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Although approved BRAF inhibitors have transformed the treatment of patients with certain BRAF V600 mutant cancers, their long-term efficacy is thought to be limited by poor brain penetration. As a result, disease progression in the brain is a significant cause of morbidity and mortality. PF-07284890 (ARRY-461) is an orally bioavailable, brain penetrant, potent, small molecule inhibitor targeting BRAF V600 mutant tumors and is in clinical development in patients with BRAF V600 metastatic melanoma with progression to the brain. In biochemical in vitro studies, PF-07284890 inhibits BRAF and CRAF with IC50's of 5.8 and 4.1 nM, respectively. Additionally, PF-07284890 inhibits the clinically relevant kinase domain BRAF V600 mutants (V600E and V600K) (IC50 = 24-25 nM). In cell-based systems, PF-07284890 potently inhibits phosphorylation of ERK, a downstream marker of BRAF inhibition, and potently inhibits proliferation of BRAF V600E/K mutant melanoma cell lines (IC50 18-38 nM). PF-07284890 was designed to distribute to the brain and, as such, in vitro experiments indicate that PF-07284890 has high cellular membrane permeability and is not a substrate for human P glycoprotein (P-gp). After oral administration of PF-07284890 to mice and rats, PF-07284890 distributes to the brain where the free fraction adjusted exposure in the brain was approximately proportional to the free fraction adjusted exposure in plasma (i.e., Cbrain,u/Cplasma,u approaches 1.0). In vivo, PF-07284890 inhibits phosphorylation of ERK in A375 BRAF V600E tumors, achieving maximal target inhibitions at a dose of 10 mg/kg. PF-07284890 has been evaluated for its ability to control BRAF V600E cell line and patient-derived melanoma xenograft tumor growth in nude mice when implanted both subcutaneously and intracranially. Dose-related tumor growth inhibition was demonstrated at dose levels ranging from 1 to 30 mg/kg, BID in both subcutaneous and intracranial xenograft models. In all models, regardless of tumor location, maximal efficacy was observed in dose ranges of 10-30 mg/kg BID. In the intracranial A375-luc BRAF V600E melanoma xenograft model, significant and durable tumor regressions were seen at doses of 10 and 30 mg/kg/day which translated to a profound survival benefit in animals receiving PF-07284890 (median survival > 55 days post-implantation) compared to control animals (median survival = 15 days). GLP safety studies demonstrated a good safety profile for PF-07284890. PF-07284890 is fully brain penetrant, with the potential to address this key unmet medical need and thereby defines a new class of brain penetrant, potent and selective BRAF inhibitors. Citation Format: Karyn Bouhana, Deborah Anderson, Walter DeWolf, Suzy Brown, Lance Williams, Li Ren, David Moreno, Ross Wallace, Jay Brad Fell, Dylan Hartley, Patrice Lee. Nonclinical development of PF-07284890 (ARRY-461), a potent, brain-penetrant, small molecule inhibitor of BRAF V600-mutation-driven tumors in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1473.

HER2 is a transmembrane tyrosine kinase receptor that mediates cell growth, differentiation, and survival. HER2 is overexpressed in approximately 20% of breast cancers and in subsets of gastric, colorectal, and esophageal cancers. Both antibody and small-molecule drugs that target HER2 and block its tyrosine kinase activity are effective in treating HER2-driven cancers. In this article, we describe the preclinical properties of tucatinib, an orally available, reversible HER2-targeted small-molecule tyrosine kinase inhibitor. In both biochemical and cell signaling experiments, tucatinib inhibits HER2 kinase activity with single-digit nanomolar potency and provides exceptional selectivity for HER2 compared with the related receptor tyrosine kinase EGFR, with a >1,000-fold enhancement in potency for HER2 in cell signaling assays. Tucatinib potently inhibits signal transduction downstream of HER2 and HER3 through the MAPK and PI3K/AKT pathways and is selectively cytotoxic in HER2-amplified breast cancer cell lines in vitro. In vivo, tucatinib is active in multiple HER2+ tumor models as a single agent and shows enhanced antitumor activity in combination with trastuzumab or docetaxel, resulting in improved rates of partial and complete tumor regression. These preclinical data, taken together with the phase-I tucatinib clinical trial results demonstrating preliminary safety and activity, establish the unique pharmacologic properties of tucatinib and underscore the rationale for investigating its utility in HER2+ cancers.

Current therapies for treating skeletal pain have significant limitations as available drugs (non-steroidal anti-inflammatory drugs and opiates) have significant unwanted side effects. Targeting nerve growth factor (NGF) or its cognate receptor tropomysin receptor kinase A (TrkA) has recently become an attractive target for inhibition of adult skeletal pain. Here we explore whether sustained administration of a selective small molecule Trk inhibitor that blocks TrkA, TrkB and TrkC kinase activity with nanomolar affinity reduces skeletal pain while allowing the maintenance of sensory and sympathetic neurons in the adult mouse. Twice-daily administration of a Trk inhibitor was begun 1 day post fracture and within 8 h of acute administration fracture pain-related behaviors were reduced by 50% without significant sedation, weight gain or inhibition of fracture healing. Following administration of the Trk inhibitor for 7 weeks, there was no significant decline in the density of unmyelinated or myelinated sensory nerve fibers, sympathetic nerve fibers, measures of acute thermal pain, acute mechanical pain, or general neuromuscular function. The present results suggest that sustained administration of a peripherally selective TrkA, B and C inhibitor significantly reduces skeletal pain without having any obvious detrimental effects on adult sensory and sympathetic nerve fibers or early fracture healing. As with any potential therapeutic advance, understanding whether the benefits of Trk blockade are associated with any risks or unexpected effects will be required to fully appreciate the patient populations that may benefit from this therapeutic approach.

Tumor cells extensively utilize the pentose phosphate pathway for the synthesis of ribose. Transketolase is a key enzyme in this pathway and has been suggested as a target for inhibition in the treatment of cancer. In a pharmacodynamic study, nude mice with xenografted HCT-116 tumors were dosed with 1 ('N3'-pyridyl thiamine'; 3-(6-methyl-2-amino-pyridin-3-ylmethyl)-5-(2-hydroxy-ethyl)-4-methyl-thiazol-3-ium chloride hydrochloride), an analog of thiamine, the co-factor of transketolase. Transketolase activity was almost completely suppressed in blood, spleen, and tumor cells, but there was little effect on the activity of the other thiamine-utilizing enzymes alpha-ketoglutarate dehydrogenase or glucose-6-phosphate dehydrogenase. Synthesis and SAR of transketolase inhibitors is described.

Transketolase, a key enzyme in the pentose phosphate pathway, has been suggested as a target for inhibition in the treatment of cancer. Compound 5a ('N3'-pyridyl thiamine'; 3-(6-methyl-2-amino-pyridin-3-ylmethyl)-5-(2-hydroxy-ethyl)-4-methyl-thiazol-3-ium chloride hydrochloride), an analog of the transketolase cofactor thiamine, is a potent transketolase inhibitor but suffers from poor pharmacokinetics due to high clearance and C(max) linked toxicity. An efficient way of improving the pharmacokinetic profile of 5a is to prepare oxidized prodrugs which are slowly reduced in vivo yielding longer, sustained blood levels of the drug. The synthesis of such prodrugs and their evaluation in rodent models is reported.

Introduction: Monocyte/macrophage lineage cells have been reported to provide a supportive signal in a variety of neoplastic settings. Tumor-associated macrophages (TAMs) have been shown to provide microenvironmental support that maintains tumor cell viability and growth for a variety of solid tumor types, and these TAMs have been shown to depend on the receptor tyrosine kinase, CSF1R (M-CSFR). Monocyte/macrophage lineage cells have also been implicated in the microenvironment of CLL and are termed nurse-like cells in this setting. However, the role of CSF1R in CLL including potential maintenance of these nurse-like cells has not been explored. Using an ex vivo, functional screening platform applied directly to primary specimens from CLL patients, we have identified recurrent sensitivity to CSF1R inhibitors as well as decreased viability after depletion of CSF1R-expressing monocytes, thereby depriving the CLL cells of an important, microenvironmental growth/survival signal. We also have seen synergistic anti-tumor activity when combining CSF1R inhibitors with idelaisib and ibrutinib which inhibit b-cell receptor (BCR) activated pathways. Methods: We evaluated the impact on cell viability of hundreds of CLL patient specimens against panels of targeted small molecule inhibitors. These panels include two small-molecules with exquisite specificity for CSF1R (GW-2580; ARRY-382). In addition, we evaluated the impact of antibody depletion of monocytes (CD14-depletion) on ex vivo CLL cell viability as well as the effect of monocyte cell depletion on response to CSF1R inhibitors. We also evaluated the combination of GW-2580 or ARRY-382 with idelalisib (PI3kδ inhibitor) and ibrutinib (BTK inhibitor). Results: We found that 20-30% of CLL specimens showed sensitivity to inhibition of CSF1R with good concordance between GW-2580 and ARRY-382. Analysis of clinical and demographic features of these patients failed to reveal correlation of CSF1R with any prominent disease subsets. Flow cytometry analysis revealed that CSF1R was not expressed on CLL cells but only on a subpopulation of CD14-expressing monocytes. Depletion of these monocytes with CD14 antibody had little to no impact on samples not exhibiting ex vivo sensitivity to CSF1R inhibitors, however, samples showing strong sensitivity to CSF1R inhibitors were also quite sensitive to depletion of this CD14-positive monocyte population. After CD14 depletion, the remaining CLL cells showed no further sensitivity to CSF1R inhibitors. Finally, when combined with ibrutinib or idelalisib synergy was seen. Conclusions: These results show that CSF1R is a potential therapeutic target in CLL and suggest that CLL supporting monocytes (nurse-like cells) express CSF1R and depend on CSF1R for viability in a similar manner as TAMs depend on CSF1R in a variety of solid tumor settings. As such, CSF1R inhibitors may deprive CLL cells of this supportive microenvironmental signal resulting in CLL cell death. Therefore, we propose that CSF1R inhibitors, such as ARRY-382, possibly in combination with ibrutinib, idelalisib, or other approved agents, may be a promising new line of therapy to target CLL cells by impacting the tumor microenvironment. Disclosures Spurgeon: Genentech: Honoraria; Acerta Pharma: Research Funding; Bristol Meyers Squibb: Research Funding; Gilead sciences: Honoraria, Research Funding; Janssen: Research Funding; Pharmacyclics: Honoraria. Tyner:Janssen Pharmaceuticals: Research Funding; Incyte: Research Funding; Aptose Biosciences: Research Funding; Constellation Pharmaceuticals: Research Funding; Array Biopharma: Research Funding. Agarwal:CTI BioPharma: Research Funding. Lee:Array Biopharma: Employment. Chantry:Array Biopharma: Employment. Druker:Fred Hutchinson Cancer Research Center: Research Funding; Bristol-Myers Squibb: Research Funding; Henry Stewart Talks: Patents & Royalties; Millipore: Patents & Royalties; Sage Bionetworks: Research Funding; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy; Novartis Pharmaceuticals: Research Funding; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; CTI Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncotide Pharmaceuticals: Research Funding; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties; ARIAD: Research Funding; Aptose Therapeutics, Inc (formerly Lorus): Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Purpose: The Ras-Raf-mitogen-activated protein kinase kinase (MEK) pathway is overactive in many human cancers and is thus a target for novel therapeutics. We have developed a highly potent and selective inhibitor of MEK1/2. The purpose of these studies has been to show the biological efficacy of ARRY-142886 (AZD6244) in enzymatic, cellular, and animal models. Experimental Design: The ability of ARRY-142886 to inhibit purified MEK1 as well as other kinases was evaluated. Its effects on extracellular signal-regulated kinase (ERK) phosphorylation and proliferation in several cell lines were also determined. Finally, the inhibitor was tested in HT-29 (colorectal) and BxPC3 (pancreatic) xenograft tumor models. Results: The IC50 of ARRY-142886 was determined to be 14 nmol/L against purified MEK1. This activity is not competitive with ATP, which is consistent with the high specificity of compound for MEK1/2. Basal and epidermal growth factor–induced ERK1/2 phosphorylation was inhibited in several cell lines as well as 12-O-tetradecanoylphorbol-13-acetate–induced ERK1/2 phosphorylation in isolated peripheral blood mononuclear cells. Treatment with ARRY-142886 resulted in the growth inhibition of several cell lines containing B-Raf and Ras mutations but had no effect on a normal fibroblast cell line. When dosed orally, ARRY-142886 was capable of inhibiting both ERK1/2 phosphorylation and growth of HT-29 xenograft tumors in nude mice. Tumor regressions were also seen in a BxPC3 xenograft model. In addition, tumors remained responsive to growth inhibition after a 7-day dosing holiday. Conclusions: ARRY-142886 is a potent and selective MEK1/2 inhibitor that is highly active in both in vitro and in vivo tumor models. This compound is currently being investigated in clinical studies.

The role of MEK 1,2 in cancer tumorgenesis has been clearly demonstrated preclinically, and two selective inhibitors are currently undergoing clinical evaluation to determine their role in the human disease. We have discovered 4-(4-bromo-2-fluorophenylamino)-1-methylpyridin-2(1H)-ones as a new class of ATP noncompetitive MEK inhibitors. These inhibitors exhibit excellent cellular potency and good pharmacokinetic properties and have demonstrated the ability to inhibit ERK phosphorylation in HT-29 tumors from mouse xenograft studies.

A nuclease resistant ribozyme targeting the 5′ untranslated region (5′ UTR) of hepatitis C virus (HCV) at site 195 has been identified. To investigate the therapeutic utility of this ribozyme, we evaluated the pharmacokinetics and tissue distribution with two labeled forms of this ribozyme. [32P]-labeled ribozyme was administered as a single subcutaneous (SC) or intravenous (IV) bolus at a dose of 10 mg/kg or 30 mg/kg in C57Bl/6 mice. Regardless of route of administration, peak liver concentrations achieved were greater than the concentration necessary to inhibit HCV-IRES-luciferase expression in cell culture. The ribozyme was well absorbed after SC administration (89%) and had an elimination half-life of 23 minutes. To show intracellular localization of the ribozyme in target tissue, a tetramethyl rhodamine (TMR)–labeled ribozyme was administered as a single SC or IV bolus at a dose of 30 mg/kg in C57Bl/6 mice. Mice treated SC or IV with TMR–labeled ribozyme had positive fluorescence in the liver from 15 minutes to 48 hours after dosing. Definite positive fluorescence was still present at 72 hours in the mice dosed via the IV route. At early time points (15 and 30 minutes postinjection), nuclear and possibly cytoplasmic fluorescence was present in the hepatocytes, and sinusoidal fluorescence was intense. At the later time points, fluorescence became more punctate. Abundant staining was often present in Kupffer cells. This study confirms the retention of ribozyme in liver cells and supports the potential of an anti-HCV ribozyme as a therapeutic agent for treatment of chronic hepatitis C. (Hepatology 2000;32:640-646.)

The Trk family of genes, which includes trkA/NTRK1, trkB/NTRK2 and trkC/NTRK3, encode the tyrosine kinase receptors for the neurotrophin family of nerve growth factors. Deregulated kinase activity of trk family members is associated with human cancer. Oncogenic translocations involving trkC kinase domain have been identified in AML, salivary gland carcinoma, adult secretory breast cancer, congenital fibrosarcoma, and pediatric nephroma. Oncogenic trkA translocations have been reported in papillary thyroid and colorectal cancers. We have identified orally bioavailable, potent and selective ATP-competitive inhibitors of the trk family of receptor tyrosine kinases and are developing these for the treatment of Trk-driven cancers. AR523 is a pan-Trk inhibitor which demonstrates similar activity against TrkA, TrkB and TrkC receptors in a cell based assay (IC50 ∼10 nM). In a screen at 1 µM against a panel of 230 kinases, AR523 inhibited only three additional kinases more than 50% (TNK2, Bmx and Txk). In cell culture AR523 was found to exhibit selective anti-proliferative activity toward the cell line KM12, which harbors the oncogenic TPM-TrkA translocation, while exhibiting no activity toward HT29, a cell line with no trk gene rearrangements. Studies in mice engrafted with KM12 tumors revealed significant anti-tumor and pharmacodynamic activity of AR523. Administration of a single oral dose of AR523 reduced tyrosine phosphorylation of TrkA by nearly 80% at twelve hours after dosing. Parallel analysis of Akt and Erk revealed reduced phosphorylation of these downstream effectors. Administration of 100 mg AR523 daily for two weeks to mice with KM12 xenografts produced mean tumor growth inhibition of 86% and a mean of 42% tumor regression. AR523 exhibits dose dependent inhibition of tumor growth at 10 and 30 mg/kg in KM12 xenografts with 54 and 72% TGI respectively. AR523 was well-tolerated, causing no weight loss or deaths compared with vehicle control. Increasingly Trk mutations have been shown to be activating and the use of Trk inhibitors may provide a new therapeutic strategy for targeted treatment. 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 1798. doi:1538-7445.AM2012-1798

The RAS/RAF/MEK/ERK and PI3K signaling pathways are known to play key roles in cell growth, proliferation and survival. Targeted agents against factors in these pathways have been shown to provide therapeutic benefit in the treatment of cancer. However, there exists a considerable degree of cross-talk between these pathways such that when one pathway is inhibited the other is upregulated to sustain growth and survival signaling. Therefore, it would be expected that the concomitant use of targeted agents against both of these pathways would overcome the negative effects of this crosstalk, and enhance the ability of these agents to block cancer cell growth. We sought to understand whether or not the combination of the MEK inhibitor ARRY-438162 and the TORC1 inhibitor Everolimus would combine to enhance cell killing compared to the activity of the single agents. It has been established that in the majority of cancers, constituents of one or both of these pathways are mutated conferring constitutive activation to the pathway. Thus, we investigated whether key common mutations in either of these pathways conferred particular sensitivity or resistance to the combination. The growth inhibitory activities of ARRY-438162 and Everolimus were assessed alone and in combination in 26 different cell lines from a variety of human cancers with documented mutations in the KRAS, PTEN, PI3K and BRAF genes. In this regard, we report the identification of key genotypes that are associated with antagonistic, additive, and synergistic responses to the combination. In particular, treatment of the NCI-H460 NSCLC line [KRASG12D/PIK3CA] was only modestly responsive to either ARRY-162 or Everolimus as single agents (10% and 20% growth inhibition, respectively). However, the combination significantly inhibited the growth of this line, a result that was confirmed as synergistic by Bliss analysis. In order to validate the relevance of our in vitro data, an in vivo study was performed using NCI-H460 xenografts in nude mice. Everolimus alone showed no tumor growth inhibition while ARRY-438162 at doses of 30, 100 and 300 mg/kg, BID produced dose-related modest tumor growth inhibition (up to ∼50%). Combining the agents produced enhanced activity at all dose levels of ARRY-438162 without altering the exposure of either agent. Finally, in addition to growth inhibition, the biochemical effects of the single agent and the combination treatment on S6 protein, Erk, Akt, and various apoptosis regulator proteins were assessed as well as their effects on apoptosis and autophagy. ARRY-438162 having demonstrated significant tumor growth inhibitory activity in numerous pre-clinical models and acceptable preclinical and clinical safety profiles has advanced into Phase 1 clinical development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3855.

Activation of redundant receptor tyrosine kinases (RTKs) in tumors can confer resistance to selective RTK inhibitors. In particular, activation of multiple ErbB-family kinases is associated with diminished activity of selective EGFR or ErbB2 inhibitors. ErbB-family ligands are commonly expressed in cancers and can activate multiple redundant ErbB dimers. We hypothesize that ligand activation of multiple ErbB-family kinases in tumors can confer resistance to selective ErbB inhibitors, but not pan-ErbB inhibitors, suggesting a setting where pan-ErbB inhibitors may be advantageous over selective inhibitors. One such candidate currently in Phase 1/2 clinical trials is ARRY-334543, an oral, reversible pan-ErbB inhibitor with potent in vivo activity in both EGFR- and ErbB2-dependent human tumor xenografts. We tested this hypothesis in vitro with N87 gastric carcinoma cells that express high levels of constitutively active ErbB2 and low levels of EGFR. In the absence of EGF, ErbB2 homodimers are predominant and cells are highly sensitive to ErbB2 inhibition. ARRY-334543 and the ErbB2-selective inhibitor, ARRY-380, strongly inhibited N87 proliferation (IC50 = 60 nM and 5 nM, respectively) while the EGFR-selective inhibitor erlotinib was active only at high concentrations (IC50 = 860 nM). No benefit was observed by combining ARRY-380 and erlotinib. EGF-stimulation of N87 cells caused the formation of EGFR homodimers and EGFR/ErbB2 heterodimers which significantly alters cellular responses to selective ErbB inhibitors. The cellular IC50 for ARRY-380 increased 57-fold while erlotinib activity was relatively unchanged, suggesting a loss of dependence on ErbB2 signaling alone. The dual inhibition of EGFR and ErbB2, either by ARRY-334543 or the combination of ARRY-380 and erlotinib, largely maintained potency with only a modest increase in IC50 (9- to 10-fold). These data suggest that activation of EGFR can significantly diminish the activity of selective ErbB inhibitors, but not pan-ErbB inhibitors. Dual-responsiveness was also observed in vivo where both EGFR and ErbB2 homodimers were detected in N87 xenografts and dual EGFR/ErbB2 treatment resulted in superior tumor growth inhibition (TGI). ARRY-334543 was highly active with 90% TGI (100 mg/kg BID). Treatment with the combination of ARRY-380 and erlotinib (50 mg/kg QD each, MTD when given in combination) had similar activity with 82% TGI. Treatment with either erlotinib (100 mg/kg QD) or ARRY-380 (50 mg/kg QD) alone was less efficacious. These results suggest that the pan-ErbB inhibitor, ARRY-334543, can be differentiated from selective ErbB inhibitors by targeting tumors that signal through multiple ErbB-family receptors. A wide range of tumor types has been shown to activate multiple ErbB-family members and clinical evaluation of this hypothesis is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3610.

We report here the biological effects of inhibiting Pim-1, -2 and -3 serine/threonine protein kinases in cells and in vivo using a novel, potent and selective small molecule inhibitor directed against these kinases. In vitro enzyme assays revealed potent inhibition of all three Pim kinase isoforms while maintaining selectivity against more than 220 other protein kinases. In cells, compound treatment produced biological effects consistent with Pim inhibition, including a reduction in BAD protein phosphorylation. Moreover, this biological activity corresponds well with the compound’s anti-proliferative and pro-apoptotic activity in the IL-3 dependent mouse pro-B cell line Ba/F3 and in factor-indpendent Ba/F3 cell lines dependent upon the expression of BCR-Abl or TEL-JAK2 for growth and survival. Importantly, compared to the BCR-Abl driven Ba/F3 cell line, this compound was equally effective at blocking the growth of an Imatinib-resistant BCR-Abl[ T315I]-driven Ba/F3 cell line. Finally, this compound possesses pharmacokinetic properties that predict the potential for in vivo activity. Therefore, we assessed the effect of Pim kinase inhibition on tumor growth in a mouse subcutaneous tumor xenograft model employing a cell line driven by the TEL-JAK2 oncogene. Results demonstrated that the compound significantly inhibited tumor growth in a dose-dependent manner. In terms of potential clinical utility, these data show that Pim kinase inhibition could be an effective therapeutic strategy for numerous hematologic malignancies including chronic and acute myelogenous leukemias, particularly in those patients that have become resistant to existing therapies such as Imatinib.

Multiple Myeloma (MM) is a malignancy characterized by the clonal expansion of plasma cells within the bone marrow microenvironment. Within this compartment, the proliferative capacity of MM cells is enhanced by pro-inflammatory cytokines, and there is growing recognition that bone marrow stromal cells play an important role in the support of MM growth and chemo-resistance. We have identified a series of compounds, of which ARRY-614 is representative, that are potent inhibitors (EC50

Multiple myeloma (MM) is characterized by the expansion of malignant plasma cells within the bone marrow. Their growth, survival, and migration are mediated in part via cytokines. Interleukin 6 (IL-6) is necessary for sustaining the in vitro growth of many MM cell lines and enhancing the proliferation of explanted human myeloma cells. The mitogen-activated protein kinase family member, p38, is activated by cytokines and growth factors and plays a significant role in inflammatory diseases. However, its role in the pathogenesis of multiple myeloma is poorly understood. Specific p38 inhibitors inhibit paracrine MM cell growth which is associated with IL-6 and VEGF secretion from bone marrow stromal cells (BMSCs). Furthermore, p38 inhibition blocks TNF-alpha-induced IL-6 secretion in BMSCs, thereby further inhibiting MM cell growth and survival. Although these data suggest an important role for p38 in MM, the direct effects of p38 inhibiton on MM has not been extensively explored. Therefore, we investigated the effects of p38 inhibition on in vitro and in vivo IL-6 production and MM cell growth in vivo after lipopolysaccaride (LPS) stimulation. LPS has been shown to induce various cytokines, including TNF-alpha and IL-6, via the p38 pathway. ARRY-797, an orally bioavailable, small molecule inhibitor of p38 directly inhibited LPS-induced IL-6 production from RPMI-8226 (IC50 = 100 pM) in vitro. In SCID-beige mice, LPS (3 μg/kg) induced IL-6 (7897 ± 827 pg/mL) and TNF-alpha (1922 ± 282 pg/mL) after 2 hours and these cytokines were inhibited by oral administration of ARRY-797 (30 mg/kg) by 91% and 95%, respectively. In MM xenograft models, ARRY-797 (30 mg/kg, BID, PO) inhibited RPMI 8226 tumor growth by 72% as a single agent and by 56% when LPS was administered to stimulate growth in vivo. In addition, ARRY-797 inhibited LPS-induced phosphorylation of p38 in RPMI-8226 xenografts. Together, these data support a role for p38 in IL-6-mediated growth of multiple myelomas. To our knowledge, ARRY-797 is the first small molecule p38 inhibitor to demonstrate single agent activity in a MM xenograft model and it has been advanced into preclinical development.

Kinesins are eukaryotic microtubule-associated motor proteins. One of these, kinesin spindle protein (KSP) is a mitotic-specific kinesin that plays a key role in spindle pole separation and production of the bipolar spindle, as well as centrosome separation and maturation. As KSP is expressed predominately in proliferating cells and is absent from postmitotic neurons, inhibition of KSP has the potential to provide the antitumor activity of a mitotic inhibitor while avoiding peripheral neuropathy. ARRY-429520 is a member of a series of KSP inhibitors discovered and optimized by structure-based design. It is a potent inhibitor of human KSP (IC50 6 nM), with an EC50 of 1.5 nM for cellular phosphorylation of histone H3 (a pharmacodynamic marker for accumulation of cells in mitosis), and in vitro antiproliferative IC50s ranging from subnanomolar to low single digit nanomolar across a varity of human and murine tumor cell lines. We report here the in vivo characterization of this compound. ARRY-429520 inhibits histone (H3) phosphorylation in tumor xenografts , showing that the compound has pharmacodynamic activity in vivo. As a result, it is a highly efficacious inhibitor of the growth of tumor xenografts. At its maximally tolerated dose in mice of 25 – 30 mg/kg i.p. on a Q4Dx3 schedule, ARRY-429520 caused tumor regressions, with some complete responses, in subcutaneous mouse xenograft models of leukemia: HL-60 (acute promyelocytic leukemia) and K-562 (chronic myelogenous leukemia). The compound was also highly efficacious against other tumor types, including HT-29 (colon), HCT-116 (colon), and A2780 (ovarian). ARRY-429520 also showed activity in intravenous leukemia models. The molecule possess desirable drug-like properties, including high aqueous solubility (> 4 mg/ml at physiological pH), low CYP inhibition (> 25uM for 3A4, 2C9, 1A2, 2D6, and 2C19), and pharmacokinetics favorable for an IV-infused targeted chemotherapeutic.