Decreased tissue oxygen tension is a component of many diseases. Although hypoxia can be secondary to a low inspired P02 or a variety of lung disorders, the commonest cause is ischemia due to an oxygen demand greater than the local oxygen supply. In tumors, low tissue p02 is often observed, most often due to a blood supply inadequate to meet the tumor's demands. Hypoxic tumor tissue is associated with increased resistance to therapy. In the heart tissue hypoxia is often observed in persistent low-flow states, such as hibernating myocardium. In patients with stroke, hypoxia has been associated with the penumbral region, where an intervention could preserve function. Despite the potential importance of oxygen levels in tissue, difficulty in making this measurement in vivo has limited its role in clinical decision making. A class of compounds known to undergo different intracellular metabolism depending on the availability of oxygen in tissue, the nitroimidazoles, have been advocated for imaging hypoxic tissue. When a nitroimidazole enters a viable cell the molecule undergoes a single electron reduction, to form a potentially reactive species. In the presence of normal oxygen levels the molecule is immediately reoxidized. This futile shuttling takes place for some time, before the molecule diffuses out of the cell. In hypoxic tissue the low oxygen concentration is not able to effectively compete to reoxidize the molecule and further reduction appears to take place, culminating in the association of the reduced nitroimidazole with various intracellular components. The association is not irreversible, since these agents clear from hypoxic tissue over time. Initial development of nitroimidazoles for in vivo imaging used radiohalogenated derivatives of misonidazole, such as fluoromisonidazole, some of which have recently been employed in patients. Two major problems with fluoromisonidazole are its relatively low concentration within the lesion and the need to wait several hours to permit clearance of the agent from the normoxic background tissue (contrast between lesion and background typically <2:1 at about 90 min after injection). Even with high-resolution positron emission tomographic imaging, this combination of circumstances makes successful evaluation of hypoxic lesions a challenge. Single-photon agents, with their longer halflives and comparable biological properties, offer a greater opportunity for successful imaging. In 1992 technetium-99m labeled nitroimidazoles were described that seem to have at least comparable in vivo characteristics. Laboratory studies have demonstrated preferential binding of these agents to hypoxic tissue in the myocardium, in the brain, and in tumors. These investigations indicate that imaging can provide direct evidence of tissue with low oxygen levels that is viable. In the experimental setting this information is useful to plan a more aggressive approach to treating tumors, or revascularize a heart suffering ischemic dysfunction. Even from this early vantage point the utility of measuring tissue oxygen levels with external imaging suggests that hypoxia imaging could play a major role in clinical decision making.

Nitroimidazoles and imaging hypoxia.

Evolution of Tc-99m in diagnostic radiopharmaceuticals

In ischaemic stroke, expansion of the infarct core occurs at the expense of surrounding hypoxic, metabolically compromised tissue over a period of 24 h or more in a considerable proportion of patients. It is uncertain whether hypoxic tissue observed at later times after stroke onset retains the potential for survival or whether such survival has an impact on functional outcome. These factors may determine the effectiveness of therapeutic strategies aimed at salvaging this tissue. We tested the hypotheses that metabolically compromised hypoxic tissue observed within 48 h after onset of ischaemic stroke retains the potential for spontaneous survival and that the impact of such survival on functional outcome is time dependent. Consecutive patients presenting within 48 h of ischaemic stroke were studied with [(18)F]fluoromisonidazole, a ligand binding to hypoxic but viable tissue, and PET. Subjects were grouped into two time epochs, 12 h, based on the interval from stroke onset to the time of tracer injection, and had infarct volumes measured on CT/MRI at 7 days (n = 60). The total ischaemic volume (TIV) and the proportion of the TIV that spontaneously survived (surviving hypoxic volume ratio, SHVR) were defined from the co-registered CT/MRI images. These volumetric measures were correlated with neurological outcome assessed at day 7-10 by percentage change in the National Institutes of Health Stroke Scale (DeltaNIHSS), and at 3 months by Barthel Index (BI) and modified Rankin Score (mRS). Of 66 patients investigated, hypoxic tissue occurred in 33 and outcome data was available in 27. Hypoxic tissue constituted >20% of the TIV in 60% of studies 12 h. The spontaneously surviving proportion of the TIV (median 6.9%) or hypoxic tissue (median 45.9%) was not significantly different in patient subgroups studied 12 h after stroke onset. Spontaneous survival of hypoxic tissue (surviving hypoxic volume ratio) was associated with improved neurological outcome in both time epochs: 12 h, DeltaNIHSS (r = 0.59, P < 0.01) and day 90 mRS (r = -0.46, P < 0.05). The finding that similar proportions of hypoxic tissue survived spontaneously within each time epoch suggests that its fate is not predetermined. The favourable neurological outcome associated with spontaneous survival of hypoxic tissue, even 12-48 h after stroke onset, suggests that the volume of hypoxic tissue that progressed to infarction may represent a valuable target for therapeutic intervention.

Hypoxic tissue in ischaemic stroke: persistence and clinical consequences of spontaneous survival

Background and Purpose— Nitroimidazole imaging is a promising contender for noninvasive in vivo mapping of brain hypoxia after stroke. However, there is a dearth of knowledge about the behavior of these compounds in the various pathophysiologic situations encountered in ischemic stroke. In this article we report the findings from a systematic review of the literature on the use of the nitroimidazoles to map hypoxia after stroke. Summary of Review— We describe the characteristics of nitroimidazoles as imaging tracers, their pharmacology, and results of both animal and clinical studies during and after focal cerebral ischemia. Findings in brain tumors are also presented to the extent that they enlighten results in stroke. Early results from application of kinetic modeling for quantitative measurement of tracer binding are briefly discussed. Conclusions— Based on this literature review, nitroimidazole hypoxia imaging agents are of considerable interest in stroke because they appear, both in animal models and...

Applications of Nitroimidazole In Vivo Hypoxia Imaging in Ischemic Stroke

Recent applications of high-performance liquid chromatography to the analysis of metal complexes.

Autoradiograms obtained after middle cerebral artery occlusion (MCAO) in spontaneously hypertensive rats show that the 99mTc complex of a 2-nitroimidazole-derivatized propylene amine oxime (BMS-181321) is selectively retained in acutely ischemic brain before disruption of the blood-brain barrier (BBB), but not in the ischemic infarct. BMS-181321 is therefore a marker of ischemic tissue at risk of infarction and its uptake, unlike that of x-ray and magnetic resonance contrast agents, does not require disruption of the BBB. In keeping with this conclusion, we have found that the single-pass cerebral extraction fraction of BMS-181321 is 0.67 at normal rat whole-brain blood flow. Sequential single-photon emission computed tomographic images obtained from cats after MCAO show that the initial distribution of BMS-181321 approximates regional CBF and that selective retention subsequently produces a positive image within the ischemic territory. BMS-181321 is the first Tc complex able to indicate not only ischemia, but also ischemic tissue at risk of infarction. Use of this novel Tc complex to monitor biochemical events during ischemia may contribute to the clinical management of acute stroke.

Imaging ischemic tissue at risk of infarction during stroke.

We have developed a 99mTechnetium complex for imaging of hypoxic tissue (BMS-181321). Recently, another nitroimidazole derivative, based upon a bis(amine-phenol) ligand, was described in the patent literature. To compare this compound to BMS-181321, we have synthesized the ligand, prepared its 99mTc complex, and evaluated its performance in two in vitro assays of bioefficacy: membrane permeability and uptake in normoxic and anoxic cardiocytes. In attempting to reproduce the synthesis of the ligand described in the patent application, we found that one intermediate could not be made by the method described, and alternative routes were investigated. Complexation of the bis(amine-phenol) nitroimidazole with 99mTc gave an apparent single complex; this appeared as a broad peak on HPLC analysis. Purification by a solid-phase method gave a complex with 95% radiochemical purity. This complex was not permeable to cultured bovine brain endothelial cells nor did it show preferential uptake in anoxic myocytes.

The Synthesis and in vitro Evaluation of a 99mTechnetium-Nitroimidazole Complex Based on a Bis(amine-phenol) Ligand: Comparison to BMS-181321

Synthesis of some isothiocyanatophenylboronic acids

It is well known that cardiac muscle has a high and continuous requirement for oxygen. Oxygen is primarily needed, i.e., over 95%, to maintain flux through mitochondrial oxidative phosphorylation for synthesis of ATP. Oxygen is delivered to the working cardiac myocytes at levels consistent with the prevailing metabolic demands established by the various ATP-dependent reactions, principally cycling of the contractile myofilaments. When oxygen delivery is diminished, for example during ischemia, electron flux within the respiratory chain is impeded by the lack of appropriate electron acceptor at the cytochrome oxidase reaction. Consequently, the concentration of reducing equivalents (NADH and FADH2) increases. This condition establishes the opportunity for these and other sources of biological reductants to interact with exogenously supplied molecules having high electron affinity.

Potential of Nitroimidazoles as Markers of Hypoxia in Heart

The Resolvosil BSA-7, Chiracel OD, and Chiralpak AD columns were examined to determine whether one might be suitable for the separation of enatiomers of the ligand hexamethyl-propyleneamine oxime (HM-PAO) and/or its technetium-99m complex. Using hexane/IPA as eluent, partial separation (Rs = 0.96) of the d- and l-ligand enantiomers was achieved on the OD column, but as the meso-form of the ligand was eluted midway between the d- and l-enantiomers. There was excellent separation of the technetium-99m complexes of d- and 1-HM-PAO on the OD column, and partial separation (Rs = 0.90) of the meso- and d-complexes. Only partial resolution of the Tc-99m complexes of d,l- and meso-diastereoisomers was achieved on the AD column, but this column provided baseline resolution of all three stereoisomeric forms of the ligand. Thus, the OD column could be used for HPLC analysis of Tc-99m complexes of HM-PAO stereoisomers, while the AD column is suitable for analysis of the ligand stereoisomers.

K. Ramalingam

Papers

Imaging ischemic tissue at risk of infarction during stroke.

The Synthesis and in vitro Evaluation of a 99mTechnetium-Nitroimidazole Complex Based on a Bis(amine-phenol) Ligand: Comparison to BMS-181321

Synthesis of some isothiocyanatophenylboronic acids

Potential of Nitroimidazoles as Markers of Hypoxia in Heart

Separation of the Stereoisomers of Hexamethyl-Propyleneamine Oxime (HM-PAO) by High-Performance Liquid Chromatography