A Sensitive Catalytic Wave Formed by Electrochemical Reduction of Morin in the Presence of an Oxidant KIO3

The investigation of the electrogenerated free radical of morin reacting with an oxidant is helpful in understanding its antioxidant pharmacology. In phosphate buffer (pH 5.6 ± 0.1), the reduction of morin proceeds with a one-electron transfer of the C=O double bond into a free radical intermediate, which then delivers the final primary alcohol via a one-electron reduction. When an oxidant KIO3 is present, the free radical intermediate of morin is oxidized to regenerate the original ‘C=O’ bond. Further reduction processes are effectively inhibited, resulting in a sensitive catalytic peak, with the peak current enhanced 70 times.

Activity of the Novel Ribonucleotide Reductase Inhibitor Didox on Human and Murine Models of Acute Leukemia,

Abstract 3637 Acute Myeloid Leukemia (AML) is an aggressive, genetically heterogeneous disease wherein immature myeloid progenitors crowd out the normal hematopoietic cells leading to marrow failure and ultimately death. Each year in the United States there are approximately 12,000 new cases and 9,000 deaths from AML. Combination chemotherapy of an anthracycline and cytarabine has remained the mainstay of frontline treatment despite decades of active research. Treatment will often lead to a remission; however, the majority of patients relapse with chemoresistant disease. This highlights the need for the development of new therapies for patients. The Ribonucleotide Reductase (RR) reaction facilitates the conversion of ribonucleotides to deoxyribonucleotides via a free radical intermediate and is the rate-limiting step in DNA replication. The anti-tumor activity of Hydroxyurea is due to its ability to quench the free radical intermediate generated at the M2 subunit of RR ultimately resulting in a depletion of the cell's deoxyribonucleotide pool. This chemotherapeutic is useful for the palliative treatment of AML; however, its myelosuppressive effects are dose limiting. 3,4-Dihydroxybenzohydroamic acid (Didox) is a RR inhibitor with a mechanism of action similar to that of Hydroxyurea that has been shown to be less myelosuppressive in an animal model. Didox has been examined for toxicity in Phase I and Phase II trials in the United Kingdom in several malignancies. These clinical trials have reported Didox to be well tolerated at 6 g/m2. Major dose limiting organ toxicities were hepatic and renal. Given this low toxicity and mechanism of action we tested the activity of Didox in pilot studies of human and murine leukemias. In vitro studies were done to assess the Inhibitory Concentration (IC50) against five human AML cell lines, KG1a, OCI-AML3, THP-1, HL60 and K562. Cells were exposed to a titration of Didox for 72 hours and viability determined. The average IC50 of Didox was 5.76 μM (Range 3.62–7.37 μM). Initial In vivo studies were carried out in a syngeniec, therapy resistant MLL-ENL driven AML model. Murine leukemias were injected into sublethally irradiated recipients and allowed to engraft. Animals were given five days of Didox 425 mg/kg or a control treatment via intraperitoneal injection. Response and survival data were collected. A nimals tolerated the therapy well. Treated animals demonstrated a significant survival benefit in two separately derived MLL leukemias (one expressing the Flt3-ITD and one expressing NRasG12D) with p values of 0.0094 and 0.009 respectively. To determine the efficacy of Didox against a murine Acute Lymphoblastic Leukemia (ALL) model we injected syngeniec Balb/c mice with Baf3 cells engineered to express the p210 BCR-ABL fusion protein. We found that while the animals tolerated Didox, treatment did not provide a survival benefit, p =0.3581. This suggests that the use of Didox would be more efficacious in the treatment of AML than in ALL. Didox is a novel ribonucleotide reductase inhibitor with IC50 values in the low micromolar range for a panel of human and murine cell lines. Pilot studies in our syngeneic mouse models show that Didox is well tolerated in AML and ALL. Moreover, Didox demonstrates a survival benefit in chemoresistant murine AMLs. This data would imply Didox has activity in AML and additional studies to further characterize its activity are underway. Disclosures: Elford: Molecules for Health: Equity Ownership, Patents & Royalties.

Lipoxygenase-Mediated N-Demethylation of Imipramine and Related Tricyclic Antidepressants in the Presence of Hydrogen Peroxide

In this study, we examined the ability of soybean lipoxygenase to mediate the N-demethylation of imipramine and related drugs in the presence of hydrogen peroxide. Formaldehyde generation resulting from the N-demethylation of imipramine, a prototype drug, was found to depend on incubation time, and the concentration of the enzyme, imipramine, and hydrogen peroxide. Under optimal assay conditions, Vmax values of 14 to 18 nmol formaldehyde/min/nmol enzyme or 133 to 164 nmol formaldehyde/min/mg protein were observed. An inhibition of formaldehyde and desipramine formation by nordihydroguaiaretic acid confirmed the lipoxygenase involvement. The blockade of the reaction by glutathione, dithiothreitol, butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) indicated the generation of a free radical intermediate from imipramine. Desipramine, trimipramine, clomipramine, and diltiazem, but not amitriptyline and doxepin, were also oxidized, albeit at a lower rate. Collectively, the evidence gathered in this study suggests, for the first time, that tricyclic antidepressant drugs may undergo lipoxygenase-catalyzed N-demethylation.

Time-Resolved Resonance Raman Studies of Radicals From 4-Aminoresorcinol as Models for the Active Site Radical Intermediate in Copper Amine Oxidases

Radicals formed by one-electron oxidation of 4-aminoresorcinol have been studied as models for the active site free radical intermediate which forms as a stable product during anaerobic incubation of amine oxidases with amine substrates. Pulse radiolysis shows that the radical undergoes ionisations with pKa's of 3.4 and 6.4. Although the two deprotonated forms are difficult to distinguish by absorption spectroscopy, they have clearly different resonance Raman spectra. Comparison with the resonance Raman spectrum of the enzyme intermediate shows it to be the singly deprotonated form of the radical.