Original ArticlesSynthesis, characterization, and evaluation of a novel bifunctional chelating agent for the lead isotopes 203Pb and 212Pb
Introduction
Continued interest exists in the area of synthesis of bifunctional chelating agents (BCAs), their conjugation to monoclonal antibodies (mAb) and peptides, and their subsequent use for sequestering radioactive metal ions for use in radioimmunotherapy (RIT) and radioimmunoimaging (RII) applications 1, 5, 34. Chelating agents must meet strict requirements to be useful for such applications, the most important being thermodynamic and kinetic stability of the metal-chelate complex in vivo 11, 20. The wide variety of radionuclides utilized for imaging and therapy, from lanthanides and actinides to different transition metals, requires the design of ligands suitable for the particular radionuclide or group of radionuclides 22, 36.
Isotopes of Pb(II) have been of some interest; however, the absence of a kinetically stable BCA for Pb(II) has hampered its in vivo use. 203Pb has a γ-emission that is ideal for single photon emission computerized tomography (SPECT) (31). In addition, 212Pb is a source of highly cytotoxic α-particles via its decay to its 212Bi daughter and has been proposed for RIT as an “in vivo generator” system 15, 32. Acyclic polyaminocarboxylate chelates such as ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) are clearly far too labile to be of any practical use for RIT or RII (33). Macrocyclic polyaminocarboxylate chelates such as 1,4,7,10-tetraazacyclodocane-1,4,7,10-tetraacetic acid (DOTA; Figure 1 ) have been utilized and demonstrate great in vivo stability for lanthanides such as 90Y and 177Lu 27, 29, 38. Animal studies with mAb B72.3 conjugated to a C-functionalized DOTA (Fig. 1) and labeled with 203Pb proved this complex to be adequately stable in vivo, with low accretion in the bone and kidneys. In addition, excellent tumor imaging of xenografts was obtained in athymic mice 120 h postinjection 25, 31. The exceptional kinetic stability of Pb[DOTA]2− to metal ion release, with a half-life of dissociation at pH 7 on the order of years, has been reported (17). However, acid lability of this complex may prove a source of toxicity upon internalization and metabolic processing.
Therapeutic applications for radioisotopes of lead include the use of 212Pb as an “in vivo generator” for 212Bi. 212Bi emits a highly cytotoxic α-particle; however, RIT applications with this isotope may be limited by its half-life of 60.6 min. The short half-life of 212Bi could effectively be lengthened by chelation of the parent 212Pb radionuclide (t1/2 = 10.6 h) to a mAb, thereby effectively extending the available delivery time of the 212Bi daughter to tumor cells. Development of such a system has met with many challenges. Although the metal complexes 203Pb[DOTA] and 206Bi[DOTA] were found to be stable to dissociation in the pH range of 4–10 (17), when 212Pb[DOTA] undergoes decay to 212Bi[DOTA], approximately 36% of the bismuth isotope formed has been reported lost from the ligand. This loss of bismuth ion from the ligand was attributed to the internal conversion of the γ rays emitted by the excited 212Bi nuclide (26). This observation was also demonstrated in animal studies with tumor bearing mice treated with 212Pb-labeled mAb-DOTA conjugates. Severe bone marrow toxicity that was either directly fatal or produced a very narrow therapeutic window was observed in most cases 15, 32.
Observations of a 212Pb/212Bi system with an analogous ligand 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-(methylene-phosphonic acid) (DOTMP) indicated that this complex was a bone-seeking agent and might well have applications in palliation therapy (13). However, the results of this study, coupled with one other report that examined the metal complex decay chemistry of the formed radio-bismuth ion with a similar phosphorus DOTA analog, tend to disqualify these ligands as suitable for 212Pb RIT (39). Thus, the development of a suitable BCA that can withstand the decay from 212Pb to 212Bi and maintain the complex of the daughter radionuclide remains a challenge in the use of 212Pb as an in vivo generator for 212Bi.
Our laboratory continues to be interested in the development of new BCAs for Pb(II), for both radioimmunotherapeutic and radioimmunodiagnostic applications. Although use of a C- or N-functionalized DOTA for Pb(II) has been found to be stable in vivo, the syntheses of these ligands can be very involved 8, 23, 27. Alternate entries into the use of bifunctional DOTA reagents have made use of one of the carboxylates to either directly link this ligand to protein or indirectly link it to protein by derivativation of the carboxylate to introduce a linking moiety. In either scenario, the amide formed in the process serves as the final metal ion coordination site 19, 30, 35.
One of the challenges to the use of the Pb(II) radionuclides is the issue of acid catalyzed dissociation of the radiometal from the ligand. Although this is not a problem when the mAb is bound to the exterior of the targeted cell, after internalization, the acid environment during metabolic processing of the protein may pose an additional obstacle to maintaining the Pb(II) complex (37). The two therapeutic studies with 212Pb in animal models employing internalizing mAbs have noted bone marrow toxicity 15, 32. In the case of one model system, the related experiment with 212Bi did not exhibit this type of toxicity (16), whereas the data for the other model has yet to be generated. The source of this toxicity has been proposed to originate from the release of 212Pb from the complex after internalization with the +2 ion then trafficking from the cell. At very low concentrations, Pb(II) is actually transported by red blood cells and transported to the bone 12, 32. In effect, the 212Bi effectively would continuously be delivered to the bone and serve as a source of toxicity.
The ligand based on the cyclen foundation with four carbamoyl methyl pendent arms (TCMC; Fig. 1), rather than carboxylates, has also been found to form metal complexes that are exceptionally inert to metal ion release, including Pb(II) 2, 4, 24. Replacement of the carboxylates with amides should then reduce the propensity for acid catalyzed dissociation of Pb(II) while providing a stable complex for this element. Although it seems doubtful that this ligand will have significant effect on controlling the decay event chemistry, the control or even elimination of the source of toxicity arising from 212Pb departing from cells postinternalization would be advantageous. This may then provide a potential range of therapeutic applications for this isotope; that is, rapid targeting coupled with rapid internalization would insure α-emissions occurring within the cell, thus effecting cell death.
To this end, we have chosen to investigate the potential application of such a C-functionalized cyclen-based macrocycle with carbamoyl methyl pendent arms for RII and RIT. An examination of the relative lability of the Pb(II) complex of DOTA versus that of the analogous tetraamide through an isotope exchange experiment provided the impetus to proceed to the BCA that was derivatized with a 4-nitrobenzyl group in order to produce a bifunctional ligand. In our laboratory, the chemistry for a C-functionalized DOTA derivative had been previously developed (23). Thus, the precursor substituted macrocyclic tetraamine was readily available to prepare this ligand with four carbamoyl methyl pendent groups in place of the acetate pendent groups.
Herein, we report effects of pH on the isotopic exchange of the Pb(II) DOTA complex and the tetraamide analog and the synthesis of the novel bifunctional chelate 2-(4-isothiocyanotobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamoyl methyl)-cyclododecane (4-NCS-Bz-TCMC). The bifunctional ligand obtained has been conjugated to a mAb (CC49) and radiolabeled with 203Pb. Comparative serum stability and immunoreactivity studies of the radioimmunoconjugates 203Pb[TCMC]-CC49 and 203Pb[DOTA]-CC49 have been performed.
Section snippets
General
All materials used in the ligand syntheses were of reagent grade and did not require purification. 4-NCS-Bz-DOTA was prepared as previously described (23). The ligand DOTA was purchased from Parish Chemicals (Provo, UT USA), and TCMC was a generous gift from Dr. R. Hancock. Chromatography was performed on silica gel 60, 220–440 mesh ASTM (Fluka, Ronkonkoma, NY USA). Thin-layer chromatography was performed on silica gel 60 F-254 plates (EM Reagents, Darmstadt, Germany).
1H and 13C nuclear
Results and discussion
The process of developing a new BCA that would address the acid lability issues associated with internalization of a Pb(II) radiocomplex directly hinged upon obtaining evidence that replacing the carboxylates of DOTA with primary amides resulted in a decrease in the dissociation of the Pb(II)-ligand complex. One possible method by which one might demonstrate that such a condition has been met is through evaluation of isotopic exchange over the requisite pH range.
The results of the experiment to
Conclusion
Isotopic exchange experiments with the DOTA and TCMC ligands indicated that the Pb[TCMC]2+ complex was less labile to metal ion release than Pb[DOTA]2− at pH 3.5 and below. This result prompted us to synthesize a bifunctional TCMC and further evaluate its potential for use in RIT and RII. The novel BCA for radioisotopes of Pb(II), 4-NCS-Bz-TCMC, has been synthesized in good yield from the requisite C-functionalized tetraaza macrocycle. In addition to increased stability to Pb2+ ion release at
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