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. Supplementary Information The online version contains supplementary material available at 10.1186/s13550-021-00872-w.


Background
Prostate-specific membrane antigen (PSMA) is overexpressed in nearly all primary prostate cancer [1], and PSMA expression further increases in de-differentiated, metastatic, or hormone-refractory disease [2,3]. In recent years, PSMA targeting ligands have been investigated for molecular imaging and radioligand therapy of prostate cancer. Recent developments increasingly focus on 18 F-based, rather than 68 Ga-based PSMA ligands for imaging given the advantages of 18 F-based agents in terms of availability, ease of production, and image resolution.
Several 18 F-based PSMA ligands have been developed with different kinetic profiles. The kidneys and the urinary tracts are critical organs in the development of novel PSMA ligands as intense urinary retention may interfere with accurate evaluation of the prostate and adjacent area. 18 F-labelled, urea-based inhibitors of PSMA such as 18 F-DCFBC and 18 F-DCFPyL demonstrated diagnostic capabilities in clinical trials [4,5], but they still have shortcomings due to urinary excretion as their limitation to a diagnostic use. 18 F-PSMA-1007 was developed aiming for similar structure, biodistribution and tumour uptake compared to PSMA-617 which is currently used for radioligand therapy. 18 F-PSMA-1007 is predominantly excreted via the hepatobiliary tract and has very low urinary retention but its optimal imaging timepoint (120 min) is suboptimal for routine clinical practice [6].
Radiohybrid PSMA (rhPSMA) ligands are a new class of fully theranostic agents that allow fast 18 F synthesis and labelling with radiometals. They are silicon-fluoride-acceptor (SiFA)-conjugated radiopharmaceuticals that are labeled by isotopic exchange. One example, 18 F-rhPSMA-7, has been shown to have a biodistribution typical of other established PSMA ligands [7] and its clinical performance for imaging patients with prostate cancer has been reported previously [8,9]. 18 F-rhPSMA-7 is comprised of four diastereoisomers (rhPSMA-7.1, rhPSMA-7.2, rhPSMA-7.3 and rhPSMA-7.4), of which rhPSMA-7.3 exhibits the most promising targeting characteristics for clinical translation and has been selected for further development based in preclinical results [10].
Here, we analyzed the preclinical biodistribution and dosimetry of 18 F-rhPSMA-7 and 18 F-rhPSMA-7.3 at different timepoints after a single intravenous administration in mice. We further assessed biodistribution and image quality of clinical 18 F-rhPSMA-7 and 18 F-rhPSMA-7.3 PET scans in patients with histopathologically proven prostate cancer.

Preclinical biodistribution and dosimetry
Severe combined immunodeficiency (SCID) mice were supplied by Charles River Laboratories. (Freiburg, Germany). The preclinical evaluation study was performed in accordance with the German Animal Welfare Act (Deutsches Tierschutzgesetz, approval #55.2-1-54-2532-216-2015). Male mice (≥ 6 weeks old) were used for the study after reaching sexual maturation.
Of note, to achieve a similar number of timepoints for both calculations, the 18 F-rhPSMA-7 10 min and 20 min timepoints were interpolated to a 15 min timepoint. The time-integral of activity for the accumulation in the investigated source organs (AUCs) were generated both with numerical integration and physical decay [12].
To extrapolate from preclinical data to human dosimetry, linear scaling of %ID from the mice by the ratio of the organ weights and total body weights of phantoms compared to humans was necessary [13]. Normal-organ radiation doses were estimated for the 70 kg standard adult anatomic model using time-dependent organ activity concentrations in %ID/g and total-body activities measured in the biodistribution studies in mice. Tissue activity concentrations in mice were converted to tissue fractional activities in the 70 kg standard adult using the relative fractional organ masses in the standard adult and the standard 25 g mouse. Time-dependent total-body activity was fit to an exponential function and the difference between the injected activity and the total-body activity was assumed to be excreted via the urine because activity concentrations in the liver and gastrointestinal tract were low at all timepoints studied.
Organ residence time was calculated by numerical integration using the trapezoidal rule and the rest-of-body. 18 F residence times were calculated as the difference between the total-body residence time and the sum of the organ and urine residence times. The bladder contents residence time was estimated using the dynamic voiding model in the OLINDA/EXM 1.0 dosimetry software (Vanderbilt University, Nashville, TN, USA). Finally, the standard adult mean absorbed dose to organs (in µGy/ MBq) and total effective dose (in µSv/MBq) were calculated using OLINDA/EXM 1.0 [14].

Human biodistribution Patients
Data from patients with histopathologically proven prostate cancer who underwent a clinically indicated PET/ CT with 18 F-rhPSMA-7 or 18 F-rhPSMA-7.3 between October 2017 and November 2018 were retrospectively analyzed. 18 Table S5.
All patients gave written informed consent for the original procedure. All reported investigations were conducted in accordance with the Helsinki Declaration and with national regulations. This retrospective analysis was approved by the Local Ethics Committee (permits 290/18S and 99/19S) and the need for patient consent was waived. The administration of 18 F-rhPSMA-7 and 18 F-rhPSMA-7.3 was in accordance with The German Medicinal Products Act (AMG §13 2b) and the responsible regulatory body (Government of Oberbayern).

PET/CT imaging
All the PET/CT scans were obtained from the skull base to mid-thigh using a Biograph mCT Flow scanner (Siemens Medical Solutions, Erlangen, Germany). PET scanning was operated in 3D mode with an acquisition time of 1.1 mm/s in continuous table movement, and a diagnostic CT scan (240 mAs, 120 kV, 5 mm slice thickness) was acquired in the portal venous phase 80 s after the intravenous injection of an iodinated contrast agent (Imeron 400, Bracco Imaging Deutschland GmbH, Konstanz, Germany). Reconstruction of the PET images was performed based on iterative algorithms with an ordered-subsets expectation maximization (4 iterations, 8 subsets) followed by a post-reconstruction smoothing Gaussian filter (5 mm full width at one-half maximum).

Image analysis
For both quantitative and qualitative analyses PET datasets (non-Time-of-Flight/non-True X) were used. The maximum standardized uptake value (SUV max ) and the mean standardized uptake value (SUV mean ) with an isocontour of 50% of the SUV max were determined applying circular volumes of interest (VOIs) using OsiriX MD ® 11.0.2 (Pixmeo SARL, Geneva, Switzerland).
The circular VOIs were placed over normal organs; parotid gland, submandibular gland, mediastinal aortic arch (blood pool), lungs, liver, spleen, pancreas, duodenum, kidneys, bladder, sacral promontory, and background (gluteus maximus muscle). Up to 3 lesions per patient were analyzed in decreasing order of SUV max . Organ/tumour to background ratios (ratio-SUV mean , ratio-SUV max ) were calculated.
To evaluate overall image quality, non-specific blood pool activity and background uptake in bone/marrow was analysed using 3-or 4-point scales as previously described [7]. All the analyses were performed by a board-certified nuclear medicine physician.

Statistical analysis
Prior to analysis, the Kolmogorov-Smirnov test was used to assess the normality of the data distribution. The independent Student t-test was performed to compare means between groups for the normal parameters. The Mann-Whitney U test was conducted for the non-normal parameters. The Chi-square test or the Fisher's exact test was adopted to compare differences among groups for the analyses of ordinal variables. Data were expressed as mean ± standard deviation (SD) for continuous variables and frequencies (percentages, %) for categorical variables, respectively. All statistical analyses were performed using the IBM SPSS Statistics version 25 (IBM Inc. Armonk, NY, USA) and R version 3.5.2 (http:// www.rproje ct. org). P values less than 0.05 were considered statistically significant.

Preclinical biodistribution and dosimetry
Significant accumulation of radioactivity was observed (i.e. source organs) in the kidney, spleen, lung, liver and heart for both 18 F-rhPSMA-7 and 18 F-rhPSMA-7.3. The activity distributed to individual organs is presented for the source organs and whole organs in Additional file 1: Table S1 and S2, respectively. The AUCs in the source organs are summarized in Table 1, and only results calculated from the AUCs with extrapolation and physical decay were used in the following analyses. Radioactivity distribution of both 18 F-rhPSMA-7 and 18 F-rhPSMA-7.3 was found to be the highest in the kidneys at most timepoints. With respect to activity accumulation and clearance, a rapid clearance from blood and clearance to urine but relatively slow build-up in the kidneys was present for both 18 F-rhPSMA-7 and 18 F-rhPSMA-7.3.
Extrapolating the preclinical dosimetry data to humans revealed that at 1 h and 3.5 h bladder voiding intervals, the total effective doses for humans are 12.2 and 26.6 µSv/MBq for 18 F-rhPSMA-7, and 12.8 and 21.7 µSv/MBq for 18 F-rhPSMA-7.3, respectively ( Table 2). The absorbed doses for the individual organs at 1 h and 3.5 h bladder voiding intervals are provided in Additional file 1: Table S3. On the individual organ level, the kidneys were among the organs receiving the highest absorbed doses for both 18 F-rhPSMA-7 and 18 F-rhPSMA-7.3. For both bladder voiding intervals, the absorbed dose in the kidneys was slightly higher with 18 F-rhPSMA-7.3 (71.60 and 71.80 µGy/MBq at 1 h and 3.5 h, respectively) than with 18 F-rhPSMA-7 (64.70 and 65.10 µGy/MBq at 1 h and 3.5 h, respectively; Additional file 1: Table S3).

Discussion
Here, we conducted a series of preclinical and human biodistribution and dosimetry studies to compare the profiles of two lead rhPSMA ligands for prostate cancer imaging. The results of the preclinical elements of this study indicate that the biodistribution and dosimetry profiles for both 18 F-rhPSMA-7 and its single diastereoisomer form, 18 F-rhPSMA-7.3, are favourable for prostate cancer imaging. The human clinical biodistribution results confirm and extend the preclinical data to indicate that while the profiles of 18 F-rhPSMA-7 and 18 F-rhPSMA-7.3 are similar, the significantly lower bladder uptake, and the trend towards higher uptake in tumours with 18 F-rhPSMA-7.3 suggest that it is the more promising agent for further clinical translation.
The AUCs in the mouse dosimetry studies showed that 18 F-rhPSMA-7.3 exhibited appropriate profiles for human study. Compared with 18 F-rhPSMA-7, kidney uptakes of 18 F-rhPSMA-7.3 were higher, but its accumulation speed was relatively slow. Thus, dosimetry studies of 18 F-rhPSMA-7.3 were planned to be performed at a  Table 3 Comparisons of the individual organ uptake and tumour uptake in human biodistribution Bold indicate that this are significant p-values (below 0.05) SUV mean : mean standardized uptake value; ratio-SUV mean : organ/tumour-to-background ratios with background being gluteus maximus muscle *Results of group comparisons from independent t-test or Mann-Whitney U test † Normal distribution  late timepoint (300 min) to acknowledge this slow buildup in the kidneys. This kinetic profile could be helpful for PSMA ligand imaging. Since urinary excretion of the radiotracer is slow, excretion may not contribute to the kidney uptakes at early imaging timepoints, which in turn leads to lower bladder radioactivity levels and lower levels of background. While direct conversion of preclinical dosimetry studies using mice into human data will not truly model the human profile, it is reasonable to assume that kinetic profiles are similar in humans. In our earlier work with 18 F-rhPSMA-7, we recommended an early imaging timepoint (50-70 min) for 18 F-rhPSMA-7 as lower tracer retention in the urinary bladder is an important feature for PSMA ligand PET imaging [7] and it is expected that PET/CT with 18 F-rhPSMA-7.3 could also achieve good image quality at early imaging timepoints, especially given the significantly lower bladder SUV that we observe here.
The extrapolated effective doses for humans calculated from the preclinical dosimetry show estimated effective doses of both rhPSMA ligands that are comparable with those of other 18 F-based PSMA ligands. The estimated effective doses using 1 h voiding interval were 12.2 µSv/MBq for 18 F-rhPSMA-7 and 12.8 µSv/MBq for 18 F-rhPSMA-7.3, respectively, leading to an effective dose of less than 5 mSv after injection of 370 MBq of either rhPSMA ligand. Radiation dosimetry studies revealed a mean effective dose of 19.9 µSv/MBq for 18 F-DCFBC [4], 13.9 µSv/MBq for 18 F-DCFPyL [5], and 22.0 µSv/MBq for 18 F-PSMA-1007 [6]. 18 F-rhPSMA-7 and 18 F-PSMA-7.3 also provide favourable effective doses in comparison with 18 F-FDG, which has a reported effective dose of 19.0 µSv/MBq [15]. In the present study the kidneys received among the highest doses with both rhPSMA ligands. This is in line with data previously reported for 18 F-rhPSMA-7.3 in healthy volunteers [16]. Tolvanen et al. reported the kidneys to be one of the organs with the highest mean absorbed doses (172 µGy/MBq at a 3.5 h voiding window) [16]. All PSMA ligands share similar physiological distribution; organ absorbed doses are known to differ minimally among various PSMA ligands [6] and the data for 18 F-rhPSMA-7.3 appear comparable with other agents.
Our clinical data demonstrate that, in humans, the normal biodistribution of 18 F-rhPSMA-7 and 18 F-rhPSMA-7.3 are similar, but with some slight, but notable differences that are relevant to prostate cancer imaging. Defining the extent of urinary excretion of novel PSMA ligands is imperative given that PSMA ligands that are mainly excreted via the urinary tract often show high radiotracer uptake in the bladder that can interfere with image interpretation in the prostate region [17,18]. Our data show low relative bladder uptake with both rhPSMA ligands, but, of the two ligands, bladder uptake is significantly lower with 18 F-rhPSMA-7.3 than 18 F-rhPSMA-7. Moreover, while the tumour uptake of both rhPSMA ligands is higher than the kidney uptakes, facilitating easy localization of tumours with both rhPSMA ligands via PET/CT, there was a trend towards higher tumour SUV with 18 F-rhPSMA-7.3. The qualitative PET/CT image interpretation analysis confirm the suitability of both agents for prostate imaging.
Some limitations to the present study should be acknowledged. First, direct conversion of preclinical dosimetry studies to humans may not truly reflect human dosimetry, and thus, interpretation of the estimated effective doses reported here should be performed with caution. Second, our observations of low bladder retention of both ligands may be confounded by the application of furosemide at the time of tracer injection which is routinely done at our institution. It is known from 68 Ga-PSMA-11 that this can substantially lower bladder retention due to forced bladder emptying prior to the PET scan. Third, we acknowledge a trend towards higher tumour SUV using 18 F-rhPSMA-7.3 compared with 18 F-rhPSMA-7. However, an interrather than an intra-patient comparison can be affected by several clinical variables (e.g. tumour stage, differences in PSMA-expression, prior treatments). Fourth, definitive conclusions about the uptake characteristics in tumors comparing both cohorts are limited as they base on different patient cohorts. Despite the patient cohorts are weighted for disease stage differences in the distribution of primary Gleason Scores and PSA at imaging have to be acknowledged.

Conclusion
Radiation dosimetry is favourable both for 18 F-rhPSMA-7 and 18 F-rhPSMA-7.3 with an estimated effective dose of less than 5 mSv, assuming a 1 h voiding interval after injection of 370 MBq of the radiotracer. Human biodistribution of 18 F-rhPSMA-7 and 18 F-rhPSMA-7.3 were similar to each other, but significantly lower bladder uptake and a trend towards higher uptake in tumour lesions was seen with 18 F-rhPSMA-7.3.