In vivo biodistribution and accumulation of 89Zr in mice
Introduction
Zirconium (Zr) was discovered by Klaproth in 1789 as zircon in the form of orthosilicate. It was first isolated as a metal by Berzelius in 1824. For a long time, Zr was used in an impure form, that of zircon, with industrial applications that included fabrication of fake diamonds, but at that time, Zr was of very little attention in the medical field [1].
During the atomic testing period of the 1959s and 1969s, 93Zr and 95Zr emerged as important fission products. Much of the early studies of the radiochemistry of zirconium were published in the 1960 National Academy of Sciences (NAS) report [2]. One major point in the publication was that ZrO(PO4)2 had a solubility product of 2.28×10−18 and that it could be precipitated in this form from 20% sulfuric acid. Zr has a common oxidation state of +4 and the hexa-aqua ion Zr(H20)6+4 only exists in very low Zr concentrations and in highly acidic aqueous solutions. In neutral solutions, in the absence of any complexing agents, Zr is mostly found as a polynuclear and polymeric form [3], [4].
The early interests in fission / fallout lead to animal studies to investigate the biological distribution of 93Zr and 95Zr in the 1959s and 1969s. These included the toxicity [5] and the biodistribution of 95Zr in rats or in mice [6], [7]. These works showed the high affinity of Zr to the bones by autoradiography and its low toxicity in rats.
The last decade has seen a rising interest for the use of 89Zr as a potential positron emission tomography (PET) isotope for the labeling of monoclonal antibody for in vivo cancer imaging [8], [9], [10], [11]. The long half-life of 89Zr (t1/2 = 78.41 h) is compatible with the relatively slow blood clearance of most IgGs used in radioimmunodiagnosis (t1/2=1–2 days). The slow blood clearance often means that the maximum tumor accumulation of an IgG at the tumor is around 3–5 days. The 89Zr tracer is commonly attached via a desferrioxamine (DFO) moiety conjugated to the antibody. Two main conjugation methods are in use. One deals with a succinic acid linker between the amine of the DFO and an amine of the antibody [8] and the other involves a p-isothiocyanatobenzyl-desferrioxamine derivative [12]. The latter method has the advantage of vastly simplifying the conjugation procedure using commercially available p-isothiocyanatobenzyl-desferrioxamine and a one-step method.
This advantageous half-life and conjugation strategy has led to the development and the coming clinical trials of 89Zr-DFO-J591 and 89Zr-DFO-trasuzumab at Memorial Sloan-Kettering Cancer Center [9]. Despite the interest, a number of questions have arisen regarding the stability of the Zr-DFO complex in a long term study in physiological conditions (on the order of days). An increasing contrast of the bones was observed in mice three days following the 89Zr-DFO-J591 injection (∼9% ID/g) [9], which was not detected at such extended time points with labeled 111In-DOTA-J591 or 177Lu-DOTA-J591 [13], [14]. Therefore, the postulated high stability of Zr-DFO chelate conjugated to the antibody does not match with the observed non specific uptake of 89Zr by the bones. It can be hypothesized that either the attachment of Zr-DFO to the antibody is not resistant enough after immunorecognition or that the Zr is transmetallated.
The present study attempts to address these questions using electrophoresis characterization of 89Zr solvated in different ionic conditions (in saline and in phosphate buffer saline) or 89Zr chelated by different biologically relevant species such as oxalate, citrate or DFO and looking at the biological fate of these species. The biodistribution, clearances and imaging of each injection species are presented here and discussed. A special focus is also given regarding the bone accumulation of 89Zr.
Section snippets
Materials and methods
All chemicals were purchased at Sigma-Aldrich (St. Louis, MO, USA).
Electrophoresis of the chemical solutions
Fig. 1 displays the results of electrophoresis. It can be seen that [89Zr]Zr-chloride (A) and [89Zr]Zr-phosphate (C) have hardly migrated from their initial point, showing a thin sharp peak nearby the origin. This is indicative of non-charged species. [89Zr]Zr-oxalate (B) showed a broader peak than [89Zr]Zr-chloride. The peak stretches towards the anode due to being a positively charged species. A slight migration of the activity is also apparent, towards the cathode. As expected, [89Zr]Zr-DFO
Discussion
The electrophoresis of [89Zr]Zr-chloride is in accordance with reported studies describing the polynuclear and polymeric forms of Zr, when hydrolyzed in the near neutral pH region [3], [4]. When solvated in acetate buffer at pH=6.5, Zr may be octachelated likely by hydroxides, water molecules and chlorides resulting in a colloidal form. By the effect of mass of the polymeric structure, the Zr species stagnate at the origin as if the overall charge was neutral, probably due to a low charge/mass
Conclusion
Despite the complexity of Zr coordination, the electrophoretic analyses provided detailed evidences of different zirconium preparations charges, salts or complexes.
This study also shows that weakly chelated 89Zr is a bone seeker. The observed rank order of bone uptake was chloride>oxalate>citrate≫DFO which is consistent with the denticity of the chelates and the stability of the respective complexes. The intravenous (i.v.) administration of [89Zr]Zr-phosphate resulted in little bone uptake
Acknowledgments
We would like to thank the following institutions: Geoffrey Beene Cancer Research Center of Memorial Sloan-Kettering Cancer Center (JSL); the Office of Science (BER), US Department of Energy (Award DE-SC0002456; JSL); Mr. William H. Goodwin and Mrs. Alice Goodwin and the Commonwealth Foundation for Cancer Research, the Experimental Therapeutics Center of Memorial Sloan-Kettering Cancer Center and the technical services provided by the MSKCC Small-Animal Imaging Core Facility supported in part
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