Radiolysis Studies of Oxidation and Nitration of Tyrosine and Some Other Biological Targets by Peroxynitrite-Derived Radicals.

The widespread interest in free radicals in biology extends far beyond the effects of ionizing radiation, with recent attention largely focusing on reactions of free radicals derived from peroxynitrite (i.e., hydroxyl, nitrogen dioxide, and carbonate radicals). These radicals can easily be generated individually by reactions of radiolytically-produced radicals in aqueous solutions and their reactions can be monitored either in real time or by analysis of products. This review first describes the general principles of selective radical generation by radiolysis, the yields of individual species, the advantages and limitations of either pulsed or continuous radiolysis, and the quantitation of oxidizing power of radicals by electrode potentials. Some key reactions of peroxynitrite-derived radicals with potential biological targets are then discussed, including the characterization of reactions of tyrosine with a model alkoxyl radical, reactions of tyrosyl radicals with nitric oxide, and routes to nitrotyrosine formation. This is followed by a brief outline of studies involving the reactions of peroxynitrite-derived radicals with lipoic acid/dihydrolipoic acid, hydrogen sulphide, and the metal chelator desferrioxamine. For biological diagnostic probes such as 'spin traps' to be used with confidence, their reactivities with radical species have to be characterized, and the application of radiolysis methods in this context is also illustrated.

Development and pre-clinical testing of a novel hypoxia-activated KDAC inhibitor.

Tumor hypoxia is associated with therapy resistance and poor patient prognosis. Hypoxia-activated prodrugs, designed to selectively target hypoxic cells while sparing normal tissue, represent a promising treatment strategy. We report the pre-clinical efficacy of 1-methyl-2-nitroimidazole panobinostat (NI-Pano, CH-03), a novel bioreductive version of the clinically used lysine deacetylase inhibitor, panobinostat. NI-Pano was stable in normoxic (21% O2) conditions and underwent NADPH-CYP-mediated enzymatic bioreduction to release panobinostat in hypoxia (<0.1% O2). Treatment of cells grown in both 2D and 3D with NI-Pano increased acetylation of histone H3 at lysine 9, induced apoptosis, and decreased clonogenic survival. Importantly, NI-Pano exhibited growth delay effects as a single agent in tumor xenografts. Pharmacokinetic analysis confirmed the presence of sub-micromolar concentrations of panobinostat in hypoxic mouse xenografts, but not in circulating plasma or kidneys. Together, our pre-clinical results provide a strong mechanistic rationale for the clinical development of NI-Pano for selective targeting of hypoxic tumors.

Mitochondrial Inhibitor Atovaquone Increases Tumor Oxygenation and Inhibits Hypoxic Gene Expression in Patients with Non-Small Cell Lung Cancer.

PURPOSE: Tumor hypoxia fuels an aggressive tumor phenotype and confers resistance to anticancer treatments. We conducted a clinical trial to determine whether the antimalarial drug atovaquone, a known mitochondrial inhibitor, reduces hypoxia in non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: Patients with NSCLC scheduled for surgery were recruited sequentially into two cohorts: cohort 1 received oral atovaquone at the standard clinical dose of 750 mg twice daily, while cohort 2 did not. Primary imaging endpoint was change in tumor hypoxic volume (HV) measured by hypoxia PET-CT. Intercohort comparison of hypoxia gene expression signatures using RNA sequencing from resected tumors was performed. RESULTS: Thirty patients were evaluable for hypoxia PET-CT analysis, 15 per cohort. Median treatment duration was 12 days. Eleven (73.3%) atovaquone-treated patients had meaningful HV reduction, with median change -28% [95% confidence interval (CI), -58.2 to -4.4]. In contrast, median change in untreated patients was +15.5% (95% CI, -6.5 to 35.5). Linear regression estimated the expected mean HV was 55% (95% CI, 24%-74%) lower in cohort 1 compared with cohort 2 (P = 0.004), adjusting for cohort, tumor volume, and baseline HV. A key pharmacodynamics endpoint was reduction in hypoxia-regulated genes, which were significantly downregulated in atovaquone-treated tumors. Data from multiple additional measures of tumor hypoxia and perfusion are presented. No atovaquone-related adverse events were reported. CONCLUSIONS: This is the first clinical evidence that targeting tumor mitochondrial metabolism can reduce hypoxia and produce relevant antitumor effects at the mRNA level. Repurposing atovaquone for this purpose may improve treatment outcomes for NSCLC.

Targeting IGF Perturbs Global Replication through Ribonucleotide Reductase Dysfunction.

Inhibition of IGF receptor (IGF1R) delays repair of radiation-induced DNA double-strand breaks (DSB), prompting us to investigate whether IGF1R influences endogenous DNA damage. Here we demonstrate that IGF1R inhibition generates endogenous DNA lesions protected by 53BP1 bodies, indicating under-replicated DNA. In cancer cells, inhibition or depletion of IGF1R delayed replication fork progression accompanied by activation of ATR-CHK1 signaling and the intra-S-phase checkpoint. This phenotype reflected unanticipated regulation of global replication by IGF1 mediated via AKT, MEK/ERK, and JUN to influence expression of ribonucleotide reductase (RNR) subunit RRM2. Consequently, inhibition or depletion of IGF1R downregulated RRM2, compromising RNR function and perturbing dNTP supply. The resulting delay in fork progression and hallmarks of replication stress were rescued by RRM2 overexpression, confirming RRM2 as the critical factor through which IGF1 regulates replication. Suspecting existence of a backup pathway protecting from toxic sequelae of replication stress, targeted compound screens in breast cancer cells identified synergy between IGF inhibition and ATM loss. Reciprocal screens of ATM-proficient/deficient fibroblasts identified an IGF1R inhibitor as the top hit. IGF inhibition selectively compromised growth of ATM-null cells and spheroids and caused regression of ATM-null xenografts. This synthetic-lethal effect reflected conversion of single-stranded lesions in IGF-inhibited cells into toxic DSBs upon ATM inhibition. Overall, these data implicate IGF1R in alleviating replication stress, and the reciprocal IGF:ATM codependence we identify provides an approach to exploit this effect in ATM-deficient cancers. SIGNIFICANCE: This study identifies regulation of ribonucleotide reductase function and dNTP supply by IGFs and demonstrates that IGF axis blockade induces replication stress and reciprocal codependence on ATM. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2128/F1.large.jpg.

The effects of nitric oxide or oxygen on the stable products formed from the tyrosine phenoxyl radical.

Tyrosine is a critical component of many proteins and can be the subject of oxidative posttranslational modifications. Furthermore, the oxidation of tyrosine residues to phenoxyl radicals, sometimes quite stable, is essential for some enzymatic functions. The lifetime and fate of tyrosine phenoxyl radicals in biological systems are largely driven by the availability and proximity of oxidants and reductants. Tyrosine phenoxyl radicals have extremely low reactivity with molecular oxygen whereas reactions with nitric oxide are diffusion controlled. This is in contrast to equivalent reactions with tryptophanyl and cysteinyl radicals where reactions with oxygen are much faster. Despite, the quite disparate apparent reactivity of tyrosine phenoxyl radicals with oxygen and nitric oxide being known, the products of the reactions are not well established. Changes in the levels from expected basal concentrations of stable products resulting from tyrosine phenoxyl radicals, for example naturally occurring 3,3'-dityrosine, 3-nitrotyrosine, and 3-hydroxytyrosine, can be indicative of oxidative and/or nitrosative stress. Using the radiolytic generation of specific oxidizing radicals to form tyrosine phenoxyl radicals in an aqueous solution at a known rate, we have compared the products in the absence and presence of nitric oxide or oxygen. Possible reactions of the phenoxyl radicals with oxygen remain unclear although we show evidence for a small decrease in the yield of dityrosine and loss of tyrosine in the presence of 20% oxygen. Low concentrations of nitric oxide in anoxic conditions react with tyrosine phenoxyl radicals, by what is most probably through the formation of an unstable intermediate, regenerating tyrosine and forming nitrite.

Results of a phase II clinical trial of 6-mercaptopurine (6MP) and methotrexate in patients with BRCA-defective tumours.

BACKGROUND: Tumour cells with BRCA1/2 gene mutations demonstrate increased sensitivity to platinum and poly (ADP-ribose) polymerase (PARP) inhibitors. 6-mercaptopurine (6MP) was found to selectively kill BRCA-defective cells in a xenograft model as effectively as the PARP inhibitor AG014699, even after these cells acquired resistance to a PARP inhibitor or cisplatin. METHODS: This phase II single-arm trial investigated the activity of 6MP 55-75 mg/m2 per day, and methotrexate 15-20 mg/m2 per week in advanced breast or platinum-resistant ovarian cancer patients with a BRCA1/2 germline mutation, who had progressed after ≥1 previous line of chemotherapy. The primary outcome was objective response including stable disease (SD) as an assessment of clinical benefit rate (CBR), at 8 weeks, by RECIST v1.1. Secondary outcomes included overall survival (OS) and progression-free survival (PFS). RESULTS: In total, 67 evaluable patients were recruited; 55 ovarian and 11 breast cancer patients. In total, 21 patients had SD (31%), one had a partial response (1.5%); CBR was 33% at 8 weeks. In total, 12/67 patients (18%) had SD at 16 weeks. In total, five ovarian cancer patients had SD for over 200 days. Median OS was 10.3 months (95% CI 6.9-14.5), median PFS 1.9 months (1.7-2.8). CONCLUSIONS: The overall activity of 6MP and methotrexate in these patients was low; however, there was a small group of patients who appeared to derive longer-term clinical benefit. TRIAL REGISTRATION: NCT01432145 http://www.ClinicalTrials.gov.

Probing the limits of Q-tag bioconjugation of antibodies.

Site-selective labelling of antibodies (Abs) can circumvent problems from heterogeneity of conventional conjugation. Here, we evaluate the industrially-applied chemoenzymatic 'Q-tag' strategy based on transglutaminase-mediated (TGase) amide-bond formation in the generation of 89Zr-radiolabelled antibody conjugates. We show that, despite previously suggested high regioselectivity of TGases, in the anti-Her2 Ab Herceptin™ more precise native MS indicates only 70-80% functionalization at the target site (Q298H), in competition with modification at other sites, such as Q3H critically close to the CDR1 region.

An essential role for dNTP homeostasis following CDK-induced replication stress.

Replication stress is a common feature of cancer cells, and thus a potentially important therapeutic target. Here, we show that cyclin-dependent kinase (CDK)-induced replication stress, resulting from Wee1 inactivation, is synthetic lethal with mutations disrupting dNTP homeostasis in fission yeast. Wee1 inactivation leads to increased dNTP demand and replication stress through CDK-induced firing of dormant replication origins. Subsequent dNTP depletion leads to inefficient DNA replication, DNA damage and to genome instability. Cells respond to this replication stress by increasing dNTP supply through histone methyltransferase Set2-dependent MBF-induced expression of Cdc22, the catalytic subunit of ribonucleotide reductase (RNR). Disrupting dNTP synthesis following Wee1 inactivation, through abrogating Set2-dependent H3K36 tri-methylation or DNA integrity checkpoint inactivation results in critically low dNTP levels, replication collapse and cell death, which can be rescued by increasing dNTP levels. These findings support a 'dNTP supply and demand' model in which maintaining dNTP homeostasis is essential to prevent replication catastrophe in response to CDK-induced replication stress.

Focused Ultrasound Hyperthermia for Targeted Drug Release from Thermosensitive Liposomes: Results from a Phase I Trial.

Purpose To demonstrate the feasibility and safety of using focused ultrasound planning models to determine the treatment parameters needed to deliver volumetric mild hyperthermia for targeted drug delivery without real-time thermometry. Materials and Methods This study was part of the Targeted Doxorubicin, or TARDOX, phase I prospective trial of focused ultrasound-mediated, hyperthermia-triggered drug delivery to solid liver tumors ( ClinicalTrials.gov identifier NCT02181075). Ten participants (age range, 49-68 years; average age, 60 years; four women) were treated from March 2015 to March 2017 by using a clinically approved focused ultrasound system to release doxorubicin from lyso-thermosensitive liposomes. Ultrasonic heating of target tumors (treated volume: 11-73 cm3 [mean ± standard deviation, 50 cm3 ± 26]) was monitored in six participants by using a minimally invasive temperature sensor; four participants were treated without real-time thermometry. For all participants, CT images were used with a patient-specific hyperthermia model to define focused ultrasound treatment plans. Feasibility was assessed by comparing model-prescribed focused ultrasound powers to those implemented for treatment. Safety was assessed by evaluating MR images and biopsy specimens for evidence of thermal ablation and monitoring adverse events. Results The mean difference between predicted and implemented treatment powers was -0.1 W ± 17.7 (n = 10). No evidence of focused ultrasound-related adverse effects, including thermal ablation, was found. Conclusion In this 10-participant study, the authors confirmed the feasibility of using focused ultrasound-mediated hyperthermia planning models to define treatment parameters that safely enabled targeted, noninvasive drug delivery to liver tumors while monitored with B-mode guidance and without real-time thermometry. Published under a CC BY 4.0 license. Online supplemental material is available for this article. See also the editorial by Dickey and Levi-Polyachenko in this issue.

A phase 1 study to assess the safety, tolerability, and pharmacokinetics of CXD101 in patients with advanced cancer.

BACKGROUND: In the current study, the authors sought to determine the maximum tolerated dose (MTD) of the novel class 1 selective histone deacetylase inhibitor CXD101 in a dose escalation study in patients with advanced solid tumors or recurrent/refractory lymphoma. METHODS: The authors escalated the dose of CXD101 from 1 mg twice daily orally for 5 days in a 21-day cycle (3+3 design). RESULTS: A total of 39 patients were enrolled, 36 of whom received CXD101. Of the 30 patients in the escalation cohort, 29 were evaluable for determination of the dose-limiting toxicity (DLT). DLTs were noted at doses of 16 mg twice daily (1 of 6 patients), 20 mg twice daily (1 of 6 patients), and 24/25 mg twice daily (2 of 5 patients, both of whom developed neutropenic fever). The MTD was 20 mg twice daily, which achieved maximal plasma concentrations (±standard deviation) of 231±76 nM to 342±126 nM, which was within the biologically active range. Six patients received 20 mg twice daily in an expansion cohort. The most frequent adverse events were fatigue, nausea, and reversible cytopenia. Key grade 3 to 4 adverse events (according to Common Terminology Criteria for Adverse Events criteria [version 4.03]) included thrombocytopenia (11%), neutropenia (17%), and neutropenic fever (2%) across the 133 CXD101 cycles given. The toxicity profile was similar to that of licensing studies with other histone deacetylase inhibitors. In 22 evaluable patients receiving a dose of ≥16 mg twice daily (17 of whom had lymphoma and 5 of whom had solid tumors), 3 partial responses (2 in patients with classic Hodgkin lymphoma after allogenic stem cell transplantation and 1 in a patient with angioimmunoblastic T-cell lymphoma) and 1 complete response (in a patient with follicular lymphoma) were noted (overall response rate of 18%) in addition to 9 patients who achieved durable stable disease. Responses were noted predominantly among patients with lymphoma (tumor reduction noted in 63% of patients on standard computed tomography). CONCLUSIONS: The MTD in the current study was found to be 20 mg twice daily. Encouraging and durable activity was observed in patients with Hodgkin lymphoma, T-cell lymphoma, and follicular lymphoma.

Safety and feasibility of ultrasound-triggered targeted drug delivery of doxorubicin from thermosensitive liposomes in liver tumours (TARDOX): a single-centre, open-label, phase 1 trial.

BACKGROUND: Previous preclinical research has shown that extracorporeal devices can be used to enhance the delivery and distribution of systemically administered anticancer drugs, resulting in increased intratumoural concentrations. We aimed to assess the safety and feasibility of targeted release and enhanced delivery of doxorubicin to solid tumours from thermosensitive liposomes triggered by mild hyperthermia, induced non-invasively by focused ultrasound. METHODS: We did an open-label, single-centre, phase 1 trial in a single UK hospital. Adult patients (aged ≥18 years) with unresectable and non-ablatable primary or secondary liver tumours of any histological subtype were considered for the study. Patients received a single intravenous infusion (50 mg/m2) of lyso-thermosensitive liposomal doxorubicin (LTLD), followed by extracorporeal focused ultrasound exposure of a single target liver tumour. The trial had two parts: in part I, patients had a real-time thermometry device implanted intratumourally, whereas patients in part II proceeded without thermometry and we used a patient-specific model to predict optimal exposure parameters. We assessed tumour biopsies obtained before and after focused ultrasound exposure for doxorubicin concentration and distribution. The primary endpoint was at least a doubling of total intratumoural doxorubicin concentration in at least half of the patients treated, on an intention-to-treat basis. This study is registered with ClinicalTrials.gov, number NCT02181075, and is now closed to recruitment. FINDINGS: Between March 13, 2015, and March 27, 2017, ten patients were enrolled in the study (six patients in part I and four in part II), and received a dose of LTLD followed by focused ultrasound exposure. The treatment resulted in an average increase of 3·7 times in intratumoural biopsy doxorubicin concentrations, from an estimate of 2·34 µg/g (SD 0·93) immediately after drug infusion to 8·56 µg/g (5·69) after focused ultrasound. Increases of two to ten times were observed in seven (70%) of ten patients, satisfying the primary endpoint. Serious adverse events registered were expected grade 4 transient neutropenia in five patients and prolonged hospital stay due to unexpected grade 1 confusion in one patient. Grade 3-4 adverse events recorded were neutropenia (grade 3 in one patient and grade 4 in five patients), and grade 3 anaemia in one patient. No treatment-related deaths occurred. INTERPRETATION: The combined treatment of LTLD and non-invasive focused ultrasound hyperthermia in this study seemed to be clinically feasible, safe, and able to enhance intratumoural drug delivery, providing targeted chemo-ablative response in human liver tumours that were refractory to standard chemotherapy. FUNDING: Oxford Biomedical Research Centre, National Institute for Health Research.

A first-in-human phase I study to determine the maximum tolerated dose of the oral Src/ABL inhibitor AZD0424.

BACKGROUND: Src is involved in cancer invasion and metastasis. AZD0424, an oral inhibitor of Src and ABL1, has shown evidence of anti-tumour activity in pre-clinical studies. METHODS: A phase Ia, dose escalation study was performed to assess the safety of continuous oral dosing with AZD0424 in advanced solid tumours. Secondary objectives included investigation of AZD0424 pharmacokinetics, effect on Src activity using markers of bone turnover, and anti-tumour activity. RESULTS: 41 patients were treated; 34 received AZD0424 once-daily at doses ranging from 5 mg to 150 mg, and 7 received 40 mg bi-daily 41.5% of patients experienced at least one AZD0424-related adverse event that was Grade 3-5 in severity, with patients treated at doses above 60 mg per day experiencing multiple treatment-related toxicities. The most commonly observed AZD0424-related adverse events were nausea, fatigue, anorexia and alopecia. Cmax and AUC increased linearly with dose and the mean±standard deviation t1/2 was 8.4±2.8 h. Clear evidence of Src target inhibition was seen at doses ⩾20 mg per day. No responses were observed and 7 patients (17.1%) achieved stable disease lasting 6 weeks or more. CONCLUSIONS: AZD0424 displayed no evidence of efficacy as monotherapy despite a clear pharmacodynamic effect. Further evaluation of AZD0424 monotherapy in patients with solid tumours is not recommended.

CYP450 Enzymes Effect Oxygen-Dependent Reduction of Azide-Based Fluorogenic Dyes.

Azide-containing compounds have broad utility in organic synthesis and chemical biology. Their use as powerful tools for the labeling of biological systems in vitro has enabled insights into complex cellular functions. To date, fluorogenic azide-containing compounds have primarily been employed in the context of click chemistry and as sensitive functionalities for hydrogen sulfide detection. Here, we report an alternative use of this functionality: as fluorogenic probes for the detection of depleted oxygen levels (hypoxia). Oxygen is imperative to all life forms, and probes that enable quantification of oxygen tension are of high utility in many areas of biology. Here we demonstrate the ability of an azide-based dye to image hypoxia in a range of human cancer cell lines. We have found that cytochrome P450 enzymes are able to reduce these probes in an oxygen-dependent manner, while hydrogen sulfide does not play an important role in their reduction. These data indicate that the azide group is a new bioreductive functionality that can be employed in prodrugs and dyes. We have uncovered a novel mechanism for the cellular reduction of azides, which has implications for the use of click chemistry in hypoxia.

The anti-malarial atovaquone increases radiosensitivity by alleviating tumour hypoxia.

Tumour hypoxia renders cancer cells resistant to cancer therapy, resulting in markedly worse clinical outcomes. To find clinical candidate compounds that reduce hypoxia in tumours, we conduct a high-throughput screen for oxygen consumption rate (OCR) reduction and identify a number of drugs with this property. For this study we focus on the anti-malarial, atovaquone. Atovaquone rapidly decreases the OCR by more than 80% in a wide range of cancer cell lines at pharmacological concentrations. In addition, atovaquone eradicates hypoxia in FaDu, HCT116 and H1299 spheroids. Similarly, it reduces hypoxia in FaDu and HCT116 xenografts in nude mice, and causes a significant tumour growth delay when combined with radiation. Atovaquone is a ubiquinone analogue, and decreases the OCR by inhibiting mitochondrial complex III. We are now undertaking clinical studies to assess whether atovaquone reduces tumour hypoxia in patients, thereby increasing the efficacy of radiotherapy.

Identification of vitamin B1 metabolism as a tumor-specific radiosensitizing pathway using a high-throughput colony formation screen.

Colony formation is the gold standard assay for determining reproductive cell death after radiation treatment, since effects on proliferation often do not reflect survival. We have developed a high-throughput radiosensitivity screening method based on clonogenicity and screened a siRNA library against kinases. Thiamine pyrophosphokinase-1 (TPK1), a key component of Vitamin B1/thiamine metabolism, was identified as a target for radiosensitization. TPK1 knockdown caused significant radiosensitization in cancer but not normal tissue cell lines. Other means of blocking this pathway, knockdown of thiamine transporter-1 (THTR1) or treatment with the thiamine analogue pyrithiamine hydrobromide (PyrH) caused significant tumor specific radiosensitization. There was persistent DNA damage in cells irradiated after TPK1 and THTR1 knockdown or PyrH treatment. Thus this screen allowed the identification of thiamine metabolism as a novel radiosensitization target that affects DNA repair. Short-term modulation of thiamine metabolism could be a clinically exploitable strategy to achieve tumor specific radiosensitization.

DNA damage induced by nitric oxide during ionizing radiation is enhanced at replication.

Nitric oxide (NO) is a very effective radiosensitizer of hypoxic mammalian cells, at least as efficient as oxygen in enhancing cell death in vitro. NO may induce cell death through the formation of base lesions which are difficult to repair, and if they occur within complex clustered damage common to ionizing radiation, they may lead to replication-induced DNA strand breaks. It has previously been shown that 8-azaguanine and xanthine result from the reaction of guanine radicals with nitric oxide. We have now shown that adenine radicals also react with NO to form hypoxanthine and 8-azaadenine. Cells irradiated in exponential growth in the presence of NO are twice as radiosensitive compared to those irradiated in anoxia alone, whereas confluent cells are less radiosensitive to (•)NO. In addition, the numbers of DNA double strand breaks observed as γH2AX staining following radiosensitization by NO, are higher in exponential cells than in confluent cells. DNA damage, detected as 53BP1 foci, is also higher in HF-19 cells expressing Cyclin A, a marker for cells in S and G2 phases of the cell cycle, following radiosensitization by NO. RAD51 foci are highest in V79-4 cells irradiated in the presence of NO compared to in anoxia, 24h after radiolysis. This work presents evidence that radiosensitization of cells by NO is in part through the formation of specific DNA damage, difficult to repair, which in dividing cells may induce the formation of stalled replication forks and as a consequence replication-induced DNA strand breaks which may lead to cell death.

Modification of DNA damage mechanisms by nitric oxide during ionizing radiation.

Nitric oxide ((•)NO) is a very effective radiosensitizer of hypoxic mammalian cells. In vivo (•)NO may have effects on tumor vasculature and hence on tumor oxygenation and it may also interact with radiation-produced radicals to modify DNA lesions. Few studies have addressed this last aspect, and we report here specific base modifications that result from reaction of (•)NO with radicals in DNA bases and in plasmid DNA after irradiation. 2'-Deoxyxanthosine monophosphate and 2'-deoxy-8-azaguanosine monophosphate (8azadGMP) are formed upon γ-irradiation of 2'-deoxyguanosine monophosphate (dGMP) in the presence of micromolar levels of (•)NO in anoxia. In addition, the presence of (•)NO at physiological pH inhibits the formation of the well-described (•)OH-induced oxidation product of dGMP, 8-oxo-2'-deoxyguanosine monophosphate. Single-strand breaks are induced in plasmid DNA when γ-irradiated in anoxia, whereas in the presence of (•)NO the number of breaks is reduced by approximately threefold, and evidence is shown for the formation of 8azadGMP in these plasmids. The consequence of the base modifications by (•)NO are as yet unknown although additional breaks are revealed in irradiated plasmid DNA after treatment with glycosylases involved in base excision repair. V79-4 cells irradiated in anoxia show an enhancement in the number of γH2AX foci when (•)NO is present, particularly evident a few hours postirradiation, indicative of the formation of replication-induced DNA damage. We propose that the consequence of (•)NO-induced base modifications in anoxia contributes to its radiosensitization of cells.

Quantitative determination of the anticancer prodrug combretastatin A1 phosphate (OXi4503, CA1P), the active CA1 and its glucuronide metabolites in human urine and of CA1 in plasma by HPLC with mass spectrometric detection.

Validated methods for the determination of CA1, the active agent derived from the prodrug CA1P, in human plasma and urine, and of CA1P and three glucuronides CA1G1, CA1G2 and CA1DG in human urine were developed using LC-MS. Plasma CA1 was extracted using solid phase extraction and validated over the range 5-1000 nM. Urine samples were analysed without extraction, and the assays validated over the range 50-2000 nM (CA1P), 25-2000 nM (CA1), 50-40,000 nM (CA1G1 and CA1G2) and 25-4000 nM (CA1DG). The mean correlation coefficient (r²) was ≥ 0.997 for all assays. The intra-day and inter-day accuracy and precision were within the generally accepted criteria for bioanalytical methods (<15%). Mean recovery of CA1 from plasma was 101%, and 97% from urine. Mean urine recovery of CA1P was 98%, CA1G1 96%, CA1G2 93% and CA1DG 93%. The method was applied to plasma and urine samples from a recently completed clinical trial of the prodrug. Peak plasma concentrations of up to 470 nM CA1 were seen. The majority of drug-related material measured in urine comprised of the two monoglucuronides; CA1 and the diglucuronide were about 10-fold lower. No CA1P was detectable in urine.

Kinetics of oxidation of tyrosine by a model alkoxyl radical.

Oxidation of tyrosine moieties by radicals involved in lipid peroxidation is of current interest; while a rate constant has been reported for reaction of lipid peroxyl radicals with a tyrosine model, little is known about the reaction between tyrosine and alkoxyl radicals (also intermediates in the lipid peroxidation chain reaction). In this study, the reaction between a model alkoxyl radical, the tert-butoxyl radical and tyrosine was followed using steady-state and pulse radiolysis. Acetone, a product of the ß-fragmentation of the tert-butoxyl radical, was measured; the yield was reduced by the presence of tyrosine in a concentration- and pH-dependent manner. From these data, a rate constant for the reaction between tert-butoxyl and tyrosine was estimated as 6 ± 1 × 10(7) M(-1) s(-1) at pH 10. Tyrosine phenoxyl radicals were also monitored directly by kinetic spectrophotometry following generation of tert-butoxyl radicals by pulse radiolysis of solutions containing tyrosine. From the yield of tyrosyl radicals (measured before they decayed) as a function of tyrosine concentration, a rate constant for the reaction between tert-butoxyl and tyrosine was estimated as 7 ± 3 × 10(7) M(-1) s(-1) at pH 10 (the reaction was not observable at pH 7). We conclude that reaction involves oxidation of tyrosine phenolate rather than undissociated phenol; since the pK(a) of phenolic hydroxyl dissociation in tyrosine is ≈ 10.3, this infers a much lower rate constant, about 3 × 10(5) M(-1) s(-1), for the reaction between this alkoxyl radical and tyrosine at pH 7.4.

A validated HPLC method with fluorescence detection for the glucuronides of Combretastatin A1 in human plasma, and studies on their cis-trans isomerisation.

Two monoglucuronides (CA1G1 and CA1G2) of the catecholic cis-stilbene Combretastatin A1 (CA1, OXi4500), have been identified in a clinical trial of the bisphosphate prodrug of OXi4500, OXi4503. A validated assay for the two glucuronides in human plasma using HPLC with fluorescence detection after post-column photolysis is described. The assay was linear over the range 25 nM (CA1G1) or 50 nM (CA1G2) - 5000 nM, R(2)≥ 0.996. The intra-day precision for CA1G1 was better than 8.7% RSD (19.4% at the LLOQ), and the inter-day precision was better than 5.5% RSD (7.6% at the LLOQ). The intra- and inter-day accuracies were better than ± 12.6% relative error (14.8% at the LLOQ) and 4.8% (5.4% at the LLOQ) respectively. For CA1G2, the intra-day precision was better than 5.7% RSD (7.5% at the LLOQ), and the inter-day precision was better than 4.8% RSD (11.9% at the LLOQ). The intra- and inter-day accuracies were better than ± 10.1% relative error (12.6% at the LLOQ) and 2.2% (3.8% at the LLOQ) respectively. Recovery from plasma was measured at three concentrations (125, 625 and 2500nM). Mean recovery of CA1G1 was 94.5% and ranged from 94.4 to 99.2%. Mean recovery of CA1G2 was 90.7%, range 88-92%. During the validation process, one of the isomers was unexpectedly found to be unstable. CA1G1, substituted ortho to the stilbene, was relatively stable, but the meta-substituted CA1G2 readily converted from the cis-stilbene conformation to the trans isomer. This was catalysed by acid and heavy metals, and could be inhibited by antioxidants such as ascorbic acid. Isomerisation could also be induced by one-electron oxidation processes such as horseradish peroxidase and azide radicals.