Ga-68-Trivehexin

68Ga-Trivehexin is a radiotracer for positron emission tomography (PET), obtained by labeling the peptide conjugate Trivehexin (INN: relitegatide brexetan) with the positron emitting radionuclide gallium-68 (68Ga). 68Ga-Trivehexin targets (i.e., binds to) the cell surface receptor αvβ6-integrin and accumulates in αvβ6-integrin-abundant tissues after intravenous (i.v.) application. 68Ga-Trivehexin is thus applied for PET imaging of medical conditions associated with elevated αvβ6-integrin expression. αvβ6-Integrin, the biological target of 68Ga-Trivehexin, is a heterodimeric transmembrane cell adhesion receptor whose primary natural ligand is latency associated peptide (LAP) in its complex with transforming growth factor beta 1 (TGF-β1). Binding of αvβ6-integrin to LAP releases and thus, activates TGF-β1. In early-stage cancer, TGF-β1 acts as a tumor suppressor but can turn into a tumor promoter as cancers develop, and furthermore induces fibrosis, particularly of the lung. As the likely most important activator of TGF-β1, αvβ6-integrin is often found overexpressed in tumors and fibrosis, which is why 68Ga-Trivehexin PET imaging is primarily relevant in this medical context.

Chemistry

Trivehexin precursor Like most precursors used for radiolabeling with radioactive metal cations, Trivehexin is composed of a dedicated complex ligand (a so-called chelator) for kinetically inert binding of the 68GaIII ion, and the bioligand(s) for binding to αvβ6-integrin. The chelator comprised in Trivehexin is a triazacycloalkane with 3 phosphinic acid substituents, with the basic structure 1,4,7-triazacyclononane-1,4,7-triphosphinate (frequently abbreviated TRAP). The αvβ6-integrin binding molecular unit is a cyclic nonapeptide with the amino acid sequence cyclo(YRGDLAYp(NMe)K) (INN: relitegatide). In the Trivehexin molecule, three of these cyclopeptides are attached by covalent bonds to a single TRAP chelator core. Since TRAP possesses three equivalent carboxylic acids for conjugation of other molecular units via amide formation, Trivehexin is a C3-symmetrical molecule with its three peptide bioligands being fully equivalent. The peptides are attached to the chelator core via the terminal amine group of the side chains of N-methyl lysine. Actually, the conjugation is not done by amide bonding directly, but involves prior functionalization of the peptide with a short molecular extension (a linker) bearing a terminal alkyne, and of TRAP with three linkers bearing terminal azides. These components are assembled by means of copper(I) catalyzed alkyne-azide cycloaddition (CuAAC, also known as Huisgen reaction, a Click chemistry reaction), giving rise to the three 1,3-triazole linkages in the 68Ga-Trivehexin structure. Trivehexin is manufactured and distributed by the German company TRIMT GmbH.

68Ga radiolabeling 68Ga-Trivehexin is a radioactive drug. The radioactive atom, gallium-68 (68Ga), decays with a half-life of approximately 68 min to the stable isotope zinc-68 (68Zn), to 89% by β+ decay whereby a positron with a maximum kinetic energy of 1.9 MeV is emitted (the remaining 11% are EC decays). Due to the short half-life, 68Ga-Trivehexin can not be manufactured long before use but the 68Ga has to be introduced into the molecule shortly before application. This process is referred to as radiolabeling, and is done by complexation of the trivalent cation 68GaIII by the TRAP chelator in Trivehexin. 68GaIII is usually obtained from a dedicated mobile radionuclide source, a Gallium-68 generator, in form of a solution in dilute (0.04–0.1 M) hydrochloric acid (frequently and imprecisely referred to as "68Ga chloride solution in HCl" despite it contains no species with a Ga–Cl bond but [68Ga(H2O)6]3+ complex hydrate cations). For radiolabeling, the pH of the 68Ga containing generator eluate has to be raised from its initial value (depending on HCl concentration, pH 1–1.5) to pH 2–3.5 using suitable buffers, such as sodium acetate. Then, Trivehexin (5–10 nmol) is added to the buffered 68Ga-containing solution, and the mixture is briefly heated to 50–100 °C (usually 2–3 min) to finalize the complexation reaction.

Use as medical imaging agent

αvβ6-integrin target The abundance of αvβ6-integrin on most adult human cell types and respective tissues is low. It is however overexpressed in the context of several medical conditions, such as cancer or fibrosis, particularly idiopathic pulmonary fibrosis. In line with the finding that αvβ6-integrin is expressed by epithelial cells, an elevated density of the protein is observed on the cell surfaces of many carcinomas (synonymous to cancers of epithelial origin). Hence, 68Ga-Trivehexin can be used for PET imaging of αvβ6-integrin positive cancers (i.e., those whose cells possess a sufficiently high density of αvβ6 on their surface), including but not limited to pancreatic ductal adenocarcinoma, non-small cell lung cancer, squamous cell carcinomas (SCC) of different origin (most notably, oral and esophageal SCC), as well as breast, ovarian, and bladder cancer. In colorectal cancer, expression of αvβ6-integrin is higher in the more aggressive forms and correlated with reduced overall survival. 68Ga-Trivehexin has a high binding affinity to αvβ6-integrin (IC50 = 0.047 nM). Its affinity to other RGD-binding integrins is much lower (IC50 for αvβ3, αvβ8, and α5β1 are 2.7, 6.2, and 22 nM, respectively; note that for IC50, higher values mean lower affinity), resulting in a high selectivity for αvβ6-integrin.

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