A Phase Ib trial of CA4P (combretastatin A-4 phosphate), carboplatin, and paclitaxel in patients with advanced cancer.

BACKGROUND: The vascular disrupting agent combretastatin A4 phosphate (CA4P) causes major regression of animal tumours when given as combination therapy. METHODS: Patients with advanced cancer refractory to standard therapy were treated with CA4P as a 10-min infusion, 20 h before carboplatin, paclitaxel, or paclitaxel, followed by carboplatin. RESULTS: Combretastatin A4 phosphate was escalated from 36 to 54 mg m(-2) with the carboplatin area under the concentration curve (AUC) 4-5, from 27 to 54 mg m(-2) with paclitaxel 135-175 mg m(-2), and from 54 to 72 mg m(-2) with carboplatin AUC 5 and paclitaxel 175 mg m(-2). Grade 3 or 4 neutropenia was seen in 17%, and thrombocytopenia only in 4% of 46 patients. Grade 1-3 hypertension (26% of patients) and grade 1-3 tumour pain (65% of patients) were the most typical non-haematological toxicities. Dose-limiting toxicity of grade 3 hypertension or grade 3 ataxia was seen in two patients at 72 mg m(-2). Responses were seen in 10 of 46 (22%) patients with ovarian, oesophageal, small-cell lung cancer, and melanoma. CONCLUSION: The combination of CA4P with carboplatin and paclitaxel was well tolerated in the majority of patients with adequate premedication and had antitumour activity in patients who were heavily pretreated.

Ischemia reperfusion injury in tumors: the role of oxygen radicals and nitric oxide.

Oxidative stress is a key process involved in the action of several therapeutic modalities used in cancer treatment. Ischemia reperfusion insult provides a model system for investigating the processes involved in determining the sensitivity of tumor tissue to oxidative stress. We have investigated the response of the murine CaNT tumor to ischemia reperfusion injury and the role that oxygen radicals and nitric oxide may play in this phenomenon. Our results show that little or no cell kill is detected in tumors exposed to up to 3 h of ischemia if the tumors are excised immediately before reperfusion. However, if reperfusion is permitted, then extensive cell kill is evident 24 h later. i.v. administration of superoxide dismutase or catalase, at the time when vascular reperfusion occurred, resulted in a significant protection against tumor cell kill, suggesting that the damage was mediated by oxygen radicals. Conversely, administration of an inhibitor of nitric oxide synthase, N omega-nitro-L-arginine, resulted in potentiation of tumor cell damage. Administration of a nitric oxide (NO) donor, diethylamine NO, at the time when vascular reperfusion occurred resulted in significant protection against tumor damage. These results suggest that nitric oxide is a potent mediator in determining tumor damage after ischemia reperfusion injury. The role of intrinsic NO production by murine tumors was investigated by measuring the accumulation of nitrate in the medium of tumor explants cultured in vitro in two tumors with differing sensitivity to ischemia reperfusion damage. The clamp-insensitive tumor SaS showed a greater nitrate accumulation than the clamp-sensitive tumor CaNT, which may confer a greater capacity for preventing tumor and endothelial cell damage after oxidative stress.

Combretastatin A-4 phosphate as a tumor vascular-targeting agent: early effects in tumors and normal tissues.

The potential for tumor vascular-targeting by using the tubulin destabilizing agent disodium combretastatin A-4 3-0-phosphate (CA-4-P) was assessed in a rat system. This approach aims to shut down the established tumor vasculature, leading to the development of extensive tumor cell necrosis. The early vascular effects of CA-4-P were assessed in the s.c. implanted P22 carcinosarcoma and in a range of normal tissues. Blood flow was measured by the uptake of radiolabeled iodoantipyrine, and quantitative autoradiography was used to measure spatial heterogeneity of blood flow in tumor sections. CA-4-P (100 mg/kg i.p.) caused a significant increase in mean arterial blood pressure at 1 and 6 h after treatment and a very large decrease in tumor blood flow, which-by 6 h-was reduced approximately 100-fold. The spleen was the most affected normal tissue with a 7-fold reduction in blood flow at 6 h. Calculations of vascular resistance revealed some vascular changes in the heart and kidney for which there were no significant changes in blood flow. Quantitative autoradiography showed that CA-4-P increased the spatial heterogeneity in tumor blood flow. The drug affected peripheral tumor regions less than central regions. Administration of CA-4-P (30 mg/kg) in the presence of the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine methyl ester, potentiated the effect of CA-4-P in tumor tissue. The combination increased tumor vascular resistance 300-fold compared with less than 7-fold for any of the normal tissues. This shows that tissue production of nitric oxide protects against the damaging vascular effects of CA-4-P. Significant changes in tumor vascular resistance could also be obtained in isolated tumor perfusions using a cell-free perfusate, although the changes were much less than those observed in vivo. This shows that the action of CA-4-P includes mechanisms other than those involving red cell viscosity, intravascular coagulation, and neutrophil adhesion. The uptake of CA-4-P and combretastatin A-4 (CA-4) was more efficient in tumor than in skeletal muscle tissue and dephosphorylation of CA-4-P to CA-4 was faster in the former. These results are promising for the use of CA-4-P as a tumor vascular-targeting agent.

Cofactor Strap regulates oxidative phosphorylation and mitochondrial p53 activity through ATP synthase.

Metabolic reprogramming is a hallmark of cancer cells. Strap (stress-responsive activator of p300) is a novel TPR motif OB-fold protein that contributes to p53 transcriptional activation. We show here that, in addition to its established transcriptional role, Strap is localised at mitochondria where one of its key interaction partners is ATP synthase. Significantly, the interaction between Strap and ATP synthase downregulates mitochondrial ATP production. Under glucose-limiting conditions, cancer cells are sensitised by mitochondrial Strap to apoptosis, which is rescued by supplementing cells with an extracellular source of ATP. Furthermore, Strap augments the apoptotic effects of mitochondrial p53. These findings define Strap as a dual regulator of cellular reprogramming: first as a nuclear transcription cofactor and second in the direct regulation of mitochondrial respiration.

Development of an enzymatic pretargeting strategy for dual-modality imaging.

A pretargeted imaging strategy based on the HaloTag dehalogenase enzyme is described. Here, a HaloTag-Trastuzumab conjugate has been used as the primary agent targeting HER2 expression, and three new radiolabelled HaloTag ligands have been used as secondary agents, two of which offer dual-modality (SPECT/optical) imaging capability.

A novel biosensor for quantitative monitoring of on-target activity of paclitaxel.

This study describes a system for quantifying paclitaxel activity using the C-terminus of α-tubulin as a biomarker. Following stabilization of microtubules with paclitaxel, a specific detyrosination reaction occurs at the C-terminus of α-tubulin which could be used to assess efficacy. A fluorescence resonance energy transfer (FRET) based biosensor was synthesized comprising a short peptide that corresponded to the C-terminus of α-tubulin, a fluorophore (Abz), and a quencher (Dnp). The fluorophore added to the end of the peptide can be released upon enzymatic detyrosination. In addition, a single fluorophore-tagged peptide was also conjugated to mesoporous silica nanoparticles to examine the feasibility of combining the drug with the peptide biomarker. As a proof of concept, we found that the degree of peptide cleavage, and therefore enzymatic activity, was directly correlated with exogenous bovine carboxypeptidase (CPA) an enzyme that mimics endogenous detyrosination. In addition, we show that cell lysates obtained from paclitaxel-treated cancer cells competed with exogenous CPA for biosensor cleavage in a paclitaxel dose-dependent manner. Our work provides strong evidence for the feasibility of combining paclitaxel with a novel biosensor in a multi-load nanoparticle.

Free hydroxyl radicals are formed on reaction between the neutrophil-derived species superoxide anion and hypochlorous acid.

Superoxide anion reacts with hypochlorous acid to yield free hydroxyl radicals, as shown by the hydroxylation of benzoate. This reaction is analogous to the Haber-Weiss reaction but in the absence of metal ions is at least six orders of magnitude faster.

Oxidative denitrification of the antitumour drug hydroxyguanidine.

The oxidative denitrification of the antitumour agent hydroxyguanidine (HOG) has been investigated by radiolysis methods and EPR spectroscopy. The azide radical (N3.), a model one-electron oxidant, reacts with HOG with the rate constant 5.1 x 10(9) dm3 mol(-1) s(-1) to yield the guanidino carbon-centred radical (HOG.) which rapidly eliminates nitric oxide (k = 3.1 x 10[3] s[-1]) with the concomitant formation of urea. The HOG. undergoes conjugation with molecular oxygen to form a peroxyl radical (HOGOO.) with a rate constant 8.8 x 10(8) dm3 mol(-1) s(-1). The HOGOO. radical also eliminates nitric oxide but may act as a precursor to the peroxynitrite (ONOO-) ion. The oxidation of HOG by the dibromide radical (Br2.-) was found to release nitric oxide with a yield of 95% relative to Br2.- as determined from the combined yields of inorganic nitrite, nitrate and a HOG/nitric oxide-adduct. This study provides a possible mechanistic basis for the oxidative denitrification of HOG which may contribute to the observed toxicity of the drug both in vitro and in vivo and for the oxidation of nonphysiological hydroxyguanidines to NO. via nitric oxide synthase-independent pathways.

Radicals from one-electron reduction of nitro compounds, aromatic N-oxides and quinones: the kinetic basis for hypoxia-selective, bioreductive drugs.

Drugs based on nitroarene, aromatic N-oxide or quinone structures are frequently reduced by cellular reductases to toxic products. Reduction often involves free radicals as intermediates which react rapidly with oxygen to form superoxide radicals, inhibiting drug reduction. The elevation of cellular oxidative stress accompanying oxygen inhibition of reduction is generally less damaging than drug reduction to toxic products, so the drugs offer selective toxicity to hypoxic cells. Since such cells are resistant to radiotherapy, these bioreductive drugs offer potential in tumour therapy. The basis for the selectivity of action entails kinetic competition involving the contesting reaction pathways. The reduction potential of the drug, radical pKa and nature of radical/radical decay kinetics all influence drug activity and selectivity, including the range of oxygen tensions over which the drug offers selective toxicity. These properties may be quantified using generation of radicals by pulse radiolysis, presenting a physicochemical basis for rational drug design.

Phase I study of intravenous 4-hydroxyanisole.

4-Hydroxyanisole is a depigmenting agent which has been shown to have activity against malignant melanoma when given intra-arterially in man. An intravenous dose escalation study has been carried out with the aim of obtaining maximum plasma concentrations in a 5 day schedule. 8 patients entered this study which was stopped because of drug toxicity after 3 patients had been treated at the third dose escalation of 15 g/m2. 2 patients had WHO grade 4 liver and one also grade 4 renal toxicity and another had grade 4 haemoglobin toxicity. Extrapolated plateau plasma levels between 112 and 860 mumol/l were obtained, which in vitro studies suggested would be cytotoxic. Hopefully, newer analogues will have a greater specificity for the melanin pathway with less toxicity.

Measurement of incorporation of bromodeoxyuridine into DNA by high performance liquid chromatography using a novel fluorescent labelling technique.

5'-Bromo-2-deoxyuridine (BUdR) is a halogenated pyrimidine analogue that is an efficient radiosensitizer through its incorporation into DNA in place of thymidine. Radiosensitization is proportional to percentage replacement and we present here a novel derivatization technique that specifically labels the thymidine and BUdR with 4-bromomethyl-7-methoxycoumarin (BrMMC) to give the highly fluorescent coumarin derivatives which are quantitated using high performance liquid chromatography (HPLC). This allows for a simple single-stage DNA hydrolysis and sensitive peak detection. Data are presented showing the incorporation with time of BUdR into the DNA of Chinese hamster V79 cells. Attention is also drawn to the care needed in the selection of enzymes required for DNA digestion.

A comparison of tumor and normal tissue levels of acidic, basic and neutral 2-nitroimidazole radiosensitizers in mice.

Pharmacokinetic studies were carried out in mice after co-administration of 2-nitroimidazoles with differing prototropic properties: Ro 03-8799, misonidazole, SR 2508, and azomycin. In addition, the distribution of sensitizers in different areas of individual tumors, as well as several normal tissues were measured after intravenous dosing of 0.5 mmol kg-1 (145-56 mg/kg). Although there appeared to be no significant pharmacokinetic interaction between sensitizers after co-administration, there was a significant difference in the distribution of sensitizers within tumors of differing type and size. In small, fast growing SaFa and large, slow growing CaRH tumors, the levels of all sensitizers was relatively constant throughout. However, large fast growing SaFa tumors demonstrated the high degree of variability in the Ro 03-8799 concentration that is seen in the clinic. This may be important in the modelling of human tumors with regard to sensitizer distribution.

Extracellular: intracellular and subcellular concentration gradients of thiols.

Chinese hamster V79 cells in Eagle's minimum essential medium in vitro at room temperature were incubated with the aminothiol, WR-1065, or glutathione (GSH) at extracellular concentrations of approximately 1 mmol dm-3. Average intracellular concentrations of GSH, cysteine, and WR-1065 were measured by high performance liquid chromatography, and the effective reducing environment near DNA probed by staining the cells with acridine orange (AO) and measuring the delayed fluorescence. Exposure to either thiol resulted in a rapid, 10-fold increase in average intracellular cysteine concentrations (to about 1 mmol dm-3). Adding extracellular GSH after prior depletion of GSH by treatment with L-buthionine sulfoximine (BSO) did not restore intracellular GSH, but intracellular cysteine was elevated 10-fold. These results are ascribed to thiol/disulfide exchange with cystine in the medium. WR-1065 slowly concentrated intracellularly to approximately 160% of the extracellular concentration. Chemical conjugation of GSH in cells decreased the reducing environment near DNA, but BSO treatment altered the uptake of AO. The electrostatic attraction of WR-1065 toward isolated DNA was markedly affected by ionic strength.

In vivo testing of a 2-nitroimidazole radiosensitizer (Ro 03-8799) using repeated administration.

The radiosensitizing efficiency of a novel 2-nitroimidazole (Ro 03-8799) has been compared with that of misonidazole. Each compound was assessed by constructing dose response curves for regrowth delay using a range of drug doses. The concentration of each compound was measured in tumor homogenates with high performance liquid chromatography (HPLC). When compared on the basis of administered dose the new compound was no more efficient than misonidazole. Comparison on the basis of measured tumor concentrations showed that Ro 03-8799 was 3--4 times more efficient than misonidazole, but only at very high drug levels. Previous in vitro studies had shown a constant 10-fold difference in potency. In order to eliminate possible artifacts caused by the short half life in mice, repeated injections of Ro 03-8799 were used to maintain a constant tumor concentration for two hours before irradiation. No increase in effectiveness was observed with prolonged exposure. This is a charged compound whose distribution is pH dependent; gross tumor levels should therefore be interpreted with caution.

Glutathione-reactive nitro compounds as radiosensitizers: mechanistic and therapeutic implications.

The "extra" radiosensitization seen with GSH-reactive nitro compounds is too large to be accounted for by GSH-depletion acting independently--there must be competition. The GS-conjugate leaks out of cells slowly and is trapped at high concentrations. Its properties, such as concentration trapped and reduction potential, must be considered. Limited therapeutic exploitation of the glutathione conjugate trapping and concomitant GSH depletion may be possible if intratumor injection is permitted.

The effect of alpha-tocopherol and alpha-tocopheryl quinone on the radiosensitivity of thiol-depleted mammalian cells.

The effect of hypoxic cell radiosensitizers is increased when mammalian cells are depleted of endogenous glutathione by buthionine sulphoximine pre-treatment in vitro; a similar gain has not been observed in tumors in vivo despite evidence of glutathione depletion in vivo following buthionine sulphoximine treatment. However, concentrations of biological reducing agents other than glutathione were not measured in the in vivo experiments. Other reducing agents found in tumors include alpha-tocopherol, which reduces the sensitizing efficiency of nitro-aromatic sensitizers in thiol-depleted mammalian cells. These data suggest that the failure to observe large gains in misonidazole sensitizing efficiency in thiol-depleted tumors in vivo may be due, in part, to the presence of biological reducing agents such as alpha-tocopherol.

Radiosensitizer-DNA interactions in relation to intracellular uptake.

We have studied the intracellular uptake of a number of neutral, acidic, and basic radiosensitizers. For neutral sensitizers, we observed a correlation between the measured intracellular concentration and sensitization, but for bases, a large change in average intracellular concentration results in only a small change in sensitization. In addition, by modifying the intralysosomal pH, we have altered the measured average intracellular concentration of the weak base pimonidazole by a factor of two, although this had no detectable effect upon sensitization. Using spin filtration of solutions of sensitizers with naked calf thymus DNA or chromatin we have assessed the affinity of DNA for sensitizers with different prototropic and lipophilic properties. We have also shown that this anomalous behavior of the basic sensitizers could be partly explained on the basis of intracellular localization adjacent to the DNA due to ionic interactions. Thus, intracellular localization needs to be considered when interpreting average intracellular uptake data.

The uptake of the radiosensitizing compound Ro 03-8799 (Pimonidazole) in human tumors.

The nitroimidazole, Ro 03-8799, has proved unique among the drugs tested as chemical hypoxic cell radiosensitizers because of the preferential concentration which has been observed in tumors. Our accumulation of experience has allowed new analyses to be performed upon 127 samples from 39 patients; 47 samples of normal tissue were also obtained from 26 of these patients. Tissue sampling was performed usually between 20 and 30 minutes after initiation of infusion of Ro 03-8799. By expressing results as tumor: plasma ratios, difficulties in comparison because of differing doses and body sizes, together with a variation in the actual time of sampling, have been avoided. A small portion of each specimen which was analyzed for drug concentration was also examined histologically to give an impression of the percentage of the specimen occupied by tumor cells. Analyses have shown that the average tumor concentration is approximately twice that of normal tissues which have been sampled and four times that in plasma. In 38 breast tumor samples, the concentration of drug varied directly as the proportion occupied by tumor cells. The highest tumor: plasma ratios were seen in samples taken from some samples of malignant melanoma. These findings confirm that a greater potency can be expected for this drug as a radiosensitizer because of its ability to enter tumor cells in high concentration. In drug development programs for chemical sensitizing and cytotoxic agents, drugs which show this phenomenon should be explored.

Nitroaryl compounds as potential fluorescent probes for hypoxia. I. Chemical criteria and constraints.

Cellular reduction of nitroaryl compounds is efficiently inhibited by oxygen, and detection of products characteristic of reduction could form the basis for diagnostic tests for the presence of hypoxic cells in tumors. The criteria for suitable compounds include a high sensitivity and selectivity of detection response between oxic and hypoxic cells, which can be provided using fluorescence detection and suitable nitroaryl compounds which have very low fluorescence until reduced. Examples described include a nitroacridine and nitronaphthalimides. Although the intercalating ability of these ring systems lead to high sensitivity for detection of reduced metabolites in vitro by flow cytometry, poor bioavailability is an unwanted consequence of intercalation. The application of several model reducing systems for reduction of potential fluorescent probes for hypoxia is described, and the absorption and fluorescence spectral characteristics of other examples of structures which could form the basis for useful probes are outlined.

Nitroaryl compounds as potential fluorescent probes for hypoxia. II. Identification and properties of reductive metabolites.

Nitroakridin 3582 (NA) and a nitronaphthalimide (DM113), which fluoresce only upon reduction, have been studied by HPLC. V79-379A cells incubated with NA under 20% or 2% O2 and N2 gave increasing amounts of the fluorescent amine with an hypoxic:oxic differential of 160. Measurement of the uptake of NA showed that it was concentrated within the cell by over 1000-fold. Studies in 3 different cell lines of reduction under hypoxia showed a 7-fold range in amine production. DM113 yields more than one fluorescent product, which show different absorption and fluorescence spectra. Chemical reduction of NA or DM113 using a variety of methods gave, depending on conditions, amine and/or (what was presumed to be) hydroxylamine; the latter was non-fluorescent. In vivo, NA is toxic at greater than 0.19 mumol g-1. At this dose much of the drug is found in the liver and kidneys. Plasma levels at 30 minutes are only 2 microM while tumor concentrations are 10 microM compared to 600 microM in the liver. However, the half life is greater than 1 hr and amine was detectable in these tumors.