Tissue Distribution of 125I-human Nonspecific Polyclonal IgG in Normal and Induced Inflammation Mice

Authors

Abstract

Many different radiolabeled antibodies have been used for radioimmunotherapy and radioimmunoscintigraphy of human diseases in animal experiments. In order to study the in vivo tissue distribution of antibody, we labeled human nonspecific polyclonal IgG with Na125I using chloramine-T method. An animal model was developed by injecting turpentine in the posterior left thigh of Balb/c mice. Tissue distribution of 125I-IgG was assessed in normal and induced inflammation mice. Although labeled IgG accumulated in normal tissues such as liver, spleen and blood, its localization in inflammatory left thigh was significantly different from normal right thigh. Thus, by using the radiolabeling methods of IgG in order to increase the target-to-background ratio, antibody scintigraphy could be suggested as a powerful diagnostic tool for inflammation.

Keywords


Tissue Distribution of 125I-human Nonspecific Polyclonal IgG in Normal and Induced Inflammation Mice

Iranian Journal of Pharmaceutical Research 2002, 1:55-59
Received: April 2002
Accepted: July 2002

Original Article

Tissue Distribution of 125I-human Nonspecific Polyclonal IgG in Normal and Induced Inflammation Mice

Soraya Shahhosseini*a, Mohammad Hossein Babaeib, Reza Najafib

aDepartment of Pharmacetical Chemistry, School of Pharmacy, Shaheed Beheshti University of Medical Sciences and Health Services, Tehran, Iran. bRadioisotopes Division, Nuclear Research Center, Atomic Energy Organization of Iran, Tehran, Iran.
* sshahhosseini@irimc.org

Abstract

Many different radiolabeled antibodies have been used for radioimmunotherapy and radioimmunoscintigraphy of human diseases in animal experiments. In order to study the in vivo tissue distribution of antibody, we labeled human nonspecific polyclonal IgG with Na125I using chloramine-T method. An animal model was developed by injecting turpentine in the posterior left thigh of Balb/c mice. Tissue distribution of 125I-IgG was assessed in normal and induced inflammation mice. Although labeled IgG accumulated in normal tissues such as liver, spleen and blood, its localization in inflammatory left thigh was significantly different from normal right thigh. Thus, by using the radiolabeling methods of IgG in order to increase the target-to-background ratio, antibody scintigraphy could be suggested as a powerful diagnostic tool for inflammation.

Keywords: Immunoscintigraphy; Turpentine; Immunoglobulin (Ig); Percent Injected Dose per gram tissue (% ID/g).

Introduction

Various antibodies have been labeled with different radionuclides and used for radioimmunoimaging (1-6). The most important limitation of this application is that the labeled antibodies accumulate not only in the target but also in normal tissues (nontarget). To overcome this, several methods have been proposed (7-11). Most of the investigations are focused on improving radiolabeling methods (12) resulting in reduction of background activity and enhancement of target-to-background ratio. Radiolabeled leukocytes and a few radiopharmaceuticals such as 67Ga-citrate and 99mTc-labeled antigranulocytes antibody preparations can be used to detect infectious and inflammatory foci (13). Since human nonspecific polyclonal IgG accumulates in inflammatory and infectious foci, in this paper, we studied the tissue distribution of whole IgG labeled with Na125I using chloramine-T method in normal and induced inflammation mice.

Experimental

125I as Na125I with specific activity of 100 m ci/m l in NaOH was purchased from Amersham. IgG was prepared from human plasma (14) and was iodinated by Chloramin-T method (15). Briefly, 100 m g of IgG was incubated with 2 mci of Na125I and 25 m g of chloramin-T for 1min. Free radioactive iodine was separated by Sephadex G-50 column chromatography. Specific activity of 125I- IgG was estimated to be between 5-10 mci/mg.

 

An animal model was developed injecting 40 m l of turpentine in the posterior left thigh of Balb/c mice weighing approximately 25g. Mice were left for 48 h in normal condition to develop the inflammation foci. 100 m ci / 0.1 ml / 20 m g 125I -IgG / mouse was injected through tail veins into mice. Six mice from each group were killed with ether for determination of tissue radioactivity at times 2, 4, 6, 24, 30, and 48 h post administration of 125I-IgG. Selected organs (blood, spleen, liver, stomach, intestine, kidney, left thigh, right thigh, and bone) were removed and placed into pre weighed tubes and radioactivity was measured. The percent of injected dose per gram tissue (% ID/g tissue) was determined. The total injected dose was calculated by measuring syringes before and after injection to each animal.

Statistical analysis

All values were expressed as mean ± standard deviation (Mean ± SD) and data were compared using student T-test. Statistical significant was defined as P< 0.05.

 

Results and Discussion

The result of separation of free radioactive iodine from labeled IgG by Gel filtration chromatography is shown in figure 1. Labeling efficiency and specific activity were 75% and 5-10 m ci / m g respectively.

 Figure 1. Gel filtration of labeled protein ( Sephadex G-25)

The results of tissue distribution in normal and induced inflammation mice at 2, 4, 6, 24, 30 and 48 h post injection of 125I-IgG (n=6 per time point) are shown in figures 2 and 3.

Figure 2. Histograms showing biodistribution (% ID/g) at 2, 4, 6, 24, 30, and 48 hours post injection of 125I -IgG in normal mice

Figure 3. Histograms showing biodistribution (% ID/g) at 2, 4, 6, 24, 30, and 48 hours post injection of 125I -IgG in induced inflammation mice

There was no significant difference between the radioactivity of left and right thigh in mice without inflammation after injection of 125I -IgG (Table1). In inflammation bearing mice, radioactivity values of left and right thigh were significantly different (P<0.05) at definite times (6, 24, 30, and 48 h) after injection of 125I -IgG (Table 2).

Table 1. %ID/g in normal mice at 2, 4, 6, 24, 30, 48 h post injection of 125I ?IgG

Organ

2 h

4 h

6 h

24 h

30 h

48 h

Blood

14.40± 4.48

26.7 ± 4.3

17 ± 2.94

8.8 ± 0.1

2.3 ± 0.5

1.32± 0.72

Spleen

4.2 ± 0.36

5.23± 0.76

3.4 ± 0.71

2.03± 0.5

0.48± 0.13

0.52± 0.16

Liver

6.6 ± 1.15

7.3 ± 0.8

5.85± 0.68

3.1 ± 0.85

1.16± 0.57

1.25± 0.4

Stomach

6.13± 0.98

13.77± 4.81

5.48± 1.95

2.03± 0.29

1.34± 0.39

0.51± 0.2

Intestine

3.15± 1.19

5.95± 0.35

2.85± 0.5

2.08± 0.37

0.66± 0.22

0.53± 0.12

Kidney

7.05± 0.57

23.85± 3.85

6.8 ± 1.06

3.2 ± 0.29

2.2 ± 0.57

5.42± 0.11

Left thigh

1.43± 0.45

2 ± 0.5

1.53± 0.28

1.35± 0.05

0.43± 0.18

0.28± 0.1

Right thigh

1.5 ± 0.31

2.3 ± 0.36

1.25± 0.18

1.25± 0.05

0.56± 0.19

0.22± 0.05

Bone

2.1 ± 0.54

2.67± 0.19

1.57± 0.21

1.4 ± 0.25

0.31± 0.07

0.28± 0.03

Data are expressed as percent injected dose per gram. Mean ± s.d. for 6 mice 

 

Table 2. %ID/g in induced inflammation mice at 2, 4, 6, 24, 30, 48 h post injection of 125I -IgG

Organ

2 h

4 h

6 h

24 h

30 h

48 h

Blood

18.57± 3.04

28.82± 3.53

12.75± 1.64

7.58± 0.8

2.13± 0.32

1.93± 0.64

Spleen

3.33± 1.08

13.17± 1.72

2.63± 0.62

1.83± 0.33

0.49± 0.26

0.49± 0.21

Liver

5.15± 1.31

12.1 ± 2.72

4.6 ± 1.37

1.97± 0.7

1.44± 0.59

1.47± 0.12

Stomach

4.68± 1.11

24.63± 5.75

2.63± 0.7

2.2 ± 0.57

0.92± 0.45

0.82± 0.4

Intestine

3.5 ± 0.54

8.42± 1.81

1.9 ± 0.3

1.32± 0.33

0.68± 0.32

0.68± 0.24

Kidney

5.8 ± 0.73

18.1 ± 3.82

6.8 ± 1.74

4.53± 0.78

2.18± 0.84

5.27± 1.86

Left thigh

2 ± 0.54

6.87± 0.69

6 ± 1.22

5.35± 0.65

1.2 ± 0.28

1.03± 0.69

Right thigh

1 ± 0.48

2.69± 1.12

2 ± 0.52

1.77± 0.06

0.35± 0.06

0.39± 0.15

Bone

2.81± 0.29

3.28± 0.4

1.83± 0.35

2.83± 0.66

0.34± 0.07

0.36± 0.1

Data are expressed as percent injected dose per gram. Mean ± s.d. for 6 mice

The maximum radioactivity of normal tissues and inflammatory (left) thigh was achieved 4 h after injection of 125I -IgG, and reduced dramatically after 4 h (e.g. the % ID/g blood felled from ~29% at 4 h to 13% at 6 h and 7.5% at 24 h). The reduction of radioactivity in inflammatory (left) thigh was not as much as the reduction of radioactivity in normal tissues (the % ID/g dropped from 7% at 4 h to 6% at 6 h and 5% at 24 h). The best inflammatory thigh-to-normal tissue ratios were obtained at 24hr post administration of 125I -IgG (Table 3).

Table 3. Inflammatory (left) thigh-to-normal tissue ratios in inflammation bearing mice after injection of 125I ?IgG

Organ

2 h

4 h

6 h

24 h

30 h

48 h

Blood

0.1

0.23

0.47

0.7

0.56

0.53

Spleen

0.6

0.52

2.28

2.9

2.4

2.1

Liver

0.3

0.56

1.3

2.7

0.83

0.7

Stomach

0.4

0.27

2.28

2.43

1.3

1.25

Intestine

0.57

0.81

3.15

4.05

1.76

1.5

Kidney

0.34

0.37

0.88

1.18

0.55

0.19

Right thigh

2

2.55

3

3.02

3.42

2.66

Bone

0.7

2.09

3.27

1.89

3.5

0.9

Many studies using antibodies have utilized radioiodine (125I, 123I, 131I) as the tracer to follow the distribution of antibodies in animals (16-21). The beta emission and poor imaging characteristics of 131I have limited its use. Although 123I with half-life of 13 h and single gamma with 159 Kev (83-85 %) is the radionuclide of choice for most imaging tracer studies, transportation problems and cost have limited the use of 123I radionuclide. 125I, a cheap, widely available, and easily detectable radionuclide, has been used mainly as a label for biodistribution studies (22). In this study, we labeled human nonspecific polyclonal IgG with Na125I by chloramine-T method, which is an efficient method for the direct substitution of 125I into the tyrosyl residues of protein (23).

As shown in Figure 3, labeled IgG is accumulated in the inflammation site. There was significant difference at definite times between the radioactivity of left and right thigh after injection of 125I-IgG in induced inflammation mice. Levels of activity in most normal tissues, were high and based on the results, inflammation can be visualized within 24 h after injection of 125I-IgG (Table1). Following administration of radioiodinated antibodies, in vivo deiodination occurs. Deiodination increases radioactivity in blood, stomach, intestine, kidney, and decreases the target-to-nontarget radioactivity ratios (12). In spite of localization of 125I-IgG in normal tissues, its accumulation in inflammatory site is high enough to be recognized from background. Investigations have shown that the accumulation of IgG in inflammatory and infectious foci results from increased vascular permeability (24). The clinical results for immunoscintigraphy with radiolabeled human nonspecific polyclonal IgG indicate the potential value of this method for the detection of infectious and inflammatory processes (25-26). However, a general difficulty in the application of radiolabeled antibodies is the excessive accumulation in normal organs such as liver and the slow clearance of the label from circulation. To overcome this, several methods have been proposed such as splitting up the antibodies into bivalent or monovalent fragments or reducing to just the hypervariable region (7), administration of a second antibody (8), pretargeting concept (9-11), and altering the radiolabeling methods.

Therefore, due to the high localization of labeled IgG in inflammatory sites, if radiolabeling methods of antibodies are improved, background radioactivity decreases, then inflammation would be more recognizable from background in images. Although, much remains to be done to insure the clinical usefulness of this approach, it is suggested that radiolabeled antibody is more a suitable method compared to other methods as radiopharmaceutical for the localization of inflammation.

References

  1. Sakahara H, Endo K, Nakashima T, et al. Radioimmunoimaging of osteogenic sarcoma xenografts in nude mice using monoclonal antibodies to osteogenic sarcoma. J. Nucl. Med. (1985) 26: 113
  2. Stern P, Hagan P, Halpern S, et al. The effect of radiolabel on the kinetics of monoclonal anti-CEA in a nude mouse-human colon tumor model. In: Mitchell M S, Oettgen H F (Eds.) Hybridomas in Cancer Diagnosis and Treatment. Raven Press, New York, (1982) 245-253
  3. Wahl RL, Parker CW, Philpott GW. Improved radioimaging and tumor localization with monoclonal F(ab')2. J. Nucl. Med. (1983) 24: 316-325
  4. Colcher D, Zalutsky M, Kaplan W., et al. Radiolocalization of human mammary tumors in athymic mice by a monoclonal antibody. Cancer Res. (1983) 43: 736-742
  5. Khaw BA, Straus HW, Cahill S L, et al. Sequential imaging of indium-111-labeled monoclonal antibody in human mammary tumors hosted in nude mice. J. Nucl. Med. (1984) 25: 592-603
  6. Brown BA, Comeau RD, Jones PL, et al. Comparison of the pharmacokinetics of 125I and 111In labeled intact and proteolytic fragments of a monoclonal antibody. J. Nucl. Med. (1985) 26: 45
  7. Fazio F and Giovanni P. Antibody-guided scintigraphy: targeting of the "magic bullet". Eur. J. Nucl. Med. (1993) 20: 1138-1140.
  8. Goodwin D, Mears C, Diamanti C, McCall M, Lai C, Torti F and Mc Tigue M. Use of specific antibody for rapid clearance of circulating blood background from radiolabeled tumor-imaging proteins. Eur. J. Nucl. Med. (1984) 9: 209-215
  9. Goodwin DA, Mears CF, Mc Call MJ, McTigue M and Chaovapong W. Pre-targeted immunoscintigraphy of murine tumors with indium-111-labeled bifunctional haptens. J. Nucl. Med. (1988) 29: 226-234
  10. Kalofonos HP, Rusckiwski M, Siebecker DA, Sivolapenko GB, Snook D, Lavender JP, Epenetos AA and Hnatowich DJ. Imaging of tumor in-patients with indium-111-labeled biotin and streptavidin-conjugated antibodies: preliminary communication. J. Nucl. Med. (1990) 31: 1791-1796
  11. Goldenberg DM. Monoclonal antibodies in cancer detection and therapy. Am. J. Med. (1993) 94: 297-312
  12. Zimmer AM. New approaches to radiolabeling monoclonal antibodies. In: Henkin RE, Boles MA, Dillehay GL, Halama JR, Karesh SM, Wagner R and Zimmer AM. (Eds.) Nuclear Medicin. Masby-Year Book Inc., (1996) 511-515
  13. Boerman OC, Rennen H, Oyen W J and Corstens FH. Radiopharmaceuticals to image infection and inflammation. Semin Nucl. Med. (2001) 31: 286-295
  14. Johnston A and Thorpe R. Immunochemistry in Practice. 3rd ed., Blackwell Science, Oxford, (1996) 62-65
  15. Johnston A and Thorpe R. Immunochemistry in Practice. 3rd ed., Blackwell Science, Oxford, (1996) 134-136
  16. Pimm MV, Perkins AC, Baldwin RW. Difference in tumor and normal tissue concentrations of iodine and indium labeled monoclonal antibody. II. Biodistribution studies in mice with human tumor xenografts. Eur. J. Nucl. Med. (1985) 11: 300-304
  17. Herlyn D, Powe J, Alavi A, et al. Radioimmunodetection of human tumor xenografts by monoclonal antibodies. Cancer Res. (1983) 43: 2731-2735
  18. Zimmer AM, Rosen ST, Spies SM, et al. Radioimmunoimaging of human small cell lung carcinoma with 131I tumor specific monoclonal antibody. Hybridoma (1985) 4: 1-11
  19. Ghose T, Ferrone S, Imai K, et al. Imaging of human melanoma xenografts in nude mice with a radiolabeled monoclonal antibody. J. Natl. Cancer Inst (1981) 69: 823-826
  20. Moshakis V, Mcllhinney RAJ, Raghavan D, et al. Localization of human tumor xenografts after Iv administration of radiolabeled monoclonal antibodies. Br. J. Cancer (1981) 44: 91-99
  21. Shah SA, Gallagher BM and Sands H. Radioimmunodetection of small tumor xenografts in spleen of athymic mice by monoclonal antibodies. Cancer Res. (1985) 45: 5824-5829
  22. Otsuka FL and Welch MJ. Methods to label monoclonal antibodies for use in tumor imaging. Nucl. Med. Biol. (1987) 14: 243-249
  23. Chatal JF, Peltier P, Bardies M, Chetanneau A, Thedrez P and Faivre-Chauvet A. Does immunoscintigraphy serve clinical needs effectively? Is there a future for radioimmunotherapy? Eur. J. Nucl. Med. (1992) 19: 205-213
  24. Corstens FHM and Claessens RAMJ. Imaging inflammation with human polyclonal immunoglobulin: Not looked for but discovered. Eur. J. Nucl. Med. (1992) 19: 155-158
  25. Corstens FHM, Oyen WJG and Becker WS. Radiommunoconjugates in the detection of infection and inflammation. Semin. Nucl. Med. (1993) 23: 148-164
  26. Lind P, Langsteger W, Koltringer P, Dimai HP, Passl R and Eber O. Immunoscintigraphy of inflammatory processes with a technetium-99m-labeled monoclonal antigranulocyte antibody (Mab BE 250/183). J. Nucl. Med. (1990) 31: 417-423