Monitoring vascular normalization induced by antiangiogenic treatment with (18)F-fluoromisonidazole-PET.

BACKGROUND: Rationalization of antiangiogenics requires biomarkers. Vascular re-normalization is one widely accepted mechanism of action for this drug class. The interstitium of tumors with abnormal vasculature is hypoxic. We sought to track vascular normalization with (18)F-misonidazole ([18F]-FMISO, a probe that detects hypoxia) PET, in response to window-of-opportunity (WoO) treatment with the antiangiogenic dovitinib. METHODS: Two patient-derived pancreas xenografts (PDXs; Panc215 and Panc286) and the spontaneous breast cancer model MMTV-PyMT were used. Animals were treated during 1 week of WoO treatment with vehicle or dovitinib, preceded and followed by [18F]-FMISO-PET, [18F]-FDG-PET, and histologic assessment (dextran extravasation, hypoxia and microvessel staining, and necrosis, cleaved caspase-3 and Ki67 measurements). After WoO treatment, gemcitabine (pancreas)/adriamycin (breast) or vehicle was added and animals were treated until the humane endpoint. Tumor growth inhibition (TGI) and survival were the parameters studied. RESULTS: [18F]-FMISO SUV did not change after dovitinib-WoO treatment compared to vehicle-WoO (0.54 vs. 0.6) treatment in Panc215, but it decreased significantly in Panc286 (0.58 vs. 1.18; P < 0.05). In parallel, 10-KDa perivascular dextran extravasation was not reduced with dovitinib or vehicle-WoO treatment in Panc215, but it was reduced in Panc286. Whereas the addition of dovitinib to gemcitabine was indifferent in Panc215, it increased TGI in Panc286 (TGI switched from -59% to +49%). [18F]-FMISO SUV changes were accompanied by an almost 100% increase in interstitial gemcitabine delivery (665-1260 ng/mL). The results were validated in the PyMT model. CONCLUSIONS: [18F]-FMISO accurately monitored vascular re-normalization and improved interstitial chemotherapy delivery.

Characterization of hypoxia in malignant pleural mesothelioma with FMISO PET-CT.

OBJECTIVES: Malignant pleural mesothelioma (MPM) is a chemotherapy resistant tumor with a poor prognosis. Hypoxia is increasingly recognized as an important factor in tumor aggressiveness and cellular resistance to chemotherapy and radiation treatment. This prospective pilot study was performed with [F-18] fluoromisonidazole (FMISO) PET-CT to characterize hypoxia in patients with MPM. MATERIALS AND METHODS: Twenty prospectively recruited patients with histologically or cytologically confirmed MPM not currently receiving systemic or local treatment underwent both FMISO and fluorodeoxyglucose (FDG) PET-CT scans within 2 weeks. FMISO and FDG PET-CT scans were independently analyzed visually and semi-quantitatively using SUVmax and tumor to background ratio (TBR) in order to assess tumor hypoxia and metabolic activity. Lesion by lesion analysis was performed in sites of measurable pleural masses. RESULTS: Visual analysis demonstrated tumor FMISO activity in 17 of 20 patients, and tumor FDG activity in 19 of 20 patients. Focal areas of bulky tumor were most likely to demonstrate hypoxia. In 19 patients suitable for semi-quantitative analysis the median FDG SUVmax was 6.4 (range 1.9-19.1), median FMISO SUVmax was 2.5 (range 1.4-3.7) and median FMISO TBR was 1.8 (1.1-2.5). There was a positive correlation between intensity of metabolic activity and hypoxia (r=0.72, p=0.001). Lesion by lesion analysis demonstrated a positive correlation between tumor thickness and FMISO activity (r=0.77, p<0.001). CONCLUSION: This pilot study confirms that MPM is a tumor with significant areas of hypoxia, particularly in dominant tumor masses. The relationship of tumor hypoxia to effectiveness of chemotherapy and/or radiation therapy warrants prospective assessment.

Characterization of fluoromisonidazole binding in stroke.

BACKGROUND AND PURPOSE: [18F]fluoromisonidazole (FMISO) positron emission tomography has been used to image hypoxia early after human stroke. To further study the role of hypoxia in stroke and the binding characteristics of FMISO, we aimed to develop [3H]FMISO autoradiography in an animal stroke model. We hypothesized that [3H]FMISO binding is prolonged, allowing correlation with 24-hour histology, and that there is no FMISO binding after effective reperfusion. METHODS: Temporary middle cerebral artery (MCA) occlusion was performed in rats, followed by [3H]FMISO administration. Tissue preparation for autoradiography and histology (from the same sections) was performed 2.5 hours after MCA occlusion (MCAo; replicating [18F]FMISO studies). Then, otherwise identical cohorts with tissue preparation at 2.5 or 24 hours were prepared. For reperfusion studies, animals had 1-hour MCAo, with [3H]FMISO administered 1 hour after reperfusion. RESULTS: [3H]FMISO autoradiography provided a high-resolution image of hypoxia throughout the ischemic territory. Delaying animal death from 2.5 to 24 hours allowed histological changes of stroke to develop, without significantly altering either relative intensity (1.88+/-0.06 and 2.02+/-0.11, respectively) or volume (25+/-6 mm3 and 28+/-5 mm3, respectively) of hypoxic binding. [3H]FMISO binding did not occur after effective reperfusion, despite histological injury from the preceding MCAo. CONCLUSIONS: [3H]FMISO autoradiography of hypoxia in experimental stroke offers several advantages. Bound FMISO is retained in tissues long term, enabling direct correlation with 24-hour histology. It is not bound after effective reperfusion. Therefore, positive [18F]FMISO positron emission tomography studies in stroke patients are indicative of ongoing tissue hypoxia, not merely recent tissue injury.

Fully automated synthesis of [18F]fluoromisonidazole using a conventional [18F]FDG module.

We developed a new fully automated method for the synthesis of [18F]fluoromisonidazole ([18F]FMISO) by modifying a commercial FDG synthesizer and its disposable fluid pathway. A three-step procedure was used to prepare the tosylate precursor, 1-(2'-nitro-1'-imidazolyl)-2-O-tetrahydrofuranyl-3-O-toluenesulfonylpropanediol. Using glycerol as the starting material, the precursor was synthesized with a yield of 21%. The optimal labeling conditions for the automated synthesis of [18F]FMISO was 10 mg of precursor in acetonitrile (2 ml heated at 105 degrees C for 360 s, followed by heating at 75 degrees C for 280 s and hydrolysis with 1 N HCl at 105 degrees C for 300 s. Using 3.7 GBq of [18F]F- as a starting activity, [18F]FMISO was obtained with high end-of-synthesis (EOS) radiochemical yields of 58.5+/-3.5% for 60.0+/-5.2 min with high-performance liquid chromatography (HPLC) purification. When solid-phase purification steps were added, the EOS radiochemical yields were 54.5+/-2.8% (337+/-25 GBq/micromol) for 70.0+/-3.8 min (n=10 for each group, decay-corrected). With a high starting radioactivity of 37.0 GBq, we obtained radiochemical yields of 54.4+/-2.9% and 52.8+/-4.2%, respectively (n=3). The solid-phase purification removed unreacted [18F]fluoride and polar impurities before the HPLC procedure. Long-term tests showed a good stability of 98.2+/-1.5%. This new automated synthesis procedure combines high and reproducible yields with the advantage of using a disposable cassette system.

Imaging the ischemic penumbra with 18F-fluoromisonidazole in a rat model of ischemic stroke.

BACKGROUND AND PURPOSE: The ischemic penumbra is a major focus of stroke research. 18F-fluoromisonidazole (18F-FMISO), a positron emission tomography (PET) marker of hypoxic cells, has shown promise as a technique to image the penumbra in humans. Our aim was to delineate the pattern of 18F-FMISO binding in a rat middle cerebral artery transient thread-occlusion model, and correlate this with tissue outcome at 24 hours. We hypothesized that the pattern of 18F-FMISO binding would mimic that seen in humans. METHODS: Thirty-eight rats underwent 2 hours transient middle cerebral artery (MCA) occlusion, and then received 18F-FMISO at time points from 0.5 to 22 hours post-MCA occlusion and were killed 2 hours later. Autoradiographic assessment of 18F-FMISO binding and assessment (triphenyltetrazolium chloride) of the area of infarction were performed on tissue slices. RESULTS: Until 1 hour after MCA occlusion, 18F-FMISO binding was increased in the entire MCA territory, with little or no infarction visible. Over the next 5 hours, the pattern of binding evolved to a small rim of intensely binding tissue surrounding the infarct core, which itself showed reduced binding compared with the contralateral hemisphere. By 24 hours, there was minimal accumulation of 18F-FMISO binding and a large area of infarction. CONCLUSIONS: The pattern of 18F-FMISO binding rats reproduced the pattern seen in humans, consistent with this tracer being a marker of the ischemic penumbra in both species. This technique may have application in studying the ischemic penumbra in animal models, and correlating this with similar studies in humans.

Splanchnic tissues undergo hypoxic stress during whole body hyperthermia.

Exposure of conscious animals to environmental heat stress increases portal venous radical content. The nature of the observed heat stress-inducible radical molecules suggests that hyperthermia produces cellular hypoxic stress in liver and intestine. To investigate this hypothesis, conscious rats bearing in-dwelling portal venous and femoral artery catheters were exposed to normothermic or hyperthermic conditions. Blood gas levels were monitored during heat stress and for 24 h following heat exposure. Hyperthermia significantly increased arterial O2 saturation, splanchnic arterial-venous O2 difference, and venous PCO2, while decreasing venous O2 saturation and venous pH. One hour after heat exposure, liver glycogen levels were decreased approximately 20%. Two hours after heat exposure, the splanchnic arterial-venous O2 difference remained elevated in heat-stressed animals despite normal Tc. A second group of rats was exposed to similar conditions while receiving intra-arterial injections of the hypoxic cell marker [3H]misonidazole. Liver and intestine were biopsied, and [3H]misonidazole content was quantified. Heat stress increased tissue [3H]misonidazole retention 80% in the liver and 29% in the small intestine. Cellular [3H]misonidazole levels were significantly elevated in intestinal epithelial cells and liver zone 2 and 3 hepatocytes and Kupffer cells. This effect was most prominent in the proximal small intestine and small liver lobi. These data provide evidence that hyperthermia produces cellular hypoxia and metabolic stress in splanchnic tissues and suggest that cellular metabolic stress may contribute to radical generation during heat stress.

Calibration of misonidazole labeling by simultaneous measurement of oxygen tension and labeling density in multicellular spheroids.

To correlate misonidazole concentrations and oxygen pressures (Po2) at identical locations within EMT6/Ro multi-cell spheroids (mean diameters +/- SD: 867 +/- 20 microns), Po2 measurements were performed with oxygen-sensitive microelectrodes during incubation of these spheroids with tritiated misonidazole (10 mg/I; 445 microCi/mg). In each individual spheroid, Po2 profiles were correlated with the corresponding spatial distribution of misonidazole as quantified by conventional autoradiography and grain counting. To compare the oxygenation status of spheroids in the measuring chamber with that of spheroids in spinner culture, misonidazole labeling was performed in both environments following the same protocol. All experiments were conducted in 20% oxygen and BME or in 5% oxygen and DMEM to obtain spheroids with different degrees of oxygenation. Labeled misonidazole was fairly evenly distributed in the outer, better oxygenated regions of EMT6 spheroids. In contrast, there was an accumulation of the labeled substance near central necrosis where low oxygen tensions were measured. Grain densities were similar at corresponding oxygen pressures under both environmental conditions. Except for some scatter, grain density as a function of oxygen pressure showed little variation in the Po2 range of 20-60 mm Hg, but exhibited a steep increase below 10 mm Hg. The findings imply that a substantial rise in local misonidazole labeling indicates a metabolically active tissue region at low Po2 that is not necessarily identical with the radiobiologically hypoxic cell fraction. A comparison of the labeling densities of spheroids in spinner flasks and in the Po2 measuring chamber indicates that oxygenation of spheroids is better in rotation culture than during microelectrode measurements.

New techniques in the pharmacokinetic analysis of cancer drugs. III. Nuclear magnetic resonance.

Nuclear magnetic resonance spectroscopy and imaging are non-invasive methods for monitoring the metabolism and distribution of anti-cancer drugs in tumours or other tissues. 19F NMR provides the easiest approach but requires a "built-in" fluorine atom in the drug of interest (eg 5-fluorouracil) or the incorporation of fluorine with minimal perturbance of a drug's properties. 2H NMR has also been used but has an effective sensitivity about 10- to 100-fold less than 19F. 1H and 13C are likely to be useful as non-perturbing NMR probes for future pharmacokinetic studies. The method is intrinsically insensitive, with a detection limit in the range of 0.01 to 0.1 mmol for fluorinated drugs. Techniques for absolute quantitation and for reliable localization to specific tissues and tumours are now available but have only rarely been applied in pharmacokinetic studies in the current literature. The incorporation of in vivo NMR measurements in a drug development programme should help to explain the differences found between drug activity in vitro, in solid animal tumour models and in cancer patients. It may also be possible to optimize drug selection or mode of administration for an individual patient.

Pharmacokinetic contribution to the improved therapeutic selectivity of a novel bromoethylamino prodrug (RB 6145) of the mixed-function hypoxic cell sensitizer/cytotoxin alpha-(1-aziridinomethyl)-2-nitro-1H-imidazole-1-ethanol (RSU 1069).

RB 6145 is a novel hypoxic cell sensitizer and cytotoxin containing both an essential bioreductive nitro group and a bromoethylamino substituent designed to form an alkylating aziridine moiety under physiological conditions. In mice, RB 6145 is 2.5 times less toxic but only slightly less active than the aziridine analogue RSU 1069, giving rise to an improved therapeutic index. However, the mechanism for the enhanced selectivity is not clear. Reasoning that this may lie in a more beneficial pharmacokinetic profile, we investigated the plasma pharmacokinetics, tissue distribution and metabolism of RB 6145 in mice using a specially developed reversed-phase HPLC technique. An i.p. dose of 190 mg kg-1 (0.5 mmol kg-1) RB 6145 produced peak plasma concentrations of about 50 micrograms ml-1 of the pharmacologically active target molecule RSU 1069 as compared with levels of around twice this value that were obtained using an equimolar i.p. dose of RSU 1069 itself. The plasma AUC0-infinity value for administered RSU 1069 was ca. 47 micrograms ml-1 h and that for the analogue RSU 1069 was ca. 84 micrograms ml-1 h. No prodrug was detectable. Another major RB 6145 metabolite in plasma was the corresponding oxazolidinone, apparently formed on interaction of the drug with hydrogen carbonate. The oxazolidinone initially occurred at higher concentrations than did RSU 1069, with the levels becoming very similar from 30 min onwards. Post-peak plasma concentrations of both RB 6145 metabolites declined exponentially, displaying an elimination t1/2 of ca. 25 min, very similar to the 30-min value observed for injected RSU 1069. The plasma AUC0-infinity value for the metabolite RSU 1069 was about 1.3 and 1.6 times higher following i.p. injection of 95 mg kg-1 (0.25 mmol kg-1) of the prodrug as compared with administration via the oral and i.v. routes, respectively. After i.v. injection, peak levels of the oxazolidinone metabolite were twice those observed following both i.p. and oral dosing and possibly contributed to the acute toxicity. After an i.p. dose of 190 mg kg-1 RB 6145, concentrations of RSU 1069 and the oxazolidinone metabolites rose to 40% and 33%, respectively, of the ambient plasma level in i.d. KHT tumours. The peak level of metabolite RSU 1069 was ca. 6 micrograms g-1 as compared with 10 micrograms g-1 following an equimolar dose of RSU 1069 itself; the tumour AUC0-infinity value for the metabolite RSU 1069 was some 35% lower.(ABSTRACT TRUNCATED AT 400 WORDS)

Radiolabelled fluoromisonidazole as an imaging agent for tumor hypoxia.

Fluoromisonidazole labeled with H-3 or F-18 has been tested as a quantitative probe for hypoxic cells in vitro and in rodent and spontaneous dog tumors in vivo. In V-79, EMT-6(UW), RIF-1, and canine osteosarcoma cells in vitro, the binding of 50 microM [H-3]Fluoromisonidazole was 50% inhibited by 1000-2000 ppm O2, relative to binding under anoxic conditions. After a 3 hr incubation with labeled drug, the anoxic/oxic binding ratios ranged from 12 to 27 for the four cell types. Retention of [H-3]fluoromisonidazole 4 hr after injection was greater in large KHT tumors (400-600 mm3) with an estimated hypoxic fraction greater than 30%, than in smaller tumors (50-200 mm3) with an estimated hypoxic fraction of 7-12%. RIF-1 tumors, with an estimated hypoxic fraction of 1.5%, retained the least label, with tumor: blood ratios ranging from 1.7 to 1.9. Spontaneous dog osteosarcomas were imaged with a time of flight positron emission tomograph for up to 5 hr following injection of [F-18] fluoromisonidazole. Analysis of regions of interest in images allowed creation of dynamic tissue time activity curves and calculation of tissue uptake in cpm/gram. These values were compared to radioactivity in plasma. In all cases, retention in some tumor regions exceeded that in plasma and in normal tissue, such as muscle or brain, by 3 to 5 hr post injection. Uptake of fluoromisonidazole in tumors was heterogeneous, with ratios of maximum to minimum uptake as high as 4 in different regions of interest in the same tumor. Tumor:plasma values ranged from 0.28 to 2.02. The oxygen dependency of fluoromisonidazole retention was similar in a variety of cell types and was 50% inhibited by O2 levels in the transition between full radiobiological hypoxia and partial sensitization. The quantitative regional imaging of [F-18] fluoromisonidazole in spontaneous canine tumors at varying times post-injection lays the basis for imaging and modeling of oxygen-dependent drug retention in different regions of human neoplasms.

Demonstration of tumor-selective retention of fluorinated nitroimidazole probes by 19F magnetic resonance spectroscopy in vivo.

We have evaluated two fluorinated misonidazole analogues, Ro 07-0741 and CCI-103F, as potential probes for the non-invasive identification of hypoxic tumor cells by 19F magnetic resonance spectroscopy (MRS) in vivo. The equipment used was a 1.9 T Oxford Research Systems TMR-32 spectrometer, fitted with a 15 mm diameter surface coil. Signal was readily detectable, with similar intensity from EMT6 tumor, liver, and brain at early times (1-2 hr) after i.v. injection in BALB/c mice, indicative of an initial uniform biodistribution of parent probes. At later times (5-10 hr) there was a progressive reduction in signal intensity from brain and liver, but tumor levels remained constant or declined more slowly. This is illustrated by tumor/brain ratios at 6-7 hr of 2.9 (Ro 07-0741) and 4.2 (CCI-103F). In 4/5 mice analyzed at 20-24 hr after Ro 07-0741, and 1/2 following CCI-103F, tumor signal remained detectable. This occurred in the absence of parent probe as measured by HPLC, suggesting the involvement of a product of nitroreductive bioactivation. Studies with KHT and RIF-1 tumors in C3H/He mice showed a similar trend but retention in RIF-1 was less dramatic, and this was consistent with the known hypoxic fractions and comparative in vivo nitroreductase activities. These promising results support the continuing development of 19F nitroimidazole probes for non-invasive identification of hypoxic cells in vivo.

Effects of elevated temperature on misonidazole O-demethylation by mouse liver microsomes: kinetic and stability studies of a model mixed-function oxidase reaction.

We investigated the effects of a range of temperatures (33 degrees-44 degrees C) on the stability and kinetics of C3H mouse liver microsomal misonidazole (MISO) O-demethylase in vitro. Microsomal O-demethylase activity was stable for 60 min at 37 degrees C and for 30 min at 41 degrees C but was steadily inactivated with longer incubation times. Inactivation at 44 degrees and 47 degrees C was exponential, with half-lives of 41 and 11 min, respectively. MISO O-demethylation followed Michaelis-Menten kinetics from 33 degrees to 44 degrees C. The apparent Vmax for desmethylmisonidazole (Ro 05-9963) formation was decreased by 32% (from 2.14 to 1.47 nmol min-1 mg-1 protein) with a 4 degrees decrease from 37 degrees to 33 degrees C. An increase of 4 degrees from 37 degrees to 41 degrees C enhanced the Vmax by 47%, but there was only an additional 9% increase for a further 3 degrees rise to 44 degrees C. Apparent Km values were unaltered at about 1.6 mM. These results show that elevated temperatures in the clinically relevant hyperthermia range (41 degrees-44 degrees C) can enhance a model mixed-function oxidase reaction in vitro. Such effects may be important for the metabolism, activity and toxicity of anticancer drugs combined with hyperthermia in vivo.

Measurements of glutathione and other thiols in cells and tissues: a simplified procedure based on the HPLC separation of monobromobimane derivatives of thiols.

Although there is intense interest in the role of thiols in controlling the efficiency of radiosensitizers, and in developing thiols (or pro-drugs liberating thiols) as radioprotectors, there is little information regarding the concentration of specific thiols in cells, tumors and normal tissues. Details are presented of a modified procedure using the thiol binding agent monobromobimane with separation using paired-ion reverse phase high performance liquid chromatography (HPLC). This method has been extended to include measurements of the radiosensitizer misonidazole and its desmethylated metabolite Ro 05-9963 in tissues.

Systemic, bladder wall, and bladder tumor concentration of misonidazole following intravesical administration in the rat.

The hypoxic-cell radiosensitizer misonidazole was administered intravesically to normal and to bladder tumor-bearing female Fischer rats. Drug concentration was measured in the bladder wall, the tumor and in the serum using high pressure liquid chromatography at different times following administration. The data shows that misonidazole is readily adsorbed by the bladder wall and the tumor with tissue levels reaching up to 10 times those measured in the serum.

Intravesical administration of misonidazole in bladder carcinoma.

Using 11C labelled material, the diffusion of the hypoxic cell sensitizer misonidazole was tested in 19 patients with carcinoma of the bladder following its intravesical administration. Increasing concentration gradients were tested. Evidence was provided that a 20 ml solution containing 200 mmol/l of misonidazole is followed by high concentrations of the drug in the superficial as well as in deep parts of the tumour. The corresponding serum concentration was extremely low. It was concluded that this route of administration provides high local concentrations of the drug that may result in a significant therapeutic enhancement if utilized as an adjuvant to external irradiation.

Monitoring salivary misonidazole in man: a possible alternative to plasma monitoring.

Concentrations of misonidazole and its O-demethylated metabolite Ro 05-9963 in the plasma and saliva of 10 patients with malignant disease have been determined. A good linear correlation was established between plasma and saliva misonidazole concentration, and salivary sampling was found to be suitable for the estimation of a number of pharmacokinetic parameters. Data are also presented for serial tumour cencentrations of misonidazole and Ro 05-9963 in 3 of the 10 patients. Monitoring of salivary misonidazole concentration appears to be a useful alternative to plasma monitoring, particularly for those patients in whom plasma sampling is unsuitable or impossible.