ИСТИНА

Aim: Alpha-emitting radionuclides are increasingly being considered as part of the novel radiopharmaceuticals due to their much higher linear energy transfer (LET) and hence more cytotoxicity than β-emitters.[1] Ac-225 and Bi-213 are among the most commonly used radionuclides for TAT: preparations based on them are already undergoing phase II clinical trials.[2] Although the commonly used DOTA-based conjugates with peptides has already shown great results, the quantitative binding of the metal cation with heat-sensitive targeting biomolecules remains a challenge. Generally, the complexation with DOTA requires heating up to 100 °C, which complicates preparation procedure.[3] Moreover, some in vivo instability of the AcDOTA was discovered which supposed to be due to the thermodynamic preference of DOTA for smaller ions than the largest trivalent ion in periodic table.[4] Our aim was conducting the detailed research from thermodynamic stability of Bi(III) and Ac(III) complexes with new benzoazacrown ligand BATA together with kinetic study to biodistribution in normal mice. Method: Stability constants were determined by potentiometric titration as well as FISRE method (free-ion selective radiotracer extraction) with HDEHP (di-(2-ethylhexyl)phosphoric acid). Radiolabelling was conducted with Bi-207 and Ac-228 radioisotopes of Bi(III) and Ac(III) in mQ water and radiochemical yield was determined by TLC (thin layer chromatography) on aluminum-backed TLC plates (cellulose, Sigma-Aldrich) and the eluent 0.9 % NaCl – 0.01 M NaOH. Kinetic study of acid-assisted dissociation and formation of BiBATA was conducted by UV absorption spectroscopy in I = 0.6 M (H,K)Cl to prevent hydroxides and colloid formation of the free Bi(III). Complex stability in fetal bovine serum was determined by precipitation of serum peptide and measuring radioactivity of supernatant by gamma-spectroscopy. Biodistribution study was conducted with Bi-207 and Ac-225 labelled complexes in accordance with EU Directive 2010/63/EU for animal experiments and metabolic chamber was used in case of AcBATA complex to collect excrements for further measurements. Results: The absolute values of the stability constants of BiBATA forms determined by potentiometric titration were very high, so the FISRE method was used to obtain reliable values. We used the same method for the complex with Ac(III) as well as potentiometric titration with Ac(III) lanthanide analogue La(III). Obtained stability constants of BATA complexes were higher than those of DOTA indicating a great affinity of Bi(III) and Ac(III) to BATA. Radiolabelling of BATA occurred immediately at room temperature moreover BATA was labeled with Ac(III) at a lower ligand concentration than DOTA. A comparison was made of the formation and dissociation rates of the BiBATA complex with the BiDOTA and BiDTPA complexes in an acidic medium. Under these conditions, BiBATA and BiDTPA complexes formed quickly, while the complex of Bi(III) with DOTA did not form at all. The dissociation of the BiBATA occurred at pH < 1, while at pH 2 and above the complex was stable which is explained by the different stability of diprotonated and monoprotonated species. The mechanism of acid-assisted dissociation of Bi(III) complex with BATA consist in fast protonation of a coordinated carboxylate group in the first step, then the transfer of this proton from the carboxylate group to a nitrogen atom of macrocycle which is the rate-determining step and finally the dissociation of the double-N-protonated complex. Such inspiring results prompted us to study the stability in media with competing cations and blood serum. BATA labelled with Bi-207 and Ac-228 demonstrated stability in vitro at least for 2 days in case of Bi(III) and at least for 1 day in case of Ac(III). Hence biodistribution of BATA complexes in normal mice was studied 1 and 6 h after administration of the Bi(III) complex and 6 h after administration of the Ac(III) complex. The BiBATA complex had a very similar biodistribution profile and is eliminated from the body just as quickly as the complex BiDOTA. The organ with the highest Bi(III) accumulation was kidneys and % ID/g was the same for BiBATA and BiDOTA complexes. Almost all AcBATA was cleared from the body after 6 h via urinary excretion and the accumulation in each organ did not exceeded 0.5% ID/g indicating very high stability in vivo. Conclusions: In course of our research of Bi(III) and Ac(III) complexes with BATA we obtained high thermodynamic stability constants as well as high stability of the complex in vivo. High stability in vivo of BiBATA is associated with the high kinetic inertness of mono- and deprotonated forms of the complex. In addition BATA was found to be superior to DOTA for the rapid chelation of Bi(III) and Ac(III) due to the less rigid structure of the 18-crown-6. Taken together it seems that the 18-crown-6 macrocycle is more preferable for such large cations as Bi(III) and Ac(III).