Synthesis and preclinical evaluation of 68Ga-labeled collagelin analogs for imaging and quantification of fibrosis.

OBJECTIVES: Fibrosis affecting functionality of vital organs such as liver, lung, heart, and kidney, is involved in many chronic diseases. Positron emission tomography (PET) would not only provide precise localization and extent of the affected tissue but also allow the accurate quantification of the fibrotic process for the subsequent prognosis. METHODS: A cyclic peptide c[CPGRVMHGLHLGDDEGPC] conjugated either to 2-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)acetic acid (NOTA(tBu)2) or 4-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazacyclononan-1-yl)-5-(tert-butoxy)-5-oxopentanoic acid (NODAGA(tBu)3) via polyethylene glycol link (PEG2) was synthesized and labeled with (68)Ga. Non-specific organ distribution, blood clearance, and excretion were investigated ex vivo in healthy rats. The binding specificity of the radioligands was assessed in vitro using autoradiography on cryosections of dog fibrotic heart tissue. RESULTS: The yield of NOTA-PEG2-c[CPGRVMHGLHLGDDEGPC] and NODAGA-PEG2-c[CPGRVMHGLHLGDDEGPC] was 56% and 41%, respectively. Non-decay-corrected radiochemical yield was 80 ± 5% with radiochemical purity of 95 ± 4%. Pharmacokinetic studies in healthy male Sprague-Dawley rats showed fast blood clearance and renal excretion. Lower uptake in liver, spleen, and kidney was found for [[(68)Ga]Ga-NOTA](+1)-PEG2-c[CPGRVMHGLHLGDDEGPC] as compared to [[(68)Ga]Ga-NODAGA](0)-PEG2-c[CPGRVMHGLHLGDDEGPC]. Histologic evaluation of the left ventricle (LV) myocardium from a dog with severe mitral regurgitation (MR), revealed mild to moderate perivascular and subendocardial, and mild diffuse interstitial fibrosis. The tracer binding to the cryosections of the tissue was specific with the equilibrium Kd of 2.3 ± 0.8 µM and 2.1 ± 0.9 µM, respectively for [(68)Ga]Ga-NO2A-Col and [(68)Ga]Ga-NODAGA-Col. CONCLUSIONS: Two novel peptide based agents for the imaging of fibrosis by PET were developed. Moderation of the biodistribution could be achieved by variation of the charge on the complex moiety of the agents. The combination of the fast clearance from non-target organs as well as organs of interest such as lung, heart, and liver and binding specificity to the target tissue suggests the potential of the analogs for the imaging of fibrosis.

The importance of high specific radioactivity in the performance of 68Ga-labeled peptide.

The use of (68)Ga-labeled peptides in diagnosis, dosimetry, therapy planning and follow-up of response to chemo- and radiotherapy requires accurate quantification of tracer binding characteristics in vivo, which may be influenced by the specific radioactivity (SRA) of the tracer. Systematic study of the complexation reaction of DOTA-D-Phe(1)-Tyr(3)-Octreotide (DOTATOC, where DOTA is the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) with (67)Ga, (68)Ga, (69,71)Ga and in the presence of competing metal cations [Al(III), Fe(III), In(III)] was performed using conventional and microwave heating techniques and assessed by mass spectrometry. Saturation binding of (68)Ga-DOTATOC to Rhesus monkey brain slices was performed using frozen section autoradiography. High SRA was necessary in order to characterize the saturation binding of (68)Ga-DOTATOC to somatostatin receptors in Rhesus monkey brain sections. The complexation of Ga(III) with DOTATOC suggested more favorable formation compared to Fe(III) and In(III). The microwave heating mode might influence the selectivity of the complexation reaction, especially when comparing the behavior of Ga(III) and In(III). Al(III) was less critical with contamination and could be tolerated up to a concentration equal to that of the peptide bioconjugate. The SRA of (67)Ga-DOTATOC and (67)Ga-NODAGA-TATE (NODAGA-Tyr(3)-Octreotate, where NODAGA is 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid) exceeded literature data by a factor of 7 and 5-15, respectively. High SRA was critical for providing sufficient contrast and accurate quantification of PET images. Microwave heating mode apart from the acceleration of the labeling reaction also improved the selectivity of the complexation reaction towards gallium. Fe(III) was shown to be the most critical competitor deteriorating the (68)Ga-labeling efficiency.

Preparation and evaluation of (68)Ga-DOTA-hEGF for visualization of EGFR expression in malignant tumors.

UNLABELLED: Detection of epidermal growth factor receptor (EGFR) overexpression in many carcinomas provides important diagnostic information, which can influence patient management. The use of PET may enable such detection in vivo by a noninvasive procedure with high sensitivity. The aim of this study was to develop a method for preparation of a positron-emitting tracer based on a natural ligand to EGFR, the recombinant human epidermal growth factor (hEGF), and to perform a preclinical evaluation of the tracer. METHODS: DOTA-hEGF (DOTA is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) was prepared by coupling of a N-sulfosuccinimide ester of DOTA to hEGF. The conjugate was labeled with a generator-produced positron-emitting nuclide, (68)Ga (half-life = 68 min), using microwave heating. Binding specificity, affinity, internalization, and retention of (68)Ga-DOTA-hEGF was studied in 2 EGFR-expressing cell lines, U343 glioma cells and A431 cervical carcinoma cells. Biodistribution and microPET visualization studies were performed in BALB/c nu/nu mice bearing A431 carcinoma xenografts. RESULTS: A 1-min-long microwave-assisted labeling provided radioactivity incorporation of 77% +/- 4%. Both cell lines demonstrated receptor-specific uptake of the conjugate, rapid internalization of the tracer, and good retention of radioactivity. Binding to both cell lines occurred with high affinity, approximately 2 nmol/L. The biodistribution study demonstrated accumulation of radioactivity in xenografts and in EGFR-expressing organs. The microPET imaging study enabled visualization of tumors and demonstrated quick--within 5 min--localization of radioactivity in tumors. CONCLUSION: (68)Ga-DOTA-hEGF has potential for imaging EGFR overexpression in tumors.