The Effect of Deuterium Labeling on the Oxidative Metabolism and Subsequent Alkylation Kinetics of Cyclophosphamide-\316\261,\316\261,\316\261',\316\261'-d4 and Ifosfamide-\316\261,\316\261,\316\261',\316\261'-d4
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Issue Date
2014
Authors
D'Alessandro, Michael Anthony '14
Degree
MS in Pharmaceutical Sciences
Advisor
Ludeman, Susan M
Committee Members
Dolan, Eileen
Hass, Martha A.
Dearborn, Richard
Hass, Martha A.
Dearborn, Richard
Journal Title
Journal ISSN
Volume Title
Abstract
The prodrug cyclophosphamide (CP) is the most widely used alkylating agent and it is clinically effective against more than half of all cancers. CP and its clinically useful structural isomer, ifosfamide (IF), exhibit parallel oxidative metabolisms that lead to the desired alkylating agents phosphoramide mustard (PM) and isophosphoramide mustard (IPM), respectively. Competing with this activation pathway is an undesired metabolism that generates N-dechloroethyl metabolites as well as the neurotoxic and often dose-limiting metabolite, chloroacetaldehyde (CAA). Numerous isozymes of cytochrome P450 play a role in these metabolic pathways, with CYP2B6, CYP3A4 and CYP3A5 being the most notable. Chemical modifications to the chloroethyl side chains of CP and IF can reduce the undesired metabolism but these changes often negatively affect the alkylation efficiencies of the therapeutically active metabolites. In an attempt to suppress N-dechloroethylation without interfering with alkylation rates of the theraputic metabolites, the protons at the sites of undesired metabolism were replaced with deuterium. These α- and α'-perdeuterated analogs of CP and IF were individually incubated with CYP2B6, CYP3A4 and CYP3A5 and the K<sub>m</sub> and <italic>V</italic><sub>max</sub> of each were compared to the unlabeled counterparts. The observed primary kinetic isotope effects (KIE) showed a decrease in rates of side chain oxidation and an increase in 4-hydroxylation, especially noticeable in IF. Synthesized α- and α'-perdeuterated analogs of the active PM and IPM metabolites were used in proton NMR studies to investigate if the secondary KIE during alkylation would be significant. Deuterated analogs of IPM had a half-life of 666 minutes, which showed no significant variation from the literature value of 648 minutes for unlabeled IPM. However, the same experiments with a deuterated analogs of PM (half-life = 181 minutes) showed a significantly increased alkylation rate when compared to the half-life of 321 minutes for unlabeled PM. The results of the studies on the metabolic oxidation of deuterated analogs of CP and IF could help to predict outcomes of treatment based on a patient's varying expressions of the P450 isozymes. Our deuterated analogs of IF could prove especially useful for patients exhibiting dose-limiting toxicity to unlabeled IF by reducing exposure to the undesired, neurotoxic metabolite CAA.
Citation
D'Alessandro MA. The effect of deuterium labeling on the oxidative metabolism and subsequent alkylation kinetics of cyclophosphamide-\316\261,\316\261,\316\261',\316\261'-d4 and ifosfamide-\316\261,\316\261,\316\261',\316\261'-d4 [thesis]. Ann Arbor (MI): Proquest/UMI; 2014. 60 p.
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