Danute E. Nitecki

Pharmacokinetics and Metabolic Fate of Two Nitroxides Potentially Useful as Contrast Agents for Magnetic Resonance Imaging

Paramagnetic nitroxyl-containing compounds have been useful as contrast agents in magnetic resonance imaging (MRI) experiments in animals. Preliminary information on the metabolic fate, pharmacokinetic behavior, stability in tissues, and chemical reduction of two prototypic nitroxides, PCA and TES, is presented. In the dog TES was eliminated more rapidly than PCA. More than 80 % of the dose of both nitroxides was recovered in urine within 6 hours. Nitroxides were reduced in vivo to their corresponding hydroxylamines. No other metabolite was observed. Measured reducing activity in tissue homogenates was greater in liver or kidney than in brain, lung or heart. In each tissue PCA was more stable than TES. PCA was also more resistant to reduction by ascorbic acid at physiologic pH. These preliminary results favor the use of PCA, a pyrrolidinyl nitroxide, over TES, a piperidinyl nitroxide, for MRI contrast enhancement.

A water-soluble, monitorable peptide and protein crosslinking agent

A novel, freely water-soluble, heterobifunctional crosslinking reagent, N-maleimido-6-aminocaproyl ester of 1-hydroxy-2-nitro-4-benzenesulfonic acid (mal-sac-HNSA), was synthesized and used for conjugation of sulfhydryl (cysteine)-containing peptides to carrier proteins. Reaction with amino groups releases the dianion phenolate, HNSA, which allows convenient spectrophotometric quantitation of the reaction in progress. Since mal-sac-HNSA is completely water soluble, its concentration can be adjusted to maximize the rate of amine reaction and to minimize hydrolysis. Conjugates of peptides to appropriate carriers have elicited peptide-specific antibody and did not elicit detectable antibody specific to the crosslink.

Characterization of polyethylene glycol modified proteins using charge-reversed capillary electrophoresis

Abstract A capillary electrophoretic method employing a charge reversal technique [J. E. Wiktorowicz and J. C. Colburn, Electrophoresis, 11 (1990) 769] has been developed to characterize polyethylene glycol (PEG)—proteins. A removable coating is applied to a standard fused-silica capillary in order to change the negative charge of the capillary surface to a positive charge. This prevents the adsorption of basic and PEG—proteins. The positive electrode is positioned at the detector end of the capillary, orienting electroosmotic flow towards the detector. Automated reconditioning procedures prior to each analysis give relative standard deviations in migration time and area of less than 2%, with most analysis times under 20 min. As the number of PEGs conjugated to a protein increases, the net positive charge and migration time of the copolymer decrease. Resulting peak widths are broad, reflecting the broad molecular mass distribution of PEGs and the heterogeneous nature of the PEG conjugates. This method can be used to monitor process steps, optimize reaction conditions, determine extent of modification, or assess product quality and consistency. Examples of PEG derivatized molecules characterized in our laboratory by charge-reversed capillary electrophoresis are tryptophan, Lys-Trp-Lys, lysozyme, myoglobin, RNase and immunoglobulin G; the size of the attached monomethoxy-PEG molecules varied from 0.15 kDa to 10 kDa (103 dalton).

Polymeric Reagents for Protein Modification

Protein modification with polymeric reagents is carried out to extend in vivo circulation, to reduce antigenicity or to improve physical properties, such as solubility. We have developed polymeric reagents for such modifications. One group is based on polyproline. Polyproline exists in aqueous solution as a relatively rigid structure, polyproline II. Its single amino group allows derivatization to yield reagents for either amino groups or sulfhydryl groups on the protein. Development of various polyethylene glycol active esters allowed us to compare the reativities of those esters in aqueous buffers, since polyethylene glycol imparted water solubility to esters which are not normally water soluble. We investigated the influence on relative reactivity of polyethylene glycol esters by comparing leaving groups (i.e., the hydroxyl component) and by comparing relative electrophilicity of carbonyl moiety.