Biomedical subjects

M F Dennis

Publications and source records attributed to M F Dennis.

At least 37 records · Page 2Linked to original sources

PubMed1996

Chemical properties which control selectivity and efficacy of aromatic N-oxide bioreductive drugs.

Pulse radiolysis was used to generate radicals from one electron reduction of 1,2,4-benzotriazine-1,4-dioxides (derivatives of tirapazamine), and of imidazo [1,2-a]quinoxaline-4-oxides (analogues of RB90740), which have selective toxicity towards hypoxic cells. Radicals from the mono N-oxides (from the latter compounds) react with oxygen approximately 10-40 times faster than does the tirapazamine radical. Radicals from the tirapazamine analogues studied react with oxygen up to approximately 10 times slower than tirapazamine radicals. The quinoxaline N-oxide radicals are involved in prototropic equilibria with pK(a) values (5.5 to 7.4) spanning that reported for tirapazamine (6.0). Generation of radicals radiolytically in the presence of H donors (formate, 2-propanol, deoxyribose) indicate a chain reaction ascribed to H abstraction by the drug radical. The protonated drug radical is much more reactive than the radical anion (H abstraction rate constant approximately equal to 10(2) - 10(3) dm3 mol-1 s-1). Chain termination is ascribed to drug radical-radical reactions, i.e. radical stability in anoxia, with rate constants 2k approximately equal to 1 x 10(7) to 2 x 10(8) dm3 mol-1 s-1 at pH approximately 7.4. Estimates of the reduction potentials of the drug-radical couples in water at pH 7 for two of the mono-N-oxides were in the range-0.7 to 0.8 V vs NHE at pH 7.

Antineoplastic Agents↗

PubMed1996

Nitric oxide involvement in the toxicity of hydroxyguanidine in leukaemia HL60 cells.

The free-radical intermediates and the stable products formed on one-electron oxidation of hydroxyguanidine (HOG) were investigated in order to suggest a mechanistic basis for HOG-induced cytotoxicity and cytostasis in leukaemia HL60 cells. The azide radical (generated radiolytically) reacted with HOG to produce a carbon-centred radical which in the absence of oxygen decays by a first-order process (k = 3.2 x 10(3) s-1) to yield nitric oxide (NO) and urea. Although the HOG radical reacts rapidly with oxygen (rate constant for O2 addition, k = 4.2 x 10(8) dm3 mol-1 s-1) this neither prevented the elimination of NO. nor generated alternative nitrogen oxides (e.g. peroxynitrite) capable of contributing to cellular oxidative stress. The detection of NO. in HL60 cells corroborated mechanistic studies that oxidative denitrification of HOG does not require catalysis by nitric oxide synthase. Quantitation of NO. by electron paramagnetic resonance (EPR) spectroscopy (utilising a NO. -selective probe) shows higher amounts of NO. under anoxic conditions, reflecting competition for NO. with molecular oxygen in oxic cells. Inhibition of cytochrome P450 and myeloperoxidase activity decreased NO. production thereby identifying these enzyme systems as capable of oxidizing HOG in vitro. A correlation exists between the intracellular levels of NO. with both the cytotoxic and cytostatic effects of HOG within HL60 cells. A higher toxicity was observed with hypoxic than with oxic cells. The lower levels of NO. associated with aerobic conditions caused a G1 --> S block in the cell cycle which under anoxia potentiated NO. -induced apoptotic cell death.

Antineoplastic Agents↗

PubMed1995

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.

Adenocarcinoma↗

PubMed1995

Administration of nicotinamide during chart: pharmacokinetics, dose escalation, and clinical toxicity.

PURPOSE: To determine nicotinamide pharmacokinetics in patients undergoing accelerated radiotherapy with the CHART regimen (continuous, hyperfractionated, accelerated radiotherapy) and given nicotinamide on a daily basis. The aim was to establish the pharmacokinetic profiles and their reproducibility during repeated administration, the maximum tolerated dose with fractionated radiotherapy, whether such a dose achieves sufficiently high plasma levels for radiosensitization, the optimal time interval between nicotinamide and irradiation, and toxic side effects. METHODS AND MATERIALS: Nicotinamide plasma concentrations were determined using high performance liquid chromatography in 11 patients with advanced carcinomas of the head and neck and rectum being treated with CHART (36 fractions in 12 days). Kinetic profiles on the first day of radiotherapy and residual 24-h values were obtained in 10 patients; in four of these, full profiles were repeated two or three times during the course of treatment. In one other, a single sample per day was taken four times over the 12-day period. Doses of 80, 90, or 100 mg/kg/day were given 90 min prior to the second radiotherapy fraction on each day. RESULTS: A dose of 80 mg/kg/day was well tolerated by all the patients. However, an increase of 10-25% in dose led to significant drug accumulation and major clinical toxicity, and none of the patients in the dose-escalation arm completed the planned regimen. Large interpatient variations in absolute peak concentrations were seen from 0.4 to 1.4 mumol/ml (mean 0.9 +/- 0.3; standard deviation (SD)). Of the five samples with the lowest peak levels, four were obtained from one patient. The time taken to peak concentration was also very variable from 0.8 to 4 h (mean 2.1 +/- 1.3 h; SD). In 70% of the samples, absolute plasma levels > or = 0.7 mumol/ml were reached within 1-2 h after administration and maintained for up to 6 h (mean 2.8 +/- 1.8 h; SD). There was a small but nonsignificant increase in the half-life of nicotinamide when the dose was increased from 80 to 90 or 100 mg/kg (7.1 h and 8.6 h, respectively). CONCLUSIONS: In an accelerated regimen such as CHART, 80 mg/kg/day of oral nicotinamide is feasible and clinically tolerated, giving no or few side effects, and a 2-h interval between its oral administration and radiotherapy should achieve effective plasma levels in most patients.

Aged↗

PubMed1995

Analysis of the acidic microenvironment in murine tumours by high-performance ion chromatography.

High-performance ion chromatography (HPIC) has been utilised to probe the biochemistry associated with changes in tumour pH following total vascular occlusion. Samples from the tumour extracellular compartment were obtained by insertion of a microdialysis probe and analysed by HPIC with conductivity detection. Separations were carried out by ion-exclusion chromatography using an IonPac ICE AS1 weak-acid column. The eluent (0.5 mM octanesulphonic acid) was chemically suppressed with 5 mM tetrabutylammonium hydroxide through a micromembrane suppressor. After complete vascular occlusion induced by a clamp, lactate levels increased in the extracellular compartment.

Animals↗

PubMed1995

Nitric oxide in biological fluids: analysis of nitrite and nitrate by high-performance ion chromatography.

The analysis of nitric oxide-derived nitrite and nitrate ions in biological fluids represents a proven strategy for determining nitric oxide participation in a diverse range of physiological and pathophysiological processes in vivo. In this article we describe a versatile method for the simultaneous measurement of NO2- and NO3- anions in both plasma and isolated tumour models based on anion-exchange chromatography with spectrophotometric detection (214 nm). This method compares well with the capillary electrophoresis technique, exhibiting an equivalent sensitivity for NO2-/NO3- anions and short run-times, i.e. not greater than 4 min. Comparisons are also made with two alternative but less satisfactory methods which employ ion-exchange or reversed-phase ion-pair chromatography with conductimetric as well as spectrophotometric detection. Technical problems associated with each method, particularly those arising from nitrate contamination, have been addressed.

Animals↗

PubMed1995

Glutathione determination by the Tietze enzymatic recycling assay and its relationship to cellular radiation response.

Large fluctuations in glutathione content were observed on a daily basis using the Tietze enzyme recycling assay in a panel of six human cell lines of varying radiosensitivity. Glutathione content tended to increase to a maximum during exponential cell proliferation, and then decreased at different rates as the cells approached plateau phase. By reference to high-performance liquid chromatography and flow cytometry of the fluorescent bimane derivative we were able to verify that these changes were real. However, the Tietze assay was occasionally unable to detect glutathione in two of our cell lines (MGH-U1 and AT5BIVA), although the other methods indicated its presence. The existence of an inhibitory activity responsible for these anomalies was confirmed through spiking our samples with known amounts of glutathione. We were unable to detect a direct relationship between cellular glutathione concentration and aerobic radiosensitivity in our panel of cell lines.

Aerobiosis↗

PubMed1995

Pharmacokinetics and binding of the bioreductive probe for hypoxia, NITP: effect of route of administration.

The novel compound 7(-)[4'-(2-nitroimidazol-l-yl)-butyl]-theophylline (NITP) can be used as an immunologically detectable probe for hypoxic cells. Because of the limited water solubility of NITP, it has been administered dissolved in peanut oil with 10% dimethylsulphoxide (DMSO). A new aqueous formulation has been devised, based on a 50% solution of a modified beta-cyclodextrin (Molecusol HPB), which increases the water solubility of NITP 10-fold. The pharmacokinetics of NITP in plasma and tumours have been compared following oral and intraperitoneal (i.p.) administration of the NITP in Molecusol, i.p. administration of NITP dissolved in peanut oil + 10% DMSO and injection of a near-saturated aqueous solution of the drug intravenously via the tail vein or i.p. or directly into the tumours. Binding of the marker to hypoxic cells within tumours was also measured after the different routes of administration. The Molecusol vehicle was unexpectedly toxic when administered i.p., but there was no toxicity from NITP dissolved in Molecusol when administered orally. Binding of the drug within tumours was seen for both the peanut oil + 10% DMSO and Molecusol formulations and for both oral and intraperitoneal routes. Binding of NITP within tumours has also been observed following direct injection of the drug, with minimal whole-body exposure to NITP. However, the bound metabolites of NITP within tumours were localised to the injection site, suggesting that direct injection is unlikely to be a useful method of administering bioreductive hypoxia markers. The data in this paper demonstrate that bound metabolites of the hypoxia marker NITP can be detected in tumours following oral administration of an aqueous formulation of NITP, and suggest that oral administration could be a satisfactory administration route for clinical studies with NITP.

2-Hydroxypropyl-beta-cyclodextrin↗

PubMed1995

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.

Animals↗

PubMed1994

Determination of adenine nucleotides by fluorescence detection using high-performance liquid chromatography and post-column derivatisation with chloroacetaldehyde.

A novel rapid method for the analysis of adenine nucleotides in cells and tissues using post-column derivatisation with chloroacetaldehyde (CAA) followed by fluorescence detection is described. The CAA is incorporated in the eluent, but only reacts post-column when the temperature is elevated to 100 degrees C. Samples are chromatographed following neutralisation of acid extracts. Examples are given using both trichloroacetic acid extraction for cells in culture, and perchloric acid for murine liver.

Acetaldehyde↗

PubMed1994

Pharmacokinetics and biochemistry studies on nicotinamide in the mouse.

Nicotinamide sensitizes murine tumours to the effect of radiation, but the pharmacokinetics are not well characterized at doses that are achievable in humans. In the mouse, nicotinamide given i.p. at doses of 100-500 mg/kg showed biphasic elimination with dose-dependent changes in half-life. The initial half-life increased significantly (P < 0.05) from 0.8 to 2 h and the terminal half-life increased from 3.4 to 5.6 h over the dose range studied. Clearance, however, decreased significantly from 0.3 to 0.24 l kg-1 h-1 only at the highest dose. Peak concentrations increased in a dose-dependent manner from 1,000 to 4,800 nmol/ml. The main plasma metabolite in the mouse is nicotinamide N-oxide, the peak concentration of which increased only from 80 to 160 nmol/ml. The N-oxide, which is also a weak radiosensitizer, is subject to reduction to the parent nicotinamide following administration at a dose of 276 mg/kg; peak concentrations of the N-oxide of 1900 nmol/ml were reached in 10 min, whereas concentrations of nicotinamide produced by reduction reached a maximum of 144 nmol/ml at 1 h. Elimination of the N-oxide was also biphasic, with initial and terminal half-lives being 0.39 and 1.8 h, respectively. The bioavailability of both drugs given via the i.p. as compared with the i.v. route was close to 100%. Tumour concentrations of nicotinamide paralleled those in the plasma after a short lag. Tumour nicotinamide adenine dinucleotide (NAD) concentrations were elevated by factors of 1.5 and 1.8 following doses of 100 and 500 mg/kg nicotinamide, respectively. Maximal concentrations were seen after 3-6 h, but levels remained elevated for 16 h. No change in tumour energy charge or in plasma 5-hydroxytryptamine was detected following a dose of 500 mg/kg nicotinamide.

Animals↗

PubMed1993

Flavone acetic acid (FAA) with recombinant interleukin-2 (rIL-2) in advanced malignant melanoma. IV: Pharmacokinetics and toxicity of flavone acetic acid and its metabolites.

Flavone acetic acid (FAA) was administered at a dose of 4.8 g m-2 over 1 h to patients with advanced malignant disease in combination with Interleukin II. A new high performance liquid chromatography method is described to determine both the parent compound and eight drug-related products, and the conditions required to determine these components in plasma are discussed. The half-life over the first 8 h was 2.3 h, but the terminal clearance of the drug was extremely slow. Severe (WHO Grade 4) hypotension was observed in some patients. However, incidence of this did not appear to be associated with any differences in FAA plasma concentrations, nor were there differences in FAA clearance between those patients whose tumour responded to the drug combination and those who did not.

Antineoplastic Agents↗

PubMed1993

Pharmacokinetics of varying doses of nicotinamide and tumour radiosensitisation with carbogen and nicotinamide: clinical considerations.

Plasma concentrations, after administration of varying doses of nicotinamide, were measured in CBA male mice using a newly-developed high performance liquid chromatography assay. In all dose groups, peak levels were observed within the first 15 min after an i.p. administration of 0.1, 0.2, 0.3 or 0.5 mg g-1 of nicotinamide. There was a clear dose-dependent increase in plasma concentration with increasing dose, with almost a five-fold lower concentration (1.0 vs 4.9 mumol ml-1) achieved with a dose of 0.1 mg g-1 compared with 0.5 mg g-1, respectively. The half-life of nicotinamide increased from 1.4 h to 2.2 h over the dose range (P < 0.01). Comparisons with previous pharmacokinetic data in humans show that clinically-relevant oral doses of 6 and 9 g in humans give plasma levels slightly higher than those achieved at 1 h with doses of 0.1 to 0.2 mg g-1 in mice. Tumour radiosensitisation with carbogen alone, and with carbogen combined with varying doses of nicotinamide (0.05 to 0.5 mg g-1), was investigated using a 10-fraction in 5 days X-ray schedule. Relative to air-breathing mice, a statistically significant increase in sensitisation was observed with both a local tumour control and with an in vivo/in vitro excision assay (P < or = 0.007). With the local control assay, a trend was observed towards lower enhancement ratios (ERs) with decreasing nicotinamide dose (from 1.85 to 1.55); carbogen alone was almost as effective as when combined with 0.1 mg g-1 of nicotinamide. With the excision assay, ERs for carbogen combined with nicotinamide increased with decreased levels of cell survival. At a surviving fraction of 0.02, enhancement ratios of 1.39-1.48 were obtained for carbogen plus 0.1 to 0.3 mg g-1 of nicotinamide. These were lower than those seen with the two higher doses of 0.4 to 0.5 mg g-1 (ERs = 1.63-1.69).

Adenocarcinoma↗

PubMed1992

High-performance liquid chromatographic determination of nicotinamide and its metabolites in human and murine plasma and urine.

A high-performance liquid chromatographic method is described which enables the determination of nicotinamide and eight of its possible metabolites in human and murine plasma and urine, using ion-pairing on a base-deactivated reversed-phase column. Calibration curves were linear up to 2 mumol/ml for nicotinamide and 200 nmol/ml for the metabolites; both the intra- and inter-assay relative standard deviations ranged between 1 and 8%. In murine plasma, the N-oxide was the major nicotinamide metabolite, but in man, formation of 1-methylnicotinamide and the 2- and 4-pyridones was also significant. In urine, nicotinuric acid was seen in the mouse, but no nicotinic acid metabolites were seen in man.

Animals↗

PubMed1992

Pharmacokinetics of nicotinamide and its effect on blood pressure, pulse and body temperature in normal human volunteers.

The pharmacokinetics of nicotinamide were studied in four human volunteers after oral doses of 1-6 g. Plasma concentrations and clearance rates of the vitamin were found to be dose-dependent, with a half-life of approximately 7-9 h for the two highest doses administered (4 and 6 g), approximately 4 h with 2 g and approximately 1.5 h with a 1-g dose. Peak concentrations ranged from 0.7 to 1.1 mumol.ml-1 after a 6-g dose. The time to reach peak plasma concentration was dose independent with a broad range from 0.73 to 3 h. In this study, nicotinamide had no detectable effect on blood pressure, pulse or body temperature.

Administration, Oral↗

PubMed1992

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.

Animals↗

PubMed1992

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.

Animals↗

PubMed1990

Radiosensitization by misonidazole, pimonidazole and azomycin and intracellular uptake in human tumour cell lines.

The radiosensitization of two human tumour in vitro cell lines, HT-1080 and LoVo, has been compared with that of the Chinese hamster cell line V79-379A. Although the two human tumour cell lines were more radiosensitive than the V79 cell line sensitizer, enhancement ratios for misonidazole, pimonidazole and azomycin were similar (relative to extracellular concentration) for all three cell lines. Average intracellular concentrations of radiosensitizer were measured by high-performance liquid chromatography. In all three cell lines the uptake of misonidazole and azomycin was extremely rapid whereas that of pimonidazole was initially much slower before reaching a plateau. The ratios of intracellular concentration of radiosensitizer to extracellular concentration (Ci to Ce) for misonidazole were 0.8 (HT-1080) and 0.7 (LoVo and V79); for azomycin 0.9 (HT-1080 and LoVo) and 0.8 (V79). In contrast Ci/Ce for pimonidazole varied with cell line, the values being 1.8 (LoVo), 2.6 (HT-1080) and 3.3 (V79). Intracellular amounts of non-protein sulphydryl (NPSH) varied between cell lines by about a factor of three. However, when the average cell volume was taken into consideration the concentrations of NPSH were very similar, being 4.2 (HT-1080), 5.6 (LoVo) and 5.7 (V79) mmol dm-3. NPSH levels expressed as nmol per mg protein were also similar.

Animals↗