Browsing by Subject "Hyperthermia"

Traditional small molecule chemotherapeutics show limited effectiveness in the clinic as their poor pharmacokinetics lead to rapid clearance from circulation and their exposure to off-target tissues results in dose-limiting toxicity. The objective of this dissertation is to exploit a class of recombinant chimeric polypeptides (CPs) to actively target drugs to tumors as conjugation to macromolecular carriers has demonstrated improved efficacy by increasing plasma retention time, reducing uptake by healthy tissues, and enhancing tumor accumulation by exploiting the leaky vasculature and impaired lymphatic drainage characteristic of solid tumors. CPs consist of two principal components: (1) a thermally responsive elastin-like polypeptide (ELP) that displays a soluble-to-aggregate phase transition above a characteristic transition temperature (Tt); and (2) a cysteine-rich peptide fused to one end of the ELP to which small molecule therapeutics can be covalently attached (the conjugation domain). This work describes the development of CP drug-loaded nanoparticles that can be targeted to solid tumors by the external application of mild regional hyperthermia (39-43°C).

Highly repetitive ELP polymers were assembled by Plasmid Reconstruction Recursive Directional Ligation (PRe-RDL), in which two halves of a parent plasmid, each containing a copy of an oligomer, were ligated together to dimerize the oligomer and reconstitute the functional plasmid. Chimeric polypeptides were constructed by fusing the ELP sequence to a (CGG)8 conjugation domain, expressed in Escherichia coli, and loaded with small molecule hydrophobes through site specific attachment to the conjugation domain. Drug attachment induced the assembly of nanoparticles that retained the thermal responsiveness of the parent ELP in that they experienced a phase transition from soluble nanoparticles to an aggregated phase above their Tt. Importantly, the Tt of these nanoparticles was near-independent of the CP concentration and the structure of the conjugated molecule as long as it displayed an octanol-water distribution coefficient (LogD) > 1.5.

A series of CP nanoparticles with varying ratios of alanine and valine in the guest residue position was used to develop a quantitative model that described the CP transition temperature in terms of three variables - sequence, chain length, and concentration - and the model was used to identify CPs of varying molecular weights that displayed transition temperatures between 39°C and 43°C. A murine dorsal skin fold window chamber model using a human tumor xenograft was used to validate that only the thermoresponsive CP nanoparticles (and not the controls) exhibited a micelle-to-aggregate phase transition between 39-43°C in vivo. Furthermore, quantitative analysis of the biodistribution profile demonstrated that accumulation of these thermoresponsive CP nanoparticles was significantly enhanced by applying heat in a cyclical manner. It is hoped that this work will provide a helpful resource for the use of thermoresponsive CP nanoparticles in a variety of biomedical applications.

Numerous studies have shown that the combination of radiation therapy and hyperthermia, when delivered at moderate temperatures (40°-45°C) for sustained times (30-90 minutes), can help to provide palliative relief and augment tumor response, local control, and survival. However, the dependence of treatment success on achieved temperature highlights the need for accurate thermal dosimetry, so that the prescribed thermal dose can be delivered to the tumor. This can be achieved noninvasively with MR thermometry. However, there are many challenges to performing MR thermometry in the breast, where hyperthermia of locally advanced breast cancer can provide a benefit. These include magnetic field system drift, fatty tissue, and breathing motion.

The purpose of this research was to develop a system for the hyperthermia treatment of LABC while performing MR thermometry. A hardware system was developed for performing the hyperthermia treatment within the MR bore. Methods were developed to correct for magnetic field system drift and to correct for breath hold artifacts in MR thermometry of the tumor using measurement of field changes in fat references. Lastly, techniques were developed for measuring temperature in the fatty tissue using multi-echo fat water separation methods, reducing the error of performing MR thermometry in such tissues. All of these methods were characterized with phantom and in vivo experiments in a 1.5T MR system.

The results of this research can provide the means for successful hyperthermia treatment of LABC with MR thermometry. With this thermometry, accurate thermal doses can be obtained, potentially providing improved outcomes. However, these results are not only applicable in the breast, but can also be used for improved MR thermometry in other areas of the body, such as the extremities or abdomen.

Traditionally, the goal of nanoparticle-based chemotherapy has been to decrease normal tissue toxicity by improving drug specificity to tumor. Relying on the EPR effect (Enhanced Permeability and Retention), a host of nanoparticles (from micelles and dendrimers to liposomes and lipidic nanoparticles) have been developed and tested for passive accumulation into tumor interstitium. Unfortunately, most nanoparticles achieve only suboptimal drug delivery to tumors, due to heterogeneity of tumor vessel permeability, limited nanoparticle penetration, and relatively slow drug release. However, recent developments in nanoparticle technology have occurred with the design and testing of a fast drug-releasing liposome triggered by local heat. This temperature-sensitive liposome formulation loaded with doxorubicin (Dox-TSL) has already shown substantial anti-tumor efficacy and is currently in clinical trials.

Previous pre-clinical work to understand the mechanism of efficacy has illustrated increases in overall drug concentration in the tumor, and an anti-vascular effect not observed with heat alone. These initial studies have also suggested that these liposomes may be the most efficacious when they are injected into a pre-heated tumor, with the hypothesis that in this treatment scheme the liposomes may be releasing inside the tumor vasculature. However, whether intravascular release is indeed occurring, and the subsequent implications this paradigm change in drug delivery could have are still unanswered questions.

The experiments presented herein aimed to investigate two effects: the existence and influence of intravascular drug release on drug delivery and distribution within the tumor, and the effect of drug delivery on subsequent anti-vascular effects. To investigate drug delivery, two mouse models were used. Dorsal window chambers implanted with FaDu human squamous carcinomas were used with real-time intravital confocal microscopy to evaluate time-resolved delivery of doxorubicin and liposome extravasation over the first 20 minutes of treatment. As a complimentary mouse model, flank FaDu tumors were also treated with Dox-TSL or treatment controls (doxorubicin with and without heat and Doxil with heat), and subsequently sectioned and histologicaly imaged to evaluate drug delivery and penetration depth, as well as impact on hypoxia and perfusion parameters. To investigate vascular effects, a GFP-eNos transgenic mouse model was used, also with window chamber confocal microscopy, to evaluate morphological changes occurring in the tumor vasculature following treatment.

The results presented herein demonstrate that contrary to the traditional liposome paradigm of extravasation and subsequent drug release, thermally sensitive liposomes release drug inside the tumor vasculature, and that the released free drug diffuses into the tumor interstitium. Real-time confocal imaging of doxorubicin delivery to murine tumor window chambers illustrates that intravascular drug release provides a mechanism to increase both the time that tumor cells are exposed to maximum drug levels and the penetration distance achievable by free drug diffusion. Histological analysis further confirms this finding, illustrating that drug delivered with Dox-TSL intravascular release can result in drug penetration levels up to 80 µm from vessels, in comparison with 40 µm achievable with free drug with heat. Further, Dox-TSL delivers drug to a higher percentage of a tumor's hypoxic area than possible with free drug with or without heat. Endothelial cells display marked morphological changes apparent immediately following treatment, with significant vascular destruction at 6 hours. However, heat had a similar influence on vascular morphology, underscoring the complexity of the anti-vascular effect, particularly in the more sensitive vasculature of a mouse model compared with reported human vascular heat tolerances. This work establishes intravascular release as a new paradigm in drug delivery to solid tumors, resulting in improved drug bioavailability, penetration depth, and enhanced delivery of drug to hypoxic regions of tumors.

Mild hyperthermia (39°C-43°C) has numerous therapeutic benefits as an adjuvant therapy in the treatment of a variety of tumor types. Hyperthermia increases tumor blood flow and vascular permeability, promoting drug delivery and tumor oxygenation. Hyperthermia enhances the uptake and efficacy of numerous chemotherapeutic agents, including cisplatin, resulting in increased cytotoxicity. In addition to these biological responses, hyperthermia can be used as a drug-release trigger for temperature-sensitive nanoparticles, resulting in an improved and more targeted drug delivery system. Cisplatin was chosen because 1) it shows broad spectrum activity against a wide range of heatable cancers (i.e., those in sites such as the pancreas, colon and rectum, cervix and bladder, and 2) the same hyperthermic temperatures that enable temperature-sensitive lipsome-drug release also enhance cisplatin-induced cytotoxicity.

The role of hyperthermia in enhancing cisplatin delivery and cytotoxicity was investigated at both the cellular and tissue levels. While hyperthermia treatment is applicable to a variety of tumor types, the focus of this work was on bladder cancer. The synergistic effects of hyperthermia and cisplatin were investigated, along with the role of copper transport protein 1 (Ctr1) in this process. In addition, cisplatin was encapsulated within temperature-sensitive liposomes, which were used in combination with hyperthermia for targeted drug delivery. These studies demonstrated that the combination of cisplatin and hyperthermia improved drug delivery, and potentially anti-tumor efficacy, and that targeted delivery was enhanced through incorporation of temperature-sensitive liposomes. As many current methods for administering bladder hyperthermia have drawbacks, such as invasiveness and regional heating, the final aim of this study was to develop and test a less-invasive and more focused preclinical bladder heating device in a rat model.

Hyperthermia sensitizes cells to the cytotoxic effects of the commonly used chemotherapeutic agent cisplatin by increasing drug accumulation and subsequent platinum-DNA adduct formation. However, the molecular mechanisms underlying this enhancement remain unclear. Understanding the fundamental mechanisms involved in the synergistic interaction is necessary to increase the therapeutic benefits of this combination in the clinic. The synergism between the anti-cancer benefits of cisplatin and the drug delivery benefits of hyperthermia may offer a novel and more effective treatment for many cancer patients. We hypothesized that hyperthermia increases cisplatin accumulation and efficacy in part by modulating the function of Ctr1, a major regulator of cellular cisplatin uptake. To test this hypothesis, we examined the significance of Ctr1 during combined hyperthermia and cisplatin therapies and assessed the importance of cisplatin- and hyperthermia-induced Ctr1 multimerization in enhancing cisplatin cytotoxicity. We observed increased Ctr1 multimerization following hyperthermia treatment (41°C) in vitro, compared to normothermic controls (37°C), suggesting that this may be a mechanism for increased cisplatin uptake in heat-treated cells. The impact of increased Ctr1 multimerization was evaluated by measuring platinum accumulation in wild-type (WT) and Ctr1-/- cells. WT cells contained greater levels of platinum compared to Ctr1-/- cells. A further increase in platinum was observed following hyperthermia treatment, but only in the WT cells. Hyperthermia enhanced cisplatin-mediated cytotoxicity in WT cells with a dose-modifying factor (DMF) of 1.8 compared to 1.4 in Ctr1-/- cells. Our data suggest that heat increases Ctr1 activity by increasing multimerization, resulting in enhanced drug accumulation. Although we recognize that the effect of heat on cells is multi-factorial, our results support the hypothesis that Ctr1 is, in part, involved in the synergistic interaction observed with cisplatin and hyperthermia treatment.

In addition to assessing cisplatin delivery at the cellular level, we evaluated cisplatin delivery at the tissue level, using novel cisplatin-loaded temperature-sensitive liposomes. We hypothesized that delivering cisplatin encapsulated in liposomes under hyperthermic conditions would improve the pharmacokinetic profiles of cisplatin, increase drug delivery to the tumor, decrease normal tissue toxicity, and enhance the anti-tumor activity of cisplatin. We successfully prepared temperature-sensitive liposomes loaded with cisplatin and demonstrated that heat (42°C) sensitizes cisplatin-resistant cells to the cytotoxic effects of cisplatin in vitro.

Decreased toxicity was observed in animals treated with the cisplatin liposome (± heat) compared to the free drug treatments. A pharmacokinetic study of cisplatin-loaded temperature-sensitive liposomes and free drug was performed in tumor-bearing mice under normothermic and hyperthermic conditions. Cisplatin half-life in plasma was increased following liposome treatment compared to free cisplatin, and cisplatin delivery to the tumors was greatest in mice that received liposomal cisplatin under hyperthermia. These initial in vivo data demonstrate the potential effectiveness of this cisplatin-loaded liposome formulation in the treatment of certain types of cancer. To assess the anti-cancer efficacy of the liposome treatment, a tumor growth delay study was conducted and demonstrated equivalent efficacy for the cisplatin-loaded temperature-sensitive liposome compared to free drug.

In addition to the liposome work, we developed and evaluated a novel heating device for the bladder. Despite the evidence that hyperthermia is an effective adjuvant treatment strategy, current clinical heating devices are inadequate, warranting the development of a new and improved system. We induced hyperthermia using ferromagnetic nanoparticles and an alternating magnetic field device developed by Actium Biosystems. Initial preclinical studies in a rat model demonstrated preferential bladder heating. However, our preliminary studies show severe toxicity with the direct instillation of the nanoparticles in the bladder, and further studies are needed to potentially modify the nanoparticle coating, the catheterization procedure, as well as to develop a different animal model.

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Imbalance between oxygen consumption and supply often makes tumors hypoxic (Bristow and Hill 2008). Tumor hypoxia is significantly correlated with aggressive tumor growth, ineffective response to radiation and chemotherapy, and as a result, poor patient prognosis. Hyperthermia (HT) is a strong adjuvant treatment to overcome these challenges of tumor hypoxia because it causes tumor reoxygenation at temperatures lower than 43ºC (Song, Park, and Griffin 2001). However, the detailed molecular mechanisms of how HT enhances tumor oxygenation have not been elucidated. Here we determine that 1 hour HT activates hypoxia-inducible factor-1 (HIF-1) and its downstream targets, vascular endothelial growth factor (VEGF), lactate dehydrogenase A (LDHA), and pyruvate dehydrogenase kinase 1 (PDK1) in tumors. Consistent with HIF-1 activation and upregulation of its downstream genes, HT also enhances tumor perfusion/vascularization and decreases oxygen consumption rates. As a result, tumor hypoxia is reduced after HT suggesting that these physiological changes contribute to HT-induced tumor reoxygenation. Since HIF-1 is a potent regulator of tumor vascularization and metabolism, our findings suggest that HIF-1 plays a role in HT-induced tumor reoxygenation by transactivating its downstream targets. Mechanistically, we demonstrate that NADPH oxidase-mediated reactive oxygen species (ROS) production upregulates HIF-1 after HT. Further, we determine that this pathway is initiated by increased transcription of NADPH oxidase-1 (NOX1) through the ERK pathway.

A major research effort at Duke focuses on combinations of HT and doxorubicin in the treatment of locally advanced breast and other cancers. Thus, we investigated whether there are HIF-1 responses to doxorubicin treatment. We reveal that doxorubicin also activates HIF-1. Unlike HT, doxorubicin-induced HIF-1 promotes persistent tumor angiogenesis. We also reveal that the signal transducer and activator of transcription 1 (STAT1)/inducible nitric oxide synthase (iNOS) pathway causes HIF-1α accumulation in an oxygen-independent manner. We show that activated STAT1 upregulates iNOS expression and promotes nitric oxide (NO) production in tumor cells resulting in HIF-1α stabilization. We further determine that both iNOS inhibitor, 1400W and STAT1 inhibitor, epigallocatechin-3-gallate (EGCG) significantly decrease intracellular NO production and suppress doxorubicin-induced normoxic HIF-1α accumulation.

HIF-1 is often considered a promising therapeutic target because of its role in tumor progression (Semenza 2003) and therapy resistance (Moeller et al. 2004). However, our findings suggest that HIF-1 plays a pleiotropic role in response to HT and chemotherapy. Therefore, to preferentially take advantage of HT-induced HIF-1 activation and also to suppress its deleterious effects induced by chemotherapy or as we have previously reported, by radiation (Moeller et al. 2004), HIF-1 inhibition needs to be carefully regulated in a time-sensitive manner to achieve optimal therapeutic effects.

Each year, over 13,000 people in the United States die from a primary malignant brain tumor. Currently, primary BTs are treated most commonly by surgery, radiotherapy, and systemic chemotherapy, though each of these methods carries a risk of complications or acute side effects.

Ultrasound hyperthermia has been investigated as way to open the blood-brain barrier for improved chemotherapeutic drug delivery, but previous methods have involved either invasively removing skull bone via surgery or non-invasively dealing with the high ultrasound attenuation, reflection, and phase aberration resulting from the skull and its variable thickness. Dual-mode ultrasound transducers for image-guided therapy have also been investigated for several applications; in some instances, phased arrays are ideal, allowing control over the ultrasound energy deposition pattern and inherent spatial registration between imaging, treatment, and monitoring.

Additionally, thermosensitive liposomes can be configured to encapsulate drugs and actively target regions of tumor angiogenesis. When used in combination with localized hyperthermia, thermosensitive liposomes can provide targeted control of drug release that may enhance chemotherapeutic efficacy in many clinical settings. Meanwhile, catheter devices and endovascular techniques are used by interventional neuroradiologists to treat various intracranial diseases, including intracranial aneurysm and dural venous sinus thrombosis. These procedures can be extended to the treatment of intracranial tumors (advancement of a 5 Fr catheter as far as the frontal portion of the superior sagittal sinus has been demonstrated).

The objective of the work presented in this dissertation was the realization of a dual-mode catheter transducer for a minimally-invasive, vascular approach to deliver localized, image-guided ultrasound hyperthermia to an intracranial tumor target. Toward this end, a series of prototype ultrasound transducers were designed, simulated, built, and tested for imaging and therapeutic potential.

Two 14-Fr phased-array prototypes were built with PZT-5H ceramic and tested for real-time 3D intracranial imaging and focused-beam hyperthermia capability. These were able to visualize the lateral ventricles and Circle of Willis in a canine model, and generate a temperature rise over 4°C at a 2-cm focal distance in excised tissue.

Single-channel intravascular ultrasound (IVUS) coronary imaging catheters as small as 3.5 Fr were then considered as a construction template; several possible transducer apertures were simulated before fabricating prototypes with PZT-4. The transducers exhibited a dual-frequency response, due to the presence of thickness-mode and width-mode resonances. A thermal model was developed to estimate the +4°C thermal penetration depth for a given transducer aperture, predicting an effective therapeutic range of up to 12 mm with a 5 × 0.5 mm aperture.

A 3.5-Fr commercial mechanical IVUS catheter was retrofitted with a PZT-4 transducer and tested for 9-MHz imaging performance in several animal studies, successfully visualizing anatomical structures in the brain and navigating a minimally-invasive vascular pathway toward the brain. An identical PZT-4 transducer was used to build a 3.3-MHz therapy prototype, which produced a temperature rise of +13.5°C at a depth of 1.5 mm in live xenograft brain tumor tissue in the mouse model.

These studies indicate that a minimally-invasive catheter transducer can be made capable of visualizing brain structures and generating localized hyperthermia to trigger drug release from thermosensitive liposomes in brain tumor tissue.