorta of LDLR knockout mice. CONCLUSIONS: P2Y(6) receptor augments pro-inflammatory responses in macrophages and exhibits a pro-atherogenic role in hematopoietic cells. However, the overall impact of whole body P2Y(6) deficiency on atherosclerosis appears to be modest and could reflect additional roles of P2Y(6) in vascular disease pathophysiologies, such as aneurysm formation.

Insulin receptor (IR) pathway hyperactivity in IGF-IR null cells and suppression of downstream growth signaling using the dual IGF-IR/IR inhibitor, BMS-754807.

The biology of IGF-IR/IR signaling was studied in normal mouse embryonic fibroblasts (MEFs) that were either wild type (wt), heterozygous (het), or null for the IGF-IR. The ability of IGF-I, IGF-II, or insulin to stimulate serum-starved MEFs was characterized by gene expression profiling and biochemical analyses for activation of downstream signals. Each genotypic group of MEFs exhibited distinct patterns of expression both while resting and in response to stimulation. The insulin receptor (IR) pathway in IGF-IR null MEFs was hypersensitive to insulin ligand stimulation resulting in greater AKT phosphorylation than in wt or het MEFs stimulated with the same ligand. Interestingly, the IR pathway hypersensitivity in IGF-IR null MEFs occurred with no observed changes in the levels of IR isoforms A or B. A new small molecule IGF-IR inhibitor (BMS-754807), having equipotent activity against both IGF-IR and IR, proved effective in suppressing both AKT and ERK phosphorylation from both the IGF-IR and IR pathways by all three ligands tested in wt, het, and null MEFs. The use of a dual IGF-IR/IR inhibitor addresses concerns about the use of growth inhibiting therapies directed against the IGF-IR receptor in certain cancers. Lastly, comparison of the antiproliferative effects (IC(50)s) of various compounds in wt vs. null MEFs demonstrates that genetically characterized MEFs provide a simple and inexpensive tool with which to define compounds as having mostly on-target or off-target IGF-IR activities because off-target compounds affect both wt and null MEFs equally.

Antisense oligonucleotides selectively regulate aspartyl beta-hydroxylase and its truncated protein isoform in vitro but distribute poorly into A549 tumors in vivo.

Alternative splicing of the human beta-aspartyl (asparaginyl) hydroxylase (BAH) gene results in the expression of humbug, a truncated form of BAH that lacks the catalytic domain of the enzyme. Overexpression of BAH and humbug has been associated with a variety of human cancers, and although humbug lacks enzymatic activity, it is expressed at levels comparable with that of BAH in various cancer cell lines. Phosphorothioate antisense oligonucleotides (ONs) were designed to dissect out the function of these hydroxylase protein isoforms. In A549 cells, these ONs differentially down-regulated BAH and humbug at the mRNA and protein level. Phosphorothioate ON uptake and antisense studies were conducted in parallel in nude mice bearing A549 tumor xenografts. Microscopic examination of the tumor after administration of a fluorescein-labeled ON showed strong labeling of the outer layers of the tumor connective tissue but cells within the interior of the tumor were sparsely labeled. A modest but significant effect on tumor growth was observed in animals treated with an antisense ON directed against both BAH and humbug transcripts. However, Northern analysis of tumor RNA did not indicate a down-regulation of the targeted mRNA species. These results demonstrate the successful development of antisense ONs that selectively differentiate between the closely related beta-hydroxylase protein isoforms. However, determination of the biological function of these proteins in vivo was limited by the poor uptake properties of phosphorothioate ONs in A549 tumors.

Absence of post-translational aspartyl beta-hydroxylation of epidermal growth factor domains in mice leads to developmental defects and an increased incidence of intestinal neoplasia.

The BAH genomic locus encodes three distinct proteins: junctin, humbug, and BAH. All three proteins share common exons, but differ significantly based upon the use of alternative terminal exons. The biological roles of BAH and humbug and their functional relationship to junctin remain unclear. To evaluate the role of BAH in vivo, the catalytic domain of BAH was specifically targeted such that the coding regions of junctin and humbug remained undisturbed. BAH null mice lack measurable BAH protein in several tissues, lack aspartyl beta-hydroxylase activity in liver preparations, and exhibit no hydroxylation of the epidermal growth factor (EGF) domain of clotting Factor X. In addition to reduced fertility in females, BAH null mice display several developmental defects including syndactyly, facial dysmorphology, and a mild defect in hard palate formation. The developmental defects present in BAH null mice are similar to defects observed in knock-outs and hypomorphs of the Notch ligand Serrate-2. In this work, beta-hydroxylation of Asp residues in EGF domains is demonstrated for a soluble form of a Notch ligand, human Jagged-1. These results along with recent reports that another post-translational modification of EGF domains in Notch gene family members (glycosylation by Fringe) alters Notch pathway signaling, lends credence to the suggestion that aspartyl beta-hydroxylation may represent another post-translational modification of EGF domains that can modulate Notch pathway signaling. Previous work has demonstrated increased levels of BAH in certain tumor tissues and a role for BAH in tumorigenesis has been proposed. The role of hydroxylase in tumor formation was tested directly by crossing BAH KO mice with an intestinal tumor model, APCmin mice. Surprisingly, BAH null/APCmin mice show a statistically significant increase in both intestinal polyp size and number when compared with BAH wild-type/APCmin controls. These results suggest that, in contrast to expectations, loss of BAH catalytic activity may promote tumor formation.