Summary

In oncology, receptor-targeted molecular imaging using Single Photon Emission Computed Tomography (SPECT) or γ-scintigraphy has utility in diagnosis, disease staging and clinical decision-making. One class of radiotracer used for this purpose consists of a peptide attached to a chelator, which in turn is complexed to a radioactive metal ion. Two of the most prominent SPECT radiotracers employed for this purpose are ¹¹¹In-DTPA-octreotide and ^99mTc-EDDA/HYNIC-octreotide: both target the somatostatin receptor 2 that is overexpressed in neuroendocrine cancers. Whilst SPECT/γ-scintigraphy procedures with these radiotracers have been superseded in some healthcare settings by more sensitive Positron Emission Tomography (PET) imaging coupled with the PET radiotracer ⁶⁸Ga-DOTA-octreotate, they are still widely used in many clinics where PET is not available.
There are several factors that have led to the prevalence of SPECT/γ-scintigraphy imaging procedures. First, worldwide, there is simply more SPECT and γ-scintigraphy infrastructure than PET infrastructure, including in lower and middle income countries. Second, ^99mTc (t_½ = 6 h; 90% γ, 140 keV) is widely distributed and available from bench-top ⁹⁹Mo/^99mTc generators in the form of ^99mTcO₄⁻ in aqueous saline solution. Third, ^99mTc radiotracers, which are routinely used in 30–40 million procedures globally each year, are produced using simple one- or two-step kits in near-quantitative radiochemical yields.
Kits for the preparation of ^99mTc radiopharmaceuticals typically contain buffering salts, a reducing agent to reduce ^99mTc^VIIO₄⁻, a chelator derivative that ultimately complexes the ^99mTc metal ion to form the desired radiopharmaceutical, and other reagents including weak chelators, which serve to stabilise ^99mTc intermediates. The majority of ^99mTc radiopharmaceuticals are for imaging perfusion or anatomical processes. Molecular imaging using ^99mTc-EDDA/HYNIC-octreotide is not as prevalent, in part because of the low incidence of neuroendocrine cancer.
However, recognising the utility and availability of molecular SPECT/γ-scintigraphy infrastructure, in recent years, several new ^99mTc-labelled peptides have been clinically evaluated for receptor-targeted molecular imaging of the prostate-specific membrane antigen (PSMA) overexpressed in prostate cancer. These include ^99mTc-MIP-1404 (also known as ^99mTc-Trofolastat), ^99mTc-PSMA-I&S and ^99mTc-EDDA/HYNIC-iPSMA, which have been shown to be viable alternatives to efficacious PET diagnostic agents that similarly target PSMA.
There is an additional incentive for development of ^99mTc radiotracers: in some cases, chemically analogous Re complexes are accessible, providing access to pairs of “theranostic” ^99mTc and ¹⁸⁸Re radiopharmaceuticals for diagnosis and therapy, respectively. The rhenium radionuclide, ¹⁸⁸Re (t_½ = 17 h; 100% β⁻, E_max = 2.12 MeV; 15% γ, 155 keV), which can also be produced from a bench-top generator like ^99mTc, emits cytotoxic β⁻ particles. ¹⁸⁸Re radiopharmaceuticals can be effective therapeutics. For example, the lipophilic ¹⁸⁸Re-labelled radiopharmaceutical ¹⁸⁸Re-lipiodol is not only clinically efficacious for treatment of inoperable liver cancer, but is also economically viable in lower and middle income countries where supplies of other β⁻-emitting radiopharmaceuticals are limited due to economical and/or geographical barriers. Indeed, a newly developed pair of ^99mTc/¹⁸⁸Re-labelled PSMA-GCK01 theranostic agents has shown favourable properties in preclinical and initial first-in-human studies, demonstrating the feasibility of molecular imaging and therapeutic ^99mTc/¹⁸⁸Re pairs.
The authors have recently explored diphosphine derivatives as potential platforms for radiolabelling receptor-targeted biomolecules. These diphosphines include 2,3-bis(diphenylphosphino)maleic anhydride (DP^Ph) and 2,3-bis(di-p-tolylphosphino)maleic anhydride (DP^Tol) (Fig. 1) which react with the primary amine groups of peptides to furnish diphosphine-peptide (DP-peptide) conjugates. The DP-peptide derivatives coordinate [TcO₂]⁺ or [ReO₂]⁺ motifs to yield complexes of the type cis/trans-[MO₂(DP-peptide)₂]⁺ (M = Tc, Re); radiolabelled ^99mTc and ¹⁸⁸Re isotopologues are also synthetically accessible. They have also demonstrated that the resulting ^99mTc and ¹⁸⁸Re radiotracers retain affinity for their cognate target receptors in vitro and in vivo, and have favourable biodistribution pathways including rapid clearance from the bloodstream via a renal pathway. The authors note that these derivatives also coordinate the PET imaging isotope, ⁶⁴Cu (t_½ = 12.7 h; 18% β⁺, E_max = 653 keV), rapidly at room temperature, furnishing radiotracers of formula [Cu(DP-peptide)₂]⁺ that show high stability in serum.
However, DP-peptide derivatives of DP^Ph do not provide ^99mTc radiotracers in sufficiently high radiochemical yield to enable preparation using a one-step kit without subsequent purification to remove unreacted ^99mTc precursors. Furthermore, radiochemical yields of ¹⁸⁸Re derivatives are relatively low (<50%). DP-peptide derivatives of DP^Tol (which contains p-tolyl substituents in place of phenyl groups) have shown increased electron donor capacity and concomitant increased radiochemical yields compared to DP^Ph derivatives. However, even with the improved reactivity of DP^Tol, the authors have not been able to obtain radiochemical yields of [^99mTcO₂(DP-peptide)₂]⁺ compounds above ∼85–90%. This is a barrier to clinical translation.
To address this, the authors have synthesised two novel diphosphine maleic anhydrides, DP^An and DP^MEP (Fig. 1), which possess either p-anisyl or p-(2-methoxyethoxy)phenyl groups on the phosphines, respectively. Bioconjugates of these diphosphine maleic anhydride platforms enable near-quantitative radiochemical syntheses of ^99mTc radiotracers. In their most comprehensive report yet, they have explored the scope of possible bioconjugation reactions with both DP^An and DP^MEP using a range of biological targeting vectors. They have attached DP^An and DP^MEP to a PSMA-targeted dipeptide (PSMAt), enabling comparison of DP^An-PSMAt and DP^MEP-PSMAt with our prior conjugate, DP^Ph-PSMAt, including formation of ^99mTc, ¹⁸⁸Re and ⁶⁴Cu complexes. Lastly, our novel ^99mTc radiotracers have been assessed in murine models of prostate cancer using SPECT/CT imaging. The authors have therefore demonstrated the full utility of our novel and improved DP^An and DP^MEP platforms for enabling economical and accessible production of pairs of receptor-targeted theranostic radiotracers.

Results from nanoScan® SPECT/CT

SPECT/CT scanning and biodistribution studies: The 99mTc radiotracers (3.9–4.5 MBq, 3 mice per group, containing DPAn-PSMAt = 3.0 µg or DPMEP-PSMAt = 3.5 µg) were administered via tail vein injection under isoflurane anaesthesia. Following this, SPECT/CT scanning was carried out in one of two methods outlined below, to maintain 2 h biodistribution timepoints:

• The mouse was positioned on a single heated bed in a nanoScan SPECT/CT 80W scanner (Mediso Ltd., Budapest, Hungary) calibrated for 99mTc. A helical CT scan was acquired (50 kV X-ray source, 170 ms exposure time in 360 projections over 3 min). At 15 min post-injection, whole body SPECT scans were acquired (1 × 15 min, 3 × 30 min, conducted sequentially) with a frame time of 13 s and 26 s (using a 4-head scanner with 4 × 9 [1.4 mm] pinhole collimators. in helical scanning mode). At 2 h post-injection, the animals were euthanized by cervical dislocation, organs/tissues harvested and weighed, and radioactivity counted using a gamma counter.
• The mouse remained under isoflurane anaesthesia on a heated bed, followed by euthanasia by pentobarbital injection at 2 h post-injection. The mouse was then scanned using a nanoScan SPECT/CT 80W scanner (Mediso Ltd., Budapest, Hungary) calibrated for 99mTc. A helical CT scan was acquired (50 kV X-ray source, 170 ms exposure time in 360 projections over 3 min). Subsequently, a 30 min whole-body SPECT scan was acquired with a frame time of 45 s (using a 4-head scanner with 4 × 9 [1.4 mm] pinhole collimators in helical scanning mode). After scanning, the organs/tissues were harvested, weighed and radioactivity counted using a gamma counter.

Quantification of SPECT/CT images (acquired at 15–30 min, 30–60 min, 60–90 min and 90–120 min post-injection, Fig. 8) indicated that tumour uptake of [^99mTcO₂(DP^An-PSMAt)₂]⁺ was higher than that of [^99mTcO₂(DP^MEP-PSMAt)₂]⁺, consistent with in vitro uptake studies and ex vivo biodistribution data (vide infra). Additionally, for the mouse administered [^99mTcO₂(DP^An-PSMAt)₂]⁺, the ^99mTc tumour concentration noticeably increased from 30 min until 2 h post-injection.
SPECT/CT indicated that ^99mTc residualised in salivary glands for animals administered either [^99mTcO₂(DP^An-PSMAt)₂]⁺ or [^99mTcO₂(DP^MEP-PSMAt)₂]⁺. Two well-documented mechanisms of radiotracer uptake likely account for salivary gland uptake:

• PSMA is expressed at low levels in the salivary glands, kidneys and small intestine, with PSMA-targeted radiotracers showing uptake in the salivary glands, in both mice and human patients. Indeed, the salivary glands are considered dose-limiting organs, and uptake of radiotherapeutic PSMA-targeted radiopharmaceuticals can lead to xerostomia in prostate cancer patients.

• TcO₄⁻, bearing a single negative charge and with a similar polyatomic radius to that of the iodide anion, acts as a substrate for the sodium iodide symporter in vivo, and therefore accumulates in organs expressing the sodium iodide symporter – in mice, this includes the thyroid, salivary glands and stomach. SPECT imaging quantification (consistent with biodistribution data, vide infra) indicated that ^99mTc concentrations were higher in the salivary glands for the mouse administered [^99mTcO₂(DP^MEP-PSMAt)₂]⁺ compared to the mouse administered [^99mTcO₂(DP^An-PSMAt)₂]⁺. Indeed, ^99mTc concentrations in the salivary glands increased over time for the former subject. This suggests that [^99mTcO₂(DP^MEP-PSMAt)₂]⁺ could possess lower in vivo stability than [^99mTcO₂(DP^An-PSMAt)₂]⁺, with complex dissociation and oxidation of the [^99mTc^VO₂]⁺ motif resulting in formation of ^99mTcO₄⁻in vivo, and increased ^99mTc accumulation in the salivary glands for animals administered [^99mTcO₂(DP^MEP-PSMAt)₂]⁺.

  The biodistribution of [^99mTcO₂(DP^An-PSMAt)₂]⁺ and [^99mTcO₂(DP^MEP-PSMAt)₂]⁺ was further studied in (i) male SCID/Beige mice bearing DU145-PSMA+ prostate cancer xenografts (Fig. 8) and (ii) male nude mice bearing PSMA-expressing LNCaP prostate cancer xenografts (see SI).

For all animals, significant concentrations of ^99mTc radioactivity (2–5 %ID g⁻¹) were measured in PSMA-expressing tumours 2 h post-injection, and although ^99mTc tumour concentrations were consistently higher for animals administered [^99mTcO₂(DP^An-PSMAt)₂]⁺, there were no statistically significant differences between [^99mTcO₂(DP^An-PSMAt)₂]⁺ and [^99mTcO₂(DP^MEP-PSMAt)₂]⁺ in either mouse model. For both tracers, there were also significant concentrations of ^99mTc radioactivity in the spleen, which is known to express low levels of PSMA and accumulate PSMA-targeted radiotracers, and the liver. Importantly, clearance from the blood pool and subsequent excretion was largely via a renal route, with concentrations of 40–90 %ID g⁻¹ measured in kidneys.
The authors also noted that for both SCID/beige and nude mice studies, higher ^99mTc concentrations were observed in the stomach and salivary glands for animals administered [^99mTcO₂(DP^MEP-PSMAt)₂]⁺ compared to animals administered [^99mTcO₂(DP^An-PSMAt)₂]⁺, although these differences were not statistically significant. This observation is consistent with observations from SPECT/CT imaging studies, and further supports our conjecture that [^99mTcO₂(DP^MEP-PSMAt)₂]⁺ has lower in vivo stability than [^99mTcO₂(DP^An-PSMAt)₂]⁺, with in vivo formation of ^99mTcO₄⁻ from dissociation of [^99mTcO₂(DP^MEP-PSMAt)₂]⁺ resulting in higher levels of ^99mTc activity in organs expressing the sodium iodide symporter.
Overall, the in vivo biodistribution demonstrates that both [^99mTcO₂(DP^An-PSMAt)₂]⁺ and [^99mTcO₂(DP^MEP-PSMAt)₂]⁺ are useful SPECT or γ-scintigraphy imaging agents for PSMA expression in prostate cancer.

• In summary, the authors' new and highly versatile bis(phosphino) maleic anhydride platforms, DP^An and DP^MEP, enable facile preparation of diphosphine bioconjugates that can be simply radiolabelled with ^99mTc for SPECT imaging, ⁶⁴Cu for PET imaging and ¹⁸⁸Re for systemic radiotherapy, leading to the possibility of theranostic radiotracers.
• Importantly, these ^99mTc radiotracers can be prepared in high radiochemical yields (≥95%) using a single step kit. This is a critical advance upon our prior DP^Ph and DP^Tol technology, as it allows for simple, one-step formulation of peptide-based ^99mTc radiotracers, and presents new opportunities for economical molecular imaging using widely available ^99mTc production infrastructure and γ-scintigraphy/SPECT cameras.
• The authors are currently expanding the application of DP^An and DP^MEP chemistry to other therapeutically relevant receptor targets, to develop a versatile suite of molecular SPECT, PET and radiotherapeutic tracers.

Full article on pubs.rsc.org