Ajay Bhargava

1-Phenyl-5-pyrazolyl ureas: potent and selective p38 kinase inhibitors

Inhibitors of the MAP kinase p38 are potentially useful for the treatment of arthritis and osteoporosis. Several 2,3-dichlorophenyl ureas were identified as small-molecule inhibitors of p38 by a combinatorial chemistry effort. Optimization for cellular potency led to the discovery of a new class of potent and selective p38 kinase inhibitors, exemplified by the 1-phenyl-5-pyrazolyl urea 7 (IC50 = 13 nM).

Discovery of a new class of p38 kinase inhibitors

The MAP kinase p38 has been implicated in cytokine signaling, and its inhibitors are potentially useful for the treatment of arthritis and osteoporosis. Novel small-molecule inhibitors of p38 kinase were derived from a combinatorial chemistry effort and exhibit activity in the nanomolar range. Very steep structure-activity relationships are observed within this class.

Regorafenib (BAY 73‐4506): Antitumor and antimetastatic activities in preclinical models of colorectal cancer

Regorafenib, a novel multikinase inhibitor, has recently demonstrated overall survival benefits in metastatic colorectal cancer (CRC) patients. Our study aimed to gain further insight into the molecular mechanisms of regorafenib and to assess its potential in combination therapy. Regorafenib was tested alone and in combination with irinotecan in patient‐derived (PD) CRC models and a murine CRC liver metastasis model. Mechanism of action was investigated using in vitro functional assays, immunohistochemistry and correlation with CRC‐related oncogenes. Regorafenib demonstrated significant inhibition of growth‐factor‐mediated vascular endothelial growth factor receptor (VEGFR) 2 and VEGFR3 autophosphorylation, and intracellular VEGFR3 signaling in human umbilical vascular endothelial cells (HuVECs) and lymphatic endothelial cells (LECs), and also blocked migration of LECs. Furthermore, regorafenib inhibited proliferation in 19 of 25 human CRC cell lines and markedly slowed tumor growth in five of seven PD xenograft models. Combination of regorafenib with irinotecan significantly delayed tumor growth after extended treatment in four xenograft models. Reduced CD31 staining indicates that the antiangiogenic effects of regorafenib contribute to its antitumor activity. Finally, regorafenib significantly delayed disease progression in a murine CRC liver metastasis model by inhibiting the growth of established liver metastases and preventing the formation of new metastases in other organs. In addition, our results suggest that regorafenib displays antimetastatic activity, which may contribute to its efficacy in patients with metastatic CRC. Combination of regorafenib and irinotecan demonstrated an increased antitumor effect and could provide a future treatment option for CRC patients.

p38 kinase inhibitors for the treatment of arthritis and osteoporosis: thienyl, furyl, and pyrrolyl ureas.

Inhibitors of the MAP kinase p38 are potentially useful for the treatment for osteoporosis, arthritis, and other inflammatory diseases. A series of thienyl, furyl, and pyrrolyl ureas has been identified as potent p38 inhibitors, displaying in vitro activity in the nanomolar range.

Discovery and Parallel Synthesis of a New Class of Cathepsin K Inhibitors

Peptidomimetic aminomethyl ketones have been identified as a new class of cathepsin K inhibitors. Traditional and high-speed parallel synthesis techniques were applied to investigate this series. Structure-activity relationships were established, and certain analogues were characterized with IC(50) values in the range 200-500 nM.

Macrocyclic MEK1/2 inhibitor with efficacy in a mouse model of cardiomyopathy caused by lamin A/C gene mutation

Signaling mediated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) is involved in numerous cellular processes. Mitogen-activated protein kinase kinases (MEK1/2) catalyze the phosphorylation of ERK1/2, converting it into an active kinase that regulates the expression of numerous genes and cellular processes. Inhibitors of MEK1/2 have demonstrated preclinical and clinical efficacy in certain cancers and types of cardiomyopathy. We report the synthesis of a novel, allosteric, macrocyclic MEK1/2 inhibitor that potently inhibits ERK1/2 activity in cultured cells and tissues of mice after systemic administration. Mice with dilated cardiomyopathy caused by a lamin A/C gene mutation have abnormally increased cardiac ERK1/2 activity. In these mice, this novel MEK1/2 inhibitor is well tolerated, improves left ventricular systolic function, decreases left ventricular fibrosis, has beneficial effects on skeletal muscle structure and pathology and prolongs survival. The novel MEK1/2 inhibitor described herein may therefore find clinical utility in the treatment of this rare cardiomyopathy, other types of cardiomyopathy and cancers in humans.

Pharmacologic activity and pharmacokinetics of metabolites of regorafenib in preclinical models

Regorafenib is an orally administered inhibitor of protein kinases involved in tumor angiogenesis, oncogenesis, and maintenance of the tumor microenvironment. Phase III studies showed that regorafenib has efficacy in patients with advanced gastrointestinal stromal tumors or treatment‐refractory metastatic colorectal cancer. In clinical studies, steady‐state exposure to the M‐2 and M‐5 metabolites of regorafenib was similar to that of the parent drug; however, the contribution of these metabolites to the overall observed clinical activity of regorafenib cannot be investigated in clinical trials. Therefore, we assessed the pharmacokinetics and pharmacodynamics of regorafenib, M‐2, and M‐5 in vitro and in murine xenograft models. M‐2 and M‐5 showed similar kinase inhibition profiles and comparable potency to regorafenib in a competitive binding assay. Inhibition of key target kinases by all three compounds was confirmed in cell‐based assays. In murine xenograft models, oral regorafenib, M‐2, and M‐5 significantly inhibited tumor growth versus controls. Total peak plasma drug concentrations and exposure to M‐2 and M‐5 in mice after repeated oral dosing with regorafenib 10 mg/kg/day were comparable to those in humans. In vitro studies showed high binding of regorafenib, M‐2, and M‐5 to plasma proteins, with unbound fractions of ~0.6%, ~0.9%, and ~0.4%, respectively, in murine plasma and ~0.5%, ~0.2%, and ~0.05%, respectively, in human plasma. Estimated free plasma concentrations of regorafenib and M‐2, but not M‐5, exceeded the IC50 at human and murine VEGFR2, suggesting that regorafenib and M‐2 are the primary contributors to the pharmacologic activity of regorafenib in vivo.

Inhibition of hypoxia-induced gene transcription by substituted pyrazolyl oxadiazoles: initial lead generation and structure-activity relationships.

The transcription factors hypoxia‐inducible factor‐1 and ‐2 (HIF‐1 and HIF‐2) orchestrate a multitude of processes that allow tumor cells to survive under conditions of low oxygen and nutrients, and that lead to resistance to some apoptotic pathways and facilitate invasion and metastasis. Therefore, inhibition of transactivation by HIF has become an attractive target in cancer research. Herein we present the results of a cell‐based screening approach that led to the discovery of substituted 1H‐pyrazole‐3‐carboxamides. Chemical optimization of the hit class with respect to potency and metabolic stability is described; it resulted in novel 5‐(1H‐pyrazol‐3‐yl)‐1,2,4‐oxadiazoles that inhibit the hypoxia‐induced accumulation of HIF‐1α and HIF‐2α. The HIF inhibitory potency in the screening cell system was improved from IC50 190 to 0.7 nM, and significant parts of the SAR are disclosed. For a key compound, the ability to suppress the hypoxia‐induced expression of HIF target genes was studied in A549 human lung adenocarcinoma cells. The same compound shows a favorable pharmacokinetic profile in rats after i.v. and p.o. administration.

Abstract 2337: Regorafenib (BAY 73-4506): Anti-metastatic activity in a mouse model of colorectal cancer

Regorafenib is an oral multi-kinase inhibitor which has recently been reported to extend overall survival in a Phase III trial in patients with metastatic colorectal tumors who have failed standard therapies (CORRECT trial) when given with best supportive care (BSC) vs placebo plus BSC. It is also examined in a Phase III trial for the treatment of patients with metastatic and/or unresectable gastrointestinal stromal tumors who have failed imatinib and sunitinib therapy (GRID trial). Regorafenib deactivates tumors across three dimensions (angiogenesis, oncogenesis, and stromatogenesis) by inhibition of angiogenic receptor tyrosine kinases (RTK) (e.g., VEGFR1-3, TIE2), oncogenic (e.g., KIT, RET) and stromal RTKs (e.g., PDGFR, FGFR). In preclinical studies it was previously shown that regorafenib inhibits a broad spectrum of different tumor types including colorectal cancer (CRC) models. Regorafenib was efficacious as a single agent and in combination with irinotecan in CRC models and demonstrated anti-metastatic activities in preclinical models of breast cancer. VEGFR-3, a member of the VEGF receptor family, is thought to play an important role in tumor lymphangiogenesis and metastasis. Regorafenib potently inhibited (4-8 nM) VEGF-C stimulated VEGFR-3 receptor autophosphorylation in human dermal lymphatic endothelial cells (LECs) and inhibited VEGF-C stimulated LEC cell migration in a scratch assay at concentrations below 100 nM. In vivo regorafenib9s antimetastatic potential was examined in a syngeneic CRC liver metastasis model. In this model murine MC38 CRC cells were injected into the mouse spleen with following ablation of the organ. Significant survival of mice treated orally at a daily dose of 10 mg/kg regorafenib was observed compared to vehicle-treated control mice. Liver weights, which were used as read-out for the degree of metastases formation, was reduced in mice treated with regorafenib for thirteen days vs vehicle treated animals. This was accompanied by significantly reduced number of metastases observed outside the liver. Finally, the direct effects of regorafenib in CRC cell lines was evaluated in vitro in a panel of 25 different human CRC cell lines where it inhibited the proliferation of 19 cell lines with IC50 values between 1 and 10µM and no correlation with mutated KRAS or BRAF was observed. Effects of regorafenib on intracellular MAPK signalling in CRC cell lines was analyzed by western blotting. Regorafenib, which also inhibits Raf kinases, reduced pERK by 50% at concentrations of about 500-2000 nM in HT29, HCT116 and Colo-205 CRC cell lines. In summary, these results suggest that regorafenib may exert its anti-tumor activity in colorectal cancer by inhibition of multiple kinases across its three dimensions within the tumor environment. Additional analyses in particular of clinical tumor samples are necessary to more clearly understand regorafenib9s molecular mechanisms of action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2337. doi:1538-7445.AM2012-2337

Abstract 742: Comparison of commercially available phosphorylated ERK antibodies for immunohistochemical biomarker analysis

Immunohistochemical (IHC) analysis of ERK phosphorylation is commonly performed on pre- and post-treatment cancer biopsies as part of pharmacodynamic biomarker assessments for targeted oncology therapeutics. The current study was performed to assess the specificity and sensitivity of four commercially-available pERK antibodies for IHC analysis, which included three monoclonal antibodies (clones 20G11, MAPK-YT, D13.14.4E) and one polyclonal antibody. To facilitate this analysis, the HEPG2 liver cancer cell line was grown in the presence or absence of inhibitors of the RAF/MEK/ERK pathway (U0126 or sorafenib). Cell lysates were prepared for analysis via Western blotting and formalin-fixed, paraffin-embedded cell pellets were prepared for analysis via IHC. All antibodies produced bands at 44 and 42 kDa, the expected molecular weights of ERK1/2, when used for Western analysis of untreated cells and demonstrated a reduction or ablation of these bands following treatment of cells with inhibitors of the RAF/MEK/ERK pathway. When examined via IHC, the monoclonals demonstrated nuclear and cytoplasmic staining in HEPG2 cells with increased sensitivity relative to the polyclonal antibody. The monoclonals also demonstrated greatly reduced staining in HEPG2 cells treated with inhibitors of the RAF/MEK/ERK pathway. These antibodies were further characterized for reactivity in human tumor specimens representing breast cancer, colon cancer, lung cancer, melanoma, and ovarian cancer. Melanoma tissues were stained with a red chromogen; all others were stained with DAB. Differences in the frequency of expression, percentage of expression, and protein localization were identified between the three monoclonals. In summary, this work has identified commercially-available antibodies that appear to be of suitable specificity and sensitivity for use in IHC assays. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 742. doi:1538-7445.AM2012-742