J.A. Raleigh

Fluorescence immunohistochemical detection of hypoxic cells in spheroids and tumours.

Polyclonal antibodies have been raised in rabbits to a haemocyanin adduct of a reductively-activated, fluorinated analogue of misonidazole. Fluorescence immunohistochemical studies show that the polyclonal antibodies bind to spheroid sections and tumour sections in patterns similar to those revealed by autoradiographic studies with a tritium-labelled derivative of the fluorinated misonidazole analogue.

Binding of 3H-misonidazole to solid human tumors as a measure of tumor hypoxia☆

Treatment-resistant, chronically hypoxic tumor cells have been assumed to exist in some solid human tumors, limiting their curability. To date, six patients with different types of tumors have been studied using radioactive labelled electron affinic compounds that bind to hypoxic cells. Although the gross clinical appearance of the tumors in all six patients was of a large and fixed mass which might on clinical grounds be expected to contain hypoxic cells, we have observed drug binding to hypoxic regions in only two, a rapidly growing small cell lung cancer (SCLC) and a malignant melanoma. The hypoxic fraction of the malignant melanoma was found to be 6% and the SCLC tumor approximately 10%. We have observed that areas of maximum adduct formation can be found in tumor cells immediately adjacent to blood vessels, suggesting that blood flow over the labelling interval was restricted. These preliminary studies suggest that sensitizer adduct formation in human tumor tissue may be a useful measure of tissue pO2 at the cellular level and that tumor hypoxia might be more related to the rate of tumor growth and histological grading than to tumor size.

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.

A hexafluorinated 2-nitroimidazole (CCI-103F) has been synthesized and its properties as a hypoxic cell binder investigated. The drug has a plasma half-life of 90 min in Balb/C mice and a tumor-to-plasma ratio of 0.8. Following sustained exposure to the drug and a washout period for the unbound drug, liver and tumor samples were excised. NMR investigation of the excised tissue in a 2.35 T animal sized NMR facility revealed the presence of 19F bound to both tissues. Further improvement in sensitivity is required to make 19F NMR detection of binding in situ feasible.

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

The binding of ring-labelled and side-chain labelled misonidazole to hypoxic cells in monolayer and spheroid cultures of mammalian cells has been compared. The kinetics and patterns of binding for the two labelled compounds are indistinguishable. This finding has implications for the mechanism of binding and for the design of misonidazole analogues which might be used to identify hypoxic zones in tumours.

A novel technique for measuring human tissue pO2 at the cellular level.

Some electron-affinic drugs, developed as hypoxic cell radiosensitizers, become selectively bound to the molecules of hypoxic cells by metabolism. This technique has been used to identify zones of chronically hypoxic cells in multicellular spheroids and animal tumours. Tritiated-misonidazole was administered to a patient with advanced melanoma 22 h prior to the surgical resection of a large metastatic s.c. lesion growing on the face. Autoradiographic analysis of histological sections revealed zones of intense labelling by the radioactive drug, indicative of tumour cells which were chronically hypoxic. This technique appears to provide an indirect measurement of tissue pO2 at the cellular level from which estimates of the tumour hypoxic fraction can be made. These data are encouraging as regards the development of sensitizer-adduct procedures for the invasive and non-invasive measurement of hypoxia in both tumours and normal tissues.

Reductive fragmentation of 2-nitroimidazoles: amines and aldehydes.

Glyoxal has been identified as a product in the fragmentation of reduced 2-nitroimidazole radiosensitizers. Quantitative analysis of glyoxal as its bis (2,4-dinitrophenylhydrazone) derivative shows that it is formed in good yield (9-23%) in a variety of 2-nitroimidazoles. In addition to glyoxal a second as-yet-unidentified carbonyl compound, and a series of amines are formed when reduced 2-nitromidazoles fragment in the presence of water. One of the amines derived from misonidazole is identified as 1-amino-3-methoxypropan-2-ol, the product of extensive ring cleavage. Radiation chemical reduction of the 2-nitromidazoles proceeds with the consumption of 3 electrons for each molecule reduced. This could imply that a radical disproportionation or dimerization step is involved in the reductive degradation of 2-nitroimidazoles.

Nitroreductase-induced binding of nitroaromatic radiosensitizers to unsaturated lipids: Nitroxyl adducts

Abstract The nitrobenzene radiosensitizer, 3,5-dinitrobenzonitrile (DNBN), reacted with dioleoylphosphatidyl choline (DOL) in the presence of cytochrome c reductase and NADH to produce a stable free radical characteristic of a nitroxyl radical. The product was indistinguishable from that formed when nitrobenzene reacted with DOL and from that formed when DNBN is reduced by xanthine oxidase in the presence of DOL as reported previously (J. A. Raleigh and F. Y. Shum, Proceedings of an International Conference on Oxygen and Oxy-radicals in Chemistry and Biology , Austin, TX, May 1980, in press). It is concluded that a nitroso intermediate formed in the enzyme reduction of DNBN was trapped in an “ene” reaction with unsaturated lipid. Neither nitroimidazole nor nitropyrrole radiosensitizers exhibited this reaction even though electron transfer to the heterocyclic nitroaromatics could be demonstrated.

Metabolic binding of misonidazole to mouse tissues: Comparison between labels on the ring and side chain, and the production of tritiated water

The 2-nitroimidazole, misonidazole, is of current interest as an imaging agent for hypoxic regions in tumors and in vascular disease such as stroke. The basis of this technique is the reductive activation and binding of nitroheterocycles which is much more efficient in the absence of oxygen. The appropriate molecular location for an active isotope on the nitroheterocyclic probe depends on the nature of the metabolites retained in tissues after the parent drug has been cleared. Previous studies with tumor cells in vitro indicated that a ring label (2-14C) and a side-chain label (3H) were retained equally efficiently in the acid-insoluble fraction, whereas 1.5 to 3 times more side-chain label was retained in the total pool (acid soluble plus acid insoluble) of metabolites in several normal murine tissues. We show here that the excess side-chain label in six normal tissues, plasma and EMT6 tumors was found entirely in the acid-soluble fraction as a volatile component. This volatile component was tentatively identified as tritiated water. It appeared that, in general, molecular products of misonidazole metabolism were retained in mouse tissues, with the exceptions that a small excess of ring label was found in liver and heart and that tritiated water appeared in the acid-soluble fraction of all tissues. Tritiated water would not be important in imaging studies but could be a factor in studies in which scintillation counting of tritiated nitroheterocyles is used.

Quantitation of hypoxia in multicellular spheroids by video image analysis

Polyclonal antibodies to a series of radiation chemically produced 2-nitroimidazole-protein adducts have been used in the indirect immunofluorescent detection of hypoxic cells in EMT6/Ed spheroids. The spheroids were incubated with the radioactive 2-nitrioimidazoles under atmospheres of air, several intermediate oxygen concentrations, and nitrogen. The fluorescence intensity of the marker across radii of spheroids labeled at various oxygen tensions, as measured by video image analysis, paralleled the autoradiographic grain density. Thus, we conclude that the immunohistochemical approach provides a quantitative measure of the distribution of 2-nitroimidazole adducts in spheroids. The advantages of speed, technical simplicity, economy, and independence from the requirement of radiolabeled precursor render the fluorescent marker of potential use in the clinical detection of cellular hypoxia and tissue ischemia.

Misonidazole with dexamethasone rescue: an escalating dose toxicity study.

Neurotoxicity induced by misonidazole (MISO) and desmethylmisonidazole (DMM) has become the dose limiting factor in clinical work. In 1981, we reported a preliminary study suggestive that Dexamethasone (DEXA) does have a protective effect against peripheral neuropathies (PN) resulting from toxicity of misonidazole. Furthermore, in that study we have observed that DEXA did not alter the plasma pharmacokinetics of misonidazole. We are presently investigating the use of DEXA (2 mg t.i.d. during treatment), with escalating doses of MISO in an attempt to modify its neurotoxicity. To date, 16 patients have been registered to receive total doses of MISO ranging from 13.5 to 17.5 gm/M2 given in 9 equally divided doses over 3 weeks. DEXA, 2 mg t.i.d. is given 3 days prior to the first dose and continues for the duration of therapy. All patients receive palliative radiation. No toxicity was seen at the total dose of 13.5 gm/M2. One grade I PN occurred in the first four patients receiving 15.5 gm/M2. Six additional patients were entered at this dose level and no further incidence of PN was observed. Patients are being entered at the next step of 17.5 gm/M2.