Dual-Nuclide Radiopharmaceuticals for Positron Emission Tomography Based Dosimetry in Radiotherapy.

Improvement of the accuracy of dosimetry in radionuclide therapy has the potential to increase patient safety and therapeutic outcomes. Although positron emission tomography (PET) is ideally suited for acquisition of dosimetric data because PET is inherently quantitative and offers high sensitivity and spatial resolution, it is not directly applicable for this purpose because common therapeutic radionuclides lack the necessary positron emission. This work reports on the synthesis of dual-nuclide labeled radiopharmaceuticals with therapeutic and PET functionality, which are based on common and widely available metal radionuclides. Dual-chelator conjugates, featuring interlinked cyclen- and triazacyclononane-based polyphosphinates DOTPI and TRAP, allow for strictly regioselective complexation of therapeutic (e.g., 177 Lu, 90 Y, or 213 Bi) and PET (e.g., 68 Ga) radiometals in the same molecular framework by exploiting the orthogonal metal ion selectivity of these chelators (DOTPI: large cations, such as lanthanide(III) ions; TRAP: small trivalent ions, such as GaIII ). Such DOTPI-TRAP conjugates were decorated with 3 Gly-urea-Lys (KuE) motifs for targeting prostate-specific membrane antigen (PSMA), employing Cu-catalyzed (CuAAC) as well as strain-promoted (SPAAC) click chemistry. These were labeled with 177 Lu or 213 Bi and 68 Ga and used for in vivo imaging of LNCaP (human prostate carcinoma) tumor xenografts in SCID mice by PET, thus proving practical applicability of the concept.

Molar Activity of Ga-68 Labeled PSMA Inhibitor Conjugates Determines PET Imaging Results.

Radiopharmaceuticals targeting the enzyme prostate-specific membrane antigen (PSMA; synonyms: glutamate carboxypeptidase II, NAALADase; EC 3.4.17.21) have recently emerged as powerful agents for diagnosis and therapy (theranostics) of prostate carcinoma (PCa). The radiation doses for therapeutic application of such compounds are limited by substantial uptakes in kidneys and salivary glands, with excess doses reportedly leading to radiotoxicity-related adverse effects, such as kidney insufficiency or xenostomia. On the basis of the triazacyclononane-triphosphinate (TRAP) chelator, monomeric to trimeric conjugates of the PSMA inhibitor motif lysine-urea-glutamic acid (KuE) were synthesized by means of Cu(I)-mediated (CuAAC) or 5-aza-dibenzocyclooctyne (DBCO)-driven, strain-promoted click chemistry (SPAAC), which were labeled with gallium-68 for application in positron emission tomography (PET), and characterized in terms of PSMA affinity (determined in cellular displacement assays against I-125-BA) and lipophilicity (expressed as log D). Using subcutaneous murine LNCaP (PSMA-positive human prostate carcinoma) xenografts, the influence of ligand multiplicity, affinity, polarity, and molar activity (i.e., mass dose) on the uptakes in tumor, kidney, salivary, and background (muscle) was analyzed by means of region-of-interest (ROI) based quantification of small-animal PET imaging data. As expected, trimerization of the KuE motif resulted in high PSMA affinities (IC50 ranging from 6.0-1.5 nM). Of all parameters, molar activity/cold mass had the most pronounced influence on PET uptakes. Because accumulation in nontumor tissues was effected to a larger extent than tumor uptakes, lower molar activities resulted in substantially better tumor-to-organ ratios. For example, for one trimer, 68Ga-AhxKuE3 (IC50 = 1.5 ± 0.3 nM, log D = -3.8 ± 0.1), a higher overall amount of active compound (12 pmol vs 2 nmol, equivalent to molar activities of 1200 and 8 MBq/nmol) resulted in a remarkable reduction of the kidney-to-tumor ratio from 11.4 to 1.4, respectively, at 60 min p.i. Our study suggests that, for PSMA-targeting radiopharmaceuticals, molar activity has a more pronounced influence on small-animal PET imaging results than structural or in vitro parameters.

Preclinical comparison of [177Lu]Lu-rhPSMA-10.1 and [177Lu]Lu-rhPSMA-10.2 for endoradiotherapy of prostate cancer: biodistribution and dosimetry studies.

BACKGROUND: Radiohybrid PSMA-targeted ligands (rhPSMA) have been introduced as a novel platform for theranostic applications. Among a variety of rhPSMA-ligands developed for radioligand therapy, two stereoisomers [177Lu]Lu-rhPSMA-10.1 and -10.2 have been synthesized and initially characterized in preclinical experiments with the aim to provide an optimized binding profile to human serum albumin, a reduction of charge, and thus accelerated kidney excretion, and unaffected or even improved tumor uptake. As both isomers showed similar in vitro characteristics and tumor uptake at 24 h post injection in tumor bearing mice and in order to identify the isomer with the most favorable pharmacokinetics for radioligand therapy, we carried out in-depth biodistribution and dosimetry studies in tumor-bearing and healthy mice. RESULTS: rhPSMA-10.1 and -10.2 were radiolabeled with lutetium-177 according to the established procedures of other DOTA-based PSMA ligands and displayed a high and comparable stability in all buffers and human serum (> 97%, 24 h). Biodistribution studies revealed fast clearance from the blood pool (0.3-0.6%ID/g at 1 h) and other background tissues within 48 h. Distinctive differences were found in the kidneys, where [177Lu]Lu-rhPSMA-10.1 displayed lower initial uptake and faster excretion kinetics compared to [177Lu]Lu-rhPSMA-10.2 expressed by a 1.5-fold and ninefold lower uptake value at 1 h and 24 h in healthy animals, respectively. Tumor uptake was comparable and in the range of 8.6-11.6%ID/g for both isomers over 24 h and was maintained up to 168 h at a level of 2.2 ± 0.8 and 4.1 ± 1.4%ID/g for [177Lu]Lu-rhPSMA-10.1 and [177Lu]Lu-rhPSMA-10.2, respectively. CONCLUSION: Our preclinical data on biodistribution and dosimetry indicate a more favorable profile of [177Lu]Lu-rhPSMA-10.1 compared to [177Lu]Lu-rhPSMA-10.2 for PSMA-targeted radioligand therapy. [177Lu]Lu-rhPSMA-10.1 shows fast kidney clearance kinetics resulting in excellent tumor-to-organ ratios over a therapy relevant time course. Meanwhile, [177Lu]Lu-rhPSMA-10.1 is currently being investigated in clinical phase I/II studies in patients with mCRPC (NCT05413850), in patients with high-risk localized PC (NCT06066437, Nautilus Trial) and after external beam radiotherapy (NCT06105918).

Albumin-Mediated Size Exclusion Chromatography: The Apparent Molecular Weight of PSMA Radioligands as Novel Parameter to Estimate Their Blood Clearance Kinetics.

A meticulously adjusted pharmacokinetic profile and especially fine-tuned blood clearance kinetics are key characteristics of therapeutic radiopharmaceuticals. We, therefore, aimed to develop a method that allowed the estimation of blood clearance kinetics in vitro. For this purpose, 177Lu-labeled PSMA radioligands were subjected to a SEC column with human serum albumin (HSA) dissolved in a mobile phase. The HSA-mediated retention time of each PSMA ligand generated by this novel 'albumin-mediated size exclusion chromatography' (AMSEC) was converted to a ligand-specific apparent molecular weight (MWapp), and a normalization accounting for unspecific interactions between individual radioligands and the SEC column matrix was applied. The resulting normalized MWapp,norm. could serve to estimate the blood clearance of renally excreted radioligands by means of their influence on the highly size-selective process of glomerular filtration (GF). Based on the correlation between MW and the glomerular sieving coefficients (GSCs) of a set of plasma proteins, GSCcalc values were calculated to assess the relative differences in the expected GF/blood clearance kinetics in vivo and to select lead candidates among the evaluated radioligands. Significant differences in the MWapp,norm. and GSCcalc values, even for stereoisomers, were found, indicating that AMSEC might be a valuable and high-resolution tool for the preclinical selection of therapeutic lead compounds for clinical translation.

Synthesis and Preclinical Evaluation of 177Lu-Labeled Radiohybrid PSMA Ligands for Endoradiotherapy of Prostate Cancer.

The prostate-specific membrane antigen (PSMA)-targeted radiohybrid (rh) ligand [177Lu]Lu-rhPSMA-7.3 has recently been assessed in a pretherapeutic dosimetry study on prostate cancer patients. In comparison to [177Lu]Lu-PSMA I&T, application of [177Lu]Lu-rhPSMA-7.3 resulted in a significantly improved tumor dose but also higher kidney accumulation. Although rhPSMA-7.3 has been initially selected as the lead compound for diagnostic application based on the characterization of its gallium complex, a systematic comparison of the most promising 177Lu-labeled rhPSMA ligands is still missing. Thus, this study aimed to identify the rhPSMA ligand with the most favorable pharmacokinetics for 177Lu-radioligand therapy. Methods: The 4 isomers of [177Lu]Lu-rhPSMA-7 (namely [177Lu]Lu-rhPSMA-7.1, -7.2, -7.3, and -7.4), along with the novel radiohybrid ligands [177Lu]Lu-rhPSMA-10.1 and -10.2, were compared with the state-of-the-art compounds [177Lu]Lu-PSMA I&T and [177Lu]Lu-PSMA-617. The comparative evaluation comprised affinity studies (half-maximal inhibitory concentration) and internalization experiments on LNCaP cells, as well as lipophilicity measurements. In addition, we determined the apparent molecular weight (AMW) of each tracer as a parameter for human serum albumin (HSA) binding. Biodistribution studies and small-animal SPECT imaging were performed on LNCaP-tumor bearing mice at 24 h after injection. Results: 177Lu labeling of the radiohybrids was performed according to the established procedures for the currently established PSMA-targeted ligands. All ligands showed potent binding to PSMA-expressing LNCaP cells, with affinities in the low nanomolar range and high internalization rates. Surprisingly, the most pronounced differences regarded the HSA-related AMW. Although [177Lu]Lu-rhPSMA-7 isomers demonstrated the highest AMW and thus strongest HSA interactions, [177Lu]Lu-rhPSMA-10.1 showed an AMW lower than for [177Lu]Lu-rhPSMA-7.3 but higher than for the 177Lu-labeled references PSMA I&T and PSMA-617. In biodistribution studies, [177Lu]Lu-rhPSMA-10.1 exhibited the lowest kidney uptake and fastest excretion from the blood pool of all rhPSMA ligands while preserving a high tumor accumulation. Conclusion: Clinical investigation of [177Lu]Lu-rhPSMA-10.1 is highly warranted to determine whether the favorable pharmacokinetics observed in mice will also result in high tumor uptake and decreased absorbed dose to kidneys and other nontarget tissues in patients.

Validation of 18F-rhPSMA-7 and 18F-rhPSMA-7.3 PET Imaging Results with Histopathology from Salvage Surgery in Patients with Biochemical Recurrence of Prostate Cancer.

18F-rhPSMA-7, and its single diastereoisomer form, 18F-rhPSMA-7.3, are prostate-specific membrane antigen (PSMA)-targeting radiopharmaceuticals. Here, we investigated their accuracy for the assessment of lymph node (LN) metastases validated by histopathology. Methods: Data from 58 patients with biochemical recurrence of prostate cancer after radical prostatectomy receiving salvage surgery after PET imaging with 18F-rhPSMA-7 or 18F-rhPSMA-7.3 were retrospectively reviewed. Two nuclear medicine physicians reviewed all PET scans and morphologic imaging in consensus. Readers were masked from the results of histopathology. PET and morphologic imaging were correlated with histopathology from resected LNs. Results: In 75 of 150 resected regions in 54 of 58 patients, tumor lesions were present in histopathology. The template-based specificity of PET (18F-rhPSMA-7 and 18F-rhPSMA-7.3 combined) and morphologic imaging was 93.3% and 100%, respectively. However, 18F-rhPSMA-7 and 18F-rhPSMA-7.3 PET detected metastases in 61 of 75 histopathologically proven metastatic LN fields (81.3%) whereas morphologic imaging was positive in only 9 of 75 (12.0%). The positive predictive value was 92.4% for 18F-rhPSMA-7 and 18F-rhPSMA-7.3 PET and 100% for morphologic imaging. 18F-rhPSMA-7 and 18F-rhPSMA-7.3 PET performance was significantly superior to morphologic imaging (difference in the areas under the receiver-operating-characteristic curves, 0.222; 95% CI, 0.147-0.298; P < 0.001). The mean size of PET-positive and histologically confirmed LN metastases was 6.3 ± 3.1 mm (range, 2-15 mm) compared with a mean size of 9.8 ± 2.5 mm (range, 7-15 mm) on morphologic imaging. Conclusion: 18F-rhPSMA-7 and 18F-rhPSMA-7.3 PET offer a high positive predictive value comparable to that reported for 68Ga-PSMA-11 and represent a valuable tool for guiding salvage lymphadenectomy.

The Influence of Specific Activity on the Biodistribution of 18F-rhPSMA-7.3: A Retrospective Analysis of Clinical PET Data.

We investigated whether the time between synthesis and injection and the resulting decrease in specific activity affects the normal-organ and tumor uptake of the PSMA ligand 18F-rhPSMA-7.3 in patients with prostate cancer. Methods: The biodistribution of 18F-rhPSMA-7.3 on PET/CT scans obtained with a high specific activity (median, 178.9 MBq/µg; n = 42) and a low specific activity (median, 19.3 MBq/µg; n = 42) was compared. Results: Tracer uptake by the parotid gland, submandibular gland, and spleen was moderately but significantly lower in the low-specific-activity group than in the high-specific-activity group (median SUVmean, 16.7 vs. 19.2; 18.1 vs. 22.3; and 7.8 vs. 9.6, respectively). No other statistically significant differences were found for normal organs or tumor lesions. Conclusion: A 10-fold decrease in specific activity has only minor effects on the biodistribution of 18F-rhPSMA-7.3. These findings suggest that 18F-labeled PSMA ligands can be centrally produced and shipped to PET clinics in a similar way to 18F-FDG.

Preclinical biodistribution and dosimetry and human biodistribution comparing 18F-rhPSMA-7 and single isomer 18F-rhPSMA-7.3.

BACKGROUND: Radiohybrid prostate-specific membrane antigen (rhPSMA) ligands such as 18F-rhPSMA-7 are a new class of theranostic agents in clinical development for prostate cancer. We compared preclinical dosimetry and human biodistribution of 18F-rhPSMA-7 with that of single diastereoisomer form, 18F-rhPSMA-7.3. METHODS: Preclinical dosimetry was performed with SCID-mice sacrificed at multiple timepoints (10-300 min) post-injection of 25.6 ± 3.6 MBq 18F-rhPSMA-7 or 28.5 ± 4.8 MBq 18F-rhPSMA-7.3 (n = 3-6 mice per timepoint). Heart, lung, liver, spleen, pancreas, fat, stomach, small intestine, large intestine, kidney, muscle, bone, bladder, testicles, tail, and brain tissue were harvested, and urine and blood samples collected. Percentage of injected dose per gram was calculated. Absorbed doses were estimated with OLINDA/EXM 1.0. 18F-rhPSMA-7 (n = 47) and 18F-rhPSMA-7.3 (n = 33) PET/CT exams were used to estimate human biodistribution. Mean (range) injected activities were 324 (236-424) MBq versus 345 (235-420) MBq, and acquisition times were 84 (42-166) versus 76 (59-122) minutes for 18F-rhPSMA-7 versus 18F-rhPSMA-7.3, respectively. SUVmean was determined for background (gluteal muscle), normal organs (salivary glands, blood pool, lung, liver, spleen, pancreas, duodenum, kidney, bladder, bone) and up to three representative tumour lesions. Qualitative analyses assessed image quality, non-specific blood pool activity, and background uptake in bone/marrow using 3/4-point scales. RESULTS: Preclinical dosimetry revealed that at 3.5 h and 1 h bladder voiding intervals, the extrapolated total effective doses were 26.6 and 12.2 µSv/MBq for 18F-rhPSMA-7 and 21.7 and 12.8 µSv/MBq for 18F-rhPSMA-7.3 respectively. Human biodistribution of both agents was typical of other PSMA-ligands and broadly similar to each other; SUVmean were 16.9 versus 16.2 (parotid gland), 19.6 versus 19.9 (submandibular gland), 2.0 versus 1.9 (blood pool, p < 0.005), 0.7 versus 0.7 (lungs), 7.0 versus 7.3 (liver), 9.1 versus 8.4 (spleen), 32.4 versus 35.7 (kidney), 2.5 versus 2.8 (pancreas), 10.9 versus 11.0 (duodenum), 1.1 versus 1.3 (bone) and 4.6 versus 2.0 (bladder; p < 0.001) for 18F-rhPSMA-7 versus 18F-rhPSMA-7.3, respectively. Tumour SUVmean was higher for 18F-rhPSMA-7.3 (32.5 ± 42.7, n = 63 lesions) than for 18F-rhPSMA-7 (20.0 ± 20.2, n = 89 lesions). CONCLUSIONS: Radiation dosimetry is favourable for both agents. Radiation exposure, assuming a 1 h voiding interval, is less than 5 mSv after injection of 370 MBq. 18F-rhPSMA-7.3 showed significantly lower bladder uptake, and a higher uptake trend in tumours compared with 18F-rhPSMA-7.

Pretherapeutic Comparative Dosimetry of 177Lu-rhPSMA-7.3 and 177Lu-PSMA I&T in Patients with Metastatic Castration-Resistant Prostate Cancer.

Radiohybrid prostate-specific membrane antigen (rhPSMA) ligands allow for labeling with 18F and radiometals for endoradiotherapy. rhPSMA-7.3 has been designated as a lead compound with promising preclinical data for 177Lu-rhPSMA-7.3, which has shown higher tumor uptake than 177Lu-PSMA I&T. In this retrospective analysis, we compared pretherapeutic clinical dosimetry data of both PSMA ligands. Methods: Six patients with metastatic castration-resistant prostate cancer underwent both 177Lu-rhPSMA-7.3 and 177Lu-PSMA I&T pretherapeutic dosimetry. Whole-body scintigraphy was performed at 1 h, 4 h, 24 h, 48 h, and 7 d after injection. Regions of interest covering the whole body, organs, bone marrow, and tumor lesions were drawn for each patient. Absorbed doses for individual patients and pretherapeutic applications were calculated using OLINDA/EXM. To facilitate the comparison of both ligands, we introduced the therapeutic index (TI), defined as the ratio of mean pretherapeutic doses to tumor lesions over relevant organs at risk. Results: Mean whole-body pretherapeutic effective doses for 177Lu-rhPSMA-7.3 and 177Lu-PSMA I&T were 0.12 ± 0.07 and 0.05 ± 0.03 Sv/GBq, respectively. Mean absorbed organ doses for 177Lu-rhPSMA-7.3 and 177Lu-PSMA I&T were, for example, 1.65 ± 0.28 and 0.73 ± 0.18 Gy/GBq for the kidneys, 0.19 ± 0.09 and 0.07 ± 0.03 Gy/GBq for the liver, 2.35 ± 0.78 and 0.80 ± 0.41 Gy/GBq for the parotid gland, and 0.67 ± 0.62 and 0.30 ± 0.27 Gy/GBq for the bone marrow, respectively. Tumor lesions received mean absorbed doses of 177Lu-rhPSMA-7.3 and 177Lu-PSMA I&T of 6.44 ± 6.44 and 2.64 ± 2.24 Gy/GBq, respectively. The mean TIs for the kidneys were 3.7 ± 2.2 and 3.6 ± 2.2 for 177Lu-rhPSMA-7.3 and 177Lu-PSMA I&T, respectively, and those for the bone marrow were 15.2 ± 10.2 and 15.1 ± 10.2 for 177Lu-rhPSMA-7.3 and 177Lu-PSMA I&T, respectively. Conclusion: Pretherapeutic clinical dosimetry confirmed preclinical results of mean absorbed doses for tumors that were 2-3 times higher for 177Lu-rhPSMA-7.3 than for 177Lu-PSMA I&T. Absorbed doses to normal organs also tended to be higher for 177Lu-rhPSMA-7.3, resulting overall in similar average TIs for both radiopharmaceuticals with considerable interpatient variability. 177Lu-rhPSMA-7.3 has promise for a therapeutic efficacy similar to that of 177Lu-PSMA I&T at smaller amounts of injected activity, simplifying radiation safety measurements (especially for large patient numbers or dose escalation regimens).

Utility of 18F-rhPSMA-7.3 PET for Imaging of Primary Prostate Cancer and Preoperative Efficacy in N-Staging of Unfavorable Intermediate- to Very High-Risk Patients Validated by Histopathology.

18F-rhPSMA-7.3, the lead compound of a new class of radiohybrid prostate-specific membrane antigen (rhPSMA) ligand, is currently in phase III trials for prostate cancer (PCa) imaging. Here, we describe our experience in primary PCa staging. Methods: We retrospectively identified 279 patients with primary PCa who underwent 18F-rhPSMA-7.3 PET/CT (staging cohort). A subset of patients (83/279) subsequently underwent prostatectomy with lymph node (LN) dissection without prior treatment (efficacy cohort). The distribution of tumor lesions was determined for the staging cohort and stratified by National Comprehensive Cancer Network risk score. Involvement of pelvic LNs was assessed retrospectively by 3 masked independent central readers, and a majority rule was used for analysis. Standard surgical fields were rated on a 5-point scale independently for PET and for morphologic imaging. Results were compared with histopathologic findings on a patient, right-vs.-left, and template basis. Results: For the staging cohort, 18F-rhPSMA-7.3 PET was positive in 275 of 279 (98.6%), 106 of 279 (38.0%), 46 of 279 (16.5%), 65 of 279 (23.3%), and 5 of 279 (1.8%) patients for local, pelvic nodal, extrapelvic nodal, metastatic bone, and visceral metastatic disease, respectively. In the efficacy cohort, LN metastases were present in 24 of 83 patients (29%) and were located in 48 of 420 (11%) resected templates and in 33 of 166 (19.9%) hemipelvic templates in histopathology. The majority vote results showed that patient-level sensitivity, specificity, and accuracy for pelvic nodal metastases were 66.7% (95% CI, 44.7%-83.6%), 96.6% (95% CI, 87.3%-99.4%), and 88.0% (95% CI, 78.5%-93.8%), respectively, for 18F-rhPSMA-7.3 PET and 37.5% (95% CI, 19.6%-59.2%), 91.5% (95% CI, 80.6%-96.8%), and 75.9% (95% CI, 65.0%-84.3%), respectively, for morphologic imaging. 18F-rhPSMA-7.3 showed higher interobserver agreement than morphologic imaging (patient-level Fleiss κ = 0.54 [95% CI, 0.47-0.62] vs. 0.24 [95% CI, 0.17-0.31]). A mean SUV ratio of 6.6 (95% CI, 5.2-8.1) documented a high image contrast between local tumors and adjacent low urinary tracer retention. Conclusion: 18F-rhPSMA-7.3 PET offers diagnostic performance superior to morphologic imaging for primary N-staging of newly diagnosed PCa, shows lower interreader variation, and offers good distinction between primary-tumor activity and bladder background activity. With increasing National Comprehensive Cancer Network risk group, an increasing frequency of extraprostatic tumor lesions was observed.

18F-rhPSMA-7 PET for the Detection of Biochemical Recurrence of Prostate Cancer After Curative-Intent Radiation Therapy: A Bicentric Retrospective Study.

This bicentric, retrospective analysis investigated the efficacy of PET/CT with a novel theranostic prostate-specific membrane antigen (PSMA)--targeting ligand, 18F-rhPSMA-7, in patients with biochemical recurrence (BCR) of prostate cancer after curative-intent primary radiotherapy. Methods: Datasets from patients with BCR of prostate cancer after external-beam radiation therapy or brachytherapy who underwent 18F-rhPSMA-7 PET/CT at either Technical University Munich or Ludwig-Maximilians-University Munich were retrospectively reviewed by experienced nuclear medicine physicians and radiologists at both centers. The median injected activity was 299 MBq (range, 204-420 MBq), and the median uptake time was 77 min (range, 46-120 min). All lesions suggestive of recurrent prostate cancer were noted. Detection rates were correlated with patients' prostate-specific antigen (PSA) level, primary Gleason score, and prior use of androgen-deprivation therapy (ADT). Results: Ninety-seven patients were included (65 at Technical University Munich and 32 at Ludwig-Maximilians-University Munich). The median prescan PSA was 4.19 ng/mL (range, 0.1-159 ng/mL). The primary Gleason score was ≤6 in 19 patients, 7 in 25, ≥8 in 33, and unknown in 20. Thirty patients received ADT in the 6 mo preceding PET/CT. 18F-rhPSMA-7 identified lesions in 91 of 97 (94%) patients. Detection rates stratified by PSA were 88% (22/25), 97% (30/31), 90% (19/21), and 100% (20/20) for a PSA of <2, 2-<5, 5-<10, and ≥10 ng/mL, respectively. Detection rates in the subgroup of patients not meeting the Phoenix criteria for BCR were 80% (4/5), 90% (9/10), 100% (4/4), and 83% (5/6) for a PSA of <0.5, 0.5-<1, 1-<1.5, and 1.5-2 ng/mL, respectively. There were no significant differences in detection rates between patients with and without prior ADT (100% vs. 91%, P = 0.173) or patients with a Gleason score of ≤7 and a Gleason score of ≥8 (98% vs. 91%, P = 0.316).18F-rhPSMA-7 revealed local recurrence in 80% (78/97); pelvic lymph node metastases in 38% (37/97); retroperitoneal and supradiaphragmatic lymph node metastases in 9% (9/97) and 4% (4/97), respectively; bone metastases in 27% (26/97); and visceral metastases in 3% (3/97). In the subgroup of patients with a PSA of <2 ng/mL above nadir, local recurrence occurred in 76% (19/25) and pelvic lymph node metastases in 36% (9/25). Conclusion:18F-rhPSMA-7 PET/CT demonstrates high detection rates in prostate cancer patients with BCR after primary radiation therapy, even at low PSA values. Its diagnostic efficacy is comparable to published data for other PSMA ligands.

Comparative Preclinical Biodistribution, Dosimetry, and Endoradiotherapy in Metastatic Castration-Resistant Prostate Cancer Using 19F/177Lu-rhPSMA-7.3 and 177Lu-PSMA I&T.

Radiohybrid prostate-specific membrane antigen (rhPSMA) ligands are applicable as radiochemical twins for both diagnostic PET imaging and endoradiotherapy. On the basis of preliminary data as a diagnostic ligand, the isomer rhPSMA-7.3 is a promising candidate for potential endoradiotherapy. The aim of this preclinical evaluation was to assess the biodistribution, dosimetry, and therapeutic efficacy of 19F/177Lu-rhPSMA-7.3 in comparison to the established therapeutic agent 177Lu-PSMA I&T (imaging and therapy). Methods: The biodistribution of 19F/177Lu-rhPSMA-7.3 and 177Lu-PSMA I&T was determined in LNCaP tumor-bearing severe combined immunodeficiency (SCID) mice after sacrifice at defined time points up to 7 d (n = 5). Organs and tumors were dissected, percentage injected dose per gram (%ID/g) was determined, and dosimetry was calculated using OLINDA/EXM, version 1.0. The therapeutic efficacy of a single 30-MBq dose of 19F/177Lu-rhPSMA-7.3 (n = 7) was compared with that of 177Lu-PSMA I&T in treatment groups (n = 7) and control groups (n = 6-7) using C4-2 tumor-bearing SCID mice by evaluating tumor growth and survival over 6 wk after treatment. Results: The biodistribution of 19F/177Lu-rhPSMA-7.3 revealed fast blood clearance (0.63 %ID/g at 1 h after injection), and the highest activity uptake was in the spleen and kidneys, particularly in the first hour (33.25 %ID/g and 207.6 %ID/g, respectively, at 1 h after injection), indicating a renal excretion pathway. Compared with 177Lu-PSMA I&T, 19F/177Lu-rhPSMA-7.3 exhibited an initial (1 h) 2.6-fold higher tumor uptake in LNCaP xenografts and a longer retention (4.5 %ID/g vs. 0.9 %ID/g at 168 h). The tumor dose of 19F/177Lu-rhPSMA-7.3 was substantially higher (e.g., 7.47 vs. 1.96 µGy/MBq at 200 mm3) than that of 177Lu-PSMA I&T. In most organs, absorbed doses were higher for 177Lu-PSMA I&T. A significantly greater tumor size reduction was shown for a single dose of 19F/177Lu-rhPSMA-7.3 than for 177Lu-PSMA I&T at the end of the experiment (P = 0.0167). At the predefined termination of the experiment at 6 wk, 7 of 7 and 3 of 7 mice were still alive in the 19F/177Lu-rhPSMA-7.3 and 177Lu-PSMA I&T groups, respectively, compared with the respective control groups, with 0 of 7 and 0 of 6 mice. Conclusion: Compared with 177Lu-PSMA I&T, 19F/177Lu-rhPSMA-7.3 can be considered a suitable candidate for clinical translation because it has similar clearance kinetics and a similar radiation dose to healthy organs but superior tumor uptake and retention. Preliminary treatment experiments showed a favorable antitumor response.

Automated synthesis of [18F]Ga-rhPSMA-7/ -7.3: results, quality control and experience from more than 200 routine productions.

INTRODUCTION: The radiohybrid (rh) prostate-specific membrane antigen (PSMA)-targeted ligand [18F]Ga-rhPSMA-7 has previously been clinically assessed and demonstrated promising results for PET-imaging of prostate cancer. The ligand is present as a mixture of four stereoisomers ([18F]Ga-rhPSMA-7.1, - 7.2, - 7.3 and - 7.4) and after a preclinical isomer selection process, [18F]Ga-rhPSMA-7.3 has entered formal clinical trials. Here we report on the establishment of a fully automated production process for large-scale production of [18F]Ga-rhPSMA-7/ -7.3 under GMP conditions (EudraLex). METHODS: [18F]Fluoride in highly enriched [18O]H2O was retained on a strong anion exchange cartridge, rinsed with anhydrous acetonitrile and subsequently eluted with a solution of [K+ ⊂ 2.2.2]OH- in anhydrous acetonitrile into a reactor containing Ga-rhPSMA ligand and oxalic acid in DMSO. 18F-for-19F isotopic exchange at the Silicon-Fluoride Acceptor (SiFA) was performed at room temperature, followed by dilution with buffer and cartridge-based purification. Optimum process parameters were determined on the laboratory scale and thereafter implemented into an automated synthesis. Data for radiochemical yield (RCY), purity and quality control were analyzed for 243 clinical productions (160 for [18F]Ga-rhPSMA-7; 83 for [18F]Ga-rhPSMA-7.3). RESULTS: The automated production of [18F]Ga-rhPSMA-7 and the single isomer [18F]Ga-rhPSMA-7.3 is completed in approx. 16 min with an average RCY of 49.2 ± 8.6% and an excellent reliability of 98.8%. Based on the different starting activities (range: 31-130 GBq, 89 ± 14 GBq) an average molar activity of 291 ± 62 GBq/µmol (range: 50-450 GBq/µmol) was reached for labeling of 150 nmol (231 µg) precursor. Radiochemical purity, as measured by radio-high performance liquid chromatography and radio-thin layer chromatography, was 99.9 ± 0.2% and 97.8 ± 1.0%, respectively. CONCLUSION: This investigation demonstrates that 18F-for-19F isotopic exchange is well suited for the fast, efficient and reliable automated routine production of 18F-labeled PSMA-targeted ligands. Due to its simplicity, speed and robustness the development of further SiFA-based radiopharmaceuticals is highly promising and can be of far-reaching importance for future theranostic concepts.

Matched-Pair Comparison of 68Ga-PSMA-11 and 18F-rhPSMA-7 PET/CT in Patients with Primary and Biochemical Recurrence of Prostate Cancer: Frequency of Non-Tumor-Related Uptake and Tumor Positivity.

Radiohybrid prostate-specific membrane antigen (rhPSMA) ligands are a new class of prostate cancer theranostic agents. 18F-rhPSMA-7 offers the advantages of 18F labeling and low urinary excretion compared with 68Ga-PSMA-11. Here, we compare the frequency of non-tumor-related uptake and tumor positivity with 68Ga-PSMA-11 and 18F-rhPSMA-7 in patients with primary or recurrent prostate cancer. Methods: This retrospective matched-pair comparison matched 160 18F-rhPSMA-7 with 160 68Ga-PSMA-11 PET/CT studies for primary staging (n = 33) and biochemical recurrence (n = 127) according to clinical characteristics. Two nuclear medicine physicians reviewed all scans, first identifying all PET-positive lesions and then differentiating lesions suggestive of prostate cancer from those that were benign, on the basis of known pitfalls and ancillary information from CT. For each region, the SUVmax of the lesion with the highest PSMA ligand uptake was noted. Tumor positivity rates were determined, and SUVmax was compared separately for each tracer. Results:18F-rhPSMA-7 and 68Ga-PSMA-11 PET revealed 566 and 289 PSMA ligand-positive lesions, respectively. Of these, 379 and 100 lesions, equaling 67.0% and 34.6%, respectively, of all PSMA-positive lesions, were considered benign. The distribution of their etiology was similar (42%, 24%, and 25% with 18F-rhPSMA-7 vs. 32%, 24%, and 38% with 68Ga-PSMA-11 for ganglia, bone, and unspecific lymph nodes, respectively). All primary tumors were positive with both agents (n = 33 each), whereas slightly more metastatic lesions were observed with 68Ga-PSMA-11 in both disease stages (113 for 18F-rhPSMA-7 and 124 for 68Ga-PSMA-11). The SUVmax of 18F-rhPSMA-7 and 68Ga-PSMA-11 did not differ (P > 0.05) in local recurrence or primary prostate cancer; however, the tumor-to-bladder ratio was significantly higher with 18F-rhPSMA-7 (4.9 ± 5.3 vs. 2.2 ± 3.7, P = 0.02, for local recurrence; 9.8 ± 9.7 vs. 2.3 ± 2.6, P < 0.001, for primary prostate cancer). Conclusion: The tumor positivity rate was consistently high for 68Ga-PSMA-11 and 18F-rhPSMA-7. Both tracers revealed a considerable number of areas of uptake that were reliably identified as benign by trained physicians making use of corresponding morphologic imaging and known PSMA pitfalls. These were more frequent with 18F-rhPSMA-7. However, the matched-pair comparison could have introduced a source of bias. Adequate reader training can allow physicians to differentiate benign uptake from disease and be able to benefit from the logistical and clinical advantages of 18F-rhPSMA-7.

Positive Predictive Value and Correct Detection Rate of 18F-rhPSMA-7 PET in Biochemically Recurrent Prostate Cancer Validated by Composite Reference Standard.

The objective of this retrospective study was to assess the detection rate (DR), positive predictive value (PPV), and correct detection rate (CDR) of 18F-rhPSMA-7 PET/CT in biochemical recurrence (BCR) of prostate cancer (PCa) after radical prostatectomy (RP) using composite validation. Methods:18F-rhPSMA-7 PET/CT scans of patients with BCR between July 2017 and June 2018 were retrospectively reviewed. All suspicious lesions were recorded. The reference standard was histopathology or combinations of histopathology, imaging, or prostate-specific antigen (PSA) follow up, defined as composite reference standard. DR was calculated as the proportion of PSMA PET-positive patients to all patients independent of the reference standard, whereas the CDR was the percentage of patients who had at least 1 true-positive PSMA PET lesion localized that corresponded with the reference standard. The PPV was defined as the proportion of patients who had true-positive to all positive findings. The correlation between DR and patient characteristics was evaluated. Results: A total of 532 patients with a median PSA level of 0.97 ng/mL (interquartile range: 0.41-2.46 ng/mL) were included. Of these, 162 patients had composite follow-up at a median duration of 5.6 mo (range: 1.1-14.2 mo). The proportion of patients who had no lesion visualized on PET/CT, localized disease, and any distant metastases (M1) were 20%, 43%, and 37%, respectively. PET DR among all patients was 80%. On a per-patient basis, the PPV of 18F-rhPSMA-7 PET/CT in the composite cohort was 88%, and the CDR was 70%. The PPV in the histopathology-proven cohort was 91%, and the CDR in this subgroup was 73%. In patients with PSA levels ≥ 1 ng/mL the DR and PPV were 90% and 91%, respectively, resulting in a CDR of 82%. In patients with PSA levels < 1 ng/mL, the DR and PPV were 69% and 85%, respectively, resulting in a CDR of 59%. There was a significant positive correlation between 18F-rhPSMA-7 PET/CT detection efficacy and stratified PSA levels (P = 0.005), as well as PSA nadir after prostatectomy (P < 0.001). Conclusion:18F-rhPSMA-7 PET/CT offers high PPV in BCR after RP. Its CDR is dependent on the prescan PSA value with excellent CDR in patients with PSA ≥ 1 ng/mL.

Radiohybrid Ligands: A Novel Tracer Concept Exemplified by 18F- or 68Ga-Labeled rhPSMA Inhibitors.

When we critically assess the reason for the current dominance of 68Ga-labeled peptides and peptide-like ligands in radiopharmacy and nuclear medicine, we have to conclude that the major advantage of such radiopharmaceuticals is the apparent lack of suitable 18F-labeling technologies with proven clinical relevance. To prepare and to subsequently perform a clinical proof-of-concept study on the general suitability of silicon-fluoride-acceptor (SiFA)-conjugated radiopharmaceuticals, we developed inhibitors of the prostate-specific membrane antigen (PSMA) that are labeled by isotopic exchange (IE). To compensate for the pronounced lipophilicity of the SiFA unit, we used metal chelates, conjugated in close proximity to SiFA. Six different radiohybrid PSMA ligands (rhPSMA ligands) were evaluated and compared with the commonly used 18F-PSMA inhibitors 18F-DCFPyL and 18F-PSMA-1007. Methods: All inhibitors were synthesized by solid-phase peptide synthesis. Human serum albumin binding was measured by affinity high-performance liquid chromatography, whereas the lipophilicity of each tracer was determined by the n-octanol/buffer method. In vitro studies (IC50, internalization) were performed on LNCaP cells. Biodistribution studies were conducted on LNCaP tumor-bearing male CB-17 SCID mice. Results: On the laboratory scale (starting activities, 0.2-9.0 GBq), labeling of 18F-rhPSMA-5 to -10 by IE was completed in < 20 min (radiochemical yields, 58% ± 9%; radiochemical purity, >97%) with molar activities of 12-60 GBq/µmol. All rhPSMAs showed low nanomolar affinity and high internalization by PSMA-expressing cells when compared with the reference radiopharmaceuticals, medium-to-low lipophilicity, and high human serum albumin binding. Biodistribution studies in LNCaP tumor-bearing mice revealed high tumor uptake, sufficiently fast clearance kinetics from blood, low hepatobiliary excretion, fast renal excretion, and very low uptake of 18F activity in bone. Conclusion: The novel 18F-rhPSMA radiopharmaceuticals developed under the radiohybrid concept show equal or better targeting characteristics than the established 18F-PSMA tracers 18F-DCFPyL and 18F-PSMA-1007. The unparalleled simplicity of production, the possibility to produce the identical 68Ga-labeled 19F-68Ga-rhPSMA tracers, and the possibility to extend this concept to true theranostic radiohybrid radiopharmaceuticals, such as F-Lu-rhPSMA, are unique features of these radiopharmaceuticals.

Preclinical comparison of four [18F, natGa]rhPSMA-7 isomers: influence of the stereoconfiguration on pharmacokinetics.

INTRODUCTION: Radiohybrid (rh) ligands, a novel class of prostate-specific membrane antigen (PSMA)-targeted radiopharmaceuticals, can be labeled either with [18F]fluorine via isotopic exchange or with radiometals (such as [68Ga]Gallium, [177Lu]Lutetium, [225Ac]Actinium). Among these, [18F, natGa]rhPSMA-7 has recently entered clinical assessment. AIM: Since [18F, natGa]rhPSMA-7 is composed of four stereoisomers ([18F, natGa]rhPSMA-7.1, -7.2, -7.3 and -7.4), we initiated a preclinical selection process to identify the isomer with the most favorable pharmacokinetics for further clinical investigation. METHODS: A synthetic protocol for enantiopure [19F, natGa]rhPSMA-7 isomers has been developed. The comparative evaluation of the four isomers comprised human serum albumin binding, lipophilicity, IC50, internalization and classical biodistribution studies and competition experiments in LNCaP tumor-bearing CB-17 SCID mice. In addition, a radio high-performance liquid chromatography-based method was developed allowing quantitative, intraindividual comparison of [18F, natGa]rhPSMA-7.1 to -7.4 in LNCaP tumor-bearing mice. RESULTS: Cell studies revealed high PSMA affinity and internalization for [18/19F, natGa]rhPSMA-7.2, -7.3 and -7.4, whereas [18/19F, natGa]rhPSMA-7.1 showed approximately twofold lower values. Although the biodistribution profile obtained was typical of PSMA inhibitors, it did not allow for selection of a lead candidate for clinical studies. Thus, an intraindividual comparison of all four isomers in LNCaP tumor-bearing mice was carried out by injection of a diastereomeric mixture, followed by analysis of the differential uptake and excretion pattern of each isomer. Based on its high tumor accumulation and low uptake in blood, liver and kidneys, [18F, natGa]rhPSMA-7.3 was identified as the preferred isomer and transferred into clinical studies. CONCLUSION: [18F, natGa]rhPSMA-7.3 has been selected as a lead compound for clinical development of a [18F]rhPSMA-based candidate. The intraindividual differential uptake and excretion analysis in vivo allowed for an accurate comparison and assessment of radiopharmaceuticals.

Histologically Confirmed Diagnostic Efficacy of 18F-rhPSMA-7 PET for N-Staging of Patients with Primary High-Risk Prostate Cancer.

18F-rhPSMA-7 (radiohybrid prostate-specific membrane antigen [PSMA]) is a novel ligand for PET imaging. Here, we present data from a retrospective analysis using PET/CT and PET/MRI examinations to investigate the efficacy of 18F-rhPSMA-7 PET for primary N-staging of patients with prostate cancer (PC) compared with morphologic imaging (CT or MRI) and validated by histopathology. Methods: Data from 58 patients with high-risk PC (according to the D'Amico criteria) who were staged with 18F-rhPSMA-7 PET/CT or PET/MRI at our institution between July 2017 and June 2018 were reviewed. The patients had a median prescan prostate-specific antigen value of 12.2 ng/mL (range, 1.2-81.6 ng/mL). The median injected activity of 18F-rhPSMA-7 was 327 MBq (range, 132-410 MBq), with a median uptake time of 79.5 min (range, 60-153 min). All patients underwent subsequent radical prostatectomy and extended pelvic lymph node dissection. The presence of lymph node metastases was determined by an experienced reader independently for both the PET and the morphologic datasets using a template-based analysis on a 5-point scale. Patient-level and template-based results were both compared with histopathologic findings. Results: Lymph node metastases were present in 18 patients (31.0%) and were located in 52 of 375 templates (13.9%). Receiver-operating-characteristic analyses showed 18F-rhPSMA-7 PET to perform significantly better than morphologic imaging on both patient-based and template-based analyses (areas under curve, 0.858 vs. 0.649 [P = 0.012] and 0.765 vs. 0.589 [P < 0.001], respectively). On patient-based analyses, the sensitivity, specificity, and accuracy of 18F-rhPSMA-7 PET were 72.2%, 92.5%, and 86.2%, respectively, and those of morphologic imaging were 50.0%, 72.5%, and 65.5%, respectively. On template-based analyses, the sensitivity, specificity, and accuracy of 18F-rhPSMA-7 PET were 53.8%, 96.9%, and 90.9%, respectively, and those of morphologic imaging were 9.6%, 95.0%, and 83.2%, respectively. Conclusion:18F-rhPSMA-7 PET is superior to morphologic imaging for N-staging of high-risk primary PC. The efficacy of 18F-rhPSMA-7 is similar to published data for 68Ga-PSMA-11.

18F-rhPSMA-7 PET for the Detection of Biochemical Recurrence of Prostate Cancer After Radical Prostatectomy.

18F-labeled prostate-specific membrane antigen (PSMA) PET tracers are increasingly used in preference to 68Ga-PSMA-11 for restaging biochemical recurrence (BCR) of prostate cancer. They are associated with longer half-lives, larger-scale production, and lower positron range than their 68Ga-labeled counterparts. Here, we describe the efficacy of an 18F-labeled radiohybrid PSMA, rhPSMA-7, a novel theranostic PSMA-targeting agent for imaging BCR of prostate cancer. Methods: Datasets from 261 consecutive patients with noncastrate BCR after radical prostatectomy who underwent 18F-rhPSMA-7 PET/CT at our institution between June 2017 and March 2018 were reviewed retrospectively. All lesions suspected of being recurrent prostate cancer were recorded. The detection rate for sites of presumed recurrence was correlated with patients' prostate-specific antigen (PSA) level, primary Gleason score, and prior therapy (androgen deprivation therapy and external-beam radiation therapy). Results: The 261 patients had a median PSA level of 0.96 ng/mL (range, 0.01-400 ng/mL). The median injected activity of 18F-rhPSMA-7 was 336 MBq, with a median uptake time of 76 min. In total, 211 patients (81%) showed pathologic findings on 18F-rhPSMA-7 PET/CT. The detection rates were 71% (42/59), 86% (44/51), 86% (42/49), and 95% (76/80) at PSA levels of 0.2 to <0.5 ng/mL, 0.5 to <1 ng/mL, 1 to <2 ng/mL, and ≥2 ng/mL, respectively. In 32% patients (7/22) with a PSA of less than 0.2 ng/mL, suggestive lesions were present. 18F-rhPSMA-7 PET/CT revealed local recurrence in 43% of patients (113). Lymph node metastases were present in the pelvis in 42% of patients (110), in the retroperitoneum in 17% (45), and in a supradiaphragmatic location in 8.0% (21). Bone and visceral metastases were detected in 21% (54) and 3.8% (10), respectively. Detection efficacy was not influenced by prior external-beam radiation therapy (79.1% vs. 82.1%, P = 0.55), androgen deprivation therapy within the 6 mo preceding imaging (80.6% vs. 80.9%, P = 0.54), or primary Gleason score (77.9% for ≤7 vs. 82.6% for ≥8, P = 0.38). Conclusion:18F-rhPSMA-7 PET/CT offers high detection rates in early BCR after radical prostatectomy, especially among patients with low PSA values.

Quantitative and Qualitative Analyses of Biodistribution and PET Image Quality of a Novel Radiohybrid PSMA, 18F-rhPSMA-7, in Patients with Prostate Cancer.

Radiohybrid PSMA (rhPSMA) ligands, a new class of theranostic prostate-specific membrane antigen (PSMA)-targeting agents, feature fast 18F synthesis and utility for labeling with radiometals. Here, we assessed the biodistribution and image quality of 18F-rhPSMA-7 to determine the best imaging time point for patients with prostate cancer. Methods: In total, 202 prostate cancer patients who underwent a clinically indicated 18F-rhPSMA-7 PET/CT were retrospectively analyzed, and 12 groups based on the administered activity and uptake time of PET scanning were created: 3 administered activities (low, 222-296 MBq; moderate, 297-370 MBq; and high, 371-444 MBq) and 4 uptake time points (short, 50-70 min; intermediate, 71-90 min; long, 91-110 min; and extra long, ≥111 min). For quantitative analyses, SUVmean and organ- or tumor-to-background ratio were determined for background, healthy organs, and 3 representative tumor lesions. Qualitative analyses assessed overall image quality, nonspecific blood-pool activity, and background uptake in bone or marrow using 3- or 4-point scales. Results: In quantitative analyses, SUVmean showed a significant decrease in the blood pool and lungs and an increase in the kidneys, bladder, and bones as the uptake time increased. SUVmean showed a trend to increase in the blood pool and bones as the administered activity increased. However, no significant differences were found in 377 tumor lesions with respect to the administered activity or uptake time. In qualitative analyses, the overall image quality was stable along with the uptake time, but the proportion rated to have good image quality decreased as the administered activity increased. All other qualitative image parameters showed no significant differences for the administered activities, but they showed significant trends with increasing uptake time: less nonspecific blood activity, more frequent background uptake in the bone marrow, and increased negative impact on clinical decision making. Conclusion: The biodistribution of 18F-rhPSMA-7 was similar to that of established PSMA ligands, and tumor uptake of 18F-rhPSMA-7 was stable across the administered activities and uptake times. An early imaging time point (50-70 min) is recommended for 18F-rhPSMA-7 PET/CT to achieve the highest overall image quality.