31P magnetic resonance spectroscopy as a predictor of efficacy in photodynamic therapy using differently charged zinc phthalocyanines.

Photodynamic therapy (PDT) is a developing approach to the treatment of solid tumours which requires the combined action of light and a photosensitizing drug in the presence of adequate levels of molecular oxygen. We have developed a novel series of photosensitizers based on zinc phthalocyanine which are water-soluble and contain neutral (TDEPC), positive (PPC) and negative (TCPC) side-chains. The PDT effects of these sensitizers have been studied in a mouse model bearing the RIF-1 murine fibrosarcoma line studying tumour regrowth delay, phosphate metabolism by magnetic resonance spectroscopy (MRS) and blood flow, using D2O uptake and MRS. The two main aims of the study were to determine if MRS measurements made at the time of PDT treatment could potentially be predictive of ultimate PDT efficacy and to assess the effects of sensitizer charge on PDT in this model. It was clearly demonstrated that there is a relationship between MRS measurements during and immediately following PDT and the ultimate effect on the tumour. For all three drugs, tumour regrowth delay was greater with a 1-h time interval between drug and light administration than with a 24-h interval. In both cases, the order of tumour regrowth delay was PPC > TDEPC = TCPC (though the data at 24 h were not statistically significant). Correspondingly, there were greater effects on phosphate metabolism (measured at the time of PDT or soon after) for the 1-h than for the 24-h time interval. Again effects were greatest with the cationic PPC, with the sequence being PPC > TDEPC > TCPC. A parallel sequence was observed for the blood flow effects, demonstrating that reduction in blood flow is an important factor in PDT with these sensitizers.

Combination of photodynamic therapy (PDT) and melphalan in experimental tumors.

PURPOSE: To investigate whether application of "early" photodynamic therapy (PDT) using a disulphonated aluminium phthallocyanine photosensitizer can potentiate the action of melphalan in experimental RIF-1 tumors in vivo. METHODS AND MATERIALS: Tumors were irradiated with laser light of wavelength 675 nm 60 min after treatment with the photosensitizer and 15 min after melphalan. Melphalan pharmacokinetics were measured using high performance liquid chromatography with optical detection. RESULTS: Melphalan and PDT when given alone, caused a significant delay in tumor growth. This was increased for the combined treatment. Pharmacokinetic analyses showed that levels of free, unreacted melphalan in freely circulating blood are unaffected by combined treatment. However, significant differences in tumor levels were observed between treatment with melphalan alone or in combination. Whereas in the former, melphalan is still present in tumors after 2 h, it was not detectable even at the earliest time of 15-23 min for the combined treatment. CONCLUSION: The antitumor effects were additive with no evidence of significant potentiation.

Magnetic resonance spectroscopic studies on 'real-time' changes in RIF-1 tumour metabolism and blood flow during and after photodynamic therapy.

Magnetic resonance spectroscopy (MRS) in situ was used to study changes in 31P metabolism occurring during and after treatment of murine RIF-1 tumours with photodynamic therapy (PDT). Tumours were irradiated using a fibreoptic light delivery system while the mice were in position within the magnet. Changes in 31P-MRS were observable during and immediately after treatments of several minutes' duration. Both the extent and duration of the increase in the Pi/total ratio were light dose dependent. The effect on the metabolism was also affected by the time interval (TL) between administering the photosensitiser disulphonated phthalocyanine, (A1S2Pc) and the light. With a dose of 50 J the increase in Pi/total was much faster when TL was 1 h than when TL was 24 h. This difference in rate probably reflects differences in the distribution of A1S2Pc within the tumour. Significant decreases in pH were only seen after a light dose of 50 J when TL was 1 h. Blood flow measurements using deuterium uptake were also carried out using MRS. These experiments showed that for a dose of 50 J the level of blood flow was reduced by approximately 90% of the control value within 10 min from the end of the 8 min light treatment. This occurred irrespective of the value of TL. The data indicate that it is possible to observe very early changes in 31P metabolism that can be attributed to direct cellular damage as opposed to the later changes indicative of overall tumour hypoxia caused by vascular damage.

The accuracy and reproducibility of measuring blood flow in murine tumours by the D2O uptake and clearance techniques.

The use of D2O as an NMR visible tracer to monitor murine tumour blood flow (TBF) by both the wash-in and wash-out methods has been investigated. The factors that influence the models used to fit the data and the error on the measurement of the clearance and uptake rates have been assessed. The study concentrates on the uptake method which allows TBF to be measured without the need to use anaesthetic. Also, administering the D2O remotely to the mouse means it can remain undisturbed, in the magnet bore, between control and post-treatment readings. The uptake method in KHT and RIF-1 transplanted murine tumours has been investigated in a series of control experiments and after modifying TBF by hydralazine (5 mg/kg) and photodynamic therapy. These studies showed that four uptake measurements could be made on the same mouse at 20 min intervals without affecting TBF, control values were the same for anaesthetized and unanaesthetized mice and the values obtained for RIF-1 tumours were marginally higher than those obtained for the KHT tumours. The decrease in TBF seen after modification was in good agreement with published data where TBF results were obtained by using D2O clearance, radioactive tracers or laser Doppler flowmetry.

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.

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.

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.

Assessing the bioreductive effectiveness of the nitroimidazole RSU1069 and its prodrug RB6145: with particular reference to in vivo methods of evaluation.

The nitroimidazole, RSU1069, has been shown to have a very high differential toxicity towards hypoxic cells compared to oxic cells both in in vitro and in vivo experimental conditions. However, in the clinic it was found to cause severe emesis and had to be withdrawn. After an extensive drug development programme an analogue of RSU1069, RB6145, which acts as a pro-drug for RSU1069, was found to be the most suitable candidate for further investigation. In in vivo studies with murine tumour models, when RB6145 was used in combination with X-rays it was shown to produce a similar level of toxicity towards hypoxic cells as that observed for RSU1069. Its activity was the same whether it was administered interperitoneally or orally and the same level of anti-tumour effect was observed if the drug was given before or after X-rays. RB6145 is better tolerated systemically in mice than RSU1069 and canine studies have shown that it is less emetic than the parent drug. Bioreductive drugs can also be used in combination with treatments that preferentially increase tumour hypoxia. Photodynamic therapy (PDT) causes extensive vascular damage in tumours. If either RSU1069 or RB6145 are administered during PDT, very large increases in the growth delay induced by PDT alone are seen for the RIF-1 murine tumour. RB6145 has been accepted for clinical toxicity trials with the prospect of using it in combination with X-rays. In the future it may also be of clinical use with treatments such as PDT.

Increasing the effect of photodynamic therapy on the RIF-1 murine sarcoma, using the bioreductive drugs RSU1069 and RB6145.

The effect of combining photodynamic therapy (PDT) and bioreductive drugs has been investigated using the RIF-1 experimental murine tumour. Light was delivered interstially to the tumour at 675 nm using a single optical fibre attached to an argon-ion dye laser. The photosensitizer was disulphonated aluminium phthalocyanine (AlS2Pc) and the bioreductive drugs were the dual function nitroimidazole RSU1069 and its pro-drug RB6145. Varying the time between administration of the photosensitizer and light delivery (TL) from 30 min to 24 h had little influence on the extent of the anti-tumour effect of PDT alone, as measured by the regrowth delay endpoint. When the bioreductive drug was included in the treatment, administered 20 min before light irradiation, regrowth delay was greatly increased. The effectiveness of the combined treatment was optimum for short values of TL (about 1 h). Fluorescence microscopy was used to investigate the distribution of the photosensitizer within the tumours. This showed that the compound was mainly confined to the tumour vasculature over the first few hours post-treatment. The high efficacy of the combined treatment of PDT and bioreductive drugs for short values of TL suggest that photodynamic action, during the period when the photosensitizer AlS2Pc is confined to the vasculature, enhances the severity of tumour hypoxia which is sufficient to induce activation of the bioreductive drugs.

Bioreductive drugs as post-irradiation sensitizers: comparison of dual function agents with SR 4233 and the mitomycin C analogue EO9.

Various bioreductive drugs that are potent hypoxic cell cytotoxins can also function as effective potentiators of radiation action when administered in vivo post irradiation. There is evidence that a contributory mechanism to this potentiation is enhanced sensitivity to the bioreductive drugs exhibited by cells that are damaged sublethally by radiation.

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.

In vivo 31P nuclear magnetic resonance spectroscopy of experimental murine tumours and human tumour xenografts: effects of blood flow modification.

The effect of hydralazine on tumours appears to vary depending on tumour type. Blood flow and radiation sensitivity decrease more in murine tumours than human tumour xenografts. In this study a comparison between various tumour types has been made using in vivo 31P nuclear magnetic resonance spectroscopy (NMRS) to follow the metabolic responses occurring after clamping or intravenous administration of hydralazine (5 mg kg-1). Large increases in the Pi/total phosphate ratio were found with the murine sarcomas, KHT and RIF-1 implanted into C3H/He mice. However little or no effect was seen for the two human xenografted tumours, HX118 and HT29 implanted in MFI nu/nu/01a mice. An intermediate response was observed for KHT tumours grown in nu/nu mice. All tumours showed a large response to clamping. The anaesthetic Hypnorm/Hypnovel has a great influence on the response of the tumour metabolism to hydralazine appearing to both prolong and increase the changes induced. There is evidence to support the theory that the changes in 31P spectra are related to the oxygen status of the tumours.

Induction of hypoxia in the KHT sarcoma by tumour necrosis factor and flavone acetic acid.

The interaction of FAA or TNF with radiation was studied in the murine KHT sarcoma. When used alone both agents showed a dose- and time-dependent toxicity towards the tumour cells and significantly reduced tumour blood flow within 1 h of treatment. When used in combination with radiation, both TNF and FAA caused an increase in the fraction of hypoxic cells in the KHT tumour. This was assessed by an in vivo/in vitro clonogenic assay and by a comparison with the radioprotection provided by clamping tumours prior to and during irradiation. When TNF was given at a dose of 2.5 X 10(5) U/kg an increase in tumour hypoxia was seen after 30 min. Close to 100% radiobiological hypoxia was reached by 1 h after treatment, lasting for up to 16 h. Doses of TNF below 0.25 X 10(5) U/kg did not induce levels of hypoxia comparable to clamping when administered 3 h prior to irradiation. Similarly, FAA produced a rapid increase in tumour hypoxia: a dose of 200 mg/kg induced close to 100% radiobiological hypoxia when give 1 h prior to irradiation. Complete tumour hypoxia was still apparent 18 h after treatment with FAA. Administered doses of FAA below 100 mg/kg did not produce close to 100% radiobiological hypoxia when administered 3 h prior to irradiation.

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.

Bioreductive drugs and the selective induction of tumour hypoxia.

In this work tumour hypoxia is induced by physically occluding the tumour vascular supply by clamping, or by giving mice 5 mg kg-1 hydralazine. These methods have previously been shown to increase the radiobiological hypoxic fraction in tumours close to 100%. Their effectiveness in potentiating the bioreductive toxicity of: misonidazole (800 mg kg-1), RSU1069 (80 mg kg-1), mitomycin C (5 mg kg-1) and SR4233 (50 mg kg-1) is assessed in the RIF-1 and KHT tumours using regrowth delay as an assay. Clamping alone for 120 min gives little or no response, but when RSU1069 is administered 15 min before clamping, large growth delays result. RIF-1 tumours clamped for 90 or 120 min with RSU1069 give cure rates of 12.5% and 37.5% respectively. Less effect with clamping is seen for the other bioreductive agents. The effect of hydralazine with RSU1069 although significant in the RIF-1 tumour, is modest compared to that for clamping. Small enhancements of toxicity are seen with hydralazine in combination with misonidazole in the RIF-1 tumour and mitomycin C in both tumours. The varying effectiveness of these treatments is attributed to several factors which include the level and duration of hypoxia, concentration and contact time of the bioreductive drugs, the microenvironment of the tumour and the nature of the reductive metabolic pathways available in the different tumour cell types.

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.