Ga-68-Trivehexin

⁶⁸Ga-Trivehexin is a radiotracer for positron emission tomography (PET), obtained by labeling the peptide conjugate Trivehexin 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 ⁶⁸Ga^III 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(''N''Me)K).

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 M eV 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 ⁶⁸Ga^III by the TRAP chelator in Trivehexin.

⁶⁸Ga^III 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 sup>68Ga(H₂O)₆sup>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.

Imaging procedure

Since ⁶⁸Ga is a positron emitter, ⁶⁸Ga-Trivehexin is applicable for PET imaging. However, PET is rarely used as a standalone imaging technique these days. Most clinics use PET/CT or even PET/MRI systems that acquire morphological and functional images in a single workflow and thus, provide more detailed and useful medical information to the physician.

For clinical PET/CT diagnostics, an activity in the range of 80–150 MBq ⁶⁸Ga-Trivehexin is injected intravenously (i.v.). The tracer then distributes with the blood flow and moves into tissues by diffusion, where it specifically binds to its target αvβ6-integrin, while an excess is excreted via the kidneys and the urine. As a result, ⁶⁸Ga-Trivehexin and, therefore, the positron-emitting radionuclide ⁶⁸Ga, is preferably accumulated by αvβ6-integrin abundant tissues (for example, tumor tissue). Next, a PET/CT scanner is used to detect the ''gamma radiation'' which is generated by the annihilation of the positrons emitted by ⁶⁸Ga (''not'' the actual positrons, which do not leave the body but travel only a few millimetres through the tissue). The spatial distribution of the annihilation events is reconstructed from the raw detector data (referred to as listmode data), which eventually delivers a 3-dimensional data set of radioactivity distribution in the body. These data allow the visualization of αvβ6-integrin positive tissues as 2-dimensional tomographic images or 3-dimensional volume rendering. Typically, the PET/CT imaging is performed 45–60 minutes after the i.v. administration of ⁶⁸Ga-Trivehexin.

Cancers imaging

⁶⁸Ga-Trivehexin has not yet obtained a marketing approval. It is used for clinical imaging of αvβ6-integrin expression in experimental settings.

Pancreatic cancer

First-in-human application of different αvβ6-integrin radiotracers has demonstrated that ⁶⁸Ga-Trivehexin performed especially well in detecting pancreatic cancer, showing high uptake in tumor lesions and low background in the gastrointestinal tract (GI tract) (see image). Since its introduction, ⁶⁸Ga-Trivehexin has been used predominantly for PET/CT imaging of pancreatic ductal adenocarcinoma (PDAC), for example, in single cases and two cohorts (12 and 44 patients, respectively) of suspected or known PDAC.

Parathyroid adenoma (PTA)

In the context of primary hyperparathyroidism (PHPT), ⁶⁸Ga-Trivehexin PET/CT was successfully applied for PET imaging of disseminated parathyroid adenoma with a detection rate of 94.1%, and furthermore for delineation of osteolysis-associated brown tumors which occur as a result of persistent hyperparathyroidism.

Head-and-neck cancer

In a cohort of 20 suspected (19 confirmed) head-and-neck squamous cell carcinoma (HNSCC) cases, ⁶⁸Ga-Trivehexin PET had a higher sensitivity (92.5%), positive predictive value (PPV, 100%), and accuracy (93%) than the standard ¹⁸F-FDG PET, for which sensitivity, PPV, and accuracy were 90%, 93.1%, and 84.3%, respectively.

Breast cancer

The feasibility of ⁶⁸Ga-Trivehexin PET imaging of breast cancer (BC) was demonstrated in a case of triple-negative BC. Furthermore, in progesterone- and estrogen-receptor negative BC with elevated Ki67 proliferation index and strong E-cadherin, ⁶⁸Ga-Trivehexin PET identified several ¹⁸F-FDG-avid lymph nodes as false positives. In a patient with lobular BC and pancreatic neuroendocrine tumor (PNET), ⁶⁸Ga-Trivehexin selectively showed a PET signal only in the lobular carcinoma, while the metabolic tracer ¹⁸F-FGD and the neuroendocrine tumor tracer ⁶⁸Ga-DOTATATE yielded PET signals for both the BC and PNET lesions.

Other cancers

⁶⁸Ga-Trivehexin was also applied in cases of tonsillar carcinoma metastasized to the brain, of bronchial mucoepidermoid carcinoma and mucinous lung adenocarcinoma, recurrent parathyroid carcinoma, and papillary thyroid carcinoma.

Fibrosis imaging

In accordance with the known expression of αvβ6-integrin in early lung fibrosis, ⁶⁸Ga-Trivehexin was used for PET/CT imaging of idiopathic pulmonary fibrosis (IPF). In explorative studies, ⁶⁸Ga-Trivehexin could generate an IPF-specific PET signal while the same tissue areas were PET-negative using the standard metabolic PET tracer ¹⁸F-FDG. ⁶⁸Ga-Trivehexin PET scans displayed an uptake of SUV_max = 5.53 in fibrotic lung areas and thus enabled clear differentiation of fibrotic from non-fibrotic lung tissue.

Safety

Like for other radioactive imaging agents in medicine, the applied amounts of radioactivity are so low that radiation-related adverse effects are very unlikely to occur, and have not been observed in practice. Consistent with the "tracer principle", the amount of pharmacologically active compound injected to a patient in the course of such an examination is extremely low. Adverse events, such as toxicity or allergic reactions, are thus highly improbable. No adverse or clinically detectable pharmacologic effects were observed following intravenous administration of ⁶⁸Ga-Trivehexin when administered to cancer patients, and there were no significant changes in vital signs, laboratory study results, or electrocardiograms. In a study involving healthy volunteers, researchers again reported no adverse or clinically detectable pharmacologic effects and no significant changes in vital signs.

References

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Positron emission tomography
Amides
Chelating agents
Nine-membered rings
Gallium
Phosphinates
Isotopes of gallium
Nonapeptides
Triazoles