Current clinical investigations of focused ultrasound blood-brain barrier disruption: A review.

The blood-brain barrier (BBB) presents a formidable challenge in delivering therapeutic agents to the central nervous system. Ultrasound-mediated BBB disruption has emerged as a promising non-invasive technique to enhance drug delivery to the brain. This manuscript reviews fundamental principles of ultrasound-based techniques and their mechanisms of action in temporarily permeabilizing the BBB. Clinical trials employing ultrasound for BBB disruption are discussed, summarizing diverse applications ranging from the treatment of neurodegenerative diseases to targeted drug delivery for brain tumors. The review also addresses safety considerations, outlining the current understanding of potential risks and mitigation strategies associated with ultrasound exposure, including real-time monitoring and assessment of treatment efficacy. Among the large number of studies, significant successes are highlighted thus providing perspective on the future direction of the field.

Nanoparticle-Epoxy Composite Molding for Undeformed Acoustic Holograms With Tailored Acoustic Properties.

Acoustic hologram (AH) lenses are typically produced by high-resolution 3-D printing methods, such as stereolithography (SLA) printing. However, SLA printing of thin, plate-shaped lens structures has major limitations, including vulnerability to deformation during photocuring and limited control of acoustic impedance. To overcome these limitations, we demonstrated a nanoparticle-epoxy composite (NPEC) molding technique, and we tested its feasibility for AH lens fabrication. The characterized acoustic impedance of the 22.5% NPEC was 4.64 MRayl, which is 55% higher than the clear photopolymer (2.99 MRayl) used by SLA. Simulations demonstrated that the improved pressure transmission by the higher acoustic impedance of the NPEC resulted in 21% higher pressure amplitude in the region of interest (ROI, -6-dB pressure amplitude pixels) than the photopolymer. This improvement was experimentally demonstrated after prototyping NPEC lenses through a molding process. The NPEC lens showed no significant deformation and 72% lower thickness profile errors than the photopolymer, which otherwise experienced deformed edges due to thermal bending. Beam mapping results using the NPEC lens validated the predicted improvement, demonstrating 24% increased pressure amplitude on average and 10% improved structural similarity (SSIM) with the simulated pressure pattern compared to the photopolymer lens. This method can be used for AH lens applications with improved pressure output and accurate pressure field formation.

Effect of Anesthetic Carrier Gas on In Vivo Circulation Times of Intravenously Administered Phospholipid Oxygen Microbubbles in Rats.

OBJECTIVE: For the treatment of tumor hypoxia, microbubbles comprising oxygen as a majority component of the gas core with a stabilizing shell may be used to deliver and release oxygen locally at the tumor site through ultrasound destruction. Previous work has revealed differences in circulation half-life in vivo for perfluorocarbon-filled microbubbles, typically used as ultrasound imaging contrast agents, as a function of anesthetic carrier gas. These differences in circulation time in vivo were likely due to gas diffusion as a function of anesthetic carrier gas, among other variables. This work has motivated studies to evaluate the effect of anesthetic carrier gas on oxygen microbubble circulation dynamics. METHODS: Circulation time for oxygen microbubbles was derived from ultrasound image intensity obtained during longitudinal kidney imaging. Studies were constructed for rats anesthetized on inhaled isoflurane with either pure oxygen or medical air as the anesthetic carrier gas. RESULTS: Results indicated that oxygen microbubbles were highly visible via contrast-specific imaging. Marked signal enhancement and duration differences were observed between animals breathing air and oxygen. Perhaps counterintuitively, oxygen microbubbles disappeared from circulation significantly faster when the animals were breathing pure oxygen compared with medical air. This may be explained by nitrogen counterdiffusion from blood into the bubble, effectively changing the gas composition of the core, as has been observed in perfluorocarbon core microbubbles. CONCLUSION: Our findings suggest that the apparent longevity and persistence of oxygen microbubbles in circulation may not be reflective of oxygen delivery when the animal is anesthetized breathing air.

Overcoming biological barriers to improve treatment of a Staphylococcus aureus wound infection.

Chronic wounds frequently become infected with bacterial biofilms which respond poorly to antibiotic therapy. Aminoglycoside antibiotics are ineffective at treating deep-seated wound infections due to poor drug penetration, poor drug uptake into persister cells, and widespread antibiotic resistance. In this study, we combat the two major barriers to successful aminoglycoside treatment against a biofilm-infected wound: limited antibiotic uptake and limited biofilm penetration. To combat the limited antibiotic uptake, we employ palmitoleic acid, a host-produced monounsaturated fatty acid that perturbs the membrane of gram-positive pathogens and induces gentamicin uptake. This novel drug combination overcomes gentamicin tolerance and resistance in multiple gram-positive wound pathogens. To combat biofilm penetration, we examined the ability of sonobactericide, a non-invasive ultrasound-mediated-drug delivery technology to improve antibiotic efficacy using an in vivo biofilm model. This dual approach dramatically improved antibiotic efficacy against a methicillin-resistant Staphylococcus aureus (MRSA) wound infection in diabetic mice.

Loading Intracranial Drug-Eluting Reservoirs Across the Blood-Brain Barrier With Focused Ultrasound.

OBJECTIVE: Efficient, sustained and long-term delivery of therapeutics to the brain remains an important challenge to treatment of diseases such as brain cancer, stroke and neurodegenerative disease. Focused ultrasound can assist movement of drugs into the brain, but frequent and long-term use has remained impractical. Single-use intracranial drug-eluting depots show promise but are limited for the treatment of chronic diseases as they cannot be refilled non-invasively. Refillable drug-eluting depots could serve as a long-term solution, but refilling is hindered by the blood-brain barrier (BBB), which prevents drug refills from accessing the brain. In this article, we describe how focused ultrasound enables non-invasive loading of intracranial drug depots in mice. METHODS: Female CD-1 mice (n = 6) were intracranially injected with click-reactive and fluorescent molecules that are capable of anchoring in the brain. After healing, animals were treated with high-intensity focused ultrasound and microbubbles to temporarily increase the permeability of the blood-brain barrier and deliver dibenzocyclooctyne (DBCO)-Cy7. The mice were perfused, and the brains were imaged via ex vivo fluorescence imaging. RESULTS: Fluorescence imaging indicated small molecule refills are captured by intracranial depots as long as 4 wk after administration and are retained for up to 4 wk based on fluorescence imaging. Efficient loading was dependent on both focused ultrasound and the presence of refillable depots in the brain as absence of either prevented intracranial loading. CONCLUSION: The ability to target and retain small molecules at predetermined intracranial sites with pinpoint accuracy provides opportunities to continuously deliver drugs to the brain over weeks and months without excessive BBB opening and with minimal off-target side effects.

Optimization and Scale Up of Spray Dried CPZEN-45 Aerosol Powders for Inhaled Tuberculosis Treatment.

PURPOSE: Tuberculosis (TB) remains one of the most serious diseases caused by a single organism. Multiple (MDR) and extensively (XDR) drug resistant disease poses a threat to global health and requires new drugs and/or innovative approaches to treatment. A number of drugs have been proposed as inhaled therapy for TB, frequently prepared by spray drying. CPZEN-45 is a novel anti-tubercular drug that has poor oral bioavailability but has shown promise when administered via inhalation. METHODS: Excipient-free CPZEN-45 HCl has been spray dried into a powder with physicochemical characteristics, aerodynamic particle size distribution, and delivered dose suitable for consideration as an inhaled product. RESULTS: The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of the powder delivered using a RS01 inhaler were 2.62 ± 0.04 µm and 1.76 ± 0.09, respectively. Additionally, the powder was physically and chemically stable after storage at ambient conditions for >1.5 years with particle size similar to freshly manufactured product. Overages in spray dried powder were recycled the powder and resprayed into drug product likewise resulting in negligible change in quality thus allowing for further preclinical characterization as necessary. CPZEN-45 was scaled up using pilot-scale manufacturing equipment where the density of the powder was increased to facilitate larger delivered doses without affecting the aerodynamic performance properties. CONCLUSION: The spray dried powders were suitable for pharmacokinetics, efficacy and preclinical toxicology studies. The final method of manufacture may be used directly for CGMP particle manufacture to support IND and Phase I clinical trials and beyond.

Low-Intensity Focused Ultrasound Produces Immune Response in Pancreatic Cancer.

Pancreatic adenocarcinoma is an aggressive malignancy with limited therapeutic treatments available and a 5-y survival less than 10%. Pancreatic cancers have been found to be immunogenically "cold" with a largely immunosuppressive tumor microenvironment. There is emerging evidence that focused ultrasound can induce changes in the tumor microenvironment and have a constructive impact on the effect of immunotherapy. However, the immune cells and timing involved in these effects remain unclear, which is essential to determining how to combine immunotherapy with ultrasound for treatment of pancreatic adenocarcinoma. We used low-intensity focused ultrasound and microbubbles (LoFU + MBs), which can mechanically disrupt cellular membranes and vascular endothelia, to treat subcutaneous pancreatic tumors in C57BL/6 mice. To evaluate the immune cell landscape and expression and/or localization of damage-associated molecular patterns (DAMPs) as a response to ultrasound, we performed flow cytometry and histology on tumors and draining lymph nodes 2 and 15 d post-treatment. We repeated this study on larger tumors and with multiple treatments to determine whether similar or greater effects could be achieved. Two days after treatment, draining lymph nodes exhibited a significant increase in activated antigen presenting cells, such as macrophages, as well as expansion of CD8+ T cells and CD4+ T cells. LoFU + MB treatment caused localized damage and facilitated the translocation of DAMP signals, as reflected by an increase in the cytoplasmic index for high-mobility-group box 1 (HMGB1) at 2 d. Tumors treated with LoFU + MBs exhibited a significant decrease in growth 15 d after treatment, indicating a tumor response that has the potential for additive effects. Our studies indicate that focused ultrasound treatments can cause tumoral damage and changes in macrophages and T cells 2 d post-treatment. The majority of these effects subsided after 15 d with only a single treatment, illustrating the need for additional treatment types and/or combination with immunotherapy. However, when larger tumors were treated, the effects seen at 2 d were diminished, even with an additional treatment. These results provide a working platform for further rational design of focused ultrasound and immunotherapy combinations in poorly immunogenic cancers.

Low-boiling Point Perfluorocarbon Nanodroplets as Dual-Phase Dual-Modality MR/US Contrast Agent.

Detection of bare gas microbubbles by magnetic resonance (MR) at low concentrations typically used in clinical contrast-ultrasound studies was recently demonstrated using hyperCEST. Despite the enhanced sensitivity achieved with hyperCEST, in vivo translation is challenging as on-resonance saturation of the gas-phase core of microbubbles consequently results in saturation of the gas-phase hyperpolarized 129 Xe within the lungs. Alternatively, microbubbles can be condensed into the liquid phase to form perfluorocarbon nanodroplets, where 129 Xe resonates at a chemical shift that is separated from the gas-phase signal in the lungs. For ultrasound applications, nanodroplets can be acoustically reverted back into their microbubble form to act as a phase-change contrast agent. Here, we show that low-boiling point perfluorocarbons, both in their liquid and gas form, generate phase-dependent hyperCEST contrast. Magnetic resonance detection of ultrasound-mediated phase transition demonstrates that these perfluorocarbons could be used as a dual-phase dual-modality MR/US contrast agent.

Polyvinyl Alcohol Cryogels for Acoustic Characterization of Phase-Change Contrast Agents.

Phase-change contrast agents (PCCAs) consisting of lipid-encapsulated low-boiling-point perfluorocarbons can be used in conjunction with ultrasound for diagnostic and therapeutic applications. One benefit of PCCAs is site-specific activation, whereby the liquid core is acoustically vaporized into a bubble detectable via ultrasound imaging. For further evaluation of PCCAs in a variety of applications, it is useful to disperse these nanodroplets into an acoustically compatible stationary matrix. However, many traditional phantom preparations require heating, which causes premature thermal activation of low-boiling-point PCCAs. Polyvinyl alcohol (PVA) cryogels do not require heat to set. Here we propose a simple method for the incorporation of the low-boiling-point PCCAs using octafluoropropane (OFP) and decafluorobutane (DFB) into PVA cryogels for a variety of acoustic characterization applications. We determined the utility of the phantoms by activating droplets with a focused transducer, visualizing the lesions with ultrasound imaging. At 1 MHz, droplet activation was consistently observed at 2.0 and 4.0 MPa for OFP and DFB, respectively.

Harnessing ultrasound-stimulated phase change contrast agents to improve antibiotic efficacy against methicillin-resistant Staphylococcus aureus biofilms.

Bacterial biofilms, often associated with chronic infections, respond poorly to antibiotic therapy and frequently require surgical intervention. Biofilms harbor persister cells, metabolically indolent cells, which are tolerant to most conventional antibiotics. In addition, the biofilm matrix can act as a physical barrier, impeding diffusion of antibiotics. Novel therapeutic approaches frequently improve biofilm killing, but usually fail to achieve eradication. Failure to eradicate the biofilm leads to chronic and relapsing infection, is associated with major financial healthcare costs and significant morbidity and mortality. We address this problem with a two-pronged strategy using 1) antibiotics that target persister cells and 2) ultrasound-stimulated phase-change contrast agents (US-PCCA), which improve antibiotic penetration. We previously demonstrated that rhamnolipids, produced by Pseudomonas aeruginosa, could induce aminoglycoside uptake in gram-positive organisms, leading to persister cell death. We have also shown that US-PCCA can transiently disrupt biological barriers to improve penetration of therapeutic macromolecules. We hypothesized that combining antibiotics which target persister cells with US-PCCA to improve drug penetration could improve treatment of methicillin resistant S. aureus (MRSA) biofilms. Aminoglycosides alone or in combination with US-PCCA displayed limited efficacy against MRSA biofilms. In contrast, the anti-persister combination of rhamnolipids and aminoglycosides combined with US-PCCA dramatically improved biofilm killing. This novel treatment strategy has the potential for rapid clinical translation as the PCCA formulation is a variant of FDA-approved ultrasound contrast agents that are already in clinical practice and the low-pressure ultrasound settings used in our study can be achieved with existing ultrasound hardware at pressures below the FDA set limits for diagnostic imaging.

Magnetic Resonance Detection of Gas Microbubbles via HyperCEST: A Path Toward Dual Modality Contrast Agent.

Gas microbubbles are an established clinical ultrasound contrast agent. They could also become a powerful magnetic resonance (MR) intravascular contrast agent, but their low susceptibility-induced contrast requires high circulating concentrations or the addition of exogenous paramagnetic nanoparticles for MR detection. In order to detect clinical in vivo concentrations of raw microbubbles via MR, an alternative detection scheme must be used. HyperCEST is an NMR technique capable of indirectly detecting signals from very dilute molecules (concentrations well below the NMR detection threshold) that exchange hyperpolarized 129 Xe. Here, we use quantitative hyperCEST to show that microbubbles are very efficient hyperCEST agents. They can accommodate and saturate millions of 129 Xe atoms at a time, allowing for their indirect detection at concentrations as low as 10 femtomolar. The increased MR sensitivity to microbubbles achieved via hyperCEST can bridge the gap for microbubbles to become a dual modality contrast agent.

A Spray-Dried Combination of Capreomycin and CPZEN-45 for Inhaled Tuberculosis Therapy.

Tuberculosis (TB) remains the single most serious infectious disease attributable to a single-causative organism. A variety of drugs have been evaluated for pulmonary delivery as dry powders: capreomycin sulfate has shown efficacy and was safely delivered by inhalation at high doses to human volunteers, whereas CPZEN-45 is a new drug that has also been shown to kill resistant TB. The studies here combine these drugs-acting by different mechanisms-as components of single particles by spray-drying, yielding a new combination drug therapy. The spray-dried combination powder was prepared in an aerodynamic particle size range suitable for pulmonary delivery. Physicochemical storage stability was demonstrated for a period of 6 months. The spray-dried combination powders of capreomycin and CPZEN-45 have only moderate affinity for mucin, indicating that delivered drug will not be bound by these mucins in the lung and available for microbicidal effects. The pharmacokinetics of disposition in guinea pigs demonstrated high local concentrations of drug following direct administration to the lungs and subsequent systemic bioavailability. Further studies are required to demonstrate the in vivo efficacy of the combination to confirm the therapeutic potential of this novel combination.

Efficacy of pyrazinoic acid dry powder aerosols in resolving necrotic and non-necrotic granulomas in a guinea pig model of tuberculosis.

New therapeutic strategies are needed to treat drug resistant tuberculosis (TB) and to improve treatment for drug sensitive TB. Pyrazinamide (PZA) is a critical component of current first-line TB therapy. However, the rise in PZA-resistant TB cases jeopardizes the future utility of PZA. To address this problem, we used the guinea pig model of TB and tested the efficacy of an inhaled dry powder combination, referred to as Pyrazinoic acid/ester Dry Powder (PDP), which is comprised of pyrazinoic acid (POA), the active moiety of PZA, and pyrazinoic acid ester (PAE), which is a PZA analog. Both POA and PAE have the advantage of being able to act on PZA-resistant Mycobacterium tuberculosis. When used in combination with oral rifampicin (R), inhaled PDP had striking effects on tissue pathology. Effects were observed in lungs, the site of delivery, but also in the spleen and liver indicating both local and systemic effects of inhaled PDP. Tissue granulomas that harbor M. tuberculosis in a persistent state are a hallmark of TB and they pose a challenge for therapy. Compared to other treatments, which preferentially cleared non-necrotic granulomas, R+PDP reduced necrotic granulomas more effectively. The increased ability of R+PDP to act on more recalcitrant necrotic granulomas suggests a novel mechanism of action. The results presented in this report reveal the potential for developing therapies involving POA that are optimized to target necrotic as well as non-necrotic granulomas as a means of achieving more complete sterilization of M. tuberculosis bacilli and preventing disease relapse when therapy ends.

Disposable Dosators for Pulmonary Insufflation of Therapeutic Agents to Small Animals.

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver drugs locally and systemically. Evaluation of dry powder aerosols necessitates an efficient dispersion mechanism to maintain high concentrations in an exposure chamber or for direct endotracheal administration. While solutions exist to expose animals by passive inhalation to dry powder aerosols, most require masses of powder in large excess of the dose delivered. This precludes conducting early feasibility studies as insufficient drug is available at the research or early development stage to support the dose delivery requirements for conventional aerosol delivery systems. When designing an aerosol drug product, aerodynamic performance can relate directly to delivery efficiency and efficacy. Dispersion of powder into an aerosol requires energy input sufficient to overcome interparticulate forces, and particle engineering approaches can substantially improve aerosol performance. We have developed a dispersion system (dosator) which can aerosolize engineered dry powder aerosols efficiently for the purpose of direct pulmonary insufflation, dispersion into an exposure system or generation for analytical purposes.

In vitro dry powder inhaler formulation performance considerations.

It has long been desired to match airflow conditions during formulation evaluation to those of relevance to lung deposition. In this context several strategies have been adopted involving sampling at different: flow rate (without consideration of flow conditions, e.g. shear, Reynolds number, work function); pressure drop (with and without consideration of flow conditions) and; flow rate and pressure drop. Performance testing has focused on the influence of these sampling conditions on delivered dose uniformity and aerodynamic particle size distribution. However, in order to be physiologically relevant it is also important to know when the drug was delivered with respect to initiation of airflow as variation in this parameter would influence lung deposition. A light obscuration method of detecting the dose delivered from a dry powder inhaler while sampling for aerodynamic particle size distributions (APSD) by inertial impaction has been developed. Four formulations of albuterol sulfate and budesonide in sieved and milled lactose, respectively, were dispersed and their rate of delivery monitored. The differences observed have the potential to impact the site of delivery in the lungs. The rate of delivery of drug is clearly an important companion measurement to delivered dose and APSD if the intent is to predict the similarity of in vivo performance of dry powder inhaler products.