Focused Ultrasound: Crossing Barriers to Treat Alzheimer‘s Disease

Keywords::

• Alzheimer‘s disease
• blood–brain barrier
• drug delivery
• focused ultrasound
• microbubbles

Alzheimer‘s disease (AD) is the leading cause of dementia in the elderly and the seventh leading cause of death in North America [101]. It is a progressive and irreversible neurodegenerative disease that has no cure. At the molecular and cellular levels, AD is characterized by the presence of plaques composed of amyloid-β (Aβ) peptides, neurofibrillary tangles, neuronal loss and deficits in neurotransmitters [1]. In people living with AD, this pathology translates into symptoms such as a decline in reasoning, loss of memory and general deterioration of cognitive capacities. Current AD treatments are limited to drugs that improve neurotransmitter function and provide symptomatic relief [2]. The development of novel therapeutics for curing AD focuses on either preventing amyloid aggregation or initiating break-up of amyloid plaques [1]. A limiting factor in targeting the brain with novel therapeutics is the presence of the blood–brain barrier (BBB) [3]. The BBB is a specialized structure that restricts passage of molecules, including most drugs, from the blood into the brain, presenting a challenge for the pharmaceutical treatment of brain diseases.

Regarding the delivery of therapeutics to eradicate AD pathology, technological advances in the field of focused ultrasound (FUS) hold great promise to bypass the BBB and target the brain without involving surgical approaches. It is now established that transcranial FUS can increase the permeability of the BBB and allow the passage of molecules from the blood to the brain in an efficient manner [4,5]. The process of FUS concentrates acoustic energy and deposits it in a small target volume in the body with minimal or no consequences to the surrounding tissue. There are two major effects, thermal and nonthermal, which can be observed at the target. For thermal applications, the ultrasound energy is converted to heat, which can be used to activate heat-sensitive drugs or to ablate tumors at localized targets [6]. For nonthermal applications, the ultrasound energy induces cavitation (oscillation of the bubbles in the tissue), which produces mechanical changes in the tissue [7]. Over the last 10 years, it has been demonstrated that FUS energy combined with microbubble contrast agents can be used to increase the permeability of the BBB, temporarily and reversibly, to allow access of therapeutic agents to the brain [8]. These findings provide a major breakthrough for treatment of brain diseases such as AD.

Targeting the brain with FUS: overcoming the skull challenge

Delivery of therapeutics to the brain using FUS has been complicated by the skull, which strongly attenuates energy and causes distortion of the ultrasound field. Therefore, it was long believed that FUS would have limited relevance for the treatment of brain diseases [9]. Early work by the Fry brothers demonstrated that FUS could induce changes in the brain, but at that time the technique was only possible after a craniotomy [10]. Recent production of phased-array transducers to distribute energy over the entire skull surface, in combination with high-performance computers to correct ultrasound wave distortions caused by the skull, has made transcranial therapy possible [11]. Similarly, improvements in the ability to target small, specific structures with ultrasound have been addressed by combining FUS with guidance by MRI (termed MRIgFUS) [12,13]. This significant progress in the field of transcranial FUS led to a clinical trial for the treatment of glioblastoma [14] and chronic pain [15], and to intense research aimed at promoting drug delivery to the brain by transiently and reversibly opening the BBB [5,7,8].

The power of microbubbles

Stable microbubbles have been used as contrast agents for medical imaging for decades, but the use of microbubbles for BBB disruption with ultrasound is a relatively recent discovery [4]. Prior to the use of microbubbles, FUS-induced BBB opening was almost always associated with hemorrhage and tissue damage due to the induction of unpredictable, inertial cavitation [16–18]. It was hypothesized that the preformed microbubbles injected into the bloodstream would act as energy-concentrators when exposed to ultrasound and would expand and contract at the frequency of the propagating acoustic wave, leading to BBB disruption [4]. Indeed, it was found that the presence of microbubbles in the bloodstream allows the BBB to be opened using a significantly reduced acoustic power, over 100-times less than that required to produce thermal damage in the tissue [8]. Recently, a passive cavitation detector was used to verify that a FUS-disrupted BBB was probably due to oscillations of microbubbles with the blood vessel, but was not dependent on violent collapse of bubbles (inertial cavitation), which is often associated with tissue damage [19,20]. Studies using electron microscopy have identified four potential methods of transport of circulating molecules across the BBB after transcranial FUS with microbubbes: transcytosis via endothelial cell receptors, between endothelial cells following mechanical disruption of the tight junctions, through the endothelial cells themselves by opening of the cell cytoplasm and by way of injury to the endothelial lining [21,22]. Regardless of the mechanism of action, it has been repeatedly observed that enhanced permeability of the BBB by FUS and microbubbles is safe and transient. Contrast-enhanced MRI has demonstrated that the efficacy of the BBB is restored at 6 h following disruption, and the BBB remained impenetrable 2 days, 5 days and 4 weeks later [23,24].

The FUS advantage for BBB opening

A variety of strategies have been used to improve distribution of therapeutics to the brain. Direct delivery into the brain via surgery or through an implanted device can result in targeted delivery, but these surgical procedures carry severe risks. The systemic administration of osmotic solutions [24], mannitol [25] and various chemicals including vasodilators [26] and solvents [27,28], have also been used to increase BBB permeability, but many of these agents are toxic and can cause neuronal damage. Since the incorporation of microbubbles into the FUS procedure, there has been no evidence of ischemic or apoptotic regions after BBB disruption [23,29,30]. Overall, the effects to the brain from FUS appear minimal and certainly less than what has been observed from other methods for drug delivery to the brain. In addition, systemic administration of chemical agents results in widespread permeabilization of the BBB. Such extensive opening of the BBB can have serious side effects since the drugs and other potentially cytotoxic compounds present in the blood will have direct access to the entire CNS for long periods of time. These side effects are eliminated with MRIgFUS as the BBB opening is carefully controlled; for example, the BBB could be opened in a small region of the substantia nigra or the striatum, which may be used to improve drug delivery for Parkinson‘s disease without affecting the rest of the brain. Alternatively, in the case of AD where pathology is extensive and widespread disruption of the BBB is desirable, the selectivity of MRIgFUS still presents an advantage over indiscriminate chemical agents. FUS allows the BBB to be disrupted sequentially over a large timescale: one brain region is allowed to recover prior to the disruption of the next region. In conjunction with the briefness with which the BBB is disrupted with FUS, this sequential approach is able to treat the entire brain with minimal exposure to other potentially toxic blood components.

Currently, systemic administration of therapeutics for treatment of neurodegenerative disease involves multiple doses of high concentrations of drug, delivered systematically over several months or years [31]. FUS can be used to overcome the low penetration of drugs into the brain and the unwanted side effects associated with high levels of circulating drugs by increasing the permeability of the BBB. Using FUS to improve drug penetration will also decrease the cost of the therapy and reduce the number of injections, making treatments more practical and easier on the patients and their caregivers.

Drug delivery using FUS is not limited to treatment of neurodegenerative disease. The disruption of the BBB using FUS has many applications in the brain, such as improving access of chemotherapy agents to brain tumors, targeting gene therapy and delivering antibodies for breast cancer metastases [32].

The potential of FUS to improve AD therapies

Immunotherapy with Aβ is based on the ability of Aβ antibodies to clear plaques from the brain and decrease toxicity to remaining neuronal populations. In mouse models of AD, systemic administration of Aβ antibodies causes a marked reduction in amyloid burden and an improvement in cognitive function [33]. Recently, it was shown that direct delivery of low doses of Aβ antibodies to the brain via intracranial injection was more efficient at reducing Aβ plaques than peripheral injections of high doses of antibody [34,35]. Furthermore, the direct-delivery method minimized the unwanted side effects of excess antibody on the vasculature [34]. Despite the efficiency of the treatment, intracranial injections represent obvious challenges for translational application in AD patients. The use of FUS to open the BBB offers the potential to benefit from the increased efficiency and efficacy of direct drug injection without the need for an invasive surgery.

The first evidence that FUS may be applicable for delivery of antibodies to the brain came in 2006 when Kinoshita and colleagues demonstrated that antibodies circulating in the bloodstream can be detected in the brain after BBB opening by FUS in young mice [36,37]. Despite these findings, administration of antibodies to AD brains was uncertain since the amyloid pathology, which is present in the brain and the vasculature, is suggested to significantly impact the properties and function of the BBB [38]. Initial studies were aimed at addressing the safety of opening the BBB in AD mice. The results demonstrated that the properties of BBB disruption by transcranial FUS were not significantly different in AD transgenic mice (PS1/APP), compared with wild-type controls [39]. Furthermore, in a similar AD mouse model (PDAPP), it was found that the opening of the BBB was comparable in aged mice (12 vs 26 months), despite an increased brittleness of the skull and altered vasculature observed in the older transgenic animals [40]. Raymond and colleagues also showed that Aβ antibodies given in the bloodstream were co-localized with trypan blue-stained plaques in the brain following BBB opening with FUS [40]. These studies demonstrated the feasibility of targeting amyloid plaques present in the brain with Aβ antibodies administered in the bloodstream that crosses the BBB after transcranial FUS.

The landmark study demonstrating the efficacy of FUS in reducing amyloid plaque pathology was published in 2010 [41]. TgCRND mice that develop extensive amyloid deposits and display cognitive deficits were used in this study. A total of 40 µg of BAM-10 (anti-Aβ) antibodies were injected into the circulation through a tail-vein catheter. Immediately after this, the BBB was opened at four locations in the right hemisphere using a custom-built FUS system. At all time points after BBB opening (4 h, 2 and 4 days), BAM-10 antibody was found to specifically bind to plaques only on the right, MRIgFUS-targeted side of the brain. No BAM-10 antibody was detected in the left hemisphere, indicating the antibody could be delivered from the peripheral circulation to targeted areas of the brain. Stereology was performed 4 days after treatment and revealed that there were 12% fewer plaques in the cortex on the MRIgFUS-targeted side compared with the control side. In addition, the mean plaque size was reduced by 12% and the surface area of the plaques was reduced by 23%, indicating significant overall reduction in AD pathology. This study is the first evidence that FUS can be used to target drug delivery and reduce AD pathology.

Clinical trials using Aβ antibodies in AD patients have entered Phase III with hopes of success somewhat dampened by cautionary notes on safety and efficacy [42,43]. Bapineuzumap, a fully humanized Aβ antibody, raised safety concerns after 12 of 238 patients developed vasogenic edema [31]. These results highlight the potential human safety issues associated with peripheral Aβ clearance. In our opinion, targeting the delivery of the antibody to the brain could benefit the passive-immunization approach to AD by reducing the levels of circulating antibody and by improving the penetration of the antibody to the brain regions where it is needed most.

The future of FUS in the treatment of AD

Bowman et al., suggested that the presence of amyloid in the AD brain can impair the structure and function of the BBB and that increased degrees of BBB dysfunction are associated with increased rates of neurodegeneration [44]. The status of the BBB in AD is variable between patients, but it remains clear that drug delivery to the brain in AD could be improved by FUS. Importantly, the BBB seems able to restore its integrity even under deleterious circumstances. For example, following a stroke or brain trauma, physical damage to the blood vessels at the site of injury temporarily disrupts normal BBB function and allows compounds present in the blood to gain direct access to brain tissue. Still, the BBB can return to normal function after only hours [45]. The plasticity of the BBB in other damaging conditions as well as some preliminary evidence [39,40] suggests that even in the AD brain, the BBB could sustain some disruption with MRIgFUS and be capable of restoring its integrity and function over time. Careful evaluation of the effect of FUS on the BBB in mouse models of AD is underway.

Using FUS to open the BBB will significantly broaden the type of therapeutics being developed for treatment of AD. FUS will improve the capacity for several drug categories to pass the BBB, related to the amyloid cascade and directed onto novel targets, which may be curative in addition to providing symptomatic relief for AD patients.

The transient opening of the BBB by FUS has therapeutic implications that extend beyond traditional drug delivery to the brain. Cell replacement and gene therapy to the brain and spinal cord could be envisioned using MRIgFUS. Experiments are necessary to determine whether FUS can open the BBB enough to allow cell entry and to facilitate gene delivery to the brain without causing damage to the tissue.

Finally, the use of FUS could potentially increase neuronal activity and perhaps be of benefit by targeting specific neuronal pathways in AD and in other neurodegenerative disorders. This type of research is in its infancy and much remains to be understood on the effects of FUS to the brain, but some studies have shown promise for the use of FUS to stimulate neuronal activity. Indeed, low-frequency ultrasound can activate sodium- and calcium-gated channels leading to increased neuronal activity [46]. Furthermore, Tufail and colleagues demonstrated that ultrasound stimulation could increase the electrical activity of neurons in the motor cortex without inducing BBB opening or causing any damage to the tissue [47]. The authors also found that FUS stimulation of the hippocampus, a structure relevant to learning and memory and which is affected in AD, increased electrical activity and the levels of brain-derived neurotrophic factor, an important regulator of memory consolidation. These studies raise the interesting possibility that FUS alone may be able to stimulate brain circuits and enhance levels of neurotrophins and neurotransmitter release, all of which could have beneficial effects on the AD brain.

Conclusions

Focused ultrasound with microbubbles holds great promise for targeted delivery of therapeutics to treat neurodegenerative diseases such as AD. It has already been demonstrated that FUS improves delivery of Aβ antibodies to the brain, resulting in decreased amyloid pathology in a mouse model of AD. The effects of FUS on the brain and optimal antibody delivery need to be further evaluated to ensure the safety of the procedure before human patients are treated. However, slight modifications of the technology currently tested in clinical trials for thermal ablation of tumors could make MRIgFUS applications to the AD brain possible. Targeting specific brain regions of interest with MRIgFUS holds great promise for the development of treatments for AD and other brain disorders.

Financial & competing interests disclosure

Kullervo Hynynen is an investor in patents related to focusing ultrasound through skull and disrupting the blood–brain barrier with ultrasound. These patents are owned by Brigham and Women‘s Hospital, Boston, MA, USA. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.