History of Hypoxyprobe Development
Molecular oxygen is essential for life and elaborate systems for collecting, distributing and sensing oxygen exist in animals. A balance between oxygen supply and oxygen consumption produces oxygen gradients in normal and malignant tissue with regions of low oxygen concentration (hypoxia) developing distal to blood vessels. These gradients are part of normal physiology and their disruption by disease can have serious consequences.
Pimonidazole HCl (Hypoxyprobe-1)
In 1976, Varghese et al. reported that the 2-nitroimidazoles form adducts in hypoxic cells in vitro and in vivo (1). It was subsequently found that adducts form with thiol groups in proteins, peptides and amino acids in a way that all atoms of the ring and side-chain of the 2-nitroimidazole are retained (2-5). Hypoxia (pO2 < 10 mmHg) is required for binding. The binding is dependent on the presence of redox enzymes in hypoxic cells but pimonidazole binding is not dependent on the presence of any particular redox enzyme. Furthermore, wide variations in NADH and NADPH levels do not change the oxygen dependence of binding (6,7).
In 1981, Chapman et al. showed that the oxygen dependence of 2-nitroimidazole binding was close to that for radiation resistance and proposed 2-nitroimidazole binding as a hypoxia marker in normal and malignant tissue (8). Clinical feasibility of the hypoxia marker idea was demonstrated by autoradiographic analyses of 3H-misonidazole binding in human tumors (9). 3H-Misonidazole had limited clinical utility but it spurred the development of a variety of assays for tissue hypoxia based on 2-nitroimidazole binding. These included single photon electron capture tomography (SPECT), positron emission tomography (PET), nuclear medicine analysis and magnetic resonance spectroscopy (MRS) of suitably labeled 2-nitroimidazole analogues (for early review see ref. (10).
In 1986, during 19F MRS investigations of tumor hypoxia with the hexafluorinated 2-nitroimidazole, CCI-103F, Raleigh et al. concluded that a non-radioactive, histological assessment of hypoxia would be a useful complement to non-invasive assays (11-13)and they invented the immunochemical hypoxia marker technique based on monoclonal and polyclonal antibodies raised against protein adducts of reductively activated 2-nitroimidazoles such as CCI-103F and pimonidazole (14,15).
The reasons for choosing pimonidazole HCl as our signature hypoxia marker included its chemical stability; high water solubility; wide tissue distribution; and the availability of toxicity data from earlier radiosensitizer studies that allowed a safe dosage to be chosen for hypoxia marking in preclinical and clinical studies. Lastly, the relatively complex chemical structure of the side chain in pimonidazole produced a strong immune response during the creation of antibody reagents and strong binding of those reagents to pimonidazole adducts in vivo.
Stability. Solid pimonidazole HCl is very stable being unchanged after storage for 4 years at 4oC in subdued light. Saline solutions of pimonidazole HCl used for the initial clinical studies (34 millimolar in 0.9% saline, pH 3.9 ± 0.1) were extremely stable being unchanged after 1.5 years at 4oC in subdued light.
Solubility and Pharmacokinetics. High water solubility (400 millimolar; 116 grams per 1000 mL) facilitated intravenous marker infusion and produced short plasma half-lives of 25 minutes in mice; 45 minutes in rats; 1.5 hours in dogs; and 5.1 hours in humans.
Tissue Distribution. In spite of the high water solubility of pimonidazole HCl (pKa 8.7), pimonidazole itself has an octanol-water partition coefficient of 8.5 and diffuses readily into tumors and normal tissues including brain. Consistent with a large, 155 liter volume of distribution in humans, pimonidazole concentrates approximately 3 fold above plasma levels in tumors and normal tissues thereby increasing hypoxia marking sensitivity.
Safety. At a clinical dose of 0.5 g/m2 used for hypoxia marking, pimonidazole HCl causes neither central nervous system toxicity nor sensation (e.g., flushing). Central nervous system toxicity was dose limiting for pimonidazole HCl at higher, multiple doses used in radiosensitizer trials. The LD50 (7 days) in mice is 728 mg/Kg making pimonidazole a non-hazardous compound with respect to handling in the lab and giving wide latitude with respect to animal dosages. A dose of 60 mg/Kg is recommended for rodent studies as a reasonable balance between cost and effectiveness.
Antibody Reagents. Protein adducts of reductively-activated pimonidazole proved to be effective immunogens for the production of polyclonal and monoclonal antibodies. The antibodies have now been used in a variety of immunochemical analyses including
Antibodies to pimonidazole adducts are very robust. For example, aqueous solutions of the IgG1 monoclonal antibody against pimonidazole adducts (clone 22.214.171.124) are stable indefinitely when stored at -20oC and are stable for at least 4 months at 4oC when supplemented with 10 mg/mL of bovine serum albumin and 10 millimolar sodium azide. Pimonidazole adducts in vivo are robust lasting for days in hypoxic tissue. This feature provides flexibility in the timing of tissue harvesting which is an advantage in clinical studies.
a) immunoperoxidase analysis of formalin fixed, paraffin embedded sections;
b) immunofluorescence analysis of frozen fixed sections;
c) cytometry with directly labeled or secondary fluorescent antibodies;
d) flow cytometry with directly labeled or secondary fluorescent antibodies;
e) Western blotting;
f) enzyme linked immunosorbent (ELISA)assays.
Both mouse monoclonal and rabbit affinity purified IgG antibodies have been prepared. Mouse monoclonal antibodies conjugated to FITC, Dylight549, APC and Biotin are available.
Validation of Pimonidazole HCl as a Hypoxia Marker
In 1999, Raleigh et al. reported that pimonidazole immunostaining was strongly correlated with oxygen electrode measurements in mouse tumors in which the level of oxygenation was intentionally manipulated (44).
In 2000, Bussink et al. reported a strong correlation between a luminescent pO2 sensing and pimonidazole binding in 3 different xenograft models (45).
In 2000, Lungkvist et al. confirmed that the extent of pimonidazole binding in human tumor xenografts decreased in carbogen breathing animals and established the dual marker approach to following changes in tumor oxygenation (46).
In 2001, Pogue et al. showed that pimonidazole staining patterns could be used to predict pO2 electrode measurements (47).
Numerous preclinical and clinical studies show that pimonidazole staining increases at distances from blood vessels that are completely consistent with the expected location of tumor tissue hypoxia.
In addition to providing quantitative measures of hypoxia, immunohistochemical assays for pimonidazole binding provide insights into microregional relationships between hypoxia and factors such as necrosis, proliferation, differentiation, apoptosis, and oxygen regulated gene expression in a way that non-invasive imaging techniques cannot do. An example of the unique value of the immunohistochemical marker approach is the surprising clinical observation that there is very little correlation either spatially or quantitatively between pimonidazole binding and the expression of oxygen regulated proteins indicating that the expression of HIF-1, Glut-1, CAIX, VEGF, metallothionein, etc. in human tissues is probably controlled by factors in addition to hypoxia.
Pimonidazole HCl is widely used as a hypoxia marker in preclinical and clinical studies of both normal and malignant tissues. A scan of the literature in November 2012 revealed 548 publications, 69 of which were based on clinical studies. Other sections of the Knowledge Center break the references down into methods of detection, species studied, malignant tissues studied, normal tissues studied and oxygen regulated protein studies. Detailed information about pimonidazole as a hypoxia marker can be found in other sections of the Knowledge Center.
- Varghese, A. J., Gulyas, S., and Mohindra, J. K. Hypoxia-dependent reduction of 1-(2-nitro-1-imidazolyl)-3-methoxy-2- propanol by Chinese hamster ovary cells and KHT tumor cells in vitro and in vivo, Cancer Res. 36: 3761-3765, 1976.
- Chacon, E., Morrow, C. J., Leon, A. A., Born, J. L., and Smith, B. R. Regioselective formation of a misonidazole-glutathione conjugate as a function of pH during chemical reduction., Biochem. Pharmacol. 37: 361-363, 1988.
- Raleigh, J. A., Franko, A. J., Koch, C. J., and Born, J. L. Binding of misonidazole to hypoxic cells in monolayer and spheroid culture: evidence that a side-chain label is bound as efficiently as a ring label, Br. J. Cancer. 51: 229-235, 1985.
- 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.
- Varghese, A. J. Glutathione conjugates of misonidazole, Biochem Biophys Res Commun. 112: 1013-1020, 1983.
- Arteel, G. E., Thurman, R. G., Yates, J. M., and Raleigh, J. A. Evidence that hypoxia markers detect oxygen gradients in liver: pimonidazole and retrograde perfusion of rat liver, Br J Cancer. 72: 889-895, 1995.
- Arteel, G. E., Thurman, R. G., and Raleigh, J. A. Reductive metabolism of the hypoxia marker pimonidazole is regulated by oxygen tension independent of the pyridine nucleotide redox state., Eur. J. Biochem. 253: 743-750, 1998.
- Chapman, J. D., Franko, A. J., and Sharplin, J. A marker for hypoxic cells in tumours with potential clinical applicability, Br. J. Cancer. 43: 546-550, 1981.
- Urtasun, R. C., Koch, C. J., Franko, A. J., Raleigh, J. A., and Chapman, J. D. A novel technique for measuring human tissue pO2 at the cellular level, Br. J. Cancer. 54: 453-457, 1986.
- Raleigh, J. A., Dewhirst, M. W., and Thrall, D. E. Measuring tumor hypoxia., Sem. Radiat. Oncol. 6: 37-45, 1996.
- 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 rsonance spectroscopy, Int. J. Radiat. Biol. 58: 1025-1034, 1990.
- 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 2.35T, Int. J. Radiat. Oncol. Biol. Phys. 12: 1243-1245, 1986.
- Raleigh, J., Franko, A., Kelly, D., Trimble, L., and Allen, P. Development of an in vivo 19F magnetic resonance method for measuring oxygen deficiency in tumors., Magn. Res. Med. 22: 451-466, 1991.
- 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.
- Raleigh, J. A., Miller, G. G., Franko, A. J., and Chapman, J. D. Immunochemical detection of hypoxia in normal and tumor tissue. USA patent 5,086,068, February,1992.
- Azuma, C., Raleigh, J. A., and Thrall, D. E. Longevity of pimonidazole adducts in spontaneous canine tumors as an estimate of hypoxic cell lifetime., Radiat. Res. 148: 35-42, 1997.
- 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.
- 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.
- Cline, J., Rosner, G., Raleigh, J., and Thrall, D. Quantification of CCI-103F labeling heterogeneity in canine solid tumors, Int. J. Radiat. Oncol. Biol. Phys. 37: 655-662, 1997.
- 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.
- 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: 171-6, 1997.
- Evans, S. M., Hahn, S., Pook, D. R., Jenkins, W. T., Chalian, A. A., Zhang, P., Stevens, C., Weber, R., Weinstein, G., Benjamin, I., Mirza, N., Morgan, M., Rubin, S., McKenna, W. G., Lord, E. M., and Koch, C. J. Detection of hypoxia in human squamous cell carcinoma by EF5 binding, Cancer Res. 60: 2018-2024, 2000.
- Kennedy, A. S., Raleigh, J. A., Perez, G. M., Calkins, D. P., Thrall, D. E., Novotny, D. B., and Varia, M. A. Proliferation and hypoxia in human squamous cell carcinoma of the cervix: first report of combined immunohistochemical assays, Int J Radiat Oncol Biol Phys. 37: 897-905, 1997.
- Raleigh, J. A., Calkins-Adams, D. P., Rinker, L. H., Ballenger, C. A., Weissler, M. C., Fowler, W. C., Jr., Novotny, D. B., and Varia, M. A. Hypoxia and vascular endothelial growth factor expression in human squamous cell carcinomas using pimonidazole as a hypoxia marker, Cancer Res. 58: 3765-3768, 1998.
- Raleigh, J., Chou, S.-C., Calkins-Adams, D., Ballenger, C., Rinker, L., Novotny, D., and Varia, M. A clinical study of hypoxia and metallothionein protein expression in squamous cell carcinomas, Cl. Cancer Res. 6: 855-862, 2000.
- Varia, M. A., Calkins-Adams, D. P., Rinker, L. H., Kennedy, A. S., Novotny, D. B., Fowler, W. C., Jr., and Raleigh, J. A. Pimonidazole : a novel hypoxia marker for complementary study of tumor hypoxia and cell proliferation in cervical carcinoma., Gynecol. Oncol. 71: 270-277, 1998.
- Wijffels, K., Kaanders, J., Rijken, P., Bussink, J., Van den Hoogen, F., Marres, H., de Wilde, P., Raleigh, J., and Van der Kogel, A. Vascular architecture and hypoxic profiles in human head and neck squamous cell carcinomas., Br. J. Cancer. 83: 674-683, 2000.
- Carmeliet, P., Dor, Y., Herbert, J. M., Fukumura, D., Brusselmans, K., Dewerchin, M., Neeman, M., Bono, F., Abramovitch, R., Maxwell, P., Koch, C. J., Ratcliffe, P., Moons, L., Jain, R. K., Collen, D., and Keshet, E. Role of HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis, Nature. 394: 485-90, 1998.
- Hodgkiss, R. J. Use of 2-nitroimidazoles as bioreductive markers for tumour hypoxia, Anticancer Drug Des. 13: 687-702, 1998.
- Koch, C. J., Chasan, J. E., Jenkins, W. T., Chan, C. Y., Laughlin, K. M., and Evans, S. M. Co-localization of hypoxia and apoptosis in irradiated and untreated HCT116 human colon carcinoma xenografts, Adv Exp Med Biol. 454: 611-618, 1998.
- 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.
- Raleigh, J. A., Chou, S.-C., Tables, L., Suchindran, S., Varia, M. A., and Horsman, M. R. Relationship of hypoxia to metallothionein expression in murine tumors., Int. J. Radiat. Oncol. Biol. Phys. 42: 727-730, 1998.
- Waleh, N. S., Brody, M. D., Knapp, M. A., Mendonca, H. L., Lord, E. M., Koch, C. J., Laderoute, K. R., and Sutherland, R. M. Mapping of the vascular endothelial growth factor-producing hypoxic cells in multicellular tumor spheroids using a hypoxia-specific marker., Cancer Res. 55: 6222-6226, 1995.
- Leith, J. T. and Michelson, S. Secretion rates and levels of vascular endothelial growth factor in clone A or HCT-8 human colon tumour cells as a function of oxygen concentration, Cell Prolif. 28: 415-430, 1995.
- Murphy, B. J., Andrews, G. K., Bittel, D., Discher, D. J., McCue, J., Green, C. J., Yanovsky, M., Giaccia, A., Sutherland, R. M., Laderoute, K. R., and Webster, K. A. Activation of metallothionein gene expression by hypoxia involves metal response elements and MTF-1., Cancer Res. 59: 1315-1322, 1999.
- Durand, R. E. and Raleigh, J. A. Identification of nonproliferating but viable hypoxic cells in vivo., Cancer Res. 58: 3547-3550, 1998.
- Webster, L., Hodgkiss, R. J., and Wilson, G. D. Simultaneous triple staining for hypoxia, proliferation, and DNA content in murine tumours, Cytometry. 21: 344-351, 1995.
- Smithen, C., Clarke, E., Dale, J., Jacobs, R., Wardman, P., Watts, M., and Woodcock, M. Novel (nitro-1-imidazoyl)-alkanolamines as potential radiosensitizers with improved therapeutic properties. In: L. Brady (ed.) Radiation Sensitizers. Their Use in the Clinical Management of Cancer., pp. 22-32. New York: Masson, 1980.
- Saunders, M. I., Anderson, P. J., Bennett, M. H., Dische, S., Minchinton, A. I., and Stratford, M. R. L. The clinical testing of Ro 03-8799 -- pharmacokinetics, toxicology, tissue and tumor concentrations., Int. J. Radiat. Oncol. Biol. Phys. 10: 1759-1763, 1984.
- Arteel, G. E., Iimuro, Y., Yin, M., Raleigh, J. A., and Thurman, R. G. Chronic enteral ethanol treatment causes hypoxia in rat liver tissue in vivo, Hepatology. 25: 920-926, 1997.
- Bussink, J., Kaanders, J. H., Rijken, P. F., Raleigh, J. A., and Van Der Kogel, A. J. Changes in blood perfusion and hypoxia after irradiation of a human squamous cell carcinoma xenograft tumor line., Radiat. Res. 153: 398-404, 2000.
- Rijken, P., Bernsen, H., Peters, J., Hodgkiss, R., Raleigh, J., and van der Kogel, A. Spatial relationship between hypoxia and the (perfused) vascular network in human glioma xenografts: a quantitative multi-parameter analysis., Int.J. Radiat. Oncol. Biol. Phys. 48: 571-582, 2000.
- Bernsen, H. J., Rijken, P. F., Peters, H., Raleigh, J. A., Jeuken, J. W., Wesseling, P., and van der Kogel, A. J. Hypoxia in a human intracerebral glioma model, J Neurosurg. 93: 449-454, 2000.
- Raleigh JA, Chou SC, Arteel GE, Horsman MR. Comparisons among pimonidazole binding, oxygen electrode measurements, and radiation response in C3H mouse tumors. Radiat Res 1999; 151: 580-9.
- Bussink J, Kaanders JH, Strik AM, van der Kogel AJ. Effects of nicotinamide and carbogen on oxygenation in human tumor xenografts measured with luminescense based fiber-optic probes. Radiother Oncol 2000; 57: 21-30.
- Ljungkvist AS, Bussink J, Rijken PF, Raleigh JA, Denekamp J, Van Der Kogel AJ. Changes in tumor hypoxia measured with a double hypoxic marker technique. Int J Radiat Oncol Biol Phys 2000; 48: 1529-38.
- Pogue BW, Paulsen KD, O'Hara JA, Wilmot CM, Swartz HM. Estimation of oxygen distribution in RIF-1 tumors by diffusion model- based interpretation of pimonidazole hypoxia and eppendorf measurements. Radiat Res 2001; 155: 15-25.