Lyophilized ligand kits: from left to right are 62Cu kits for H2ATSM, H2ETS and H2PTSM, respectively.

BTS compounds are a class of molecules that share the same bis(thiosemicarbazone) backbone in their structures and simliar avid copper chelation chemistry. This table lists the molecular structures and organ uptake parameters for some common BTS compounds. Bis(thiosemicarbazone) compounds are avid and very selective chelators for Cu²⁺ (log K > 17) and have been utilized extensively in coordination of radiocoppers as PET agents in imaging studies.  These ligands display very high first-pass cellular extraction owing to their lipophilicity.  An additional advantage of these compounds is that they react essentially instantaneously with Cu²⁺ without the necessity for heating.  Once inside the cell, Cu(II) is reduced to Cu(I), most likely through a thiol-mediated mechanism, and dissociates from the ligand, trapping ⁶²Cu intracellularly.  Ligand dissociation is not significant in plasma.  Uptake is rapid and constant after around 5 minutes, and the cellular washout half-life exceeds one hour in most tissues. This trapping mechanism works for most of BTS family but selective trapping can also be achieved according to different reduction potential of the molecules. A generic structure of Cu bis(thiosemicarbazone) compounds is shown below.  Depending on the molecular structure, quite diverse cellular uptake properties and biodistribution can be observed.  For example the ligands H₂PTSM (R1 = R3 = R5 = CH3, R2 = R4 = R6 =H) and H₂ETS (R1 = CH2CH3, R2 – R6 = H) are blood flow perfusion agents with differential uptake properties based upon their level of plasma protein binding.  In contrast, the ligand H₂ATSM (R1 = R2 = R3 = R5 = CH3, R4 = R6 = H) displays selectivity for localization of radiocopper in hypoxic tissues.  PTI has developed ‘instant synthesis’ kit-based formulations for all three of these agents and has utilized them in clinical trials in concert with our ⁶²Cu generator system.

PTI has successfully demonstrated the utility of its ⁶²Cu generator technology in the development of several ⁶²Cu bis(thiosemicarbazone) compounds for use in diagnostic PET imaging.  These agents are rapidly synthesized in high radiochemical purity  (typical HPLC radio-chromatogram) using the ligand in a kit-based form. Following reconstitution of the kit and immediate combination with generator eluent, the ⁶²Cu labeled bis(thiosemicarbazone) compounds are instantly produced in a sterile, isotonic solution that is ready for patient injection.  The components in each injectable solution are listed here: 

Proportional Technologies, Inc. has currently developed three ligand kits available for both non-human studies and clinical investigation. These ligands include H₂PTSM, H₂ETS and H₂TASM, structures of which are shown below:

Two of the ⁶²Cu agents (Cu-PTSM and Cu-ETS) are perfusion agents. Both Cu-PTSM and Cu-ETS are quite lipophilic (log P:1.35 for ETS and 1.92 for PTSM) and readily crosses cell membranes.  In rats, they are rapidly cleared from the blood pool after injection, and radiocopper activity is localized in the kidneys, heart, liver, lungs and other tissue, consistent with the same trend observed in human volunteers.  Compared to ⁶²Cu-PTSM, ⁶²Cu-ETS exhibits reduced serum albumin binding in humans, making it more suitable for quantitation of perfusion in high blood flow environments.

Professor Mark Green and his group have extensively evaluated various copper(II) ketoaldehyde bis(thiosemicarbazone) complexes for use as radiopharmaceuticals for PET perfusion imaging (see references below). Among the compounds tested, ethylglyoxal bis(thiosemi-carbazone) (Cu-ETS), and pyruvaldehyde bis(N4-methylthiosemicarbazone) (Cu-PTSM) were identified as exhibiting high first-pass extraction followed by prolonged tissue retention of the radiolabel via a trapping process that is well understood at the molecular level. The figure below illustrates the thiol-mediated intracellular decomposition of Cu(II)-ETS/PTSM, believed to account for the prolonged tissue retention of the ⁶²Cu-radiolabel following intravenous administration.  The uncharged lipophilic copper(II) complex readily diffuses across the cell membrane, whereupon it is susceptible to reductive decomposition by reaction with ubiquitous intracellular thiols, such as glutathione. Electron transfer from the thiol sulfur to the Cu(II)-ketoaldehyde bis(thiosemicarbazone) complex leads to formation of Cu(I) and dissociation of the metal from the bis(thiosemicarbazone) ligand.  The liberated Cu is eventually reoxidized and ends up bound to (and effectively trapped by) intracellular macromolecules.

Schematic diagram illustrating the thiol-mediated intracellular decomposition of Cu(II)-ETS/PTSM believed to account for the prolonged “microsphere-like” tissue retention of the ⁶²Cu-radiolabled following intravenous administration of this and related ⁶²Cu-bis(thiosemicarbazone) complexes.

Although Cu-ATSM shares the same structural backbone as Cu-PTSM/Cu-ETS and is quite lipophilic (log P=2.26), it has significantly different properties when utilized in PET imaging.  Cu-ATSM readily crosses cell membranes but is rapidly washed out from normal tissue. In the molecular structure compared to Cu-PTSM, Cu-ATSM has an additional methyl group on the ligand backbone, which lowers the reduction potential by 100 mV.   Cu-ATSM’s high redox potential allows trapping in both normoxic and hypoxic tissue through a reduction from Cu (II) to Cu (I).  However, Cu-ATSM cannot be reduced by the electron transport system of normal mitochondria due to its lower redox potential.  As a result, it does not trap effectively in normal tissue but shows preference for hypoxic tissue where it is more easily reduced by the oxygen-depleted mitochondria. The localization of Cu-ATSM in hypoxic tissues is especially important for PET applications in oncology, since hypoxic tissue is well known to be resistant to radiotherapy and many forms of chemotherapy.  WashingtonUniversity has performed studies utilizing ⁶⁰Cu-ATSM in PET imaging of approximately 100 patients having a variety of cancers, including non-small cell lung, head and neck, cervical, breast, brain, and rectal cancers.  

PTI has made significant progress toward obtaining FDA clearance for all three kit-based ⁶²Cu agents.  All three compounds have active Investigational New Drug (IND) applications filed with the FDA. 

• An NDA for ⁶²Cu-PTSM was granted approvable status from the FDA (NDA N021401).  This NDA is eligible for continuation following addressing of concerns in the approvable letter, many of which were related to manufacturing issues associated with the previous ⁶²Cu generator system.  These issues have been subsequently addressed through modification of the current “micro-generator” design. 
• A Phase I dosimetry and biodistribution study for ⁶²Cu-ETS was recently completed and submitted to the FDA (IND75018).  A Phase II study protocol has been submitted and is currently under review by the FDA. 
• A Phase I dosimetry and biodistribution study for ⁶²Cu-ATSM has received clearance from the FDA and will be completed within the first quarter of the coming year (IND76897).

For more information, pricing, and to place an order, please call toll-free 800-759-7325, or email us at This e-mail address is being protected from spambots, you need JavaScript enabled to view it .

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Microgenerator Investigator’s brochure

Funding

This technology has been funded by the following grants-

NIH under the project name “Improved Production of ⁶²Zn for PET Imaging with ⁶²Cu" – HHSN261-200622026C.

NIH under the project name "Instant Kit Synthesis of ⁶²Cu Radiopharmaceuticals" - CA110154.

NIH under the project name "Regional Renal Perfusion Quantification with ⁶²Cu PET" - DK58466

M.A. Green, A potential copper radiopharmaceutical for imaging the heart and brain:  copper-labeled pyruvaldehyde bis(N4-methylthiosemicarbazone).  Nucl. Med. Biol. 14: 59-61, 1987. (PubMed)

M.A. Green, D.L. Klippenstein, J.R. Tennison, Copper(II) bis(thiosemicarbazone) complexes as potential tracers for evaluation of cerebral and myocardial blood flow with PET.  J. Nucl. Med. 29:1549‑1557, 1988. (PubMed)

M.A. Green, C.J. Mathias, M.J. Welch, A.H. McGuire, D. Perry, F. Fernandez-Rubio, J.S. Perlmutter, M.E. Raichle, and Bergmann S.R., [62Cu]-labeled pyruvaldehyde bis(N4-methylthiosemicarbazonato) copper(II): synthesis and evaluation as a positron emission tomography tracer for cerebral and myocardial perfusion.  J. Nucl. Med. 31:1989-1996, 1990. (PubMed)