Hypoxyprobe-F6 (CCI-103F) for the non-invasive [19F] MRS and MRI detection of hypoxia in normal and malignant disease.

 

 

 

Hypoxyprobe-F6 also known as CCI-103F is a hexafluorinated derivative of 2-nitroimidazole that has six magnetically equivalent fluorine atoms.  CCI-10F was developed specifically for the non-invasive detection of normal and malignant tissue hypoxia by means of 19F magnetic resonance spectroscopy (MRS) and 19F magnetic resonance imaging (MRI) (1-5).  During the development of CCI-103F it became clear that a histological correlate with 19F MRS measurements would be useful and the technique of immunohistochemical hypoxia markers was invented (6, 7).  CCI-103F has been widely used as an immunochemical hypoxia marker in rodents and dogs including dual marker studies in combination with Hypoxyprobe (pimonidazole HCl) (6, 8-25).  A comparison of Hypoxyprobe (pimo) and Hypoxyprobe-F6 (F6) in canine tumors is shown in Figure 1.

 

Immunoperoxidase
staining for pimonidazole and CCI-103F in contiguous sections from a canine adenocarcinoma. 

Figure 1.  Immunoperoxidase staining for pimonidazole (Pimo; Hypoxyprobe) and CCI-103F (F6; Hypoxyprobe-F6) in contiguous sections from a canine adenocarcinoma.  Non-crossreacting rabbit antisera to pimonidazole and CCI-103F adducts were used in this study.

 

 

The mechanism of CCI-10F bioreductive activation and binding is the same as that for other 2-nitroimidazoles as shown in Figure 2.

The mechanism of CCI-10F
bioreductive activation and binding is the same as that for other
2-nitroimidazoles

Figure 2.  Bioreductive activation, binding and fragmentation of Hypoxyprobe-F6 (CCI-103F).  CCI-103F adducts to protein and small molecules consititute the hypoxia signal while hydrolytic fragments have no binding properties and are merely a source of “noise”.

 

19F MRS measurement of tissue hypoxia with [19F] labeled 2-nitroimidazoles involves five more or less, independent processes: 1) build up of hypoxia marker protein adducts; 2) build up of [19F] small molecule adducts of hypoxia markers that include cysteine and glutathione adducts; 3) catabolism of protein and small molecule adducts and wash out of these metabolites and hydrolytic fragmentation products (26, 27); 4) wash in of unmetabolized [19F] hypoxia marker molecules; and 5) wash out of unmetabolized [19F] hypoxia marker molecules.  Approximately 80% of bioreductively activated 2-nitroimidazole hypoxia markers are fragmented by hydrolysis.  Fragmentation produces non-binding [19F] metabolites that make a major contribution to background noise but add nothing to the hypoxia signal.  Approximately 20% of bioreductively activated 2-nitroimidazole hypoxia markers produce the hypoxia signal – 10% from adducts with proteins and 10% from small, thiol containing compounds like glutathione (28).  While the dynamics of CCI-103F metabolism complicate the non-invasive analysis of tissue hypoxia, mathematical models have been designed to isolate the [19F] MRS hypoxia signal (i.e., protein and glutathione adducts) from background noise (i.e., unbound hypoxia marker and its non-binding metabolites and hydrolytic fragments) (3).

 

Alternatively, hypoxia measurements can be delayed to allow for background signals to diminish.   

 

Each additional F atom doubles signal intensity and magnetic equivalence makes sure that the increase is confined in a single MRS peak in CCI-103F adducts making CCI-103F an ideal marker for tissue hypoxia. 

 

Hypoxyprobe-F6 can be purchased at Hypoxyprobe Store.

 

References

 

1.         Raleigh, J. A., Franko, A. J., Treiber, E. O., Lunt, J. A., and Allen, P. S. Covalent binding of a fluorinated 2-nitroimidazole to EMT-6 tumors in Balb/C mice: detection by F-19 nuclear magnetic resonance at 2.35 T. Int J Radiat Oncol Biol Phys, 12: 1243-1245, 1986.

2.         Maxwell, R. J., Workman, P., and Griffiths, J. R. Demonstration of tumor-selective retention of fluorinated nitroimidazole probes by 19F magnetic resonance spectroscopy in vivo. Int J Radiat Oncol Biol Phys, 16: 925-929, 1989.

3.         Jin, G. Y., Li, S. J., Moulder, J. E., and Raleigh, J. A. Dynamic measurements of hexafluoromisonidazole (CCI-103F) retention in mouse tumours by 1H/19F magnetic resonance spectroscopy. Int J Radiat Biol, 58: 1025-1034, 1990.

4.         Li, S. J., Jin, G. Y., and Moulder, J. E. Prediction of tumor radiosensitivity by hexafluoromisonidazole retention monitored by [1H]/[19F] magnetic resonance spectroscopy. Cancer Commun, 3: 133-139, 1991.

5.         Kwock, L., Gill, M., McMurry, H. L., Beckman, W., Raleigh, J. A., and Joseph, A. P. Evaluation of a fluorinated 2-nitroimidazole binding to hypoxic cells in tumor-bearing rats by 19F magnetic resonance spectroscopy and immunohistochemistry. Radiat Res, 129: 71-78, 1992.

6.         Raleigh, J. A., Miller, G. G., Franko, A. J., Koch, C. J., Fuciarelli, A. F., and Kelly, D. A. Fluorescence immunohistochemical detection of hypoxic cells in spheroids and tumours. Br J Cancer, 56: 395-400, 1987.

7.         Raleigh, J. A., Miller, G. G., Franko, A. J., and Chapman, J. D. Immunochemical detection of hypoxia in normal and tumor tissue. USA: Alberta Cancer Board, 1992.

8.         Miller, G. G., Best, M. W., Franko, A. J., Koch, C. J., and Raleigh, J. A. Quantitation of hypoxia in multicellular spheroids by video image analysis. Int J Radiat Oncol Biol Phys, 16: 949-952, 1989.

9.         Cline, J. M., Thrall, D. E., Page, R. L., Franko, A. J., and Raleigh, J. A. Immunohistochemical detection of a hypoxia marker in spontaneous canine tumours. Br J Cancer, 62: 925-931, 1990.

10.       Moore, D. H., Rouse, M. B., Massenburg, G. S., and Zeman, E. M. Description of a spheroid model for the study of radiation and chemotherapy effects on hypoxic tumor cell populations. Gynecol Oncol, 47: 44-47, 1992.

11.       Raleigh, J. A., Zeman, E. M., Rathman, M., LaDine, J. K., Cline, J. M., and Thrall, D. E. Development of an ELISA for the detection of 2-nitroimidazole hypoxia markers bound to tumor tissue. Int J Radiat Oncol Biol Phys, 22: 403-405, 1992.

12.       Zeman, E. M., Calkins, D. P., Cline, J. M., Thrall, D. E., and Raleigh, J. A. The relationship between proliferative and oxygenation status in spontaneous canine tumors. Int J Radiat Oncol Biol Phys, 27: 891-898, 1993.

13.       Cline, J. M., Thrall, D. E., Rosner, G. L., and Raleigh, J. A. Distribution of the hypoxia marker CCI-103F in canine tumors. Int J Radiat Oncol Biol Phys, 28: 921-933, 1994.

14.       Thrall, D. E., McEntee, M. C., Cline, J. M., and Raleigh, J. A. ELISA quantification of CCI-103F binding in canine tumors prior to and during irradiation. Int J Radiat Oncol Biol Phys, 28: 649-659, 1994.

15.       Raleigh, J. A., La Dine, J. K., Cline, J. M., and Thrall, D. E. An enzyme-linked immunosorbent assay for hypoxia marker binding in tumours. Br J Cancer, 69: 66-71, 1994.

16.       Raleigh, J. A., Zeman, E. M., Calkins, D. P., McEntee, M. C., and Thrall, D. E. Distribution of hypoxia and proliferation associated markers in spontaneous canine tumors. Acta Oncol, 34: 345-349, 1995.

17.       Thrall, D. E., Rosner, G. L., Azuma, C., McEntee, M. C., and Raleigh, J. A. Hypoxia marker labeling in tumor biopsies: quantification of labeling variation and criteria for biopsy sectioning. Radiother Oncol, 44: 171-176, 1997.

18.       Cline, J. M., Rosner, G. L., Raleigh, J. A., and Thrall, D. E. Quantification of CCI-103F labeling heterogeneity in canine solid tumors. Int J Radiat Oncol Biol Phys, 37: 655-662, 1997.

19.       Bussink, J., Kaanders, J. H., Strik, A. M., and van der Kogel, A. J. Effects of nicotinamide and carbogen on oxygenation in human tumor xenografts measured with luminescense based fiber-optic probes. Radiother Oncol, 57: 21-30, 2000.

20.       Ljungkvist, A. S., Bussink, J., Rijken, P. F., Raleigh, J. A., Denekamp, J., and Van Der Kogel, A. J. Changes in tumor hypoxia measured with a double hypoxic marker technique. Int J Radiat Oncol Biol Phys, 48: 1529-1538, 2000.

21.       Bennewith, K. L., Raleigh, J. A., and Durand, R. E. Orally administered pimonidazole to label hypoxic tumor cells. Cancer Res, 62: 6827-6830, 2002.

22.       Bennewith, K. L. and Durand, R. E. Quantifying transient hypoxia in human tumor xenografts by flow cytometry. Cancer Res, 64: 6183-6189, 2004.

23.       Ljungkvist, A. S., Bussink, J., Kaanders, J. H., Rijken, P. F., Begg, A. C., Raleigh, J. A., and van der Kogel, A. J. Hypoxic cell turnover in different solid tumor lines. Int J Radiat Oncol Biol Phys, 62: 1157-1168, 2005.

24.       Ljungkvist, A. S., Bussink, J., Kaanders, J. H., Wiedenmann, N. E., Vlasman, R., and van der Kogel, A. J. Dynamics of hypoxia, proliferation and apoptosis after irradiation in a murine tumor model. Radiat Res, 165: 326-336, 2006.

25.       Kleiter, M. M., Thrall, D. E., Malarkey, D. E., Ji, X., Lee, D. Y., Chou, S. C., and Raleigh, J. A. A comparison of oral and intravenous pimonidazole in canine tumors using intravenous CCI-103F as a control hypoxia marker. Int J Radiat Oncol Biol Phys, 64: 592-602, 2006.

26.       Raleigh, J. A. and Liu, S. F. Reductive fragmentation of 2-nitroimidazoles: amines and aldehydes. Int J Radiat Oncol Biol Phys, 10: 1337-1340, 1984.

27.       Raleigh, J. A. and Liu, S. F. Reductive fragmentation of 2-nitroimidazoles in the presence of nitroreductases--glyoxal formation from misonidazole. Biochem. Pharmacol., 32: 1444-1446, 1983.

28.       Raleigh, J. A. and Koch, C. J. Importance of thiols in the reductive binding of 2-nitroimidazoles to macromolecules. Biochem Pharmacol, 40: 2457-2464, 1990.