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Targeted delivery of antibodies through the blood–brain barrier by MRI-guided focused ultrasound
Abstract
The blood-brain barrier (BBB) is a persistent obstacle for the local delivery of macromolecular therapeutic agents to the central nervous system (CNS). Many drugs that show potential for treating CNS diseases cannot cross the BBB and there is a need for a non-invasive targeted drug delivery method that allows local therapy of the CNS using larger molecules. We developed a non-invasive technique that allows the image-guided delivery of antibody across the BBB into the murine CNS. Here, we demonstrate that subsequent to MRI-targeted focused ultrasound induced disruption of BBB, intravenously administered dopamine D(4) receptor-targeting antibody crossed the BBB and recognized its antigens. Using MRI, we were able to monitor the extent of BBB disruption. This novel technology should be useful in delivering macromolecular therapeutic or diagnostic agents to the CNS for the treatment of various CNS disorders.
... The ability to observe BBB disruption in vivo is conferred by CE-T1 MRI, without requiring postmortem histological analysis. Multiple investigations have shown correlative relationships between the extravasation of the MRI contrast agent and that of histological tracers [61,62] and some therapeutic agents, including Herceptin [31], doxorubicin [63], and nanoparticles [23,38]. A few studies have employed the use of T2/T2* weighted MRI to track the uptake of superparamagnetic iron oxide (SPIO)-labelled drug molecules into sonicated brain parenchyma. ...
... Reported histopathological outcomes have been identified at a variety of endpoints, from immediately following [115] to months after initial sonication [123], highlighting both the potential acuteness and chronicity at which ultrasound-mediated BBB disruption may exert unwanted biological effects. Histopathological assessments generally reinforce pathological findings on MRI, but in some studies [62,92,93], they appear to highlight pathological change in the absence of any on MRI, despite equivalent timing of data acquisition, suggesting higher sensitivity for adverse pathological change. ...
... Additionally, significant patient neurological comorbidity in these trials makes it difficult to directly attribute adverse events to ultrasound-mediated BBB disruption. X [76] X [60] X X [77] X X [68] X [78] X X X [61] X X X X (PCR + WB) [31] X X X X [62] X X X X X X [54] X X X [52] X X X X [9] X X [10] X X X X [11] X X [53] X ...
Ultrasound-mediated blood–brain barrier (BBB) disruption has garnered focus as a method of delivering normally impenetrable drugs into the brain. Numerous studies have investigated this approach, and a diverse set of ultrasound parameters appear to influence the efficacy and safety of this approach. An understanding of these findings is essential for safe and reproducible BBB disruption, as well as in identifying the limitations and gaps for further advancement of this drug delivery approach. We aimed to collate and summarise protocols and parameters for achieving ultrasound-mediated BBB disruption in animal and clinical studies, as well as the efficacy and safety methods and outcomes associated with each. A systematic search of electronic databases helped in identifying relevant, included studies. Reference lists of included studies were further screened to identify supplemental studies for inclusion. In total, 107 articles were included in this review, and the following parameters were identified as influencing efficacy and safety outcomes: microbubbles, transducer frequency, peak-negative pressure, pulse characteristics, and the dosing of ultrasound applications. Current protocols and parameters achieving ultrasound-mediated BBB disruption, as well as their associated efficacy and safety outcomes, are identified and summarised. Greater standardisation of protocols and parameters in future preclinical and clinical studies is required to inform robust clinical translation.
... Microbubbles of 1-5 µm (reF. 278 ) oscillate during exposure to Fus, resulting in transient opening of the BBB at Fus-targeted sites, allowing blood-borne therapeutics and imaging agents to enter the brain at the targeted regions [279][280][281] . Fus parameters, such as burst duration and repetition frequency, pressure amplitude and exposure time, have to be optimized to achieve transient BBB opening and minimize Fus-MB-related brain damage 275,281 . ...
... Fus parameters, such as burst duration and repetition frequency, pressure amplitude and exposure time, have to be optimized to achieve transient BBB opening and minimize Fus-MB-related brain damage 275,281 . Fus-MB-induced BBB opening lasts approximately 1-4 hours and is recovered after about 24 hours 278 . in mice, Fus-MB allowed molecules of 3-2,000 kDa -such as doxorubicin, trastuzumab, rNa molecules and contrast imaging agents -to enter the brain 279,280,[282][283][284][285] , and was also found to increase transcytosis and fenestration of BBB cells in sonicated regions 286 . a pilot study using transcranial Mri-guided Fus (MrgFus) for site-specific BBB opening in patients with brain tumours intravenously infused with doxorubicin or temozolomide 287 showed MrgFus was well tolerated without adverse events. ...
... this trial was based on promising preclinical results 289,290 , and the results are yet to be published. safety is a major concern for Fus-MB-induced BBB opening as Fus has been shown to cause microhaemorrhages in the mouse brain 279,280,291 , and mechanical effects caused by Fus forces and microbubbles are known to damage blood vessels 292 . Furthermore, dark neurons and microvacuolated sites were observed 30 minutes after sonication, as were the functional effects of reduced blood oxygen level-dependent responses and functional connectivity in sonicated regions 291 . ...
Achieving sufficient delivery across the blood–brain barrier is a key challenge in the development of drugs to treat central nervous system (CNS) disorders. This is particularly the case for biopharmaceuticals such as monoclonal antibodies and enzyme replacement therapies, which are largely excluded from the brain following systemic administration. In recent years, increasing research efforts by pharmaceutical and biotechnology companies, academic institutions and public–private consortia have resulted in the evaluation of various technologies developed to deliver therapeutics to the CNS, some of which have entered clinical testing. Here we review recent developments and challenges related to selected blood–brain barrier- crossing strategies — with a focus on non- invasive approaches such as receptor- mediated transcytosis and the use of neurotropic viruses, nanoparticles and exosomes — and analyse their potential in the treatment of CNS disorders.
... Microbubbles of 1-5 µm (reF. 278 ) oscillate during exposure to Fus, resulting in transient opening of the BBB at Fus-targeted sites, allowing blood-borne therapeutics and imaging agents to enter the brain at the targeted regions [279][280][281] . Fus parameters, such as burst duration and repetition frequency, pressure amplitude and exposure time, have to be optimized to achieve transient BBB opening and minimize Fus-MB-related brain damage 275,281 . ...
... Fus parameters, such as burst duration and repetition frequency, pressure amplitude and exposure time, have to be optimized to achieve transient BBB opening and minimize Fus-MB-related brain damage 275,281 . Fus-MB-induced BBB opening lasts approximately 1-4 hours and is recovered after about 24 hours 278 . in mice, Fus-MB allowed molecules of 3-2,000 kDa -such as doxorubicin, trastuzumab, rNa molecules and contrast imaging agents -to enter the brain 279,280,[282][283][284][285] , and was also found to increase transcytosis and fenestration of BBB cells in sonicated regions 286 . a pilot study using transcranial Mri-guided Fus (MrgFus) for site-specific BBB opening in patients with brain tumours intravenously infused with doxorubicin or temozolomide 287 showed MrgFus was well tolerated without adverse events. ...
... this trial was based on promising preclinical results 289,290 , and the results are yet to be published. safety is a major concern for Fus-MB-induced BBB opening as Fus has been shown to cause microhaemorrhages in the mouse brain 279,280,291 , and mechanical effects caused by Fus forces and microbubbles are known to damage blood vessels 292 . Furthermore, dark neurons and microvacuolated sites were observed 30 minutes after sonication, as were the functional effects of reduced blood oxygen level-dependent responses and functional connectivity in sonicated regions 291 . ...
Achieving sufficient delivery across the blood–brain barrier is a key challenge in the development of drugs to treat central nervous system (CNS) disorders. This is particularly the case for biopharmaceuticals such as monoclonal antibodies and enzyme replacement therapies, which are largely excluded from the brain following systemic administration. In recent years, increasing research efforts by pharmaceutical and biotechnology companies, academic institutions and public–private consortia have resulted in the evaluation of various technologies developed to deliver therapeutics to the CNS, some of which have entered clinical testing. Here we review recent developments and challenges related to selected blood–brain barrier-crossing strategies — with a focus on non-invasive approaches such as receptor-mediated transcytosis and the use of neurotropic viruses, nanoparticles and exosomes — and analyse their potential in the treatment of CNS disorders. The blood–brain barrier is a perennial challenge for the delivery of therapeutics to the central nervous system. In their Review, Terstappen and colleagues discuss non-invasive approaches to brain delivery, particularly for biopharmaceuticals, some of which are now in clinical testing.
... In addition to delivery of anticancer chemotherapeutic agents, MB-ultrasound BBB opening has been shown to successfully deliver therapeutic proteins or monoclonal antibodies for brain tumor treatment. BBB opening can be used to deliver large molecular therapeutic monoclonal antibodies including herceptin [166], dopamine D-4 receptortargeting antibody [167], bevacizumab [168], and trastuzumab [169]. Kinoshita et al. demonstrated the possibility of delivering various large molecular monoclonal antibodies with penetration through the BBB and into the brain region, including dopamine D-4 receptor-targeting antibody [166] and humanized anti-human epidermal growth factor receptor 2 (HER2/c-erbB2) monoclonal antibody [167]. ...
... BBB opening can be used to deliver large molecular therapeutic monoclonal antibodies including herceptin [166], dopamine D-4 receptortargeting antibody [167], bevacizumab [168], and trastuzumab [169]. Kinoshita et al. demonstrated the possibility of delivering various large molecular monoclonal antibodies with penetration through the BBB and into the brain region, including dopamine D-4 receptor-targeting antibody [166] and humanized anti-human epidermal growth factor receptor 2 (HER2/c-erbB2) monoclonal antibody [167]. In addition, it has been reported that enhanced bevacizumab delivery with BBB opening led to a significant increase in animal survival compared to the bevacizumab treatment group alone [168]. ...
Ultrasonic technologies show great promise for diagnostic imaging and drug delivery in theranostic applications. The development of functional and molecular ultrasound imaging is based on the technical breakthrough of high frame–rate ultrasound. The evolution of shear wave elastography, high-frequency ultrasound imaging, ultrasound contrast imaging, and super-resolution blood flow imaging are described in this review. Recently, the therapeutic potential of the interaction of ultrasound with microbubble cavitation or droplet vaporization has become recognized. Microbubbles and phase-change droplets not only provide effective contrast media, but also show great therapeutic potential. Interaction with ultrasound induces unique and distinguishable biophysical features in microbubbles and droplets that promote drug loading and delivery. In particular, this approach demonstrates potential for central nervous system applications. Here, we systemically review the technological developments of theranostic ultrasound including novel ultrasound imaging techniques, the synergetic use of ultrasound with microbubbles and droplets, and microbubble/droplet drug-loading strategies for anticancer applications and disease modulation. These advancements have transformed ultrasound from a purely diagnostic utility into a promising theranostic tool.
... Tremendous effort has been invested in improving antibody delivery into the brain [161][162][163][164]. For instance, Kinoshita et al. introduced a technique to increase antibody brain delivery through transiently disrupting BBB by ultrasound, opening up tight cellular junctions, and facilitating the antibody penetration in the brain [164]. ...
... Tremendous effort has been invested in improving antibody delivery into the brain [161][162][163][164]. For instance, Kinoshita et al. introduced a technique to increase antibody brain delivery through transiently disrupting BBB by ultrasound, opening up tight cellular junctions, and facilitating the antibody penetration in the brain [164]. RMT-based antibody delivery has gained momentum as a viable method to treat central nervous system (CNS) disorders [78,[165][166][167][168]. ...
With more than 90 approved drugs by 2020, therapeutic antibodies have played a central role in shifting the treatment landscape of many diseases, including autoimmune disorders and cancers. While showing many therapeutic advantages such as long half-life and highly selective actions, therapeutic antibodies still face many outstanding issues associated with their pharmacokinetics (PK) and pharmacodynamics (PD), including high variabilities, low tissue distributions, poorly-defined PK/PD characteristics for novel antibody formats, and high rates of treatment resistance. We have witnessed many successful cases applying PK/PD modeling to answer critical questions in therapeutic antibodies’ development and regulations. These models have yielded substantial insights into antibody PK/PD properties. This review summarized the progress, challenges, and future directions in modeling antibody PK/PD and highlighted the potential of applying mechanistic models addressing the development questions.
... A combination of modulating the ID of MB and acoustic parameters has allowed researchers to deliver mAbs to the brain using FUS-MB. Numerous preclinical studies in mice demonstrate the ability of FUS-MB to effectively deliver antibodies to the brain with minimal tissue damage, including an anti-HER2 mAb [103] and a mAb against the dopamine D4 receptor [104]. Antibody delivery with FUS-MB has shown promise in enhancing the therapeutic efficacy of mAbs for brain diseases. ...
Antibodies (mAbs) are attractive molecules for their application as a diagnostic and therapeutic agent for diseases of the central nervous system (CNS). mAbs can be generated to have high affinity and specificity to target molecules in the CNS. Unfortunately, only a very small number of mAbs have been specifically developed and approved for neurological indications. This is primarily attributed to their low exposure within the CNS, hindering their ability to reach and effectively engage their potential targets in the brain. This review discusses aspects of various barriers such as the blood–brain barrier (BBB) and blood–cerebrospinal fluid (CSF) barrier (BCSFB) that regulate the entry and clearance of mAbs into and from the brain. The roles of the glymphatic system on brain exposure and clearance are being described. We also discuss the proposed mechanisms of the uptake of mAbs into the brain and for clearance. Finally, several methods of enhancing the exposure of mAbs in the CNS were discussed, including receptor-mediated transcytosis, osmotic BBB opening, focused ultrasound (FUS), BBB-modulating peptides, and enhancement of mAb brain retention.
... Kinoshita et al. used FUS to target delivery of polyclonal antibodies against the extracellular domain of the dopamine D4 receptor to the brain. Immunohistochemistry confirmed a positive signal of anti-rabbit IgG in the sonicated area in the ipsilateral hemisphere [82]. Alzheimer's disease is another application where opening of the blood brain barrier can be exploited for either drug delivery or to enhance clearance of beta amyloid [86,96]. ...
The blood-brain barrier (BBB) is a sophisticated structure whose full functionality is required for maintaining the executive functions of the central nervous system (CNS). Tight control of transport across the barrier means that most drugs, particularly large size, which includes powerful biologicals, cannot reach their targets in the brain. Notwithstanding the remarkable advances in characterizing the cellular nature of the BBB and consequences of BBB dysfunc-tion in pathology (brain metastasis, neurological diseases), it remains challenging to deliver drugs to the CNS. Herein, we outline the basic architecture and key molecular constituents of the BBB. In addition, we review the current status of approaches that are being explored to temporarily open the BBB in order to allow accumulation of therapeutics in the CNS. Undoubtedly, the major concern in field is whether it is possible to open the BBB in a meaningful way without causing negative consequences. In this context, we have also listed few other important key considerations that can improve our understanding about the dynamics of the BBB.
... Significant effort has been focused on improving drug delivery and distribution to benefit pancreatic cancer patients [60][61][62][63][64][65][66]. We described successful use of focused ultrasound, coupled with co-administered cavitation nuclei consisting of gas stabilizing SonoTran Particles, to improve the in situ deposition of clinically relevant drugs for pancreatic cancer. ...
Citation: Imran, K.M.; Tintera, B.; Morrison, H.A.; Tupik, J.D.; Nagai-Singer, M.A.; Ivester, H.; Council-Troche, M.; Edwards, M.; Coutermarsh-Ott, S.; Byron, C.; et al. Abstract: Pancreatic tumors can be resistant to drug penetration due to high interstitial fluid pressure , dense stroma, and disarrayed vasculature. Ultrasound-induced cavitation is an emerging technology that may overcome many of these limitations. Low-intensity ultrasound, coupled with co-administered cavitation nuclei consisting of gas-stabilizing sub-micron scale SonoTran Particles, is effective at increasing therapeutic antibody delivery to xenograft flank tumors in mouse models. Here, we sought to evaluate the effectiveness of this approach in situ using a large animal model that mimics human pancreatic cancer patients. Immunocompromised pigs were surgically engrafted with human Panc-1 pancreatic ductal adenocarcinoma (PDAC) tumors in targeted regions of the pancreas. These tumors were found to recapitulate many features of human PDAC tumors. Animals were intravenously injected with the common cancer therapeutics Cetuximab, gemcitabine, and paclitaxel, followed by infusion with SonoTran Particles. Select tumors in each animal were targeted with focused ultrasound to induce cavitation. Cavitation increased the intra-tumor concentrations of Cetuximab, gemcitabine, and paclitaxel by 477%, 148%, and 193%, respectively, compared to tumors that were not targeted with ultrasound in the same animals. Together, these data show that ultrasound-mediated cavitation, when delivered in combination with gas-entrapping particles, improves therapeutic delivery in pancreatic tumors under clinically relevant conditions.
... Drugs that have been studied include bevacizumab [81], TMZ [82,83], trastuzumab [84,85], doxorubicin [86][87][88], methotrexate [89], and carboplatin [90]. Additionally, this technique has been used to promote the migration of immunoglobulins [91][92][93][94][95], viruses [96], and cells across the BBB (Table 1). In animal models, BBB disruption occurs immediately and resolves within 6-8 h. ...
Malignant brain tumors are the leading cause of cancer-related death in children and remain a significant cause of morbidity and mortality throughout all demographics. Central nervous system (CNS) tumors are classically treated with surgical resection and radiotherapy in addition to adjuvant chemotherapy. However, the therapeutic efficacy of chemotherapeutic agents is limited due to the blood-brain barrier (BBB). Magnetic resonance guided focused ultrasound (MRgFUS) is a new and promising intervention for CNS tumors, which has shown success in preclinical trials. High-intensity focused ultrasound (HIFU) has the capacity to serve as a direct therapeutic agent in the form of thermoablation and mechanical destruction of the tumor. Low-intensity focused ultrasound (LIFU) has been shown to disrupt the BBB and enhance the uptake of therapeutic agents in the brain and CNS. The authors present a review of MRgFUS in the treatment of CNS tumors. This treatment method has shown promising results in preclinical trials including minimal adverse effects, increased infiltration of the therapeutic agents into the CNS, decreased tumor progression, and improved survival rates.
... This opening size extends brain therapeutic options to include viral vectors (H. Li et al. 2021;Lin et al. 2016;Felix et al. 2021), nanoparticles (Ohta et al. 2020), neurotrophic factors , and antibodies (Kinoshita et al. 2006). Transport of even larger molecules (2,000 kDa) has been achieved but may pose risk of permanent damage . ...
Focused ultrasound blood-brain barrier (BBB) opening is a promising tool for targeted delivery of therapeutic agents into the brain. The volume of opening determines the extent of therapeutic administration and sets a lower bound on the size of targets which can be selectively treated. We tested a custom 1 MHz array transducer optimized for cortical regions in the macaque brain with the goal of achieving small volume openings. We integrated this device into a magnetic resonance image guided focused ultrasound system and demonstrated twelve instances of small volume BBB opening with average opening volumes of 59 ± 37 mm ³ and 184 ± 2 mm ³ in cortical and subcortical targets, respectively. We developed real-time cavitation monitoring using a passive cavitation detector embedded in the array and characterized its performance on a bench-top flow phantom mimicking transcranial BBB opening procedures. We monitored cavitation during in-vivo procedures and compared cavitation metrics against opening volumes and safety outcomes measured with FLAIR and susceptibility weighted MR imaging. Our findings show small BBB opening at cortical targets in macaques and characterize the safe pressure range for 1 MHz BBB opening. Additionally, we used subject-specific simulations to investigate variance in measured opening volumes and found high correlation (R ² = 0.8577) between simulation predictions and observed measurements. Simulations suggest the threshold for 1 MHz BBB opening was 0.53 MPa. This system enables BBB opening for drug delivery and gene therapy to be targeted to more specific brain regions.
... The size of BBB opening is dependent on both the acoustic pressure and microbubble size. This procedure has been shown to enhance the delivery of a large variety of therapeutic agents, from small-molecular-weight drugs [34] to monoclonal antibodies [35] or cells [36]. Loaded liposomes and microbubbles [37] have been used to optimize local drug delivery. ...
Brain and spinal tumors affect 1 in 1000 people by 25 years of age, and have diverse histological, biological, anatomical and dissemination characteristics. A mortality of 30–40% means the majority are cured, although two-thirds have life-long disability, linked to accumulated brain injury that is acquired prior to diagnosis, and after surgery or chemo-radiotherapy. Only four drugs have been licensed globally for brain tumors in 40 years and only one for children. Most new cancer drugs in clinical trials do not cross the blood–brain barrier (BBB). Techniques to enhance brain tumor drug delivery are explored in this review, and cover those that augment penetration of the BBB, and those that bypass the BBB. Developing appropriate delivery techniques could improve patient outcomes by ensuring efficacious drug exposure to tumors (including those that are drug-resistant), reducing systemic toxicities and targeting leptomeningeal metastases. Together, this drug delivery strategy seeks to enhance the efficacy of new drugs and enable re-evaluation of existing drugs that might have previously failed because of inadequate delivery. A literature review of repurposed drugs is reported, and a range of preclinical brain tumor models available for translational development are explored.
... A recent systematic review of such parameters and their effects on the extent and safety of BBB opening across pre-clinical and clinical studies was conducted by Gandhi et al. [54]. Using appropriate parameters, therapeutic agents ranging from low-molecular-weight drugs [59,[200][201][202], larger molecular weight molecules such as monoclonal antibodies [139,203], gene vectors (both non-viral and viral) [204][205][206] and nanoparticles to even stem cells [207] and natural killer cells [208], have been successfully delivered to brain cancer sites in pre-clinical studies. The emerging evidence from these studies provides promising therapeutic potential in advancing the pharmacological management of brain cancers. ...
Despite significant advances in developing drugs to treat brain tumours, achieving therapeutic concentrations of the drug at the tumour site remains a major challenge due to the presence of the blood–brain barrier (BBB). Several strategies have evolved to enhance brain delivery of chemotherapeutic agents to treat tumours; however, most approaches have several limitations which hinder their clinical utility. Promising studies indicate that ultrasound can penetrate the skull to target specific brain regions and transiently open the BBB, safely and reversibly, with a high degree of spatial and temporal specificity. In this review, we initially describe the basics of therapeutic ultrasound, then detail ultrasound-based drug delivery strategies to the brain and the mechanisms by which ultrasound can improve brain tumour therapy. We review pre-clinical and clinical findings from ultrasound-mediated BBB opening and drug delivery studies and outline current therapeutic ultrasound devices and technologies designed for this purpose.
... However, side effects, such as seizures and headaches, were noted to occur [54]. Most of these strategies come with drawbacks and risk factors for patients such as nontargeted delivery [54][55][56][57][58]. Accordingly, alternative strategies for transient opening of the BBB to enable delivery of drugs are needed to better treat patients. ...
Despite the availability of numerous therapeutic substances that could potentially target CNS disorders, an inability of these agents to cross the restrictive blood–brain barrier (BBB) limits their clinical utility. Novel strategies to overcome the BBB are therefore needed to improve drug delivery. We report, for the first time, how Tumor Treating Fields (TTFields), approved for glioblastoma (GBM), affect the BBB’s integrity and permeability. Here, we treated murine microvascular cerebellar endothelial cells (cerebEND) with 100–300 kHz TTFields for up to 72 h and analyzed the expression of barrier proteins by immunofluorescence staining and Western blot. In vivo, compounds normally unable to cross the BBB were traced in healthy rat brain following TTFields administration at 100 kHz. The effects were analyzed via MRI and immunohistochemical staining of tight-junction proteins. Furthermore, GBM tumor-bearing rats were treated with paclitaxel (PTX), a chemotherapeutic normally restricted by the BBB combined with TTFields at 100 kHz. The tumor volume was reduced with TTFields plus PTX, relative to either treatment alone. In vitro, we demonstrate that TTFields transiently disrupted BBB function at 100 kHz through a Rho kinase-mediated tight junction claudin-5 phosphorylation pathway. Altogether, if translated into clinical use, TTFields could represent a novel CNS drug delivery strategy.
... FUS BBB opening (BBBO) has enhanced transport of molecules up to 20 nm across the BBB [2]. This opening size extends brain therapeutic options to include viral vectors [3] [4], nanoparticles [5], neurotrophic factors [6], antibodies [7], and others. Sonication parameters This required for transport of molecules 2,000 kDa or larger may pose risk of permanent damage [8]. ...
p>This work describes the design and testing of a therapeutic ultrasound array for transcranial blood brain barrier opening in macaques. The design leverages transcranial simulations to optimize for small opening volumes targetted at single brain regions. We also provide details on integrating the system into magnetic resonance image guided procedures.</p
... FUS BBB opening (BBBO) has enhanced transport of molecules up to 20 nm across the BBB [2]. This opening size extends brain therapeutic options to include viral vectors [3] [4], nanoparticles [5], neurotrophic factors [6], antibodies [7], and others. Sonication parameters This required for transport of molecules 2,000 kDa or larger may pose risk of permanent damage [8]. ...
p>This work describes the design and testing of a therapeutic ultrasound array for transcranial blood brain barrier opening in macaques. The design leverages transcranial simulations to optimize for small opening volumes targetted at single brain regions. We also provide details on integrating the system into magnetic resonance image guided procedures.</p
... MRgFUS oscillates microbubbles and mechanically disrupts the BBB in a targeted, transient, and non-invasive manner, increasing the vascular permeability to large molecules into the brain (12)(13)(14). In preclinical studies, MRgFUS-induced opening of the BBB has been used to deliver chemotherapeutic agents (15,16), antibodies (17,18), stem cells (19), and therapeutic genes (20). Extensive preclinical research has shown that FUS with microbubbles can lead to a more than 4-fold increase in the delivery and penetration of a range of intravenously administered anticancer agents in brain tumors (21). ...
An orthotopically xenografted mouse GL26 glioma model (Ccr2RFP/wt-Cx3cr1GFP/wt) was used to evaluate the effect of transient, focal opening of the Blood Brain Barrier (BBB) on the composition of tumor-associated macrophages and microglia (TAMs). Opening of the BBB was induced by Magnetic Resonance Imaging (MRI)-guided focused ultrasound (MRgFUS) combined with systemically-administered microbubbles. CX3CR1-GFP cells and CCR2-RFP cells in brain tumors and in peritumoral tissue were quantified utilizing cell counting in fluroscent, microscopic images. Tumors in animals treated with a single session of MRgFUS did not show significant changes in CX3CR1-GFP or CCR2-RFP cell numbers when compared to tumors in animals not receiving FUS. However, tumors that received two or three sessions of MRgFUS showed significantly increased amounts of both CX3CR1-GFP and CCR2-RFP cells.
The effect of MRgFUS on immune cell compositon of tumors and the brain parenchyma was also characterized and quantified utilizing flow cytometry. Glioma implantation resulted in increased amounts of monocytes, blood-derived macrophages, and microglia-derived macrophages in the brain parenchyma. Tumors administered MRgFUS showed increased numbers of monocytes and blood-derived macrophages. In addition, MRgFUS-treated tumors exhibited more CD80+CD206- cells in monocytes, blood-derived macrophages, microglia, and microglia-derived macrophages, and fewer CD80-CD206+ cells in monocytes and microglia. This signature is indicative of a shift toward a more pro-inflammatory polarization.
In summary, transient, focal opening of the BBB using MRgFUS combined with microbubbles can activate the homing and differentiation of monocytes, and induce a shift towards a more proinflammatory status of the immune environment in glioblastoma.
... Ultrasound-based blood-brain barrier disruption (US-BBBD) allows for increased penetration of systemically administered small and large molecule drug therapies into the brain [13][14][15]. Strikingly, US-BBBD alone has been shown to reduce β-amyloid and tau pathologies, stimulate neurogenesis, and improve cognitive performance in mouse AD models [16][17][18] and can be further coupled with drug therapies to improve their brain penetration and efficacy [13,19,20]. ...
Background
Temporary disruption of the blood-brain barrier (BBB) using pulsed ultrasound leads to the clearance of both amyloid and tau from the brain, increased neurogenesis, and mitigation of cognitive decline in pre-clinical models of Alzheimer’s disease (AD) while also increasing BBB penetration of therapeutic antibodies. The goal of this pilot clinical trial was to investigate the safety and efficacy of this approach in patients with mild AD using an implantable ultrasound device.
Methods
An implantable, 1-MHz ultrasound device (SonoCloud-1) was implanted under local anesthesia in the skull (extradural) of 10 mild AD patients to target the left supra-marginal gyrus. Over 3.5 months, seven ultrasound sessions in combination with intravenous infusion of microbubbles were performed twice per month to temporarily disrupt the BBB. ¹⁸ F-florbetapir and ¹⁸ F-fluorodeoxyglucose positron emission tomography (PET) imaging were performed on a combined PET/MRI scanner at inclusion and at 4 and 8 months after the initiation of sonications to monitor the brain metabolism and amyloid levels along with cognitive evaluations. The evolution of cognitive and neuroimaging features was compared to that of a matched sample of control participants taken from the Alzheimer’s Disease Neuroimaging Initiative (ADNI).
Results
A total of 63 BBB opening procedures were performed in nine subjects. The procedure was well-tolerated. A non-significant decrease in amyloid accumulation at 4 months of − 6.6% (SD = 7.2%) on ¹⁸ F-florbetapir PET imaging in the sonicated gray matter targeted by the ultrasound transducer was observed compared to baseline in six subjects that completed treatments and who had evaluable imaging scans. No differences in the longitudinal change in the glucose metabolism were observed compared to the neighboring or contralateral regions or to the change observed in the same region in ADNI participants. No significant effect on cognition evolution was observed in comparison with the ADNI participants as expected due to the small sample size and duration of the trial.
Conclusions
These results demonstrate the safety of ultrasound-based BBB disruption and the potential of this technology to be used as a therapy for AD patients. Research of this technique in a larger clinical trial with a device designed to sonicate larger volumes of tissue and in combination with disease-modifying drugs may further enhance the effects observed.
Trial registration
ClinicalTrials.gov, NCT03119961
... selection and controlling the extent of BBB opening, and this strategy has been widely explored for a variety of CNS diseases including brain tumours and Alzheimer's disease [162][163][164][165][166][167]. Increased permeability has been reported to reverse within several hours, so the window of drug administration should also be carefully decided [168]. ...
Brain tumours have a poor prognosis and lack effective treatments. The blood–brain barrier (BBB) represents a major hurdle to drug delivery to brain tumours. In some locations in the tumour, the BBB may be disrupted to form the blood–brain tumour barrier (BBTB). This leaky BBTB enables diagnosis of brain tumours by contrast enhanced magnetic resonance imaging; however, this disruption is heterogeneous throughout the tumour. Thus, relying on the disrupted BBTB for achieving effective drug concentrations in brain tumours has met with little clinical success. Because of this, it would be beneficial to design drugs and drug delivery strategies to overcome the ‘normal’ BBB to effectively treat the brain tumours. In this review, we discuss the role of BBB/BBTB in brain tumour diagnosis and treatment highlighting the heterogeneity of the BBTB. We also discuss various strategies to improve drug delivery across the BBB/BBTB to treat both primary and metastatic brain tumours. Recognizing that the BBB represents a critical determinant of drug efficacy in central nervous system tumours will allow a more rapid translation from basic science to clinical application. A more complete understanding of the factors, such as BBB–limited drug delivery, that have hindered progress in treating both primary and metastatic brain tumours, is necessary to develop more effective therapies.
Abstract
... With this technique, microbubbles consisting of a lipid or protein shell and a heavy gas core (diameter 1-10 μm) are injected into the bloodstream along with the therapeutic agents of interest. Pulses of ultrasound are then applied to the targeted brain region, driving the microbubbles to oscillate, which ultimately allows the delivery of drugs across the BBB and into the brain [24,25]. In vivo studies have demonstrated that liposomes can be delivered into the brain using this focused ultrasound technique [26][27][28][29][30][31][32][33][34][35][36][37][38][39][40]. ...
Liposomes are clinically used drug carriers designed to improve the delivery of drugs to specific tissues while minimising systemic distribution. However, liposomes are unable cross the blood-brain barrier (BBB) and enter the brain, mostly due to their large size (ca. 100 nm). A noninvasive and localised method of delivering liposomes across the BBB is to intravenously inject microbubbles and apply long pulses of ultrasound (pulse length: >1 ms) to a targeted brain region. Recently, we have shown that applying rapid short pulses (RaSP) (pulse length: 5 μs) can deliver drugs with an improved efficacy and safety profile. However, this was tested with a relatively smaller 3-kDa molecule (dextran).
In this study, we examine whether RaSP can deliver liposomes to the murine brain in vivo. Fluorescent DiD-PEGylated liposomes were synthesized and injected intravenously alongside microbubbles. The left hippocampus of mice was then sonicated with either a RaSP sequence (5 μs at 1.25 kHz in groups of 10 ms at 0.5 Hz) or a long pulse sequence (10 ms at 0.5 Hz), with each pulse having a 1-MHz centre frequency (0.35 and 0.53 MPa). The delivery and distribution of the fluorescently-labelled liposomes were assessed by fluorescence imaging of the brain sections. The safety profile of the sonicated brains was assessed by histological staining.
RaSP was shown to locally deliver liposomes across the BBB at 0.53 MPa with a more diffused and safer profile compared to the long pulse ultrasound sequence. Cellular uptake of liposomes was observed in neurons and microglia, while no uptake within astrocytes was observed in both RaSP and long pulse-treated brains. This study shows that RaSP allows a targeted and safe delivery of liposomal drugs into the murine brain with potential to deliver drugs into neuronal and glial targets.
... Ultrasound-based blood-brain barrier disruption (US-BBBD) allows for increased penetration of systemically administered small and large molecule drug therapies into the brain (8-10). Strikingly, US-BBBD alone has been shown to reduce β-amyloid and tau pathologies, stimulate neurogenesis, and improve cognitive performance in mouse AD models (11)(12)(13) and can be further coupled with drug therapies to improve their brain penetration and e cacy (8, 14,15). ...
Background: Temporary disruption of the blood-brain barrier (BBB) using pulsed ultrasound leads to the clearance of both amyloid and tau from the brain, increased neurogenesis, and mitigation of cognitive decline in pre-clinical models of Alzheimer’s disease (AD) while also increasing BBB penetration of therapeutic antibodies. The goal of this pilot clinical trial was to investigate the safety and the efficacy of this approach in patients with mild AD using an implantable ultrasound device.
Methods: An implantable, 1 MHz ultrasound device (SonoCloud-1) was extradurally implanted under local anesthesia in the skull of 10 mild AD patients to target the left supra-marginal gyrus. Over 3.5 months, seven ultrasound sessions in combination with intravenous infusion of microbubbles were performed twice per month to temporarily disrupt the BBB. ¹⁸F-Florbetapir and ¹⁸F-fluorodeoxyglucose positron emission tomography (PET) imaging were performed on a combined PET/MRI scanner at inclusion and at four and eight months after initiation of sonications to monitor brain metabolism and amyloid levels along with cognitive evaluations. Evolution of cognitive and neuroimaging features were compared to that of a matched sample of control participants.
Results: A total of 63 BBB opening procedures were performed in nine subjects. The procedure was well-tolerated. A non-significant decrease in amyloid accumulation at four months of -6.6% (SD=7.2%) on ¹⁸F-Florbetapir PET imaging in the sonicated gray matter targeted by the ultrasound transducer was observed compared to baseline in six subjects that completed treatments and who had evaluable imaging scans. No differences in longitudinal change in glucose metabolism were observed compared to neighboring or contralateral regions or to the change observed in the same region in ADNI participants. No significant effect on cognition evolution was observed in comparison to the ADNI participants as expected due to the small sample size and duration of the trial.
Conclusions: These results demonstrate the safety of ultrasound-based BBB disruption and potential of this technology to be used as a therapy for AD patients. They support further research of this technique in a larger clinical trial with a device designed to sonicate larger volumes of tissue and in combination with disease modifying drugs to further enhance the effects observed.
Trial registration: NCT03119961 (clinicaltrials.gov)
... Thus, by applying different acoustic pressures, the two target areas showed comparable degrees of BBBD, comparable patterns of DTI parameters, and histological results. Since the FUS parameter in group-0.65 MPa has been widely used in the rat brain to safely disrupt the BBB and deliver therapeutics in several previous studies (Kinoshita et al., 2006b;Cho et al., 2016;Jung et al., 2019), we selected the FUS parameter to determine whether water transport could be facilitated in the BBBD brain area. We additionally applied higher sonication conditions in group-0.80 ...
Interstitial solutes can be removed by various overlapping clearance systems, including blood–brain barrier (BBB) transport and glymphatic clearance. Recently, focused ultrasound (FUS)-induced BBB disruption (BBBD) has been applied to visualize glymphatic transport. Despite evidence that FUS–BBBD might facilitate glymphatic transport, the nature of fluid movement within the sonication region is yet to be determined. In this study, we sought to determine whether FUS–BBBD may facilitate the local movement of water molecules. Two different FUS conditions (0.60–0.65 MPa and 0.75–0.80 MPa) were used to induce BBBD in the caudate-putamen and thalamus regions of healthy Sprague–Dawley rats. The water diffusion caused by FUS–BBBD was analyzed using the apparent diffusion coefficient (ADC), axial diffusivity, radial diffusivity (RD), and fractional anisotropy, obtained at 5 min, 24 and 48 h, as well as the water channel expression of aquaporin-4 (AQP-4) immunostaining at 48 h after FUS-induced BBBD. In addition, hematoxylin and eosin histopathology and Fluoro-Jade C (FJC) immunostaining were performed to analyze brain damage. The signal changes in ADC and RD in the sonication groups showed significant and transient reduction at 5 min, with subsequent increases at 24 and 48 h after FUS-induced BBBD. When we applied higher sonication conditions, the ADC and RD showed enhancement until 48 h, and became comparable to contralateral values at 72 h. AQP-4 expression was upregulated after FUS-induced BBBD in both sonication conditions at 48 h. The results of this study provide preliminary evidence on how mechanical forces from FUS alter water dynamics through diffusion tensor imaging (DTI) measures and AQP4 expression.
... The delivery of large molecular drugs across the BBB has also been demonstrated, including IL-12 (70 kDa) [67], dopamine D-4 receptor-targeting antibody [68], and HER2/c-erbB2 (humanized antihuman epidermal growth factor receptor 2) monoclonal antibody [69]. We have demonstrated the enhanced delivery of bevacizumab (149 kDa) in a mouse glioma model [45]. ...
The blood–brain barrier (BBB) protects the central nervous system (CNS) from invasive pathogens and maintains the homeostasis of the brain. Penetrating the BBB has been a major challenge in the delivery of therapeutic agents for treating CNS diseases. Through a physical acoustic cavitation effect, focused ultrasound (FUS) combined with microbubbles achieves the local detachment of tight junctions of capillary endothelial cells without inducing neuronal damage. The bioavailability of therapeutic agents is increased only in the area targeted by FUS energy. FUS with circulating microbubbles is currently the only method for inducing precise, transient, reversible, and noninvasive BBB opening (BBBO). Over the past decade, FUS-induced BBBO (FUS-BBBO) has been preclinically confirmed to not only enhance the penetration of therapeutic agents in the CNS, but also modulate focal immunity and neuronal activity. Several recent clinical human trials have demonstrated both the feasibility and potential advantages of using FUS-BBBO in diseased patients. The promising results support adding FUS-BBBO as a multimodal therapeutic strategy in modern CNS disease management. This review article explores this technology by describing its physical mechanisms and the preclinical findings, including biological effects, therapeutic concepts, and translational design of human medical devices, and summarizes completed and ongoing clinical trials.
... Focused ultrasound (FUS)-mediated disruption of the BBB is an example of a therapeutic strategy that could be influenced by such findings, given that this application relies on the presence of intravascular MBs. FUS BBB opening is rapidly advancing as a non-invasive, safe, and repeatable strategy 26 to treat a wide variety of neurological pathologies [25][26][27][28][29][30][31][32][33][34][35][36][37][38] . ...
Abstract Microbubbles (MB) are widely used as contrast agents to perform contrast-enhanced ultrasound (CEUS) imaging and as acoustic amplifiers of mechanical bioeffects incited by therapeutic-level ultrasound. The distribution of MBs in the brain is not yet fully understood, thereby limiting intra-operative CEUS guidance or MB-based FUS treatments. In this paper we describe a robust platform for quantification of MB distribution in the human brain, allowing to quantitatively discriminate between tumoral and normal brain tissues and we provide new information regarding real-time cerebral MBs distribution. Intraoperative CEUS imaging was performed during surgical tumor resection using an ultrasound machine (MyLab Twice, Esaote, Italy) equipped with a multifrequency (3–11 MHz) linear array probe (LA332) and a specific low mechanical index (MI
... Focused ultrasound techniques concentrate acoustic energy on a focal spot in the brain measuring a few millimeters in diameter and, combined with magnetic resonance imaging (MRI), have been used in the clinic for thermal coagulation of tumors in human patients with real-time monitoring [44,45]. MRI-guided focused ultrasound (MRgFUS) used to induce cavitation of intravenously (IV) administered microbubbles has been reported to reliably cause temporary physical disruption of the BBB in small animal models and has been used for delivery of BBB-impermeably compounds such as antibody drugs [46,47]. Airan et al. delivered nanoemulsions encapsulating a BBB-permeable small molecule anesthetic using MRgFUS to enable potent intracranial drug delivery with high spatiotemporal control in an acute rate seizure model without brain parenchymal damage [48], demonstrating the fine level of control that can be exerted with this neuromodulatory technique. ...
Glioblastoma is one of the deadliest forms of primary adult tumors, with median survival of 14.6 months post-diagnosis despite aggressive standard of care treatment. This grim prognosis for glioblastoma patients has changed little in the past two decades, necessitating novel treatment modalities. One potential treatment modality is cancer immunotherapy, which has shown remarkable progress in slowing disease progression or even potentially curing certain solid tumors. However, the transport barriers posed by the blood–brain barrier and the immune privileged status of the central nervous system pose drug delivery obstacles that are unique to brain tumors. In this review, we provide an overview of the various physiological, immunological, and drug delivery barriers that must be overcome for effective glioblastoma treatment. We discuss chemical modification strategies to enable nanomedicines to bypass the blood–brain barrier and reach intracranial tumors. Finally, we highlight recent advances in biomaterial-based strategies for cancer immunotherapy that can be adapted to glioblastoma treatment.
... The BBB is very challenging for the clinical diagnosis and treatment of brain tumors. To overcome this problem, focused ultrasound sonication (FUS) with drug-loaded microbubble, opens the rigid protective barrier of the brain and allows the penetration of therapeutics agent across the BBB into the brain [112,113]. The focused ultrasonic waves break the microbubbles in nanosized, which is more effective in the therapeutic delivery of medicine (Fig. 14). ...
... It has been demonstrated that the concentration of methotrexate in the rabbit brain can be increased by more than 3 times (251). In pre-clinical studies, ultrasound has been shown to enhance delivery of chemo-, immuno-and gene-therapy with subsequent improvement in treatment efficacy (127,(252)(253)(254) (Figure 9). While the Sonocloud™ operates without imaging guidance, Exablate neuro brain system™ (Insightec, Israel) uses MRI to guide the focal beam. ...
... Especially intriguing is the ability to deliver neural stem cells (125) or natural killer cells (69), which may provide a platform for a variety of therapeutic approaches. Other therapeutic agents delivered include antiamyloid beta antibodies (126), antidopamine receptor D4 antibodies (127), Herceptin (128), interleukin-12 (129), erythropoietin (130), and brain-derived neurotrophic factor (131). In addition to characterizing the delivery and efficacy of FUS+MB-mediated therapeutic agent delivery, further work in the field should focus on evaluating the movement of substances that have crossed the BBB. ...
Specialized features of vasculature in the central nervous system greatly limit therapeutic treatment options for many neuropathologies. Focused ultrasound, in combination with circulating microbubbles, can be used to transiently and noninvasively increase cerebrovascular permeability with a high level of spatial precision. For minutes to hours following sonication, drugs can be administered systemically to extravasate in the targeted brain regions and exert a therapeutic effect, after which permeability returns to baseline levels. With the wide range of therapeutic agents that can be delivered using this approach and the growing clinical need, focused ultrasound and microbubble (FUS+MB) exposure in the brain has entered human testing to assess safety. This review outlines the use of FUS+MB-mediated cerebrovascular permeability enhancement as a drug delivery technique, details several technical and biological considerations of this approach, summarizes results from the clinical trials conducted to date, and discusses the future direction of the field.
Expected final online publication date for the Annual Review of Biomedical Engineering, Volume 23 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... An alternative technique for BBB opening is based on systemic injection of ultrasound contrast agents. This method was successfully used to increase the delivery of Herceptin to brain parenchyma in a mice model (Hynynen et al., 2001Kinoshita et al., 2006). ...
Blood brain barrier (BBB) is formed by the brain microvascular endothelial cells (BMVECs) lining the wall of brain capillaries. Its integrity is regulated by multiple mechanisms, including up/downregulation of tight junction proteins or adhesion molecules, altered Ca2 + homeostasis, remodeling of cytoskeleton, that are confined at the level of BMVECs. Beside the contribution of BMVECs to BBB permeability changes, other cells, such as pericytes, astrocytes, microglia, leukocytes or neurons, etc. are also exerting direct or indirect modulatory effects on BBB. Alterations in BBB integrity play a key role in multiple brain pathologies, including neurological (e.g. epilepsy) and neurodegenerative disorders (e.g. Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis etc.). In this review, the principal Ca2 + signaling pathways in brain microvascular endothelial cells are discussed and their contribution to BBB integrity is emphasized. Improving the knowledge of Ca2 + homeostasis alterations in BMVECa is fundamental to identify new possible drug targets that diminish/prevent BBB permeabilization in neurological and neurodegenerative disorders.
Ultrasound-mediated cavitation shows great promise for improving targeted drug delivery across a range of clinical applications. Cavitation nuclei—sound-sensitive constructs that enhance cavitation activity at lower pressures—have become a powerful adjuvant to ultrasound-based treatments, and more recently emerged as a drug delivery vehicle in their own right. The unique combination of physical, biological, and chemical effects that occur around these structures, as well as their varied compositions and morphologies, make cavitation nuclei an attractive platform for creating delivery systems tuned to particular therapeutics. In this review, we describe the structure and function of cavitation nuclei, approaches to their functionalization and customization, various clinical applications, progress toward real-world translation, and future directions for the field.
The efforts to use novel nanotechnologies in medicine and cancer have been widespread. In order to understand better the focus areas of cancer nanomedicine research to date, we conducted a survey of nanomedicine developmental and clinical research in conjunction with treatment of various cancers. The survey has been performed based on number of publications, rate of citations, entry into clinical trials, and funding rates by the National Cancer Institute. Our survey indicates that breast and brain cancers are the most and one of the least studied by nanotechnology researchers, respectively. Breast cancer nano‐therapies seem to also be most likely to achieve clinical translation as the number of publications produced, amount of funding, total citations, and clinical trials (active and completed) are the highest when compared with research in other cancers. Brain cancer, despite its low survival, has capture much less attention of nanomedicine research community as survey indicated, although nanotechnology can offer novel approaches which can address brain cancer challenges.
This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease
The local fluid dynamics experienced by circulating microbubbles vary across different anatomical sites, which can influence ultrasound-mediated therapeutic delivery efficacy. This study aimed to elucidate the effect of fluid flow rate in combination with repeated short-pulse ultrasound on microbubble-mediated endothelial cell permeabilization. Here, a seeded monolayer of human umbilical (HUVEC) or brain endothelial cells (HBEC-5i) was co-perfused with a solution of microbubbles and propidium iodide (PI) at either a flow rate of 5 or 30 ml/min. Using an acoustically coupled inverted microscope, cells were exposed to 1 MHz ultrasound with 20-cycle bursts, 1 ms PRI, and 2 s duration at a peak negative pressure of 305 kPa to assess the role of flow rate on ultrasound-stimulated endothelial cell permeability, as well as Ca2+ modulation. In addition, the effect of inter-pulse delays (∆t = 1s) on the resulting endothelial permeability was investigated. Our results demonstrate that under an identical acoustic stimulus, fast-flowing microbubbles resulted in a statistically significant increase in cell membrane permeability, at least by 2.3-fold, for both endothelial cells. Likewise, there was a substantial difference in intracellular Ca2+ levels between the two examined flow rates. In addition, multiple short pulses rather than a single pulse ultrasound, with an equal number of bursts, significantly elevated endothelial cell permeabilization, at least by 1.4-fold, in response to ultrasound-stimulated microbubbles. This study provides insights into the design of optimal, application-dependent pulsing schemes to improve the effectiveness of ultrasound-mediated local therapeutic delivery.
Focused ultrasound (FUS) can temporarily open the blood brain barrier (BBB) and increase the delivery of chemotherapeutics, viral vectors, and other agents to the brain parenchyma. To limit FUS BBB opening to a single brain region, the transcranial acoustic focus of the ultrasound transducer must not be larger than the region targeted. In this work, we design and characterize a therapeutic array optimized for BBB opening at the frontal eye field in macaques. We used 115 transcranial simulations in four macaques varying f-number and frequency to optimize the design for focus size, transmission, and small device footprint. The design leverages inward steering for focus tightening, a 1 MHz transmit frequency, and can focus to a simulation predicted 2.5 ± 0.3 mm lateral and 9.5 ± 1.0 mm axial full-width at half maximum spot size at the frontal eye field without aberration correction. The array is capable of steering axially 35 mm outward, 26 mm inward, and laterally 13 mm with > 50% the geometric focus pressure. The simulated design was fabricated, and we characterized the performance of the array using hydrophone beam maps in a water tank and through an
ex vivo
skull cap to compare measurements with simulation predictions, achieving a 1.8 mm lateral and 9.5 mm axial spot size with a transmission of 37 % (transcranial, phase corrected). The transducer produced by this design process is optimized for BBB opening at the frontal eye field in macaques.
An orthotopically allografted mouse GL26 glioma model (Ccr2RFP/wt-Cx3cr1GFP/wt) was used to evaluate the effect of transient, focal opening of the blood-brain barrier (BBB) on the composition of tumor-associated macrophages and microglia (TAMs). BBB opening was induced by magnetic resonance imaging (MRI)-guided focused ultrasound (MRgFUS) combined with microbubbles. CX3CR1-GFP cells and CCR2-RFP cells in brain tumors were quantified in microscopic images. Tumors in animals treated with a single session of MRgFUS did not exhibit significant changes in cell numbers when compared with tumors in animals not receiving FUS. However, tumors that received two or three sessions of MRgFUS had significantly increased amounts of both CX3CR1-GFP and CCR2-RFP cells. The effect of MRgFUS on immune cell composition was also characterized and quantified using flow cytometry. Glioma implantation resulted in increased amounts of lymphocytes, monocytes and neutrophils in the brain parenchyma. Tumors administered MRgFUS exhibited increased numbers of monocytes and monocyte-derived TAMs. In addition, MRgFUS-treated tumors exhibited more CD80+ cells in monocytes and microglia. In summary, transient, focal opening of the BBB using MRgFUS combined with microbubbles can activate the homing and differentiation of monocytes and induce a shift toward a more pro-inflammatory status of the immune environment in glioblastoma.
Drug delivery into the peripheral nerves and nerve roots has important implications for effective local anesthesia and treatment of peripheral neuropathies and chronic neuropathic pain. Similar to drugs that need to cross the blood‐brain barrier (BBB) and blood‐spinal cord barrier (BSCB) to gain access to the central nervous system (CNS), drugs must cross the peripheral nerve barriers (PNB), formed by the perineurium and blood‐nerve barrier (BNB) to modulate peripheral axons. Despite significant progress made to develop effective strategies to enhance BBB permeability in therapeutic drug design, efforts to enhance drug permeability and retention in peripheral nerves and nerve roots are relatively understudied. Guided by knowledge describing structural, molecular and functional similarities between restrictive neural barriers in the CNS and peripheral nervous system (PNS), we hypothesize that certain CNS drug delivery strategies are adaptable for peripheral nerve drug delivery. In this review, we describe the molecular, structural and functional similarities and differences between the BBB and PNB, summarize and compare existing CNS and peripheral nerve drug delivery strategies, and discuss the potential application of selected CNS delivery strategies to improve efficacious drug entry for peripheral nerve disorders. This article is protected by copyright. All rights reserved
The human brain is central, not only to normal biological function, but also to personal identity. Diseases and injuries to the brain can erase this sense of self. Delivering drugs to the brain is a critical challenge in addressing diseases and injuries that degrade human interaction. While delivering drugs to the brain can play a role in addressing a host of degenerative diseases of the central nervous system diseases like Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS), it is perhaps closer to being broadly clinically realized in the context of acute injuries or attacks. The subsequent review examines polymeric nanocarriers as a means to deliver drugs to the brain while using ischemic stroke as a specific example of an application. The explicit intravenous (IV) nature of ischemic stroke provides an opportunity to illustrate some of the opportunities and limits of nanocarrier drug delivery.KeywordsIschemic strokeTissue plasminogen activatorPoly(lactic-co-glycolic acid) nanocarriersMiddle cerebral artery occlusionBlood-brain barrier permeabilityFocused ultrasoundNear-infrared femtosecond-pulsed laser irradiationCarrier-mediated transcytosisTargeted deliveryEnzyme-responsive polymeric nanocarriersTriggered deliveryCore-shell nanocarriers
Neurosonology is non-invasive, portable, and has excellent temporal resolution, making it a valuable and increasingly popular tool for the diagnosis and monitoring of neurological conditions when compared to other imaging techniques. This guide looks beyond the use of neurovascular ultrasound in stroke to encompass a wide range of other neurological diseases and emergencies. It offers a practical approach to the examination of patients, interpretation of ultrasound studies, and the application of neurosonology to the development of management and treatment strategies. Each chapter incorporates a thorough and clear procedural methodology alongside scanning tips for trainees; this step-by-step approach is further enhanced by example images and focused diagnostic questions. Authored and edited by international experts, this practical manual of neurosonology is an invaluable resource for neurologists, neurosurgeons, intensivists, radiologists and ultrasonographers.
Interventional oncology has emerged as an important specialty within cancer care, providing targeted therapy and palliative benefits without the side effects of chemotherapy, surgery and radiation. Covering the principles of current and emerging interventional oncology techniques and detailed diagnosis, staging and treatment algorithms, this book outlines the ways in which these image-guided therapies can inform cancer management strategies. Access to the most current information is vital in this rapidly growing and evolving area of practice. This new edition reflects the most recent clinical data on interventional oncology procedures. Chapters on image guidance and targeting, tumor ablation, embolotherapy, and response assessment have been updated to reflect major technological advances, and new material on microwave ablation and irreversible electroporation has been added. This invaluable resource for interventional radiologists provides essential education and guidance on the full range of minimally invasive image-guided procedures and their integration into comprehensive cancer care.
The blood-brain barrier (BBB) presents a major obstacle in developing specific diagnostic imaging agents for many neurological disorders. In this study we aimed to generate single domain anti-mouse transferrin receptor antibodies (anti-mTfR VHHs) to mediate BBB transcytosis as components of novel MRI molecular contrast imaging agents. Anti-mTfR VHHs were produced by immunizing a llama with mTfR, generation of a VHH phage display library, immunopanning, and in vitro characterization of candidates. Site directed mutagenesis was used to generate additional variants. VHH fusions with neurotensin (NT) allowed rapid, hypothermia-based screening for VHH-mediated BBB transcytosis in wild-type mice. One anti-mTfR VHH variant was fused with an anti-amyloid-beta (Aβ) VHH dimer and labeled with fluorescent dye for direct assessment of in vivo target engagement in a mouse model of AD-related Aβ plaque pathology. An anti-mTfR VHH called M1 and variants had binding affinities to mTfR of
The blood-brain barrier (BBB) is a protective and semipermeable border of endothelial cells that prevents toxins and foreign bodies to enter and damage the brain. Unfortunately, the BBB also hampers the development of pharmaceuticals targeting receptors, enzymes, or other proteins that lie beyond this barrier. Especially large molecules, such as monoclonal antibodies (mAbs) or nanoparticles, are prevented to enter the brain. The limited passage of these molecules partly explains why nanomedicines - targeting brain diseases - have not made it into the clinic to a great extent. As nanomedicines can target a wide range of targets including protein isoforms and oligomers or potentially deliver cytotoxic drugs safely to their targets, a pathway to smuggle nanomedicines into the brain would allow to treat brain diseases that are currently considered ‘undruggable’. In this review, strategies to transport nanomedicines over the BBB will be discussed. Their challenges and opportunities will be highlighted with respect to their use for molecular imaging or therapies. Several strategies have been explored for this thus far. For example, carrier-mediated and receptor-mediated transcytosis (RMT), techniques to disrupt the BBB, nasal drug delivery or administering nanomedicines directly into the brain have been explored. RMT has been the most widely and successfully explored strategy. Recent work on the use of focused ultrasound based BBB opening has shown great promise. For example, successful delivery of mAbs into the brain has been achieved, even in a clinical setting. As nanomedicines bear the potential to treat incurable brain diseases, drug delivery technologies that can deliver nanomedicines into the brain will play an essential role for future treatment options.
Glioblastoma (GBM) is the most frequent and the most aggressive primary brain cancer in adults. The current standard of care of newly diagnosed GBM includes maximal safe surgery followed by concurrent temozolomide (TMZ) and radiotherapy followed by adjuvant TMZ. Despite this intensive therapeutic regimen, the median overall survival is below 18 months. One of the main limitations of current anti-tumor treatments is the blood-brain barrier and the local immunosuppressed microenvironment in the setting of brain cancers.This thesis is focused on overcoming the blood-brain barrier to modulate local immunity to better anti-tumor treatments. Firstly, we reviewed the role of ABC proteins superfamily members expressed in the BBB cells and their role in GBM cells' resistance to chemotherapy. Secondly, we have studied the expression of immune checkpoint proteins CD80 and CD86 and their impacts on outcome in newly diagnosed glioblastoma patients treated with the standard of care. CD86 overexpression is associated with a better prognosis. Finally, we have shown dramatic efficacy of immune checkpoint inhibitors targeting PLD1 when combined with ultrasound-mediated BBB opening in glioblastoma-bearing mice compared to anti-PDL1 alone. BBB and immunity modulations are promising strategies to improve our therapeutic arsenal against glioblastoma.
Although great progress has been made in recent years and more than 7000 small-molecule drugs are available, few effective treatments and no cures of the central nervous system (CNS) diseases are currently available. This is mainly due to the impermeability of the blood-brain barrier (BBB) that allows only 5% of those drugs to diffuse to the brain parenchyma thereby allowing treatment of only a tiny fraction of these diseases. Safe and localized opening of the BBB has been proven to pose an equally significant challenge. Focused ultrasound (FUS), in conjunction with microbubbles, remains the sole technique that can induce localized BBB opening noninvasively. In this chapter, we demonstrate how the microbubble diameter and peak negative pressure can be optimized in order to dictate the BBB opening volume and permeability in small and large animals. We subsequently demonstrate that neuroprotection and neurorestoration in the dopaminergic neurons in the nigrostriatal pathway at the early stages of Parkinson’s disease as well as amyloid and tau reduction at the early stages of Alzheimer’s disease can be achieved at therapeutic levels safely.
The blood-brain barrier (BBB) greatly limits therapeutic treatment options for many diseases of the brain. The use of focused ultrasound (FUS) in conjunction with circulating microbubbles (MBs) provides a noninvasive means of transiently increasing BBB permeability with a high level of spatial precision. Drugs can be administered systemically to extravasate in the targeted brain regions and exert a therapeutic effect. In the hours following sonication, BBB permeability returns to baseline levels. The flexibility of this approach in facilitating the delivery of a wide range of therapeutic agents to either precise locations or large volumes, combined with efficacious results in preclinical models of disease, has motivated clinical trials to assess safety. This chapter will describe the development of FUS and MB-mediated BBB permeability enhancement as a drug delivery technique, detail several technical and biological considerations of this approach, and summarize results from the clinical trials conducted to date.KeywordsAcoustic feedback controlBlood-brain barrierClinical trialsDrug deliveryFocused ultrasoundMicrobubblesUltrasound
Survival outcomes for patients with glioblastoma multiforme (GBM) have remained poor for the past 15 years, reflecting a clear challenge in the development of more effective treatment strategies. The efficacy of systemic therapies for GBM is greatly limited by the presence of the blood-brain barrier (BBB), which prevents drug penetration and accumulation in regions of infiltrative tumour, as represented in a consistent portion of GBM lesions. Focused ultrasound (FUS) – a technique that uses low-frequency ultrasound waves to induce targeted temporary disruption of the BBB – promises to improve survival outcomes by enhancing drug delivery and accumulation to infiltrating tumour regions. In this review we discuss the current state of preclinical investigations using FUS to enhance delivery of systemic therapies to intracranial neoplasms. We highlight critical methodological inconsistencies that are hampering clinical translation of FUS and we provide guiding principles for future preclinical studies. Particularly, we focus our attention on the importance of the selection of clinically relevant animal models and to the standardization of methods for FUS delivery, which will be paramount to the successful clinical translation of this promising technology for treatment in GBM patients. We also discuss how preclinical FUS research can benefit the development of GBM immunotherapies.
An emerging approach with potential in improving the treatment of neurodegenerative diseases and brain tumors is the use of focused ultrasound (FUS) to bypass the blood–brain barrier (BBB) in a non-invasive and localized manner. A large body of pre-clinical work has paved the way for the gradual clinical implementation of FUS-induced BBB opening. Even though the safety profile of FUS treatments in rodents has been extensively studied, the histological and behavioral effects of clinically relevant BBB opening in large animals are relatively understudied. Here, we examine the histological and behavioral safety profile following localized BBB opening in non-human primates (NHPs), using a neuronavigation-guided clinical system prototype. We show that FUS treatment triggers a short-lived immune response within the targeted region without exacerbating the touch accuracy or reaction time in visual-motor cognitive tasks. Our experiments were designed using a multiple-case-study approach, in order to maximize the acquired data and support translation of the FUS system into human studies. Four NHPs underwent a single session of FUS-mediated BBB opening in the prefrontal cortex. Two NHPs were treated bilaterally at different pressures, sacrificed on day 2 and 18 post-FUS, respectively, and their brains were histologically processed. In separate experiments, two NHPs that were earlier trained in a behavioral task were exposed to FUS unilaterally, and their performance was tracked for at least 3 weeks after BBB opening. An increased microglia density around blood vessels was detected on day 2, but was resolved by day 18. We also detected signs of enhanced immature neuron presence within areas that underwent BBB opening, compared to regions with an intact BBB, confirming previous rodent studies. Logistic regression analysis showed that the NHP cognitive performance did not deteriorate following BBB opening. These preliminary results demonstrate that neuronavigation-guided FUS with a single-element transducer is a non-invasive method capable of reversibly opening the BBB, without substantial histological or behavioral impact in an animal model closely resembling humans. Future work should confirm the observations of this multiple-case-study work across animals, species and tasks.
Background Focused ultrasound combined with microbubbles has been used in clinical studies for blood-brain barrier (BBB) opening in conjunction with MRI. However, the impact of the static magnetic field generated by an MRI scanner on the BBB opening outcome has not been evaluated. Purpose To determine the relationship of the static magnetic field of an MRI scanner on focused ultrasound combined with microbubble-induced BBB opening. Materials and Methods Thirty wild-type mice were divided into four groups. Mice from different groups were sonicated with focused ultrasound in different static magnetic fields (approximately 0, 1.5, 3.0, and 4.7 T), with all other experimental parameters kept the same. Focused ultrasound sonication was performed after intravenous injection of microbubbles. Microbubble cavitation activity, the fundamental -physical mechanism underlying focused ultrasound BBB opening, was monitored with passive cavitation detection. After sonication, contrast-enhanced T1-weighted MRI was performed to assess BBB opening outcome. Intravenously injected Evans blue was used as a model agent to evaluate trans-BBB delivery efficiency. Results The microbubble cavitation dose decreased by an average of 2.1 dB at 1.5 T (P = .05), 2.9 dB at 3.0 T (P = .01), and 3.0 dB at 4.7 T (P = .01) compared with that outside the magnetic field (approximately 0 T). The static magnetic field of an MRI scanner decreased BBB opening volume in mice by 3.2-fold at 1.5 T (P < .001), 4.5-fold at 3.0 T (P < .001), and 11.6-fold at 4.7 T (P <.001) compared with mice treated outside the magnetic field. It also decreased Evans blue trans-BBB delivery 1.4-fold at 1.5 T (P = .009), 1.6-fold at 3.0 T (P < .001), and 1.9-fold at 4.7 T (P < .001). Conclusion Static magnetic fields dampened microbubble cavitation activity and decreased trans-blood-brain barrier (BBB) delivery by focused ultrasound combined with microbubble-induced BBB opening. © RSNA, 2021.
Extracranial ultrasound assessment of the cervical arteries provides real-time, bedside evaluation of blood flow and vessel walls. Ultrasound examination is a non-invasive imaging modality of atherosclerosis in the extracranial arteries. For better yield of microembolic signals, power motion Doppler is superior to single-channel transcranial Doppler ultrasonography (TCD). TCD yields good-to-excellent results in detecting steno-occlusive intracranial lesions in the setting of acute cerebral ischemia when compared against computed tomographic angiography (CTA) or magnetic resonance angiography (MRA). Waveform pattern yields more information about thrombus location, hemodynamic significance of occlusion, and resistance in the distal vessels compared to velocity difference by itself. TCD is more sensitive than transthoracic echocardiogram for right-to-left shunt detection. Despite initial promising results of sonothrombolysis, the efficacy of ultrasound enhancement of thrombolysis has not been proven. Novel applications of ultrasound are molecular imaging, opening of the blood-brain barrier, and targeted drug delivery.
Much evidence indicates that abnormal processing and extracellular deposition
of amyloid-
To test the feasibility of noninvasive magnetic resonance (MR) imaging-guided focused ultrasound surgery (FUS) of benign fibroadenomas in the breast.
Eleven fibroadenomas in nine patients under local anesthesia were treated with MR imaging-guided FUS. Based on a T2-weighted definition of target volumes, sequential sonications were delivered to treat the entire target. Temperature-sensitive phase-difference-based MR imaging was performed during each sonication to monitor focus localization and tissue temperature changes. After the procedure, T2-weighted and contrast material-enhanced T1-weighted MR imaging were performed to evaluate immediate and long-term effects.
Thermal imaging sequences were improved over the treatment period, with 82% (279 of 342) of the hot spots visible in the last seven treatments. The MR imager was used to measure temperature elevation (12.8 degrees -49.9 degrees C) from these treatments. Eight of the 11 lesions treated demonstrated complete or partial lack of contrast material uptake on posttherapy T1-weighted images. Three lesions showed no marked decrease of contrast material uptake. This lack of effective treatment was most likely due to a lower acoustic power and/or patient movement that caused misregistration. No adverse effects were detected, except for one case of transient edema in the pectoralis muscle 2 days after therapy.
MR imaging-guided FUS can be performed to noninvasively coagulate benign breast fibroadenomas.
To determine if focused ultrasound beams can be used to locally open the blood-brain barrier without damage to surrounding brain tissue and if magnetic resonance (MR) imaging can be used to monitor this procedure.
The brains of 18 rabbits were sonicated (pulsed sonication) in four to six locations, with temporal peak acoustic power ranging from 0.2 to 11.5 W. Prior to each sonication, a bolus of ultrasonographic (US) contrast agent was injected into the ear vein of the rabbit. A series of fast or spoiled gradient-echo MR images were obtained during the sonications to monitor the temperature elevation and potential tissue changes. Contrast material-enhanced MR images obtained minutes after sonications and repeated 1-48 hours later were used to depict blood-brain barrier opening. Whole brain histologic evaluation was performed.
Opening of the blood-brain barrier was confirmed with detection of MR imaging contrast agent at the targeted locations. The lowest power levels used produced blood-brain barrier opening without damage to the surrounding neurons. Contrast enhancement correlated with the focal signal intensity changes in the magnitude fast spoiled gradient-echo MR images.
The blood-brain barrier can be consistently opened with focused ultrasound exposures in the presence of a US contrast agent. MR imaging signal intensity changes may be useful in the detection of blood-brain barrier opening during sonication.
Getting drugs and genes into the brain is a tall order. This is because the presence of the blood-brain barrier prevents many molecules from crossing into the brain. Overcoming this problem will have a profound effect on the treatment of many neurological disorders, allowing larger water-soluble molecules to pass into the brain. Transport vectors, such as endogenous peptides, modified proteins or peptidomimetic monoclonal antibodies, are one way of tricking the brain into allowing these molecules to pass. This article will review such molecular Trojan Horses, and the progress that has been made in the delivery of drugs and genes to the brain.
Brain drug development of either small molecule or large molecule (recombinant proteins, gene medicines) neurotherapeutics has been limited, owing to the restrictive transport properties of the brain microvasculature, which forms the blood-brain barrier (BBB) in vivo. Widespread drug delivery to the brain, while not feasible via craniotomy and intracerebral injection, is possible if the drug is delivered to brain via the transvascular route through the BBB. Novel brain drug delivery and drug targeting strategies can be developed from an understanding of the molecular and cellular biology of the brain microvascular and BBB transport processes.
The feasibility and safety of magnetic resonance (MR) imaging-guided focused ultrasound surgery for uterine leiomyomas is reported. Sequential sonications were delivered to nine targets. Temperature-sensitive phase-difference MR imaging monitored the location of the focus and measured tissue temperature elevations, ensuring therapeutic dose. MR images and hysterectomy specimens were evaluated. Six leiomyomas received full therapeutic doses, and 98.5% of the sonications were visualized. MR thermometry was successful in all sonications and cases. Focal necrotic lesions were seen in all cases at MR, and five were pathologically confirmed. MR imaging-guided focused ultrasound causes thermocoagulation and necrosis in uterine leiomyomas and is feasible and safe, without serious consequences.
Blood-brain barrier (BBB) genomics enables the rapid discovery of novel transporters that are expressed at the brain capillary endothelium. The BBB transporters are potential conduits to the brain that therapeutic drugs may use to gain passage across the BBB. Due to the small volume of brain occupied by the endothelium (10(-3) parts of the brain), it is necessary to build a BBB genomics program that is separate from a whole-brain genomics analysis. It is estimated that approximately 15% of all genes selectively expressed at the BBB encode for transporter proteins, and that only approximately 50% of BBB transporters are currently known. The development of a BBB genomics program and the discovery of novel BBB transporters could lead to the invention of new approaches to solving the BBB drug delivery problem.
One of the most clinically advanced forms of experimental disease-modifying treatment for Alzheimer disease is immunization against the amyloid beta protein (Abeta), but how this may prevent cognitive impairment is unclear. We hypothesized that antibodies to Abeta could exert a beneficial action by directly neutralizing potentially synaptotoxic soluble Abeta species in the brain. Intracerebroventricular injection of naturally secreted human Abeta inhibited long-term potentiation (LTP), a correlate of learning and memory, in rat hippocampus in vivo but a monoclonal antibody to Abeta completely prevented the inhibition of LTP when injected after Abeta. Size fractionation showed that Abeta oligomers, not monomers or fibrils, were responsible for inhibiting LTP, and an Abeta antibody again prevented such inhibition. Active immunization against Abeta was partially effective, and the effects correlated positively with levels of antibodies to Abeta oligomers. The ability of exogenous and endogenous antibodies to rapidly neutralize soluble Abeta oligomers that disrupt synaptic plasticity in vivo suggests that treatment with such antibodies might show reversible cognitive deficits in early Alzheimer disease.
Most lymphomas that involve the central nervous system are B-cell neoplasms that express the cell surface molecule CD20. After intravenous administration, rituximab can be reproducibly measured in the cerebrospinal fluid (CSF) in patients with primary central nervous system lymphoma; however, the CSF levels of rituximab are approximately 0.1% of serum levels associated with therapeutic activity in patients with systemic non-Hodgkin lymphoma. Because lymphomatous meningitis is a frequent complication of non-Hodgkin lymphoma, we have conducted an analysis of the safety and pharmacokinetics of direct intrathecal administration of rituximab using cynomolgus monkeys. No significant acute or delayed toxicity, neurologic or otherwise, was detected. Pharmacokinetic analysis suggests that drug clearance from the CSF is biphasic, with a terminal half-life of 4.96 hours. A phase 1 study to investigate the safety and pharmacokinetics of intrathecal rituximab in patients with recurrent lymphomatous meningitis will be implemented based on these findings.
Delivery of therapeutic proteins into tissues and across the blood-brain barrier is severely limited by the size and biochemical
properties of the proteins. Here it is shown that intraperitoneal injection of the 120-kilodalton β-galactosidase protein,
fused to the protein transduction domain from the human immunodeficiency virus TAT protein, results in delivery of the biologically
active fusion protein to all tissues in mice, including the brain. These results open new possibilities for direct delivery
of proteins into patients in the context of protein therapy, as well as for epigenetic experimentation with model organisms.
The aim of this study was to determine the safety and efficacy of intraarterial chemotherapy with osmotic opening of the blood-brain barrier (BBB) for the treatment of malignant brain tumors when administered across multiple centers. Patients with primary central nervous system lymphoma (PCNSL), primitive neuroectodermal tumor (PNET), germ cell tumor, cancer metastasis to the brain, or low or high grade glioma were eligible. Prior to entry, magnetic resonance imaging or computed tomography brain scan, medical history, neurologic status, and Karnofsky performance status were reviewed at the coordinating center. Standardized anesthesia and intraarterial catheterization guidelines were followed by a multidisciplinary team at each center. Between March 1994 and November 1997, 5 universities treated 221 adult patients with intraarterial chemotherapy with or without osmotic opening of the BBB (2464 procedures). Of evaluable patients with PCNSL, 40 of 53 (75%) achieved complete response (CR). All evaluable patients with PNET (n = 17), metastatic disease (n = 12), or germ cell tumor (n = 4) achieved stable disease (SD) or better. Of 57 evaluable patients with glioblastoma multiforme, 45 (79%) achieved SD or better. Asymptomatic subintimal tear occurred in 11of 221 patients (5%), pulmonary embolism in 6 of 221 (2.7%), and renal toxicity in 4 of 221 (1.8%). One patient with extensive glioma expired within 48 hours after treatment. Using standard guidelines and protocols, intraarterial chemotherapy with or without osmotic opening of the BBB is feasible across multiple centers with a low incidence of catheter-related complications. In patients with chemotherapy-sensitive tumors, such as PCNSL, PNET, germ cell tumor, and cancer metastasis to the central nervous system, enhanced delivery results in a high degree of tumor response, with an efficacy profile that is reproducible across multiple centers. Cancer 2000;88:637–47.
In this study, the threshold for subharmonic emission during in vivo sonication of rabbit brain was investigated. In addition, the histologic effects of pulsed sonication above this threshold were studied. Two spherically curved focused ultrasound transducers with a diameter of 80 mm and a radius of curvature of 70 mm were used in the sonications. The operating frequencies of the transducers were 0.936 and 1.72 MHz. The sonication duration was varied between 0.001 and 1 s and the repetition frequency between 0.1 and 5 Hz. The threshold for subharmonic emission at the frequency of 0.936 MHz was found to be approximately 2000 W cm-2 and 3600 W cm-2 for pulse durations of 1 s and 0.001 s, respectively. The threshold was approximately 1.5-fold as high at a frequency of 1.72 MHz. However, there was considerable variation from experiment to experiment. The multiple pulse experiments at a frequency of 1.72 MHz and an intensity of 7000 W cm-2 showed that the histologic effects ranged from no observable damage of the tissue, to blood-brain barrier breakage, to local haemorrhagia, to local destruction of the tissue, to gross hemorrhage resulting in the death of the animal. The severity of the tissue damage increased as the pulse duration, number of pulses and their repetition frequency increased. The results indicate that the end point of the tissue damage may be controlled by selecting the sonication parameters. Such control over tissue effects can have several different applications when brain disorders are treated.
In 1996, we are half-way through the Decade of the Brain, yet we still have few effective treatments for major disorders of the central nervous system. These include affective disorders, epilepsy, neurodegenerative disorders, brain tumours, infections and HIV encephalopathy; sufferers far outnumber the morbidity of cancer or heart disease. Increased understanding of the pharmacology of the brain and its blood supply, and methods for rational drug design, are leading to potential new drug therapies based on highly specific actions on particular target sites, such as neurotransmitter receptors and uptake systems. These methods are capable of reducing the side effects that are common with more general treatments. However, all these treatments and potential treatments meet a formidable obstacle--the blood-brain barrier. In this article, we review the properties of this barrier that complicate drug delivery to the brain, and some of the most hopeful strategies for overcoming or bypassing the barrier in humans.
The distribution of the dopaminergic D4 receptor in rat brain was studied employing site directed polyclonal antibodies. Antisera were raised in rabbits to two oligopeptides corresponding to amino acids 160-172 of the second extracellular loop (P1) and amino acids 260-273 of the third intracellular loop (P2) of the D4 receptor sequence. Affinity-purified antibodies (anti-P1 and anti-P2) specifically recognized two major bands of 42-45 and 95 kDa in Western blots of denatured preparations of various rat brain areas. Immunocyto-chemistry studies showed that D4 receptor is widely distributed in rat central nervous system (CNS) showing higher labelling in the hippocampus (CA1, CA2, CA3 and dentate gyrus) frontal cortex, entorhinal cortex, caudate putamen, nucleus accumbens, olfactory tubercle, cerebellum, supraoptic nucleus and sustancia nigra pars compacta. In addition, anti-P1 decreased the binding of the antagonist [3H]YM-09151-2 selective for D2, D3 and D4 receptors but did not modify the binding of [3H]raclopride an antagonist selective for D2 and D3, in striatal synaptosomes. Anti-P2 did not modify the binding of these ligands. These results confirm the selectivity of the antibodies towards the D4 receptor and suggest that the binding site for the antagonists might be located at or close to the second extracellular loop of the protein sequence. D4 receptor protein is mainly expressed in plasma membranes and in the peripheral cytoplasm of neurons and is more widely distributed than was originally proposed based on mRNA localization, since it is present both in limbic, diencephalic and motor areas of rat brain.
To determine the feasibility of the use of temperature-sensitive magnetic resonance (MR) imaging for the detection of local temperature elevations at the focus of a low-power ultrasound beam in the brain.
The brains in 28 rabbits were sonicated at acoustic power levels of 3.5-17.5 W. Four to five different locations were sonicated at different acoustic power levels in each rabbit. MR images were obtained 2 hours, 48 hours, 10 days, and 23 days after the sonications, depending on when the animals were sacrificed. Histologic evaluation of whole brain was performed.
Forty of 43 (93%) of the lowest-power (3.5-W) sonications were visible on temperature-sensitive MR images and did not result in any short- or long-term histologic or MR imaging evidence of tissue damage. A contrast-to-noise ratio of approximately 6 and a temperature elevation of 7 degrees-8 degrees C were observed.
Temperature elevations induced by means of focused ultrasound exposures that do not cause damage in the in vivo rabbit brain can be detected at temperature-sensitive MR imaging.
We investigated regional therapy of recurrent malignant brain tumors with transferrin-CRM107, a conjugate of human transferrin (Tf) and a genetic mutant of diphtheria toxin (CRM107) that lacks native toxin binding. Physiological barriers to delivering proteins to tumor and surrounding infiltrated brain were circumvented with high-flow interstitial microinfusion. At least a 50% reduction in tumor volume on magnetic resonance imaging (MRI) occurred in 9 of 15 patients who could be evaluated (60%), including two complete responses. Peritumoral toxicity developed 1-4 weeks after treatment in three of three patients at 1.0 microg/ml, but in zero of nine patients treated at lower concentrations. No symptomatic systemic toxicity occurred. Regional perfusion with Tf-CRM107 produces tumor responses without systemic toxicity in patients with malignant brain tumors refractory to conventional therapy. Direct interstitial infusion can be used successfully to distribute a large protein in the tumor and infiltrated brain surrounding the tumor.
This article reviews historical aspects of the blood-brain barrier (BBB) and recent advances in mechanisms to deliver therapeutic agents across the BBB for the treatment of intracerebral tumors and other neurological diseases.
The development of the osmotic BBB disruption procedure as a clinically useful technique is described. Osmotic BBB disruption is contrasted with alternative methods for opening or bypassing the BBB, including pharmacological modification of the BBB with bradykinin and direct intracerebral infusion.
Laboratory studies have played a fundamental role in advancing our understanding of the BBB and delivery of agents to brain. Preclinical animal studies will continue to serve an integral function in our efforts to improve the diagnosis and treatment of a number of neurological disorders. Techniques involving the modification of the BBB and/or blood-tumor barrier to increase delivery of therapeutic agents have been advanced to clinical trials in patients with brain tumors with very favorable results.
Improving delivery of agents to the brain will play a major role in the therapeutic outcome of brain neoplasms. As techniques for gene therapy are advanced, manipulation of the BBB also may be important in the treatment of central nervous system genetic disorders.
Significant advances have been made in the application of monoclonal antibody-based therapies to the treatment of patients with lymphoma. The most promising areas appear to be the use of unconjugated monoclonal antibodies and the use of radiolabeled monoclonal antibodies. The recent approval by the US Food and Drug Administration (FDA) of rituximab (Rituxan), an unconjugated chimeric antibody against the CD20 antigen for the treatment of relapsed low-grade or follicular B-cell non-Hodgkin's lymphoma marked a milestone in the development of these antibody-based treatments. Other new drug applications to the FDA are pending using both unconjugated and radiolabeled monoclonal antibodies, and it is anticipated that further new treatment options based on monoclonal antibody technology will soon be available for the treatment of patients with non-Hodgkin's lymphoma. Forthcoming clinical trial results combining these new agents with current therapies are needed to determine if the addition of these new biologic agents to our armamentarium against lymphoma will alter the natural history of this disease for our patients. The most promising of these treatments and the comparison of these strategies are reviewed here.
Delivery of therapeutic proteins into tissues and across the blood-brain barrier is severely limited by the size and biochemical properties of the proteins. Here it is shown that intraperitoneal injection of the 120-kilodalton beta-galactosidase protein, fused to the protein transduction domain from the human immunodeficiency virus TAT protein, results in delivery of the biologically active fusion protein to all tissues in mice, including the brain. These results open new possibilities for direct delivery of proteins into patients in the context of protein therapy, as well as for epigenetic experimentation with model organisms.
The aim of this study was to determine the safety and efficacy of intraarterial chemotherapy with osmotic opening of the blood-brain barrier (BBB) for the treatment of malignant brain tumors when administered across multiple centers.
Patients with primary central nervous system lymphoma (PCNSL), primitive neuroectodermal tumor (PNET), germ cell tumor, cancer metastasis to the brain, or low or high grade glioma were eligible. Prior to entry, magnetic resonance imaging or computed tomography brain scan, medical history, neurologic status, and Karnofsky performance status were reviewed at the coordinating center. Standardized anesthesia and intraarterial catheterization guidelines were followed by a multidisciplinary team at each center. Between March 1994 and November 1997, 5 universities treated 221 adult patients with intraarterial chemotherapy with or without osmotic opening of the BBB (2464 procedures).
Of evaluable patients with PCNSL, 40 of 53 (75%) achieved complete response (CR). All evaluable patients with PNET (n = 17), metastatic disease (n = 12), or germ cell tumor (n = 4) achieved stable disease (SD) or better. Of 57 evaluable patients with glioblastoma multiforme, 45 (79%) achieved SD or better. Asymptomatic subintimal tear occurred in 11 of 221 patients (5%), pulmonary embolism in 6 of 221 (2.7%), and renal toxicity in 4 of 221 (1.8%). One patient with extensive glioma expired within 48 hours after treatment.
Using standard guidelines and protocols, intraarterial chemotherapy with or without osmotic opening of the BBB is feasible across multiple centers with a low incidence of catheter-related complications. In patients with chemotherapy-sensitive tumors, such as PCNSL, PNET, germ cell tumor, and cancer metastasis to the central nervous system, enhanced delivery results in a high degree of tumor response, with an efficacy profile that is reproducible across multiple centers.
Using polyclonal antibody against dopamine D4 receptor we investigated cortical distribution of D4 receptors, with the special emphasis on regions of the prefrontal cortex. Prefrontal cortex is regarded as a target for neuroleptic drugs, and engaged in the regulation of the psychotic effects of various substances used in the experimental modeling of schizophrenia. Western blot analysis performed on samples from the rat cingulate, parietal, piriform cortices and also striatum revealed that antibody recognized one main band of approximately 40 kD, which corresponds to the predicted molecular weight of D4 receptor protein. In immunocytochemical studies we found D4 receptor-positive neurons in all regions of prefrontal cortex (cingulate, agranular/insular and orbital cortices) and all cortical regions adjacent to prefrontal cortex, such as frontal, parietal and piriform cortex. Substantial number of D4 receptor-positive neurons has also been observed within the striatum and nucleus accumbens. In general, a clear stratification of the D4 receptor-positive neurons was observed in the cortex with the highest density seen in layers II/III and V/VI. D4 immunopositive material was also found in the dendritic processes, particularly clearly visible in the layer II/III. At the cellular level D4 receptor immunoreactivity was seen predominantly on the periphery of the cell body, but a certain population of neurons with clear cytoplasmatic localization was also identified. In addition to cortical distribution of D4 receptor-positive neurons we tried also to define types of neurons expressing D4 receptor protein. In double-labeling experiments, D4 receptor protein was found in nonphosphorylated neurofilament H-positive, calbindin-D28k-positive, as well as parvalbumin-positive cells. Since, used proteins are markers of certain populations of pyramidal neurons and GABA-ergic interneurons, respectively, our data indicate that D4 receptors are located on cortical pyramidal output neurons and their dendritic processes as well as on interneurons. Above localization indicates that D4 receptors are not only directly influencing excitability of cortical inter- and output neurons but also might be engaged in dendritic spatial and temporal integration, required for the generation of axonal messages. Additionally, our data show that D4 receptors are widely distributed throughout the cortex of rat brain, and that their cortical localization exceeds the localization of dopaminergic terminals.
Much evidence indicates that abnormal processing and extracellular deposition of amyloid-beta peptide (A beta), a proteolytic derivative of the beta-amyloid precursor protein (betaAPP), is central to the pathogenesis of Alzheimer's disease (reviewed in ref. 1). In the PDAPP transgenic mouse model of Alzheimer's disease, immunization with A beta causes a marked reduction in burden of the brain amyloid. Evidence that A beta immunization also reduces cognitive dysfunction in murine models of Alzheimer's disease would support the hypothesis that abnormal A beta processing is essential to the pathogenesis of Alzheimer's disease, and would encourage the development of other strategies directed at the 'amyloid cascade'. Here we show that A beta immunization reduces both deposition of cerebral fibrillar A beta and cognitive dysfunction in the TgCRND8 murine model of Alzheimer's disease without, however, altering total levels of A beta in the brain. This implies that either a approximately 50% reduction in dense-cored A beta plaques is sufficient to affect cognition, or that vaccination may modulate the activity/abundance of a small subpopulation of especially toxic A beta species.
Herceptin (trastuzumab), an anti-HER2 monoclonal antibody, is the first oncogene-targeted therapy to be developed for the treatment of metastatic breast cancer. The Herceptin clinical trial program has demonstrated that treatment with Herceptin provides substantial clinical benefits when used either as monotherapy or in combination with a number of chemotherapeutic agents. Of note, accurate assessment of HER2 status is essential to ensure that the patients most likely to benefit from Herceptin are identified: patients with immunohistochemistry (IHC) 3+ or fluorescence in-situ hybridization (FISH)-positive disease gain the greatest clinical benefits. In addition, clinical benefits appear to be greater the earlier Herceptin is used, although there is currently no direct clinical evidence to indicate whether an initial strategy of combination therapy is better than monotherapy or vice versa. Herceptin has been shown to be generally well tolerated. The most severe adverse events are rare serious infusion-related reactions and cardiotoxicity. These adverse events can be managed by standard care and patients at risk can often be identified prior to the initiation of Herceptin treatment. Currently, Herceptin should be given until disease progression, but there could be benefit in continuing treatment beyond disease progression.
MRI-guided focused ultrasound was tested in the brains of rhesus monkeys. Locations up to 4.8 cm deep were targeted. Focal heating was observed in all cases with MRI-derived temperature imaging. Subthreshold heating was observed at the focus when the ultrasound beam was targeted with low power sonications, and in the ultrasound beam path during high-power exposures. Lethal temperature values and histologically confirmed tissue damage were confined to the focal zone (e.g., not in the ultrasound beam path), except when the focus was close to the bone. In that case, damage to the neighboring brain tissue was observed. Focal lesions were observed on histological examination and, in some cases, in MR images acquired immediately after the ultrasound exposures. The capabilities demonstrated in this study will be of benefit for clinical ultrasound therapies in the brain.
The authors review the preclinical and clinical results of the ligand-targeted toxin conjugate Transferrin-CRM107 (Tf-CRM107), for the treatment of malignant gliomas. Tf-CRM107 is a conjugate protein of diphtheria toxin with a point mutation (CRM107) linked by a thioester bond to human transferrin (Tf). This conjugate exhibits potent cytotoxicity in vitro against mammalian cells expressing the transferrin receptor with activity at picomolar concentrations. Phase I clinical trial results demonstrated that Tf-CRM107, delivered via a high-flow convection method utilizing stereotactically placed catheters, produced tumor response in patients with malignant brain tumors refractory to conventional therapy without severe neurologic or systemic toxicity. The results of a Phase II study are also summarized. Tf-CRM107 treatment results in complete and partial tumor response without severe toxicity in 35% of the evaluable patients. These data warrant a Phase III study as well as continued research in the field of targeted toxin therapy. Future directions of research include optimizing Tf-CRM107 delivery to targeted brain regions, and improving the treatment efficacy by combining with other toxin conjugates targeted to different receptors.
Blood brain barrier (BBB) is a major barrier for delivering therapeutic agents in the brain. In this study we investigated the feasibility of open the BBB by using focused ultrasound. Rabbit brains were exposed to pulsed focused ultrasound while injecting ultrasound contrast agent containg microbubbles intravenously. The BBB opening was measured after the sonications by injecting MRI contrast agent i.v. and evaluating the local enhancement in the brain. Low ultrasound powers and pressure amplitudes were found to cause focal enhancement. Before sacrificing the animals trypan blue was also injected i.v.. After the sacrifice of the animals blue spots were found in the brain in the sonicated locations. This method may have potential for targeted delivery of macromolecules in the brain.
The ability of MRI-derived thermometry to predict thermally induced tissue changes in the brain was tested, and the thermal thresholds for blood-brain barrier (BBB) disruption and brain tissue damage were estimated. In addition, the ability of standard MRI to detect threshold-level effects was confirmed. These safety thresholds are being investigated to provide guidelines for clinical thermal ablation studies in the brain. MRI-monitored focused ultrasound heating was delivered to 63 locations in 26 rabbits. Tissue changes were detected in T(2)-weighted imaging and T(1)-weighted imaging (with and without contrast) and with light microscopy. The probability for tissue damage as a function of the accumulated thermal dose, the peak temperature achieved, the applied acoustic energy, and the peak acoustic power was estimated with probit regression. The discriminative abilities of these parameters were compared using the areas under the receiver operator characteristic (ROC) curves. In MRI, BBB disruption was observed in contrast-enhanced T(1)-weighted imaging shortly after the ultrasound exposures, sometimes accompanied by changes in T(2)-weighted imaging. Two days later, changes in T(2)-weighted imaging were observed, sometimes accompanied by changes in T(1)-weighted imaging. In histology, tissue damage was seen at every location where MRI changes were observed, ranging from small (diameter <1.0 mm) areas of tissue necrosis to severe vascular damage and associated hemorrhagic infarct. In one location, small (diameter: 0.8 mm) damage was not detected in MRI. The thermal dose and peak temperature thresholds were between 12.3-40.1 equivalent min at 43 degrees C and 48.0-50.8 degrees C, respectively, and values of 17.5 equivalent min at 43 degrees C and 48.4 degrees C were estimated to result in tissue damage with 50% probability. Thermal dose and peak temperature were significantly better predictors than the applied acoustic energy and peak acoustic power (P < 0.01). BBB disruption was always accompanied by tissue damage. The temperature information was better than the applied acoustic power or energy for predicting the damage than the ultrasound parameters. MRI was sensitive in detecting threshold-level damage.
Local blood-brain barrier (BBB) opening is an advantageous approach for targeted drug delivery to the brain. Recently, it has been shown that focused ultrasound (US) exposures (sonications), when applied in the presence of preformed gas bubbles, caused magnetic-resonance (MR) proven reversible opening of the BBB in targeted locations. The cellular mechanisms of such transient barrier disruption are largely unknown. We investigated US-induced changes in endothelial cell fine morphology that resulted in the BBB opening in rabbits. To obtain evidence for the passage of blood-borne macromolecules through the opened transvascular routes, an immunocytochemical procedure for endogenous immunoglobulinG (IgG) was performed, in addition to the routine electron microscopy. An increased number of vesicles and vacuoles, fenestration and channel formation, as well as opening of some tight junctions, were seen in capillaries after low-power (0.55 W) sonication. Immunosignals presented in some of the vesicles and vacuoles, in the cytoplasmic channels and, so rarely, in intercellular clefts; immunosignals could also be seen in neuropil around the blood vessels. Damage to the cellular ultrastructure was not seen in these areas. However, cell destruction and leakage of IgG through defects of the endothelial lining took place at 3 W sonications. The data reveals that several mechanisms of transcapillary passage are possible after such sonications: 1. transcytosis; 2. endothelial cell cytoplasmic openings - fenestration and channel formation; 3. opening of a part of tight junctions; and 4. free passage through the injured endothelium (with the higher power sonications). These findings could be considered in further development of the strategy for drug delivery to brain parenchyma. (E-mail: [email protected]
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The purpose of this study was to test the hypothesis that burst ultrasound in the presence of an ultrasound contrast agent can disrupt the blood-brain barrier (BBB) with acoustic parameters suitable for completely noninvasive exposure through the skull. The 10-ms exposures were targeted in the brains of 22 rabbits with a frequency of 690 kHz, a repetition frequency of 1 Hz, and peak rarefactional pressure amplitudes up to 3.1 MPa. The total exposure (sonication) time was 20 s. Prior to each sonication, a bolus of ultrasound contrast agent was injected intravenously. Contrast-enhanced MR images were obtained after the sonications to detect localized BBB disruption via local enhancement in the brain. Brain sections were stained with H&E, TUNEL, and vanadium acid fuchsin (VAF)-toluidine blue staining. In addition, horseradish peroxidase (HRP) was injected into four rabbits prior to sonications and transmission electron microscopy was performed. The MRI contrast enhancement demonstrated BBB disruption at pressure amplitudes starting at 0.4 MPa with approximately 50%; at 0.8 MPa, 90%; and at 1.4 MPa, 100% of the sonicated locations showed enhancement. The histology findings following 4 h survival indicated that brain tissue necrosis was induced in approximately 70-80% of the sonicated locations at a pressure amplitude level of 2.3 MPa or higher. At lower pressure amplitudes, however, small areas of erythrocyte extravasation were seen. The electron microscopy findings demonstrated HRP passage through vessel walls via both transendothelial and paraendothelial routes. These results demonstrate that completely noninvasive focal disruption of the BBB is possible.
Short interfering RNA (siRNA) has attracted much attention for clinical use in various diseases. However, its delivery, especially through the cell membrane, continues to present a challenge. Advances in ultrasound- and ultrasound contrast-agent technologies have made it possible to change transiently the permeability of the cell membrane and, using a focused ultrasound transducer, to narrow and focus the ultrasound energy on a small target, thereby avoiding damage to surrounding tissue. In this in vitro study, we demonstrate that it is possible to deliver siRNA intracellularly via microbubble-enhanced focused ultrasound. Although further optimization is necessary, our novel method for siRNA transduction represents a powerful tool for using siRNA in vivo and possibly in the clinical setting.
Drug and gene targeting to the brain with molecular Trojan horses
- Pardridge