Preparation and biodistribution of 99mTc-IgG-HYNIC in normal rats

ORIGINAL PAPER NUKLEONIKA 2009;54(4):279−284 Preparation and biodistribution of 99mTc-IgG-HYNIC in normal rats Saeed Rajabifar, Mehdi Akhlaghi, Amir R. Jalilian, Fateme Bolourinovin, Behbood Maashkar, Mahbobe Talebimehrdar, Mahdieh Ghafouri Abstract. Human gamma globulin can be labeled by a direct or indirect method of radiotracer incorporated in a protein molecule. In this indirect method hydrazinonicotinic acid (HYNIC) is used which saves the structure and biological activity of the protein. Our goal was the efficient labeling of the human gamma globulin and evaluation of its biodistribution in different organs which can be used on experimentally induced infection causing inflammation. Immune globulin is mixed with s-hynic and IgG-hynic is developed using sidle A-lyzer and stored at –20°C which can be used at least for six months and then Sn-tricine kit is prepared which is used for 99mTc labeling. Efficiency of 99mTc-IgG-hynic labeling at pH 6.4 was very much dependent on ligand (hynic) and coligand (tricine) presence in the reaction mixture. Radiochemical purity was more than 90% in the kits prepared. Serum stability study showed no decomposition of 99mTc from the complex. The biodistribution studies showed the highest percentage ID/organ in the blood, liver and kidney, respectively. A human gamma globulin was successfully labeled through hynic to 99mTc by an indirect method with high radiochemical purity. Key words: 99mTc • HIG • HYNIC • IgG-hynic • infection • inflammation Introduction S. Rajabifar , M. Akhlaghi, A. R. Jalilian, F. Bolourinovin Nuclear Medicine Research Group, Agricultural, Medical and Industrial Research School (AMIRS), Moazen Blvd., Rajaeeshahr, P. O. Box 31485-498, Karaj, Iran, Tel.: +98 261 443 6397, Fax: +98 261 446 4053, E-mail: B. Maashkar Engineering College, Research and Science Unit, Azad University, Tehran, Iran M. Talebimehrdar Payam Noor University, Karaj, Iran M. Ghafouri Arak University, Arak, Iran Received: 28 April 2008 Accepted: 22 June 2009 Over the last decades several radiopharmaceuticals have been developed for the detection of infection and inflammation and some have found their way into clinical practice and are routinely used for evaluation of infectious and inflammatory diseases [7, 28, 38, 41]. Radiopharmaceuticals currently used for infection and inflammation are, 67galium citrate [23, 39, 50], radiolabeled leucocytes [17, 31, 40], labeled anti-granulocyte antibodies [8, 14, 49], radiolabeled non-specific human immunoglobulin G(HIG) [15, 26, 37, 45, 47, 50], chemotactic peptides [2–5, 18, 19, 21, 55], interleukines [13, 43, 48, 51–54], radiolabeled liposomes [16, 46] and Platelet factor 4 [34]. Initially, it was believed that human immunoglobulin was retained in inflammatory foci due to interaction with Fc gamma receptors [22], but studies have shown that radiolabeled HIG is retained in infectious foci by non-specific extravasation because of locally enhanced vascular permeability [20]. HIG has been tested for localization of musculoskeletal infection [35], in pulmonary infection [36], and abdominal inflammation [32]. In a comparative study it was shown that labeling HIG through hydrazinonicotinic acid (hynic) by using 99mTc has a similar characteristic to that of 111In-HIG which in most cases can replace 111In-labeled compound [15]. The radioisotope 99mTc is the radionuclide of choice in diagnostic nuclear medicine. Current design 280 Fig. 1. Conversion of 6 hydrazinonicotinic acid in presence of 99mTc and tricine. of new 99mTc diagnostic agents has concentrated on the development of target specific via the bifunctional approach, this design approach involves the 99mTc labeling of target or receptor specific molecules via bifunctional chelator which enables a rapid and efficient labeling of targeting molecules such as proteins [10] antibodies [26] peptides [9, 18] or other biomolecules with 99mTc via hynic. The 6-hydrazinonicotinic acid (Fig. 1) is a bifunctional molecule capable of bonding to lysine residues of peptides or proteins at one end and to 99mTc on the other, but modification of peptides or proteins is involved with succinimidyl 6-hydrazinonicotinate hydrochloride. The resulting modified molecule can be rapidly labeled with 99m technetium with a high radiolabeling yield. Experimental Human immunoglobulin G was obtaind from Baxter (Austria), slide A-lyzer dialysis cassette Pierce #66453 (USA), succinimidyl hydrazinonicotinic acid (s-hynic) from Sololink (USA), Millipore filter (USA), ITLC silica gel strips from Pal Gelman (USA) and the rest of the chemicals used were either from Merck (Germany) or Fluka (Switzerland). IgG conjugation The conjugation was done according to the method described by Abrams [1], briefly HIG is dissolved in a final concentration of 50 mg/ml, followed by extensive dialysis (sidle A-lyzer cassette, molecular weight cut off, 10 KD, this device is used for dialysing samples). Low molecular weight contaminant removal, buffer exchange, desalting and concentration can be accomplished and also maintaining the highest sample retention with this device for 24 h in a sterile container against 0.9% NaCl at 4°C under constant stirring, refreshing it 4 times and then 3 ml of the content with final concentration of 30 mg/ml is adjusted to 5 ml and then 1/10 vol. of 1.0 M NaHCO3 is added to IgG and sterilized by membrane filtration. 4 molar excess of freshly dissolved s-hynic was solubilized in 100 μl of dry DMSO and added dropwise to stirred IgG in 10 portions (portion/min) after incubation in the dark for 30 min. 3 ml of the solution is taken and the pH is adjusted to 6.4 using 0.15 M Na/acetate and injected into another sidle A-lyzer and kept over night against S. Rajabifar et al. Na/acetate buffer pH 6.4 at 4°C as described above. After drawing up the content, it is passed through a 0.22 μm filter and pH is adjusted to 6.4 using the same buffer and the final concentration is made to 4 mg/ml. The solution is divided into 0.5 ml aliqoutes and stored frozen at –20°C until used. Tricine-SnSO4 kits were prepared containing 100 mg tricine which is dissolved in sterile distilled water and oxygen is removed by applying N2/argon through it. Then 10 mg of SnSO4 in 0.5 ml of 2.0 M HCl is added to the tricine solution applying N2/argon again through it, the pH is then adjusted to 4.5 as this is the best pH to avoid turbidity and the total volume is made to 10 ml with 0.9% NaCl which is passed through 0.22 μm filter and divided into 10 vials and stored frozen at –20°C until used. Methods of 99mTc-IgG-hynic preparation An IgG-hynic vial is thawed and Sn-tricine kit is dissolved in 5 mL of sterile 0.9% saline solution. 50–60 μl of the freshly dissolved Sn-tricine is added to the IgG-hynic vial and then 1110 MBq of freshly eluted 99mTc eluate is added and incubated for 15 min at room temperature in the dark. Radiochemical purity of 99mTc-IgG-hynic After the incubation period 2–4 μl of the r (...truncated)