The radiochemical investigations with copper(II)-64 were performed at the Institute of Radiopharmaceutical Cancer Research of the Helmholtz Zentrum Dresden-Rossendorf (HZDR). Radiolabeling experiments, in vitro competition assays and the determination of the partition coefficients log Do/w were carried out in the group of Dr. Holger STEPHAN by Karin LANDROCK and Janine PARTSCH. The biodistribution studies were performed by Prof. Dr. Jens PIETZSCH and coworkers.
Radiolabeling experiments
Prior to the experiments, the radiochemical purity of the 64CuII-radiolabeled bispa ligands
B1a and B1b was determined by radio-high performance liquid chromatography (radio-
HPLC) to be > 99 %. The radiolabeling efficiency of B1a and B1b with copper(II)-64 was
investigated as a function of time and concentration. Therefore, sample solutions with different amounts of the respective ligand and 64CuCl
2 were prepared and allowed to radiolabel at ambient temperature. If not stated otherwise, the ligands were used in their protonated form with one trifluoroacetate (TFA) as the counterion. The experimental setup was carried out in a way to obtain final ligand concentrations of ~10-4 to 10-6 M and the studies were performed with starting activities of 10 and 100 MBq 64CuCl
2, respectively. Depending on the molar activity of different 64CuII production runs, and the time of 64CuII use after the end of bombardment (EOB), different aliquots of 64CuCl
2 were added to obtain the desired starting activity. The degree of radiolabeling was assessed by radio- thin layer chromatography (radio-TLC) at 5, 30 and 60 minutes after addition of 64CuCl
2. The radio-TLC conditions described in the Experimental Section allowed for the clear separation of “free” copper(II)-64 and the two radiolabeled bispa ligands. While 64CuCl
2 remains at the origin, both 64CuII-bispa complexes move with an R
radiochemical yield (RCY) was determined by integrating the peaks in the radio-thin layer chromatograms. In the case of B1a and B1b a total amount of 0.5 µg ligand per 100 µL
(~510-6 M) could be radiolabeled quantitatively (RCY > 99%) within 5 minutes at room
temperature using 100 MBq 64CuCl
2. After decreasing the amount of ligand to 0.1 µg (~10-6 M) under the same conditions, only partial radiolabeling was observed, i.e. 15 % RCY for 64CuII-B1a and 30 % RCY for 64CuII-B1b.
The specific or molar activity of a compound is a measure of its ability to be radiolabeled by a certain amount of radioactivity and is given in detected activity per gram or mole of the substance.[135] Radiolabeling of B1a and B1b with copper(II)-64 yielded molar activities of ~100 GBq/µmol and ~200 GBq/µmol, respectively. Such high activities are desirable for nuclear medicine applications, as this means that a small quantity of ligand can be radiolabeled with a high amount of activity. As a consequence, the drug dose required for the patient is minimized. Altogether, a very efficient and rapid 64CuII complexation of the bispa ligands was found under mild conditions. The other bispidine systems studied so far also displayed fast radiolabeling kinetics with 64CuII, but they were not investigated with activities as high as 100 MBq.[172,173] For this reason the maximum molar activity of these compounds could not be determined.
Competition assays
The stability of a radiolabeled metal complex towards transchelation is usually tested in challenge experiments with competing compounds or mixtures thereof. The standard procedure involves labeling of the ligand with the respective radiometal ion followed by incubation with the chosen competitor. After a certain time period, the amount of stable complex is then determined using a suitable analytical method.
The first challenge experiments with the 64CuII-bispa complexes were performed in the presence of the competing ligands cyclam (L3) and EDTA (L9). Complete radiolabeling of B1a and B1b with copper(II)-64 (RCY > 99 %) was initially checked by radio-TLC
analysis. The radiolabeled complexes were then incubated with a 100-fold molar excess of cyclam and EDTA, respectively. After incubation at room temperature for 2 and 24 hours, the samples were assayed by radio-HPLC. The radio-HPLC protocol applied revealed well-separated signals for the metal complexes, hence providing information on the degree of transchelation. As expected, both 64CuII-B1a and 64CuII-B1b were stable by more than 99 % after 2 and 24 hours in the presence of cyclam as well as of EDTA.
To further study the kinetic stability of the 64CuII-bispa complexes, superoxide dismutase (SOD) challenge experiments were carried out. The copper / zinc enzyme SOD is mostly abundant in kidney and liver cells as well as erythrocytes. The major function of SOD is the cellular defense against oxidative damage, as it catalyzes the disproportionation of superoxide radicals to oxygen and hydrogen peroxide. This catalytic conversion takes place at the active site of the enzyme, which embeds the metal ions copper and zinc.[402,403] Several stability studies of 64CuII-labeled BFC-protein conjugates have shown transchelation of 64CuII to SOD.[207,208] For this reason, STEPHAN and coworkers developed a new SOD stability assay,[182] which was also used in the study presented here. Briefly, a 3-fold excess of human SOD was added to the bispa complexes 64CuII-B1a and
64CuII-B1b and the samples were then incubated for 1 hour at 37 °C. The subsequent
analysis was performed using non-reducing and non-denaturing polyacrylamide gel electrophoresis (native PAGE). After electrophoresis, the gel was examined by electronic autoradiography with a radioluminography laser scanner. The different blackening intensities of the bands correlate with the amount of copper-64, which dissociates from the ligand upon incubation with SOD and is subsequently bound to the enzyme.[182] Following autoradiography, the gels were stained with colloidal Coomassie G-250 to visualize the protein bands. A representative gel is depicted in Figure 51 and the results obtained for
64CuII-B1a and 64CuII-B1b are given in Table 14. The results are compared to prominent
literature-known copper(II) chelators studied by STEPHAN et al. to show the potential of the newly-developed SOD assay.[182] The corresponding experiments were carried out under the same conditions applied in this study and the results can hence be directly compared. The 64CuII-bispa complexes displayed high resistance towards transchelation to human SOD with less than 1 % of incorporation. These values are in the same range as those observed for N2py4 (B3) and all macrocyclic ligands under investigation. However,
64CuII-(p-MeO)B4 shows 4.3 ± 0.1 % transchelation ratio, which means that it is slightly
less stable against SOD than the bispa complexes. In contrast, the acyclic radiolabeled chelator EDTA underwent an almost complete transchelation (85.4 ± 13.6 %) in the presence of SOD.[182]
Figure 51. Analysis of 64CuII transchelation to human erythrocyte superoxide dismutase (SOD) for 64CuII-B1a. Colloidal Coomassie stained native polyacrylamide gel (left) and autoradiography showing 64CuII-labeled bands of SOD (right). The respective analysis for
64CuII-B1b is shown in Appendix H.
Table 14. 64CuII transchelation [% of control] to human erythrocyte superoxide dismutase
(SOD) for various 64CuII-labeled ligands after 1 h. Each value is given as mean ± standard deviation of three measurements.
complex transchelation [% of control] ref 64CuII-B1a 0.2 ± 0.1 64CuII-B1b 0.4 ± 0.1 64CuII-B3 0.7 ± 0.3 [173,177] 64CuII-(p-MeO)B4 4.3 ± 0.1 [172,177] 64CuII-TETA 1.3 ± 0.6 [182] 64CuII-DOTA 1.7 ± 1.3 [182] 64CuII-NOTA 1.4 ± 0.7 [182] 64CuII-cyclam 1.0 ± 0.5 [182] 64CuII-DIAMSAR 0.6 ± 0.1 [182] 64CuII-EDTA 85.4 ± 13.6 [182]
The ultimate in vitro stability test for radiometal ion complexes is the incubation of the compound in the presence of blood serum. In addition to the SOD challenge experiment described above, STEPHAN et al. also developed a complementary serum stability assay.[182] Instead of SOD, the radiolabeled ligands B1a and B1b were incubated with aliquots of human serum and the samples were analyzed by non-reducing sodium dodecyl sulfate (SDS) PAGE. The stained gels as well as the autoradiographic scans for
64CuII-B1a and 64CuII-B1b are shown in Figure 52 and Appendix H. The prominent protein
band in the size range of approximately 65 kDa most likely corresponds to 64CuII-labeled serum albumin. Other serum proteins with high affinity for copper(II) are not visible,
possibly due to their predominantly low abundance in human serum and the rather low activities of 64CuCl
2 used in the experiments.[182] Quantitative analysis of the band intensities revealed 17.8 ± 1.1 % transchelation to human serum proteins for 64CuII-B1a and 6.0 ± 1.1 % for 64CuII-B1b. These high values were surprising, considering that the stability of radiolabeled ligands in SOD is usually observed to be in the same range as that in human serum.[182] Furthermore, the bands in the autoradiography were considerably smeared and this made the integration difficult. The autoradiography corresponding to the serum stability assay of 64CuII-B1b additionally shows a band at the top of the scan that cannot be assigned.
Figure 52. Analysis of 64CuII transchelation to human serum proteins for 64CuII-B1a.
Colloidal Coomassie stained native polyacrylamide gel (left) and autoradiography showing 64CuII-labeled bands of human serum proteins (right).
To validate the serum stability of 64CuII-B1a and 64CuII-B1b the respective experiments were repeated and then analyzed by radio-size exclusion chromatography (SEC) instead of gel electrophoresis. In both cases, the completely intact radiometal complexes (> 99 %) were eluted from the SEC column and no serum bound activity could be detected. This contradicts the results of the above-described serum challenge assays evaluated by gel electrophoresis. Finally, biodistribution studies were performed with the bispa-based complexes to provide clarity about their stability (see below).
Lipophilicity studies
Information about the lipophilicity of the 64CuII-labeled bispa ligands B1a and B1b were obtained using the shake-flask procedure.[189,190] The distribution ratio D
o/w was determined as the concentration ratio of the respective 64CuII-bispa complexes between 1-octanol and
an aqueous phase at different pH. The distribution ratios are given as log Do/w values in
Table 15. Similar to previously investigated bispidine systems,[172,173] the complexes
64CuII-B1a and 64CuII-B1b are quite hydrophilic. Therefore, comparative biodistribution
behavior, i.e. fast blood clearance and renal excretion, are expected for the 64CuII-labeled bispa ligands. The log Do/w values of 64CuII-B1b with a positive overall charge of one and of the twice positively charged 64CuII-B3 are in the same range.[173] Reduction of the C9 keto group leads to a slight increase of hydrophilicity, which can be seen by comparison of the 64CuII-bispidine complexes based on B3 and B6.[173] The para-methoxy substitution of the pyridine groups in (p-MeO)B4, on the other hand, decreases the hydrophilicity.[172] Note that an efficient QSPR model was developed by COMBA et al. for the prediction of lipophilicities / hydrophilicities.[188,404]
The distribution ratios of 64CuII-B1a are clearly more negative than those of 64CuII-B1b. This can be explained by the structural differences of the two copper(II) complexes, thoroughly discussed in Chapter 4.1. The lipophilic behavior of compounds is correlated to the respective charge distributions. The copper(II)-carboxylate bond length in the solid state structure of CuII-B1a is 2.3127(12) Å and therefore significantly larger than the respective distance in CuII-B1b with 2.0119(11) Å. In several spectroscopic studies it could be shown that the structures determined by X-ray analysis are preserved in solution. The positive charge at the central copper(II) ion and the negative charge of the carboxylate are apparently more separated in CuII-B1a resulting in a more hydrophilic complex.
Table 15. Distribution ratios log Do/w of 64CuII-bispidine complexes at different pH.
bispidine ligand log Do/w at pH ref
7.2 7.4 7.6
Hbispa1a (B1a) (a) -3.79 ± 0.02 -3.78 ± 0.02 -3.77 ± 0.02 Hbispa1b (B1b) (a) -2.70 ± 0.02 -2.74 ± 0.02 -2.74 ± 0.02
N2py4 (B3) -2.73 -2.77 -2.69 [173]
B6 -2.89 -2.88 -2.85 [173]
(p-MeO)B4 -2.49 -2.44 -2.42 [172]
Biodistribution
The stability of the 64CuII-bispa complexes 64CuII-B1a and 64CuII-B1b was additionally tested in biodistribution studies on male Wistar Kyoto rats. The radiolabeled bispa ligands were injected into the tail vain of the rats and the animals were sacrificed 5 minutes, 30 minutes, and 24 hours post injection (p.i.). Organs and tissues of interest were rapidly excised, weighed, and the accumulated radioactivity determined using a γ counter. When uncomplexed copper(II) ions in the form of 64CuCl
2 are injected, an accumulation of radioactive species is observed in the liver, intestine and kidneys.[173] The biodistribution pattern of 64CuII-B1a and 64CuII-B1b, in contrast, show fast elimination via the kidney and no significant accumulation in other organs or tissues. The 64CuII radioactivity concentration was calculated as either the percentage of the injected dose per gram (%ID/g) or, for stomach, intestine, feces and urine, the percentage of injected dose (%ID). The biodistribution and elimination data of 64CuII-B1a and 64CuII-B1b are depicted in
Figure 53 and Figure 54, and additionally given in Appendix H. Except for the kidneys,
the activity uptake was less than 1 %ID/g tissue and this activity was eliminated after 24 hours. As expected from the lipophilicity studies the 64CuII-bispa complexes are rapidly excreted via the renal pathway, i.e. kidneys and urine. Furthermore, the biodistribution shows no signs for demetalation of the complexes in vivo. On the contrary, they appear to be highly stable and this is in agreement with the results of the serum assays analyzed by radio-SEC.
Figure 53. Biodistribution pattern of the 64CuII complex based on B1a at 5 min, 60 min,
and 24 h p.i. (four Wistar Kyoto rats per time point and compound, mean value ± standard deviation); graphs provided by Prof. Dr. Jens PIETZSCH.
Bloo d
HeartLungMuscleWAT BATBonSplee en Adren als Kidn eysLiverBrain Pancrea s ThymusThyroid
Harde rian
Testes Skin 0 1 2 3 4 5 6 7 8 9 10 5 min 60 min 24 h % ID /g 64CuII-B1a Stom ach Intes tine Feces Urine 0 20 40 60 80 100 %ID
Figure 54. Biodistribution pattern of the 64CuII complex based B1b at 5 min, 60 min, and 24 h p.i. (four Wistar Kyoto rats per time point and compound, mean value ± standard
deviation); graphs provided by Prof. Dr. Jens PIETZSCH. Bloo
d
HeartLungMuscleWAT BATBonSplee en Adren als Kidn eysLiverBrain Pancrea s ThymusThyroid
Harde rian
Testes Skin 0 1 2 3 4 5 6 7 8 9 10 5 min 60 min 24 h % ID /g 64CuII-B1b Stom ach Intes
tine Feces Urine
0 20 40 60 80 100 %ID