DMSO Increases Radioiodination Yield of Radiopharmaceuticals

A high-yield radioiodination method for various types of molecules is described. The approach employs DMSO as precursor solvent, a reaction ratio of 2–5 precursor molecules per iodine atom, 5–10 μ g oxidant, and a 10–25-μ l reaction volume. The solution is vortexed at room temperature for 1–5 min and progress of the reaction is assessed by HPLC. Radioiodinated products are obtained in ≥ 95% yield and meet the requirements for radiotracer imaging, biodistribution studies, and molecular and cellular biology research.


Introduction
Radioactive isotopes of iodine possess excellent characteristics for noninvasive imaging ( 123 I, 124 I), radiotracer studies ( 125 I), tumor therapy ( 125 I, 131 I), and molecular biology research.For many types of molecules, radioiodination presents problems of yield, purification, and stability of the radiolabeled product (Prusoff, 1959;Mannan et al., 1991;Tjuvajev et al., 1993;Van den Abbeele et al., 1996;Kumar et al., 2005).In a standard procedure, radioiodide is oxidized and added to the molecule to be labeled.The iodine cation then reacts with the active position (e.g.phenol, hydroxyl, tributyltin, benzamino or vinyl moiety) on the molecule.
During our studies, in which radioiodination of water-insoluble precursors in aqueous medium often produces low yields, we observed that dissolving these compounds in DMSO improves the efficiency of radioiodination.While the original purpose for the addition of DMSO was the solublization of water-insoluble compounds, we have since realized that the presence of DMSO dramatically increases the radioiodination yields of various water-soluble as well as water-insoluble organic and non-organic molecules.

General methods
Reagents were obtained from Sigma Aldrich Chemical Company.HPLC separations were performed on a reversed-phase Zorbax SB C 18 column, 9.4 × 250 mm (Agilent Technology) at a flow rate of 3 mL/min with UV absorption (Waters 486 detector) and Y-ray detection *Corresponding author.Department of Radiology, Armenise Building Room D2-137, 200 Longwood Avenue, Harvard Medical School, Boston, MA 02115.E-mail: amin_kassis@hms.harvard.edu;Telephone: (617) 432-7777; Fax: (617) 432-2419;.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form.Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.A stock solution of tributylstannyl-2′-deoxyuridine (SnUdR, 20 μg/μL DMSO) was prepared.Phosphate buffered saline (PBS, 10 μL, 0.01 M, pH 7.4), SnUdR (1 μL, 1 μg/μL DMSO, methanol, or water), and Na 125 I (0.5-3 μL) were placed in a reaction vial coated with 1, 3,4,10 μg).When dichloromethane was used as precursor solvent, SnUdR (1 μg/20 μL solvent) also adhered to the vial, and the rest of the conditions were the same.The mixture was vortex mixed at ambient temperature for 1 min.Progress of the reaction was checked by C 18 HPLC with isocratic elution (3 mL/min) using 75% buffer A (phosphate buffer, 0.05 M, pH 2.5) and 25% buffer B (methanol) for 16 min.
In our rapid DMSO synthesis, the surface of the reaction vial is coated with the oxidant Iodogen; the precursor, 5-tributylstannyl-2′-deoxyuridine is dissolved in DMSO (1 μg/μL); the reaction medium is 0.01 M PBS; and the total reaction volume is maintained within 12 μL to keep concentrations high.Figure 1 shows the HPLC profiles of the reaction and Table 1 presents the reaction conditions and radioiodination yields.For 125 IUdR, the retention time (t R ) of 8:24 min (gamma counter) (Fig. 1A) is consistent with that of 8:16 min (UV detector) for standard nonradiolabeled IUdR (Fig. 1D); the difference of 8 sec represents the transition time between the two detector cells.The radioiodination yield is greater than 99.8% (Fig. 1A).In this reaction, the amount of precursor should be twice that of radioisotope, 123 I/ 125 I (as NaI).For example, 1 μg SnUdR (1.9 × 10 −9 mol) and Na 125 I (74 MBq, 2 mCi, 0.9 × 10 −9 mol) give 99.8% 125 IUdR.Only 0.5μg SnUdR precursor (0.95 × 10 −9 mol) and 0.3 μg tributyltin (0.95 × 10 −9 mol) are left as impurities in the system, and the Iodogen (10 μg) remains attached to the surface of the vial.The whole process is simple and clean with no purification necessary to remove other radioiodinated products/species.Using this method, 125 IUdR has been prepared successfully for molecular biology, cell biology, genetic and imaging research. 125IUdR has also been synthesized in the presence of methanol (yield 90.5% -Fig.1B), dichloromethane (yield 82.1% -data not shown) and water (yield 18.9% -Fig.1C), all other conditions remaining the same (Fig. 1B and C).These lower yields may be a consequence of the incomplete solubility of the tin precursor during the synthesis.The radioiodinated quinazolinone derivative, ammonium 2-(2′-phosphoryloxyphenyl)-6iodo-4-(3H)-quinazolinone has been developed as a new antitumor radiopharmaceutical (Ho et al., 2002;Chen et al., 2006;Chen et al., 2007;Pospisil et al., 2007;Wang et al., 2007).This is a water-soluble compound that is hydrolyzed by various phosphatases (overexpressed extracellularly by tumor cells) to a water-insoluble compound, 2-(2′-hydroxyphenyl)-6-iodo-4-(3H)-quinazolinone, which is trapped within the extracellular spaces of solid tumors.The precursor SnQ 2-P is dissolved in DMSO (20 μg/μL) and diluted to 2μg/μL in DMSO, methanol, or water.When DMSO is the precursor solvent, the 125 I-labeled product IQ 2-P (t R = 9.23 min) is obtained in 99.3% yield (Fig. 2A).Similar high yields are obtained when the quinazolinone precursor is radioiodinated with 123 I or 131 I in the presence of DMSO (Table 1).With methanol as solvent, the labeling yield is only 11.6% (Fig. 2B) and, with water as solvent, the yield is 18.8% (Fig. 2C).Unlike SnUdR, the SnQ 2-P derivative is soluble in methanol-water and appears to be a true solution; the explanation for the consequent low labeling yield is still under investigation.A turbid suspension is obtained when SnQ 2-P is in water, and the low yield is, therefore, most likely due to the unequal distribution of the reactants.

Synthesis of radiolabeled ( 125 I) rhodamine 123 ( 125 I-Rhod)
Rhodamine 123, a lipophilic, permeant cationic fluorescent dye, has been used extensively as a mitochondrial stain in the study of cellular function.It is relatively nontoxic to healthy cells but displays selective toxicity in certain carcinoma cells in vitro and in vivo.These observations have prompted its radioiodination and evaluation of its tumor-targeting potential in vivo.Moonen et al. (Moonen et al., 1987) first radioiodinated rhodamine 123 with Iodogen for biodistribution studies, and the overall radiochemical yield of the reaction was 20%.Kinsey et al. (Kinsey et al., 1987) found that electrophilic radioiodination of rhodamine 123 using Iodogen or ChT leads to formation of the iodide salt in which the radioiodine is not covalently bound to the xanthene ring of the dye, since electrophilic iodination is greatly inhibited by the presence of the delocalized positive charge on the ring.Harapanhalli et al. (Harapanhalli et al., 1998) selected peracetic acid as the oxidizing agent and optimized the conditions for synthesis, including buffer, pH, time, and temperature.This group reported radiolabeling yields of 125 Irhodamine 123 between 40% and 45%.
Having analyzed the possible reasons for low radiolabeling yields of rhodamine 123, we have used Cu 2+ to protect the amine groups and to inhibit salt formation and ChT as oxidant.When the precursor I-Rhod is dissolved in DMSO, 125 I-Rhod (t R = 11.0 min) is obtained in 97.3% yield (Fig. 3A).Since the rhodamine molecule has two aryl rings, iodine labeling is favored at either or both positions that is/are ortho to the amine.During radioiodination, however, the concentration of procursor is much greater than that of radioisotope.Consequently, the probability for di-iodination, i.e.I 2 -Rho, is very low and the monoiodo-derivative is produced (Fig. 3A).Here again, when the solvents for the precurser are methanol and water, the labeling yields are only 29.9% (Fig. 3B) and 27.1% (Fig. 3C), respectively.Since methanol is a good solvent for I-Rhod, the cause of low yield with this solvent is unknown.Water is a poor solvent for I-Rhod, and this may have contributed to the low labeling yield.Activation of the enzyme telomerase (hTERT), a reverse transcriptase responsible for annealing hexanucleotide telomeric repeats (TTAGGG) to the ends (telomeres) of chromosomes, has been shown to be an important step in human epithelial-tumor evolution.There is a burgeoning interest in the use of polycyclic acridines to stabilize the G-rich singlestranded telomeric overhang in G-quadruplex DNA polymorphic forms; these agents have also been shown to inhibit telomerase at the micromolar level (Heald et al., 2002).Acridine, therefore, is a potential antitumor agent, and its radioiodination provides a useful tool for the study of its biologic function and radiation efficiency in vitro and in vivo.

Synthesis of radioiodinated (
When iodination of SnAcr is carried out in the presence of DMSO, 125 I-Acr (t R = 8.46 min) is obtained in 98.0% yield (Fig. 4A).Similar high yields (95.6% and 96.5%) are obtained when SnAcr is radiolabeled respectively with 131 I and 131 I (Table 1).Under similar conditions with methanol as solvent, the yield is 79.7% (Fig. 4B), and with water, the yield is only 40.1% (Fig. 4C).

Synthesis of radiolabeled ( 125 I) Bolton-Hunter reagent ( 125 I-BH)
Bolton and Hunter (Bolton, Hunter, 1973) developed a method of indirectly labeling proteins by conjugating them to a 125 I-containing acylating agent.This approach avoids contact between the protein and the oxidizing agent and preserves the biologic activity of the former.The method includes two steps: the first is radioactive labeling of BH and the second is the conjugation of BH and protein.The yield reported for the first step, however, is only 30% to 75% and a final absolute labeling yield of 13% to 53%.
When the BH reagent is dissolved in DMSO and the precursor solution is added to the radioiodination system, HPLC shows a t R peak of 9.23 min and the yield is 97.3% (Fig. 5A).DMSO is, therefore, an excellent solvent for radioiodination of the reagent.With methanol as solvent, the yield is 66.3% (Fig. 5B).This low yield is not, however, a consequence of the presence of free iodine since the t R of free iodine under these elution conditions is 3.22 min.
The radioiodination yield in the presence of dichloromethane is somewhat higher (86.3%) but not as high as that obtained in the presence of DMSO (Table 1).

Radioiodination ( 125 I) of immunoglobulin G (IgG)
Radioiodinated IgG has many uses in preclinical and clinical research.Various radiolabeling yields have been reported and this is usually ascribed to the radiolabeling conditions and/or the physical and chemical characteristics of the antibody molecule.In our current studies, we decided to determine whether the radioiodination yield of these proteins can also be improved by the presence of DMSO.
When mouse IgG is radioiodinated in the presence and absence of a small amount of DMSO (Fig. 6A and B), the radioiodination yield increases from 49.5% (no DMSO) to 99.7% (with DMSO).However, when we use the anti-mucin monoclonal antibody B72.3, known to target colon-adenocarcinoma cells grown subcutaneously in mice (Kassis et al., 1996), the radiolabeling yields with and without DMSO are both over 99% (Fig. 7A and B).The presence of DMSO during the radioiodination does not compromise the in vitro immunoreactivity of this antibody (data not shown).

Conclusion
Various types of precursors, including those with hydroxyl, tributylbenzene, and aminobenzene groups, were radioiodinated using DMSO, methanol, and water as alternative solvents.The data demonstrate that the addition of DMSO consistently led to quantitative radioiodination yields (95%-100%).