Summary                         

Radioligand therapy with [¹⁷⁷Lu]Lu‑PSMA‑617 can be used to prolong life and reduce tumor burden
in terminally ill castration resistant prostate cancer patients. Still, - as PSMA is also expressed in non-prostate tissue, e.g. the proximal tubule of the kidney, the small intestines, and the salivary glands - accumulation in healthy tissue limits the activity that can be administered. Therefore, fractionated therapy is used to lower toxicity. However, there might be a need to reduce toxicity even further with e.g. radioprotectors. The aim of this study was to (i). establish a preclinical mouse model with fractionated high activity therapy of three consecutive doses of 200 MBq [¹⁷⁷Lu]Lu‑PSMA‑617 in which the aim was to (ii). achieve measurable hematotoxicity and nephrotoxicity and to (iii). analyze the potential protective effect of co‑injecting recombinant α1‑microglobulin (rA1M), a human antioxidant previously shown to have radioprotective effects. In both groups, three cycles resulted in increased albuminuria for each cycle, with large individual variation. Another marker of kidney injury, serum blood urea nitrogen (BUN), was only significantly increased compared to control animals after the third cycle. The number of white and red blood cells decreased significantly and did not reach the levels of control animals during the experiment. rA1M did reduce absorbed dose to kidney but did not show significant protection here, but future studies are warranted due to the recent clinical studies showing a significant renoprotective effect in patients.

Results from nanoScan® SPECT/CT

Every 5 weeks, Male BALB/cAnNRj nude mice in the [¹⁷⁷Lu]Lu-PSMA-617 groups were injected i.v. first with either rA1M or PBS and then directly with approximately 200 MBq, [¹⁷⁷Lu]Lu-PSMA-617 with repeated administration of rA1M or corresponding volume of PBS again 24 h later. Animals were given a total of three cycles (approximately 600 MBq). An additional group of 6 animals were given 50 MBq, 0.8 nmol, 25 µL [¹⁷⁷Lu]Lu-PSMA-617 each i.v. Three of these were sacrificed at 2 weeks p.i. and three at 3 weeks p.i. Kidneys were excised, weighed and the activity measured in a well counter.

For every cycle, 4 animals from the [¹⁷⁷Lu]Lu-PSMA-617 + rA1M group and 4 animals from the [¹⁷⁷Lu] Lu-PSMA-617 + PBS group were selected for imaging for dosimetry.

These animals were anesthetized with 2–3% Isoflurane in a O₂ and N₂O
mix, placed in the animal bed of the SPECT/CT (NanoSPECT/CT Plus, Mediso; Budapest, Hungary), and immediately a CT scan was performed. The field-of-view for SPECT imaging was set to the location of the kidneys on the CT image. Injections were performed, and SPECT imaging started approximately half a minute post ¹⁷⁷Lu injection on a protocol of dynamic imaging with 90–100 frames of 1 min length each. A 1h static SPECT image of the whole animal was also acquired at 7 h p.i. and at 48 h p.i. For the first cycle, an additional static SPECT image was taken at 96 h p.i. but it was calculated that this could be substituted by correcting the final mean absorbed dose per injected activity (Gy/MBq) result by a factor of 0.9019 when only using 7 and 48 h p.i. static scans, introducing only a mean error of 1.2%. Also, during the first cycle, a standard of known ¹⁷⁷Lu activity was included in the field-of-view to ensure that the high injected activity did not affect the detector efficiency. All SPECT images were reconstructed using HiSPECT software and the “Standard” pre-set. SPECT images were quantitatively analysed using VivoQuant 3.0 software. Any activity measured on the static scans as remaining in the tail of the animal was subtracted from the injected activity when calculating accumulation in each image, and decay corrected from the 7 h p.i. scan to also be included for calculations of the dynamic scan.

Results show:

• In the initial kidney accumulation, all animals exhibited a very quick accumulation and excretion when imaged during cycle 1, while some exhibited higher and slower accumulation during the second cycle imaging, and many more did so during the third cycle

Figure 1. Uptake characteristics of [¹⁷⁷Lu]Lu-PSMA-617 changes after each cycle of treatment. Representative SPECT images and graphs of [¹⁷⁷Lu]Lu-PSMA-617 activity in male BALB/cAnNRj nude mice. Maximum intensity projections using the NIH color scale windowed to best display kidneys with the top activity noted. Note that the Cycle 2 animal is the one with an outlier kidney. Annotations: k = kidney, and s = standard. (A) Sum of all frames of the dynamic scans immediately p.i. with 90–100 frames of 1 min. (B) Examples of [¹⁷⁷Lu] Lu-PSMA-617 kinetics in kidneys as measured using dynamic SPECT imaging directly after injection. Percent of injected activity per gram of kidney (%IA/g) plotted. Chosen animals have typical distribution for their cycles except for the second cycle animal where the high uptake kidney is an outlier.

• The absorbed dose per injected activity value calculated for each group was applied to the injected activities and the resulting absorbed doses for each cycle can be seen in Table 1

• The post-cycle 3 imaging session at 135 days after the first injection revealed that the [¹⁷⁷Lu]LuPSMA-617 + PBS group had higher mean and median accumulation than [¹⁷⁷Lu]Lu- SMA-617 + rA1M, see Fig. 2D, at both time points. This indicates a slight protection effect from rA1M, but the difference was not statistically significant
• significant increase of albumin/creatinine ratio in the [¹⁷⁷Lu]Lu-PSMA-617 + PBS group compared to control group
• The white blood cell counts (WBC) showed a significant drop 2 weeks after the first cycle in the [¹⁷⁷Lu]LuPSMA-617 + PBS group that recovered after 3.5 weeks (Fig. 3A, supplementary S3–S4). The drop after the second cycle was significant in both groups compared to control mice. The WBC values did not reach the control levels again, neither after the second cycle nor after the third cycle
• The third cycle resulted in a significantly lower levels of RBC after six weeks (Fig. 3B, D) in both groups receiving [¹⁷⁷Lu]Lu-PSMA-617

In conclusion, treating mice with three cycles of 200 MBq of [¹⁷⁷Lu]Lu-PSMA-617 gives detectable and non-transient nephrotoxicity and hematotoxicity. The high activity, multi treatment cycle design may provide a template for future studies of different types of toxicity and interventions designed to mitigate them. This study did not show a significant protective effect of rA1M but there are some indications of a preservative effect on pharmacokinetics that should be further investigated.

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