Potent Inhibitor Of Ribonucleotide Reductase Research Articles

Potent ribonucleotide reductase inhibitors: Thiazole-containing thiosemicarbazone derivatives.

The antioxidant, antimalarial, antibacterial, and antitumor activities of thiosemicarbazones have made this class of compounds important for medicinal chemists. In addition, thiosemicarbazones are among the most potent and well-known ribonucleotide reductase inhibitors. In this study, 24 new thiosemicarbazone derivatives were synthesized, and the structures and purity of the compounds were determined by IR, 1 H NMR, 13 C NMR, mass spectroscopy, and elemental analysis. The IC50 values of these 24 compounds were determined with an assay for ribonucleotide reductase inhibition. Compounds 19, 20, and 24 inhibited ribonucleotide reductase enzyme activity at a higher level than metisazone as standard. The cytotoxic effects of these compounds were measured on the MCF7 (human breast adenocarcinoma) and HEK293 (human embryonic kidney) cell lines. Similarly, compounds 19, 20, and 24 had a selective effect on the MCF7 and HEK293 cell lines, killing more cancer cells than cisplatin as standard. The compounds (especially 19, 20, and 24 as the most active ones) were then subjected to docking experiments to identify the probable interactions between the ligands and the enzyme active site. The complex formation was shown qualitatively. The ADME (absorption, distribution, metabolism, and excretion) properties of the compounds were analyzed using in-silico techniques.

Design, Synthesis and biological evaluation of diazeno-thiazole derivatives as ribonucleotide reductase inhibitors

Ribonucleotide reductase(RNR) is a metalloenzyme that catalyses the rate limiting step in DNA synthesis and repair. It causes the reduction of ribonucleotide to 2’-deoxyribonuclotides which are used as precursors for DNA synthesis, thus offering a good target for inhibition of cell synthesis. Experimental results have been proven that RNR inhibitors can be used as antiviral, anticancer or antibacterial agents. Here we report the synthesis of a novel class of diazeno-thiazole derivatives as potent RNR inhibitors. A series of forty molecules were synthesized and evaluated for their RNR inhibitory properties. All compounds were found to be good inhibitors of the RNR. Compound 3iwas found to be most active showing an IC50 value of 0.8 µm. The established SAR study indicated the presence of a polar bridge with an adjacent flexible aromatic ringprerequisite for RNR inhibitory activity. Moreover, compounds having an additional 4-chloro phenyl ring were found to be most potent.

Didox suppresses IgE-mediated mast cell response (HYP4P.317)

Abstract Mast cells play a central role in allergic reactions through high-affinity IgE receptor (FcεRI)-mediated responses. FcεRI cross-linking on mast cells activates multiple signaling pathways that lead to sustained inflammation through degranulation, prolonged generation of reactive oxygen species, and production of various cytokines and chemokines. These cellular responses are involved in inflammatory diseases including asthma, rheumatoid arthritis, multiple sclerosis and heart disease. As a synthetic anti-oxidant and a potent ribonucleotide reductase inhibitor capable of suppressing gene expression, Didox (3,4-Dihyroxybenzohydroxamic acid) has become an attractive therapeutic for treating inflammatory diseases. Here we investigated the anti-inflammatory abilities of Didox as a therapeutic approach to attenuate the allergic response. Our results show that Didox treatment yields a dose-dependent reduction in IgE-stimulated cytokine secretion among bone marrow-derived mast cell (BMMC). These effects are not directly linked to reduced receptor expression, or an increase in cell death and suggest a decrease in signaling associated with cytokine secretion. Together these data could indicate a role for Didox in the treatment of MC-associated inflammatory and autoimmune diseases.

Abstract 2637: Terminal dimethyl substitution of Triapine leads to activation of CHOP and induction of apoptosis via ER stress in human colon cancer cells

Abstract The α-N-heterocyclic thiosemicarbazone Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone) has been studied in several phase I & II clinical trials and it is known as a potent ribonucleotide reductase inhibitor. We synthesized Triapine (3-AP) and the corresponding terminally N4-dimethylated derivative (3-AP-Me2). Previously, we have shown that dimethylation of the terminal amino group leads to significant enhancement of cytotoxicity. In this study we utilized the intrinsic fluorescence properties of these compounds to investigate their intracellular distribution and studied the ability of both compounds to induce endoplasmic reticulum (ER) stress in SW480 colon carcinoma cells. Fluorescence microscopy was performed on viable SW480 cells (colon carcinoma) treated with 3-AP and 3-AP-Me2, showing a strong affinity of the compounds to the nuclear membrane and to cytosolic structures. Co-localization studies with organelle-specific staining revealed that both agents are associated with structures of ER and mitochondria. Therefore, we hypothesized an interference with ER functions. Immunoblotting showed that treatment with the dimethylated compound (3-AP-Me2) results in increased expression of BIP/GRP78 and induction of CHOP. Both are relevant stress markers for unfolded protein response (UPR), and CHOP indicates induction of apoptosis via the mitochondrial pathway. In contrast, treatment with 3-AP leads to an inhibition of BIP/GRP78 expression and does not induce CHOP expression. However, both compounds cause splicing of XBP-1 mRNA indicating activation of the IRE1-α pathway, which triggers apoptosis by activation of the ASK1-JNK pathway. In accordance with these findings 3-AP as well as 3-AP-Me2 activates c-Jun N-terminal kinase (JNK), which is commonly associated with cellular stress such as UPR. A dichlorofluorescein assay showed no generation of reactive oxygen species (ROS) neither by 3-AP nor by 3-AP-Me2, suggesting that the initiation of the ER stress pathway is ROS independent. In conclusion, dimethylation of the terminal amino group leads to up regulation of ER stress mechanism by increase of BIP/GRP79 expression and induction of CHOP. Activation of CHOP indicates induction of apoptosis and may be crucial for the higher cytotoxicity of dimethylated derivates of thiosemicarbazones. XBP-1 splicing and phosphorylation of JNK suggest an activation of the IRE1α-ASK1-JNK signaling pathway, which is connected to cell death involving proteins of the BCL-2 family. The induction of ER stress in a ROS independent manner is a novel mechanism of action relevant for the anti-neoplastic activity of this class of compounds. 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 2637. doi:10.1158/1538-7445.AM2011-2637

Didox (3,4-Dihydroxybenzohydroxamic acid) is a potent anti-oxidant and anti-inflammatory compound (35.17)

Abstract Didox (3,4-Dihydroxybenzohydroxamic acid) is a synthetic antioxidant and one of the most potent ribonucleotide reductase inhibitors developed to date. Little is known about the potential for Didox as an anti-inflammatory agent. After an analysis of more than 200 oxidative stress- and inflammatory-associated genes in mouse RAW264.7 macrophages we show that Didox significantly lowers LPS-induced expression of inflammatory genes (iNOS, IL-1α, IL-1β, IL-6, TNF-α, NF-KB, Cox-2, p38α, TLR4, GM-CSF, G-CSF, CCL2, CCL4, others), while upregulating expression levels of anti-oxidant and anti-inflammatory genes (Catalase, glutathione peroxidase, IL-10, superoxide dismutase, and peroxiredoxins, others). In addition, Didox dose-dependently inhibits LPS-induced IL-6 secretion and nitric oxide production. Buthionine Sulfoximine- and LPS-induced ROS production are also suppressed by Didox. We also show that phagocytosis of yeast particles is decreased as well. In terms of cellular signaling, Didox prevents NF-KB activation and translocation of NF-kappa beta p65 subunits in LPS-treated cells. These results suggest that the mechanism behind anti-oxidant and anti-inflammatory properties of Didox may be mediated, at least in part, via modulation of NF-kappa beta signaling. Didox may, therefore, may hold promise in managing chronic diseases exacerbated by LPS-induced oxidative stress and inflammation in macrophage-mediated inflammation.

Inhibitory mechanisms of heterocyclic carboxaldehyde thiosemicabazones for two forms of human ribonucleotide reductase

Two forms of ribonucleotide reductase (RR), consisting of M1 with M2 subunits and M1 with p53R2 subunits, are involved in DNA replication and damage repair, respectively. 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone (3AP), one of the heterocyclic carboxaldehyde thiosemicabazones (HCTs), is a potent RR inhibitor in clinical trial for cancer treatment. In this study, 3AP and its 7 derivatives showed 100–1000-fold higher inhibitory potency on KB nasopharyngeal carcinoma cells than hydroxyurea and were fully active against hydroxyurea- and gemcitabine-resistant KB cells. In vitro RR assays using two recombinant RRs showed that all 8 HCTs decreased the activity of both RRs in a dose-dependent manner and the efficiency was compatible with that on cell proliferation inhibition. Iron has different impact on the behavior of the compounds toward RRs. In the absence of iron, the HCTs showed more selective inhibition for p53R2–M1 than M2–M1, while addition of iron increased their activity but reduced their selectivity for two RRs. Radioligand binding assays showed that [ 3H]3AP directly bounded to the small subunits. Electron paramagnetic resonance measurements demonstrated that these HCTs generated reactive oxygen species with ferrous iron, which quenched the diiron-tyrosyl radical co-factor of the small subunits and hence the enzyme activity. While the ROS may be a common mediator responsible for the potent activity of the HCTs, the different characteristics of the small subunit proteins are probably associated with the subunit-selectivity of inhibition. Better understanding of the mechanism of action of RR inhibition may improve design of new potent and subunit-selective RR inhibitors for cancer therapy.

Electron paramagnetic resonance study of peripheral blood mononuclear cells from patients with refractory solid tumors treated with Triapine®

The metal chelator Triapine®, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone, is a potent inhibitor of ribonucleotide reductase. EPR spectra consistent with signals from Fe–transferrin, heme, and low-spin iron or cupric ion were observed in peripheral blood mononuclear cells (PBMCs) obtained from patients treated with Triapine®. One signal that is unequivocally identified is the signal for Fe–transferrin. It is hypothesized that Fe uptake is blocked by reactive oxygen species generated by FeT2 or CuT that damage transferrin or transferrin receptor. A potential source for the increase in the heme signal is cytochrome c released from the mitochondria. These results provide valuable insight into the in vivo mechanism of action of Triapine®.

Phase I and pharmacokinetic study of Triapine ®, a potent ribonucleotide reductase inhibitor, in adults with advanced hematologic malignancies

Triapine ®, a potent inhibitor of ribonucleotide reductase, has demonstrated anti-leukemia activity in pre-clinical models. We conducted a Phase I study of Triapine administered as a 2 h infusion for 5 days in 25 adults with advanced leukemias. We established that Triapine at 96 mg/m 2 once a day can be given safely on days 1–5 and 15–19 or 1–5 and 8–12 of a 4-week cycle. When administered twice a day on days 1–5 and 8–12, the maximum tolerated dose of Triapine appears to be 64 mg/m 2, although the true criteria for DLT were not met by protocol definition. No CR or PR were observed, but 76% of patients had a >50% reduction in white blood cell counts. At all dose levels, the peak plasma concentration of Triapine (2.2–5.5 μM) was above levels required to achieve in vitro/ in vivo leukemia growth inhibition. Based on these data, we conclude that Triapine warrants further investigation in hematologic malignancies.

The synergistic interaction of gemcitabine and cytosine arabinoside with the ribonucleotide reductase inhibitor triapine is schedule dependent

Gemcitabine and ara-C have multiple mechanisms of action: DNA incorporation and for gemcitabine also ribonucleotide reductase (RNR) inhibition. Since dCTP competes with their incorporation into DNA, dCTP depletion can potentiate their cytotoxicity. We investigated whether additional RNR inhibition by Triapine (3-AP), a new potent RNR inhibitor, enhanced cytotoxicity of gemcitabine and ara-C in four non-small-cell-lung-cancer (NSCLC) cell lines, using the multiple-drug-effect analysis. Simultaneous and sequential exposure (preexposure to 3-AP for 24 h) in a constant molar ratio of 3-AP and gemcitabine was antagonistic/additive in all cell lines. Preexposure to 3-AP at an IC 25 concentration for 24 h before variable concentrations of gemcitabine was synergistic. RNR inhibition by 3-AP resulted in a more synergistic interaction in combination with ara-C, which does not inhibit RNR. Two cell lines with pronounced synergism (SW1573) or antagonism (H460) for gemcitabine/3-AP, were evaluated for accumulation of the active metabolites, dFdCTP and ara-CTP. Simultaneous exposure induced no or a small increase, but ara-CTP increased after pretreatment with 3-AP, 4-fold in SW1573 cells, but not in H460 (<1.5 fold). Ara-C and gemcitabine incorporation into DNA were more pronounced (about 2-fold increased) for sequential treatment in SW1573 compared to H460 cells (<1.5 fold). This was not related to the activity and expression of deoxycytidine kinase and the M2 subunit of RNR. In conclusion, RNR inhibition by 3-AP prior to gemcitabine or ara-C exposure stimulates accumulation of the active metabolites and incorporation into DNA. The combination 3-AP/Ara-C is more synergistic than 3-AP/gemcitabine possibly because gemcitabine already inhibits RNR, but ara-C does not.

Preventive Effect of Trimidox on Oxidative Stress in U937 Cell Line

Trimidox (3,4,5-trihydroxybenzamidoxime) is one of the most potent ribonucleotide reductase inhibitors, revealing an antitumor effect in several experimental studies. We have examined the effect of trimidox on the induction of cytotoxicity and apoptosis via oxidative stress by typical free radical inducers, hydrogen peroxide (H(2)O(2)), tert-butylhydroperoxide (tBuOOH) or ultraviolet (UV) irradiation in a human diffuse histiocytic lymphoma U937 cell line. Trimidox showed strong radical scavenging activity by the DPPH reduction assay. The 50% rate inhibited the DPPH reduction concentration of trimidox, and its derivates didox, or gallic acid were 8.8 microM, 117.5 microM, or 41.8 microM, respectively. Induction of cytotoxicity by H(2)O(2) (500 microM) or tBuOOH (100 microM) was concentration-dependently attenuated by incubation with Trimidox (10-150 microM). Trimidox also prevented the effect of UV-induced apoptosis estimated by both nuclear morphological change and DNA fragmentation. This effect was due to inhibition of the production of reactive oxygen species. Moreover, the activity and mRNA expression of catalase, an antioxidant enzyme, was significantly increased by trimidox. These results indicate that trimidox has radical scavenging activity and prevents exogenous oxidative stress and increase in catalase; therefore, trimidox is suggested as an anticancer agent exhibiting potent antioxidant properties in this study.

Effects of hydroxyurea and aphidicolin on phosphorylation of ataxia telangiectasia mutated on Ser 1981 and histone H2AX on Ser 139 in relation to cell cycle phase and induction of apoptosis

DNA replication stress often induces DNA damage. The antitumor drug hydroxyurea (HU), a potent inhibitor of ribonucleotide reductase that halts DNA replication through its effects on cellular deoxynucleotide pools, was shown to damage DNA inducing double-strand breaks (DSBs). Aphidicolin (APH), an inhibitor of alpha-like DNA polymerases, was also reported to cause DNA damage, but the evidence for induction of DSBs by APH is not straightforward. Histone H2AX is phosphorylated on Ser 139 in response to DSBs and one of the protein kinases that phosphorylate H2AX is ataxia telangiectasia mutated (ATM); activation of ATM is through its phosphorylation of Ser 1981. The present study was undertaken to reveal whether H2AX is phosphorylated in cells exposed to HU or APH and whether its phosphorylation is mediated by ATM. HL-60 cells were treated in cultures with 0.1-5.0 mM HU or 1-4 muM APH for up to 5 h. Activation of ATM and H2AX phosphorylation was detected immunocytochemically using Ab specific to Ser1981-ATM or Ser 139-H2AX epitopes, respectively, concurrent with measurement of cellular DNA content. While exposure of cells to HU led to H2AX phosphorylation selectively during S phase and the cells progressing through the early portion of S (DI = 1.1-1.4) were more affected than late-S phase (DI = 1.6-1.9) cells, ATM was not activated by HU. In fact, the level of constitutive ("programmed") ATM phosphorylation was distinctly suppressed, in all phases of the cell cycle, at 0.1-5.0 mM HU. Cells' exposure to APH also resulted in H2AX phosphorylation at Ser139 with no evidence of ATM activation, and as in the case of HU, the early-S cells were more affected than the late-S phase cells. The rise in frequency of apoptotic cells became apparent after 2 h of exposure to HU or APH, and all apoptotic cells had markedly elevated levels of both H2AX-Ser139 and ATM-Ser1981 phosphorylation. The lack of correlation between H2AX phosphorylation and ATM activation indicates that protein kinase(s) other than ATM (ATR and/or DNA-dependent protein kinase) are activated by DSBs induced by replication stress. Interestingly, HU inhibits the constitutive ("programmed") level of ATM phosphorylation in untreated cells. However, DNA fragmentation during apoptosis activates ATM and dramatically increases level of H2AX phosphorylation.

A Ferrous-triapine complex mediates formation of reactive oxygen species that inactivate human ribonucleotide reductase

Ribonucleotide reductase plays a central role in cell proliferation by supplying deoxyribonucleotide precursors for DNA synthesis and repair. The holoenzyme is a protein tetramer that features two large (hRRM1) and two small (hRRM2 or p53R2) subunits. The small subunit contains a di-iron cluster/tyrosyl radical cofactor that is essential for enzyme activity. Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP) is a new, potent ribonucleotide reductase inhibitor currently in phase II clinical trials for cancer chemotherapy. Ferric chloride readily reacts with Triapine to form an Fe(III)-(3-AP) complex, which is reduced to Fe(II)-(3-AP) by DTT. Spin-trapping experiments with 5,5-dimethyl-1-pyrroline-N-oxide prove that Fe(II)-(3-AP) reduces O2 to give oxygen reactive species (ROS). In vitro activity assays show that Fe(II)-(3-AP) is a much more potent inhibitor of hRRM2/hRRM1 and p53R2/hRRM1 than Triapine. Electron paramagnetic resonance measurements on frozen solutions of hRRM2 and p53R2 show that their tyrosyl radicals are completely quenched by incubation with Fe(II)-(3-AP). However, the enzyme activity is maintained in protein samples supplemented with catalase alone or in combination with superoxide dismutase. Furthermore, catalase alone or in combination with superoxide dismutase markedly decreases the antiproliferative effect of Triapine in cytotoxicity assays. These results indicate that Triapine-induced inhibition of ribonucleotide reductase is caused by ROS. We suggest that ROS may ultimately be responsible for the pharmacologic effects of Triapine in vivo.

Triapine and cytarabine is an active combination in patients with acute leukemia or myelodysplastic syndrome

Triapine ®, an iron chelator and a potent inhibitor of ribonucleotide reductase, has significant anti-leukemia activity. A phase I study of Triapine in combination with ara-C was conducted in 32 patients with refractory acute leukemia and high-risk MDS. Triapine (105 mg/m 2/day 6-h infusion) was followed immediately by ara-C [100 ( n = 4), 200 ( n = 6), 400 ( n = 7), or 800 ( n = 8) mg/m 2/day] as an 18-h infusion for 5 consecutive days. Dose-limiting toxicities (DLTs) were observed at the 800 mg/m 2 ara-C dose level (one patient each with grade 4 mucositis; grade 4 neutropenic colitis, sepsis; grade 4 neuropathy; and grade 4 hyperbilirubinemia). Therefore, the study was amended to include an ara-C dose level of 600 mg/m 2/day, no DLTs occurred in seven patients treated at this dose level. Mean Triapine C max and AUC were 1.13 μg/mL and 251.5 min μg/mL. Of 31 evaluable patients, 4 (13%) (3 AML, 1 Ph + ALL) achieved a CR (1 at a dose of 800 mg/m 2; 2 at 600 mg/m 2; 1 at 200 mg/m 2). The recommended phase II regimen is Triapine 105 mg/m 2/day followed by ara-C 600 mg/m 2/day for 5 consecutive days every 3–6 weeks.

Triapine (3-aninopyridine-2-carboxaldehyde thiosemicarbazone) Induces Apoptosis in Ovarian Cancer Cells

Triapine (Vion Pharmaceuticals, New Haven, CT) is a potent ribonucleotide reductase inhibitor which exerts its antineoplastic activity by inhibiting DNA synthesis and repair. The objectives of this study were: (1) to determine whether Triapine has cytotoxic effects on epithelial ovarian cancer (EOC) cells; (2) to characterize the apoptotic cascade induced in response to this agent; and (3) to determine its utility in combination treatment with carboplatin and paclitaxel. Five EOC cell lines were treated with tenfold dilutions of Triapine (0.1 to 100 microM) for 24 and 48 hours. Cell viability was determined by the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega Corp, Madison, WI) and the morphologic features of apoptosis were observed using Hoechst staining. The apoptotic cascade was characterized by Western blot analyses. All EOC cell lines treated with Triapine showed decreased cell viability in a time- and dose-dependent manner. Hoechst staining revealed nuclear shrinkage and chromatin condensation and fragmentation, which correlated with the occurrence of apoptosis. Western blots demonstrated that Bid activation was one of the initiating signals involved in the cascade. In addition, cleavage of XIAP and down-regulation of Akt were observed. We also demonstrated that Triapine enhances the cytotoxic effects of carboplatin and paclitaxel. The present findings demonstrate that Triapine induces cell death through the induction of apoptosis. The initial activation of Bid indicates the involvement of the mitochondrial pathway. The demonstration that Triapine is an effective addition to a carboplatin regimen suggests the possibility of a new combination therapy for ovarian cancer.

Phase I Study of Triapine® and Cytarabine (ara-C) in Patients with Relapsed or Refractory Acute Leukemias and High-Risk Myelodysplastic Syndrome (MDS).

Triapine is a potent inhibitor of ribonucleotide reductase (RNR), which is required for the conversion of ribonucleotides to deoxyribonucleotides. Two phase I trials in patients with hematologic malignancies demonstrated that Triapine could reduce circulating blasts in the majority of patients without significant non-hematologic toxicity (Giles et al. Leuk Res 2003; 27:1077–1083; Karp et al. Blood 2002; 100:560a). Preclinical studies have shown that the combination of Triapine and ara-C produces additive or synergistic cytotoxicity against several tumor cell lines, potentially by increasing intracellular ara-CTP levels when Triapine inhibits RNR. Therefore, a phase I study of Triapine in combination with ara-C was conducted in 32 patients with relapsed or refractory acute leukemia and high-risk MDS (1 MDS, 28 AML, 2 Ph+ ALL, and 1 Ph- ALL) in order to determine the tolerability, safety, and maximum tolerated dose (MTD). Triapine® was administered at a dose of 105 mg/m2/d as a 6-hour infusion for 5 consecutive days (D1–5) followed immediately by ara-C [100 (n=4), 200 (n=6), 400 (n=7), or 800 (n=8) mg/m2/d] as an 18-hour infusion for 5 consecutive days (D1–5). Median age was 59 years (range, 15–83 years). Median ECOG status 1 (range, 0–2). Median number of prior therapies was 2 (range 1–9), including prior autologous (n= 2) and allogeneic stem cell transplant (SCT) (n=4). Dose-limiting toxicities (DLTs) were observed at the 800 mg/m2 ara-C dose level (1 patient each with grade 4 mucositis; grade 4 neutropenic colitis, sepsis and death; grade 4 neuropathy; and grade 4 hyperbilirubinemia). Therefore, the study was amended to include an ara-C dose level of 600 mg/m2/d. Seven patients were treated at this dose level without development DLTs. Thirty-one patients received at least 1 course, 2 received 2 courses, and 4 received 3 courses; 1 patient withdrew prior to completion of 1 course of therapy. Of the 31 evaluable patients, 4 (13%) patients (3 AML, 1 Ph+ ALL) achieved a CR (1 at an ara-C dose of 800 mg/m2; 2 at 600 mg/m2; 1 at 200 mg/m2). Two of the responders also achieved a complete cytogenetic remission (including 1 Ph+ ALL). All responses occurred after 1 induction course. One patient went on to receive an allogeneic (SCT) and continues in CR. Duration of responses was 9, 20, 52+, and 73+ weeks. Mean Cmax and AUC achieved for Triapine were 1.13 μg/mL and 251.5 min•μg/mL. The most frequent toxicities included nausea &/or vomiting (66%), elevated liver enzymes (50%; gr. 3 in 22%), fever (47%), diarrhea (41%; gr. 3 in 6%), rash (31%; gr. 3 in 6%), mucositis (28%), hand-foot syndrome (16%; gr. 3 in 3%), and peripheral edema (16%; gr. 3 in 9%). Triapine and cytarabine has activity in patients with relapsed or refractory acute leukemias. The recommended phase II dose is Triapine 105 mg/m2/day for 5 consecutive days (D1–5) followed by ara-C 600 mg/m2/d for 5 consecutive days (D1–5).

Inhibition of Ribonucleotide Reductase Reduces Neointimal Formation following Balloon Injury

Percutaneous transluminal coronary angioplasty (PTCA) has greatly benefited patients with occluded coronary arteries, but its benefits have been undermined by a high incidence of restenosis. The introduction of coronary stents has significantly improved the short and long term outcome but restenosis still occurs in approximately 15 to 30% of patients within 6 months. Research efforts are now being directed toward combination stenting and drug delivery. Among the therapeutic targets being pursued are agents that can impede smooth muscle cell migration and proliferation, as these processes are critical components of restenosis injury. We propose that inhibiting the conversion of ribonucleotides to deoxyribonucleotides will impede cell proliferation and, as such, limit the degree of restenosis. Therefore, we tested whether the potent ribonucleotide reductase inhibitors Didox (3,4-dihydroxybenzohydraxamic acid) and Imidate (ethyl-3,4,5-hydroxybenzimidate) can limit the neointimal proliferation associated with restenosis using a rat carotid model of balloon dilatation injury. Results demonstrated that both Didox and Imidate significantly reduced intimal thickening, resulting in a 71 and 62% decrease in the intima/media ratio, respectively. Similar efficacy was seen with the commercially available ribonucleotide reductase inhibitor hydroxyurea, demonstrating the importance of this enzyme in vascular remodeling. Results from cell proliferation studies suggest that the mechanism of protection is inhibition of smooth muscle cell (SMC) proliferation. In addition, Didox and Imidate (100 microM) are potent inhibitors of SMC migration, which may also contribute to their vascular protective effects. These results suggest that inhibition of ribonucleotide reductase may provide a potent strategy to prevent post-PTCA restenosis.

Results of a phase 1-2 study of clofarabine in combination with cytarabine (ara-C) in relapsed and refractory acute leukemias

AbstractClofarabine (2-chloro-2′-fluoro-deoxy-9-β-D-arabinofuranosyladenine) is a second-generation nucleoside analog with activity in acute leukemias. As clofarabine is a potent inhibitor of ribonucleotide reductase (RnR), we hypothesized that clofarabine will modulate ara-c triphosphate accumulation and increase the antileukemic activity of cytarabine (ara-C). We conducted a phase 1-2 study of clofarabine plus ara-C in 32 patients with relapsed acute leukemia (25 acute myeloid leukemia [AML], 2 acute lymphoblastic leukemia [ALL]), 4 high-risk myelodysplastic syndrome (MDS), and 1 blast-phase chronic myeloid leukemia (CML).1 Clofarabine was given as a 1-hour intravenous infusion for 5 days (days 2 through 6) followed 4 hours later by ara-C at 1 g/m2 per day as a 2-hour intravenous infusion for 5 days (days 1 through 5). The phase 2 dose of clofarabine was 40 mg/m2 per day for 5 days. Among all patients, 7 (22%) achieved complete remission (CR), and 5 (16%) achieved CR with incomplete platelet recovery (CRp), for an overall response rate of 38%. No responses occurred in 3 patients with ALL and CML. One patient (3%) died during induction. Adverse events were mainly less than or equal to grade 2, including transient liver test abnormalities, nausea/vomiting, diarrhea, skin rashes, mucositis, and palmoplantar erythrodysesthesias. Plasma clofarabine levels generated clofarabine triphosphate accumulation, which resulted in an increase in ara-CTP in the leukemic blasts. The combination of clofarabine with ara-C is safe and active. Cellular pharmacology data support the biochemical modulation strategy.

Clofarabine plus cytarabine (ara-C) is an active induction regimen for newly diagnosed patients (pts) ≥ age 50 with acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (MDS)

6609 Background: Clofarabine is a new-generation nucleoside analog with activity in acute leukemias. As a potent inhibitor of ribonucleotide reductase, it is also suited for biochemical modulation strategies with other nucleoside analogs. In a phase I/II study of clofarabine + ara-C in pts with relapsed/refractory AML/MDS, we reported a response rate of 41% (24% CR, 17% CRp) (Blood 102:615a, 2003). Cellular pharmacology studies suggested that accumulation of intracellular clofarabine triphosphate resulted in higher ara-CTP accumulation of the leukemia blasts. Methods: We are conducting a phase II study of clofarabine + ara-C to evaluate efficacy and safety of this combination in pts ≥ 50 years (yrs) with newly diagnosed AML and high-risk MDS (≥ 10% marrow blasts). Pts with good prognosis karyotypes [t(8;21), inv(16), t(15;17)], ECOG PS > 2, and age ≥ 75 are excluded. Clofarabine is given at 40mg/m2/d as a 1-hour (hr) i.v. infusion for 5 days (d2–6) followed 4 hrs later by ara-C at 1g/m2/d as a 2-hr i.v. infusion for 5 days (d1–5). Results: 31 (28 AML, 3 MDS) of 37 pts are evaluable. Median age 61 yrs (range 50–72). Cytogenetics: diploid in 16 and abnormal in 15 pts. Median WBC 9.1 x 109/L (1–135.8). Median platelet count 44 x 109/L (8–871). 16 pts (52%) achieved CR after one induction course; another 4 pts (13%) achieved CRp. Of 6 pts who received a second induction course, one pt (CRp) responded. The CR rate was 63% (10/16 pts) in the diploid and 40% (6/15) in the abnormal group. Median time to CR: 28 days (23–56). 4 pts (13%) died on study: 1 pt died on d35 of complications due to sepsis; the remaining 3 pts died following a second induction course (d 47, 52, 78) due to prolonged cytopenias and sepsis. Clofarabine infusion related adverse events included facial flushing and headaches (< gr. 3) and were transient. Other toxicities included hyperbilirubinemia (gr. 3 in 3 pts) and pancreatitis (1 pt). Conclusions: The combination of clofarabine + ara-C is active in older pts with newly diagnosed AML/high-risk MDS. The toxicity profile is acceptable. An update of response and toxicity data will be presented. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration ILEX ILEX Oncology ILEX Oncology

A phase II trial of 3-Aminopyridine-2-Carboxaldehyde Thiosemicarbazone (3-AP) in patients with metastatic breast cancer

864 Background: 3-Aminopyridine-2-Carboxaldehyde Thiosemicarbazone (3-AP, Triapine), a potent inhibitor of ribonucleotide reductase, demonstrates both in vitro and in vivo antitumor activity. This cytotoxic effect seems to be maximized by chronic, intermittent enzymatic inhibition. Methods: Patients(Pts) with measurable metastatic breast cancer(MBC) refractory to at least one regimen were eligible for enrollment. Pts were required to have an ECOG PS of 0–1, adequate organ function, no active brain metastases. Treatment: 2-hour IV infusion of 3-AP daily × 5 days every other week at an initial dose of 96 mg/m2/d. Four courses were considered 1 cycle of treatment. Based on interval and residual toxicities, dose modifications to 84mg/m2 or 72mg/m2 were employed. Dose escalation to 105mg/m2 was also permitted. Full staging to be completed every 8 weeks. Results: 9 pts with a median age of 58 were enrolled (range 36–68). Median # of prior chemotherapy regimens was 6 (range 4–10) and 44% of pts (4/9) were refractory to 6 or more chemotherapy regimens. Three of nine pts demonstrated stable disease and the remainder had progression of disease (2/9) or discontinued treatment (4/9) before restaging was required/performed. A total of 42 courses were administered and all pts completed at least 2 courses of treatment (range 2–12). Seven of nine (77%) pts required a cycle 2 dose reduction of 84mg/m2 and an additional two required a cycle 3 dose reduction to 72mg/m2. Dominant toxicity was hematologic with thirty Grade 3 or 4 toxicities documented. All pts experienced Grade 4 neutropenia (4/9 hospitalized with nadir fevers) and 3/9 had Grade 4 thrombocytopenia. Conclusions: Thirty-three percent of pts achieved stable disease following administration of 3-AP(Triapine®) on a daily for five days schedule. Hematological toxicity was significant and managed with dose reductions. Future studies are planned with a more convenient dosing schedule. No significant financial relationships to disclose.

Clofarabine plus cytarabine (ara-C) is an active induction regimen for newly diagnosed patients (pts) ≥ age 50 with acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (MDS)

6609 Background: Clofarabine is a new-generation nucleoside analog with activity in acute leukemias. As a potent inhibitor of ribonucleotide reductase, it is also suited for biochemical modulation strategies with other nucleoside analogs. In a phase I/II study of clofarabine + ara-C in pts with relapsed/refractory AML/MDS, we reported a response rate of 41% (24% CR, 17% CRp) (Blood 102:615a, 2003). Cellular pharmacology studies suggested that accumulation of intracellular clofarabine triphosphate resulted in higher ara-CTP accumulation of the leukemia blasts. Methods: We are conducting a phase II study of clofarabine + ara-C to evaluate efficacy and safety of this combination in pts ≥ 50 years (yrs) with newly diagnosed AML and high-risk MDS (≥ 10% marrow blasts). Pts with good prognosis karyotypes [t(8;21), inv(16), t(15;17)], ECOG PS > 2, and age ≥ 75 are excluded. Clofarabine is given at 40mg/m2/d as a 1-hour (hr) i.v. infusion for 5 days (d2–6) followed 4 hrs later by ara-C at 1g/m2/d as a 2-hr i.v. i...