Protein binding of copper-64-bis(thiosemicarbazone)copper(II) radiopharmaceuticals

Nathan E Basken, Purdue University

Abstract

The purpose of this project was to characterize bis(thiosemicarbazone) copper(II) positron emission tomography (PET) radiopharmaceutical binding to serum albumins. PET is a nuclear medicine imaging modality that has the unique capability to map the regional uptake of radiotracers which directly participate in biochemical and metabolic processes. These [62/64/67 Cu]Cu-labeled radiopharmaceuticals have shown promise in diagnostic applications, including: myocardial, cerebral, renal tissue perfusion imaging, and selective uptake in hypoxic tumor tissue. Plasma protein binding, especially to serum albumin, directs the in vivo distribution and kinetics of each chelate. It is a well-recognized principle that only free (non-protein bound) radiotracer is available to diffuse out of the vasculature to facilitate nuclear medicine imaging procedures. In addition, the effect of protein binding on tracer pharmacokinetics across species is an important consideration for predictive interpretation of animal model studies prior to human clinical trials. Though the consequences of protein binding are well documented, there is a knowledge gap understanding the structural basis for these interactions. Ultrafiltration binding assays were used to characterize various aspects of tracer binding to serum albumins. Ultrafiltration devices have a micropartition membrane which mechanically separates protein-bound chelate from the free fraction by centrifugation. The binding of pyruvaldehyde bis(N4-methylthiosemi-carbazone) [64Cu]-copper(II) (Cu-PTSM), diacetyl bis(N4-methylthiosemi-carbazone) [64Cu]-copper(II) (Cu-ATSM), and ethylglyoxal bis(thiosemicarbazone) [64Cu]-copper(II) (Cu-ETS) to serum albumins from twelve mammalian species was measured using this technique. Additional assays used competing drugs, such as ibuprofen and warfarin, which bind at specific sites on human serum albumin (HSA), to indirectly probe the site-specific binding of tracers such as Cu-PTSM and Cu-ATSM. To determine if differences in serum albumin binding between species could be directly explained by variation(s) in primary protein structure, serum albumin sequence homology was investigated. Finally, the serum albumin binding of sixteen, structurally diverse, chelates was measured to elucidate details of the tracer structure-protein binding relationship. [64Cu]Cu-PTSM and [64Cu]Cu-ATSM were highly bound to HSA (∼4.0% free), and not extensively bound to canine serum albumin (CSA) (∼40% free). CSA binding for all chelates in this project was limited to non-specific interactions, which increased with tracer lipophilicity. The competitive drug binding assays showed Cu-PTSM and Cu-ATSM binding to human and rat serum albumins was disrupted by drugs known to bind a high affinity drug site on subdomain IIA of HSA. Protein structure comparisons did not reveal any single amino acid substitutions between species that universally explained the interspecies variation in tracer binding. Chelates having one or two methyl groups in the diimine backbone, such as pyruvaldehyde bis(N4-dimethylthiosemicarbazone) copper(II) (Cu-PTSM2), Cu-PTSM, and Cu-ATSM, had common high affinities for HSA (<6% free). Chelates with bulkier substituent groups at this location such as ethylglyoxal bis(N4-methylthiosemicarbazone) copper(II) (Cu-ETSM), and Cu-ETS had HSA binding that was dominated by non-specific interactions that were roughly a function of tracer lipophilicity, similar to the non-specific binding observed with CSA. Completion of this project illustrated the highly species- and compound-dependent nature of bis(thiosemicarbazone)copper(II) chelate serum albumin binding. Competitive drug binding studies suggested that high affinity tracer binding to HSA may be localized to the IIA binding site. The structure-activity studies further indicated that protein binding was especially sensitive to chelate diimine alkylation. These insights are important for animal model studies, and ultimately, may be used to develop strategies to direct protein binding and improve the clinical utility of radiopharmaceuticals in humans.

Degree

Ph.D.

Advisors

Green, Purdue University.

Subject Area

Nuclear chemistry|Organic chemistry

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