The Scientific Core will measure cyanide in red blood cells (RBCs), and thiocyanate in plasma and urine of animals and humans. It will provide support for all three scientific projects, because assessing changes in cyanide and thiocyanate concentrations provides a quantitative measure of the efficacy of cobinamide and 3- mercaptopyruvate dithiane (3-MPDT) in cyanide-treated animals and cigarette smokers. The core also will measure cobinamide in serum and urine of animals and humans to determine the drug's pharmacokinetics. Cobinamide binds cyanide with extremely high affinity, and 3-MPDT serves as a substrate for the cyanide detoxifying enzyme mercaptopyruvate sulfurtransferase. Both drugs lower RBC cyanide, but, because of their different mechanisms of action, they have opposing effects on thiocyanate concentrations: cobinamide lowers plasma and urinary thiocyanate levels, whereas 3-MPDT raises them. Thus, in studies of administering cobinamide and 3-MPDT singly to animals and humans, we will follow RBC cyanide and plasma and urinary thiocyanate concentrations as indicators of drug efficacy. However, when the drugs are used together, we will follow RBC cyanide levels only, because it is difficult to predict thiocyanate levels in the presence of both drugs. Both cyanide and thiocyanate will be measured by highly sensitive and specific colorimetric assays that have rapid sample throughput. As part of Dr. Boss'UO1 grant, formal pharmacokinetic studies of cobinamide will be performed by SRI International, a pre-clinical drug development institute in Menlo Park, CA. Similarly, pharmacokinetic studies of 3-MPDT will be performed as part of Dr. Patterson's UO1 grant. In the proposed work, we plan to measure cobinamide concentrations during the animal studies, and the Phase I and lla clinical studies. The latter studies will provide information about the pharmacokinetics of cobinamide in humans. Cobinamide has a distinctive spectrum in the ultraviolet and visible range, with peaks at 348, 491, and 519 nm. Since only a small number of physiological compounds absorb at wavelengths over 500 nm, cobinamide can be detected easily in animal and human samples by high performance liquid chromatography (HPLC), monitoring absorbance at two wavelengths.
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