Receptor-Targeted Peptide Conjugates Based on Diphosphines Enable Preparation of 99mTc and 188Re Theranostic Agents for Prostate Cancer

Visual Abstract

The PSMAt peptide, which targets the prostate-specific membrane antigen (PSMA), has had clinical impact as a vector for delivering radionuclides to prostate cancer for diagnostic imaging and systemic peptide receptor radionuclide therapy (PRRT).The radiopharmaceutical [ 177 Lu]Lu-PSMA-617 (1,2) has recently been approved by the Food and Drug Administration for PRRT of metastatic castration-resistant prostate cancer.Diagnostic PET imaging with [ 68 Ga]Ga-PSMA-11 can inform clinical decision-making for treatment of prostate cancer, and [ 68 Ga]Ga-PSMA-11 is widely used as a diagnostic companion to [ 177 Lu]Lu-PSMA-617 (3).PSMAtargeted radiopharmaceuticals for SPECT/g-scintigraphy imaging, such as [ 99m Tc]Tc-MIP-1404 (4,5), [ 99m Tc]Tc-PSMA-I&S (6,7), and [ 99m Tc]Tc-EDDA/HYNIC-iPSMA (8,9), have been developed as alternatives to 68 Ga to enhance access to PSMA scanning when 68 Ga and PET are less accessible but generator-based 99m Tc and SPECT/ g-scintigraphy cameras are available.Although these 99m Tc (half-life of 6 h, 90% g, 140 keV) radiotracers exhibit lower sensitivity than PSMA-targeted PET radiotracer alternatives, particularly in the case of biochemical recurrence of prostate cancer at low PSA levels or low tumor volumes, they have potential utility in providing useful diagnostic information at high tumor volumes, assessing suitability for and response to PRRT and radioguided surgery for which detection of every site of small volume of disease is less critical (4,7,8).
A range of radiometal ions with therapeutically efficacious emission profiles (e.g., 225 Ac, 227 Th, 212 Pb, and 161 Tb) has been used as alternatives to 177 Lu.However, in lower-and middle-income countries (LMICs), the availability of PRRT is limited by cost.The batchproduced radiopharmaceuticals that prevail in high-income countries are prohibitively expensive and are available only to the wealthiest patients in LMICs.Additionally, the availability of PET infrastructure, including cyclotrons and scanners, in LMICs is limited (10).
The chemistry of rhenium is closely similar to that of its lighter congener technetium.Importantly, b 2 -emitting 188 Re (half-life of 17 h, 100% b 2 , 2.12 MeV, 15% g, 155 keV) is available from a benchtop 188 W/ 188 Re generator, offering hospitals an economical and routinely accessible source of a therapeutic radionuclide.Radiopharmaceuticals based on the theranostic 99m Tc/ 188 Re pair offer a potentially viable solution to economic and geographic barriers posed by existing theranostic PRRT (11)(12)(13).Traditional 99m Tc-radiopharmaceuticals are used in 30 million scans worldwide per year, including in LMICs, in combination with g-scintigraphy (10,14).Additionally, international consortia have identified 188 Re as a highly promising basis for systemic radiotherapy in LMICs: recognizing the affordability of 188 Re, the International Atomic Energy Agency has sponsored multinational clinical trials of 188 Re-labeled Lipiodol for treatment of inoperable liver cancer in LMICs (11). 188Re-labeled Lipiodol proved effective and inexpensive. 188Re-labeled bisphosphonates have also been extremely beneficial in palliative treatment of bone metastases in LMICs (15).There is high potential for the combination of 99m Tc with 188 Re to provide economical, population-wide access to stratified molecular imaging and PRRT in LMICs, provided that suitable chemical platforms are available to enable radiochemical production of well-defined pairs of theranostic 99m Tc/ 188 Re agents.
[ 188 Re]Re-DP1-PSMAt and [ 188 Re]Re-DP2-PSMAt were subsequently isolated using reverse-phase HPLC, lyophilized, and then reconstituted in phosphate-buffered saline to yield the radiotracers in more than 95% radiochemical purity.The purified 188 Re compounds were analyzed by reverse-phase radio-HPLC.Each 188 Re compound coeluted with its nonradioactive nat Re isotopolog and radioactive 99m Tc analog (17), confirming not only the chemical identity of these new 188 Re agents but also that they are isostructural and isoelectronic with their 99m Tc analogs (Fig. 1).Each rhenium and technetium compound consists of 2 closely eluting isomers, cis-and trans-[M V O 2 (DPX-PSMAt) 2 ] 1 (M 5 Re or Tc, X 5 1 or 2) (17).The cis and trans designations denote the relative positions of the PSMAt moieties.
Solutions of each new 188 Re radiotracer were added to human serum and incubated at 37 C for 24 h followed by analysis by reverse-phase analytic radio-HPLC, which demonstrated that [ 188 Re]Re-DP1-PSMAt and [ 188 Re]Re-DP2-PSMAt are stable, with more than 95% each radiotracer, respectively, observed intact in human serum over this time frame (Supplemental Fig. 2).
The cellular localization of [ 99m Tc]Tc-DP1-PSMAt and [ 99m Tc]Tc-DP2-PSMAt was also evaluated in DU145-PSMA1 and LNCaP cells over time (Fig. 2).Uptake of both radiotracers increased over 2 h, and most cell-associated radioactivity was found in the internalized cell fraction at all measured time points for both PSMA-expressing cell lines.Uptake of [ 99m Tc]Tc-DP1-PSMAt (both surface-bound and internalized radioactivity) was slightly higher than that of [ 99m Tc]Tc-DP2-PSMAt.
In SPECT/CT scans of animals administered either [ 99m Tc]Tc-DP1-PSMAt or [ 99m Tc]Tc-DP2-PSMAt only, tumors could be clearly delineated at both 2 h (Fig. 3) and 24 h (Supplemental Fig. 4) after injection.The kidneys and bladder were also clearly visible across these time points, consistent with ex vivo biodistribution data.SPECT/CT also showed negligible tumor uptake for animals either coadministered PMPA or bearing DU145 tumors that do not express PSMA receptor (Fig. 3).For all animals administered either [ 99m Tc]Tc-DP1-PSMAt only or [ 99m Tc]Tc-DP2-PSMAt only, the spleen was also identified in SPECT/CT scans acquired at 2 h after injection.Coadministration of PMPA decreased spleen uptake of both radiotracers.

Biodistribution of 188 Re Radiotracers in Mice Bearing Prostate Cancer Tumors
The biodistributions of the 188 Re radiotracers, [ 188 Re]Re-DP1-PSMAt and [ 188 Re]Re-DP2-PSMAt, were first assessed in SCID/ beige mice bearing DU145-PSMA1 tumors (Fig. 3).Radioactivity concentration in the tumors of animals administered [ 188 Re]Re-DP1-PSMAt measured 27.7 6 6.4 %ID/g at 2 h after injection, whereas that of animals given [ 188 Re]Re-DP2-PSMAt measured 19.2 6 8.6 %ID/g.Both compounds cleared the circulation via a renal pathway, as evidenced by high concentrations of radioactivity measured in the kidneys ([ 188 Re]Re-DP1-PSMAt measured 88.3 6 18.8 %ID/g, [ 188 Re]Re-DP2-PSMAt measured 96.8 6 16.3 %ID/g).Biodistribution data also indicated that both compounds had low retention in nontarget, healthy organs and tissues, except for the spleen.In this experiment, there were no notable significant differences between the biodistribution profiles of [ 188 Re]Re-DP1-PSMAt and [ 188 Re]Re-DP2-PSMAt at 2 h after injection.As expected, coadministration of PMPA significantly inhibited uptake of both 188 Re radiotracers in the tumor and spleen.
Urine was collected from mice administered either [ 188 Re]Re-DP1-PSMAt or [ 188 Re]Re-DP2-PSMAt at 2 h after injection and was analyzed by reverse-phase radio-HPLC.Radiochromatograms showed that both [ 188 Re]Re-DP1-PSMAt and [ 188 Re]Re-DP2-PSMAt were highly stable and were cleared from the blood pool and excreted chemically intact (Supplemental Fig. 5).
With a view to developing a molecular [ 188 Re]Re-labeled agent for PSMA PRRT, we further characterized the biodistribution profiles of [ 188 Re]Re-DP1-PSMAt and [ 188 Re]Re-DP2-PSMAt in male athymic nude mice bearing LNCaP xenografts (Fig. 4), which recapitulate clinical metastatic prostate cancer more closely than DU145-PSMA1 xenografts.Importantly, the 188 Re agents exhibited significant retention in tumors up to at least 1 d after administration.In mice administered [ 188 Re]Re-DP1-PSMAt, LNCaP tumor uptake measured 7.0 6 2.3 %ID/g at 2 h after injection and 2.9 6 0.8 %ID/g at 24 h after injection.For [ 188 Re]Re-DP2-PSMAt, LNCaP tumor uptake measured 9.8 6 2.8 %ID/g at 2 h after injection and 7.6 6 4.4 %ID/g at 24 h after injection.With the exception of the kidneys, radioactivity concentrations in other organs were similar to what was observed in prior experiments with male SCID/beige mice.

DISCUSSION
Theranostic PSMA-targeted radiopharmaceuticals have had an extraordinary impact on prostate cancer care in health care settings where they are available.We have developed 2 pairs of chemically analogous theranostic agents based on the generatorproduced radionuclides 99m Tc and 188 Re.Radio-HPLC alongside careful chemical characterization of nonradioactive or long-lived isotopologs (17) demonstrates that technetium and rhenium pairs are chemical analogs and isostructural.Consequently, each pair exhibits highly similar biologic behavior in in vitro and in vivo models of prostate cancer.Importantly, both 188 Re-and 99m Tc-labeled complexes of DP1-PSMAt and DP2-PSMAt show a high accumulation in PSMA-expressing tumors and prostate cancer cells and, in vivo, rapidly clear from the circulation via a renal pathway, with minimal retention in healthy tissues.Furthermore, in vivo, these 4 radiotracers all demonstrate significant retention in prostate cancer tumors up to 24 h after injection.
In a clinical context, the favorable and near-identical biologic behaviors of chemically analogous 99m Tc and 188 Re radiotracers bring about the possibility of using 99m Tc molecular imaging to predict the biodistribution, accumulation, and dosimetry of a complementary 188 Re PRRT agent.Our novel theranostic pairs, M-DP1-PSMAt and M-DP2-PSMAt (M 5 [ 99m Tc]Tc, [ 188 Re]Re), which use economical generator-produced isotopes, have strong potential utility for this purpose in prostate cancer treatment, particularly in LMICs.Additionally, the DP chemical platform underpinning these theranostic radiotracers is an excellent candidate for development of other peptide-based radiotracers: this technology could increase clinical use of receptor-targeted 99m Tc and 188 Re radiopharmaceuticals and widen patient access to the benefits of molecular theranostic agents.
yielding radiotracers of extremely high specific activity; that is, with the exception of decay products, there is minimal unlabeled DP1-PSMAt or DP2-PSMAt present in final radiotracer formulations.This culminates in an extremely high accumulation in PSMAexpressing prostate cancer cells in vitro and tissue in vivo (tumors, spleen, and in the case of DP1-PSMAt derivatives, kidneys).
We elected to use a 2-step protocol to prepare 188 Re-radiolabeled agents, in which a [ 188 Re]Re V -citrate precursor is reacted with either DP1-PSMAt or DP2-PSMAt to yield the desired 188 Re radiotracers.This procedure is similar to prior radiosyntheses of [ 188 Re][Re V O 2 ] 1 -labeled phosphine-containing P 2 S 2 and P 2 N 2 tetradentate chelator derivatives (20,21).However, complexes of the latter chelators were obtained in more than 90% radiochemical yield; the highest radiochemical yields obtained for [ 188 Re]Re-DP1-PSMAt and [ 188 Re]Re-DP2-PSMAt were about 70% and 50%, respectively.We also note that the [ 188 Re][ReO V ] 31 -based complex, [ 188 Re]Re-PSMA-GCK01, can be obtained in 78% radiochemical yield and 96% radiochemical purity, using a one-pot radiochemical procedure (16).This procedure involves heating an aqueous solution of precursor GCK01, [ 188 Re]ReO 4 2 , citrate, and ascorbic acid at pH 2.0-3.5 at high temperature for 1 h, followed by neutralization, further heating for 5 min, and final C18 Sep-Pak purification.We are currently optimizing kit-based formulations to increase the radiochemical yields of DP-based 99m Tc and 188 Re radiotracers and to obviate postsynthetic purification procedures to remove unreacted 99m Tc and 188 Re precursors.

CONCLUSION
We have developed new PSMA-targeting 99m Tc/ 188 Re theranostic agents, using versatile diphosphine chemical platforms.These radiotracers can be prepared using eluate from bench-top 99m Tc and 188 Re generators and chemical kits.The resulting isostructural 99m Tc and 188 Re pairs show near-equivalent biologic behaviors in models of prostate cancer.We are further developing optimized kit-based formulations to enable near-quantitative radiochemical yields to obviate purification steps after radiolabeling.Our new, generator-based theranostic agents have potential to provide access to the benefits of PSMA-targeted diagnostic imaging and systemic radiotherapy in health care settings that do not routinely have access to either reactorproduced 177 Lu radiopharmaceuticals or PET/CT infrastructure.

DISCLOSURE
This research was supported by a Cancer Research UK.Career Establishment Award (C63178/A24959), an EPSRC program grant (EP/S032789/1), the Cancer Research UK.National Cancer Imaging Translational Accelerator Award (C4278/A27066), and the Wellcome Trust (212885/Z/18/Z).The authors have submitted a patent application describing the intellectual property described in this article.No other potential conflict of interest relevant to this article was reported.

ACKNOWLEDGMENTS
We are grateful for research assistance with animal work from James Cormack, Harmony Blythin, and Hagen Schmidt.PERTINENT FINDINGS: Pairs of chemically analogous 99m Tc/ 188 Re radiotracers show equivalent uptake in PSMA-expressing prostate cancer cells and favorable, highly similar biodistribution profiles in mouse models of prostate cancer.
IMPLICATIONS FOR PATIENT CARE: These generator-based theranostic agents have potential to provide access to the benefits of PSMA-targeted diagnostic imaging and systemic radiotherapy in health care settings that do not routinely have access to either cyclotron-or reactor-produced radionuclides.

FIGURE 2 .
FIGURE 2. (A) Uptake of radiotracers in PSMA-positive and PSMA-negative prostate cancer cells.(B) Time course uptake and localization of 99m Tc radiotracers in DU145-PSMA cells and LNCaP cells.Data are presented as mean 6 SD; n 5 3-4 biologic repeats performed in triplicate.
Are molecular theranostic radiotracers based on 99m Tc and 188 Re feasible for diagnostic imaging and systemic radiotherapy of prostate cancer?
), which was heated at 90 C for 30 min.
SPECT/CT Scanning and Biodistribution Studies in Mice