A standardised study to compare prostate cancer targeting efficacy of five radiolabelled bombesin analogues

Abstract

Purpose: Prostate-specific antigen (PSA)-based screening for prostate cancer (PC) has dramatically increased early diagnosis. Current imaging techniques are not optimal to stage early PC adequately. A promising alternative to PC imaging is peptide-based scintigraphy using radiolabelled bombesin (BN) analogues that bind to gastrin-releasing peptide receptors (GRPR) being overexpressed in PC. When labelled to appropriate radionuclides BN targeting of GRPRs may also provide applications for peptide radionuclide receptor therapy (PRRT). Assessment studies under identical experimental conditions allowing a reliable comparison of the potential of such analogues are lacking. This study was performed to evaluate and directly compare five promising radiolabelled BN analogues for their targeting efficacy for PC under standardised conditions.

Methods: The BN agonists [(111)In]DOTA-PESIN, [(111)In]AMBA, [(111)In]MP2346 and [(111)In]MP2653 and one antagonist [(99m)Tc]Demobesin-1 were evaluated in GRPR-overexpressing human PC-3 tumour-bearing mice to determine peptide stability in vivo, biodistribution and GRPR targeting potential by animal SPECT/CT imaging and ex vivo autoradiography.

Results: HPLC analysis of blood showed intact Demobesin-1 at 5 and 15 min after injection (64.1 +/- 1.6% and 41.0 +/- 01%, respectively) being much less for the other compounds. AMBA, the second most stable analogue, showed 36.1 +/- 2.7% and 9.8 +/- 1.1% intact peptide after 5 and 15 min. PC-3 tumour uptake at 1 h was comparable for Demobesin-1, AMBA, PESIN and MP2346 (3.0 +/- 0.4, 2.7 +/- 0.5, 2.3 +/- 0.5 and 2.1 +/- 0.9%ID/g, respectively), but very low for MP2653 (0.9 +/- 0.2%ID/g). In addition, MP2346 showed undesirably high uptake in the kidneys (7.9 +/- 1.9%ID/g) being significantly less for the other analogues. AMBA, MP2346 and PESIN revealed favourable increases in tumour to blood ratios over time while changes in tumour to kidney and pancreas ratios for Demobesin-1 from 1 to 24 h after injection were significantly better than for the other analogues. All analogues visualised PC-3 tumours by SPECT/CT and autoradiography.

Conclusion: In the present study the BN antagonist Demobesin-1 was the best performing analogue showing superior in vivo stability, highest tumour uptake and retention while pancreatic and renal clearance were rapid. PESIN and AMBA were the best GRP agonists with sufficient in vivo stabilities as well as high tumour uptake and retention. Based on these results all three analogues deserve further evaluation for clinical use in PC patients.

Fig. 1

I Amino acid sequence of native BN (14 amino acids) and the BN analogues used in this study. Chelators: N₄ = 6-R-1,4,8,11-tetraazaundecane, DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DTPA = diethylene triamine pentaacetic acid. Linkers: R = BzDig = p-aminobenzyldiglycolic acid, PEG = polyethylene glycol. Introduced amino acids: ACMpip =4-amino-carboxymethylpiperidine (non-natural amino acid), Tha = β-(2-thienyl)-alanine (non-natural amino acid), β-Ala = β-alanine (non-natural amino acid), Nle = norleucine, Pro = proline, Tyr = tyrosine, Phe = phenylalanine, *reference number from list references. II HPLC of all five radiolabelled analogues 1 h after labelling

Fig. 2

Tumour uptake for all analogues in PC-3 tumour-bearing athymic nude mice injected with 0.5 MBq ¹¹¹In or 2.5 MBq ^99mTc bound to 10 pmol of peptide conjugate, in a volume of 0.1 ml into a lateral tail vein sacrificed at 1, 4 and 24 h p.i. Results are indicated as mean ± standard deviation of four mice per analogue per time point

Fig. 3

Ratios calculated from biodistribution in PC-3 tumour-bearing athymic nude mice injected with 0.5 MBq ¹¹¹In or 2.5 MBq ^99mTc bound to 10 pmol of peptide conjugate, in a volume of 0.1 ml into a lateral tail vein sacrificed at 1, 4 and 24 h p.i. Data show the tumour to blood ratio (a), tumour to pancreas ratio (b) and tumour to kidney ratio (c) at all time points for all radiolabelled analogues

Fig. 4

I SPECT/CT image of the PC-3 tumour in athymic xenografts at 1 h after i.v. tail injection of 0.25 nmol of radiolabelled analogue (23–31 MBq for ¹¹¹In and 142 MBq for ^99mTc) in a volume of 0.1 ml into a lateral tail vein. a AMBA, b Demobesin-1, c MP2653, d PESIN, e MP2346. II Autoradiograms (right) and corresponding H&E-stained sections (left) of a PC-3 tumour derived from a mouse 4 h after injection of MP2346 (a) and AMBA (b). Ex vivo tumour sections were derived from PC-3 tumour-bearing athymic nude mice injected with 0.25 pmol peptide, labelled with 23–31 MBq for ¹¹¹In analogues in a volume of 0.1 ml into a lateral tail vein