Dose-dependent Antitumor Activity Research Articles

Abstract 845: In vitro and in vivo pharmacology of SAR125844, a potent and selective intravenous MET kinase inhibitor undergoing Phase I clinical trial

Abstract SAR125844 is a potent MET kinase inhibitor for intravenous (IV) administration, with nanomolar activity against the wild-type enzyme (IC50 = 4.2 nM) and some kinase domain mutants, such as M1250T and Y1235D. It is highly selective for MET kinase in a panel of 275 kinases tested, with only 5 other protein kinases inhibited at IC50 values below 300 nM. In cellular assays, SAR125844 inhibits MET autophosphorylation at the nanomolar range (IC50 from 1.4 to 5.1 nM), translating into antiproliferative activity selectively in MET-driven cell lines such as MET-amplified cell lines, with IC50 values in the low nanomolar range and induction of apoptosis. SAR125844 induces a G1 block in the two MET-amplified tumor cell lines tested. Moreover, the compound is also able to inhibit HGF-induced cell migration in PC-3 prostate tumor cell line. In two MET-amplified human gastric tumor xenograft models tested, SNU-5 and Hs 746T, SAR125844 intravenous treatment leads to potent impact on the MET signaling pathway in a dose and time dependent manner, with potent inhibition of MET autophosphorylation, as well as a significant effect on downstream PI3K/AKT and RAS/MAPK pathways. As a single agent, this translates into a dose-dependent antitumor activity in these two xenograft models, with tumor stasis at the lowest active dose and tumor regression at the highly active doses. High loading single dose administration of SAR125844 using a nanosuspension formulation allowed a sustained P-MET inhibition in tumors up to 7 days. In all models, antitumor activity was achieved at well tolerated doses without significant effect on body weight. The observed correlation between P-MET inhibition in the tumor and antitumor activity in preclinical settings suggests that P-MET evaluation in tumor biopsies could be a direct pharmacodynamic biomarker of SAR125844 activity in patients. In order to investigate whether a non-invasive biomarker could also be used as pharmacodynamic read-out, we measured shed-MET (soluble extracellular domain of the MET receptor) in the plasma of tumor-bearing mice and showed that its level correlates with tumor burden in 2 MET-amplified xenograft models. 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 845. doi:1538-7445.AM2012-845

AZD4547: An Orally Bioavailable, Potent, and Selective Inhibitor of the Fibroblast Growth Factor Receptor Tyrosine Kinase Family

The fibroblast growth factor (FGF) signaling axis is increasingly implicated in tumorigenesis and chemoresistance. Several small-molecule FGF receptor (FGFR) kinase inhibitors are currently in clinical development; however, the predominant activity of the most advanced of these agents is against the kinase insert domain receptor (KDR), which compromises the FGFR selectivity. Here, we report the pharmacologic profile of AZD4547, a novel and selective inhibitor of the FGFR1, 2, and 3 tyrosine kinases. AZD4547 inhibited recombinant FGFR kinase activity in vitro and suppressed FGFR signaling and growth in tumor cell lines with deregulated FGFR expression. In a representative FGFR-driven human tumor xenograft model, oral administration of AZD4547 was well tolerated and resulted in potent dose-dependent antitumor activity, consistent with plasma exposure and pharmacodynamic modulation of tumor FGFR. Importantly, at efficacious doses, no evidence of anti-KDR-related effects were observed, confirming the in vivo FGFR selectivity of AZD4547. Taken together, our findings show that AZD4547 is a novel selective small-molecule inhibitor of FGFR with potent antitumor activity against FGFR-deregulated tumors in preclinical models. AZD4547 is under clinical investigation for the treatment of FGFR-dependent tumors.

CEP-28122, a Highly Potent and Selective Orally Active Inhibitor of Anaplastic Lymphoma Kinase with Antitumor Activity in Experimental Models of Human Cancers

Anaplastic lymphoma kinase (ALK) is constitutively activated in a number of human cancer types due to chromosomal translocations, point mutations, and gene amplification and has emerged as an excellent molecular target for cancer therapy. Here we report the identification and preclinical characterization of CEP-28122, a highly potent and selective orally active ALK inhibitor. CEP-28122 is a potent inhibitor of recombinant ALK activity and cellular ALK tyrosine phosphorylation. It induced concentration-dependent growth inhibition/cytotoxicity of ALK-positive anaplastic large-cell lymphoma (ALCL), non-small cell lung cancer (NSCLC), and neuroblastoma cells, and displayed dose-dependent inhibition of ALK tyrosine phosphorylation in tumor xenografts in mice, with substantial target inhibition (>90%) for more than 12 hours following single oral dosing at 30 mg/kg. Dose-dependent antitumor activity was observed in ALK-positive ALCL, NSCLC, and neuroblastoma tumor xenografts in mice administered CEP-28122 orally, with complete/near complete tumor regressions observed following treatment at doses of 30 mg/kg twice daily or higher. Treatment of mice bearing Sup-M2 tumor xenografts for 4 weeks and primary human ALCL tumor grafts for 2 weeks at 55 or 100 mg/kg twice daily led to sustained tumor regression in all mice, with no tumor reemergence for more than 60 days postcessation of treatment. Conversely, CEP-28122 displayed marginal antitumor activity against ALK-negative human tumor xenografts under the same dosing regimens. Administration of CEP-28122 was well tolerated in mice and rats. In summary, CEP-28122 is a highly potent and selective orally active ALK inhibitor with a favorable pharmaceutical and pharmacokinetic profile and robust and selective pharmacologic efficacy against ALK-positive human cancer cells and tumor xenograft models in mice.

The Investigational Aurora A Kinase Inhibitor MLN8237 in Combination with Vincristine Demonstrates Robust Anti-Tumor Activity in Preclinical B-Cell Non-Hodgkin's Lymphoma Models

Abstract 2733Aurora A kinase (AAK) is a serine/threonine protein kinase that is essential for normal mitotic progression. MLN8237, an investigational, selective inhibitor of AAK, is being evaluated in multiple Phase 1 and 2 clinical studies in patients with solid cancers and hematological malignancies. In early clinical trials in heme-lymphatic malignancy, MLN8237 dosed as a single agent has demonstrated antitumor activity (Padmanabhan ASH 2010; Goldberg ASH 2010). In order to identify potential combination partners for MLN8237 for clinical evaluation, previous preclinical efforts demonstrated that MLN8237 combined with rituximab leads to significant antitumor activity in a panel of in vivo diffuse large B-cell lymphoma (DLBCL) models (Huck et. al. ASH 2008; Zhang et, al. AACR 2009). In the present preclinical studies, a novel combination treatment of MLN8237 and vincristine was tested in two xenograft models of DLBCL, WSU and OCI-LY19. Mice were dosed for three weeks with MLN8237 orally daily (QD) and with vincristine intravenously weekly (QW). In both tumor models, single agent dose dependent antitumor activity was observed with MLN8237 and vincristine; however, tumors gradually re-grew after the treatment. Combined MLN8237 and vincristine treatment led to additive or synergistic antitumor activity and resulted in significant tumor growth delay even after discontinuing treatment. In the OCI-Ly19 model, MLN8237 (20 mg/kg) with vincristine (0.5mg/kg or 1 mg/kg) resulted in complete cures with no tumor re-growth out to >120 day. In the WSU model, MLN8237 (20mg/kg) combined with vincristine (1mg/kg) led to tumor regression during the treatment period and significant tumor growth delay (TGD=38). Body weight loss (BWL) was observed in the combination arms in a dose dependent manner in an acceptable range (maximum ∼10%); however body weight recovered quickly after treatment termination. The plasma and tumor pharmacokinetic profiles of MLN8237 (20 and 3 mg/kg) and vincristine (1 mg/kg) in the WSU model demonstrated decreased MLN8237 exposure when co-administered with vincristine. Vincristine exposures were not affected by co-administration with MLN8237. Additional preclinical studies are planned to evaluate the antitumor activity of MLN8237 combined with vincristine and rituximab. Preclinical data showing the antitumor activity of the combination of MLN8237 and vincristine provides the basis for its clinical evaluation as a treatment option for aggressive DLBCL. Disclosures:No relevant conflicts of interest to declare.

Combining Betulinic Acid and Mithramycin A Effectively Suppresses Pancreatic Cancer by Inhibiting Proliferation, Invasion, and Angiogenesis

Both betulinic acid (BA) and mithramycin A (MIT) exhibit potent antitumor activity through distinct mechanisms of Sp1 inhibition. However, it is unknown whether a combination of these two compounds results in a synergistic inhibitory effect on pancreatic cancer growth and/or has a therapeutic advantage over gemcitabine. In xenograft mouse models of human pancreatic cancer, treatment with either BA or MIT alone showed dose-dependent antitumor activity but led to systemic side effects as measured by overall weight loss. Treatment with a nontoxic dose of either compound alone had only marginal antitumor effects. Importantly, combination treatment with nontoxic doses of BA and MIT produced synergistic antitumor activity, including inhibitory effects on cell proliferation, invasion, and angiogenesis. The treatment combination also produced less discernible side effects than therapeutic doses of gemcitabine. Moreover, combined treatment of BA and MIT resulted in drastic inhibition of Sp1 recruitment onto Sp1 and VEGF promoters, leading to transcriptional inhibition of both Sp1 and VEGF and downregulation of Sp1 and VEGF protein expression. Ectopic overexpression of Sp1 rendered tumor cells resistant to BA, MIT, and the combination of the two. Overall, our findings argue that Sp1 is an important target of BA and MIT and that their combination can produce an enhanced therapeutic response in human pancreatic cancer.

Pre-clinical characterization of CX-4945, a potent and selective small molecule inhibitor of CK2 for the treatment of cancer

In this article we describe the preclinical characterization of 5-(3-chlorophenylamino) benzo[c][2,6]naphthyridine-8-carboxylic acid (CX-4945), the first orally available small molecule inhibitor of protein CK2 in clinical trials for cancer. CX-4945 was optimized as an ATP-competitive inhibitor of the CK2 holoenzyme (Ki = 0.38 nM). Iterative synthesis and screening of analogs, guided by molecular modeling, led to the discovery of orally available CX-4945. CK2 promotes signaling in the Akt pathway and CX-4945 suppresses the phosphorylation of Akt as well as other key downstream mediators of the pathway such as p21. CX-4945 induced apoptosis and caused cell cycle arrest in cancer cells in vitro. CX-4945 exhibited a dose-dependent antitumor activity in a xenograft model of PC3 prostate cancer model and was well tolerated. In vivo time-dependent reduction in the phosphorylation of the biomarker p21 at T145 was observed by immunohistochemistry. Inhibition of the newly validated CK2 target by CX-4945 represents a fresh therapeutic strategy for cancer.

Comparative Antitumor Activity of Different Solvent Fractions from an Auricularia auricula-judae Ethanol Extract in P388D1 and Sarcoma 180 Cells

The objective of this study was to evaluate and compare the antitumor activity of different solvent fractions (ethanol, dichloromethane, ethyl acetate, butanol and water) of the Auricularia auricula-judae 70% ethanol extract on the P388D1 macrophage and sarcoma 180 cells. A dose-dependent antitumor activity of each solvent fraction (from 0.01 mg/ml to 0.3 mg/ml) was shown against both cell types. These cytotoxic effects of all the tested fractions were confirmed on the MTT and SRB assays, without statistical differences each other. IC50 value of dichloromethane fraction was 94.2 μg/ml against sarcoma 180 cells lower than any other solvent fractions. The potent antitumor effect of the dichloromethane (DCM) fraction was also found against solid tumor in BALB/c mice. The splenomegaly and higher splenic index were found in tumor-bearing mice, with the DCM fraction returning to the negative control values. Thus, the results indicated the dichloromethane fraction may have potential ingredients as antitumor candidates.

Abstract 3574: Identification and preclinical characterization of CEP-28122, a highly potent and selective orally active inhibitor of anaplastic lymphoma kinase, in lymphoma and non-small cell lung cancer models

Abstract Anaplastic lymphoma kinase (ALK) was originally identified as the oncogenic NPM (nucleophosmin)-ALK fusion protein due to a t (2;5) chromosomal translocation in anaplastic large cell lymphomas (ALCL). Many other chromosomal rearrangements or gene mutations/amplification leading to enhanced ALK activity have subsequently been identified and characterized in a number of human cancer types. The recent reports of EML4 (echinoderm microtubule-associated protein-like 4)-ALK oncogenic proteins in non-small cell lung cancer (NSCLC) and the identification of ALK activating point mutations and gene amplification in neuroblastoma have indicated ALK as a potential major therapeutic target for human cancers. Here we report the identification and preclinical characterization of CEP-28122, a highly potent and selective orally active ALK inhibitor. CEP-28122 is a potent inhibitor of recombinant ALK activity (IC50 of 3 nM) and NPM-ALK tyrosine phosphorylation in ALCL cells (IC50 of 20-30 nM). It is selective over a broad panel of protein kinases and a panel of receptors and ion channels with greater than 300-fold selectivity for insulin receptor. CEP-28122 induced concentration-dependent growth inhibition and cytotoxicity of ALK-positive ALCL and NSCLC cells with minimal activity against ALK-negative lymphoma and leukemia cells as well as ALK-negative NSCLC cells at concentrations up to 3 μM. CEP-28122 exhibited favorable oral bioavailability (F = 37-71% across species) with adequate tissue distribution in rodents. It displayed dose-dependent inhibition of NPM-ALK tyrosine phosphorylation in human ALCL tumor xenografts in mice with complete target inhibition (> 90%) for more than 12 h following single oral dosing at 30 mg/kg. Dose-dependent anti-tumor activity was observed in NPM-ALK-positive Sup-M2 and Karpas-299 ALCL tumor xenografts and EML4-ALK-positive NCI-H2228 and NCI-H3122 tumor xenografts in mice dosed with CEP-28122, bid, po, with complete or near complete tumor regressions observed following 2 weeks of treatment with CEP-28122 at 30 mg/kg or higher. Treatment of mice bearing Sup-M2 tumor xenografts or primary human ALCL tumorgrafts with CEP-28122 at 55 or 100 mg/kg bid, po, for 4 weeks led to sustained tumor regression in all mice, with no tumor re-emergence in any mouse up to 60 days post cessation of CEP-28122 treatment. On the contrary, CEP-28122 displayed marginal anti-tumor activity against ALK-negative lymphoma and NSCLC tumor xenografts under the same dosing regimens. It was well tolerated with all dosing regimens in mice and rats with no overt toxicity and no compound-related body weight loss. CEP-28122 advanced into preclinical development based on its potency, selectivity and overall favorable pharmacological, pharmaceutical and safety profiles. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3574. doi:10.1158/1538-7445.AM2011-3574

Abstract 2525: Intravenous infusions of TriN 2755, a new alkylating agent, inhibit the growth of human MDA-MB-231 breast tumor in nude rats

Abstract Background: The new cytotoxic anti-neoplastic agent TriN 2755, containing a triazene unit with alkylating properties, exhibited a potent antitumor activity in a large panel of human xenograft tumor models in mice when dosed by IV bolus injection. In rat, dog and mini pig toxicity studies, TriN 2755 demonstrated a favourable safety profile. Here, we present efficacy data of TriN 2755 intravenously injected in the MDA-MB-231 tumor bearing Nude rat model. Material and methods: The breast tumors were induced subcutaneously by injecting 2×107 of MDA-MB-231 cells in 200 µl of RPMI 1640 containing matrigel (50:50, v:v). Tumor-bearing nude rats received a single 4-hour IV infusion of TriN 2755 at 30, 80 or 240 mg/kg/infusion or IV bolus injection of TriN 2755 at 160, 240 or 360 mg/kg/injection twice weekly for 4 consecutive weeks. Moreover, two groups of rats received a single 4-hour IV infusion of TriN 2755 at 240 mg/kg/infusion twice weekly for 4 consecutive weeks in combination with repeated weekly IV bolus injection of CPT-11 at 40 mg/kg/inj for 4 consecutive weeks or Docetaxel at 2.5 mg/kg/inj for 3 consecutive weeks. The treatment started when the mean tumor volumes reached about 400-800 mm3, and the antitumor activity of TriN 2755 was assessed as tumor volume inhibition relative to a vehicle control group (T/C value in %). Tolerability was analysed by recording mortality and body weight loss. Results: Repeated IV infusions of TriN 2755 were well tolerated by female nude rats bearing subcutaneous (SC) MDA-MB-231 tumors at all tested doses (no death, no body weight loss). The histopathological analysis of liver, kidneys, and bone marrow of treated rats revealed no treatment related changes. Based upon TriN 2755 antitumor efficacy criteria at 80 and 240 mg/kg when injected by IV infusion, and at 160, 240 or 360 mg/kg when injected by IV bolus displayed a marked, and dose-dependent antitumor activity against SC MDA-MB-231 tumor (Optimal T/C% values were 37, and 7%, 21, 19 and 12%, respectively) Tumor regressions were observed after two weeks of treatment for all rats treated with TriN 2755 administered by IV bolus or IV infusion of 240mg/kg/infusion. Tumor cells derived from TriN 2755 IV bolus treated rats had an impaired colony formation compared to non-treated tumors in vitro. Moreover, an enhancement of the antitumor activity of drugs alone was observed in the SC MDA MB-231 tumor bearing nude rats model when TriN 2755 at 240 mg/kg/infusion was combined with CPT-11 at 40 mg/kg/inj or Docetaxel at 2.5 mg/kg/inj. Conclusions: These results demonstrate that TriN 2755 dosed by IV infusion and IV bolus displayed a marked antitumor activity alone, and efficacy of IV infusion was increased by combination with CPT-11 or Docetaxel in SC nude rats human breast tumor model. TriN 2755 is currently under evaluation in phase I clinical trials for the treatment of solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2525. doi:10.1158/1538-7445.AM2011-2525

Abstract 2823: TAK-441, a novel investigational small molecule hedgehog pathway inhibitor for use in cancer therapy

Abstract Dysregulation of hedgehog (Hh) signal, which is caused by Hh-ligand overexpression or patched1 (Ptch1) mutation, has been found to play an important role in tumorigenesis. TAK-441 is a potent and selective smoothened (Smo) antagonist that blocks Hh signaling. In the Gli-responsive promoter-luciferase (Gli-luc) reporter assay in NIH3T3/Gli-luc cells, TAK-441 inhibited Gli transcriptional activity with an IC50 of 4.4 nM. TAK-441 also inhibited expression of human Gli1 messenger mRNA with an IC50 of 1.9 nM in MRC5 human embryonic fibroblasts. In addition, TAK-441 inhibited binding of cyclopamine, a plant-derived well-known Smo inhibitor, to human Smo on 293T cells overexpressing human Smo with an IC50 of 8.6 nM. These data suggest that TAK-441 inhibits the Hh signaling pathway through its binding to Smo. Oral administration of TAK-441 (0.5, 1, 5, 25 mg/kg QD) in mice bearing a Ptch1 (+/-), p53(−/-) medulloblastoma allograft resulted in dose-dependent antitumor activity with concomitant reduction of Gli1 mRNA expression in the tumors. Complete remissions were observed in the 25 mg/kg QD dose cohort after two consecutive weeks of treatment. Significant (p≤0.025 by one-tailed Williams test) antitumor activity was also detected in sonic hedgehog expressing human primary pancreatic and ovarian cancer xenografts when dosed at 10 and 25 mg/kg QD for two weeks, and in a colon cancer xenograft when dosed at 6.25 and 25 mg/kg BID for three weeks. In these models, the expression of tumor-associated mouse stromal Gli1 mRNA was markedly decreased following TAK-441 treatment, suggesting that Hh signaling in these tumors was driven in a paracrine mode of action. The combination of TAK-441 and rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), demonstrated significantly better antitumor activity than either agent alone in human primary pancreatic cancer xenograft models (p≤0.05 by Students’ t-test). In addition, delayed tumor re-growth was observed in the TAK-441/rapamycin combination treatment group compared with the rapamycin single-agent treatment group after terminating treatment. As a result of these preclinical studies, TAK-441 has recently entered Phase I clinical studies in cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2823. doi:10.1158/1538-7445.AM2011-2823

Preclinical development of small-molecule CRM1 inhibitors as novel therapy for the treatment of colorectal cancer (CRC).

430 Background: CRM1 (XPO1) is a key nuclear export protein which controls the location of multiple tumor suppressor (TSP) and growth regulatory (GRP) proteins including p53, PI3K/AKT, Wnt/ß-catenin and NF-kB. Forced nuclear expression of TSP and GRP by CRM1 inhibition can lead to apoptosis in cancer cells while sparing normal cells. Methods: Novel small-moleculeCRM1 inhibitors were synthesized and nuclear distribution studies were performed in cells transfected with HIV-rev GFP proteins. Cell proliferation studies were performed in 16 CRC cell lines: LS-123, SW-626, Colo-201, Colo-205, Colo-320DM, Colo-320HSR, Lovo, DLD-1, HCT-15, WiDi, LS-174T, LS-180, SW-620, C2BBe1, HCT-8, HCT-116, and in human peripheral leukocytes (PBMC). Cellular distribution and apoptosis assays were performed on HCT-116. Antitumor activity is assessed in human HCT-116 xenografts in scid-mice. Results: The lead CRM1 inhibitor, KPT-0127, blocks CRM1 mediated nuclear export of HIV-Rev-GFP, FOXO, and p53 with an IC50 of ∼300 nM. KPT-0127 is cytotoxic to various CRC cell lines with EC50s of 0.07-1.1 μM; in 9 CRC lines EC50s were < 0.3 mM. In contrast, normal cell lines and PBMCs had EC50 > 5-20 μM. In HCT- 116 cells, KPT-0127 induces cell cycle arrest at both G1/S and G2/M checkpoints and dose dependently increases nuclear p53, followed by an increase in caspase 3. KTP-0127 10μM shows no significant effect on 37 proteins including several cysteine proteases. In mice, KPT-0127 given by SC injection of 30-100 mg/kg leads to serum levels exceeding the effective CRM1 inhibitory concentration for at least 4 hours and is well tolerated. KPT-0127 given SC to mice bearing HCT-116 colon xenografts results in dose-dependent antitumor activity. Conclusions: The novel small- molecule CRM1 inhibitor KTP-0127 kills CRC lines with multiple TSP, GRP, and oncogenic abnormalities, including p53 mutations/deletions and PTEN deficiency/AKT activation, while sparing normal cells. This likely reflects the ability of CRM1 inhibition to affect multiple critical and non-redundant regulatory pathways. These results support the development of CRM1 inhibitors for the treatment of CRC. IND-enabling CMC and toxicology work are in preparation. [Table: see text]

Preclinical Development of Small-Molecule CRM1 Inhibitors as Novel Therapy for the Treatment of Myeloma and Other Hematological Malignancies

AbstractAbstract 3012 Background:CRM1 (XPO1) is a key nuclear export protein which controls multiple tumor suppressor proteins (TSP) and cell proliferation pathways including p53, PI3K/AKT, Wnt/ß-catenin and NF-kB. Further, mislocalization of proteins can abrogate TSP functions and render chemotherapies ineffective. For example, multiple myeloma (MM) cells that are grown at high densities (to mimic the in vivo situation) become resistant to topoisomerase 2 (topo2) inhibitors (e.g., doxorubicin) simply because topo2α is exported from the nucleus. Forcing the nuclear expression of chemotherapy targets, TSP and growth regulatory proteins by CRM1 inhibition can restore drug sensitivity and restore checkpoint control/genome surveying functions. These events lead to apoptosis or autophagy in cancer cells while sparing normal cells. Methods:CRM1 inhibitors were synthesized and nuclear distribution studies were performed in U2OS cells transfected with HIV-rev GFP proteins. Cell proliferation studies were performed in multiple myeloma (MM), leukemia and lymphoma cell lines and in human peripheral blood mononuclear cells (PBMCs). Toxicology studies were performed in several mouse strains. Antitumor activity is assessed in a xenograft model of human MM.1 cells growing in scid-mice. Results:The lead CRM1 inhibitor, KPT-0127, blocks CRM1 mediated nuclear export of HIV-Rev-GFP, FOXO, and p53 with an IC50 of ~300 nM. KPT-0127 is selectively cytotoxic to various hematological cell lines with EC50s in the 0.02–1.0 μM range, and shows limited cytotoxicity in similar studies in normal cell lines (NIH-3T3, MRC5, HUVECs) and PBMCs (EC50 >5-20 μM). KPT-0127 increases the nuclear localization of IkB in HUT-78 leukemia cells and human PBMCs, and inhibits TNF-α secretion in LPS stimulated U937 macrophage derived cells, likely through inhibition of NF-kB signaling. In MM cells grown at high densities which actively export topo2 to the cytoplasm via CRM1, blocking CRM1-dependent transport strongly enhanced topo2 nuclear localization and augmented the apoptotic effects of doxorubicin in <24 hours showing clear synergy between KPT-0127 and the anthracycline. Combination of sublethal concentrations of bortezomib plus KPT-0127 in MM1.S and MM.1R myeloma cells (as well as in Jurkat and HS-Sultan) induced synergistic cytotoxicities. In mice, KPT-0127 given by subcutaneous (SC) injection of 30–100 mg/kg leads to serum levels exceeding the effective CRM1 inhibitory concentration for at least 4 hours. In 5 day repeated dose toxicology studies, SC administration of 100 mg/kg KPT-0127 (QD × 5) was well tolerated in mice with no cutaneous or obvious systemic clinical findings. Modest neutrophilia and mild lymphopenia were seen and no neurologic signs resulted from treatment with KPT-0127. Administration of KPT-0127 SC to mice bearing HCT-116 colon cancer results in dose-dependent antitumor activity. Xenograft studies with MM.1 myeloma cells are underway and will be presented at the meeting. Conclusions:The sensitivity of tumors with multiple TSP and oncogenic abnormalities, including p53 mutations/deletions and PTEN deficiency/AKT activation, to killing with KPT-0127 likely reflects the ability to affect multiple critical and non-redundant regulatory pathways. CRM1 inhibition also forces topo 2 to remain in the nucleus, and increases levels of nuclear IkB antagonizing NF-kB function, thereby reducing the likelihood of resistance development. These results support the development of small molecule, drug-like CRM1 inhibitors for the treatment of MM and other hematological cancers. IND-enabling CMC and toxicology work are expected to begin in early 2011. Disclosures:Shacham:Karyopharm: Employment, Equity Ownership, Patents & Royalties. Nir:Karyopharm: Consultancy. Draetta:Karyopharm: Consultancy. Sandanayaka:Karyopharm: Employment. Shechter:Karyopharm: Employment. Kauffman:Karyopharm: Consultancy, Equity Ownership, Patents & Royalties.

Preclinical Development of Triptolide Derivative MRx102 as a Therapeutic Agent for Acute Myeloid Leukemia

AbstractAbstract 3271Acute Myeloid Leukemia (AML) is the most common form of adult acute leukemia and the second most common childhood leukemia. AML has the lowest survival rate among leukemias, and the frequency is increasing as the population ages. Current therapies are inadequate, and a need exists for better therapeutic agents to treat AML, both as initial treatment for newly diagnosed patients and for those who have failed current therapy and relapsed. Natural products, such as taxol, have shown activities in a variety of disease states, including cancer. Triptolide is a natural product diterpenoid derived from Tripterygium wilfordii Hook f, and has shown anti-cancer activity in a broad range of solid tumors in preclinical models. It induces apoptosis in various leukemic cell lines and primary AML blasts (Carter, B et al, Blood 2006). Derivatives of triptolide with improved pharmacokinetics and bioavailability offer the opportunity to optimize the activity of triptolide for clinical application in AML. MRx102 is a triptolide derivative that is more hydrophobic than triptolide. It has potent in vitro cytotoxic activity with human tumor and leukemia cell lines, an unusual result for triptolide derivatives because they are usually much less active in vitro than the parent compound. Designed as a prodrug, MRx102 exerts cytotoxic activity with human AML cell lines and other human leukemia cell lines without pre-incubation with plasma esterases (IC50 of 51.0 and 37.1 nM with MV4-11 AML cells at 48 and 72 hours, respectively, ∼55% and ∼36% of the activity of triptolide, respectively). MRx102 decreases the viable CD34+ blasts of AML patient samples (a mean of 79.8 ± 8.8% specific apoptosis at 100 nM, n=3), and overcomes the apoptosis protection by co-cultivated stromal cells (with a similar mean of 74.1 ± 8.5%). MRx102 shows dose-dependent anti-tumor activity with the MV4-11 cell line in nude mouse human AML tumor xenografts. After 42 days of MRx102 dosing at 1.35 mg/kg/day i.p., tumor volume was inhibited by 99.7%. Tumors removed from several mice appeared to be Matrigel pellets rather than vascularized tumors, suggesting that many of the tumors were completely eliminated. In studies with the OCI-AML3 human AML cell line xenograft model, the group receiving MRx102 at 1.35 mg/kg/day i.p. showed similar high activity, with mean tumor volume reduced by as much as 99.2% on day 23 compared to the vehicle control group. Tumors of 7 of 10 mice were smaller than the day 0 volumes at the day 28 end of the study. As part of drug development, toxicology testing with MRx102 was initiated with an acute single dose rat toxicology study with no deaths and no adverse signs up to the top dose of 3.0 mg/kg MRx102 in DMSO/PBS administered i.v. The maximum tolerated dose (MTD) is greater than 3 mg/kg of MRx102, and the no observable adverse effect level (NOAEL) is at least 3 mg/kg. A 7-day subacute rat toxicology study of MRx102 showed no deaths and no adverse signs up to the top dose of 1.5 mg/kg/day MRx102 in DMSO/PBS administered daily i.v. for 7 days. The histopatholgy report shows no findings related to administration of the test article. The MRx102 MTD is greater than 1.5 mg/kg/day, and the NOAEL is at least 1.5 mg/kg/day. Previously observed NOAELs for related compounds have been less than 0.1 mg/kg/day. The current studies show potent anti-tumor activity as well as an unusually positive safety profile for MRx102 when compared to triptolide and other triptolide derivatives. Further MRx102 drug development is underway, with the intention of submitting an Investigational New Drug application to the Food and Drug Administration leading to clinical evaluation of MRx102 in AML patients. Updated results on current drug development activities will be presented at the meeting. This work is supported in part by NCI SBIR Contract HHSN261200900061C to MyeloRx LLC. Disclosures:Fidler:MyeloRx LLC: Employment, Equity Ownership, PI for an NCI Contract to MyeloRx LLC, Patents & Royalties. An:MyeloRx LLC: Employment, Equity Ownership, participant in research under an NCI SBIR Contract to MyeloRx LLC. Musser:MyeloRx LLC: Employment, Equity Ownership, Patents & Royalties, participant in research under an NCI SBIR Contract to MyeloRx LLC. Mak:MyeloRx LLC: participant in research under an NCI SBIR Contract to MyeloRx LLC. Carter:MyeloRx LLC: participant in research under an NCI SBIR Contract to MyeloRx LLC. Andreeff:MyeloRx LLC: Consultancy, participant in research under an NCI SBIR Contract to MyeloRx LLC.

Clinical Pharmacokinetics of CAL-101, a p110δ Isoform-Selective PI3K Inhibitor, Following Single- and Multiple-Dose Administration In Healthy Volunteers and Patients with Hematological Malignancies

AbstractAbstract 1774Phosphatidylinositol 3-kinases (PI3Ks) regulate several cellular functions including motility, proliferation, and survival. PI3K pathway signaling is mediated by the Class I PI3K isoforms, α, β, δ and γ. The PI3K p110δ isoform is preferentially expressed in cells of hematological origin and in a variety of malignant cells. CAL-101 is a potent p110δ inhibitor with an EC50 of 62 nM in a whole-blood p110δ assay and >200-fold selectivity for p110δ relative to other PI3K isoforms. Consistent with this target selectivity, nonclinical toxicology and safety pharmacology data supported initial clinical assessment of oral CAL-101 in single-dose, multiple-dose, and food-effect studies in healthy volunteers. Because CAL-101 is a CYP450 3A4 substrate, the effect of ketoconazole (a potent CYP450 3A4 inhibitor) on CAL-101 pharmacokinetics was also evaluated in healthy volunteers. Preliminary evaluation of disposition, metabolism and elimination in healthy volunteers was achieved by coadministering a trace amount of [14C]CAL-101 and unlabeled CAL-101 either orally or intravenously (IV) with samples evaluated by accelerator mass spectrometry. CAL-101 pharmacokinetics (PK) were subsequently evaluated in patients with lymphoid malignancies. In healthy volunteers, CAL-101 was well tolerated at 400 mg (the highest single dose tested) and at 200 mg BID through 7 days (the highest multiple dose tested). The drug has also been symptomatically well tolerated in patients with lymphoid malignancies receiving CAL-101 at dose levels through 350 mg/kg (the highest dose tested) over many months. Monitorable, reversible transaminase elevations have been observed in some patients, most commonly in patients with lymphoma. No maximum tolerated dose (MTD) has been apparent. Increases in Cmax and AUC are less than dose proportional, revealing minimal gains in plasma exposure at dose levels >150 mg BID. The mean volume of distribution was moderate at 57.7 L. The t1/2 was ∼8 hours across all dose levels and there was no plasma accumulation over 7 or 28 days. The collective data support BID dosing at ≥150 mg; dose levels in this range maintain steady-state trough plasma concentrations that are >10-fold above the EC50 for the in vitro whole-blood assay. [14C]CAL-101 was metabolized to only 1 metabolite in plasma and CAL-101-derived materials were primarily excreted in feces (>65% of total dose) with minimal elimination via urine (<15% of total dose). A high-fat, high-calorie meal had no effect on Cmax but slowed absorption, leading to a shift in observed median Tmax from 1.5 h to 4.5 h, and a moderate 1.4-fold increase in AUC; these data suggest that CAL-101 can be given with or without food. When administered following 4 days of ketoconazole, increases in mean CAL-101 Cmax and AUC values were 1.3- and 1.8-fold, respectively; thus, CAL-101 is not a sensitive substrate for CYP450 3A4 and coadministration of CAL-101 with CYP450 3A4 inhibitors does not appear to be contraindicated. Taken together with clinical data documenting PI3K pathway inhibition, modulation of chemokine signaling, and substantial dose-dependent antitumor activity, these findings provide a characterization of CAL-101 clinical pharmacology that supports CAL-101 development as an investigational therapy for cancer and other indications. Disclosures:Webb:Calistoga Pharmaceuticals: Employment. Chen:Calistoga Pharmaceuticals: Employment. Yu:Calistoga Pharmaceuticals: Employment. Peterman:Calistoga Pharmaceuticals: Employment. Holes:Calistoga Pharmaceuticals: Employment. Lannutti:Calistoga Pharmaceutical Inc.: Employment. Miller:Calistoga Pharmaceuticals: Employment. Ulrich:Calistoga Pharmaceuticals: Employment, Equity Ownership.

Antitumor Activity and Immune Response Induction of a Dual Agonist of Toll-Like Receptors 7 and 8

Viral and synthetic single-stranded RNAs are the ligands for Toll-like receptors 7 and 8 (TLR7 and TLR8). We have reported a novel class of synthetic oligoribonucleotides, referred to as stabilized immune-modulatory RNA compounds, which act as agonists of TLR7, TLR8, or both TLR7 and TLR8 depending on the sequence composition and the presence of specific chemical modifications. In the present study, we evaluated the antitumor activity of a dual TLR7/8 agonist in tumor-bearing mice with peritoneal disseminated CT26.CL25 colon and 3LL-C75 lung carcinomas. Peritoneal administration of dual TLR7/8 agonist in mice bearing CT26.CL25 colon carcinomas had potent dose-dependent antitumor activity, which was associated with a marked decrease in CD4(+)CD25(+)Foxp3(+) T regulatory cells and a significant increase in tumor antigen-specific IFN-gamma-secreting effector cell responses in splenocytes and local tumor-infiltrating cells. In 3LL-C75 lung carcinoma, dual TLR7/8 agonist induced strong immune responses and antitumor effects in C57BL/6 and TLR9(-/-) mice, but not in TLR7(-/-) and MyD88(-/-) mice, indicating that the agonist induces immune responses via TLR7 and through the MyD88-dependent signaling pathway. TLR8 is not functional in mice. Additionally, s.c. administration of TLR7/8 agonist effectively prevented lung metastasis of tumors in the CT26.CL25 pulmonary metastasis model. These studies show that the dual TLR7/8 agonist induced Th1-type immune responses and potent antitumor activity in mice via TLR7 and through the MyD88-dependent pathway.

Abstract 571: PK and antitumor activity of DEBIO 0507, a new platinum derivative, in preclinical tumor models

Abstract Background: Since the introduction of cisplatin, carboplatin and oxaliplatin, several new generations of platinum analogues have been developed as candidates for chemotherapy. DEBIO 0507/NC-4016 is a DACH-Platin Polymeric Micelle. The main objectives of this project were to assess pharmacokinetic (PK) profile of DEBIO 0507 and evaluate its efficacy in preclinical studies against a broad spectrum of experimental tumors including syngenic, xenogenic tumor models and a cisplatin-resistant cell line. Methods: PK and biodistribution studies were conducted in multiple matrices (blood, tumor, liver, spleen, kidneys and pancreas) collected in mice bearing C38 mouse colon carcinoma and treated with a single IV injection of DEBIO 0507 at 2 mg Pt/kg. Antitumor activity of DEBIO 0507 intravenously injected every 4, 7 or 14 days at different doses was investigated in C38 mouse colon carcinoma bearing C57BL/6 mice. Antitumor effect of DEBIO 0507 was also assessed in a panel of human tumor xenografted in nude mice, Calu-6 and A-549 human lung carcinoma, HT-29 human colon carcinoma, PC-3 human prostatic tumor, FaDu human pharynx carcinoma, NIH:OVCAR3 human ovarian tumor, BxPC-3 human pancreas tumor and HepG2 human hepatocarcinoma. Results: A concentration of 61 µg Pt/ml was detected in plasma from 5 minutes after injection and persisted during two hours. Platinum was very slowly cleared from plasma as it was still detected 14 days after injection (t1/2α : 40h). An accumulation was observed in tumor from 5 minutes to 8 days after injection and terminal elimination phase was very long since platinum was also detected 14 days after injection. The same PK and BD profiles were observed for other tissues. DEBIO 0507 was not active in the models of NIH: OVCAR3 human ovarian tumor, BxPC-3 human pancreas tumor and HepG2 human hepatocarcinoma. DEBIO 0507 exhibited a significant and dose-dependent antitumor activity against C38 mouse colon carcinoma when administered every 4, 7 or 14 days (optimal T/C of 0, 0 and 5%, respectively). We also demonstrated growth inhibitory effects of DEBIO 0507 in human PC-3 prostate, Calu-6 lung, FaDu head and neck and A-549 lung tumors xenografted in nude mice when injected with similar doses and schedules (optimal T/C of : 26, 29, 39 and 40%, for each model respectively). Conclusion: DEBIO 0507 displayed a marked antitumor activity against a large spectrum of tumor models associated with a long exposure of tumor to Pt. These preclinical results provide useful data to support the design of clinical trials for DEBIO 0507. DEBIO 0507/NC-4016 was produced by Nanocarrier Co.,Ltd. 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 571.

Combined Treatment of Pancreatic Cancer with Mithramycin A and Tolfenamic Acid Promotes Sp1 Degradation and Synergistic Antitumor Activity

Mithramycin (MIT) and tolfenamic acid (TA) inhibit the activity of the transcription factor Sp1. In the present study, we investigated whether pancreatic cancer treatment with a combination of these compounds has a synergistic effect on Sp1 activity, tumor growth, and their underlying response mechanisms. Treatment of pancreatic tumor xenografts with MIT and TA produced dose-dependent antitumor activity, and significant antitumor activity of either compound alone was directly associated with systemic side effects. Combination treatment with nontoxic doses of both compounds produced synergistic antitumor activity, whereas treatment with a nontoxic dose of either compound alone lacked a discernible antitumor effect. Synergistic therapeutic effects correlated directly with synergistic antiproliferation and antiangiogenesis in vitro. Moreover, combination treatment resulted in Sp1 protein degradation, drastically downregulating expression of Sp1 and vascular endothelial growth factor. Our findings established that Sp1 is a critical target of TA and MIT in human pancreatic cancer therapy, rationalizing clinical studies to determine the effect of existing pancreatic cancer therapy regimens on Sp1 signaling in tumors and normal pancreatic tissue, and the ability of Sp1-targeting strategies to modify cancer responses.

Antiproliferative activity, mechanism of action and oral antitumor activity of CP-4126, a fatty acid derivative of gemcitabine, in in vitro and in vivo tumor models

SummaryGemcitabine is a deoxycytidine (dCyd) analog with activity in leukemia and solid tumors, which requires phosphorylation by deoxycytidine kinase (dCK). Decreased membrane transport is a mechanism of resistance to gemcitabine. In order to facilitate gemcitabine uptake and prolong retention in the cell, a lipophilic pro-drug was synthesized (CP-4126), with an elaidic fatty acid esterified at the 5′position. CP-4126 was tested in cell lines resistant to cytarabine, another dCyd analog or gemcitabine. Activity of gemcitabine and the derivative was comparable in the parent cell lines, while in dCK deficient cells all compounds were inactive. However, inhibition of nucleoside transport increased the IC50 for gemcitabine up to 200-fold, but not for CP-4126, underlining the independence of a nucleoside transporter. For in vivo evaluation, nude mice bearing a human xenograft were treated intraperitoneally every third day for five doses at the maximal tolerated dose. In melanoma, sarcoma, lung, prostate, pancreatic and breast cancer xenografts, gemcitabine and CP-4126 were equally and highly effective; in four other xenografts moderately but equally active. In contrast to gemcitabine, CP-4126 could be administered orally, with a schedule and dose dependent toxicity and antitumor activity. In a colon cancer xenograft, antitumor activity of orally administered CP-4126 was equal to the intraperitoneally administered drug. In conclusion, CP-4126 is membrane transporter independent. Intraperitoneally administered CP-4126 was as effective as gemcitabine in several xenografts and CP-4126 is tolerated when orally administered. CP-4126 seems to be a promising new anticancer drug.

Pharmacology and Antitumor Activity of ABC294640, a Selective Inhibitor of Sphingosine Kinase-2

Sphingolipid-metabolizing enzymes control the dynamic balance of the cellular levels of important bioactive lipids, including the apoptotic compound ceramide and the proliferative compound sphingosine 1-phosphate (S1P). Many growth factors and inflammatory cytokines promote the cleavage of sphingomyelin and ceramide leading to rapid elevation of S1P levels through the action of sphingosine kinases (SK1 and SK2). SK1 and SK2 are overexpressed in a variety of human cancers, making these enzymes potential molecular targets for cancer therapy. We have identified an aryladamantane compound, termed ABC294640 [3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide], that selectively inhibits SK2 activity in vitro, acting as a competitive inhibitor with respect to sphingosine with a K(i) of 9.8 muM, and attenuates S1P formation in intact cells. In tissue culture, ABC294640 suppresses the proliferation of a broad panel of tumor cell lines, and inhibits tumor cell migration concomitant with loss of microfilaments. In vivo, ABC294640 has excellent oral bioavailability, and demonstrates a plasma clearance half-time of 4.5 h in mice. Acute and chronic toxicology studies indicate that ABC294640 induces a transient minor decrease in the hematocrit of rats and mice; however, this normalizes by 28 days of treatment. No other changes in hematology parameters, or gross or microscopic tissue pathology, result from treatment with ABC294640. Oral administration of ABC294640 to mice bearing mammary adenocarcinoma xenografts results in dose-dependent antitumor activity associated with depletion of S1P levels in the tumors and progressive tumor cell apoptosis. Therefore, this newly developed SK2 inhibitor provides an orally available drug candidate for the treatment of cancer and other diseases.

Abstract A238: The effects of foretinib on MET phosphorylation, gene expression, cell proliferation and tumor growth in MET-amplified tumor cells in vitro and in vivo

Abstract The MET receptor tyrosine kinase (RTK) is frequently deregulated in many types of cancer and is a promising target for cancer drug development. Foretinib is a multikinase inhibitor with potent activity against MET and is currently in phase II clinical trials. In these studies, we determined the anti-tumor activity of foretinib in MET-amplified tumor cells and the MKN45 gastric xenograft tumor model in nude mice. We also measured the effect of foretinib on MET phosphorylation by immunoprecipitation and western blot and evaluated gene expression by Affymetrix microarrays in these models. In vitro foretinib demonstrated the most potent cell growth inhibitory activity in MET-amplified tumor cell lines MKN45 (IC50 = 9 ± 2 nM), HS746T (IC50 = 8 ± 2 nM), SNU5 (IC50 = 2 ± 0.2 nM) and NCI-H1993 (IC50 = 69 ± 5 nM). Foretinib inhibited the phosphorylation of MET, suppressed the phosphorylation of AKT and ERK, and reduced the expression of cyclin D1 in a dose dependent manner in these cells. Furthermore, foretinib given orally showed durable and near complete inhibition of MET phosphorylation (&amp;gt;90%) and dose dependent anti-tumor activity against MKN45 MET-amplified gastric xenograft tumors in nude mice. Tumor stasis and significant tumor growth delay were observed for foretinib dosed at 10 mg/kg and PF-02341066, a MET selective inhibitor, dosed at 50 mg/kg in the same study. In addition, foretinib or PF-02341066 showed similar gene alterations on both MKN45 cells and xenograft tumors. These genes are involved in RTK cross-talk and MET signaling pathways (e.g., HER2, HER3, MET, PIK3IP1, DUSP4, DUSP6), cell motility (e.g., CDH1, TIMP2, CLDN3, MMP1), cell proliferation (e.g., CCND1, TP53INP1, TYMS, CCNB1) and apoptosis (e.g., TNFSF10, CASP8). These results demonstrate that foretinib is a potent MET inhibitor with associated changes in gene expression which correlate with anti-tumor activity in MET-amplified tumor cells both in vitro and in vivo. This gene signature warrants further investigation for its relevance as a MET-inhibitor mediated pharmacodynamic marker. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A238.