Betty K. Samulitis

Phase I Trial of Imexon in Patients With Advanced Malignancy

PURPOSEnImexon, a pro-oxidant small molecule, has antitumor activity in preclinical models. The drug induces apoptosis through accumulation of reactive oxygen species. The purpose of this trial was to define the maximum-tolerated dose (MTD), toxicities, pharmacokinetics, and pharmacodynamics of imexon in patients with advanced cancers.nnnPATIENTS AND METHODSnForty-nine patients with metastatic cancer received intravenous imexon over 30 to 45 minutes for 5 consecutive days (one course) every other week (days 1 through 5 and 15 through 19) monthly. Doses were initially escalated using an accelerated trial design and then a modified Fibonacci method. Plasma imexon levels and six different thiols were measured by high-performance liquid chromatography assays.nnnRESULTSnThere were 13 dose levels evaluated, from 20 mg/m2/d to 1,000 mg/m2/d. The MTD recommended for phase II studies was 875 mg/m2/d for 5 days every 2 weeks (n = 9 patients). The two dose-limiting toxicities at 1,000 mg/m2/d involved grade 3 abdominal pain and fatigue and grade 4 neutropenia, which occurred in one patient each. Other common toxicities included nausea and vomiting (58%) and constipation (63%); both were managed well with prophylactic medications. One partial response was obtained in a heavily pretreated patient with non-Hodgkins lymphoma. Pharmacokinetic studies showed dose-independent clearance, with a 95-minute mean half-life. Plasma thiol studies showed a dose- and area under the curve-dependent decrease in cystine levels 8 hours after dosing at 750 mg/m2/d.nnnCONCLUSIONnThe phase II recommended dose of imexon is 875 mg/m2/d for 5 days every other week. A decrease in plasma thiols did correlate with imexon exposure.

Gemcitabine resistant pancreatic cancer cell lines acquire an invasive phenotype with collateral hypersensitivity to histone deacetylase inhibitors

Gemcitabine based treatment is currently a standard first line treatment for patients with advanced pancreatic cancer, however overall survival remains poor, and few options are available for patients that fail gemcitabine based therapy. To identify potential molecular targets in gemcitabine refractory pancreatic cancer, we developed a series of gemcitabine resistant (GR) cell lines. Initial drug exposure selected for an early resistant phenotype that was independent of drug metabolic pathways. Prolonged drug selection pressure after 16 weeks, led to an induction of cytidine deaminase (CDA) and enhanced drug detoxification. Cross resistance profiles demonstrate approximately 100-fold cross resistance to the pyrimidine nucleoside cytarabine, but no resistance to the same in class agents, azacytidine and decitabine. GR cell lines demonstrated a dose dependent collateral hypersensitivity to class I and II histone deacetylase (HDAC) inhibitors and decreased expression of 3 different global heterochromatin marks, as detected by H4K20me3, H3K9me3 and H3K27me3. Cell morphology of the drug resistant cell lines demonstrated a fibroblastic type appearance with loss of cell-cell junctions and an altered microarray expression pattern, using Gene Ontology (GO) annotation, consistent with progression to an invasive phenotype. Of particular note, the gemcitabine resistant cell lines displayed up to a 15 fold increase in invasive potential that directly correlates with the level of gemcitabine resistance. These findings suggest a mechanistic relationship between chemoresistance and metastatic potential in pancreatic carcinoma and provide evidence for molecular pathways that may be exploited to develop therapeutic strategies for refractory pancreatic cancer.

Characterization of novel diaryl oxazole-based compounds as potential agents to treat pancreatic cancer.

A series of diaryl- and fluorenone-based analogs of the lead compound UA-62784 [4-(5-(4-methoxyphenyl)oxazol-2-yl)-9H-fluoren-9-one] was synthesized with the intention of improving upon the selective cytotoxicity of UA-62784 against human pancreatic cancer cell lines with a deletion of the tumor suppressor gene deleted in pancreas cancer locus 4 (DPC-4, SMAD-4). Over 80 analogs were synthesized and tested for antitumor activity against pancreatic cancer (PC) cell lines (the PC series). Despite a structural relationship to UA-62784, which inhibits the mitotic kinesin centromere protein E (CENP-E), none of the analogs was selective for DPC-4-deleted pancreatic cancer cell lines. Furthermore, none of the analogs was a potent or selective inhibitor of four different mitotic kinesins (mitotic kinesin-5, CENP-E, mitotic kinesin-like protein-1, and mitotic centromere-associated kinesin). Therefore, other potential mechanisms of action were evaluated. A diaryl oxazole lead analog from this series, PC-046 [5-(4-methoxyphenyl)-2-(3-(3-methoxyphenyl)pyridin-4-yl) oxazole], was shown to potently inhibit several protein kinases that are overexpressed in human pancreatic cancers, including tyrosine receptor kinase B, interleukin-1 receptor-associated kinase-4, and proto-oncogene Pim-1. Cells exposed to PC-046 exhibit a cell cycle block in the S-phase followed by apoptotic death and necrosis. PC-046 effectively reduced MiaPaca-2 tumor growth in severe combined immunodeficiency mice by 80% compared with untreated controls. The plasma half-life was 7.5 h, and cytotoxic drug concentrations of >3 μM were achieved in vivo in mice. The diaryl oxazole series of compounds represent a new chemical class of anticancer agents that inhibit several types of cancer-relevant protein kinases.

Correlates of imexon sensitivity in human multiple myeloma cell lines

Imexon (NSC-714597) is an aziridine-containing imminopyrolidone in Phase I clinical trials. The current studies compared biological indices of cytotoxicity in 7 human multiple myeloma (MM) cell lines to develop a correlative model for imexon sensitivity. In the MM cell lines there was a wide range of sensitivity to imexon measured by standard cytotoxicity assays (MTT) and by viability/apoptosis/necrosis (Annexin-V-FITC/PI) measurements. The following sensitivity pattern was observed in order of decreasing sensitivity: IM-9 > 8226/S > MM.1S, ARH-77, H929 > 8226/I > U266. The same descending rank order was seen for loss of mitochondrial membrane potential (MMP), generation of reactive oxygen species (ROS) and, at high drug concentrations, thiol depletion. Cell cycle analysis showed imexon sensitive cells accumulate at the G2/M interphase. Although there was a positive correlation between increasing CuZnSOD levels and imexon resistance, no relationship was found for catalase, Bcl-2, mitochondrial thioredoxin or MnSOD levels. These findings suggest consistent phenotypes for imexon sensitivity and resistance in human MM cell lines exposed to drug for 48 h, with a combination of apoptosis and necrosis. Resistance is correlated with CuZnSOD expression, reduced drug accumulation, lack of ROS generation and maintenance of MMP. Oxidation of cellular thiols occurs only at high (supra-cytotoxic) drug levels and is, therefore, weakly correlated with cytotoxicity. This unique mechanism involving oxidation and the previously reported absence of myelosuppression suggests that imexon may be rationally combined with existing cytotoxic agents to improve therapeutic activity in MM.

Imexon-based combination chemotherapy in A375 human melanoma and RPMI 8226 human myeloma cell lines

PurposeThis study evaluated the cytotoxic effects of imexon (NSC-714597) in tumor cells when combined with a broad panel of chemotherapeutic drugs.MethodsThe sulforhodamine B (SRB) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assays were used to analyze the degree of growth inhibition for the combination studies in the A375 human malignant melanoma and RPMI 8226 human multiple myeloma cell lines, respectively. Cells were continuously exposed to both drugs at a constant molar ratio for 4–5xa0days. Combination effects were analyzed using the Median Effect method. Statistical significance was inferred if the 95% confidence interval for the combination interaction (C.I.) values for a particular two-drug combination did not include 1.0 (additivity). Synergy was inferred for C.I. valuesxa0<xa01.0 and antagonism for CI valuesxa0>xa01.0.ResultsImexon was synergistic when combined with DNA-binding agents (cisplatin, dacarbazine, melphalan) and pyrimidine-based antimetabolites (cytarabine, fluorouracil, gemcitabine) in both cell lines. Antagonistic combinations with imexon included methotrexate and the topoisomerase I (TOPO I) and II (TOPO II) inhibitors irinotecan, doxorubicin, mitoxantrone and etoposide. Docetaxel was synergistic with imexon in both cell lines whereas paclitaxel and fludarabine showed a mixed result. Dexamethasone and the proteasome inhibitor bortezomib showed synergy in myeloma cells and additivity in the melanoma cells. The vinca alkaloid, vinorelbine, and the multi-targeted antifol, pemetrexed, were additive with imexon in both cell lines.DiscussionThe consistent synergy seen for imexon and alkylating agents may relate to the sulfhydryl-lowering effect of imexon, which would render cells more sensitive to electrophilic species from the alkylators. The marked synergy noted with pyrimidine-based antimetabolites was unexpected and may relate to the induction of cell cycle arrest in S-phase. The strong antagonism noted for imexon with topoisomerase I and II inhibitors may be due to the effect of imexon at increasing oxidant levels which are known to antagonize the cytotoxic effects of topoisomerase poisons. In contrast, the synergy seen with bortezomib in myeloma cells may be related to an increase in reactive oxygen species (ROS) from both drugs. These results suggest that combinations of imexon with alkylating agents and pyrimidine-based antimetabolites are rational to pursue in therapeutic studies in vivo.

Imexon Induces an Oxidative Endoplasmic Reticulum Stress Response in Pancreatic Cancer Cells

Oxidative protein folding in the endoplasmic reticulum (ER) requires strict regulation of redox homeostasis. Disruption of the lumenal redox balance induces an integrated ER stress response that is associated with reduced protein translation, increased chaperone activity, and ultimately cell death. Imexon is a small-molecule chemotherapeutic agent that has been shown to bind glutathione (GSH) and induce oxidative stress in tumor cells; however, the mechanism of cytotoxicity is not well understood. In this report, we investigate the effects of imexon on the integrated ER stress response in pancreatic carcinoma cells. Acute exposure to imexon induces an ER stress response characterized by accumulation of the oxidized form of the oxidoreductase Ero1α, phosphorylation of eIF2α, and inhibition of protein synthesis. An RNA interference chemosensitization screen identified the eukaryotic translation initiation factor eIF2B5 as a target that enhanced imexon-induced growth inhibition of MiaPaCa-2 pancreatic cancer cells, but did not significantly augment the effects of imexon on protein synthesis. Concurrent reduction of intracellular thiols with N-acetyl cysteine reversed imexon activity, however cotreatment with superoxide scavengers had no effect, suggesting thiol binding may be a primary component of the oxidative effects of imexon. Moreover, the data suggest that disruption of the redox balance in the ER is a potential therapeutic target. Mol Cancer Res; 10(3); 392–400. ©2012 AACR.

Inhibition of protein synthesis by imexon reduces HIF-1α expression in normoxic and hypoxic pancreatic cancer cells

SummaryHypoxia-inducing factor-1 alpha (HIF-1α), is a major survival factor for tumor cells growing in a low oxygen environment. The anti-cancer agent imexon binds thiols and causes accumulation of reactive oxygen species (ROS) in pancreatic cancer cells. Unlike many cytotoxic agents, imexon is equi-cytotoxic in human MiaPaCa-2 and Panc-1 cells grown in normoxic (21% O2) and hypoxic (1% O2) conditions. Western blot analyses of imexon-treated cells demonstrated that imexon reduces HIF-1α protein levels in both normoxic and hypoxic conditions in a time- and concentration-dependant fashion. Gemcitabine did not similarly affect HIF-1α levels. Imexon did not reduce transcription of new HIF-1α mRNA, but did reduce the synthesis of new proteins, including HIF-1α, measured by 35S methionine/cysteine (Met/Cys) incorporation. Concurrently, the half-life of existing HIF-1α protein was increased by imexon, in association with a marked inhibition of chymotryptic activity in the 20S proteasome. The inhibition of HIF-1α translation was not specific, rather it was part of a general decrease in protein translation caused by imexon. This inhibitory effect on translation did not involve phosphorylation of eukaryotic initiation factor-2α (eIF-2α) and was not closely correlated to cell growth inhibition by imexon, suggesting that mechanisms other than protein synthesis inhibition contribute to the drug’s cytotoxic effects. In summary, imexon blocks the translation of new proteins, including HIF-1α, and this effect overcomes an increase in the stability of preformed HIF-1α due to proteasome inhibition by imexon. Because net HIF-1α levels are reduced by imexon, combination studies with other drugs affected by HIF-1α survival signaling are warranted.

Imexon enhances gemcitabine cytotoxicity by inhibition of ribonucleotide reductase

PurposeGemcitabine (GEM) is currently the standard first line treatment for pancreatic cancer; however, the overall survival of patients with this disease remains poor. Imexon is a pro-oxidant small molecule which produced a high response rate in combination with GEM in a phase I trial in pancreatic cancer. In this study, we investigate the combination of GEM with a novel redox-active agent, imexon, in vitro and in vivo.MethodsMedian effect analysis was used for in vitro combination cytotoxicity. The effect of imexon on GEM metabolism and uptake into cells and into DNA and effects on ribonucleotide reductase (RNR) were examined in vitro. The pharmacokinetics and antitumor efficacy of the imexon/GEM combination was evaluated in mouse models.ResultsIn three human pancreatic cancer lines, there was additivity for the imexon/GEM combination. There was significantly greater efficacy for the drug combination in Panc-1 xenograft tumors. A pharmacokinetic study in mice showed a near doubling in the AUC of imexon when GEM was co-administered, with no effect of imexon on GEM’s pharmacokinetic disposition. In vitro, imexon did not alter GEM’s metabolism or uptake into DNA, but significantly inhibited RNR, and this effect was greater when combined with GEM.ConclusionsThese results suggest that the interaction between imexon and GEM may be due to complimentary inhibition of RNR plus an enhanced exposure to imexon when the GEM is administered in vivo. This combination is currently being tested in a randomized phase II trial in pancreatic cancer.

Anti-tumor activity and mechanism of action for a cyanoaziridine-derivative, AMP423

PurposePreclinical studies evaluated the anti-tumor activity and mechanism of action of AMP423, a naphthyl derivative of 2-cyanoaziridine-1-carboxamide with structural similarity to the pro-oxidant anti-tumor agent imexon.MethodsThe cytotoxic potency was evaluated in vitro against a variety of human cancer cell lines. Mechanism-of-action studies were performed in the human 8226/S myeloma cell line and its imexon-resistant variant, 8226/IM10. In vivo activity was evaluated against human myeloma and lymphoma xenografts in SCID mice. Pharmacokinetics and toxicology were investigated in non-tumor-bearing mice.ResultsThe 72-h IC50s for all cell types ranged from 2 to 36xa0μM, across a wide variety of human cancer cell lines. AMP423 was active in SCID mice bearing 8226/S myeloma and SU-DHL-6 B-cell lymphoma tumors, with a median tumor growth delay (T−C) of 21xa0days (Pxa0=xa00.0002) and 5xa0days (Pxa0=xa00.004), respectively, and a median tumor growth inhibition (T/C) of 33.3% (Pxa0=xa00.03) and 82% (Pxa0=xa00.01), respectively. In non-tumor-bearing mice, AMP423 was not myelosuppressive. Mechanistic studies show that AMP423’s mode of cell death is a mixture of necrosis and apoptosis, with generation of reactive oxygen species, inhibition of protein synthesis, and a decrease in reduced sulfhydryl levels, but no alkylation of nucleophiles. Unlike its structural analog imexon, which causes cell cycle arrest in G2/M, AMP423 induces the accumulation of cells in S-phase.ConclusionsAMP423 has pro-oxidant effects similar to imexon, has greater cytotoxic potency in vitro, and has anti-tumor activity in hematologic tumors in vivo.

The diaryl oxazole PC-046 is a tubulin-binding agent with experimental anti-tumor efficacy in hematologic cancers

SummaryMicrotubule targeting agents are among the most widely used chemotherapeutics for both solid and hematological malignancies. This study characterizes the diaryl-oxazole based anticancer agent PC-046, which was originally identified for development based on selective activity in deleted in pancreas cancer locus 4 (DPC4/SMAD4) deficient tumors. PC-046 has growth inhibitory activity in a variety of tumor types in vitro, and efficacy in SCID mice was shown in human tumor xenografts of MV-4-11 acute myeloid leukemia, MM.1S multiple myeloma, and DU-145 prostate cancer. Pharmacokinetic studies demonstrated relatively high oral bioavailability (71xa0%) with distribution to both plasma and bone marrow. No myelosuppression was seen in non-tumor bearing SCID mice given a single dose just under the acute lethal dose. The COMPARE algorithm in the NCI-60 cell line panel demonstrated that PC-046 closely correlated to other known tubulin destabilizing agents (correlation coefficients ≈0.7 for vincristine and vinblastine). Mechanism of action studies showed cell cycle arrest in metaphase and inhibition of tubulin polymerization. Overall, these studies show that PC-046 is a synthetically-derived, small molecule microtubule destabilizing agent. Advantages over existing microtubule destabilizing agents include ease of synthesis, lack of MDR cross-resistance, good oral bioavailability and the lack of acute myelotoxicity.