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.

Effect of serum starvation on expression and phosphorylation of PKC-alpha and p53 in V79 cells: implications for cell death.

The effect of serum starvation on the expression and phosphorylation of PKC-alpha and p53 in Chinese hamster V79 cells was investigated. Serum starvation led to growth arrest, rounding up of cells and the appearance of new PKC-alpha and p53 bands on Western blots. Prolonged incubation (> or = 48 hr) in serum-deprived medium led to cell detachment and death. Moving cells to fresh medium containing 10% serum before, but not after, cell detachment reversed the changes observed in PKC-alpha and p53, and also prevented later cell detachment. Radiolabelling studies showed that the higher-molecular-weight PKC-alpha and p53 bands result from increased phosphorylation, while a lower-molecular-weight PKC-alpha band reflects newly synthesized protein. Immunocomplex kinase assays have shown that the increased phosphorylation of PKC-alpha is associated with its increased activity. To study the relationship between PKC-alpha, p53 and cell death, cells were treated either with TPA, to down-regulate PKC or with staurosporine, to inhibit PKC activity. Staurosporine, a potent PKC inhibitor and inducer of programmed cell death, caused the appearance of new PKC-alpha and p53 bands similar to those induced by serum starvation. If serum starvation was preceded by prolonged (48 hr) TPA treatment to down-regulate PKC-alpha, cell detachment and death did not take place within the same time frame. Intracellular fractionation of cells demonstrated that increased expression of PKC-alpha and the appearance of the associated higher and lower molecular-weight bands occurred in the nucleus. These data highlight the association of PKC-alpha and p53 with cellular events leading to cell death.

Indolequinone antitumor agents: reductive activation and elimination from (5-methoxy-1-methyl-4,7-dioxoindol-3-yl)methyl derivatives and hypoxia-selective cytotoxicity in vitro.

A series of indolequinones bearing a variety of leaving groups at the (indol-3-yl)methyl position was synthesized by functionalization of the corresponding 3-(hydroxymethyl)indolequinone, and the resulting compounds were evaluated in vitro as bioreductively activated cytotoxins. The elimination of a range of functional groups-carboxylate, phenol, and thiol-was demonstrated upon reductive activation under both chemical and quantitative radiolytic conditions. Only those compounds which eliminated such groups under both sets of conditions exhibited significant hypoxia selectivity, with anoxic:oxic toxicity ratios in the range 10-200. With the exception of the 3-hydroxymethyl derivative, radiolytic generation of semiquinone radicals and HPLC analysis indicated that efficient elimination of the leaving group occurred following one-electron reduction of the parent compound. The active species in leaving group elimination was predominantly the hydroquinone rather than the semiquinone radical. The resulting iminium derivative acted as an alkylating agent and was efficiently trapped by added thiol following chemical reduction and by either water or 2-propanol following radiolytic reduction. A chain reaction in the radical-initiated reduction of these indolequinones (not seen in a simpler benzoquinone) in the presence of a hydrogen donor (2-propanol) was observed. Compounds that were unsubstituted at C-2 were found to be up to 300 times more potent as cytotoxins than their 2-alkyl-substituted analogues in V79-379A cells, but with lower hypoxic cytotoxicity ratios.

Magnetic resonance imaging and spectroscopy of combretastatin A4 prodrug-induced disruption of tumour perfusion and energetic status.

The effects of combretastatin A4 prodrug on perfusion and the levels of 31P metabolites in an implanted murine tumour were investigated for 3 h after drug treatment using nuclear magnetic resonance imaging (MRI) and spectroscopy (MRS). The area of regions of low signal intensity in spin-echo images of tumours increased slightly after treatment with the drug. These regions of low signal intensity corresponded to necrosis seen in histological sections, whereas the expanding regions surrounding them corresponded to haemorrhage. Tumour perfusion was assessed before and 160 min after drug treatment using dynamic MRI measurements of gadolinium diethylenetriaminepentaacetate (GdDTPA) uptake and washout. Perfusion decreased significantly in central regions of the tumour after treatment. This was attributed to disruption of the vasculature and was consistent with the haemorrhage seen in histological sections. The mean apparent diffusion coefficient of water within the tumour did not change, indicating that there was no expansion of necrotic regions during the 3 h after drug treatment. Localized 31P-MRS showed that there was decline in cellular energy status in the tumour after treatment with the drug. The concentrations of nucleoside triphosphates within the tumour fell, the inorganic phosphate concentration increased and there was a significant decrease in tumour pH for 80 min after drug treatment. The rapid, selective and extensive damage caused to these tumours by combretastatin A4 prodrug has highlighted the potential of the agent as a novel cancer chemotherapeutic agent. We have shown that the response of tumours to treatment with the drug may be monitored non-invasively using MRI and MRS experiments that are appropriate for use in a clinical setting.

Hypoxia facilitates tumour cell detachment by reducing expression of surface adhesion molecules and adhesion to extracellular matrices without loss of cell viability.

The effects of acute hypoxia on integrin expression and adhesion to extracellular matrix proteins were investigated in two human melanoma cell lines, HMB-2 and DX3, and a human adenocarcinoma cell line, HT29. Exposure to hypoxia caused a significant down-regulation of cell surface integrins and an associated decrease in cell adhesion. Loss of cell adhesion and integrin expression were transient and levels returned to normal within 24 h of reoxygenation. Other cell adhesion molecules, such as CD44 and N-CAM, were also down-regulated after exposure of cells to hypoxia. Acute exposure to hypoxia of cells at confluence caused rapid cell detachment. Cell detachment preceded loss of viability. Detached HMB-2 and DX3 cells completely recovered upon reoxygenation, and floating cells re-attached and continued to grow irrespective of whether they were left in the original glass dishes or transferred to new culture vessels, while detached HT29 cells partly recovered upon reoxygenation. Cell detachment after decreased adhesion appears to be a stress response, which may be a factor enabling malignant cells to escape hypoxia in vivo, with the potential to form new foci of tumour growth.

5-substituted analogues of 3-hydroxymethyl-5-aziridinyl-1-methyl-2-[1H-indole-4,7-dione]prop-2-en- 1-ol (EO9, NSC 382459) and their regioisomers as hypoxia-selective agents: structure-cytotoxicity in vitro.

A series of regioisomeric analogues of 3-hydroxymethyl-5-aziridinyl-1-methyl-2-[1H-indole-4,7-dione]prop-2-en-1 -ol (EO9, NSC 382459) with the hydroxymethyl and hydroxypropenyl substituents situated at either the 2- or the 3-position of the indole ring were synthesized. The compound lacking the 2-hydroxypropenyl substituent (31) had similar properties to EO9 under both aerobic and hypoxic conditions against V79 cells and was more potent against a human tumour cell line (A549) than EO9. It was reduced by human DT-diaphorase (DTD) at more than double the rate of EO9, thus implicating the importance of the enzyme activation step. Compound 16 (lacking the 3-hydroxymethyl substituent) was a better substrate for human DTD than EO9, yet exhibited lesser toxicity under both aerobic and hypoxic conditions. The toxicity generated by 16 was attributed to the 5-aziridinyl moiety and suggests a greater contribution from the 3-substituent over the 2-substituent. The toxicity of EO9 was attributed to a combination of the aziridinyl group and the leaving group properties of the 3-hydroxymethyl substituent. In general, compounds with a 5-methylaziridinyl moiety, such as EO8, exhibited substantially better hypoxia-selectivity due to much slower reduction by DTD (20-fold), thus reducing aerobic potency. All compounds had similar electron affinities, as indicated by their one-electron reduction potentials.

The Klaas Breur Lecture. Radiation, hypoxia and genetic stimulation: implications for future therapies.

The cellular stress response, whereby very low doses of cytotoxic agents induce resistance to much higher doses, is an evolutionary defence mechanism and is stimulated following challenges by numerous chemical, biological and physical agents including particularly radiation, drugs, heat and hypoxia. There is much homology in the effects of these agents which are manifest through the up-regulation of various genetic pathways. Low-dose radiation stress influences processes involved in cell-cycle control, signal transduction pathways, radiation sensitivity, changes in cell adhesion and cell growth. There is also homology between radiation and other cellular stress agents, particularly hypoxia. Whereas traditionally, hypoxia was regarded mainly as an agent conferring resistance to radiation, there is now much evidence illustrating the cytokine-like properties of hypoxia as well as radiation. Stress phenomena are likely to be important in risks arising from low doses of radiation. Conversely, exploitation of the stress response in settings appropriate to therapy can be particularly beneficial not only in regard to radiation alone but in combinations of radiation and drugs. Similarly, tissue hypoxia can be exploited in novel ways of enhancing therapeutic efficacy. Bioreductive drugs, which are cytotoxically activated in hypoxic regions of tissue, can be rendered even more effective by hypoxia-induced increased expression of enzyme reductases. Nitric oxide pathways are influenced by hypoxia thereby offering possibilities for novel vascular based therapies. Other approaches are discussed.

2-Cyclopropylindoloquinones and their analogues as bioreductively activated antitumor agents: structure-activity in vitro and efficacy in vivo.

A series of 2-cycloalkyl- and 2-alkyl-3-(hydroxymethyl)-1-methylindoloquinones and corresponding carbamates have been synthesized and substituted in the 5-position with a variety of substituted and unsubstituted aziridines. Cytotoxicity against hypoxic cells in vitro was dependent upon the presence of a 5-aziridinyl or a substituted aziridinyl substituent for 3-hydroxymethyl analogues. The activity of 5-methoxy derivatives was dependent upon the presence of a 3-(carbamoyloxy)methyl substituent. Increasing the steric bulk at the 2-position reduced the compounds' effectiveness against hypoxic cells. A 2-cyclopropyl substituent was up to 2 orders of magnitude more effective than a 2-isopropyl substituent, suggesting possible radical ring-opening reactions contributing to toxicity. Nonfused 2-cyclopropylmitosenes were more effective than related fused cyclopropamitosenes reported previously. The reduction potentials of the quinone/semiquinone one-electron couples were in the range -286 to -380 mV. The semiquinone radicals reacted with oxygen with rate constants 2-8 x 10(8) dm3 mol-1 s-1. The involvement of the two-electron reduced hydroquinone in the mediation of cytotoxicity is implicated. The most effective compounds in vitro were the 2-cyclopropyl and 5-(2-methylaziridinyl) derivatives, and of these, 5-(aziridin-1-yl)-2-cyclopropyl-3-(hydroxymethyl)-1-methylindole-4 ,7-dione (21) and 3-(hydroxymethyl)-5-(2-methylaziridin-1-yl)-1,2-dimethylindole+ ++-4,7-dione (54) were evaluated in vivo. Both compounds showed antitumor activity both as single agents and in combination with radiation, with some substantial improvements over EO9 (3) at maximum tolerated doses and as single agents against the RIF-1 tumor model and comparable efficacy in the KHT tumor model.

Enhancement of bioreductive drug toxicity in murine tumours by inhibition of the activity of nitric oxide synthase.

Nitro-L-arginine inhibits the production of nitric oxide and can thereby cause vasoconstriction in vivo. One consequence of this is that nitro-L-arginine can increase hypoxia in a range of transplantable and spontaneous murine solid tumours. Bioreductive drugs such as RB6145 are more active under hypoxic conditions, and the combination of RB6145 with nitro-L-arginine in vivo shows greater anti-tumour activity than either agent individually. In mice given nitro-L-arginine at 10 mg kg(-1) i.p. up to 1 h before or after 300 mg kg(-1) i.p. RB6145, survival of KHT tumour cells was reduced by 3-4 logs when assessed by clonogenic assay 24 h after treatment. RB6145 or nitro-L-arginine alone caused no more than 20% cell kill. Similar effects were found in SCCVII tumours. The tumour response to the drug combination was tumour size dependent, with increased tumour cell sensitivity occurring when the tumour volume at the time of treatment was increased. Further, the response of KHT tumours to the combination of RB6145 and nitro-L-arginine was also dependent on the time interval between treatment and on when tumours were excised for determination of survival in vitro. The relative surviving fraction was about 0.3 for intervals less than 4 h but was reduced to 0.01 at 12 h and 0.001 at 24 h. These results were supported by histological examination of tumours, when necrosis at 2 h after treatment was less than 5% but increased to greater than 90% at 24 h. RB6145-induced normal tissue damage, as measured by CFU-A survival, was not altered by combining with nitro-L-arginine. Hence, this drug combination may provide therapeutic benefit. It is likely that the substantial anti-tumour effects are due to enhancement of bioreductive toxicity through increased tumour hypoxia, although additional mechanism(s) may also contribute to the overall response.

Changes in energy metabolism and X-ray sensitivity in murine tumours by the nitric oxide donor SIN-1.

The effect of the nitric oxide (NO) donor SIN-1 on energy metabolism was examined in three murine transplantable tumours in vivo using 31P MRS. SIN-1 at 2 mg kg-1 i.v. reduced Pi/total by 40-50% in SCCVII/Ha and KHT tumours within 5 min of injection, returning to control levels by 20 min. However, this dose of SIN-1 did not consistently alter Pi/total in RIF-1 tumours. Reduction in Pi/total in SCCVII/Ha tumours 10 min after 5 mg kg-1 i.v. SIN-1 was similar to that for 2 mg kg-1. SIN-1 at 10 mg kg-1 had no effect on Pi/total at 10 min after injection, but increased this ratio 2-fold over control at 60 min, at which time no effect of the lower doses of SIN-1 were observed. SIN-1 effects on SCCVII/Ha tumour response to X-rays were also examined, using an in vivo/in vitro clonogenic assay 24 h after treatment. SIN-1 at 0.5-2 mg kg-1 i.v. given immediately before irradiation increased tumour cell killing 2-4-fold over that for 15 Gy X-rays alone, while higher SIN-1 doses were ineffective. The results indicate that NO donors can alter tumour energy metabolism and X-ray response in a manner consistent with increased oxygenation. However, these responses are dependent upon dose, timing and tumour type.

Effect of hydralazine in spontaneous tumours assessed by oxygen electrodes and 31P-magnetic resonance spectroscopy.

Hydralazine can substantially decrease the blood flow, oxygen status and energy metabolism of transplanted tumours. In spontaneous tumours, two recent studies reported no effects of hydralazine on energy metabolism measured by 31P-magnetic resonance spectroscopy (31P-MRS), although another study saw changes in oxygen partial pressure (pO2) distributions measured with electrodes. We have now performed 31P-MRS and pO2 measurements in the same T138 spontaneous tumours, before and after intravenously (i.v.) injecting anaesthetised mice with 5 mg kg-1 hydralazine. Tumour pO2 was measured with an Eppendorf oxygen electrode and 31P-MRS spectra obtained with a 7-tesla SISCO magnet. Of 12 tumours all showed an increase in the percentage of pO2 values < or = 5 mmHg after hydralazine treatment and 10/12 showed a decrease in median pO2. The average (+/-1 s.e.) values for the percentage < or = 5 mmHg went from 45% (+/-9) to 79% (+/-5) and the median from 9 mmHg (+/-2) to 2 mmHg (+/-1). With the 31P-MRS 8/12 tumours showed an increase in the ratio of the peaks of inorganic phosphate to beta-nucleoside triphosphate with the average (+/-1 s.e.) values going from 1.1 (+/-0.2) to 2.4 (+/-0.9). Thus spontaneous tumours can respond to hydralazine and do so in a way consistent with that reported for transplanted tumours.

Induction and phosphorylation of protein kinase C-alpha and mitogen-activated protein kinase by hypoxia and by radiation in Chinese hamster V79 cells.

Protein kinase C (PKC) and mitogen-activated protein (MAP) kinase are protein-serine/threonine kinases which are important regulators of diverse cellular processes including metabolism, proliferation and differentiation. This study shows that both hypoxia and X irradiation of serum-deprived Chinese hamster V79 cells cause the induction and phosphorylation of the PKC-alpha isoform. The increased induction and phosphorylation of PKC occur mainly in the nuclear fraction. Unlike the PKC activator TPA, neither hypoxic nor radiation stress causes translocation of PKC-alpha from the cytosol to the membrane. The induction of PKC-alpha by hypoxia is accompanied by an increased expression of MAP kinase but, in contrast, this does not occur when PKC-alpha is induced by radiation. Radiation, like TPA, causes a complete redistribution of MAP kinase from the cytosol to the nucleus.

Fused pyrazine mono-n-oxides as bioreductive drugs. II Cytotoxicity in human cells and oncogenicity in a rodent transformation assay.

PURPOSE: To determine what structural moieties of the fused pyrazine mono-N-oxides are determining factors in their in vitro cytotoxicity and oncogenicity. METHODS AND MATERIALS: A new series of experimental bioreductive drugs, fused pyrazine mono-N-oxides, was evaluated in vitro for aerobic and hypoxic cytotoxicity in the HT29 human colon adenocarcinoma cell line by using clonogenic assays. The relative oncogenicities of these compounds were also determined in aerobic cultures of C3H 10T1/2 mouse embryo fibroblasts by using a standard transformation assay. RESULTS: Removal of the 4-methyl piperazine side chain from the parent compound, RB 90740, reduced the potency of the hypoxic cytotoxin. Reduction of the N-oxide function increased the aerobic cytotoxicity and eliminated most of the hypoxic/aerobic cytotoxic differential. The reduced N-oxide also had significant oncogenicity, consistent with a mechanism of genotoxicity following bioreduction of RB 90740. CONCLUSION: This new series of bioreductive compounds may be effective in cancer therapy, particularly the lead compound RB 90740. The oncogenic potential of these compounds is similar to that for other cancer therapies. Further studies should include evaluation of these compounds in vivo and the development of analogs with reduced oncogenic potential and retention of the hypoxic/aerobic cytotoxicity differential.

A comparison of the physiological effects of RSU1069 and RB6145 in the SCCVII murine tumour.

The physiological and therapeutic effects of the bioreductive agent RSU1069 (80 mg/kg i.p.) and its prodrug RB6145 (240 mg/kg i.p.) were investigated in the SCCVII tumour. Using laser Doppler flowmetry it was found that RSU1069 produced a significant 30% reduction in tumour blood flow 30 min after administration, while RB6145 had no effect. Tumour oxygenation, measured with an Eppendorf oxygen electrode, was unchanged by either agent except for a reduction in values less than 2.5 mmHg at 30 min after injection. Neither agent significantly altered tumour energy metabolism, assessed by 31P magnetic resonance spectroscopy. Both agents significantly increased tumour glucose content by a factor of 1.6-1.7 at 30 min after injection, but had no effect on glucose-6-phosphate or lactate levels. Tumour growth was significantly delayed by heating (42.5 degrees C, 60 min), and although neither RSU1069 nor RB6145 alone had any effect on tumour growth they produced a similar enhancement of the tumour response to heat. The therapeutic effects are consistent with the known conversion in vivo of one third of the pro-drug RB6145 to its active product RSU1069, however the physiological effects of the two agents in the SCCVII tumour are not identical.

Importance of P450 reductase activity in determining sensitivity of breast tumour cells to the bioreductive drug, tirapazamine (SR 4233).

P450 reductase (NADPH:cytochrome P450 reductase, EC 1.6.2.4) is known to be important in the reductive activation of the benzotriazene-di-N-oxide tirapazamine (SR 4233). Using a panel of six human breast adenocarcinoma cell lines we have examined the relationship between P450 reductase activity and sensitivity to tirapazamine. The toxicity of tirapazamine was found to correlate strongly with P450 reductase activity following an acute (3 h) exposure under hypoxic conditions, the drug being most toxic in the cell lines with the highest P450 reductase activity. A similar correlation was also observed following a chronic (96 h) exposure to the drug in air but not following acute (3 h) exposure in air. We have also determined the ability of lysates prepared from the cell lines to metabolise tirapazamine to its two-electron reduced product, SR 4317, under hypoxic conditions using NADPH as an electron donor. The rate of SR 4317 formation was found to correlate both with P450 reductase activity and with sensitivity to tirapazamine, the highest rates of SR 4317 formation being associated with the highest levels of P450 reductase activity and the greatest sensitivity to the drug. These findings indicate a major role for P450 reductase in determining the hypoxic toxicity of tirapazamine in breast tumour cell lines.

Fused pyrazine mono-N-oxides as bioreductive drugs. III. Characterization of RB 90740 in vitro and in vivo.

RB 90740 is the lead compound in a series of aromatic mono-N-oxide bioreductive drugs. The compound shows considerably greater toxicity towards hypoxic verses aerobic mammalian cells in vitro. The differential in concentration required to give the same level of cell killing under these conditions ranges from 3.5 in a human bronchio-alveolar tumor cell line up to 120 in a rodent cell line defective in the repair of DNA strand breaks. The ability of RB 90740 to cause DNA strand breaks under hypoxic conditions was confirmed by alkaline sucrose gradient and pulsed field gel electrophoresis techniques. Despite these properties demonstrated in vitro, RB 90740 was shown not to be cytotoxic to hypoxic cells in experimental murine tumors in vivo. This may be due, in part, to the level of hypoxia (< 0.02% O2) necessary to produce toxicity in vitro.

One-electron oxidation of C(2) and C(3) methyl substituted 5,6-dihydroxyindoles: model pathways of melanogenesis.

One-electron oxidation of a series of C(2)- and C(3)-methyl substituted analogues of 5,6-dihydroxyindole (DHI), in the pH range 5-9, was studied using the technique of pulse radiolysis with spectrophotometric detection. This investigation was undertaken to further our understanding of the involvement of free radical species in the polymerization processes leading to melanin formation. The optical absorption spectra of the protonated indole semiquinone radicals resulting from the one-electron oxidation of C(2)- and C(3)-methyl substituted indoles were similar to those of their corresponding hydroxylated indoles. From this similarity, it is inferred that methylation at C(2) and C(3) of DHI has little or no effect upon the initial radicals. The semiquinone radicals of these analogues subsequently decay to yield the corresponding quinone methide/imine. However, methyl substitution at C(2) and C(3) of the quinone methide derived from these analogues results in their stabilization. This stabilization contracts with the reactivity of the corresponding quinone methide from DHI. Further, these stabilized quinone methides do not interact with the azide ion (N3-), in contrast to the reaction of N3- with the quinone methide of DHI. It is concluded that methylation at C(2) and C(3) of DHI will modify the pathways so that the polymerization processes are less effective than those with DHI.

Induction of hypoxia in experimental murine tumors by the nitric oxide synthase inhibitor, NG-nitro-L-arginine.

The nitric oxide synthase inhibitor NG-nitro-L-arginine (NOARG) was examined for its ability to alter energy metabolism in three murine tumors using 31P magnetic resonance spectroscopy. NOARG (10 mg/kg, i.v.) increased the inorganic phosphate:total phosphate ratio (Pi:total) 2-3-fold in the KHT, RIF-1, and SCCVII/Ha intradermal back tumors from 30 min to 6 h after injection, but the 31P magnetic resonance spectrum from normal tissue on the mouse back was unchanged after this treatment. NOARG (10 mg/kg, i.v.) injected 30 min before X-rays increased tumor cell survival 3-5-fold in SCCVII/Ha and 50-200-fold in RIF-1, measured using an in vivo/in vitro clonogenic assay. These effects were equivalent to those obtained from clamped tumors, indicating full radiobiological hypoxia. In KHT, only a 2-fold increase in radioresistance was observed after NOARG, which was less than the response of clamped tumors. In RIF-1 tumors, NOARG induced full radiobiological hypoxia when given from 30 min to 6 h prior to X-rays, consistent with the time course for the increase in Pi:total, measured by 31P magnetic resonance spectroscopy. Pi:total after NOARG doses of 0.1-10 mg/kg, i.v., increased in a dose-dependent manner in this tumor. Increased RIF-1 tumor radioresistance was similarly dependent on NOARG dose. The combination of the bioreductive agent RB6145 (300 mg/kg, i.p.) 15 min prior to NOARG (10 mg/kg, i.v.) produced greater than 5 decades of KHT tumor cell killing at 24 h after treatment. This combination also increased Pi:total 4.5-fold over the control value at 24 h in the KHT tumor. Histological examination of tumors at this time indicated extensive necrosis.

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.