A New Unnatural Amino Acid with Unique Charge Distribution for Modular Synthesis of Peptide-Based Radiopharmaceuticals with Reduced Kidney and Healthy Tissue Uptake

Introduction: Utilizing radiolabeled peptides in both diagnosis and therapy of cancer has burgeoned recently, yet the high kidney uptake of most radiometallated peptides impedes the maximum potential of these radiopharmaceuticals. The effort to find a solution to this high kidney uptake and retention is needed. Herein, we synthesized a novel Un-natural Amino Acid with unique Charge distribution (UAAC) that we tethered to the FDA approved receptor-mediated compound DOTA-TATE using solid phase peptide synthesis (SPPS). In comparison to [⁶⁸Ga]Ga-DOTA-TATE, our tracers [⁶⁸Ga]Ga-DOTA-(UAAC)_n-Spacer-TATE showed dramatically less kidney uptake in healthy CD1 female mice. To our surprise, the tumor uptake for our tracer [⁶⁸Ga]Ga-DOTA-UAAC-Spacer-TATE was higher in comparison to [⁶⁸Ga]Ga-DOTA-TATE while maintaining lower kidney uptake in AR42J xenograft bearing mice (nude, NOD/SCI F1). Based on these promising results, further studies with xenograft bearing mice with different mice type is required to confirm that the difference in kidney uptake is independent of the mice type for formulation of injectate. Also, our UAAC is modular and can be applied to different radiolabeled peptides such as PSMA and Exendin 4 analogues to decrease kidney uptake and improve pharmacokinetics.

Methods: Gallium ([⁶⁸Ga]Ga³⁺) activity was eluted from a commercial TiO₂-based [⁶⁸Ge]Ge⁴⁺/[⁶⁸Ga]Ga³⁺ generator (Eckert & Ziegler), using 6 mL of 0.1 M HCl. Then, the eluted gallium ([⁶⁸Ga]Ga³⁺) was concentrated using SCX resin (in-box) eluted with 5.5M NaCl and 0.05M HCl solution. For radiolabeling, ~60-70 MBq of [⁶⁸Ga]Ga³⁺ solution was incubated at 95°C with 20-27 μg of DOTA-TATE and DOTA-(UAAC)_n-Spacer-TATE at pH 3.8-3.9 for 12 minutes. Purification of radiolabeled tracers was done using C-18 Sep Pak light cartridge. Dynamic PET/CT imaging was conducted (0-90 min) following administration of 5-6 MBq of labelled peptide via tail-vein catheters, followed by biodistribution at 2 hrs post injection.

Results: The peptide-conjugates were synthesized successfully utilizing ultrasonic-SPPS with yields of ~ 29%. They were radiolabelled with [⁶⁸Ga]Ga³⁺ showed RCY% of more than 95% using radio-HPLC. They remained intact in both of mouse and human serum after incubation at 37°C for 2 hrs. The biodistribution study showed lower kidney uptake of ~20 %ID/g (~3.2 fold decrease) in CD1 female healthy mice in comparison to the gold standard [⁶⁸Ga]Ga-DOTA-TATE (~65 %ID/g). Furthermore, our modified DOTA-TATE showed lower uptake in many healthy non-targeted organs by ~2 fold. Despite the faster blood clearance and lower background uptake, the AR42J tumor uptake of our UAAC containing derivative was 20.6 ± 7.6 %ID/g whereas it was 14.2 ± 4.5 %ID/g for [⁶⁸Ga]Ga-DOTA-TATE.

Conclusions: We synthesized a novel DOTA-TATE derivative embedded with our novel Un-natural Amino Acid with unique Charge distribution (UAAC) as a linker, and compared to standard DOTA-TATE using in vitro and in vivo radiochemical experiments. Our novel [⁶⁸Ga]Ga-DOTA-(UAAC)_n-Spacer-TATE showed great promise by showing ~3-fold lower kidney and ~2-fold lower non-targeted organ uptake in healthy mice relative to standard [⁶⁸Ga]Ga-DOTA-TATE. Additionally, our new UAAC-containing derivative showed slightly higher AR42J tumor uptake.

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