DOTA-TATE alternative labellings with halogens and radiometals

Full text: DOTA-D-phe-cys-tyr-D-trp-lys-thr-cys-thr (DOTA-TATE), a somatostatin analog with high affinity for somatostatin receptors expressed in cancer of neuroendocrine origin, may be labeled with different beta radionuclides for its use in targeted radiotherapy. This allows the obtanting of products with particular chemical, radiochemical and biological properties, which interact in specific way with cells that express somatostatin receptors. In this study the labelling of DOTA-TATE with 125I, 131I and 177Lu and their biological properties were investigated, with the aim of potential application as a tumour seeking agents. Radioiodine was introduced on the tyrosyl residue of the DOTA-TATE by oxidation of iodide with chloramine-T. The labeled peptide was isolated from the reaction mixture by sep-pak purification and/or by RP-HPLC. Lutetium-177 was introduced in the peptide via the DOTA ring. To 7,2μg of DOTA-TATE 1mg/mL, 2μL 177LuCl3 (7.2 mCi) and 7.2 μL of gentisic acid in CH3COONa 0.4M were added. The mixture was incubated 30 minutes at 100 deg. C. Purification by RP-HPLC was conducted on a C18 column eluted with a linear gradient from 10% to 40% B in 30 min (A: 0.1% TFA in water and B: 0.1% TFA in acetonitrile). Fractions containing the labeled peptide were pooled and most of the solvent removed by evaporation and re-suspended in 1 mL of saline. For the control of 131I-DOTA-TATE, different gradients were used in order to optimize the separation. In the case of 177Lu-DOTA-TATE analysis, gradient was from 5 to 95% B in 15 minutes. Stability at different time intervals was evaluated by RP-HPLC and by chromatography in ITLC-SG or Whatman 3MM using different mobile phases: 2-butanone (MEK); NaCl 0.9%; EtOH-HCl 0.01N 90:10 and butanol:acetic:H2O (4:1:5) for radioidinated peptides. For 177Lu-DOTA-TATE, the chromatographic systems were: CH3COONH4 10%/MeOH (30:70), BuOH-CH3COOH-H2O (5:2:1), TFA 0,1% in ACN- H2O (1:1) and Sodium citrate 0.1M, pH:5 in Whatman 3MM (Rf of radiopeptide 0.9; 0.9-1.0; 0.9-1.0; 0.0 respectively). Biological behavior of radiolabelled peptides was evaluated using AR4-2J cell which express somatostatin receptors through internalization and externalization studies. Binding to AR4-2J cells at 1, 2, 3 and 4 h were studied, using 5 x 105 cells and 200.000 cpm of purified labeled conjugates per tube. Internalization of the radioconjugates at 3 and 4 h was done by further incubation in acidic conditions in order to remove the radiactivity bound to membrane. Biodistribution studies were carried out in CD-1 mice weighing 34-45g, injected with 0,5 to 74 KBq of 125I-DOTA-TATE, or 1.85 to 5.50 MBq of 177Lu-DOTA-TATE at 1, 2 or 4 and 24 hs post-injection. Results were expressed as percent injected dose per organ (%ID) and as percent injected dose per gram of tissue (%ID/g). The results evidenced a labeling yield higher than 95% for 125I as well as for 177Lu, according with sep-pak elution profile as well as by chromatographic behavior of the reaction mixture using HPLC and ITLC-SG/saline system (radioiodine) or TFA 0,1% in ACN-H2O (1:1) (177Lu). In the case of 125I-DOTA-TATE the HPLC profile revealed that two radiochemical species were present having retention times of 23,5 and 25,0 min, while the unlabelled peptide eluted at 21,0 min, whilst for the other radiopeptides only one peak was found (11.2 min for 177Lu-DOTA-TATE). Stability of the radioconjugate (at 4 deg. C and -80 deg. C) was confirmed during more than 8 days for the peptide labelled with 125I, while with 177Lu was more than 24 hs at 4 deg. C. The binding of radiopeptides to viable AR4-2J cells increased along the time of incubation. Internalization experiments demonstrated that the radioconjugate penetrates into the cells reaching 73 ± 14 % (n=10), 32 ± 9 % (n=3) and 62 ± 18 (n=6) of the total bound activity in 3 - 4 h for the peptide labelled with 125I, 131I and 177Lu respectively. The biological uptake pattern of the radioidinated peptide indicates urinary as well as gastrointestinal excretion. In vivo metabolization of the radioidinated peptide is evidenced by the thyroid uptake which reaches 10,2 ± 8,5 % of the injected activity in 24 h. In the case of labelling with 177Lu the elimination is mainly by urinary excretion. In conclusion, DOTA-TATE labelled with radioiodine as well as with 177Lu was obtained with very high yield even prior purification. The two radiochemical species evidenced for 125I-DOTA-TATE by RP-HPLC could be interpreted as mono and diiodinated species. Their retention times are slightly higher than the intact unlabelled peptide, allowing the preparation of a labelled molecule with high specific activity. Binding experiments using AR42J cells indicated that the radioconjugates are able to recognize and bind to somatostatin receptors present in viable tumour cells. Biodistribution in normal mice shows a different pattern when labelling with radioiodide and radiolantanide. These findings opens the possibility of the use of cocktails of DOTA-TATE labelled with different radionuclides in order to minimize the radiation dose to organs not compromised with the tumour. (author)