Ultrasound-directed enzyme-prodrug therapy (UDEPT) using self-immolative doxorubicin derivatives

Background: Enzyme-activatable prodrugs are extensively employed in oncology and beyond. Because enzyme concentrations and their (sub)cellular compartmentalization are highly heterogeneous in different tumor types and patients, we propose ultrasound-directed enzyme-prodrug therapy (UDEPT) as a means to increase enzyme access and availability for prodrug activation locally. Methods: We synthesized β-glucuronidase-sensitive self-immolative doxorubicin prodrugs with different spacer lengths between the active drug moiety and the capping group. We evaluated drug conversion, uptake and cytotoxicity in the presence and absence of the activating enzyme β-glucuronidase. To trigger the cell release of β-glucuronidase, we used high-intensity focused ultrasound to aid in the conversion of the prodrugs into their active counterparts. Results: More efficient enzymatic activation was observed for self-immolative prodrugs with more than one aromatic unit in the spacer. In the absence of β-glucuronidase, the prodrugs showed significantly reduced cellular uptake and cytotoxicity compared to the parent drug. High-intensity focused ultrasound-induced mechanical destruction of cancer cells resulted in release of intact β-glucuronidase, which activated the prodrugs, restored their cytotoxicity and induced immunogenic cell death. Conclusion: These findings shed new light on prodrug design and activation, and they contribute to novel UDEPT-based mechanochemical combination therapies for the treatment of cancer

Cavitation-enhanced back projection for acoustic detection of attenuating structures

Current methodology for the detection of attenuating structures in abdominal HIFU interventions requires lengthy, elaborate image analysis, which is undesired in a clinical setting. In this work, a method for the acoustic detection of attenuating structures in the beam path of the therapeutic HIFU array is described. The proposed method is used to determine binary apodizations that can be applied to the HIFU transducer for intercostal shot positions. Such a binary apodization was determined in vivo on an anesthetized pig under controlled breathing. Validation of the proposed method was done by comparing the binary apodization based on the proposed method to a binary apodization obtained using methodology based on MR image analysis and a collision detection algorithm. The proposed acoustical method provided a binary apodization that was over 90% similar to the apodization obtained using the image analysis-based method. Additionally, the proposed method can provide a measure of the amount of attenuation that each respective transducer element encounters in its beam path towards the focus

Cavitation-enhanced back projection for acoustic detection of attenuating structures

Current methodology for the detection of attenuating structures in abdominal HIFU interventions requires lengthy, elaborate image analysis, which is undesired in a clinical setting. In this work, a method for the acoustic detection of attenuating structures in the beam path of the therapeutic HIFU array is described. The proposed method is used to determine binary apodizations that can be applied to the HIFU transducer for intercostal shot positions. Such a binary apodization was determined in vivo on an anesthetized pig under controlled breathing. Validation of the proposed method was done by comparing the binary apodization based on the proposed method to a binary apodization obtained using methodology based on MR image analysis and a collision detection algorithm. The proposed acoustical method provided a binary apodization that was over 90% similar to the apodization obtained using the image analysis-based method. Additionally, the proposed method can provide a measure of the amount of attenuation that each respective transducer element encounters in its beam path towards the focus

Spatiotemporal control of gene expression in bone-marrow derived cells of the tumor microenvironment induced by MRI guided focused ultrasound

The tumor microenvironment is an interesting target for anticancer therapies but modifying this compartment is challenging. Here, we demonstrate the feasibility of a gene therapy strategy that combined targeting to bone marrow-derived tumor microenvironment using genetically modified bone-marrow derived cells and control of transgene expression by local hyperthermia through a thermo-inducible promoter. Chimera were obtained by engraftment of bone marrow from transgenic mice expressing reporter genes under transcriptional control of heat shock promoter and inoculated sub-cutaneously with tumors cells. Heat shocks were applied at the tumor site using a water bath or magnetic resonance guided high intensity focused ultrasound device. Reporter gene expression was followed by bioluminescence and fluorescence imaging and immunohistochemistry. Bone marrow-derived cells expressing reporter genes were identified to be mainly tumor-associated macrophages. We thus provide the proof of concept for a gene therapy strategy that allows for spatiotemporal control of transgenes expression by macrophages targeted to the tumor microenvironment

Field drift correction of proton resonance frequency shift temperature mapping with multichannel fast alternating nonselective free induction decay readouts

Purpose: To demonstrate that proton resonance frequency shift MR thermometry (PRFS-MRT) acquisition with nonselective free induction decay (FID), combined with coil sensitivity profiles, allows spatially resolved B0 drift-corrected thermometry. Methods: Phantom experiments were performed at 1.5T and 3T. Acquisition of PRFS-MRT and FID were performed during MR-guided high-intensity focused ultrasound heating. The phase of the FIDs was used to estimate the change in angular frequency δωdrift per coil element. Two correction methods were investigated: (1) using the average δωdrift over all coil elements (0th-order) and (2) using coil sensitivity profiles for spatially resolved correction. Optical probes were used for independent temperature verification. In-vivo feasibility of the methods was evaluated in the leg of 1 healthy volunteer at 1.5T. Results: In 30 minutes, B0 drift led to an apparent temperature change of up to –18°C and –98°C at 1.5T and 3T, respectively. In the sonicated area, both corrections had a median error of 0.19°C at 1.5T and –0.54°C at 3T. At 1.5T, the measured median error with respect to the optical probe was –1.28°C with the 0th-order correction and improved to 0.43°C with the spatially resolved correction. In vivo, without correction the spatiotemporal median of the apparent temperature was at –4.3°C and interquartile range (IQR) of 9.31°C. The 0th-order correction had a median of 0.75°C and IQR of 0.96°C. The spatially resolved method had the lowest median at 0.33°C and IQR of 0.80°C. Conclusion: FID phase information from individual receive coil elements allows spatially resolved B0 drift correction in PRFS-based MRT

Field drift correction of proton resonance frequency shift temperature mapping with multichannel fast alternating nonselective free induction decay readouts

Purpose: To demonstrate that proton resonance frequency shift MR thermometry (PRFS-MRT) acquisition with nonselective free induction decay (FID), combined with coil sensitivity profiles, allows spatially resolved B0 drift-corrected thermometry. Methods: Phantom experiments were performed at 1.5T and 3T. Acquisition of PRFS-MRT and FID were performed during MR-guided high-intensity focused ultrasound heating. The phase of the FIDs was used to estimate the change in angular frequency δωdrift per coil element. Two correction methods were investigated: (1) using the average δωdrift over all coil elements (0th-order) and (2) using coil sensitivity profiles for spatially resolved correction. Optical probes were used for independent temperature verification. In-vivo feasibility of the methods was evaluated in the leg of 1 healthy volunteer at 1.5T. Results: In 30 minutes, B0 drift led to an apparent temperature change of up to –18°C and –98°C at 1.5T and 3T, respectively. In the sonicated area, both corrections had a median error of 0.19°C at 1.5T and –0.54°C at 3T. At 1.5T, the measured median error with respect to the optical probe was –1.28°C with the 0th-order correction and improved to 0.43°C with the spatially resolved correction. In vivo, without correction the spatiotemporal median of the apparent temperature was at –4.3°C and interquartile range (IQR) of 9.31°C. The 0th-order correction had a median of 0.75°C and IQR of 0.96°C. The spatially resolved method had the lowest median at 0.33°C and IQR of 0.80°C. Conclusion: FID phase information from individual receive coil elements allows spatially resolved B0 drift correction in PRFS-based MRT

Hyperthermia-triggered release of hypoxic cell radiosensitizers from temperature-sensitive liposomes improves radiotherapy efficacy in vitro

Hypoxia is a characteristic feature of solid tumors and an important cause of resistance to radiotherapy. Hypoxic cell radiosensitizers have been shown to increase radiotherapy efficacy, but dose-limiting side effects prevent their widespread use in the clinic. We propose the encapsulation of hypoxic cell radiosensitizers in temperature-sensitive liposomes (TSL) to target the radiosensitizers specifically to tumors and to avoid unwanted accumulation in healthy tissues. The main objective of the present study is to develop and characterize TSL loaded with the radiosensitizer pimonidazole (PMZ) and to evaluate the in vitro efficacy of free PMZ and PMZ encapsulated in TSL in combination with hyperthermia and radiotherapy. PMZ was actively loaded into TSL at different drug/lipid ratios, and the physicochemical characteristics and the stability of the resulting TSL-PMZ were evaluated. PMZ release was determined at 37 °C and 42 °C in HEPES buffer saline and fetal bovine serum. The concentration-dependent radiosensitizing effect of PMZ was investigated by exposing FaDu cells to different PMZ concentrations under hypoxic conditions followed by exposure to ionizing irradiation. The efficacy of TSL-PMZ in combination with hyperthermia and radiotherapy was determined in vitro, assessing cell survival and DNA damage by means of the clonogenic assay and histone H2AX phosphorylation, respectively. All TSL-PMZ formulations showed high encapsulation efficiencies and were stable for 30 d upon storage at 4 °C and 20 °C. Fast PMZ release was observed at 42 °C, regardless of the drug/lipid ratio. Increasing the PMZ concentration significantly enhanced the effect of ionizing irradiation. Pre-heated TSL-PMZ in combination with radiotherapy caused a 14.3-fold increase in cell death as compared to radiotherapy treatment alone. In conclusion, our results indicate that TSL-PMZ in combination with hyperthermia can assist in improving the efficacy of radiotherapy under hypoxic conditions

Tumor drug distribution after local drug delivery by hyperthermia, in vivo

Tumor drug distribution and concentration are important factors for effective tumor treatment. A promising method to enhance the distribution and the concentration of the drug in the tumor is to encapsulate the drug in a temperature sensitive liposome. The aim of this study was to investigate the tumor drug distribution after treatment with various injected doses of different liposomal formulations of doxorubicin, ThermoDox (temperature sensitive liposomes) and DOXIL (non-temperature sensitive liposomes), and free doxorubicin at macroscopic and microscopic levels. Only ThermoDox treatment was combined with hyperthermia. Experiments were performed in mice bearing a human fibrosarcoma. At low and intermediate doses, the largest growth delay was obtained with ThermoDox, and at the largest dose, the largest growth delay was obtained with DOXIL. On histology, tumor areas with increased doxorubicin concentration correlated with decreased cell proliferation, and substantial variations in doxorubicin heterogeneity were observed. ThermoDox treatment resulted in higher tissue drug levels than DOXIL and free doxorubicin for the same dose. A relation with the distance to the vasculature was shown, but vessel perfusion was not always sufficient to determine doxorubicin delivery. Our results indicate that tumor drug distribution is an important factor for effective tumor treatment and that its dependence on delivery formulation merits further systemic investigation