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Pancreatic ductal adenocarcinomas respond poorly to chemotherapy, in part due to the dense tumor stroma that hinders drug delivery. Ultrasound (US) in combination with microbubbles has previously shown promise as a means to improve drug delivery, and the therapeutic efficacy of ultrasound-mediated drug delivery is currently being evaluated in multiple clinical trials. However, most of these utilize echogenic contrast agents engineered for imaging, which might not be optimal compared to specialized formulations tailored for drug delivery. In this study, we evaluated the in vivo efficacy of phase-shifting microbubble-microdroplet clusters that, upon insonation, form bubbles in the size range of 20–30 μm. We developed a patient-derived xenograft model of pancreatic cancer implanted in mice that largely retained the stromal content of the originating tumor and compared tumor growth in mice given chemotherapeutics (nab-paclitaxel plus gemcitabine or liposomal irinotecan) with mice given the same chemotherapeutics in addition to ultrasound and acoustic cluster therapy. We found that acoustic cluster therapy significantly improved the effect of both chemotherapeutic regimens and resulted in 7.2 times higher odds of complete remission of the tumor compared to the chemotherapeutics alone.

Systemic injections of chemotherapeutics often deliver only minor fractions of the administered dose to the targeted pathology, resulting in unwanted toxicity towards healthy tissue. In brain tissue, drug delivery is impaired by the highly selective nature of the blood brain barrier impeding the influx of most substances. Acoustic Cluster Therapy (ACT®) is a platform for targeted therapeutic enhancement, facilitating increased local drug transfer. The platform is comprised of ACT® clusters, a mix of microbubbles and microdroplets and low intensity (diagnostic) ultrasound insonation. Intravenously injected ACT® clusters circulate freely through the body before activation by localised insonation at the targeted pathology. In the ultrasound field, microbubbles transfer energy to microdroplets, which undergo a liquid-to-gas phase transition, forming larger ACT® bubbles that transiently deposit in the targeted microvasculature. Further exposure to ultrasound results in controlled volume oscillation of the ACT® bubbles, inducing a range of biomechanical effects. The effects lead to enhanced extravasation across the endothelial barrier, facilitating the transport of co-administered chemotherapeutics to the targeted tissue. Proof of concept studies showed an increased therapeutic efficacy of standard of care drugs, when combined with ACT® treatment. Furthermore, ACT® treatment induced a controlled and temporal opening of the blood brain barrier observed by the extravasation of fluorescent macromolecules into brain tissue.

IntroductionAcoustic cluster therapy (ACT) comprises co-administration of a formulation containing microbubble/microdroplet clusters (PS101), together with a regular medicinal drug (e.g., a chemotherapeutic) and local ultrasound (US) insonation of the targeted pathological tissue (e.g., the tumor). PS101 is confined to the vascular compartment and, when the clusters are exposed to regular diagnostic imaging US fields, the microdroplets undergo a phase-shift to produce bubbles with a median diameter of 22 µm when unconstrained by the capillary wall. In vivo these bubbles transiently lodge in the tumor’s microvasculature. Low frequency ultrasound (300 kHz) at a low mechanical index (MI = 0.15) is then applied to drive oscillations of the deposited ACT bubbles to induce a range of biomechanical effects that locally enhance extravasation, distribution, and uptake of the co-administered drug, significantly increasing its therapeutic efficacy.MethodsIn this study we investigated the therapeutic efficacy of ACT with liposomal doxorubicin for the treatment of triple negative breast cancer using orthotopic human tumor xenografts (MDA-MB-231-H.luc) in athymic mice (ICR-NCr-Foxn1nu). Doxil® (6 mg/kg, i.v.) was administered at days 0 and 21, each time immediately followed by three sequential ACT (20 ml/kg PS101) treatment procedures (n = 7–10). B-mode and nonlinear ultrasound images acquired during the activation phase were correlated to the therapeutic efficacy.ResultsResults show that combination with ACT induces a strong increase in the therapeutic efficacy of Doxil®, with 63% of animals in complete, stable remission at end of study, vs. 10% for Doxil® alone (p < 0.02). A significant positive correlation (p < 0.004) was found between B-mode contrast enhancement during ACT activation and therapy response. These observations indicate that ACT may also be used as a theranostic agent and that ultrasound contrast enhancement during or before ACT treatment may be employed as a biomarker of therapeutic response during clinical use.

The blood-brain barrier (BBB) is a major obstacle in drug delivery for diseases of the brain, and today there is no standardized route to surpass it. One technique to locally and transiently disrupt the BBB, is focused ultrasound in combination with gas-filled microbubbles. However, the microbubbles used are typically developed for ultrasound imaging, not BBB disruption. Here we describe efficient opening of the BBB using the promising novel Acoustic Cluster Therapy (ACT), that recently has been used in combination with Abraxane® to successfully treat subcutaneous tumors of human prostate adenocarcinoma in mice. ACT is based on the conjugation of microbubbles to liquid oil microdroplets through electrostatic interactions. Upon activation in an ultrasound field, the microdroplet phase transfers to form a larger bubble that transiently lodges in the microvasculature. Further insonation induces volume oscillations of the activated bubble, which in turn induce biomechanical effects that increase the permeability of the BBB. ACT was able to safely and temporarily permeabilize the BBB, using an acoustic power 5-10 times lower than applied for conventional microbubbles, and successfully deliver small and large molecules into the brain. This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) License. See http://ivyspring.com/terms for full terms and conditions.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers with survival averaging only 3months if untreated following diagnosis. A major limitation in effectively treating PDAC using conventional and targeted chemotherapeutic agents, is inadequate drug delivery to the target location, predominantly due to a poorly vascularised, desmoplastic tumour microenvironment. Ultrasound in combination with ultrasound contrast agents, i.e., microbubbles, that flow through the vasculature and capillaries can be used to disrupt such mechanical barriers, potentially allowing for a greater therapeutic efficacy. This phenomenon is commonly referred to as sonoporation. In an attempt to improve the efficacy of sonoporation, novel microbubble formulations are being developed to address the limitation of commercially produced clinical diagnostic ultrasound contrast agents. In our work here we evaluate the ability of a novel formulation; namely Acoustic Cluster Therapy (ACT®) to improve the therapeutic efficacy of the chemotherapeutic agent paclitaxel, longitudinally in a xenograft model of PDAC. Results indicated that ACT® bubbles alone demonstrated no observable toxic effects, whilst ACT® in combination with paclitaxel can transiently reduce tumour volumes significantly, three days posttreatment (p=0.0347-0.0458). Quantitative 3D ultrasound validated the calliper measurements. Power Doppler ultrasound imaging indicated that ACT® in combination with paclitaxel was able to transiently sustain peak vasculature percentages as observed in the initial stages of tumour development. Nevertheless, there was no significant difference in tumour vasculature percentage at the end of treatment. The high vascular percentage correlated to the transient decrease and overall inhibition of the tumour volumes. In conclusion, ACT® improves the therapeutic efficacy of paclitaxel in a PDAC xenograft model allowing for transient tumour volume reduction and sustained tumour vasculature percentage.

Introduction: Acoustic Cluster Therapy (ACT) comprises coadministration of a formulation containing microbubble-microdroplet clusters (PS101) together with a regular medicinal drug and local ultrasound (US) insonation of the targeted pathological tissue. PS101 is confined to the vascular compartment and when the clusters are exposed to regular diagnostic imaging US fields, the microdroplets undergo a phase shift to produce bubbles with a median diameter of 22 µm. Low frequency, low mechanical index US is then applied to drive oscillations of the deposited ACT bubbles to induce biomechanical effects that locally enhance extravasation, distribution, and uptake of the coadministered drug, significantly increasing its therapeutic efficacy. Methods: The therapeutic efficacy of ACT with irinotecan (60 mg/kg i.p.) was investigated using three treatment sessions given on day 0, 7, and 14 on subcutaneous human colorectal adenocarcinoma xenografts in mice. Treatment was performed with three back-to-back PS101+US administrations per session with PS101 doses ranging from 0.40–2.00 ml PS101/kg body weight (n = 8–15). To induce the phase shift, 45 s of US at 8 MHz at an MI of 0.30 was applied using a diagnostic US system; low frequency exposure consisted of 1 or 5 min at 500 kHz with an MI of 0.20. Results: ACT with irinotecan induced a strong, dose dependent increase in the therapeutic effect (R2 = 0.95). When compared to irinotecan alone, at the highest dose investigated, combination treatment induced a reduction in average normalized tumour volume from 14.6 (irinotecan), to 5.4 (ACT with irinotecan, p = 0.002) on day 27. Median survival increased from 34 days (irinotecan) to 54 (ACT with irinotecan, p = 0.002). Additionally, ACT with irinotecan induced an increase in the fraction of complete responders; from 7% to 26%. There was no significant difference in the therapeutic efficacy whether the low frequency US lasted 1 or 5 min. Furthermore, there was no significant difference between the enhancement observed in the efficacy of ACT with irinotecan when PS101+US was administered before or after irinotecan. An increase in early dropouts was observed at higher PS101 doses. Both mean tumour volume (on day 27) and median survival indicate that the PS101 dose response was linear in the range investigated.

Acoustic cluster therapy (ACT) is designed to overcome some of the limitations of conventional microbubbles for ultrasound-assisted delivery of drugs to tumors. ACT consists of clusters of oil microdroplets and microbubbles. Diagnostic ultrasound imaging can be used to visualize the microbubble component and to activate (vaporize) the droplets to produce large (>20 μm) bubbles which lodge in the tumor’s microvessels. A subsequent low-frequency ultrasound field then gently pulsates the activated bubbles which are in direct contact with endothelium, enhancing vascular permeability. We present in-vivo pre-clinical imaging experiments designed to better understand ACT behavior. Conventional microbubbles were observed to wash out of tumors within 6 min whereas activated ACT failed to wash out during the observation time of 15 min, confirming activation. Stationary echoes in tumors uniquely accumulated in fundamental B-mode images after ACT activation (but not with a microbubble agent, nor in nonlinear contrast mode) providing a signature for identifying ACT activation in vivo. ACT significantly enhanced CW800 dye uptake in tumor relative to dye alone. Photoacoustic CW800 signals appeared in the tumor periphery, suggesting potential for high-resolution, three-dimensional dynamic monitoring although with lower sensitivity than fluorescence imaging.

Acoustic Cluster Therapy (ACT) employs an intravenously-injected dispersion of clusters of microbubbles and oil microdroplets, in combination with ultrasound directed at the tumor, to enhance the delivery of a co-administered drug. Diagnostic ultrasound activates the clusters, causing the droplets to change phase and become large (> 20 m) bubbles which lodge in the tumor capillaries. Delivery-enhancement is then achieved using a low-frequency (e.g. 300 kHz) ultrasound field also directed at the tumor, which causes the stationary bubbles to pulsate whilst in direct contact with the vascular endothelium. ACT has been shown to significantly increase the efficacy of various chemotherapeutics in a variety of tumor models in mice. Here we demonstrate through preclinical in-vivo experiments that the efficacy of treating human triple negative breast cancer in mice with liposomal doxorubicin (Doxil®) is significantly enhanced by ACT; for example, 63 % of animals achieved complete, stable remission, in the group treated with the drug and ACT versus only 10 % for Doxil® alone (p < 0.02). We also show in the same experiments that the ACT contrast enhancement obtained under ultrasound activation of the ACT clusters, either before or during treatment, holds promise as an imaging biomarker for predicting response.

TPS3145 Background: Response to existing chemotherapeutics (chemo) can be limited by exposure, itself limited by systemic toxicity. Interstitial fluid pressure can impede transport of drugs with, in some cases

Acoustic cluster therapy (ACT) is a novel approach for ultrasound mediated, targeted drug delivery. In the current study, we have investigated ACT in combination with paclitaxel and Abraxane® for treatment of a subcutaneous human prostate adenocarcinoma (PC3) in mice. In combination with paclitaxel (12 mg/kg given i.p.), ACT induced a strong increase in therapeutic efficacy; 120 days after study start, 42% of the animals were in stable, complete remission vs. 0% for the paclitaxel only group and the median survival was increased by 86%. In combination with Abraxane® (12 mg paclitaxel/kg given i.v.), ACT induced a strong increase in the therapeutic efficacy; 60 days after study start 100% of the animals were in stable, remission vs. 0% for the Abraxane® only group, 120 days after study start 67% of the animals were in stable, complete remission vs. 0% for the Abraxane® only group. For the ACT + Abraxane group 100% of the animals were alive after 120 days vs. 0% for the Abraxane® only group. Proof of concept for Acoustic Cluster Therapy has been demonstrated; ACT markedly increases the therapeutic efficacy of both paclitaxel and Abraxane® for treatment of human prostate adenocarcinoma in mice. © 2016. This is the authors’ accepted and refereed manuscript to the article. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/

Acoustic cluster technology (ACT) is a two-component, microparticle formulation platform being developed for ultrasound-mediated drug delivery. Sonazoid microbubbles, which have a negative surface charge, are mixed with micron-sized perfluoromethylcyclopentane droplets stabilized with a positively charged surface membrane to form microbubble/microdroplet clusters. On exposure to ultrasound, the oil undergoes a phase change to the gaseous state, generating 20- to 40-μm ACT bubbles. An acoustic transmission technique is used to measure absorption and velocity dispersion of the ACT bubbles. An inversion technique computes bubble size population with temporal resolution of seconds. Bubble populations are measured both in vitro and in vivo after activation within the cardiac chambers of a dog model, with catheter-based flow through an extracorporeal measurement flow chamber. Volume-weighted mean diameter in arterial blood after activation in the left ventricle was 22 μm, with no bubbles >44 μm in diameter. After intravenous administration, 24.4% of the oil is activated in the cardiac chambers.

Background: Pancreatic ductal adenocarcinoma (PDAC), which has a five-year survival rate of less than 9%, is one of the most lethal types of malignancies. PDAC is notoriously resistant to conventional and targeted therapeutic agents. One of the main reasons for this extreme drug resistance is the desmoplastic and poorly vascularized stroma that forms a protective barrier around the PDAC cells. Acoustic Cluster Therapy (ACT) is a novel technology that utilizes microbubbles and ultrasound guidance to create localized openings in tumor vasculature, which leads to a transient increase in vascular permeability and allows drug to better penetrate the tumor bed. In this study we investigated the activity of ACT in enhancing the therapeutic effects of clinically used chemotherapeutic regimens in patient-derived xenograft (PDX) mouse models for PDAC. Method: PDAC patient-derived tumor xenografts were implanted subcutaneously into athymic nude mice which were enrolled into studies when tumor size reached between 150–180 mm3. The mice (10 mice/group) were treated with either chemotherapeutics alone or in combination with ACT. Two chemotherapeutic regimens were tested: the nab-paclitaxel (nab-pac, 15 mg/kg, i.v., QW×3) and gemcitabine (GEM, 60 mg/kg, i.p., QW×3) combination (nab-pac/GEM) and liposomal irinotecan (ONI, 15 mg/kg, i.v., QW×3). ACT given before or after the chemotherapies was evaluated. Results: nab-pac/GEM or ONI alone treatment showed significant inhibition of the tumor growth at the doses used. However, application of ACT before or after the chemotherapy treatment dramatically enhanced the activity and markedly induced tumor shrinkage (even > 100 days after the last dose of treatment). Applying ACT following the dosing of nab-pac/GEM resulted in a 90% reduction in tumor volume compared to nab-pac/GEM alone at Day 50 after the initial drug dosing (P= 0.001). Some of mice had complete remissions. At Day 120, 50% of the mice in nab-pac/GEM + ACT group were still in complete remission vs. 10% in the nab-pac/GEM alone group. Similar activity was also observed with ONI treatment. At Day 50 after the initiation of study, ONI + ACT induced a 85% reduction in tumor volume compared to ONI alone (P=0.024). At Day 120, 60% of the mice in the ONI + ACT treated group were still in complete remission vs. 10% in the ONI alone group. Applying ACT right before the dosing of chemotherapeutics also showed improvement of efficacy, but is slightly less than applying after the chemotherapy. Overall, these findings demonstrate the potential utility of ACT in improving the therapeutic efficacy of nab-pac/GEM and ONI treatments in PDAC and provide solid preclinical evidence that supports evaluation in patients. The data also provides insights for the design of clinical trials. Citation Format: Serina Ng, Andrew Healey, Svein Kvåle, Spiros Kotopoulis, Daniel D Von Hoff, Per Christian Sontum, Haiyong Han. Acoustic Cluster Therapy enhances the efficacy of chemotherapeutic regimens in patient-derived xenograft mouse models for pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A099. doi:10.1158/1535-7163.TARG-19-A099

A novel approach for ultrasound (US) mediated drug delivery - Acoustic Cluster Therapy (ACT) - is proposed, and basic characteristics of the ACT formulation are elucidated. The concept comprises administration of free flowing clusters of negatively charged microbubbles and positively charged microdroplets. The clusters are activated within the target pathology by diagnostic US, undergo an ensuing liquid-to-gas phase shift and transiently deposit 20-30 μm large bubbles in the microvasculature, occluding blood flow for ∼5-10 min. Further application of US will induce biomechanical effects that increases the vascular permeability, leading to a locally enhanced extravasation of components from the vascular compartment (e.g. released or co-administered drugs). Methodologies are detailed for determination of vital in-vitro characteristics of the ACT compound; cluster concentration and size distribution. It is shown how these attributes can be engineered through various formulation parameters, and their significance as predictors of biological behaviour, such as deposit characteristics, is demonstrated by US imaging in a dog model. Furthermore, in-vivo properties of the activated ACT bubbles are studied by intravital microscopy in a rat model, confirming the postulated behaviour of the concept.

Proof of principle for local drug delivery with Acoustic Cluster Therapy (ACT) was demonstrated in a human prostate adenocarcinoma growing in athymic mice, using near infrared (NIR) dyes as model molecules. A dispersion of negatively charged microbubble/positively charged microdroplet clusters are injected i.v., activated within the target pathology by diagnostic ultrasound (US), undergo an ensuing liquid-to-gas phase shift and transiently deposit 20–30 μm large bubbles in the microvasculature, occluding blood flow for ~ 5–10 min. Further application of low frequency US induces biomechanical effects that increase the vascular permeability, leading to a locally enhanced extravasation of components from the vascular compartment (e.g., released or co-administered drugs). Results demonstrated deposition of activated bubbles in tumor vasculature. Following ACT treatment, a significant and tumor specific increase in the uptake of a co-administered macromolecular NIR dye was shown. In addition, ACT compound loaded with a lipophilic NIR dye to the microdroplet component was shown to facilitate local release and tumor specific uptake. Whereas the mechanisms behind the observed increased and tumor specific uptake are not fully elucidated, it is demonstrated that the ACT concept can be applied as a versatile technique for targeted drug delivery. © 2016. This is the authors’ accepted and refereed manuscript to the article. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/

Microencapsulated and microsphere products have become increasingly popular in the pharmaceutical and cosmetic industry. They are used as controlled-release drug systems as well as in diagnostic contrast agents. An example on how these kinds of products can be optimised is presented. The selected product is optimised by the use of a model based on an on-line steady state optimisation concept where the objective is based on the final product characteristic, where the product characteristic is measured in-line. The on-line steady-state optimisation algorithm used is a modified simplex where the production constraints are embedded into the optimisation routine.

A diagnostic drug's manufacture and use has been modeled and optimized. The optimization is performed by means of a total approach, i.e. , the best combination of process parameters to give an adequate biologic ( in vivo ) response is determined. The examined drug is Infoson™, a new contrast agent for ultrasonographic imaging consisting of air-filled microspheres suspension with a thin shell of heat aggregated human albumin.

A dynamic model has been developed for the size fractionation process of gas-filled microspheres by flotation. The aim of this dynamic model is to control the fractionation process in an Nycomed Imaging AS Infoson ™ production plant. A general population balance is established for the dispersed phase that is later simplified for the specific particle suspension and process equipment used. This dynamic model has been tested against experimental data and shows a good correlation between the predicted and measured values. This paper describes both the physical phenomena and the modeling method.