The purpose of this study was to determine, with a rodent tumor model, if microelectrode measurements of unmodulated tumor oxygenation predict for the avidity of hypoxic markers to tumor tissue. Methods: The rapidly growing, anaplastic variant of the Dunning rat prostate carcinoma cell line (R3327-AT) was implanted subcutaneously on the upper backs of Fischer X Copenhagen rats. Approximately 100 measurements of Po-2 were obtained from tumors of 5-10 g in animals that were restrained and then subjected to different anesthetic procedures. Values of median Po-2 (in mm Hg) and percentage of measurements (5 mm Hg obtained from individual tumors were used to define tumor oxygenation status. The radiodiagnostic hypoxic markets P-D-iodinated azomycin galactopyranoside (IAZGP) and [Tc-99m]HL-91 were simultaneously administered to 26 animals whose tumor oxygen levels had been measured. Six hours after marker administration, the animals were killed; tumor, blood, and muscle tissues were sampled; and percentage injected dose per gram (%ID/g*), tumor/blood ratio (T/B), and tumor/muscle ratio (T/M) parameters were determined. Parameters of marker avidity to individual tumors were linearly correlated with microelectrode measurements of tumor oxygenation to determine the significance of inverse associations. Results: The median Po-2 Of 41 tumors varied from 2.0 to 20.9 mm Hg, with an average value of 7.5 +/- 1.4 mm Hg. Six tumors had unusually high values; that is, >10 mm Hg, and when these were excluded from the analysis, the average median Po-2 of the remaining 35 was 4.3 +/- 0.7 mm Hg. When electrode measurements of tumor oxygenation were obtained under conditions of halothane anesthesia with the animals breathing O-2, carbogen, or air, median Po-2 values increased significantly (P = 0.001). When animals were deeply anesthetized by intraperitoneal injection of ketamine-xylazine, median Po-2 values were not significantly different (P = 0.13) from those obtained while the animals were restrained and breathing air. There was no inverse correlation of significance between the electrode measurements of median Po-2 and the avidity of beta -D-IAZGP nor [99mTc]HL-91 in this tumor model. The range of median Po-2 values in these tumors was at least 3 mm Hg, and the range of hypoxic marker avidity was less than twofold. Conclusion: These data demonstrate that microelectrode measurements of rat tumor oxygenation did not correlate with the avidity of the two hypoxic markers, at least in this tumor model. The larger dynamic range of tumor oxygen measurements obtained with microelectrodes might be biased to low values by their necrotic fractions, the zones within solid tumors that contain dead cells and debris that will not be labeled by bioreducible hypoxic markers. Hypoxic marker avidity to individual tumors will have to be validated by other assays that can predict for their radiosensitivity.

Tumour oxygenation status in individual patients may be assessed using the bioreduction and linkage of 2-nitroimidazole markers to viable hypoxic cells in vivo with subsequent detection by conventional nuclear medicine techniques. Iodoazomycin arabinoside (IAZA) was radiolabelled with Iodine-123 and administered i.v. to 51 patients with newly diagnosed malignancies whose tumours were subsequently imaged by planar and single-photon emission computed tomographic (SPECT) procedures. Quantitative analyses of radiotracer avidity were performed at 24 h post-injection and tumour-normal tissue ratios of greater than 1.10 were deemed positive for tumour hypoxia. By this criterion, the frequencies of hypoxia in small-cell lung cancer, squamous cell carcinomas of head and neck and malignant gliomas were 60% (9/15), 40% (6/15) and 0% (0/11) respectively. The correlation of positive IAZA scans with tumour control and survival in patients with lung cancer and head and neck tumours is currently under study. Preliminary observations in neck metastases from squamous cell carcinoma of head and neck tumours indicates decreased local control at 3 months post-treatment in tumours with IAZA avidity. This study concludes that: (1) 123I-IAZA can be administered safely and repeatedly as an outpatient routine imaging procedure in cancer patients during initial work-up and follow-up; (2) that retained drug can be detected by conventional nuclear medicine procedures in inaccessible deep-seated tumours; and (3) that this technique could prove useful for identifying those patients for whom hypoxia-directed therapy is indicated.

1-(4-Iodo-4-deoxy-beta-L-xylopyranosyl)-2-nitroimidazole (Iodoazomycin Pyranoside; IAZP) was synthesized, labelled with radioiodine(123I, 125I) and evaluated for non-invasive assessment of tumor hypoxia. A biodistribution study with Balb/c mice bearing EMT-6 tumors showed a tumor-to-blood ratio of 13.9, representing 0.5 percent of injected dose per gram of tissue, at 24 hours post injection. This ratio is the highest for any 2-nitro-imidazole reported to date in this tumor model. Rapid elimination of radioactivity from the whole-body was noted (greater than 97% in 24 hours) and thyroid radioactivity at 24 hours was much lower than with other analogues of this series. No toxicity was observed in Balb/c mice at a dose 100 times higher than the anticipated human dose required for scintigraphic imaging. Planar, whole-body gamma scintigraphic images in the murine Balb/c EMT-6 tumor model clearly delineated tumor tissue at 24 hours post injection. These observations suggest that IAZP may be a suitable agent for non-invasive, clinical assessment of tumor hypoxia.

1-(2-Fluoro-4-iodo-2,4-dideoxy-beta-L-xylopyranosyl)-2-nitroimidazole (FIAZP) has been synthesized and labeled with radioiodine (125I). Radioiodinated FIAZP is one of a series of sugar-coupled 2-nitroimidazoles developed in our laboratory as probes for noninvasive scintigraphic assessment of tumor hypoxia. An in vivo biodistribution study with [125I]FIAZP in the murine BALB/c EMT-6 tumor model showed a tumor-to-blood ratio of 6, 24 h after injection, with 0.5% of the injected dose present per gram of tumor. These values are several times higher than the respective ratios and distribution values in any of the organs, with the exception of liver. Radioactivity from tissues other than tumor and liver declined with time, following the decline of blood radioactivity. Rapid whole-body elimination of radioactivity was observed (> 96% in 24 h). The thyroid showed little uptake of radioactivity, indicating minimal in vivo deiodination. 1-(2-Fluoro-4-iodo-2,4-dideoxy-beta-L-xylopranosyl)-2-nitroimidazo le appears to undergo hypoxia-dependent binding in tumor tissue at levels comparable to those of other sugar-coupled 2-nitroimidazoles. The potential for imaging with this compound is discussed.

Non-invasive predictive assays which can confirm the presence or absence of hypoxic cells in human tumours show promise for understanding the natural history of tumour oxygenation, and improving the selection of patient subsets for novel radiotherapeutic strategies. Sensitiser adducts have been proposed as markers for hypoxic cells. Misonidazole analogues radiolabelled with iodine-123 have been developed for the detection of tumour hypoxia using conventional nuclear medicine techniques. In this pilot study, we have investigated one such potential marker, 123I-iodoazomycin arabinoside (123I-IAZA). Patients with advanced malignancies have undergone planar and single-photon emission computed tomographic (SPECT) imaging after intravenous administration of 123I-IAZA. We have observed radiotracer avidity in three out of ten tumours studied to date. Normal tissue activity of variable extent was also seen in the thyroid and salivary glands, upper aerodigestive tract, liver, intestine, and urinary bladder. Quantitative analysis of those images showing radiotracer avidity revealed tumour/normal tissue (T/N) ratios of 2.3 (primary small cell lung carcinoma), 1.9 (primary malignant fibrous histiocytoma) and 3.2 (brain metastasis from small cell lung carcinoma) at 18-24 h post injection. These preliminary data suggest that the use of gamma-emitter labelled 2-nitroimidazoles as diagnostic radiopharmaceuticals is feasible and safe, and that metabolic binding of 123I-IAZA is observed in some, but not all tumours. The inference that tumour 123I-IAZA avidity could be a non-invasive measure of tumour hypoxia deserves independent confirmation with needle oximetry.

The effect of photodynamic therapy (PDT) on tumour perfusion in both anaplastic (R3327-AT) and well differentiated (R3327-H) Dunning prostatic tumours was studied using the radiopharmaceutical 99Technetium hexamethylpropyleneamine oxime (Tc-99m-HMPAO). Tumours in the left flanks of rats (Copenhage x Fischer, F1 hybrids) were treated with interstitial PDT when their volumes reached 2-3 cm3. Qualitative and quantitative data from pre- and post-PDT scintigraphy revealed a light-dose-dependent shut-down of tumour perfusion which was also time-dependent. Maximal shut-down, following a 1,600 J light-dose, occurred about 8 h post-PDT. Light exposure 2 h after the intravenous administration of the photosensitiser (Photofrin II) produced a greater vascular shut-down than did light exposure 24 h after the administration of the drug. Regional differences in perfusion within treated and non-treated tumours were measured by tomographic procedures. Light-dose-dependent volumes of perfusion shut-down were demonstrated in addition to the naturally occurring regional differences in tumour perfusion. This radiopharmaceutical may have future utility for monitoring the clinical treatment of solid tumours with PDT.

The effect of photodynamic therapy (PDT) on tumour perfusion in both anaplastic (R3327-AT) and well differentiated (R3327-H) Dunning prostatic tumours was studied using the radiopharmaceutical 99Technetium hexamethylpropyleneamine oxime (99mTc-HMPAO). Tumours in the left flanks of rats (Copenhage x Fischer, F1 hybrids) were treated with interstitial PDT when their volumes reached 2-3 cm3. Qualitative and quantitative data from pre- and post-PDT scintigraphy revealed a light-dose-dependent shut-down of tumour perfusion which was also time-dependent. Maximal shut-down, following a 1,600 J light-dose, occurred about 8 h post-PDT. Light exposure 2 h after the intravenous administration of the photosensitiser (Photofrin II) produced a greater vascular shut-down than did light exposure 24 h after the administration of the drug. Regional differences in perfusion within treated and non-treated tumours were measured by tomographic procedures. Light-dose-dependent volumes of perfusion shut-down were demonstrated in addition to the naturally occurring regional differences in tumour perfusion. This radiopharmaceutical may have future utility for monitoring the clinical treatment of solid tumours with PDT.

Nitroimidazoles are good quenchers of triplet state porphyrins in chemical systems, thereby inhibiting singlet oxygen formation and type II photodynamic reactions. Photobiological studies were performed with EMT-6 tumor cells in vitro utilizing Photofrin II (PII) in combination with etanidazole (ETAN), misonidazole (MISO), and trifluoromisonidazole (TF-MISO). After short-term (1 h) exposure of cells to PII, 5 mM ETAN and MISO had no effect on photoinactivation while 5 mM TF-MISO had a small but significant protective effect. When the intracellular oxygen level was equilibrated with 0.3% oxygen in the gas phase, all three nitroimidazoles produced significant photoprotection at concentrations as low as 0.3 microM. After long-term (24 h) exposure of cells to PII, all three nitroimidazoles demonstrated large photoprotective effects under both aerobic and 0.3% oxygen conditions. At equal concentrations of nitroimidazole, photoprotection was greatest for the most lipophilic compound (TF-MISO) and least effective for the most hydrophilic compound (ETAN). These studies suggest that nitroimidazoles can quench triplet state porphyrins (within cells) to reduce intracellular concentrations of singlet oxygen, the putative toxin in PII photoinactivation. In addition, after long-term exposures to PII when porphyrins have partitioned into cellular membranes and lipid environments, the lipophilicity of this class of photoprotector correlates with effectiveness in these mammalian cells.

S of the Society of Nuclear Medicine in Canada and Prairie Provinces Chapter, SNM Conjoint Meeting: PDF Only

1-(5-Iodo-5-deoxy-beta-D-arabinofuranosyl)-2-nitroimidazole (IAZA) has been synthesised and labeled with 125I. Radioiodinated IAZA was shown to undergo hypoxia-dependent binding in EMT-6 cells in vitro and to have an initial binding rate of 284 pmole/10(6) cells/hr at a substrate concentration of 30 microM. This binding rate is more than three times that of the reference compound, misonidazole (89 pmole/10(6) cells/hr). The elevated binding rate was accompanied by in vitro cytotoxicity 30-40 times greater than that observed for misonidazole. Whole-body elimination and biodistribution studies in BALB/c mice bearing implanted, subcutaneous EMT-6 tumors showed a rapid excretion (greater than 98% in 24 hr) with moderate tissue levels which, in general, declined as a function of blood clearance. Tumor-to-blood ratios of 4.6 (4 hr) and 8.7 (8 hr), with respective tumor uptake values of 2.08% and 1.22% ID/g of tissue, form a rational basis for evaluation of this and related 2-nitroimidazole analogs as radiopharmaceuticals suitable for scintigraphic evaluation of tissue (tumor) hypoxia.

A series of radioiodinated 1-substituted 2-nitroimidazoles were synthesised and evaluated as non-invasive probes for the detection of hypoxic tissues. The compounds synthesised were 2-(2-nitroimidazolyl)-3',5'-diiodo-4'-hydroxyacetophenone 4 and the 3'-monoiodo analog 5, the equivalent 3'-hydroxyacetophenone 6 and 2-(2-nitroimidazolyl)-1-(3,5-diiodo-4-hydroxyphenyl)ethanol 8. These compounds were prepared as their radioiodinated derivatives directly from the appropriate precursors, by electrophilic aromatic substitution reactions using I-125. The in vivo distribution of {I-125}-4 in male B6D2F1 mice bearing a subcutaneous Lewis lung tumor indicated very slow blood clearance (42.8% remaining at 4 hours and 22.9% at 24 hours) and excretion of only about 10% of the dose over 24 hours. Lung and tumor tissue accumulated the greatest concentrations of activity, reaching maximum organ to blood ratios of 0.52 and 0.48 respectively at 24 hours after administration of the drug. Compounds 4 and 5 exhibited no significant toxicity to EMT-6 cell cultures under either oxic or hypoxic conditions. The compounds generally showed selective binding to EMT-6 cells under hypoxic incubation with initial binding rates similar to misonidazole 1.

S of the Society of Nuclear Medicine in Canada and Prairie Provinces Chapter, SNM Conjoint Meeting: PDF Only

Nitropyrrole derivatives have been tested as hypoxic cell radiosensitizers in vitro and in vivo. Radiosensitizing potential generally increases with nitropyrrole electron affinity. N-hydroxyethyl substitution decreases toxicity relative to N--CH3, N--CH2 CH3 and N--CH2 CH2 CH3 substitution. The most effective nitropyrrole tested in vivo is N-hydroxyethyl-2-cyano-5-nitropyrrole (NP-1).

CHAPMAN, J. D., BLAKELY, E. A., SMITH, K. C., URTASUN, R. C., LYMAN, J. T., AND TOBIAS, C. A. Radiation Biophysical Studies with Mammalian Cells and a Modulated Carbon Ion Beam. Radiat. Res. 74, 101-111 (1978). Chinese hamster (V-79) and human kidney (T-l) cells were irradiated in stirred suspensions placed at various positions in the plateau and extended Bragg peak of a 400-MeV/amu carbon ion beam. The range of the ions was modulated by a lead (translational) ridge filter and a brass (spiral) ridge filter designed to produce extended peaks of -4 and 10 cm, respectively. Stationary-phase and Gi-phase populations of Chinese hamster cells were found to have different absolute radiosensitivities which, in turn, were different from that of asynchronous human kidney cells. The increase in relative biological effectiveness (RBE) observed as carbon ions were slowed down and stopped in water was similar for the three cell populations at doses greater than 400 rad. At lower doses the RBE was greater for the hamster cell populations than for the human kidney cells. The gain in RBE (at the 50% survival level) between the plateaus and the middle region of the extended peaks was -2.0 and 1.7 for the 4- and 10-cm extended peaks, respectively. Oxygen enhancement ratios (OER) were determined at the 10% survival levels with stationary-phase populations of hamster cells. Values of 2.8, 2.65, and 1.65 were obtained for the OER of 220-kV X rays, plateau carbon, and the middle region of the 4-cm carbon peak, respectively. Across the 10-cm carbon peak the OER was found to vary between values of 2.4 to 1.55 from the proximal to distal positions.

Chemicals which selectively radiosensitize hypoxic mammalian cells have been considered as adjuvants in the radiotherapy of human tumours (Adams, 1973). The screening of various nitroheterocyclic drugs indicated numerous structures which are active as sensitizers in mammalian cells in vitro (Chapman, Reuvers and Borsa, 1973). The discovery that some drugs with current clinical usage were excellent radiosensitizers of hypoxic mammalian cells (Reuvers, Chapman and Borsa, 1972; Chapman et al., 1972), generated some optimism as regards an early clinical trial. A successful application of such drugs in the radiotherapy of human tumours would depend upon favourable pharmacological properties. In the selection of metronidazole for this study, radiosensitizing effectiveness at low drug concentration was sacrificed in favour of acceptable pharmacological properties.