Experimental Study Of A Novel Inflammatory Imaging Agent Targeted To MIF

BackgroundTo quickly and exactly detect acute or subacute inflammatory lesions is a difficult subject in clinical. In most cases to determine the anatomical location of the inflammatory lesions is even more important than to identify of the type of infection pathogen. The fast and accurate positioning inflammatory lesions are very helpful to clarify the cause of inflammation and to make rapid effective treatment. Traditional diagnostic imaging technologies such as X-ray, CT and ultrasound imaging can not make accurate identification when the abscess had not yet formed. The radioisotope inflammatory imaging can detect and locate the inflammatory lesions, especially deep inflammatory lesions which is its advantage. So radioisotope inflammatory imaging is a most effective method of early diagnosis of inflammation or infection.The commonly used inflammatory imaging agents are ⁶⁷Ga-citrate, ¹¹¹In-WBC, ^99mTc-HMPAO labeled leukocytes, ^99mTc-or ¹¹¹In-IgG, radiolabeled liposome, and so on, but all of them have some shortcomings and deficiencies. For example: the commonly used radiolabeled-WBC imaging need a large number of human blood, that may have the risk of blood pollution and cross infection during isolation ,which limits its use in clinical. The radiolabeled non-specific immunoglobulin imaging is easily to use, but there are non-specific deposition in blood, liver, spleen, and kidney. In recent years, there are many new types of inflammatory imaging agent research reported along with the medicine and pharmaceutical development. For example: the radiolabeled anti-granulocyte monoclonal antibody or antibody fragment, radiolabeled IL-1, 8, radiolabeled the chemical synthesized peptides of WBC receptor and ciprofloxacin. However, some kinds of specific and sensitive deficiencies still existed, which limit their clinical application. Therefore, seeking a new, specific and sensitive inflammatory imaging agent is currently urgent in clinical.With the rapidly development of immunology and other basic medical research, people begin to know more and more about the effect of cytokines during inflammation developing. The significance of cytokines in diagnostic and treatment inflammation has been cause a highly attention. In recent years, macrophage migration inhibitory factor (macrophage migration inhibitory factor, MIF) has been confirmed to be an important inflammatory response regulator. MIF can promote macrophage accumulation, proliferation, activation, adhesion, and phagocytosis in the local of inflammation, it also can promote the formation of a variety of cytokines, can offset the immune inhibition by corticosteroid hormones for other inflammatory cytokines. MIF plays an important role in the local or systemic inflammation and immune responses, and is a key media in systemic inflammatory response syndrome. The study of anti-inflammatory drugs targeted MIF has become a hot spot in medical research at present. The purpose of this study is to provide experimental basis for clinical application of the MIF targeted inflammation imaging agent by investigating the synthesis, preparation, biodistribution and dynamic imaging using radioisotope labeled anti-MIF monoclonal antibody.Method1 .Radiolabeled anti-MIF McAb was oxidized by Iodogen with 125-I (or 131 -I) . The stability was detected by investigating the radiochemical purity of radioiodine labeled anti-MIF McAb and its control antibody in the 37℃storage at different times. The immunological activity of radioiodine labeled anti-MIF McAb was measured by ELISA.2. Mice were induced inflammation by injecting of S.aurous, E.coli and turpentine oil. When the swelling of the muscle was apparent, the radiopharmaceuticals were injected .The mice of each group were sacrificed at different time after the injection. A sample of 1ml blood was obtained. The whole infected tissue, the control lateral muscle, heart, liver, spleen, kidney, lung and the sample of bone were taken, weighted, counted for radioactivity in a gamma counter and then calculated the tissue concentrations which expressed as percentage injected dose per gram %ID/g,T/NT.3. In order to make sure that the high intake of radiolabeled anti-MIF antibody in inflammatory tissue is caused by high expression of MIF in locus; the expression of MIF in locus was analyzed by RT-PCR and immunohistochemistry.4. Whole-body images were obtained with storage phosphor screen. Serial images were performed at 24h, 48h, and 72h after injection. The anesthetized mice were placed on the storage phosphor screen plate with the ventral side facing the plate, in subdued light. The plate was exposed to a mouse for 45 minutes. At cessation of exposure, the plate was immediately covered with an opaque plastic sheet, then transferred to the scanner, and scanned by typhoon trio + (laser red 633 nm, pixel size 200 mcrons, phosphor mode: best sensitivity).Results1. Radioiodine labeled antibody was separated from free iodine using a size exclusion column (Sephadex G-25, Pharmacia). The specific activity of ¹²⁵I-anti-MIF McAb is 29.56 GBq/μmol. The radiochemical purity is 98.25% (paper chromatography). The specific activity of ¹³¹I-anti-MIF McAb is 29.56 GBq/μmol, of ¹³¹I-IgG is 30.12 GBq/μmol. The radiochemical purity of ¹³¹I-anti-MIF McAb is 97.10% (paper chromatography), and that of ¹³¹I-IgG is 97.17% (paper chromatography).The immunological activities of radioiodine labeled anti MIF McAb was proved through ELISA (Enzyme-linked immune -sorbent assay).2. ¹²⁵I-anti-MIF McAb shortly transited from the peritoneal cavity to the circulation after intraperitoneal injection, it went up and reached a zenith at 4 hours post injection, and then the activity in the blood went down quickly. The change of activity in the heart and lung was the same as blood. The biodistribution of ¹²⁵I-anti-MIF McAb showed the highest uptake and the lowest decrease in the inflammatory tissue. The activity in the blood was higher than the kidney, liver, spleen, heart, and lung. Peak uptake in the kidney, liver, spleen occurred around 30 minutes, followed by gradual clearance over time. This indicated that the product of ¹²⁵I-anti-MIF McAb was excreted from kidney or swallowed by reticuloendothelium of liver and spleen, resulting in deiodination; the activity of ¹²⁵I-anti-MIF McAb in the inflammatory tissue increased gradually for three inflammation models. The highest uptake was S. aureus group and the lowest was E. coli group. In all three groups, T/NT was >3 at 4h post injection and increased continually in the whole observed period: T/NT ratio was >7 at 48h and >9 at 72h Mice with the left thigh inflammation were injected intravenously via the tail vein by ¹³¹I-anti-MIF McAb or ¹³¹I IgG, respectively. It shows that the ¹³¹I-anti-MIF McAb group T/NT ratio was > 6 at 24h, and increased continually, T/NT ratio was > 9 until 72h, but the ¹³¹I-IgG group T/NT ratio was > 6 at 48h and > 8 at 72h. The highest uptake happened in the ¹³¹I-anti-MIF McAb group (p<0.05). The T/B ratios for the ¹³¹I-anti-MIF McAb group werel.41±0.031, 1.53±0.018, 2.58±0.025 at 24h, 48h and 72h after injection, respectively. These ratios were significantly higher than that of ¹³¹I-IgG group (p<0.05).3. Result of RT-PCR showed that the expression of MIF mRNA in normal tissues were little changes at 24h, 48h and 72h. However, there was a 3-fold increase in MIF mRNA expression in inflammatory tissues at 24h compared with normal tissues (p<0.05). There was a 2-fold increase in MIF mRNA levels in inflammatory tissues at 48h compared with normal tissues (p<0.05). Immunohistochemistry result showed that the localization of MIF protein in tissues specimens from inflammatory mouse at 24h, 48h, and 72h after focal inflammation occurred. MIF was negative expressed in normal tissues. However, there was a significant increase in MIF expression in inflammatory specimens according to the time after focal inflammation occurred in the first 48h and then decreased at 72h. MIF-expression localized among the inflammatory cells strongly after focal inflammation occurred.4. After ¹²⁵I-anti-MIF McAb were injected intravenously via the tail vein. Whole-body autoradiography showed that all inflammation foci could be visualized clearly from 24h after injection, but after 48h images were much clearer in accordance with the high T/NT ratio. The radioactivity was the highest in S. aureus lesion, average in turpentine lesion, and the lowest in E.coli lesion. The whole-body autoradiography images of the ¹³¹I-anti-MIF McAb and ¹³¹I-IgG at 24h, 48h and 72h after injection showed that both radiotracers focally increased uptake in the inflammatory muscles beginning at 24h. Compared analysis of the scintigrams of three times points showed that the ¹³¹I-anti-MIF McAb group had much more clear images than the ¹³¹I-IgG groups which is in accordance with the high T/NT ratio (p<0.05).Conclusion1. Radioiodine labeled anti-MIF McAb made from Iodogen method is of high radiochemical purity and with high stability.2. Radioiodine labeled anti-MIF McAb achieve very high target to background ratio and has relatively low level of accumulation in non-target tissues, it possess a high relations with local expression of MIF.3. Whole-body autoradiography showed that all inflammation foci could be visualized clearly by storage phosphor screen. This study demonstrates the ability of radioiodinated anti-MIF McAb to measure in vivo inflammatory events represented by high expression of MIF and suggests that radiolabeled anti-MIF McAb warrants further investigation as a potential inflammation-seeking agent for imaging to detect inflammatory disorders.