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Drug discovery research on new pain targets with human genetic validation, including the voltage-gated sodium channel NaV1.7, is being pursued to address the unmet medical need with respect to chronic pain and the rising opioid epidemic. As part of early research efforts on this front, we have previously developed NaV1.7 inhibitory peptide-antibody conjugates with tarantula venom-derived GpTx-1 toxin peptides with an extended half-life (80 h) in rodents but only moderate in vitro activity (hNaV1.7 IC50 = 250 nM) and without in vivo activity. We identified the more potent peptide JzTx-V from our natural peptide collection and improved its selectivity against other sodium channel isoforms through positional analogueing. Here we report utilization of the JzTx-V scaffold in a peptide-antibody conjugate and architectural variations in the linker, peptide loading, and antibody attachment site. We found conjugates with 100-fold improved in vitro potency relative to those of complementary GpTx-1 analogues, but pharmacokinetic and bioimaging analyses of these JzTx-V conjugates revealed a shorter than expected plasma half-life in vivo with accumulation in the liver. In an attempt to increase circulatory serum levels, we sought the reduction of the net +6 charge of the JzTx-V scaffold while retaining a desirable NaV in vitro activity profile. The conjugate of a JzTx-V peptide analogue with a +2 formal charge maintained NaV1.7 potency with 18-fold improved plasma exposure in rodents. Balancing the loss of peptide and conjugate potency associated with the reduction of net charge necessary for improved target exposure resulted in a compound with moderate activity in a NaV1.7-dependent pharmacodynamic model but requires further optimization to identify a conjugate that can fully engage NaV1.7 in vivo.

Inhibitors of the voltage-gated sodium channel NaV1.7 are being investigated as pain therapeutics due to compelling human genetics. We previously identified NaV1.7-inhibitory peptides GpTx-1 and JzTx-V from tarantula venom screens. Potency and selectivity were modulated through attribute-based positional scans of native residues via chemical synthesis. Herein, we report JzTx-V lead optimization to identify a pharmacodynamically active peptide variant. Molecular docking of peptide ensembles from NMR into a homology model-derived NaV1.7 structure supported prioritization of key residues clustered on a hydrophobic face of the disulfide-rich folded peptide for derivatization. Replacing Trp24 with 5-Br-Trp24 identified lead peptides with activity in electrophysiology assays in engineered and neuronal cells. 5-Br-Trp24 containing peptide AM-6120 was characterized in X-ray crystallography and pharmacokinetic studies and blocked histamine-induced pruritis in mice after subcutaneous administration, demonstrating systemic NaV1.7-dependent pharmacodynamics. Our data suggests a need for high target coverage based on plasma exposure for impacting in vivo end points with selectivity-optimized peptidic NaV1.7 inhibitors.

The voltage-gated sodium channel NaV1.7 has received much attention from the scientific community due to compelling human genetic data linking gain- and loss-of-function mutations to pain phenotypes. Despite this genetic validation of NaV1.7 as a target for pain, high quality pharmacological tools facilitate further understanding of target biology, establishment of target coverage requirements and subsequent progression into the clinic. Within the sulfonamide class of inhibitors, reduced potency on rat NaV1.7 versus human NaV1.7 was observed, rendering in vivo rat pharmacology studies challenging. Herein, we report the discovery and optimization of novel benzoxazine sulfonamide inhibitors of human, rat and mouse NaV1.7 which enabled pharmacological assessment in traditional behavioral rodent models of pain and in turn, established a connection between formalin-induced pain and histamine-induced pruritus in mice. The latter represents a simple and efficient means of measuring target engagement.

Because of its strong genetic validation, NaV1.7 has attracted significant interest as a target for the treatment of pain. We have previously reported on a number of structurally distinct bicyclic heteroarylsulfonamides as NaV1.7 inhibitors that demonstrate high levels of selectivity over other NaV isoforms. Herein, we report the discovery and optimization of a series of atropisomeric quinolinone sulfonamide inhibitors [Bicyclic sulfonamide compounds as sodium channel inhibitors and their preparation. WO 2014201206, 2014] of NaV1.7, which demonstrate nanomolar inhibition of NaV1.7 and exhibit high levels of selectivity over other sodium channel isoforms. After optimization of metabolic and pharmacokinetic properties, including PXR activation, CYP2C9 inhibition, and CYP3A4 TDI, several compounds were advanced into in vivo target engagement and efficacy models. When tested in mice, compound 39 (AM-0466) demonstrated robust pharmacodynamic activity in a NaV1.7-dependent model of histamine-induced pruritus (i...

Several reports have recently emerged regarding the identification of heteroarylsulfonamides as NaV1.7 inhibitors that demonstrate high levels of selectivity over other NaV isoforms. The optimization of a series of internal NaV1.7 leads that address a number of metabolic liabilities including bioactivation, PXR activation, as well as CYP3A4 induction and inhibition led to the identification of potent and selective inhibitors that demonstrated favorable pharmacokinetic profiles and were devoid of the aforementioned liabilities. The key to achieving this within a series prone to transporter-mediated clearance was the identification of a small range of optimal cLogD values and the discovery of subtle PXR SAR that was not lipophilicity dependent. This enabled the identification of compound 20, which was advanced into a target engagement pharmacodynamic model where it exhibited robust reversal of histamine-induced scratching bouts in mice.

Drug discovery research on new pain targets with human genetic validation, including the voltage-gated sodium channel Na

Deregulation of the receptor tyrosine kinase mesenchymal epithelial transition factor (MET) has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of compound 23 (AMG 337), which demonstrates nanomolar inhibition of MET kinase activity, desirable preclinical pharmacokinetics, significant inhibition of MET phosphorylation in mice, and robust tumor growth inhibition in a MET-dependent mouse efficacy model.

Efforts to improve upon the physical properties and metabolic stability of Aurora kinase inhibitor 14a revealed that potency against multidrug-resistant cell lines was compromised by increased polarity. Despite its high in vitro metabolic intrinsic clearance, 23r (AMG 900) showed acceptable pharmacokinetic properties and robust pharmacodynamic activity. Projecting from in vitro data to in vivo target coverage was not practical due to disjunctions between enzyme and cell data, complex and apparently contradictory indicators of binding kinetics, and unmeasurable free fraction in plasma. In contrast, it was straightforward to relate pharmacokinetics to pharmacodynamics and efficacy by following the time above a threshold concentration. On the basis of its oral route of administration, a selectivity profile that favors Aurora-driven pharmacology and its activity against multidrug-resistant cell lines, 23r was identified as a potential best-in-class Aurora kinase inhibitor. In phase 1 dose expansion studies with G-CSF support, 23r has shown promising single agent activity.

The voltage-gated sodium channel Na

The voltage-gated sodium channel Na

Because of its strong genetic validation, Na

Several reports have recently emerged regarding the identification of heteroarylsulfonamides as Na

Human genetic evidence has identified the voltage-gated sodium channel Na

The impact of inhibitor depletion on the determination of shifted IC₅₀ (IC₅₀ determined after 30 minutes of preincubation with inhibitor) is examined. In addition, IC₅₀-shift data are analyzed using a mechanistic model that incorporates the processes of inhibitor depletion, as well as reversible and time-dependent inhibition. Anomalies such as a smaller-than-expected shift in IC₅₀ and even increases in IC₅₀ with preincubation were explained by the depletion of inhibitor during the preincubation. The IC₅₀-shift assay remains a viable approach to characterizing a wide range of reversible and time-dependent inhibitors. However, as with more traditional time-dependent inactivation methods, it is recommended that IC₅₀-shift experimental data be interpreted with some knowledge of the magnitude of inhibitor depletion. For the most realistic classification of time-dependent inhibitors using IC₅₀-shift methods, shifted IC₅₀ should be calculated using observed inhibitor concentrations at the end of the incubation rather than nominal inhibitor concentrations. Finally, a mechanistic model that includes key processes, such as competitive inhibition, enzyme inactivation, and inhibitor depletion, can be used to describe accurately the observed IC₅₀ and shifted IC₅₀ curves. For compounds showing an IC₅₀ fold shift >1.5 based on the observed inhibitor concentrations, reanalyzing the IC₅₀-shift data using the mechanistic model appeared to allow for reasonable estimation of Ki, KI, and kinact directly from the IC₅₀ shift experiments.

Human genetic evidence has identified the voltage-gated sodium channel NaV1.7 as an attractive target for the treatment of pain. We initially identified naphthalene sulfonamide 3 as a potent and selective inhibitor of NaV1.7. Optimization to reduce biliary clearance by balancing hydrophilicity and hydrophobicity (Log D) while maintaining NaV1.7 potency led to the identification of quinazoline 16 (AM-2099). Compound 16 demonstrated a favorable pharmacokinetic profile in rat and dog and demonstrated dose-dependent reduction of histamine-induced scratching bouts in a mouse behavioral model following oral dosing.

Deregulation of the receptor tyrosine kinase mesenchymal epithelial transition factor (MET) has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of compound 23 (AMG 337), which demonstrates nanomolar inhibition of MET kinase activity, desirable preclinical pharmacokinetics, significant inhibition of MET phosphorylation in mice, and robust tumor growth inhibition in a MET-dependent mouse efficacy model.

Deregulation of the receptor tyrosine kinase c-Met has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of a structurally diverse series of carbon-linked quinoline triazolopyridinones, which demonstrates nanomolar inhibition of c-Met kinase activity. This novel series of inhibitors exhibits favorable pharmacokinetics as well as potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver pharmacodynamic model.

Compound 1, (7-methoxy-N-((6-(3-methylisothiazol-5-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)methyl)-1,5-naphthyridin-4-amine) is a potent, selective inhibitor of c-Met (mesenchymal-epithelial transition factor), a receptor tyrosine kinase that is often deregulated in cancer. Compound 1 displayed desirable pharmacokinetic properties in multiple preclinical species. Glutathione trapping studies in liver microsomes resulted in the NADPH-dependent formation of a glutathione conjugate. Compound 1 also exhibited very high in vitro NADPH-dependent covalent binding to microsomal proteins. Species differences in covalent binding were observed, with the highest binding in rats, mice, and monkeys (1100-1300 pmol/mg/h), followed by dogs (400 pmol/mg/h) and humans (144 pmol/mg/h). This covalent binding to protein was abolished by coincubation with glutathione. Together, these in vitro data suggest that covalent binding and glutathione conjugation proceed via bioactivation to a chemically reactive intermediate. The cytochrome (CYP) P450 enzymes responsible for this bioactivation were identified as cytochrome P450 3A4, 1A2, and 2D6 in human and cytochrome P450 2A2, 3A1, and 3A2 in rats. The glutathione metabolite was detected in the bile of rats and mice, thus demonstrating bioactivation occurring in vivo. Efforts to elucidate the structure of the glutathione adduct led to the isolation and characterization of the metabolite by NMR and mass spectrometry. The analytical data confirmed conclusively that the glutathione conjugation was on the 4-C position of the isothiazole ring. Such P450-mediated bioactivation of an isothiazole or thiazole group has not been previously reported. We propose a mechanism of bioactivation via sulfur oxidation followed by glutathione attack at the 4-position with subsequent loss of water resulting in the formation of the glutathione conjugate. Efforts to reduce bioactivation without compromising potency and pharmacokinetics were undertaken in order to minimize the potential risk of toxicity. Because of the exemplary pharmacokinetic/pharmacodynamic (PK/PD) properties of the isothiazole group, initial attempts were focused on introducing alternative metabolic soft spots into the molecule. These efforts resulted in the discovery of 7-(2-methoxyethoxy)-N-((6-(3-methyl-5-isothiazolyl)[1,2,4]triazolo[4,3-b]pyridazin-3-yl)methyl)-1,5-naphthyridin-4-amine (compound 2), with the major metabolic transformation occurring on the naphthyridine ring alkoxy substituent. However, a glutathione conjugate of compound 2 was produced in vitro and in vivo in a manner similar to that observed for compound 1. Furthermore, the covalent binding was high across species (360, 300, 529, 208, and 98 pmol/mg/h in rats, mice, dogs, monkeys, and humans, respectively), but coincubation with glutathione reduced the extent of covalent binding. The second viable alternative in reducing bioactivation involved replacing the isothiazole ring with bioisosteric heterocycles. Replacement of the isothiazole ring with an isoxazole or a pyrazole reduced the bioactivation while retaining the desirable PK/PD characteristics of compounds 1 and 2.

The discovery of aurora kinases as essential regulators of cell division has led to intense interest in identifying small molecule aurora kinase inhibitors for the potential treatment of cancer. A high-throughput screening effort identified pyridinyl-pyrimidine 6a as a moderately potent dual inhibitor of aurora kinases -A and -B. Optimization of this hit resulted in an anthranilamide lead (6j) that possessed improved enzyme and cellular activity and exhibited a high level of kinase selectivity. However, this anthranilamide and subsequent analogues suffered from a lack of oral bioavailability. Converting the internally hydrogen-bonded six-membered pseudo-ring of the anthranilamide to a phthalazine (8a-b) led to a dramatic improvement in oral bioavailability (38-61%F) while maintaining the potency and selectivity characteristics of the anthranilamide series. In a COLO 205 tumor pharmacodynamic assay measuring phosphorylation of the aurora-B substrate histone H3 at serine 10 (p-histone H3), oral administration of 8b at 50 mg/kg demonstrated significant reduction in tumor p-histone H3 for at least 6 h.

Deregulation of the receptor tyrosine kinase c-Met has been implicated in several human cancers and is an attractive target for small molecule drug discovery. We previously showed that O-linked triazolopyridazines can be potent inhibitors of c-Met. Herein, we report the discovery of a related series of N-linked triazolopyridazines which demonstrate nanomolar inhibition of c-Met kinase activity and display improved pharmacodynamic profiles. Specifically, the potent time-dependent inhibition of cytochrome P450 associated with the O-linked triazolopyridazines has been eliminated within this novel series of inhibitors. N-linked triazolopyridazine 24 exhibited favorable pharmacokinetics and displayed potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver PD model. Once-daily oral administration of 24 for 22days showed significant tumor growth inhibition in an NIH-3T3/TPR-Met xenograft mouse efficacy model.

In vitro intrinsic metabolic clearance (CL(int)) is used routinely for compound selection in drug discovery; however, in vitro CL(int) often underpredicts in vivo clearance (CL). Forty-one proprietary compounds and 16 marketed drugs were selected to determine whether permeability and efflux status could influence the predictability of CL from in vitro CL(int) obtained from liver microsomal and hepatocyte incubations. For many of the proprietary compounds examined, rat CL was significantly underpredicted using the well stirred model incorporating both fraction of unbound drug in blood and fraction of unbound drug in the microsomal or hepatocyte incubation. Further analysis revealed that the accuracy of the prediction was differentiated by permeability and P-glycoprotein- (P-gp) and mouse breast cancer resistance protein (mBcrp)-mediated efflux. For proprietary compounds with passive permeability greater than 5 x 10(-6) cm/s and efflux ratios less than 5 in both P-gp- and mBcrp-expressing cells, CL(int) provided reasonable prediction. The average -fold error (AFE) was 1.8 for rat liver microsomes (RLMs) and 2.3 for rat hepatocytes. In contrast, CL was dramatically underpredicted for compounds with passive permeability less than 5 x 10(-6) cm/s; AFEs of 54.4 and 29.2 were observed for RLM and rat hepatocytes, respectively. In vivo CL was also underpredicted for compounds that were good efflux substrates (permeability >5 x 10(-6) cm/s). The AFEs were 7.4 and 8.1 for RLM and rat hepatocytes, respectively. A similar relationship between permeability, efflux status, and human CL prediction reported in the literature was observed for 16 marketed drugs. These data show that permeability and efflux status are determinants for the predictability of CL from in vitro metabolic CL(int).

To predict volume of distribution at steady-state (V(ss)), empirical (e.g., allometry) and mechanistic (using physicochemical property data and plasma protein binding) methods have been used. None of these approaches has been able to predict V(ss) accurately for the total compliment of a wide range of drugs. Therefore, alternative approaches would be of value. This study evaluates the utility of in vitro nonspecific tissue-binding measurements in predicting V(ss) for a wide range of drugs in rats. Literature as well as proprietary compounds were studied. It was found that in vitro tissue-binding measurements combined with calculated effects of the pH partition hypothesis often predict V(ss) more accurately than other available mechanistic methods and that this approach can compliment existing methods. The V(ss) values for some compounds were not accurately predicted using either nonspecific tissue-binding experiments or other available mechanistic methods. The V(ss) for these drugs may not be describable by nonspecific tissue binding alone; there may be significant specific components to the mechanism of distribution for these drugs, such as pH-dependent uptake into lysosomes (primarily strongly basic drugs), active transport, and/or enterohepatic recirculation. A lack of prediction for certain drugs warrants further investigation into these mechanisms and their application to more accurate prediction of V(ss) by mechanistic means.

The overexpression of c-Met and/or hepatocyte growth factor (HGF), the amplification of the MET gene, and mutations in the c-Met kinase domain can activate signaling pathways that contribute to cancer progression by enabling tumor cell proliferation, survival, invasion, and metastasis. Herein, we report the discovery of 8-fluorotriazolopyridines as inhibitors of c-Met activity. Optimization of the 8-fluorotriazolopyridine scaffold through the combination of structure-based drug design, SAR studies, and metabolite identification provided potent (cellular IC50 < 10 nM), selective inhibitors of c-Met with desirable pharmacokinetic properties that demonstrate potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver pharmacodynamic model.

1. This study was designed to evaluate how the absence of P-glycoprotein (Pgp, Mdr1a), breast cancer-resistance protein (Bcrp, Abcg2) or both affects drug distribution into sciatic nerves, brain and cerebrospinal fluid (CSF) in rats. 2. Pgp substrate (loperamide), BCRP substrates (dantrolene and proprietary compound X) and dual substrates (imatinib and proprietary compound Y) were well distributed into sciatic nerves with comparable nerve to plasma concentration ratios between wild-type and knockout (KO) rats. 3. Brain exposure increased substantially in Mdr1a(-/-) rats for loperamide and in Mdr1a(-/-)/Abcg2(-/-) rats for imatinib and compound Y, but minimally to modestly in Abcg2(-/-) rats for dantrolene and compound X. The deletion of Mdr1a or Abcg2 alone had little effect on brain distribution of compound Y. 4. While CSF to unbound brain concentration ratio remained ≥3 in the KO animals for dantrolene, compounds X and Y, it was reduced to 1 in the Mdr1a(-/-)/Abcg2(-/-) rats for imatinib. 5. The data indicate that Pgp and Bcrp do not play significant roles in drug distribution into peripheral nerve tissues in rats, while working in concert to regulate brain penetration. Our results further support that CSF concentration may not be a good surrogate for unbound brain concentration of efflux substrates.

The impact of inhibitor depletion on the determination of shifted IC₅₀ (IC₅₀ determined after 30 minutes of preincubation with inhibitor) is examined. In addition, IC₅₀-shift data are analyzed using a mechanistic model that incorporates the processes of inhibitor depletion, as well as reversible and time-dependent inhibition. Anomalies such as a smaller-than-expected shift in IC₅₀ and even increases in IC₅₀ with preincubation were explained by the depletion of inhibitor during the preincubation. The IC₅₀-shift assay remains a viable approach to characterizing a wide range of reversible and time-dependent inhibitors. However, as with more traditional time-dependent inactivation methods, it is recommended that IC₅₀-shift experimental data be interpreted with some knowledge of the magnitude of inhibitor depletion. For the most realistic classification of time-dependent inhibitors using IC₅₀-shift methods, shifted IC₅₀ should be calculated using observed inhibitor concentrations at the end of the incubation rather than nominal inhibitor concentrations. Finally, a mechanistic model that includes key processes, such as competitive inhibition, enzyme inactivation, and inhibitor depletion, can be used to describe accurately the observed IC₅₀ and shifted IC₅₀ curves. For compounds showing an IC₅₀ fold shift1.5 based on the observed inhibitor concentrations, reanalyzing the IC₅₀-shift data using the mechanistic model appeared to allow for reasonable estimation of Ki, KI, and kinact directly from the IC₅₀ shift experiments.

AMG 900 is an orally available small molecule that is highly potent and selective as a pan-aurora kinase inhibitor. AMG 900 is currently undergoing phase 1 clinical evaluation in patients with advanced solid tumors. The metabolism of AMG 900 was investigated in both male and female rats. We conducted studies in bile-duct catheterized (BDC) rats where bile, urine and plasma were analyzed to obtain metabolism profiles for each gender. These studies identified gender differences in the metabolism profiles in bile. Bile contained the majority of the drug related material and contained little unchanged AMG 900 which indicated that metabolism was the prominent process in drug elimination. Although bile contained the same metabolites for both genders, the amount of specific metabolites differed. Male rats metabolized AMG 900 primarily through hydroxylation with subsequent sulfate conjugation on the pyrimidinyl-pyridine side-chain whereas female rats favored a different oxidation site on the thiophene ring's methyl group, which is then metabolized to a carboxylic acid with subsequent conjugation to an acyl glucuronide. CYP phenotyping identified the prominent isoforms as being gender specific or biased in the oxidative metabolism of AMG 900. The metabolism in male rats favored both CYP2C11 and CYP2A2 whereas females favored the CYP2C12. The prominent sulfate conjugate identified in the male rat bile could also be due to male biased metabolism since it has been reported that sulfate conjugation is more prevalent in male rats. All the prominent rat metabolism routes for AMG 900 either have male or female bias. These differences in the rat AMG 900 metabolism profiles in bile can be explained by gender specific P450CYP isoforms.

AMG 900 is a small molecule being developed as an orally administered, highly potent, and selective pan-aurora kinase inhibitor. The aim of the investigations was to characterize in vitro and in vivo pharmacokinetic (PK) properties of AMG 900 in preclinical species. AMG 900 was rapidly metabolized in liver microsomes and highly bound to plasma proteins in the species tested. It was a weak Pgp substrate with good passive permeability. AMG 900 exhibited a low-to-moderate clearance and a small volume of distribution. Its terminal elimination half-life ranged from 0.6 to 2.4 h. AMG 900 was well-absorbed in fasted animals with an oral bioavailability of 31% to 107%. Food intake had an effect on rate (rats) or extent (dogs) of AMG 900 oral absorption. The clearance and volume of distribution at steady state in humans were predicted to be 27.3 mL/h/kg and 93.9 mL/kg, respectively. AMG 900 exhibited acceptable PK properties in preclinical species and was predicted to have low clearance in humans. AMG 900 is currently in Phase I clinical testing as a treatment for solid tumours. Preliminary human PK results appear to be consistent with the predictions.

Colony-stimulating-factor-1 (CSF-1) signaling through cFMS receptor kinase is increased in several diseases. To help investigate the role of cFMS kinase in disease, we identified GW2580, an orally bioavailable inhibitor of cFMS kinase. GW2580 completely inhibited human cFMS kinasein vitroat 0.06 μM and was inactive against 26 other kinases. GW2580 at 1 μM completely inhibited CSF-1-induced growth of mouse M-NFS-60 myeloid cells and human monocytes and completely inhibited bone degradation in cultures of human osteoclasts, rat calvaria, and rat fetal long bone. In contrast, GW2580 did not affect the growth of mouse NS0 lymphoblastoid cells, human endothelial cells, human fibroblasts, or five human tumor cell lines. GW2580 also did not affect lipopolysaccharide (LPS)-induced TNF, IL-6, and prostaglandin E2 production in freshly isolated human monocytes and mouse macrophages. After oral administration, GW2580 blocked the ability of exogenous CSF-1 to increase LPS-induced IL-6 production in mice, inhibited the growth of CSF-1-dependent M-NFS-60 tumor cells in the peritoneal cavity, and diminished the accumulation of macrophages in the peritoneal cavity after thioglycolate injection. Unexpectedly, GW2580 inhibited LPS-induced TNF production in mice, in contrast to effects on monocytes and macrophagesin vitro. In conclusion, GW2580's selective inhibition of monocyte growth and bone degradation is consistent with cFMS kinase inhibition. The ability of GW2580 to chronically inhibit CSF-1 signaling through cFMS kinase in normal and tumor cellsin vivomakes GW2580 a useful tool in assessing the role of cFMS kinase in normal and disease processes.

Background: The aurora family of serine-threonine kinases (aurora-A, -B, -C) regulate cell-cycle progression in mammalian cells. Aurora-A and aurora-B are essential for proper chromosome congression, segregation, and cytokinesis during mitosis, whereas aurora-C function appears restricted to male meiosis. Aurora-B is responsible for the direct phosphorylation of histone H3 on serine-10. Aurora-A and aurora-B expression is elevated in a variety of human cancers and is associated with advanced clinical staging and poor prognosis. The emergence of aurora kinases as key mitotic regulators and their potential role in tumorigenesis has focused substantial interest in developing selective small molecule inhibitors for the treatment of human cancers. Objective: The aim of this study was to determine the in vitro effects of AMG 900 on a panel of tumor cell lines and profiling its antitumor activity across multiple human xenograft models. Results: AMG 900 is a novel ATP competitive small molecule inhibitor that is potent and highly selective for aurora kinases A, B, and C. In cells, AMG 900 inhibits autophosphorylation of aurora-A and aurora-B as well as the phosphorylation of histone H3. The predominant cellular response of tumor cells to AMG 900 treatment is aborted cell division, which leads to endoreduplication and cell death. The effect of AMG 900 was tested in a panel of 26 cell lines to evaluate its potential to inhibit cell proliferation across multiple tumor types. AMG 900 inhibited the proliferation of tumor cell lines at low nanomolar concentrations (EC50 values 1-6 nM), including a number of multidrug resistant (MDR) cell lines expressing ATP-binding cassette transporters (P-gp and BCRP1). In contrast, paclitaxel and three aurora kinase inhibitors (AZD1152, MK-0457, and PHA-739358) showed a loss of potency in these MDR cell lines compared to matched parental cell lines. In nude mice, oral administration of AMG 900 inhibited phosphorylation of histone H3 in tumors in a concentration- and dose-dependent manner. The effect of AMG 900 was tested in a panel of nine human xenografts representing five tumor types (breast, colon, lung, pancreatic, and uterine). Oral administration of AMG 900 at 15 mg/kg BID for two consecutive days per week or 3 mg/kg BID every day inhibited tumor growth (50-97%, P < 0.005) in all nine of the xenograft models. Importantly, three of these xenograft models are resistant to taxanes. Mice treated with efficacious doses of AMG 900 showed a transient loss of body weight and bone marrow cellularity, consistent with its on-mechanism effects on normal proliferating cells. Conclusion: Based on these preclinical activities, AMG 900 has the potential to treat advanced cancers, including tumors resistant to chemotherapeutic agents such as paclitaxel and docetaxel. AMG 900 is currently undergoing phase 1 clinical evaluation in patients with advanced solid tumors. 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 4437.

The aurora family of serine/threonine kinases (Aurora-A, -B, -C) regulate cell-cycle progression in mammalian cells. Whereas aurora kinase C function appears restricted to meiosis in males, aurora kinases A and B are essential for proper chromosome congression, segregation, and cytokinesis during mitosis. Aurora kinases A and B have been implicated in tumorigenesis, with overexpression levels correlating to clinical staging of cancers and poor prognosis. Thus, these mitotic kinases have become the subject of much interest as targets for anticancer therapy. N-(4-((3-(2-amino-4-pyrimidinyl)-2-pyridinyl)oxy)phenyl)-4-phenyl-1-phthalazinamine was a key aurora kinase inhibitor lead, possessing oral bioavailability in rats that was lacking in the anthranilamide compounds from which it was derived. This phthalazine compound possessed a key feature that was deemed important to maintain in a clinical candidate: potency against a model multidrug resistant (MDR) cell line (MES-SA Dx5) commensurate with its activity against a cell line that does not overexpress P-gp (HeLa). Improved in vivo potency was desired, as measured by suppression of the phosphorylation of the aurora kinase B substrate Histone H3 on Ser10 six hours after dosing. SAR from targeting this improvement in in vivo activity uncovered a delicate balance between protein binding, pharmacokinetic parameters, and cell potency in MES-SA Dx5 cells. AMG 900 was identified as a suitable candidate for clinical development based on its low single digit nanomolar potency against MDR cell lines, robust PD response (with complete suppression of Histone H3 phosphorylation at six hours), and high selectivity against other kinases. Oral administration of AMG 900 at a well-tolerated dose of 4 mg/kg BID inhibited tumor growth (83% TGI; p < 0.0001) in an HCT116 xenograft model. AMG 900 is currently undergoing phase 1 clinical evaluation in patients with advanced solid tumors. 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 5776.

Introduction: AMG 900 is a small molecule being developed as an orally administered, highly potent and selective pan-aurora kinase inhibitor. AMG 900 is currently undergoing phase 1 clinical evaluation in patients with advanced solid tumors. The objectives of the studies were to characterize single dose pharmacokinetics (PK) of AMG 900 in preclinical species and use the preclinical PK data to predict its human PK. Methods: Following a single intravenous and oral administration of AMG 900 to mice, rats, dogs, and monkeys, blood samples were collected and processed for plasma by centrifugation. AMG 900 plasma concentrations were determined by LC-MS/MS. Non-compartmental analysis of the concentration - time data was performed to calculate PK parameters of AMG 900. Human PK were predicted based on data from preclinical species using the simple allometric scaling method assuming a human body weight of 60 kg. Results: AMG 900 exhibited low (mouse, monkey and dog) to moderate (rat) clearance in the species evaluated. The volume of distribution at steady state was less than 0.5 L/kg across species. Following intravenous administration, the compound had terminal elimination half-life ranging from 0.6 to 2.4 hours. AMG 900 was well absorbed in fasted animals across species with an oral bioavailability ranging from 31% to 104%. The clearance and volume of distribution at steady state in humans were predicted to be 0.03 L/hr/kg and 0.1 L/kg, respectively, with the predicted half-life to be approximately 2.3 hours. Bioavailability after oral administration of AMG 900 was predicted to be 75%, calculated from the mean values observed in non-clinical species. Conclusion: AMG 900 exhibits good PK properties in preclinical species and it was predicted to have low clearance and good oral bioavailability in human. Available PK results from the on-going phase 1 trial are consistent with the allometric scaling predictions. 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 2663.

Background: The aurora family of serine-threonine kinases (Aurora-A, -B, -C) regulate cell-cycle progression in mammalian cells. Aurora kinases A and B are essential for proper chromosome congression, segregation, and cytokinesis during mitosis, whereas aurora kinase C function appears restricted to male meiosis. Aurora kinase B is responsible for the direct phosphorylation of histone H3 on serine-10 (p-histone H3). Aurora kinase A and B expression is elevated in a variety of human cancers and is associated with advanced clinical staging and poor prognosis. With the emergence of aurora kinases as key mitotic regulators and their potential role in tumorigenesis, we developed AMG 900, a novel ATP competitive small molecule inhibitor, that is highly potent and selective for aurora kinases A, B, and C. In vivo, AMG 900 inhibits the growth of multiple human tumor xenografts, including multidrug-resistant models. Objective: The aim of this study was to establish a pharmacokinetic-pharmacodynamic (PK-PD) relationship for AMG 900 in human tumor xenografts and surrogate tissues. Results: In nude mice, oral administration of AMG 900 inhibited p-histone H3 in tumors and bone marrow in a concentration- and dose-dependent manner. To further refine the AMG 900 PK-PD relationship, p-histone H3 IC50 values for bone marrow and dissociated COLO 205 tumors from mice were associated with plasma concentrations of 294 and 273 ng/mL, respectively. The duration of target coverage required for anti-tumor efficacy was determined to be > 6 hours per day above the in vivo p-histone H3 IC50 using different dosing schedules. To explore whether skin could be a suitable surrogate tissue for measuring p-histone H3 inhibition, tumor-bearing mice were administered AMG 900 at 3.75, 7.5, and 15 mg/kg for 3 hours. Significant suppression of p-histone H3 in skin was only observed at 15 mg/kg (p < 0.0001), whereas in tumor, a dose-dependent inhibition was achieved at all doses (p < 0.0015). To determine the feasibility of measuring p-histone H3 using fine-needle aspirate (FNA) tumor biopsies, tumor-bearing mice were administered AMG 900 at 15 mg/kg for 3 hours. Treatment with AMG 900 significantly inhibited p-histone H3 (> 99%, p < 0.0001) in COLO 205 tumors, suggesting that FNA biopsies may be a viable approach for assessing AMG 900 PD effects in the clinic. Conclusion: These studies demonstrate that AMG 900 inhibits p-histone H3 in tumors and surrogate tissues, although tissues such as skin may be less sensitive to assess PD effects. The use of p-histone H3 as a biomarker to determine plasma levels of AMG 900 required to inhibit aurora B activity may be useful in clinical studies. 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 3600.