Delineation of the border zone of ischemic rabbit myocardium by a technetium-labeled nitroimidazole.

Delineation of viable ischemic myocardium is an important problem in nuclear cardiology. To determine the feasibility of using a technetium-labeled nitroimidazole as an indicator of ischemic myocardium at risk of infarction, we characterized the distribution of a 2-nitroimidazole-derivatized PnAO ligand and its 99mTc complex, 99mTcO(PnAO)-1-CH2-(2NI) (BMS-181321) in the ischemic territory of the left anterior descending (LAD) coronary artery of the rabbit. In preliminary experiments, the performance of 14C-deoxyglucose (14C-2DG) and 14C-misonidazole was assessed relative to apparent regional relative myocardial blood flow (rMBF) indicated by 99mTc-teboroxime using double-label autoradiography in the rabbit LAD occlusion model. After demonstrating that 14C-2DG and 14C-misonidazole are selectively retained in the lateral border of the ischemic territory, BMS-181321 was co-injected intravenously, with either 14C-2DG or 14C-misonidazole, 20 min after LAD occlusion. In a separate experiment, 99mTcO(PnAO)-6-CH3, a complex with the same lipophilicity (log k' 0.26 vs. 0.31) as BMS-181321 but which lacks the 2NI moiety, was co-injected with 14C-2DG. After 30 min, the rabbits were sacrificed and 14C/99mTc autoradiograms were obtained from the same tissue sections. The autoradiograms revealed that BMS-181321 was retained with the same microregional distribution as both 14C-2DG and 14C-misonidazole in the border zone of the ischemic LAD territory. The selective retention of BMS-181321 depends on the presence of the nitroimidazole group, since 99mTcO(PnAO)-6-CH3 has a uniformly low myocardial distribution in contrast to the enhanced uptake of co-injected 14C-2DG. These data demonstrate that BMS-181321 is selectively retained in hypoxic myocardium and demarcates the ischemic border zone in a manner similar to 14C-2DG and 14C-misonidazole.

Relationship between in vitro transendothelial permeability and in vivo single-pass brain extraction.

UNLABELLED: In vitro transendothelial permeability was compared to in vivo rat single-pass cerebral extractions to evaluate which method would best estimate the blood-brain barrier (BBB) permeability of several SPECT imaging agents. METHOD: Six 99mTc complexes and seven non-Tc complexes were tested in vitro using monolayers of primary bovine brain microvessel endothelial cells and in vivo using the rat single-pass cerebral extraction model. In vitro transendothelial permeability indices (PI) were determined by measuring the average percent of radioactivity traversing the monolayers as a function of time. In vivo single-pass cerebral extractions were determined using an indicator fractionation method. RESULTS: A positive correlation between extraction and PI was found for the non-Tc complexes (r2 = 0.96). The CBF imaging agents 99mTc-ECD and 99mTc-PnAO have high values for E and PI, demonstrating that these agents penetrate the BBB and have a high membrane permeability, while the heart imaging agent 99mTc-sestamibi had low values for both E and PI. The low PI and E values for 99mTc-sestamibi are consistent with a low brain uptake for this agent, except in cases of disruption of the BBB. In contrast to 99mTc-ECD, 99mTc-PnAO and 99mTc-sestamibi, which had concordant values for E and PI, two highly lipophilic boronic acid adducts of technetium dioxime (BATOs), 99mTc-teboroxime and 99mTcCl(DMG)3(2)MP, had low negative values for PI, but high values for E. In addition, after 3 hr of incubation, the monolayer-to-medium concentration ratio of the BATOs was 642:1 and 744:1, respectively. This compares with values of 89:1 (99mTc-PnAO), 25:1 (99mTc-ECD) and 34:1 (99mTc-sestamibi). CONCLUSION: These data suggest that the high in vivo single-pass extraction of the BATOs may be explained by a hydrophobic interaction with the luminal surface of the capillary endothelial cell plasma membrane. We conclude that a high single-pass extraction cannot necessarily be used to infer high BBB or membrane permeability.

Imaging ischemic tissue at risk of infarction during stroke.

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.

Measurement of myocardial blood flow using a co-injection technique for technetium-99m-teboroxime, technetium-96-sestamibi and thallium-201.

We have compared apparent myocardial blood flow (MBFapparent) indicated by 99mTc-teboroxime, 96Tc-sestamibi and 201TI to true MBF indicated by radiolabeled microscopheres using a technique for the co-injection of four radionuclides in the same animal. Studies were performed using rats in a single-pass model to obtain global MBF and using dogs in a multiple-pass model to determine regional MBF. To provide a wide range of MBF, adenosine was administered intravenously and the left anterior descending coronary artery was then ligated in the dogs, or hypercapnia was induced by decreasing respiratory frequency in the rats. The microsphere formula for determining MBF was applied to all agents. When MBFapparent was plotted as a function of true MBF, the ability of each agent to measure changes in true MBF was demonstrated by the proximity of the plotted function to the line of identity. For both the single and multiple-pass studies, statistical analysis of the nonlinear relationship between MBFapparent and true MBF showed that 201TI and 99mTc-teboroxime approximate true MBF better than 96Tc-sestamibi (p less than 0.001) under the conditions used in the present studies. In the single-pass studies, 99mTc-teboroxime approximated true MBF better than 201Tl (p less than 0.05), but in the multiple-pass experiments, 201Tl approximated true MBF better than 99mTc-teboroxime in only one dog (p less than 0.01) with no difference in the other two. Determination of the permeability-surface area product, PS, for each agent shows that the higher fidelity to true MBF obtained with 201Tl and 99mTc-teboroxime is related to substantially greater PS values for these agents relative to 96Tc-sestamibi.

The response of human and rodent cells to hyperthermia.

Inherent cellular radiosensitivity in vitro has been shown to be a good predictor of human tumor response in vivo. In contrast, the importance of the intrinsic thermosensitivity of normal and neoplastic human cells as a factor in the responsiveness of human tumors to adjuvant hyperthermia has never been analyzed systematically. A comparison of thermal sensitivity and thermo-radiosensitization in four rodent and eight human-derived cell lines was made in vitro. Arrhenius plots indicated that the rodent cells were more sensitive to heat killing than the human, and the break-point was 0.5 degrees C higher for the human than rodent cells. The relationship between thermal sensitivity and the interaction of heat with X rays at low doses was documented by thermal enhancement ratios (TER's). Cells received either a 1 hr exposure to 43 degrees C or a 20 minute treatment at 45 degrees C before exposure to 300 kVp X rays. Thermal enhancement ratios ranged from 1.0 to 2.7 for human cells heated at 43 degrees C and from 2.1 to 5.3 for heat exposures at 45 degrees C. Thermal enhancement ratios for rodent cells were generally 2 to 3 times higher than for human cells, because of the fact that the greater thermosensitivity of rodent cells results in a greater enhancement of radiation damage. Intrinsic thermosensitivity of human cells has relevance to the concept of thermal dose; intrinsic thermo-radiosensitization of a range of different tumor cells is useful in documenting the interactive effects of radiation combined with heat.

Studies with bifunctional bioreductive drugs. II. Cytotoxicity assayed with A-549 lung carcinoma cells of human origin.

A lung carcinoma cell line of human origin (A-549) cultured in vitro was used to investigate the cytotoxic effect of a range of bifunctional bioreductive drugs. The drugs tested consisted of nitroimidazoles or nitrofurans with terminal aziridine rings on the side chain and are designated RSU-1069, RSU-1164, RB-7040, RB-88716, and RB-88712. Measurements of the cytotoxicity in air demonstrated that methyl and alkyl addition to the aziridine ring reduced cell killing with progressive substitution of the alkylating moiety. A comparison was made of cytotoxicity in air and hypoxia with cells exposed to drugs for a 4-h period. A direct comparison of the aerobic and hypoxic cytotoxicity of RSU-1069 in human (A-549) and rodent cells (V-79-379A) yielded similar results. The cytotoxicity factors, defined to be the ratio of drug concentrations under aerobic and hypoxic conditions which result in 10% cell survival, were found to be 40, 25, 18, and 8, respectively, for the four agents RSU-1069, RSU-1164, RB-88712, and RB-88716 tested in A-549 cells. It has been suggested that under aerobic conditions the aziridine ring is primarily responsible for aerobic toxicity, whereas under hypoxic conditions, the aziridine moiety combined with a reduced 2-nitro moiety produces a bifunctional agent (I. J. Stratford et al., Br. J. Cancer 53, 339-344, 1986).

Hyperthermia studies in polyamine-altered human lung carcinoma cells.

The effect of polyamine depletion on the survival response of human lung carcinoma cells (A-549) to acute heating at 45 degrees C and its effect on the induction and decay of thermotolerance were investigated in exponential and plateau-phase cells. A 48-h exposure to 1 mM alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, was used to deplete intracellular levels of putrescine and spermidine. Inhibition of polyamine synthesis had no effect on the survival of exponential cells to heating at 45 degrees C, but slightly enhanced the killing of slowly proliferating plateau-phase cells. While DFMO treatment did not inhibit the development of thermotolerance, it caused a reduction in the thermotolerance ratio of exponential cells from 2.6 to 1.80, and from 1.66 to 1.59 in plateau-phase cells. DFMO caused thermotolerance to decay more rapidly in polyamine-depleted cells as well. Flow cytometry demonstrated that DFMO did not alter the cell cycle distribution of plateau-phase cells (i.e., greater than 73% in G1/G0), but caused a block and time-dependent accumulation of exponential cells in G1/G0. The cytostatic properties of DFMO in exponential cells which favor its use with phase-specific agents, and its ability to alter the magnitude and decay of thermotolerance in human carcinoma cells suggest a potential role for this nontoxic agent in clinically oriented hyperthermia studies.

Thermotolerance in human cells of normal and neoplastic origin.

Thermotolerance was assayed under controlled conditions in normal and neoplastic human cells with the aim of identifying intrinsic differences in the acquisition of heat resistance. Carcinoma cells from colon (WIDR) and lung (A-549) were compared to fibroblasts (AG-1522) and primary explants of umbilical vein endothelial cells (HUVEC) in terms of their response to a 20 min heat shock at 45 degrees C. Single cell survival, heat shock protein (HSP) synthesis, and glutathione were studied as common endpoints. Production of HSPs was immediate in normal derived cells, and was evident in neoplastic cells 2 h following heat shock. Maximum translation of 70 and 90 kDa HSPs was observed at 6 h in all cell types and ceased by 24 h. Maximum cell survival peaked 4-24 h after the second heat dose showing a close association with HSP synthesis. Thermotolerance developed rapidly and decayed slowly over the next 5 days in normal and transformed cells. There was no correlation between the development of thermotolerance and endogenous glutathione content in all cells. There is little to distinguish thermotolerance in normal and neoplastic cells when compared on an equal basis as demonstrated here, indicating that therapeutic gain in vivo may be largely dependent on tumour physiology.

Oxygen dependence for chemosensitization by misonidazole.

Misonidazole (MISO) potentiates the cell killing effect of certain chemotherapy agents, but only under hypoxic conditions. The purpose of the present study was to define the range of oxygen concentrations over which chemosensitization by MISO takes place using mammalian cells cultured in vitro, and to compare this with the oxygen levels required for radiosensitization. V-79 hamster cells, attached to permanox dishes, were gassed with known concentrations of oxygen (less than 10 to 200,000 ppm) and treated with 1 and 5 mM MISO for 4 h previous to exposure to the chemotherapy agent, melphalan. In a parallel series of experiments, under the same gassing conditions, cells were irradiated with graded doses of X-rays at various oxygen concentrations. The K factor i.e. the oxygen concentration which defined half the maximum effect was found to be approximately 4776 ppm for radiosensitization and approximately 400 ppm for chemosensitization by MISO. It is evident that a significantly more stringent level of hypoxia is required for chemosensitization by MISO to take place than for radiosensitization.

A comparison of the heat and radiation sensitivity of rodent and human derived cells cultured in vitro.

A human lung and breast carcinoma cell line of epithelial origin (A-549 and MCF-7) were compared with a rodent fibroblast line (V-79) for their sensitivity to killing by X rays and heat, in addition, a correlation was sought between loss of endogenous thiols and thermosensitivity. Endogenous cellular thiols play a major role in many protective, enzymatic and synthetic processes in mammalian cells. Glutathione, a key non-protein thiol, not only protects against radiation and peroxide-induced damage, but is also a primary intracellular reductant. Thiol depletion was achieved using two agents that work by totally different mechanisms--one a substrate for glutathione-S-transferase (Diethylmaleate) and the other an inhibitor of a key enzyme in the gamma-glutamyl cycle (Buthionine-SR-Sulfoximine). The results of this study demonstrated that thiol depletion by DEM to 50% of the control values had no effect on the response of hamster cells to acute (45 degrees C) or chronic (42.5 degrees C) hyperthermia. Substantial potentiation of heat damage, however, was seen at thiol levels below 10% at 42.5 degrees C. Thiol depletion by BSO to levels of 25% of the control values had no sensitizing effect on the heat sensitivity of hamster or human lung carcinoma cells at 45 degrees C. For any given heat exposure, the human cells were markedly more resistant to killing than the hamster cells, however, they were more radiosensitive than the V79, line when exposed to 300 kVp X rays (D0's of 1.65 vs. 2.52 Gy). The results of this study indicate that thiols do not play a critical role in mammalian cell thermosensitivity at 45 degrees C and indicate that the use of human carcinoma cell lines may better predict heat inactivation in human cells in vivo.