Search

This publication-based thesis aims to add to the existing knowledge surrounding three consecutive aspects of hyperthermia: (1) the threats to human health from hyperthermia, (2) the putative mechanisms underlying the systemic damage, and (3) potential treatments. The six primary research papers forming the basis of the thesis have been published in peer-reviewed journals, using a variety of methodologies, including field studies, lab-based studies and systematic reviews. Prolonged exertion, especially if causing hyperthermia, and passive heat illness, for example in heat waves or due to adverse effects of certain medications, are common causes of multi-organ failure and death. Similarities between the various hyperthermic states suggest that the raised temperature rather than the underlying cause in itself is predominant. Two of these effects, acute kidney injury (AKI) and electrolyte disturbancesin exertional heat illness(EHI), are examined further in papers one and two. The deleterious effects of hyperthermia may include the heat rendering the intestinal tract more permeable, allowing bacteria and toxins from the intestinal tract to enter the systemic circulation. Endurance exercise also appears to stimulate a systemic inflammatory response and increase intestinal permeability. Hyperthermia, especially when associated with exertion, is further characterised in papers three and four, which found that the intestinal permeability was higher after exercise-induced hyperthermia than passive hyperthermia or exercise, respectively, alone, suggesting synergistic effects. There may therefore be the possibility of reducing the systemic damage caused by the exertional and non-exertional hyperthermia by reducing the inflammatory response or the bacterial load in the circulation. Papers five and six are systematic reviews suggesting that in animals both drugs appear to be beneficial in reducing the harm and risk of death from hyperthermia. The work presented here allows the possibility for development of future work on reducing the inflammatory response and intestinal toxin absorption, for example with steroids or antibiotics, to reduce the risk of death and disability after heatstroke and hyperthermia.

Microneedles (MN) arrays are micron-size needle arrays with length generally ranging between 50 μm and 2 mm. When applied to the skin surface, they pierce the stratum corneum creating micropores that can enhance the delivery of drugs into the viable skin layers. MN arrays offer great advantages for localised treatment of skin disorders which, in combination with skin hyperthermia can help inhibiting bacterial growth and induce apoptosis in cancer cells. In this thesis, new approaches to induce superficial and deep skin hyperthermia using gold nanorods (GnRs) were investigated. In addition, single needles, hollow and dissolving MN arrays for skin drug delivery were developed, aiming to enhance deposition of drugs in the dermis. GnRs were synthesized and loaded in hydrogels made from aqueous blends containing poly(methyl vinyl ether-co-maleic acid) and poly(ethylene glycol). These polymeric blends were used to fabricate films to heat up a thin skin model upon Near Infrared (NIR) laser irradiation. This technology circumvents skin deposition and injection of GnRs and, thereby, the uncertainty regarding their metabolism, safety and toxicity once they reach the systemic circulation. This approach was further improved in its use for skin hyperthermia, using GnRs coated on MN arrays where their efficacy heating up a thick skin model was confirmed. For intradermal drug delivery, hydrogel- forming single needles were fabricated, which were then coated with a model drug to investigate how deep these devices deliver the drug in a skin model. As the drug was coated and dried on the surface of needles, issues associated with hypodermic needles, such as stability of liquid formulations and generation of hazardous waste are avoided. When compared to a control formulation, single needles increased penetration depth of the drug significantly in the skin model, confirming the potential of coated polymeric needles for deep intradermal delivery. Finally, hollow polymeric MN arrays and dissolving MN arrays were fabricated to enhance the intradermal delivery of tofacitinib citrate, a drug for which clinical trials provide strong evidence for its effectiveness against inflammatory skin conditions. However, oral administration is not ideal due to reported side effects. Skin deposition studies showed that hollow MN arrays led to slightly higher deposition of drug in the dermis in comparison to a control cream. However, dissolving MN arrays showed superiority on this regard, providing proof of principle for intradermal delivery of tofacitinib citrate using MN arrays. Overall, the results shown in this thesis confirm the benefits of using MN arrays for skin hyperthermia and intradermal delivery and their potential future applications in the management of skin diseases.

Local mild hyperthermia stimulates the activity of immune cells including prompting leukocyte migration and trafficking, but the molecular mechanisms involved in this process are poorly understood. The Wiskott Aldrich Syndrome Protein (WASP) and the WASP interacting protein (WIP) work as a functional unit that plays a major role in assembly and disassembly of myeloid cell adhesions termed podosomes by binding both F-actin and other actin-related proteins. Besides, WIP can also regulate actin dynamics by modulating the lipid composition of the plasma membrane, as recently shown in neurons. In various organisms, the lipid composition and fluidity of the cell membrane work as a thermosensor that regulates the cellular response to changes in temperature. This study aimed to investigate whether WIP or WASP may regulate the migratory response to mild hyperthermia as in local inflammation or during a febrile event. Results showed that mild hyperthermia (40°C) promoted random cell migration correlating with the assembly of more robust and dynamic podosomes with increased matrix degradation capability in a WASP and WIP-dependent manner. We observed that this response is dependent on the actin polymerising activity and the nuclear translocation of WASP. Our data also indicate that increased membrane fluidity, like the one induced by hyperthermia, can trigger a migratory response in myeloid cells. Following hyperthermia, WIP delivers changes in monocytic cells membrane lipid composition to maintain fluidity homeostasis in the plasma membrane, particularly in podosomal areas at adhesion sites, triggering the increased invasive migratory capacity. The modulation of the lipid composition of the plasma membrane is mediated by the WIPdependent regulation of genes involved in lipid biosynthesis and rafts formation in response to hyperthermia. These genes include NSMAF, OSBPL5 and regulators of cholesterol biosynthesis. In response to hyperthermia, WIP also controls the upregulation of Heat Shock protein 90 (HSP90), a protein previously associated with podosome formation. This process is independent of WASP. Taken together, our data show a role of both WIP and WASP in the increased migratory phenotype of myeloid cells in response to mild hyperthermia.

Physical activity of moderate intensity and duration leads to healthy biological adaptations in humans. However, very intense and prolonged exercise may induce disruption in redox balance, potentially increasing oxidative stress. In addition, exposure to environmental heat stress and associated hyperthermia further increases oxidative stress and may induce the expression of heat shock proteins. However, antioxidant supplementation is believed to minimise the effect of oxidative stress and may therefore help reduce or limit the heat shock response to exercise heat stress. The first study (Chapter 4) examined whether exertional heat illness (EHI) casualties among military recruits may exhibit greater disturbances in redox balance following exercise compared to non-EHI controls. Nine (n=9) recruits were identified as having suspected EHI during the Loaded March (LM) on day 1, with a peak mean (SD) body core temperature of 40.1 (0.5) °C. Fifteen (n=15) recruits were identified as having suspected EHI during the Log Race (LR) on day 2, with a peak mean (SD) body core temperature of 39.7 (0.5) °C. A further twenty-one (n=21) recruits, which successfully finished both LM and LR events, were treated as controls (CON). Interestingly, the plasma antioxidant concentration was significantly elevated from pre to post-exercise (p < 0.001) for EHI and CON groups, during both LM and LR events, with no changes on lipid peroxide protein carbonyl concentrations. These data suggest there is no increase in lipid peroxide or protein carbonyl level damage in response to intense hyperthermic military exercise, regardless of acute heat illness. It is possible that military training augments the body's defence capabilities, thus reducing oxidative stress and damage induced by free radical production. To date there is a scarcity of data examining the effects of acute intake of antioxidant supplements on oxidative stress and heat shock response during continuous exercise in a hot environment. Hence, the aims of the second study (Chapter 5) were to examine the effects of acute ingestion of Quercetin (Q), Quercetin + vitamin C (QC) or placebo (P) 14 hours before, 2 hours before and every 20 minutes during trials on oxidative stress and heat shock response. In this randomised, crossover study 10 recreationally active males (age 21±2 y, V̇ O2max 54.9±8.4 ml.kg.min-1) completed three running trials at 70% V̇ O2max for 60 minutes in the heat (33.0±0.3°C; 28.5±1.8% relative humidity). Exercise heat stress significantly elevated plasma quercetin (p=0.02), antioxidant power (FRAP) (p < 0.001),plasma heat shock protein 70 (HSP70) (p=0.009) and plasma heat shock protein 90α(HSP90α) (p < 0.001) over time, but no differences were detected between trials. Also, no changes were observed in protein carbonyl concentration. Acute intake of quercetin significantly increased the level of plasma quercetin however, this did not affect the plasma antioxidant capacity or heat shock response to exercise heat stress. The increases in plasma HSP70 and HSP90α concentrations might act as supplementary antioxidants, reducing the oxidative damage reflected in the absence of changes in protein carbonyl. Exercise heat stress is effective in inducing both intracellular HSP70 (muscle and peripheral blood mononuclear cell (PBMC)) and extracellular HSP70 (plasma) concentrations. Thus, the third study (Chapter 6) tested the hypothesis that this acute quercetin supplementation would induce similar trends in plasma HSP70 and intracellular HSP70 concentrations 2 days following exercise heat stress. In this randomised, crossover study, 9 recreationally active males (age 22±2y, V̇ O2max 50.3±3.3ml.kg.min-1) completed three running trials at 70% V̇ O2max for 60 minutes in the heat (32.9±0.3°C; 28.3±1.2% relative humidity). This study demonstrated that there is no positive relationship between both intracellular of HSP70 (muscle and PBMC) and plasma HSP70 (eHSP70) 2 days following exercise heat stress. These data suggest that the release of eHSP70 could originate from others tissue or cells. Additionally, the absence of differences between trials in the expression of muscle HSP70, PBMC HSP70 and plasma HSP70 might indicate it is implausible that quercetin might inhibit the expression of HSP70 in plasma, muscle and PBMC 2 days following the exercise heat stress stimulus. Overall, the results from this thesis emphasise that the hyperthermia experienced in response to exercise and environmental heat stress could potentially influence the human redox response and heat shock response. Besides, there is reasonable evidence that acute quercetin co-ingestion with vitamin C has the potential to improve the bioavailability and bioactive effects of quercetin, however, the effects of quercetin supplementation in reducing oxidative stress in response to exercise heat stress remains to be elucidated. In addition, the anti-oxidative ability of acute ingestion of quercetin to suppress the intracellular and extracellular heat shock response remains uncertain and worthy for further investigation.

Core temperature (Tc) regulation is fundamental to mammalian survival, since hypothermia (Tc ≤ 35°C) and hyperthermia (Tc ≥ 40°C) are major risk factors for health and wellbeing. The purpose of this thesis was to determine if acetaminophen, an analgesic and antipyretic drug, increased the onset of hypothermia or hyperthermia during passive cold and heat stress, respectively. It was later investigated if acetaminophen induced inhibition of cyclooxygenase mediated these side-effects. In Study 1a, the plasma acetaminophen response to a dose of 20 mg·kg-1 of lean body mass was determined through enzyme linked immunosorbent assay. In Study 1b, the effect of acetaminophen administration on internal temperature (rectal; Tre) during a passive 2-hour mild cold (20°C, 40% relative humidity) exposure was examined. Study 1a showed that the plasma response was homogenous between subjects, reaching peak concentrations between 80-100 minutes (14 ± 4 μg·ml-1). In Study 1b, acetaminophen reduced Tre in all participants compared with baseline, and the average peak reduction was 0.19 ± 0.09°C. In contrast, Tre remained stable when participants ingested a sugar placebo. Study 1 is the first experiment which confirms a hypothermic side-effect of acetaminophen in healthy humans. Study 2 investigated whether acetaminophen augmented the rate of Tre rise during exposure to passive dry (45°C, 30% r.h.) and humid (45°C, 70% r.h.) heat stress for 2-hours and 45-minutes, respectively. This study showed that the rate of Tre rise in the dry (0.005 vs 0.006°C∙min-1) and humid (0.023 vs 0.021 °C∙min-1) conditions were similar between the acetaminophen and placebo trials (p > 0.05). Study 2 is the first experiment which confirms acetaminophen has no meaningful effect on thermoregulation during passive dry or humid heat exposure. Study 3 determined how the hypothermic effect of acetaminophen changes during exposure to a thermoneutral (25°C, 40% r.h.) and cold (10°C, 40% r.h.) environment for 2-hours. In summary, there was no hypothermic effect of acetaminophen in a thermoneutral environment (p > 0.05), whereas Tre fell by 0.40 ± 0.15°C compared with baseline during cold stress (p < 0.05). Compared with the placebo, Tre was ~0.35°C lower at 120 minutes, but was significantly lower from 70-minutes. Study 3 confirmed that there is a relationship between the level of cold stress and magnitude of the hypothermic effect of acetaminophen. Study 4 determined whether ibuprofen (400 mg), a cyclooxygenase inhibitor, reduced Tre during 2-hour passive cold stress (10°C, 40% r.h.) to a level comparable with acetaminophen. Ibuprofen administration did not influence Tre, vastus medialis shivering, or energy expenditure compared with a placebo throughout the cold exposure (p > 0.05). Taken together, this renders it unlikely that cyclooxygenase activity is required for thermogenesis induced by skin cooling. Study 4 provides evidence that acetaminophen induced hypothermia is not exclusively mediated by cyclooxygenase inhibition. In Summary, this series of experiments has shown that acetaminophen has a hypothermic side effect in healthy humans, which is amplified during acute cold stress. Ibuprofen had no such effect on thermoregulation during cold exposure, so it is unlikely that cyclooxygenase inhibition mediates the hypothermic side-effect of acetaminophen.

The TARDOX study is a Phase I first-in-man proof-of-concept study which aims to demonstrate the safety and feasibility of targeted delivery of a thermo-sensitive liposome in combination with mild hyperthermia induced using an extracorporeal Focused Ultrasound (FUS) device. ThermoDox® is a specially formulated long-circulating and thermosensitive liposome, activated by conditions of mild hyperthermia, to release its cytotoxic doxorubicin content. FUS offers non-invasive and non-ionising approach to tumour therapy, classically by the mechanism of thermal ablation (High Intensity Focused Ultrasound, HIFU) rather than hyperthermia. Nine patients have received intervention; a single treatment cycle of ThermoDox® combined with FUS to a single target primary or secondary liver tumour using an ultrasound-guided extracorporeal FUS device (Model JC200 Focused Ultrasound Tumor Therapeutic System, Haifu Medical). Five cases proceeded with real-time thermometry and four without. In those with thermometry, optimal hyperthermia (T > 39.5°C for over 300 cumulative and 60 continuous seconds, and CEM43 < 60 minutes) was achieved in 4/5. Core tumour biopsies were used for drug quantification studies using high performance liquid chromatography. In an interim analysis, of the evaluable tissue samples analysed to date, an average of 8.41 μg/g ± 4.37 SD of intratumoral doxorubicin was demonstrated in those receiving optimal hyperthermia under real-time thermometry (n=2) and 6.75 μg/g ± 0.13 SD in those whose intervention proceed without thermometry (n=2). Overall, an aver- age of 7.58 μg/g ± 2.70 SD of doxorubicin was seen in the included post-delivery samples analysed to date (n=4). This compares to an average of 2.48 μg/g ± 1.26 SD in evaluable samples taken immediately prior to FUS-mediated delivery (n=4). Microscopic analysis of post-delivery biopsies demonstrated intercalation of doxorubicin in the nuclei of tumour cells in 6/6 patients with adequate tissue samples, evidencing that release of bioavailable drug from the administered liposomes has been achieved. Radiological follow-up at two weeks with 18F-FDG PET-CT demonstrated unequivocal localised response in 3/4 evaluable patients receiving optimal hyperthermia and 2/4 without real-time thermometry (subjective response in 1 of the remaining 2), despite only receiving a single standard dose treatment cycle of doxorubicin. Serious adverse events were limited to transient neutropenia in 4/9 patients, in line with the adverse event profile for doxorubicin, and prolonged hospital stay due to confusion in 1/9. We have demonstrated that combined treatment with ThermoDox® and extracorporeal targeted FUS hyperthermia is feasible, safe and can enhance intratumoral drug delivery, providing localised response in human liver tumours refractory to standard chemotherapy. This study, which builds on decades of research efforts in targeted liposome delivery using FUS, is one of the earliest studies to translate ultrasound-mediated tumour targeting into the clinic. The use of chemotherapy in combination with FUS devices, as a non-invasive targeting method, has the potential to transform future clinical treatments for solid organ cancers.

Microwave thermography, tomography and hyperthermia are potentially useful clinical techniques that are currently at the development stage. They may ultimately assist in the early detection and treatment of breast cancer and other conditions causing a local temperature or tissue permittivity anomaly in the body. Accurate complex permittivity values for human tissues are needed for computer modelling to fulfil the potential of these techniques. A novel open-ended co-axial probe measurement system operating at room temperature was used to investigate the complex permittivity of many animal and human tissues at a frequency of 3 GHz. The probe was calibrated using a bilinear transform method based on an equivalent circuit model of the probe aperture. A quarter-wave choke at the probe aperture provided an area of high impedance around the open-end. This minimised the effects of surface currents, and thus greatly improved measurement repeatability and general probe performance in comparison with ordinary co-axial probes. Mixture equations were used to analyse the measured permittivity values in terms of the permittivities of the other major component materials of biological tissue. By considering all the sources contributing to the microwave permittivity, the amount of bound water in tissues was estimated, and an approximate value for its average relaxation frequency evaluated using an extrapolation technique. Further measurements on partially dehydrated tissue samples extended the range of tissue water content investigated, and permitted better extrapolation of the bound water relaxation frequency. These values were in close agreement with the estimates of previous researchers. To verify the validity of this method, aqueous gelatine solutions were used as simple tissue phantoms. Since the solutions contained comparatively few distinct components, it was possible to use mixture equations again to extrapolate the permittivity of water bound to the gelatine polymer molecules. The method gave highly consistent estimates for the bound water relaxation frequency using either or both of the real and imaginary parts of the measured solution permittivity.

The aim of this area of research is to provide techniques which would allow long-term low temperature storage and subsequent transplantation of human organs, such as the kidney. A major difficulty in organ cryopreservation is that of obtaining rapid warming of the frozen tissue, whilst maintaining a uniform temperature profile . Before cooling, the organ is perfused with a 'cryoprotectant', a solution which reduces freezing injury. Warming rates that can be achieved by thermal conduction cannot exceed about 1K/min without damaging the exterior of the organ. Experimental evidence from a range of cell and organ systems suggest that warming should be much more rapid. One proposed warming technique employs dielectric heating using electromagnetic energy at microwave frequencies. Crucial to the design of any such rapid warming system are the electrical properties of the material to be heated. This dissertation describes two measurement systems that have been developed for measuring the electrical properties of cryoprotectants and of perfused rabbit kidney tissue. Dielectric measurements were made with both time and frequency domain instrumentation, over a frequency range of 50MHz to 2.6GHz, and a temperature range of -30'C to +20'c. The more successful of the two systems uses an open-ended coaxial probe with a new, simplified, error correction and calibration technique. The measurements show that electrical properties of perfused tissue are heavily influenced by those of the cryoprotectant in this frequency range. A new low conductivity cryoprotectant has been developed which has more favourable electrical properties. There is a strong suggestion that using such a cryoprotectant, rapid warming may become a practical proposition around 430MHz.