Jane C.M. Bremner

Glutathione depletion in tissues after administration of buthionine sulphoximine

Buthionine sulphoximine (BSO) an inhibitor of glutathione (GSH) biosynthesis, was administered to mice in single and repeated doses of 0.5, 1 and 5 mmol kg-1 (i.p.). The resultant pattern of GSH depletion was studied in liver, kidney, skeletal muscle and three types of murine tumor. Liver and kidney exhibited a rapid depletion to GSH levels of ca. 20% of controls after single doses of 1-5 mmol kg-1 BSO. Muscle was depleted to a similar level, but at a slower rate after a single dose. All three tumors required repeated administration of BSO over several days to obtain a similar degree of depletion to that shown in the other tissues.

Manipulation and Exploitation of the Tumour Environment for Therapeutic Benefit

We describe aspects of the tumour microenvironment that are available as targets for manipulation. In particular, the question asked is whether hypoxia in tumours is a problem to be overcome, or a physiological abnormality to be exploited? Bioreductive drugs require metabolic reduction to generate cytotoxic metabolites. This process is facilitated by appropriate reductases and the lower oxygen conditions present in solid tumours compared with normal tissues. Because of their specificity, bioreductive drugs are used to help answer this question. Other aspects of tumour physiology and biochemistry that may be exploited include tissue dependent reductase expression, pH and angiogenesis.

The measurement of radiosensitizer-induced changes in mouse tumor metabolism by 31P magnetic resonance spectroscopy

Flunarizine and nicotinamide have previously been shown to increase blood perfusion to experimental mouse tumors and consequently, to increase their sensitivity to X rays. These agents were examined for their ability to alter metabolism, measured by 31P magnetic resonance spectroscopy, in the SCCVII/Ha carcinoma and the KHT sarcoma. Flunarizine at 5 mg/kg I.P. produced a 45% reduction in the ratio of inorganic phosphate to total phosphate (Pi/total) in the SCCVII/Ha tumor but only a 24% reduction in this ratio in the KHT tumor. These effects were seen 45 min after drug administration, and ratios returned to control levels by 90 min. In the SCCVII/Ha tumor, nicotinamide at 1000 mg/kg I.P. reduced Pi/total by 56% from 30 min to at least 2 hr after injection, and the ratio was reduced by 59% in the KHT tumor at 30 min after injection, returning to control levels by 2 hr. For the SCCVII/Ha tumor, the time course for the effects of flunarizine and nicotinamide on the inorganic phosphate ratio coincided with that previously reported for radiosensitization.

Comparing the anti-tumor effect of several bioreductive drugs when used in combination with photodynamic therapy (PDT)

PURPOSE To compare the effect on the RIF-1 murine sarcoma of nine bioreductive agents from five different classes when used in combination with either photodynamic therapy or clamping. METHODS AND MATERIALS RIF-1 tumors implanted intradermally in C3H mice were treated with either 50J photodynamic therapy or with 120 min clamping in combination with either misonidazole, pimonidazole, metronidazole, nimorazole, RB6145, RSU1069, SR4233, mitomycin-C, or RB90740. The tumors were measured 3 times-per-week until reaching 4 x their initial treatment volume. RESULTS RSU1069 produced the greatest anti-tumor activity in combination with both photodynamic therapy and clamping. RB6145 also substantially enhanced the effect of photodynamic therapy and clamping whereas misonidazole induced a smaller, but significant increase. Mitomycin-C had no effect under clamped conditions, but greatly increased the tumorcidal effect of photodynamic therapy. Mitomycin-C also induced an effect when given with light alone. None of the other agents showed any augmentation of the tumor cell killing induced by photodynamic therapy. CONCLUSION Of the bioreductive agents studied RSU1069, RB6145 and mitomycin-C showed the greatest anti-tumor response in combination with photodynamic therapy.

Magnetic resonance spectroscopy studies on experimental murine and human tumors: Comparison of changes in phosphorus metabolism with induced changes in vascular volume

The responses of two experimental murine tumors and two human tumor xenografts to the vasodilator hydralazine were compared using two magnetic resonance spectroscopy endpoints. Changes in tumor metabolism were determined using 31P MRS where inorganic phosphate levels relative to total phosphate (Pi/total) were measured, and alteration in tumor blood volume was examined using 19F MRS with perfluorooctylbromide (PFOB) as tracer. The integrated 19F signal from PFOB is dose dependent and stable for at least 2 hr after injection. The murine tumors SCCVII/Ha and KHT both showed changes in tumor metabolism after hydralazine, as an increase in Pi/total. However, hydralazine reduced vascular volume in the KHT tumor, demonstrated by reduced 19F signal from PFOB, but no such reduction was seen in the SCCVII/Ha tumor. In contrast, hydralazine had no effect on phosphorus metabolism in the HT29 and HX118 human tumor xenografts, but reduced vascular volume in both tumors. These results demonstrate that the effects of vasoactive agents such as hydralazine on tumor phosphorus metabolism are only partially consistent with changes in vascular volume, measured by the 19F MRS technique.

The Influence of Pre-treatment Temperature on the Thermal Sensitivity of a Mouse Tumour

The response of tumours to hyperthermia was tested by giving graded heat treatments and assessing local control at 90 days. Mice were divided into three groups which were pre-treated for 3 days in ambient temperatures of 4, 21 or 35 degrees C. This enabled the mean tumour resting temperature to be varied by up to 11 degrees C, before subsequent heat treatment. For the heat treatments, the tumours were clamped in order to eliminate blood flow, resulting in uniform temperature distributions and hence more uniform thermal sensitivity. TCD50 values were used to construct Arrhenius plots. For all three pre-treatment temperatures, these plots demonstrated a factor of 1.6 increase in heating time per degree Celsius reduction in heating temperature. However, tumours kept in a 4 degrees C environment before treatment were more thermally sensitive than those kept in 21 degrees C conditions, while those in a 35 degrees C environment were more resistant. Pretreatment at 4 degrees C was equivalent to an increase of either 0.5 degree C in heating temperature or 28 per cent in heating time, compared with pre-treatment at 21 degrees C. Pre-treatment at 35 degrees C was equivalent to a reduction of either 0.6 degree C in heating temperature or 25 per cent in heating time. These data indicate that the pre-treatment tumour temperature is an important parameter, but the effect of heat treatment is more closely related to absolute heating temperature rather than to the increase in temperature above the normal resting level.

The therapeutic advantage of combined X-rays and melphalan.

Early skin reactions on mouse feet and delay in growth of a mouse tumor (CA NT) were measured after combined treatments with X-rays and the cytotoxic drug Melphalan. The drug was given as a single dose (10 mg kg-1) with graded single doses of X-rays, either before or after irradiation with an interval of up to 4 days. In the mouse skin, addition of the drug increased the radiation response only slightly. The maximum Enhancement Ratio (ER) measured at a skin reaction of 1.5 (approximately 23 Gy) was 1.07 +/- 0.02 SEM for the schedule MEL 3 days before X-rays. ERs for all schedules tested were similar with a range of 1.00 to 1.07. For the tumor more enhancement was observed; the largest ERs were found when Melphalan was given before rather than after irradiation, with maximum ERs of 2.3 and 2.4 when the drug was given 3 days or 1 day before X-rays. This has been attributed to reoxygenation of hypoxic cells after drug treatment, rendering the tumor more radiosensitive. The range of ER over all schedules was 1.5-2.4. Since ER is greater for the tumor than skin for all schedules, a therapeutic advantage is indicated under these specific experimental conditions of single X-ray and drug doses.

31P MRS to monitor the induction of tumor hypoxia by the modification of the oxygen affinity of hemoglobin using BW 589C

PURPOSE BW 589C induces severe tumor hypoxia by modifying the affinity of oxyhemoglobin, causing a left shift of the oxygen-hemoglobin dissociation curve. 31P magnetic resonance spectra (MRS) was used to monitor the effects of BW 589C on tumor energy metabolism in three experimental tumor models. METHODS AND MATERIALS HT-29 colon xenograft, murine transplantable RIF-1 fibrosarcoma and KHT sarcoma were studied in unanesthetised mice. 31P MR spectra were acquired on a 4.7 Tesla magnet before administering oral BW 589C (250 mg/kg) and after 3, 6, and 24 h. Samples of tail vein blood were then taken for 2,3 DPG levels for RIF-1 and HT-29 tumors. RESULTS Doubling of inorganic phosphorus (Pi) to total phosphorus was observed 5-6 h after BW 589C for all three tumor types. Although the left shift due to BW 589C persists at 24 h, the level of Pi to total phosphorus returned to baseline with no significant difference from control values for the RIF-1 and HT-29 tumors. These results suggest that there was cellular metabolic adaptation to the reduction of oxygen delivery by BW 589C. This does not appear to involve 2,3 DPG as there was no significant alteration in tumor levels. The death of hypoxic cells may, also, have contributed to the recovery of Pi to total phosphorus. CONCLUSION The efficacy of bioreductive drugs can be enhanced by increasing the severity of tumor hypoxia. 31P MRS in conjunction with other techniques for assessing the intratumor environment could play an important role in planning cancer therapy.