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

The significance of shape and orientation in single-particle weak-scatterer models.

Particles that have a density and compressibility comparable to those of the surrounding medium (weak scatterers) are often modeled as spherical scatterers of equivalent volume. The simplicity and symmetry of the spherical model does not however account for the effect of the angle of incidence of the incident field onto the particle. By performing a comparative study of scattering models for the case of a fluid sphere, it is shown that models derived using the Born approximation closely match the exact solution for weak scatterers. This approximation is therefore applied to produce a model for the fluid disk, which is used to investigate the significance of particle shape and orientation. Scattering by a red blood cell of given volume modeled as a sphere is compared to the result obtained by approximating its shape as a disk of varying aspect ratio and orientation. The spherical model is shown to provide a good description for frequencies up to 20 MHz, beyond which particle shape becomes significant. This effect could go undetected if the scattered field is only observed at 90 degrees relative to the direction of the incident field. Nevertheless, the significance of particle shape and orientation might form the basis of a novel detection technique

Passive cavitation mapping for localization and tracking of bubble dynamics

Current acoustic techniques for studying cavitation dynamics are only readily applicable to single-bubble activity, while optical methods can only be used in transparent media. However, multi-bubble cavitation often occurs in opaque media such as biological tissue. Here, the signals received passively by each of the 64 channel of a diagnostic ultrasound array are used to localize and separate emissions from several bubble clusters cavitating in agar gel, thereby providing a method of observing cavitation dynamics. The method has a high spatiotemporal resolution and is applicable to cavitation in opaque media. (C) 2010 Acoustical Society of Americ

PAX (Passive-Active Crossing) method for sub-millimeter coregistration of passive acoustic mapping and b-mode images

Nonlinear ultrasonic emissions produced during a therapeutic ultrasound procedure can be detected, localized, and quantified through a class of methods that can be referred to as passive acoustic mapping (PAM). While a variety of PAM beamforming algorithms may be employed, they share a common limitation that a single sound speed is specified for both phase steering of array elements and for calculation of source power or energy. The specified value may be inadequate whether derived from B-mode-based metrics or literature values for constituent materials. This study employed experiments and simulations with linear and curvilinear array geometries to investigate the impact of in situ sound speed uncertainties on source localization in layered media. The data were also used to evaluate a new method for optimizing coregistration of PAM and B-mode images. Coregistration errors as large as 10 mm were observed with the curvilinear array, which also showed much greater sound speed sensitivity than the linear array. Errors with both array geometries were typically reduced to the order of 0.1 mm using the proposed optimization method regardless of beamformer choice or whether the array was calibrated. In a further step toward reliable implementation of PAM, the current work provides an approach that can help ensure that therapeutic ultrasound procedures are accurately guided by cavitation emissions

Effect of temperature on rectified diffusion during ultrasound-induced heating.

Experimental observations of delayed-onset cavitation during ultrasound insonation have been suggested as being caused by a change in the size distribution of the bubble population due to rectified diffusion. To investigate this hypothesis, a single bubble model is used here to explore the effect of heating and the subsequent elevated temperatures on the rectified diffusion process. Numerical solution of the model, which includes the temperature dependences of seven relevant physical parameters, allows quantification of the change in the pressure threshold for rectified diffusion, as well as the importance of the bulk liquid saturation concentration in determining bubble evolution. Although elevated temperatures and liquid supersaturation reduce the rectified diffusion threshold, it remains coincident with the inertial cavitation thresholds at submicron bubble sizes at all temperatures. This observation suggests that changes in the nucleation environment, rather than bubble growth due to rectified diffusion, is a more likely cause of delayed-onset cavitation events

Diffraction effects and compensation in passive acoustic mapping

Over the last decade, a variety of noninvasive techniques has been developed to monitor therapeutic ultrasound procedures in support of safety or efficacy assessments. One class of methods employs diagnostic ultrasound arrays to sense acoustic emissions, thereby providing a means to passively detect, localize and quantify the strength of nonlinear sources, including cavitation. Real array element diffraction patterns may differ substantially from those presumed in existing beamforming algorithms. However, diffraction compensation has received limited treatment in passive and active imaging, and measured diffraction data has yet to be used for array response correction. The objectives of this work were to identify differences between ideal and real element diffraction patterns, and to quantify the impact of diffraction correction on cavitation mapping beamformer performance. These objectives were addressed by performing calibration measurements on a diagnostic linear array, using the results to calculate diffraction correction terms, and applying the corrections to cavitation emission data collected from soft tissue phantom experiments. Measured diffraction patterns were found to differ significantly from those of ideal element forms, particularly at higher frequencies and shorter distances from the array. Diffraction compensation of array data resulted in cavitation energy estimates elevated by as much as a factor of five, accompanied by the elimination of a substantial bias between two established beamforming algorithms. These results illustrate the importance of using measured array responses to validate analytical field models and to minimize observation biases in imaging applications where quantitative analyses are critical for assessment of therapeutic safety and efficacy

Nonlinear acoustic properties of ex vivo bovine liver and the effects of temperature and denaturation.

Thermal ablation by high intensity focused ultrasound (HIFU) has a great potential for the non-invasive treatment of solid tumours. Due to the high pressure amplitudes involved, nonlinear acoustic effects must be understood and the relevant medium property is the parameter of nonlinearity B/A. Here, B/A was measured in ex vivo bovine liver, over a heating/cooling cycle replicating temperatures reached during HIFU ablation, adapting a finite amplitude insertion technique, which also allowed for measurement of sound-speed and attenuation. The method measures the nonlinear progression of a plane wave through liver and B/A was chosen so that numerical simulations matched the measured waveforms. To create plane-wave conditions, sinusoidal bursts were transmitted by a 100 mm diameter 1.125 MHz unfocused transducer and measured using a 15 mm diameter 2.25 MHz broadband transducer in the near field. Attenuation and sound-speed were calculated using a reflected pulse from the smaller transducer using the larger transducer as the reflecting interface. Results showed that attenuation initially decreased with heating then increased after denaturation, the sound-speed initially increased with temperature and then decreased, and B/A showed an increase with temperature but no significant post-heating change. The B/A data disagree with other reports that show a significant change and we suggest that any nonlinear enhancement in the received ultrasound signal post-treatment is likely due to acoustic cavitation rather than changes in tissue nonlinearity

Improving Clinical Outcomes in Renal HIFU Therapy

The rising incidence of small, asymptomatic renal tumours discovered using abdominal imaging during the investigation of unrelated symptoms has fuelled the desire for new therapies which avoid surgical excision. Extracorporeal High Intensity Focused Ultrasound (HIFU) was proposed as one of these modalities but so far clinical research has been inconclusive. The present work was designed to improve these clinical outcomes through the conduct of further clinical trials, laboratory based research and the translation of new technology into existing HIFU devices. A Phase II clinical trial of patients (n=13) with newly diagnosed <4cm renal tumours (clinical stage T1a) was designed, peer reviewed and received ethical approval (Ox REC 09/H0606/04). Ten of 13 patients underwent renal HIFU using a clinical HIFU device (Model JC/JC200, HAIFU, China). One patient could not be treated due to poor tumour visualisation after anaesthesia and two patients could not be treated as they became unwell before or during anaesthesia. Histological evidence of HIFU ablation in either tumour or normal renal parenchyma was seen in all ten patients. Evidence of sub-total tumour ablation was seen in 8/10 of patients. Grade 1 (<50%), 2 (50-90% & 3 (90-99%) ablation was achieved in 4/10, 3/10 & 3/10 patients respectively but complete (100%) tumour ablation was not possible. HIFU treatment caused minimal morbidity – no Grade III-V (Clavien-Dindo) complications related to HIFU treatment occurred. Grade I skin pain and induration was seen in 9/10 patients; Grade II skin pain occurred in a single patient. Patient demographics, imaging and tumour characteristics were used to design parameters to improve patient selection for renal HIFU. The tumour location, thickness of peri-nephric fat and renal nephrometry score were useful predictors of successful screening for treatment. Diligent use of these factors could limit unnecessary treatments and improve ablation outcomes. It is well known that ultrasound imaging of small renal masses can be challenging. Ultrasound imaging often deteriorates further during HIFU as the abdominal wall and fat tissues swell and cause increased attenuation. This loss of imaging quality was clearly demonstrated in this clinical trial and resulted in the early termination of treatment, before endpoints were reached, in a number of cases. The current clinical method for monitoring the success of HIFU ablation using hyperecho analysis of B-mode ultrasound images is also questionable. Laboratory based studies using ex-vivo bovine liver subjected to HIFU confirmed that hyperecho monitoring had low sensitivity, predictive values and overall accuracy. A novel method of HIFU monitoring – passive mapping of the emissions received from acoustic cavitation activity and other sources of non-linearity during HIFU treatment – is believed to represent a significant opportunity to improve feedback. This technique uses the passively received signature of cavity activity which, when time-reversed, gives high-resolution images of the precise location of the activity. Laboratory-based ex-vivo work, using a commercially available ultrasound system (z.one, Zonare, USA), demonstrates its superiority over hyperecho monitoring. Indeed, thresholds could be applied to successfully predict HIFU ablation with high sensitivity and specificity. This technique was successfully translated into the clinical setting through the design of a Passive Acoustic Mapping (PAM) device. Custom-built receiving elements were applied without limiting the function of the existing HIFU devices. Both pre-clinical and ethically-approved clinical studies demonstrated its safe integration without significant impact on the device energy output or treatment accuracy. Using similar passive beamforming algorithms, acoustic cavitation activity was successfully mapped and corresponded with the location of thermal ablation in both ex-vivo tissue phantoms and during clinical HIFU therapy. It is believed that the development of new patient selection parameters will eliminate unnecessary investigation and treatment for those who are unsuitable. The use of PAM will lead to a significant improvement in the efficacy of treatment. It can be successfully applied to existing devices and predicts the location and extent of HIFU ablation with greater accuracy that existing techniques

Enhanced viral activity in tumors using focused ultrasound and microbubbles-A long term study.

Oncolytic viruses target and kill cancer cells and self-amplify through replication. However, viral therapy in vivo is limited by insufficient systemic delivery. Here, focused ultrasound (FUS) was used in conjunction with microbubbles to produce cavitation and enhance tumor viral delivery. Human breast cancer cells (ZR75.1) were injected subcutaneously in mice (n = 8), which grew to a tumor volume of at least 30 mm(3). The tumors were then exposed to FUS (frequency: 0.5 MHz, pulse duration: 50 000 cycles, pulse repetition frequency: 0.5 Hz) for 4 min following injection of 100 μl of SonoVue microbubbles (SVM) with polymer-coated adenovirus encoding luciferase (pc-Ad-Luc). Experiments were repeated for three controls: pc-Ad-luc with neither FUS nor SVMs, FUS and SVM without pc-Ad-Luc, and buffer alone. Acoustic emissions were recorded with a passive detector, which validated the presence of inertial cavitation and ensured good SVM reperfusion kinetics. Tumor viral expression was then imaged using IVIS following luciferin injection. At 1, 2, 3, and 7 days post-injection, 3, 20.3, 30.2, and 22.9-fold increases in photons/s/cm(2) were observed when pc-Ad-Luc was used with FUS and SVM compared to pc-Ad-Luc alone. In conclusion, FUS and SVM enhanced oncolytic virus delivery resulting in amplified viral expression over time

Targeting of liposomes via PSGL1 for enhanced tumor accumulation.

PURPOSE: To improve the delivery of liposomes to tumors using P-selectin glycoprotein ligand 1 (PSGL1) mediated binding to selectin molecules, which are upregulated on tumorassociated endothelium. METHODS: PSGL1 was orientated and presented on the surface of liposomes to achieve optimal selectin binding using a novel streptavidin-protein G linker molecule. Loading of PSGL1 liposomes with luciferin allowed their binding to e-selectin and activated HUVEC to be quantified in vitro and their stability, pharmacokinetics and tumor accumulation to be tested in vivo using murine models. RESULTS: PSGL1 liposomes showed 5-fold (p 7-fold (p 3-fold enhancement in the level of delivery to tumors (p < 0.05). CONCLUSIONS: The technologies and strategies described here may contribute to clinical improvements in the selectivity and efficacy of liposomal drug delivery agents