2. Experimental
3.5. In vitro stability studies
For radioimmunotherapeutic (RIT) applications, metal radionuclides must remain associated with the targeting chelate-protein to minimize toxicity arising from their dissociation from the conjugate followed be undesired biodistribution. Thus, a critical point is examination of the complex stability under challenging conditions. To this aim, we performed in vitro challenging experiments. One of the most informative stability assays that can be readily performed in laboratory is a measurement of stability of a metal ion complex in blood serum.
[54] Whilst serum stability can be seen as a benchmark of in vivo stability in the extracellular environment (i.e. during blood perfusion), it cannot be relied on alone as it does not fully mimic the intracellular conditions that might be encountered. In addition, serum stability studies provide information on possible pathways by which the radiopharmaceutical in question can become demetallated. These studies are often informative and practical at the final stage before carrying the complexes forward for in vivo studies. Free lanthanide(III) ions have a high affinity for bone and are also taken up by liver because of Ln(OH)3 colloid formation at physiological pH. With this in mind, hydroxyapatite (HA) was selected as an in vitro model material of bone to investigate the title complexes stability. Thus, stability of the various radiometal complexes can be assessed by determining a degree of exchange of the radiometal complexes with HA.
The stabilities against either hydroxyapatite or rat serum were monitored as a function of time and are presented in Figure 8A and 8B, respectively. It was observed that 44Sc-DO3AP, 44Sc- DO3APABn and 44Sc-DO3APPrA complexes were stable over several hours in serum, similarly to 44Sc-DOTA. Therefore, there is possible to conclude that ligands with DOTA-like structure are good chelators for Sc(III) forming stable complexes. In addition, there has been shown that the 44mSc/44Sc in-vivo generator used with radiolabeled peptides is stable in biological media; it has been already observed for 44Sc-DOTATOC with generator 44Sc [6,20,21] or for
44Sc-DOTATATE with cyclotron 44m/44Sc.[3] It is well known that compounds with phosphonate group(s) exhibit high in vivo bone uptake. Therefore, the in vitro stability against
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HA, the principal mineral component of bones, was studied to check affinity of the 44m/44Sc complexes to the bone or released of free 44Sc induced by the bone. For complexes of all the ligands, there is not any uptake of radioactivity on the bone. Therefore, no in vivo bone uptake is expected for complexes of the title ligands; one coordinated phosphorus acid group on the DOTA skeleton is not sufficient for observable bone uptake. [33,34]
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4. Conclusion
To fully explore potential of 44mSc/44Sc labelled antibodies as in vivo generator of 44Sc for PET imaging, there is a clear unmet demand of new DOTA-based ligands of high complexation efficiency permitting formation of the Sc(III) complexes close to temperature compatible with labeling such biomolecules. To this aim, the phosphorus acid DOTA derivatives bearing one methylphosphonic/methylphosphinic acid pendant arm, DO3AP, DO3APPrA and DO3APABn, were evaluated as chelators for trivalent scandium.
Thermodynamic stabilities of their Sc(III) complexes are very high and similar to that of the [Sc(DOTA)]– complex. As expected, stability constants with Sc(III) are several orders of magnitude higher than those for trivalent lanthanides. The presence of hard phosphonate/phosphinate group induces unexpected behavior in acidic solutions. It leads to rather facile formation of out-of-cage complexes and it is more pronounced for the phosphonate group leading even to behavior precluding stability constant determination of the Sc(III)-DO3AP complex. At pH above 4, expected in-cage complex where all donor atoms are bound to the central metal ion is formed. This coordination mode should assure stability of the complexes in vivo. Thermodynamic stability constants were also examined by FISRE method. The method gave only qualitatively similar results but it has an advantage over the common method that, as it is based on tracer amount of metal ions, can be used for several radiometal ions where conventional methods for stability constant determination can be hardly implemented. Labeling efficiency of the DOTA and its analogues was investigated on radioscandium from two sources, generator- or cyclotron-produced 44Sc. Main difference in labeling found between the radioscandium sources is in different chelator excess over radioscandium necessary for efficient labeling being about two orders of magnitude higher for generator 44Sc. The difference might be attributed to possible cold metal impurities. As for DOTA, the best labeling conditions for DO3AP, DO3APABn and DO3APPrA were pH= 4 at 70°C for 30 min with different ligand amount according to radioscandium source. The phosphonate ligand, DO3AP, showed somewhat better labeling efficiency at low temperature.
Specific activity of the labeled chelators was higher for cyclotron produced 44m/44Sc (~10 MBq/nmol) than for generator produced 44Sc(~2 MBq/nmol). This parameter is of great importance if peptide and/or antibodie conjugates with these ligands are considered for applications in nuclear imaging. In vitro stability of the Sc(III) complexes against hydroxyapatite and in rat serum is very high as expected for complexes of macrocyclic ligands. Although all these chelators can be used as contraction of targeted radipharmaceticals, slow formation kinetics remains a main challenge in desing of chelators especially devoted for scandium radioisotopes.
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