Increased oxygenation of intracranial tumors by efaproxyn (efaproxiral), an allosteric hemoglobin modifier: In vivo EPR oximetry study.

PURPOSE: To determine quantitatively the changes in oxygenation of intracranial tumors induced by efaproxiral, an allosteric hemoglobin modifier. Efaproxiral reduces hemoglobin-oxygen binding affinity, which facilitates oxygen release from hemoglobin into surrounding tissues and potentially increases the pO(2) of the tumors. METHODS AND MATERIALS: The study was performed on 10 male Fisher 344 rats with 9L intracranial tumors. Electron paramagnetic resonance (EPR) oximetry was used to measure quantitatively the changes in the pO(2) in the tumors. Lithium phthalocyanine (LiPc) crystals were implanted in the tumors and in the normal brain tissue in the opposite hemispheres. We monitored the cerebral pO(2) starting 7 to 10 days after the tumor cells were implanted. NMR imaging determined the position and size of tumor in the brain. After an initial baseline EPR measurement, efaproxiral (150 mg/kg) was injected intravenously over 15 minutes, and measurements of tumor and normal brain oxygen tension were made alternately at 10-minute intervals for the next 60 minutes; the procedure was repeated for 6 consecutive days. RESULTS: Efaproxiral significantly increased the pO(2) of both the intracranial tumors and the normal brain tissue on all days. The maximum increase was reached at 52.9 to 59.7 minutes and 54.1 to 63.2 minutes after injection, respectively. The pO(2) returned to baseline values at 106 to 126.5 minutes after treatment. The maximum tumor and normal tissue pO(2) values achieved after efaproxiral treatment from Day 1 through Day 6 ranged from 139.7 to 197.7 mm Hg and 103.0 to 135.9 mm Hg, respectively. The maximum increase in tumor tissue pO(2) values from Day 2 to Day 5 was greater than the maximum increase in normal tissue pO(2). CONCLUSION: We obtained quantitative data on the timing and extent of efaproxiral-induced changes in the pO(2) of intracerebral 9L tumors. These results illustrate a unique and useful capability of in vivo EPR oximetry to obtain repeated noninvasive measurements of tumor oxygenation over a number of days. The information on the dynamics of tumor pO(2) after efaproxiral administration illustrates the ability of efaproxiral to increase intracranial tumor oxygenation.

"Distant spin trapping": a method for expanding the availability of spin trapping measurements.

The technique of spin trapping is used to study a wide range of free radicals in various systems, including those generated in vitro and in vivo. But unfortunately, EPR spectrometers are not always immediately accessible at the site of experimentation, and therefore it is important to find a method that can preserve a radical adduct over longer periods of time. We describe here an alternative method in which the samples can be frozen and transported for EPR measurements at another site. Various spin adducts of DEPMPO were frozen and measured at 0 degrees C at various intervals after freezing to determine their stability in the frozen state. The radical adducts were generated by established methods and stored at two different temperatures; -196 degrees C (liquid nitrogen) and -80 degrees C (dry ice). The experiments were carried out in an aqueous solution with and without a model of reducing environment (2 mM ascorbate). The results indicate that it is feasible to store and transport spin adducts for subsequent analysis. We conclude that this approach, which we term "distant spin trapping", makes it feasible to transport samples to another site for EPR measurements. This should significantly expand the ability to use spin trapping in biology and medicine.

Effect of RSR13, an allosteric hemoglobin modifier, on oxygenation in murine tumors: an in vivo electron paramagnetic resonance oximetry and bold MRI study.

PURPOSE: RSR13, an allosteric modifier of hemoglobin, reduces hemoglobin-oxygen binding affinity facilitating oxygen release from hemoglobin, resulting in increases in tissue pO(2). The purpose of this study was noninvasively to monitor the time course and effect of RSR13 on tumor oxygenation, directly using in vivo electron paramagnetic resonance (EPR oximetry), and indirectly using blood oxygen level dependent magnetic resonance imaging (BOLD MRI). METHODS AND MATERIALS: The study was performed in transplanted radiation-induced fibrosarcoma tumors (RIF-1) in 18 female C3H/HEJ mice, which had two lithium phthalocyanine (LiPc) deposits implanted in the tumor when the tumors reached about 200-600 mm(3). Baseline EPR measurements were made daily for 3 days. Then, for 6 consecutive days and after an initial baseline EPR measurement, RSR13 (150 mg/kg) or vehicle (same volume) was injected intraperitoneally, and measurements of intratumoral oxygen were made at 10-min intervals for the next 60 min. In each mouse, every third day, instead of EPR oximetry, BOLD MRI measurements were made for 60 min after administration of the RSR13. RESULTS: Based on EPR measurements, RSR13 produced statistically significant temporal increases in tumor pO(2) over the 60-min time course, which reached a maximum at 35-43 min postdose. The average time required to return to the baseline pO(2) was 70-85 min. The maximum increase in tumor tissue pO(2) values after RSR13 treatment from Day 1 to Day 5 (8.3-12.4 mm Hg) was greater than the maximum tumor tissue pO(2) value for Day 6 (4.7 mm Hg, p < 0.01). The maximum increase in pO(2) occurred on Day 2 (12.4 mm Hg) after RSR13 treatment. There was little change in R(2)*, indicating that the RSR13 had minimal detectable effects on total deoxyhemoglobin and hemoglobin-oxygen saturation. CONCLUSION: The extent of the increase in tumor pO(2) achieved by RSR13 would be expected to lead to a significant increase in the effectiveness of tumor radiotherapy. The lack of a change in the BOLD MRI signal suggests that the tumor physiology was largely unchanged by RSR13. These results illustrate a unique and useful capability of in vivo EPR oximetry and BOLD MRI to obtain repeated measurements of tumor oxygenation and physiology. The dynamics of tumor pO(2) after RSR13 administration may be useful for the design of clinical protocols using allosteric hemoglobin effectors.

Tumor pO2 assessments in human xenograft tumors measured by EPR oximetry: location of paramagnetic materials.

Radioantibody immunotherapy (RAIT) is a promising treatment modality but the effectiveness of this targeted low dose radiation varies from tumor to tumor. Since RAIT is an oxygen dependent treatment, baseline pO2 or growth-induced changes in the microenvironment may alter treatment response. In this pilot work we monitored tumor pO2 in untreated human xenograft tumors growing s.c. in nude mice. These data will be used to plan a study of the relationship between the effectiveness of RAIT and tumor pO2. Growth or treatment-induced changes in the microenvironment may alter the tumor pO2 and thus affect the response to therapy but may also affect location and microenvironment of the particulate oxygen sensor. We monitored tumor pO2 during growth and also examined the tumor histological structure overall and in the region of the paramagnetic material in the tumor at the time of necropsy.

Blood flow dynamics after photodynamic therapy with verteporfin in the RIF-1 tumor.

In the present study, the effects of photodynamic therapy (PDT) with verteporfin on tumor blood flow and tumor regrowth were compared as verteporfin distributed in different compartments within the RIF-1 tumor. Tissue distribution of verteporfin was examined by fluorescence microscopy, and blood flow measurements were taken with a laser Doppler system. It was found that, at 15 min after drug administration, when verteporfin was mainly confined within the vasculature, PDT induced a complete arrest of blood flow by 6 h after treatment. PDT treatment at a longer drug-light interval (3 h), which allowed the drug to diffuse to the tumor interstitium, caused significantly less flow decrease, only to 50% of the initial flow in 6 h. A histological study and Hoechst 33342 staining of functional tumor vasculature confirmed the primary vascular damage and the decrease in tumor perfusion. The regrowth rate of tumors treated with 15-min interval PDT was 64% of that of the control group. However, when tumors were treated with 3-h interval PDT, the regrowth rate was not significantly different from that of the control, indicating that only the 15-min interval PDT caused serious damage to the tumor vascular bed. These results support the hypothesis that temporal pharmacokinetic changes in the distribution of the photosensitizer between the tumor parenchyma and blood vessels can significantly alter the tumor target of PDT.

Spin traps: in vitro toxicity and stability of radical adducts.

We have evaluated the effects of DMPO, CMPO, EMPO, BMPO, and DEPMPO on functioning CHO cells and the stability of the radical adducts in the presence of cells. The potential toxic effects of the spin traps were measured by two estimates of cell viability (trypan blue exclusion and colony formation) and one of cell function (rate of oxygen consumption). We also studied the effects of the spin traps on colony formation in a second cell line, 9L tumor cells. Toxicity varied with the type of cell line and the parameter that was measured. In aqueous solutions the order of stability for all spin adducts was SO(3) > OH > CH(3), while in cell suspensions it was SO(3) > OH approximately CH(3). The radical adducts of the new spin traps have significantly increased stability as compared to DMPO. These results indicate that the new spin traps potentially offer increased stability of spin adducts in functioning cells. It also is clear that it is necessary to carry out appropriate studies of the stability and toxicity in the system that is to be studied for any particular use of these spin traps. It then should be feasible to select the spin trap(s) best suited for the proposed study.

Spatial heterogeneity and temporal kinetics of photosensitizer (AlPcS2) concentration in murine tumors RIF-1 and MTG-B.

In this study we compared the photosensitizer concentration in two experimental murine tumors using an in situ fluorescence detection instrument to examine temporal and spatial variations, after intravenous versus intratumor injection. Also, the variations in the estimate as detected by large area sampling and micro-region sampling are compared, in order to determine what the inter-tissue and inter-animal variations are, and how the method of sampling affects this estimate. The latter study was carried out ex vivo in the same tumors, which had been harvested and frozen after in vivo measurements were made. The photosensitizer, disulphonated aluminum phthalocyanine (AlPcS2) was injected either intravenously (IV) or directly into the tumor (ITu), using two murine models, MTG-B (mammary adenocarcinoma) and RIF-1 (radiation-induced fibrosarcoma) grown subcutaneously on the flank. An in situ microsampling fluorescence probe was used to assess photosensitizer concentration, through real-time measurement of the remitted intensity. The photosensitizer concentration was evaluated at 8 time endpoints between 15 min and 48 h post-injection. Inter-tumor and intra-tumor variations were assessed by repeated samples from the tumor tissues. The average photosensitizer level reaches a peak between 3 to 6 h in both tumor and normal tissues using IV administration, but peaks within 1 h following ITu administration. MTG-B tumors demonstrated a factor of 2 higher uptake than RIF-1 tumors. The pharmacokinetic uptake rates of the RIF-1 tumor were 3 times faster than for MTG-B, while there was no statistical difference in their clearance rates. Preferential uptake of AlPcS2 by both tumors compared to contra-lateral flank subcutaneous normal tissue was documented, with ITu injection exceeding IV injection by a factor of 10 in the tumor to normal tissue ratio. Inter-animal standard deviation in the mean fluorescence was near 76% for both routes of administration, but estimates of the variation within tumor were near 16% standard deviation when a large sampling volume was used. In contrast, microscopic intra-tumor standard deviation in the mean estimate was near 76%, with IV injection, indicating that high heterogeneity exists in the photosensitizer concentration on a smaller distance scale. The inter-tumor variation was reduced by ITu injection, but at the expense of increasing intra-tumor variation.

Photodynamic therapy with verteporfin in the radiation-induced fibrosarcoma-1 tumor causes enhanced radiation sensitivity.

Photodynamic therapy (PDT) with verteporfin (lipid form of benzoporphyrin derivative,benzoporphyrin derivative monoacid ring A) was used to treat radiation-induced fibrosarcoma tumors before X-ray treatment. When verteporfin was injected 3 h before light irradiation, the tumor partial pressure of oxygen (pO(2)) rose from a pretreatment value of 2.8 +/- 1 to 15.2 +/- 6.9 mm Hg immediately after light application was complete (P = 0.048). When the optical irradiation was given 15 min after verteporfin injection, the tumor pO(2) decreased slightly after treatment [i.e., 6.8 +/- 1.6 mm Hg (pretreatment) versus 4.1 +/- 0.3 mm Hg (posttreatment)], whereas control tumor pO(2) did not change significantly. In vitro study of the cellular oxygen consumption rate before and after PDT treatment indicated that the consumption rate decreased linearly with delivered optical dose and quantitatively matched the loss of cell viability as measured by a mitochondrial tetrazolium assay. Doppler measurements show that red cell flux is still patent immediately after treatment, indicating that oxygen should still be delivered to the tumor. Computational simulations of the oxygen supply from the vessels and the consumption from mitochondrial activity confirmed that if oxygen consumption is decreased in the presence of unhindered blood flow, the tumor oxygenation should rise, and the hypoxic fraction of the tumor should decrease. Combination treatments with PDT delivered (100 J/cm(2) optical dose, with 1 mg/kg benzoporphyrin derivative monoacid ring A injected 3 h before treatment) after radiation treatment (10 Gy from 300 keV source) were compared with PDT delivered simultaneously with radiation. Tumor regrowth assay showed that the delays to reach double the tumor volume for PDT alone and radiation alone were 2.7 +/- 1.6 and 3.2 +/- 1.7 days, respectively. When radiation was given before PDT, the delay was 5.4 +/- 1.4 days, and when PDT was given at the same time as radiation, the delay was 8.1 +/- 1.5 days. This observation indicates that the combined effect in the latter case was greater than additive (P = 0.049).

Tumor PO(2) changes during photodynamic therapy depend upon photosensitizer type and time after injection.

In this study, the vascular and tissue oxygen changes induced by photodynamic therapy in the RIF-1 tumor were examined, using electron paramagnetic resonance (EPR) oximetry. Two photosensitizers, including verteporfin (BPD-MA in a lipid-based formulation) and aminolevulinic acid-induced protoporphyrin IX (ALA-PPIX), were investigated with optical irradiation, sufficient to induce sub-curative damage in the tumor tissue, and the transient changes in PO(2) and vascular perfusion were examined. A large increase in tissue oxygenation (from 3 up to 9.5 mmHg) was observed when treated with ALA-PPIX based photodynamic therapy, which lasted during the treatment and a small residual increase that returned back to baseline levels by 48 h after treatment. With verteporfin-based photodynamic therapy, one group of animals was irradiated 15 min after injection and exhibited a small decrease in oxygenation relative to pre-irradiation levels. The second group was irradiated at 3 h after injection and exhibited a large increase in the average PO(2), (from 3 to 15 mmHg) by the end of the treatment. These observations indicate that photodynamic therapy significantly increases tissue PO(2) under certain treatment conditions, with the potential cause being either increased local blood flow or decreased local oxygen metabolic consumption due to cellular damage.