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

Use of horseradish peroxidase for gene-directed enzyme prodrug therapy with paracetamol

Gene therapy is a potential method of treating cancer with a greater degree of targeting than conventional therapies. In addition, therapy can be directed towards cells within the tumour population that are traditionally resistant to current treatment schedules. Horseradish peroxidase (HRP) can oxidise paracetamol to N-acetyl-p-benzoquinoneimine via a one-electron pathway. Incubation of human cells expressing HRP with 0.5–10 mm paracetamol reduced clonogenic survival, but had little effect on control cells. A small increase in apoptosis was seen and a decrease in the number of cells undergoing mitosis, consistent with reports in hepatocytes using higher paracetamol concentrations. The cytotoxicity was also seen under conditions of severe hypoxia (catalyst induced anoxia), indicating that the HRP/paracetamol combination may be suitable for hypoxia-targeted gene therapy

Oxidative activation of indole-3-acetic acids to cytotoxic species- a potential new role for plant auxins in cancer therapy.

Indole-3-acetic acid (IAA) and some derivatives can be oxidised by horseradish peroxidase (HRP) to cytotoxic species. Upon treatment with IAA/HRP, liposomes undergo lipid peroxidation, strand breaks and adducts are formed in supercoiled plasmid DNA, and mammalian cells in culture lose colony-forming ability. IAA is only toxic after oxidative decarboxylation; no effects are seen when IAA or HRP is incubated independently in these systems at equivalent concentrations. Toxicity is similar in both hamster fibroblasts and some human tumour cells. The effect of IAA/HRP is thought to be due in part to the formation of 3-methylene-2-oxindole, which may conjugate with DNA bases and protein thiols. Our hypothesis is that IAA/HRP could be used as the basis for targeted cancer therapy involving antibody-, polymer-, or gene-directed approaches. HRP can thus be targeted to a tumour allowing non-toxic IAA delivered systemically to be activated only in the tumour. Exposure to newly synthesised analogues of IAA shows a range of four orders of magnitude difference in cellular toxicity but no structure-activity relationships are apparent, in contrast to well-defined redox dependencies of oxidation by HRP intermediates or rates of decarboxylation of radical-cation intermediates

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

Validation of a method for the determination of the anticancer agent Combretastatin A1 phosphate (CA1P, OXi4503) in human plasma by HPLC with post-column photolysis and fluorescence detection

A validated method for the determination of Combretastatin A1 phosphate (CA1P, OXi4503), a bisphosphate prodrug of the vascular disrupting agent Combretastatin A1 in human plasma has been developed using fluorescence detection after post-column photolysis. The separation used the ion-pairing agent tetrabutylammonium hydrogen sulphate, and this agent was also required to give consistently high recovery from plasma. Initially, the range was shown to be linear (r(2)>0.995) from the LOQ of 0.025 μM to 5 μM, but as the trial progressed to much higher doses, using a lower injection volume, the assay was subsequently subject to limited revalidation to cover the range from 0.05 to 50 μM. Intra-assay precision and accuracy ranged from 2.2 to 11.8% and 1.8 to 13% respectively, and for inter-assay from 4.4 to 14.9% and 1.7 to 6.5%. Mean recovery of OXi4503 from plasma was 80.2%

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

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

Kinetics of the reaction between nitric oxide and glutathione: implications for thiol depletion in cells.

Nitric oxide in the absence of oxygen was suggested to react with 5-50 mM glutathione (GSH) over many minutes when [NO*] << [GSH] (N. Hogg et al., FEBS Lett. 382:223-228; 1996). However, Aravindakumar et al. (J. Chem. Soc. Perkin Trans. 2:663-669; 2002) provided data suggesting approximately 200-fold higher reactivity under conditions of [NO*] >> [GSH]. To help resolve these differences, the rate of loss of NO* ( approximately 9 microM) in aqueous solutions of GSH (2.5-20 mM) was measured by chemiluminescence. An apparent second-order rate constant of 0.080 +/- 0.008 M(-1) s(-1) at pH 7.4, 37 degrees C, was calculated based on the total [GSH] and "pseudo-first-order" kinetics; thiolate anion was much more reactive than undissociated thiol. These data imply a half-life of approximately 30 min for low concentrations of NO* with 5 mM GSH, 37 degrees C, pH 7.4, in the absence of oxygen. Possible kinetic schemes that can partially explain the divergent literature reports are discussed, notably an equilibrium in the reaction between NO* and GSH. Human breast carcinoma MCF-7 cells were exposed to NO* (initially approximately 18 microM) in alidded six well plate in an anaerobic chamber in vitro; intracellular GSH levels decreased by half in approximately 60 min. Aerobic exposure depletes GSH in cells in vitro much faster because of autoxidation of NO* to NO2*, >10(8) times more reactive toward GSH

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

Enhancing the efficacy of photodynamic cancer therapy by radicals from plant auxin (indole-3-acetic acid).

Indole-3-acetic acid (plant auxin) has low toxicity but dramatically enhances the killing of mammalian cells on illuminating phenothiazinium dyes with red light. Suitable dyes include toluidine blue, used in cancer diagnosis because of localization in tumors, and methylene blue, used in experimental photodynamic therapy of cancer. The photosensitized oxidation of indole acetic acid forms a free radical that fragments in microseconds, forming reactive cytotoxins. Unlike conventional photodynamic therapy, requiring excitation of oxygen to the reactive singlet state, the treatment is effective even at the low oxygen levels common in tumors and with much lower light doses than normally used