Reaction SMILES: [131I:1][131I].[CH3:3][N+:4]([CH2:7][CH2:8][O:9][P:10]([O:13][CH2:14][CH2:15][CH2:16][CH2:17][CH2:18][CH2:19][CH2:20][CH2:21][CH2:22][CH2:23][CH2:24][CH2:25][CH2:26][CH2:27][CH2:28][CH2:29][CH2:30][CH2:31][C:32]1[CH:37]=[CH:36][C:35](I)=[CH:34][CH:33]=1)([O-:12])=[O:11])([CH3:6])[CH3:5]>>[CH3:3][N+:4]([CH2:7][CH2:8][O:9][P:10]([O:13][CH2:14][CH2:15][CH2:16][CH2:17][CH2:18][CH2:19][CH2:20][CH2:21][CH2:22][CH2:23][CH2:24][CH2:25][CH2:26][CH2:27][CH2:28][CH2:29][CH2:30][CH2:31][C:32]1[CH:37]=[CH:36][C:35]([131I:1])=[CH:34][CH:33]=1)([O-:12])=[O:11])([CH3:6])[CH3:5]
Reactant: C[N+](C)(C)CCOP(=O)([O-])OCCCCCCCCCCCCCCCCCCc1ccc(I)cc1
Reactant: [131I][131I]
Product: C[N+](C)(C)CCOP(=O)([O-])OCCCCCCCCCCCCCCCCCCc1ccc([131I])cc1

Procedure:
Radioiodination of stable NM404 with 124I-sodium iodide is routinely achieved by modification of an ammonium sulfate-mediated isotope exchange reaction reported by Mangner and recently optimized for NM404 in our lab. Exchange reaction methodology has been used effectively for initial human trials with NM324, the predecessor of NM404 and is currently being used for preclinical studies and the human lung cancer trial. Briefly, a 2-ml glass vial is charged with 10 mg of ammonium sulfate dissolved in 50 μl of deionized water. Fourteen 2 mm chemical resistant glass beads are added, a Teflon lined septum and screw cap are added and the vial gently swirled. A solution of 20 μg (in 20 μl of ethanol) of stock NM404 is added followed by aqueous sodium iodide (124, 1-5 mCi) in less than 30 μl aqueous 0.01 N sodium hydroxide. The isotope syringe is rinsed with three 20-μl portions of ethanol. The reaction vile is swirled gently. A 5-ml disposable syringe containing glass wool in tandem with another 5-ml charcoal nugget filled syringe with needle outlet are attached. The glass wool syringe acts as a condensation chamber to catch evaporating solvents and the charcoal syringe traps free iodide/iodine. The reaction vessel is heated in a heating block apparatus for 45 minutes at 150° C. Four 20 ml volumes of air are injected into the reaction vial with a 25-ml disposable syringe and allowed to vent through the dual trap attachment. The temperature is raised to 160° C. and the reaction vial is heated another 30 minutes. After cooling to room temperature, ethanol (200 μl) is added and the vial swirled. The ethanolic solution is passed through a pre-equilibrated Amberlite IRA 400-OH resin column to remove unreacted iodide. The eluent volume is reduced to 50 μl via a nitrogen stream (use charcoal syringe trap) and the remaining volume injected onto an HPLC silica gel column (Perkin Elmer, 3 μm×3 cm disposable cartridge column eluted at 1 ml/min with hexane/isopropanol/water (52:40:8)) for purification. Final purity is determined by TLC (plastic backed silica gel-60 eluted with chloroform-methanol-water (65:35:4, Rf=0.1). The HPLC solvents are removed by rotary evaporation and the resulting radioiodinated NM404 is solubilized in aqueous 2% pharmaceutical grade Polysorbate-20 (0.1 μl/mg of compound). The ethanol is removed under vacuum and the residue dissolved in sterile water to give a final solution containing no more than 2-3% Polysorbate-20. Sterilization will be achieved by filtration through a sterile 0.2 μm filter unit. Each of the solutions will be tested for pyrogens using the Limulus Amebocyte Lysate test kit. This is the same procedure currently employed for the preparation, purification and sterile formulation of 1-131-labeled NM404 for lung cancer patient studies. The drug Master Formulation Card and Product Preparation Checklist are included in the supplemental section of this proposal. We have performed this radioiodination hundreds of times with either iodine-131 or 125 and typically achieve radiochemical yields ranging from 60-90% (isolated pure product) with specific activities of exceeding 130 mCi/μmol of NM404. This method has afforded sufficient pure injectable 131I-NM404 for our ongoing preclinical and human trials. We have not had a labeling failure to date. Moreover, and relevant to the current proposal, we have recently successfully radiolabeled NM404 with iodine-124 sodium iodide (5 mCi) from Eastern Isotopes in >60% isolated radiochemical yield. Although acceptance criteria require >95% radiochemical purity, results typically exceed 99% since the entire reaction mixture is subjected to preparative HPLC purification.