Novel tight-binding inhibitory factor-kappaB kinase (IKK-2) inhibitors demonstrate target-specific anti-inflammatory activities in cellular assays and following oral and local delivery in an in vivo model of airway inflammation.

Nuclear factor-kappaB (NF-kappaB) is one of the major families of transcription factors activated during the inflammatory response in asthma and chronic obstructive pulmonary disease. Inhibitory factor-kappaB kinase 2 (IKK-2) has been shown to play a pivotal role in cytokine-induced NF-kappaB activation in airway epithelium and in disease-relevant cells. Nevertheless, the potential toxicity of specific IKK-2 inhibitors may be unacceptable for oral delivery in chronic obstructive pulmonary disease. Therefore, local delivery to the lungs is an attractive alternative that warrants further exploration. Here, we describe potent and selective small-molecule IKK-2 inhibitors [8-(5-chloro-2-(4-methylpiperazin-1-yl)isonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-carboxamide (PHA-408) and 8-(2-(3,4-bis(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-5-chloroisonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo-[g]indazole-3-carboxamide (PF-184)] that are competitive for ATP have slow off-rates from IKK-2 and display broad in vitro anti-inflammatory activities resulting from NF-kappaB pathway inhibition. Notably, PF-184 has been designed to have high systemic clearance, which limits systemic exposure and maximizes the effects locally in the airways. We used an inhaled lipopolysaccharide-induced rat model of neutrophilia to address whether inhibiting NF-kappaB activation locally within the airways would show anti-inflammatory effects in the absence of systemic exposure. PHA-408, a low-clearance compound previously shown to be efficacious orally in a rodent model of arthritis, dose-dependently attenuated inhaled lipopolysaccharide-induced cell infiltration and cytokine production. Interestingly, PF-184 produced comparable dose-dependent anti-inflammatory activity by intratracheal administration and was as efficacious as intratracheally administered fluticasone propionate (fluticasone). Together, these results support the potential therapeutic utility of IKK-2 inhibition in inflammatory pulmonary diseases and demonstrate anti-inflammatory efficacy of an inhaled IKK-2 inhibitor in a rat airway model of neutrophilia.

Synthesis of pyrazoles and isoxazoles as potent alpha(v)beta3 receptor antagonists.

We describe a series of pyrazole and isoxazole analogs as antagonists of the alpha(v)beta3 receptor. Compounds showed low to sub-nanomolar potency against alpha(v)beta3, as well as good selectivity against alpha(IIb)beta3. In HT29 cells, most analogs also demonstrated significant selectivity against alpha(v)beta6. Several compounds showed good pharmacokinetic properties in rats, in addition to anti-angiogenic activity in a mouse corneal micropocket model. Compounds were synthesized in a straightforward manner from readily available glutarate precursors.

Assessment of anti-metastatic drug efficacy via localization and quantification of ex vivo murine bone tumor load using high-throughput MRI T1 parametric analysis.

MRI methods show great utility for assessing the growth of tumors metastasized to bone in clinical studies. However, preclinical MRI methods in rodents do not translate well to high-throughput studies of bone tumors, especially for early-stage tumors typically examined in pharmaceutical discovery efforts. To overcome these limitations, an ex vivo MR T1 parametric mapping method has been developed to measure metastasized bone tumor load in murine long bones. This method has been used to assess the therapeutic efficacy of SU11248, a multi-targeted inhibitor with demonstrated anti-tumor activity and reduction of bone loss, in a murine model of metastasized breast tumor cells. The results show precise localizations of relative tumor loads within the bones and reveal significant differences between SU11248-treated and untreated animal groups. The procedures were optimized for simultaneous, high-throughput parallel image acquisition of MRI data for 30 samples and included an automated segmentation method for image processing. The merits of this T1 parametric mapping method are compared with clinical T1-weighted MRI methods, histopathology and bioluminescence imaging of the same murine bone tumor model.

A small molecule antagonist of the alpha(v)beta3 integrin suppresses MDA-MB-435 skeletal metastasis.

INTRODUCTION: Breast cancer is one of the most common malignancies affecting women in the United States and Europe. Approximately three out of every four women with breast cancer develop metastases in bone which, in turn, diminishes quality of life. The alpha(v)beta3 integrin has previously been implicated in multiple aspects of tumor progression, metastasis and osteoclast bone resorption. Therefore, we hypothesized that the alpha(v)beta3-selective inhibitor, S247, would decrease the development of osteolytic breast cancer metastases. MATERIALS AND METHODS: Cells were treated in vitro with S247 and assessed for viability and adhesion to matrix components. Athymic mice received intracardiac (left ventricle) injections of human MDA-MB-435 breast carcinoma cells expressing enhanced green-fluorescent protein. Mice were treated with vehicle (saline) or S247 (1, 10, or 100 mg/kg/d) using osmotic pumps beginning either one week before or one week after tumor cell inoculation. Bones were removed and examined by fluorescence microscopy and histology. The location and size of metastases were recorded. RESULTS AND CONCLUSIONS: IC50 for S247 adhesion to alpha(v)beta3 or alpha(IIB)beta3a substrates was 0.2 nM vs. 244 nM, respectively. Likewise, S247 was not toxic at doses up to 1000 microM. However, osteoclast cultures treated with S247 exhibited marked morphological changes and impaired formation of the actin sealing zone. When S247 was administered prior to tumor cells, there was a significant, dose-dependent reduction (25-50% of vehicle-only-treated mice; P = 0.002) in osseous metastasis. Mice receiving S247 after tumor cell inoculation also developed fewer bone metastases, but the difference was not statistically significant. These data suggest that, in the MDA-MB-435 model, the alpha(v)beta3 integrin plays an important role in early events (e.g., arrest of tumor cells) in bone metastasis. Furthermore, the data suggest that alpha(v)beta3 inhibitors may be useful in the treatment and/or prevention of breast cancer metastases in bone.