The Scientific Core will measure cyanide in red blood cells (RBCs), and thiocyanate in plasma and urine ofanimals and humans. It will provide support for all three scientific projects, because assessing changes incyanide 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 willmeasure 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 cyanidedetoxifying enzyme mercaptopyruvate sulfurtransferase. Both drugs lower RBC cyanide, but, because of theirdifferent mechanisms of action, they have opposing effects on thiocyanate concentrations: cobinamide lowersplasma and urinary thiocyanate levels, whereas 3-MPDT raises them. Thus, in studies of administeringcobinamide and 3-MPDT singly to animals and humans, we will follow RBC cyanide and plasma and urinarythiocyanate concentrations as indicators of drug efficacy. However, when the drugs are used together, we willfollow 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 haverapid sample throughput.As part of Dr. Boss' UO1 grant, formal pharmacokinetic studies of cobinamide will be performed by SRIInternational, a pre-clinical drug development institute in Menlo Park, CA. Similarly, pharmacokinetic studiesof 3-MPDT will be performed as part of Dr. Patterson's UO1 grant. In the proposed work, we plan to measurecobinamide concentrations during the animal studies, and the Phase I and lla clinical studies. The latter studieswill provide information about the pharmacokinetics of cobinamide in humans. Cobinamide has a distinctivespectrum in the ultraviolet and visible range, with peaks at 348, 491, and 519 nm. Since only a small numberof physiological compounds absorb at wavelengths over 500 nm, cobinamide can be detected easily in animaland human samples by high performance liquid chromatography (HPLC), monitoring absorbance at twowavelengths.
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