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| Cyclophosphamide (CP) is a pro-drug metabolized in liver by cytochrome p450(CYP)2B6 to the active metabolite, 4-hydroxycyclophosphamide (4-OH-CP) and by CYP3A4/5 to toxic chloracetaldehyde and 2-dechloroethylcyclophosphamide (2-deCl-CP). We developed a very sensitive high-throughput LC-MS/MS assay to measure all 3 analytes (CP, 4-OH-CP, and 2-deCl-CP) from 0.2 mL blood sample. The assay is being used in several preclinical and clinical studies at Duke and in collaborative projects with other institutions (UNC, MD Anderson) investigating influence of other (liver-metabolized) drugs, race, pharmacogenomics, etc on the pharmacokinetics and outcome after CP (CTX) therapy. |
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Fludarabine levels in dose-adjusted renal-failure cancer patient
| Patient with |
Cmax [mg/L] |
Cl [L/h] |
AUCinf [mgh/L] |
| Renal disease |
549 |
5.3* |
4529 |
| Normal renal function (#1) |
788 |
6.5 |
4705 |
| Normal renal function (#2) |
1170 |
6.4 |
4718 |
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| Pharmacokinetic modeling affirmed a successful maintenance of the drug exposure (AUC, clearance) after 20% dose reduction and intensive daily dialysis of a sickle cell disease patient with renal failure undergoing allogenic stem cell transplantation. |
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Busulfan TDM (therapeutic dose monitoring) - 1/10 Dose
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| Busulfan is an important drug in bone marrow transplant regimens. A high-sensitivity liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed on Applied Biosystems/SCIEX API 4000 instrument with 1.6 ng/mL - 5 mg/mL quantification limits to be able to measure busulfan levels after both therapeutic and non-therapeutic (1/10) regimens. Providing linear pharmacokinetics behavior, the test regimen is expected to provide optimal personalized dosing in the first cycle of the busulfan therapy. Semi-log plot on the right illustrates equality of the elimination phase slopes (elim. rate, clearance) between test and therapeutic regimens. |
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Pharmacokinetics of Metalloporphyrins
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| At the same time the oxidative stress was recognized as an underlying mechanism for many biological disorders, the idea came about to use ANTIOXIDANTS as a therapy. Two main classes of compounds emerged: (1) stoichiometric scavengers (irreversibly reacting with the oxidant thus added in excess for prolonged effect) and (2) catalytic scavengers (just promoting self-distraction, i.e. “dismutation”, of the oxidants thus added in small quantities. The later are also called enzyme “mimics”. Metallo(e.g. Fe, Mn, Cu, Mo)porphyrins are low-molecular-weight superoxide dismutase (SOD) mimics intensively developed at Duke last 15 years. Meanwhile, the compounds are recognized in more general terms as potent “bio-catalysts” and “redox-mediators” capable of affecting redox-based pathways acting either as anti-or pro-oxidants depending upon the biological environment. Thus, it is not surprising that this class of molecules exhibits activity in many different models of diseases/treatments: stroke, trauma, diabetes, as radiation-protectors, as anticancer agents, etc. Our lab has developed numerous HPLC and LC-MS/MS assays for measuring cell-culture, plasma, and tissue levels as well as providing mass spectral scan services for assessment of purity during the drug development. Picture shows pharmacokinetic profiles and a noticeable accumulation in brain of a manganese porphyrin under development for stroke therapy. |
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Isoprostanes (lipid oxidation markers) in Urine
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| Free-radicals and other oxygen- and nitrogen-derived damaging agents originate from radiation (x-ray, uv light), chemicals (drugs, polluted air, water, food), and metabolism (infection, trauma, ischemia-reperfusion, genetic disorders). Most of the highly-reactive molecules involved are transient and present in low concentration thus difficult to measure. Damage itself, though often with serious consequences, is typically not apparent (especially during initial phase of disease) and assessment methods are usually invasive. Thus search for reliable “biomarkers of oxidative stress” is actively ongoing and isoprostanes (products of lipid peroxidation), are presently considered the most promising. Measurement of isoprostanes is very challenging because of the presence of numerous isomers of equal masses and even equal mass-to-mass transitions. Nevertheless, our lab has successfully developed an LC-MS/MS method superior to already published methodologies. The assay has been already utilized in several studies at Duke investigating role of oxidative stress in: diabetes, chemotherapy, exercise, cardiac interventions, and pain. The Picture illustrates the urine levels of isoprostanes during cardiac by-pass surgery and in the course of doxorubicin treatment. |
