Bidirectional Regulation between NDRG1 and GSK3ß Controls Tumor Growth and Is Targeted by Differentiation Inducing Factor-1 in Glioblastoma.

The development of potent and selective therapeutic approaches to glioblastoma (GBM), one of the most aggressive primary brain tumors, requires identification of molecular pathways that critically regulate the survival and proliferation of GBM. Previous studies have reported that deregulated expression of N-myc downstream regulated gene 1 (NDRG1) affects tumor growth and clinical outcomes of patients with various types of cancer including glioma. Here, we show that high level expression of NDRG1 in tumors significantly correlated with better prognosis of patients with GBM. Loss of NDRG1 in GBM cells upregulated GSK3ß levels and promoted cell proliferation, which was reversed by selective inhibitors of GSK3ß. In contrast, NDRG1 overexpression suppressed growth of GBM cells by decreasing GSK3ß levels via proteasomal degradation and by suppressing AKT and S6 cell growth signaling, as well as cell-cycle signaling pathways. Conversely, GSK3ß phosphorylated serine and threonine sites in the C-terminal domain of NDRG1 and limited the protein stability of NDRG1. Furthermore, treatment with differentiation inducing factor-1, a small molecule derived from Dictyostelium discoideum, enhanced NDRG1 expression, decreased GSK3ß expression, and exerted marked NDRG1-dependent antitumor effects in vitro and in vivo. Taken together, this study revealed a novel molecular mechanism by which NDRG1 inhibits GBM proliferation and progression. Our study thus identifies the NDRG1/GSK3ß signaling pathway as a key growth regulatory program in GBM, and suggests enhancing NDRG1 expression in GBM as a potent strategy toward the development of anti-GBM therapeutics. SIGNIFICANCE: This study identifies NDRG1 as a potent and endogenous suppressor of glioblastoma cell growth, suggesting the clinical benefits of NDRG1-targeted therapeutics against glioblastoma.

A Phase 2 Randomized, Double-Blind, Multicenter Trial of Imexon Plus Gemcitabine Versus Gemcitabine Plus Placebo in Patients With Metastatic Chemotherapy-naïve Pancreatic Adenocarcinoma.

BACKGROUND: Imexon is a cyanoaziridine-derived iminopyrrolidone which has synergistic cytotoxicity with gemcitabine. A phase 1 study of the combination demonstrated good tolerance with encouraging clinical activity and thus we conducted this randomized phase II study. MATERIALS AND METHODS: Patients with measurable, metastatic, treatment-naive pancreatic adenocarcinoma were randomized 1:1 to receive gemcitabine at 1000 mg/m days 1, 8, and 15 with either imexon, 875 mg/m or placebo days 1, 8, and 15 every 28 days. The primary endpoint was overall survival. Secondary endpoints included progression-free survival and response rate. RESULTS: A total of 142 patients were randomized, 72 to the imexon containing arm and 70 to the placebo arm. Patients in the imexon arm received an average of 3.6 cycles (range, 1 to 23) compared with 4.4 (range, 1 to 21) in the placebo arm. There was no increased rate of ≥grade 3 toxicity in the imexon arm. Seven patients had objective responses in the imexon arm (13.7%), whereas 9 did in the placebo arm (17%). In the imexon arm, 23 patients had ≥50% reduction in CA 19-9 from baseline (33%), whereas 22 did in the placebo arm (31.4%). The median progression-free survival was 2.8 months in the imexon arm (95% confidence interval [CI], 2.0-4.1 m) and 3.8 months in the placebo arm (95% CI, 2.2-4.7 m), P=0.504. The median overall survival time in the imexon arm was 5.2 months (95% CI, 4.2-6.7 m) as compared with 6.8 m (95% CI, 4.9-8.5 m) in the placebo arm, P=0.6822. CONCLUSIONS: The combination of imexon and gemcitabine does not result in improved outcome as initial therapy of metastatic pancreatic adenocarcinoma.

Differentiation-inducing factor-3 inhibits intestinal tumor growth in vitro and in vivo.

Differentiation-inducing factor-1 (DIF-1) produced by Dictyostelium discoideum strongly inhibits the proliferation of various types of cancer cells by suppression of the Wnt/ß-catenin signal transduction pathway. In the present study, we examined the effect of differentiation-inducing factor-3 (DIF-3), a monochlorinated metabolite of DIF-1 that is also produced by D. discoideum, on human colon cancer cell lines HCT-116 and DLD-1. DIF-3 strongly inhibited cell proliferation by arresting the cell cycle at the G0/G1 phase. DIF-3 reduced the expression levels of cyclin D1 and c-Myc by facilitating their degradation via activation of GSK-3ß in a time and dose-dependent manner. In addition, DIF-3 suppressed the expression of T-cell factor 7-like 2, a key transcription factor in the Wnt/ß-catenin signaling pathway, thereby reducing the mRNA levels of cyclin D1 and c-Myc. Subsequently, we examined the in vivo effects of DIF-3 in Mutyh(-/-) mice with oxidative stress-induced intestinal cancers. Repeated oral administration of DIF-3 markedly reduced the number and size of cancers at a level comparable to that of DIF-1. These data suggest that DIF-3 inhibits intestinal cancer cell proliferation in vitro and in vivo, probably by mechanisms similar to those identified in DIF-1 actions, and that DIF-3 may be a potential novel anti-cancer agent.

Exposure to 2,5-hexanedione is accompanied by ovarian and uterine oxidative stress and disruption of endocrine balance in rats.

2,5-Hexanedione (2,5-HD) is an aliphatic diketone identified as the main neurotoxic metabolite of the industrial chemicals n-hexane and methyl-n-butyl ketone. Considering the dearth of information on the female reproductive toxicity effects of 2,5-HD in the literature, we assessed the potential oxidative stress mechanisms of 2,5-HD in the ovary and uterus of Wistar rats. A total of 32 female rats were randomly allotted to four groups, in which rats were exposed to 2,5-HD at doses of 0% (control), 0.25%, 0.5% and 1.0% respectively in their drinking water for 21 days. The results showed that 2,5-HD significantly increased ovarian and uterine malondialdehyde (MDA) and hydrogen peroxide (H2O2) levels (p < 0.05). Additionally, while significant decreases in ovarian catalase, superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) activities occurred in all the 2,5-HD-treated groups, uterine catalase, GST, and GPx activities increased. Further, 2,5-HD increased follicle stimulating hormone, but decreased estrogen levels in all the 2,5-HD-treated groups, while prolactin increased in the 0.5, and 1.0% 2,5-HD-treated rats compared with the control (p < 0.05). Thus, these data imply that 2,5-HD exposure disrupts hormonal homeostasis and induces oxidative stress in the ovary and uterus of rats. These findings may therefore have toxicological implications in women occupationally exposed to n-hexane and methyl-n-butyl ketone.

Role of N-acetylcysteine in protecting against 2,5-hexanedione neurotoxicity in a rat model: changes in urinary pyrroles levels and motor activity performance.

The interference of N-acetylcysteine (NAC) on 2,5-hexanedione (2,5-HD) neurotoxicity was evaluated through behavioral assays and the analysis of urinary 2,5-HD, dimethylpyrrole norleucine (DMPN), and cysteine-pyrrole conjugate (DMPN NAC), by ESI-LC-MS/MS, in rats exposed to 2,5-HD and co-exposed to 2,5-HD and NAC. Wistar rats were treated with 4 doses of: 400mg 2,5-HD/kg bw (group I), 400mg 2,5-HD/kg bw+200mg NAC/kg bw (group II), 200mg NAC/kg bw (group III) and with saline (group IV). The results show a significant decrease (p<0.01) in urinary DMPN and free 2,5-HD, a significant increase (p<0.01) in DMPN NAC excretion, and a significant recovery (p<0.01) on motor activity in rats co-exposed to 2,5-HD+NAC, as compared with rats exposed to 2,5-HD alone. Taken together, our findings suggest that at the studied conditions NAC protects against 2,5-HD neurotoxicity and DMPN may be proposed as a new sensitive and specific biomarker of 2,5-HD neurotoxicity in animals treated with a toxic amount of 2,5-hexanedione.

Phase 2 study of imexon, a prooxidant molecule, in relapsed and refractory B-cell non-Hodgkin lymphoma.

Lymphoma cells are subject to higher levels of oxidative stress compared with their normal counterparts and may be vulnerable to manipulations of the cellular redox balance. We therefore designed a phase 2 study of imexon (Amplimexon/NSC-714597), a prooxidant molecule, in patients with relapsed/refractory B-cell non-Hodgkin lymphoma (NHL). Imexon was administered at 1000 mg/m(2) IV daily for 5 days in 21-day cycles. Gene expression analysis performed on pretreatment tumor specimens included 13 transcripts used to generate a redox signature score, previously demonstrated to correlate with lymphoma prognosis. Twenty-two patients were enrolled having follicular (n = 9), diffuse large B-cell (DLBCL) (n = 5), mantle cell (n = 3), transformed follicular (n = 2), small lymphocytic (n = 2), and Burkitt (n = 1) lymphoma. The most common grade 3/4 adverse events were anemia (14%) and neutropenia (9%). The overall response rate was 30%, including responses in follicular lymphoma (4 of 9) and DLBCL (2 of 5). Gene expression analyses revealed CD68 and the redox-related genes, GPX1 and SOD2, as well as a higher redox score to correlate with clinical responses. Therefore, pretreatment markers of oxidative stress may identify patients likely to respond to this therapeutic approach. This trial was registered at www.clinicaltrials.gov as #NCT01314014.

Inhibin B response to testicular toxicants hexachlorophene, ethane dimethane sulfonate, di-(n-butyl)-phthalate, nitrofurazone, DL-ethionine, 17-alpha ethinylestradiol, 2,5-hexanedione, or carbendazim following short-term dosing in male rats.

BACKGROUND: Inhibin B is a heterodimer glycoprotein that downregulates follicle-stimulating hormone and is produced predominantly by Sertoli cells. The potential correlation between changes in plasma Inhibin B and Sertoli cell toxicity was evaluated in male rats administered testicular toxicants in eight studies. Inhibin B fluctuations over 24 hr were also measured. METHODS: Adult rats were administered one of eight testicular toxicants for 1 to 29 days. The toxicants were DL-ethionine, dibutyl phthalate, nitrofurazone, 2,5-hexanedione, 17-alpha ethinylestradiol, ethane dimethane sulfonate, hexachlorophene, and carbendazim. In a separate study plasma was collected throughout a 24-hr period via an automatic blood sampler. RESULTS: Histomorphologic testicular findings included seminiferous tubule degeneration, round and elongate spermatid degeneration/necrosis, seminiferous tubule vacuolation, aspermatogenesis, and interstitial cell degeneration. There was a varying response of plasma Inhibin B levels to seminiferous tubule toxicity, with three studies showing high correlation, three studies with a response only at a certain time or dose, and two studies with no Inhibin B changes. In a receiver operating characteristics exclusion model analysis, where treated samples without histopathology were excluded, Inhibin B showed a sensitivity of 70% at 90% specificity in studies targeting seminiferous tubule toxicity. CONCLUSION: Decreases in Inhibin B correlated with Sertoli cell toxicity in the majority of studies evaluated, demonstrating the value of Inhibin B as a potential biomarker of testicular toxicity. There was no correlation between decreases in Inhibin B and interstitial cell degeneration. In addition, a pattern of Inhibin B secretion could not be identified over 24 hr.

Differentiation-inducing factor-1 enhances 5-fluorouracil action on oral cancer cells inhibiting E2F1 and thymidylate synthase mRNAs accumulation.

PURPOSE: Differentiation-inducing factor-1 (DIF-1) is a morphogen originally identified in the amoebozoan Dictyostelium discoideum. In mammalian cells, it has been shown to activate GSK3ß, which in turn is expected to reduce levels of ß-catenin and cyclin D1, thus mediating DIF-1 antiproliferative properties. Since this could alter the expression and activity of E2F1 transcription factor and consequently those of the prognostic marker/chemotherapy target thymidylate synthase (TS), we evaluated (1) whether DIF-1 could effectively regulate these genes, (2) whether it could interfere with cell viability, and (3) whether DIF-1 activity could enhance the efficacy of the TS inhibitor 5-fluorouracil (5-FU). METHODS: We investigated the effects of DIF-1 in continuous human cell lines derived from two oral tumor histotypes (corresponding to an adenosquamous and a squamous carcinoma) and a gingival epithelium. We evaluated mRNA accumulation by means of quantitative real-time PCR and efficacy of drugs on cell viability by means of MTT assay. RESULTS: DIF-1 inhibited the accumulation of E2F1 mRNA and reduces TS mRNA levels in tumor cell lines, but did not alter mRNA levels in the gingival counterpart. As a result, it inhibited proliferation preferentially of tumor cell in time- and concentration-dependent manner. Moreover, it enhanced cytotoxic effects of 5-FU only in tumor cell, whereas reduced them in the gingival counterpart. CONCLUSIONS: These findings suggest a tumor-specific action of DIF-1 on oral carcinoma cells. Thus, interfering with E2F1 and TS transcription, DIF-1 potentiates TS enzymatic inhibitors.

Imexon enhances gemcitabine cytotoxicity by inhibition of ribonucleotide reductase.

PURPOSE: Gemcitabine (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. METHODS: Median 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. RESULTS: In 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. CONCLUSIONS: These 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.

A phase 1-2 study of imexon plus dacarbazine in patients with unresectable metastatic melanoma.

BACKGROUND: Imexon (Amplimexon) is an aziridine compound that increases reactive oxygen species, disrupts mitochondrial membranes, and induces apoptosis. Preclinical studies showed activity against melanoma cell lines and models in mice, and synergy with dacarbazine. The authors evaluated standard doses of dacarbazine combined with increasing doses of imexon to determine the maximal tolerated dose (MTD), toxicities, pharmacokinetics, and efficacy. METHODS: Sixty-eight chemotherapy-naive melanoma patients (1 inoperable stage III and 67 stage IV) were treated with dacarbazine (250 mg/m2) and imexon (570-1300 mg/m2), both daily for 5 days every 3 weeks. RESULTS: There were 18 patients in the phase 1, and 50 in the phase 2 component of the study. The MTD of imexon with dacarbazine was 1000 mg/m2. Dose-limiting toxicities were pulmonary edema and hepatorenal failure. At the MTD, therapy was well tolerated. The most common toxicities (any grade) were vomiting, diarrhea, anemia, thrombocytopenia, anorexia, fever, and constipation. Among 68 patients, there were 7 treatment-related serious adverse events. Partial response and stable disease rates were 5.9% and 25% for all subjects and 2% and 30% for the phase 2 patients, respectively. Median progression-free and overall survival of all patients were 2.0 and 11.7 months and 2 and 7.5 months for the phase 2 patients, respectively. Overall survival of the 31 patients with normal lactate dehydrogenase levels was >22.5 months. Pharmacokinetics of both drugs were similar to previous reports. CONCLUSIONS: Imexon plus dacarbazine was well tolerated. The survival data suggest further evaluation in a randomized phase 2 study.

A phase I study of imexon plus gemcitabine as first-line therapy for advanced pancreatic cancer.

PURPOSE: Imexon is an aziridine-derived iminopyrrolidone which has synergy with gemcitabine in pancreatic cancer cell lines. Gemcitabine is a standard therapy for pancreatic cancer. We performed a phase I trial of imexon and gemcitabine to evaluate safety, dose-limiting toxicity (DLT), and maximum tolerated dose (MTD) in patients with advanced pancreatic cancer. METHODS: Patients with untreated locally advanced or metastatic pancreatic adenocarcinoma received therapy in sequential cohorts on regimen A (n = 19; imexon 200 or 280 mg/m(2) intravenously (IV) over 30 min days 1-5, 15-19 and gemcitabine 800 or 1,000 mg/m(2) IV over 30 min on days 1,8,15 every 28 days) or regimen B (n = 86; imexon 280-1,300 mg/m(2) IV over 30-60 min days 1, 8, and 15 and gemcitabine 1,000 mg/m(2) IV over 30 min on days 1, 8, and 15 every 28 days). RESULTS: One hundred five patients received 340 treatment cycles (median 2, range 1-16). PATIENT CHARACTERISTICS: median age 63, 61% male, ECOG PS 0/1 50%/50%, 93% metastatic. DLT was abdominal cramping and pain, often with transient, acute diarrhea. Best response was confirmed partial response (PR) in 11.4%, 8.9% unconfirmed PR, and 48.1% with stable disease. There was a dose proportional increase in imexon AUC across the doses tested with terminal half life 69 min at the MTD and no alteration of gemcitabine pharmacokinetics. CONCLUSIONS: The recommended phase II dose of imexon is 875 mg/m(2) with gemcitabine 1,000 mg/m(2). DLT was acute abdominal pain and cramping. Encouraging antitumor responses support further evaluation of this combination in advanced pancreatic cancer.

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

PURPOSE: This study evaluated the cytotoxic effects of imexon (NSC-714597) in tumor cells when combined with a broad panel of chemotherapeutic drugs. METHODS: The 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 4-5 days. 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. values<1.0 and antagonism for CI values>1.0. RESULTS: Imexon 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. DISCUSSION: The 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.

Oxidative stress and apoptosis: a new treatment paradigm in cancer.

Redox regulation has been shown to be an important component of malignant cell survival. Tipping the cellular redox balance through pharmacologic regulation in favor of increasing intracellular reactive oxygen species (ROS) and/or depleting protective reducing metabolites (such as glutathione and nicotinamide adenine dinucleotide phosphate) may lead to oxidative stress and resultant induction of apoptosis for the treatment of cancer. We review the biology and importance of ROS with regard to malignant and normal cells. Moreover, we discuss pre-clinical and clinical data regarding novel therapeutic agents that modulate the cellular redox system including buthionine sulfoximine, ascorbic acid, arsenic trioxide, imexon, and motexafin gadolinium as single-agents and in combination. Continued research is needed to better understand the mechanisms and specific apoptotic pathways involved in ROS-induced cell death, as well as, to determine the most rationale and effective combination of redox-active agents.

Differentiation-inducing factor-1 suppresses gene expression of cyclin D1 in tumor cells.

To determine the mechanism by which differentiation-inducing factor-1 (DIF-1), a morphogen of Dictyostelium discoideum, inhibits tumor cell proliferation, we examined the effect of DIF-1 on the gene expression of cyclin D1. DIF-1 strongly reduced the expression of cyclin D1 mRNA and correspondingly decreased the amount of beta-catenin in HeLa cells and squamous cell carcinoma cells. DIF-1 activated glycogen synthase kinase-3beta (GSK-3beta) and inhibition of GSK-3beta attenuated the DIF-1-induced beta-catenin degradation, indicating the involvement of GSK-3beta in this effect. Moreover, DIF-1 reduced the activities of T-cell factor (TCF)/lymphoid enhancer factor (LEF) reporter plasmid and a reporter gene driven by the human cyclin D1 promoter. Eliminating the TCF/LEF consensus site from the cyclin D1 promoter diminished the effect of DIF-1. These results suggest that DIF-1 inhibits Wnt/beta-catenin signaling, resulting in the suppression of cyclin D1 promoter activity.

Compatibility and stability of Imexon in infusion devices and its in vitro biocompatibility.

Imexon is an investigational anti-cancer agent, pharmaceutically formulated as a lyophilized solid for i.v. infusion requiring reconstitution and subsequent dilution in infusion fluid before infusion. Imexon contains a highly reactive aziridine ring in its structure, which limits its stability in aqueous solutions. In the present study, several in vitro studies were conducted to determine the administration parameters for use in the forthcoming phase I clinical trial. The stability of Imexon in the reconstituted solution and infusion solutions was investigated, including its tendency to degrade to its main degradation product BM41.209, and to its hydroxy and chloride adducts. The compatibility of the infusion solution with glass, low-density polyethylene and freeflex polyolefin containers, and its potency to cause vascular irritation and hemolysis upon i.v. infusion were investigated. Imexon was found to be stable for 8 h in the reconstituted product and for 2 h after dilution to infusion solution concentrations in normal saline. The infusion solution was compatible with the freeflex polyolefin container and polyvinylchloride infusion lining, showing no sorption of Imexon during a 15-min infusion duration and no release of the plasticizer diethylhexyl phthalate. Furthermore, Imexon infusion solution showed no indication for vascular irritation or hemolysis upon i.v. infusion, as measured with a static in vitro model with incubation with whole blood. In conclusion, Imexon should be administered using a freeflex polyolefin infusion container within 2 h after preparation and a 15-min infusion duration. The results obtained with an in vitro model show that no vascular irritation or hemolysis is expected upon i.v. infusion.

Pharmaceutical development of a lyophilised dosage form for the investigational anticancer agent Imexon using dimethyl sulfoxide as solubilising and stabilising agent.

Imexon is a member of the class of 2-cyanoaziridine derivatives, which have been of interest as immunomodulators and anticancer agents since the late 1970s. For the scheduled phase I clinical trials a stable, sterile, injectable pharmaceutical dosage form containing 100 mg Imexon was required. Despite adequate solubility, its instability in aqueous media seriously hampered the pharmaceutical development of Imexon. In this study we describe the successful use of the organic solvent dimethyl sulfoxide (DMSO) as a formulation vehicle for Imexon. DMSO is shown to provide the stability required for Imexon during manufacturing and to be a suitable vehicle for lyophilisation, which was employed to gain sufficient shelf-life for the final product. The relatively low vapour pressure of DMSO, which would theoretically result in extremely slow sublimation during lyophilisation, was shown not to limit the successful lyophilisation of Imexon from DMSO at a concentration of 25 mg/mL. The lyophilisation cycle developed for Imexon resulted in residual DMSO contents of 4.6 +/- 0.6% in the lyophilised product, limiting the amount of DMSO administered to the patient to well below the 50 mg/day acceptable in pharmaceutical products as stated in ICH guidelines. Imexon 100 mg/vial lyophilised product was shown stable for at least 12 months of storage at -20 degrees C and +5 +/- 3 degrees C in the dark.

Thermolabile hemoglobin formation by 2-cyanaziridine derivatives.

The erythrocyte toxicity of imexone, azimexone, ciamexone, and its derivatives was assessed by means of the thermolabile hemoglobin formation test. After administration of doses up to 500 mg/kg to mice, imexone had no effect, whereas in accordance with previous studies azimexone strongly and ciamexone moderately enhanced thermolabile hemoglobin formation. With regard to the derivatives (metabolites) of ciamexone, the alcohol was as active as ciamexone itself, whereas ciamexone aldehyde gave a weak reaction. Both ciamexone acid and ciamexone-cysteine had no influence. Demethyl ciamexone, however, was more toxic than ciamexone, further indicating that the metabolic oxidation of the methyl group represents a detoxifying step. In contrast to the in vivo results, after incubation with blood samples solely imexone (in the range of mg/ml blood) but none of the other 2-cyanaziridines enhanced thermolabile globin formation. Preincubation of imexone with cysteine inhibited this effect. The results are discussed in view of the hypothesis that binding of the nitrile group of the 2-cyanaziridines to mercapto groups of proteins precedes their biological effects.

Preclinical pharmacokinetics and antitumor activity of imexon.

Imexon is an aziridine compound originally studied for immune-enhancing effects on lymphocytes. The drug was well-tolerated in humans and was shown to be active in a variety of animal tumor models. Recently, imexon has demonstrated antitumor activity in human multiple myeloma cell lines in vitro. The pharmacokinetics of the compound using normal phase HPLC assay were studied in normal mice and in dogs with mast cell tumors. Doses of 100 mg/kg given intraperitoneally produced peak plasma levels over 100 micrograms/ml in mice and the drug was rapidly eliminated with half lives of 8 minutes (alpha phase) and 29 minutes (beta phase). Only 20% of an oral imexon dose was absorbed in the mouse. In dogs, the alpha and beta phase half lives ranged from 18-26 minutes and 91-110 minutes, respectively. Peak levels over 100 micrograms/ml were obtained following intravenous doses of 12.5 mg/kg and 25 mg/kg. Imexon was active in mice bearing either P-388 or L-1210 leukemia, but not in mice with B-16 melanoma. These results suggest that cytotoxic drug concentrations can be obtained in vivo and that imexon is active in lymphoproliferative tumors.

Effect of acute administration of 2,5-hexanedione on nociception in rats

Hexacarbon compounds are neurotxic to man and animals. These substance also inhibit various enzymes in vitro, including acetylcholinesterase. Since some cholinesterase inhibitor alter nociceptor we determined the effect of acute ip administration of 2,5-hexanedione on nociception in female Wistar rats (75-90 days old, 170-200g; 15-17 rats in each group) using a tail-flick apparatus. The rats were injected ip with vehicle solution (120mMNaCl containing 10 mM potassium phosphate buffer, pH 7.2) and 200, 400 or 800 mg/Kg of 2,5-hexanedione in a volume of 1 ml/Kg body weight. Tail-flick latencies were obtained 10, 30, 60 and 90 min after drug administration. All doses of 2,5-hexanedione caused antinociception (p<0.001) but the appearance and duration of the analgesia varied according to the dose of the drug. The highest dose tested (800 mg/Kg) caused analgesia from 10 to 60 min, 400 mg/Kg caused anal00 mg/Kg caused analgesia at 30 and 60 min, and 200 mg/Kg produced antinociception only at 60 min after drug injection (P < 0.05 for all the above comparisons). These results suggest that 2,5-hexanedione induces antinociception in rats. Whether this effect is mediated by a cholinergic mechanism is under inverstigation