Transcranial focused ultrasound for hypertension treatment

Abstract

A minimally invasive method for lowering hypertension using focused ultrasound is provided. Following injection of ultrasound-responsive nanodroplets containing a therapeutic agent (e.g., a barbiturate), focused ultrasound is delivered to norepinephrine-producing regions, such as the periaqueductal gray area, resulting in localized release of the therapeutic payload from the nanodroplets, achieving therapeutic blood pressure reduction. Acoustic emissions from the evaporating droplets can be used to infer one or more therapeutic parameters, for example, based on pre-established correlations. For example, changes in plasma hormone content and / or blood pressure can be inferred, thereby providing a means for real-time treatment monitoring. An exemplary method of the present invention can be used to achieve blood pressure reduction in subjects exhibiting drug-resistant hypertension.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 391,201, filed July 21, 2022, entitled "METHODS FOR THE TREATMENT OF HYPERTENSION VIA TRANSCRANIAL-FOCUSED-ULTRASOUND," the entire contents of which are incorporated herein by reference. [Background technology]

[0002] The present disclosure relates to methods of neuromodulation using focused ultrasound.

[0003] Elevated blood pressure, known as hypertension, affects one-quarter of adults worldwide, with prevalence rates continuing to rise in low-income countries (WHO, 2022). Current strategies for lowering high blood pressure generally involve pharmaceuticals. Antihypertensive drugs have been in clinical use for over 60 years and are grouped into five major classes (beta-blockers, diuretics, angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, and calcium channel blockers) that act on the central nervous system, reduce cardiac output, cause vasodilation, or alter enzyme secretion. However, many of these drugs are ineffective or unsafe for drug-resistant hypertension and during pregnancy (Laurent, 2017).

[0004] Focused ultrasound (FUS), a type of sound wave above 20 kHz whose maximum intensity is focused on a single target, can noninvasively modulate brain activity. This technique offers greater precision and deeper penetration than external electric and magnetic fields, allowing for targeting of specific anatomical brain regions (Meng et al., 2021). The U.S. Food and Drug Administration (FDA) approved the use of focused ultrasound for the treatment of essential tremor in July 2016. Precise MRI-guided ablation of the thalamus, called focused ultrasound thalamotomy, can achieve significant reductions in hand tremor and improvements in motor function (Elias et al., 2016) and is currently used to treat approximately 7,000 patients worldwide (InSightec). Similar techniques have since been utilized to treat Parkinson's disease ( Martinez-Fernandez et al., 2018 ), obsessive-compulsive disorder (OCD), and major depressive disorder ( Davidson et al., 2020 ), and have been found to be safe for treating epilepsy ( Stern et al., 2021 ).

[0005] At lower intensities, non-ablative ultrasound exposure can transiently modulate neuronal activity (Fomenko et al., 2018). This has been preclinically demonstrated in the treatment of epilepsy (Hakimova et al., 2015; Zhang, Li, Liu, et al., 2021; Zhang, Li, Lv, et al., 2021) and essential tremor (Sharabi et al., 2019). In a model of acute pain, sonication of the periaqueductal gray (PAG) demonstrated an analgesic effect (Zhang et al., 2022). In a rat model of hypertension, Li et al. demonstrated that daily delivery of low-intensity ultrasound to the ventrolateral periaqueductal gray (vlPAG) reduced systolic blood pressure, heart rate, and plasma hypertension-related proteins (Li et al., 2020). This study demonstrated how ultrasound could noninvasively achieve blood pressure reductions similar to those achieved by DBS. However, the duration of blood pressure reduction after treatment was not reported, so it remains unclear whether continuous treatment seven times daily was optimal or necessary, and whether the effect persisted for a clinically relevant time frame.

[0006] In addition to direct neuromodulation, FUS is a noninvasive method for intracerebral anesthetic release using ultrasound-responsive drug carriers, such as droplets for the delivery of propofol (Airan et al., 2017) and barbiturates (Lea-Banks et al., 2020), and microbubbles for muscimol release (Ozdas et al., 2020). Combining FUS with locally released anesthetics allows the duration of neuromodulation to be controlled to extend the effect based on the drug's kinetics. Previously, we demonstrated this offline neuromodulation using pentobarbital-loaded nanodroplets (PBNDs), demonstrating persistent sensorimotor deficits in rats 60 minutes (Lea-Banks et al., 2020) and 90 minutes (Lea-Banks et al., 2021) after sonication had ended. Furthermore, encapsulation of anesthetics prevents off-target effects, such as general sedation, and improves the reliability of suppression within the focal region ( Wang et al., 2018 ). Summary of the Invention

[0007] A minimally invasive method for lowering hypertension using focused ultrasound is provided. Following injection of ultrasound-responsive nanodroplets containing a therapeutic agent (e.g., a barbiturate), focused ultrasound is delivered to norepinephrine-producing regions, such as the periaqueductal gray area, resulting in localized release of the therapeutic payload from the nanodroplets, achieving therapeutic blood pressure reduction. Acoustic emissions from the evaporating droplets can be used to infer one or more therapeutic parameters, for example, based on pre-established correlations. For example, changes in plasma hormone content and / or blood pressure can be inferred, thereby providing a means for real-time treatment monitoring. An exemplary method of the present invention can be used to achieve blood pressure reduction in subjects exhibiting drug-resistant hypertension.

[0008] Thus, in some aspects, a method for treating hypertension using focused ultrasound is provided, the method comprising delivering focused ultrasound to norepinephrine-producing regions of the brain following intravenous injection of nanodroplets containing a therapeutic agent, the focused ultrasound being configured to promote evaporation of the nanodroplets and release of the therapeutic agent.

[0009] In some exemplary embodiments of the method, the therapeutic agent comprises a barbiturate.

[0010] In some exemplary embodiments of the method, the therapeutic agent comprises a lipophilic anesthetic, which may have a molecular weight of less than 400 Da and a logarithm of a partition coefficient approximately equal to 2.

[0011] In some exemplary embodiments of the method, the norepinephrine-producing region comprises the periaqueductal gray region.

[0012] In some exemplary embodiments of the method, the norepinephrine-producing region comprises the ventrolateral periaqueductal gray region.

[0013] In some exemplary embodiments of the method, the norepinephrine-producing region includes at least one of the locus coeruleus, the thalamus, the hypothalamus, the neocortex, and the cerebellum.

[0014] In another aspect, a method of treating hypertension using focused ultrasound is provided, the method comprising delivering focused ultrasound to a norepinephrine-producing region of the brain, the focused ultrasound configured to induce repetitive mechanical stress on tissue by forming two or more closely spaced ultrasound acoustic pressure nodes, wherein adjacent nodes are in different acoustic phases, thereby generating forces between the nodes that stimulate or inhibit neuronal activity.

[0015] In some exemplary implementations of the method, the ultrasonic field is generated by one of a phased array, a lens, a reflector, a waveguide, and multiple overlapping ultrasonic fields.

[0016] In some exemplary embodiments of the method, the norepinephrine-producing region comprises the periaqueductal gray region.

[0017] In some exemplary embodiments of the method, the norepinephrine-producing region includes at least one of the locus coeruleus, the thalamus, the hypothalamus, the neocortex, and the cerebellum.

[0018] In some exemplary embodiments of the method, the norepinephrine-producing region comprises the ventrolateral periaqueductal gray region.

[0019] In some exemplary embodiments of the method, focused ultrasound delivery is performed after intravenous injection of nanodroplets containing a therapeutic agent to promote evaporation of the nanodroplets and release of the therapeutic agent.

[0020] In some exemplary embodiments of the method, focused ultrasound delivery is combined with a systemically delivered agent, such as an anesthetic, that enhances the ultrasound impact on brain tissue.

[0021] A further understanding of the functional and advantageous aspects of the present disclosure can be realized by reference to the following detailed description and drawings.

[0022] Embodiments will now be described, by way of example only, with reference to the drawings in which: [Brief explanation of the drawings]

[0023] [Figure 1] 1 is a flow chart illustrating an exemplary method for reducing hypertension using transcranial focused ultrasound delivery to the periaqueductal gray region (PAG) following injection of drug-loaded nanodroplets. [Figure 2] 1 illustrates an exemplary transcranial focused ultrasound system. [Figure 3] 1 illustrates an exemplary focused ultrasound headset. [Figure 4]This figure shows the sustained reduction in blood pressure after a single sonication in normotensive rats. (A) Schematic of noninvasive blood pressure measurement in awake rats in a cylindrical rodent holder using an occlusion cuff and a volumetric pressure recording (VPR) cuff. Representative pre-treatment pressure measurements are shown. (B) Schematic of the focused ultrasound system targeting the ventrolateral periaqueductal gray (vlPAG). (C) Ultrasound pulse scheme. (D) Histological safety assessment showed no signs of bleeding or edema after sonication (scale bar 50 μm). (E) Systolic blood pressure, (F) diastolic blood pressure, and (G) heart rate before and after sonication. Lines indicate individual measurements from six healthy rats. Significance is assessed from baseline (*p<0.05). [Figure 5] Figure 1 shows the reduction in systolic and diastolic blood pressure in normotensive rats. Single treatment scheme: (A) treatment timeline, (B) heart rate, (C) systolic blood pressure (BP), (D) diastolic blood pressure (BP). Two consecutive treatments: (E) treatment timeline, (F) heart rate, (G) systolic blood pressure (BP), (H) diastolic blood pressure (BP). Error bars represent 1 standard deviation, N=6, *p<0.05. [Figure 6] Figure 1 shows the effect of sonication of brain regions on reducing blood pressure in hypertensive rats. Changes in systolic blood pressure measured daily from baseline, during treatment (2 h after treatment), and 6 days after treatment were shown for (A) naive, (B) PBND + FUS (active control), (C) FUS (active control), (H) PBND + FUS (vlPAG), and (I) FUS (vlPAG) rats. Plasma noradrenaline content after 2 h of sonication of (D-F) active control and (J-L) vlPAG regions was plotted against acoustic emission at subharmonic frequencies, systolic, and diastolic blood pressure. Markers represent individual rats after treatment with FUS alone (circles) or pentobarbital-encapsulated nanodroplets (PBND) with FUS (triangles). Linear correlation was assessed using Pearson's coefficient (R2). [Figure 7]Figure 1. Reduction in systolic and diastolic blood pressure in hypertensive rats. (A) Schematic of a crossover study design with five consecutive treatments. (B) Acoustic emission from activated pentobarbital-loaded nanodroplets (PBNDs) is mapped to changes in systolic blood pressure 2 hours after treatment. (C) Heart rate, (D) systolic blood pressure (BP), and (E) diastolic blood pressure (BP). Error bars represent 1 standard deviation, N=4, *p<0.05. [Figure 8] Immunohistochemical analysis showing increased neuronal activity after treatment is illustrated. (A) Immunohistochemical sections of VLPAG from three hypertensive rats (naive, FUS only, PBND+FUS) stained for c-Fos (red), NeuN (green), and DAPI (blue). Scale bars represent 100 μm. (B, C) C-Fos expression from unsonicated (right) and sonicated (left) PAG regions is shown in (D) a schematic diagram of a brain cross-section. (E, F) NeuN-positive cells in unsonicated (right) and sonicated PAG regions, and (G) the proportion of c-Fos-positive cells that were also NeuN-positive, N=1. [Figure 9] Habituation to blood pressure and heart rate measurements. (A1-A3) Normotensive rats and (B1-B3) SHR were trained for 5 days before treatment to increase tolerance, reduce tail movement, and decrease variability (standard deviation, STD) in blood pressure and heart rate (beats per minute, bpm) measurements. [Figure 10] (A) Healthy normotensive Wistar (n = 6) and (B) spontaneously hypertensive rats (n = 4) were evaluated for body weight and temperature during acclimation, treatment, and post-treatment follow-up. Error bars represent 1 standard deviation; no significance (NS) was found between groups (p > 0.05). [Figure 11] Plasma hormonal evaluation of adrenaline levels in spontaneously hypertensive rats was shown and showed no correlation with systolic or diastolic blood pressure (BP) as assessed by Pearson's coefficient (R2<0.1). [Figure 12]Representative images of histological safety assessments performed using (A) TUNEL staining and (B) H&E staining to identify hemorrhage or cell apoptosis are shown. No damage was observed within the sonicated area in normotensive or hypertensive rats. [Figure 13] 1 is a table presenting the distribution of animals across experimental groups. [Figure 14] A, B, C, and D provide experimental results demonstrating the dose-dependence of ultrasound-induced blood pressure reduction. DETAILED DESCRIPTION OF THE INVENTION

[0024] Various embodiments and aspects of the present disclosure are described with reference to the details discussed below. The following description and drawings are illustrative of the present disclosure and should not be construed as limiting the disclosure. Numerous specific details are set forth in order to provide a thorough understanding of various embodiments of the present disclosure. However, in certain instances, well-known or conventional details are not set forth in order to provide a concise discussion of the embodiments of the present disclosure.

[0025] As used herein, the terms "comprises" and "comprising" are to be construed as inclusive and open-ended, not exclusive. Specifically, when used in the specification and claims, the terms "comprises" and "comprising" and variations thereof mean that the specified features, steps, or components are included. These terms are not to be construed as excluding the presence of other features, steps, or components.

[0026] As used herein, the word "exemplary" means "serving as an example, instance, or illustration," and is not to be construed as preferred or advantageous over other configurations disclosed herein.

[0027] As used herein, the terms "about" and "approximately" are intended to encompass variations that may exist within the upper and lower limits of a range of values, including variations in properties, parameters, and dimensions. Unless otherwise specified, the terms "about" and "approximately" mean plus or minus 25 percent or less.

[0028] Unless otherwise specified, it should be understood that any specified range or group is intended as a shorthand for referring to every member of that individual range or group, as well as to every possible subrange or subgroup contained therein, and the same for every subrange or subgroup therein. Unless otherwise specified, the present disclosure relates to and expressly incorporates every specific member and combination of subranges or subgroups.

[0029] As used herein, when used in conjunction with an amount or parameter, the term "on the order of" refers to a range ranging from about one-tenth to ten times the stated amount or parameter.

[0030] Unless otherwise defined, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise indicated, the following terms are intended to have the following meanings as used herein:

[0031] As mentioned above, previous studies involving the delivery of focused ultrasound alone to the PAG have been successful in demonstrating a reduction in hypertension, but the duration of such benefits has remained unclear. As described in the Examples provided below, in a hypertensive rat model, we found that delivery of focused ultrasound alone to the PAG resulted in only a subset of treated rats exhibiting a reduction in blood pressure after focused ultrasound treatment. Furthermore, in hypertensive rats that demonstrated a reduction in blood pressure, the transient reduction lasted only 24 hours after five consecutive days of focused ultrasound delivery to the PAG.

[0032] The present inventors sought to improve the therapeutic effect and duration of focused ultrasound treatment of the brain to improve the reduction of hypertension. Instead of simply sonicating the PAG region of the brain, the inventors discovered a combined treatment modality involving the use of focused ultrasound and barbiturate-loaded nanodroplets. Subjects were injected with barbiturate-loaded nanodroplets, followed by focused ultrasound delivery to the PAG. It was found that the combined effect of focused ultrasound on the PAG region and the impact of local release of barbiturate from the nanodroplets within the PAG via focused ultrasound resulted in a dual-mechanism neuromodulation therapeutic effect.

[0033] Accordingly, various exemplary embodiments of the present disclosure use focused ultrasound (FUS) in conjunction with barbiturate-loaded nanodroplets as a minimally invasive approach to reducing hypertension by modulating central brain activity. An exemplary method for focused ultrasound-based neuromodulation of hypertension via triggered release of barbiturate-loaded nanodroplets is shown in FIG. 1. In step 100, nanodroplets loaded with a therapeutic agent (e.g., pentobarbital) are injected into a subject's bloodstream. Focused ultrasound is then delivered to the subject's periaqueductal gray region (PAG), as shown in step 110. The focused ultrasound is configured to facilitate the focused ultrasound triggering the release of the therapeutic agent from the nanodroplets.

[0034] As shown in optional step 120, the acoustic emissions resulting from the collapse of the nanodroplets can be monitored, for example, using one or more of the transducers used to deliver the focused ultrasound and / or via one or more separate transducers. For example, the detected acoustic emissions can be used to provide a feedback mechanism for controlling the delivery of the focused ultrasound, and / or as a means for verifying or determining that an effective dose has been delivered via evaporation of the nanodroplets, and / or as a means for determining or inferring the reduction in blood pressure caused by the treatment. For example, at sufficient ultrasound pressure, known as the evaporation threshold, the nanodroplets transition from a liquid to a gaseous state, releasing the therapeutic agent and emitting a unique acoustic signal. Subharmonic frequencies (half the transmission frequency) can be used as a marker of drug release (Lea-Banks et al. 2020, 2021). As described below, the inventors have demonstrated that acoustic emissions correlate poorly with a reduction in blood pressure (R 2 = 0.46 and 0.36, Pearson coefficients) (see Figure 7B), whereas the acoustic emissions detected from FUS alone showed no correlation with changes in blood pressure (R 2 =0.08 and 0.04, Pearson coefficients).

[0035] 1, steps 100 and 110 (and optional step 120) may be repeated one or more times at appropriate time intervals to provide a sustained reduction in blood pressure, as shown at 130. For example, the treatment may be repeated for two or more consecutive days. In some exemplary embodiments, the treatment is repeated for three, four, five, six, seven, eight, nine, or ten consecutive days.

[0036] As shown in step 140, after one or more treatments including steps 100 and 110, an increase in blood pressure may be detected after a time interval, such as 5, 6, 7, 8, 9, or 10 days. Steps 100-130 may then be repeated to further reduce blood pressure, as shown in 150.

[0037] Evaporation of nanodroplets can be driven by changes in pressure and / or temperature. The underlying mechanisms are referred to as mechanical or thermal (Lea-Banks et al. 2019, JCR). Mechanically driven evaporation predominates when using low ultrasound frequencies (<2 MHz), typically used during transcranial sonication. The evaporation threshold (the ultrasonic acoustic pressure required to activate nanodroplets) is directly related to frequency; low-frequency exposure (50 kHz–2 MHz) requires low ultrasonic acoustic pressures (0.2–3.0 MPa) and burst lengths between 1 ms and 100 ms. Droplets can also be evaporated with shorter bursts, but higher acoustic pressure amplitudes may be required. In a rodent model, pentobarbital-loaded nanodroplets were found to remain acoustically active in the circulation for up to 20 min after a single bolus injection (Lea-Banks et al. 2020, Theranostics). Therefore, durations of sonication between 1 and 30 min may be effectively used after bolus administration, although sonication may be extended if the nanodroplets are delivered by infusion or multiple bolus injections.

[0038] The PAG is one component of the pathway controlling cardiovascular function. The arcuate nucleus of the hypothalamus (ARC), ventrolateral periaqueductal gray matter (vlPAG), and rostral ventrolateral medulla (rVLM) are essential in governing sympathomimetic cardiovascular reflex responses. Without intending to be limited by theory, the inventors believe that these alternative brain regions may be sensitive to ultrasound-triggered therapeutic drug (e.g., barbiturate) delivery and may also affect blood pressure. Furthermore, the examples provided below demonstrate that changes in norepinephrine (noradrenaline) excretion and clearance are associated with antihypertensive effects. Therefore, exemplary embodiments of the present invention may be useful for targeting other norepinephrine-excreting centers throughout the norepinephrine system, such as the locus coeruleus, thalamus, hypothalamus, neocortex, and cerebellum.

[0039] In an exemplary embodiment of the present invention, a therapeutic drug (drug) encapsulation strategy is used that is applicable to a wide range of therapeutic drugs, including small molecule lipid-soluble drugs. In the example of nanodroplets with a lipid shell, the drug must have a molecular weight of less than 400 Da and a LogP (logarithm of the partition coefficient) close to 2 in order to be incorporated into the lipid shell and subsequently released and delivered across an intact blood-brain barrier. Non-limiting examples of suitable barbiturates with molecular weights less than 400 Da and logarithms of partition coefficients approximately equal to 2 include amobarbital, alphenal, butabarbital, butalbital, butetal, pentobarbital, phenobarbital, secobarbital, and thiopental. All barbiturates are GABAergic. A Being receptor agonists, triggering a similar inhibitory pathway using alternative barbiturates would be expected to lead to a reduction in blood pressure.

[0040] Although various examples of the present disclosure relate to barbiturate-loaded nanodroplets, it will be understood that the nanodroplets may load other therapeutic agents, such as, but not limited to, poorly lipid-soluble anesthetics, including benzocaine, bupivacaine (marcaine), ketamine, levobupivacaine, lidocaine, prilocaine, procaine, propofol, ropivacaine, and tetracaine, which have similar inhibitory effects on neuronal activity.

[0041] In some exemplary embodiments, nanodroplets can be formed with a core comprising a low-boiling-point perfluorocarbon, such as, but not limited to, decafluorobutane and octafluoropropane, and a shell comprising one or more of phospholipids, proteins (e.g., albumin), and polymers (e.g., amphiphilic copolymers). An exemplary method for nanodroplet production is disclosed in U.S. Patent Application No. US20130336891, which is incorporated herein by reference in its entirety. An exemplary method for nanodroplet production is disclosed in U.S. Patent Application No. US20200368352, which is incorporated herein by reference in its entirety. In some exemplary embodiments, focused ultrasound, when delivered to the periaqueductal gray matter region, induces repeated mechanical stress on the tissue by forming two or more closely spaced ultrasound pressure nodes (or focii), with adjacent nodes being in different (and in an optimal configuration, opposite) acoustic phases, generating forces between the nodes that stimulate or inhibit neuronal activity. Such exemplary embodiments may be performed after injection of nanodroplets containing a therapeutic agent or in the absence of injection of nanodroplets containing a therapeutic agent.

[0042] The ultrasonic field may be generated by one of a phased array, a lens, a reflector, a waveguide, and / or multiple overlapping ultrasonic fields, and / or any other method that allows for control of the ultrasonic field.

[0043] As shown in the example provided below, after a single 10-minute sonication session, blood pressure in healthy Wistar rats was reduced for 6 hours. Following five consecutive daily treatments of focused ultrasound (FUS) with pentobarbital-loaded nanodroplets (PBND), diastolic blood pressure in hypertensive rats was reduced for up to 5 days, whereas sonication of the frontal cortex did not affect blood pressure. vlPAG stimulation was confirmed by immunohistochemistry, which showed increased neuronal activity, and by enzyme-linked immunosorbent assay (ELISA), which showed changes in plasma hormone content.

[0044] The results presented herein demonstrate the use of focused ultrasound as a tool for minimally invasive treatment of hypertension using a clinically relevant timescale. Hypertension is the most common disorder in pregnancy. However, many antihypertensive drugs pose significant risks associated with restricted fetal growth, reduced uteroplacental blood flow, and fetal loss (Podymow and August, 2011). Regulatory approval and clinical use vary worldwide due to mixed evidence regarding health risks. For example, labetalol is a first-line treatment for hypertension during pregnancy in the UK but is avoided in North America (US FDA pregnancy category C).

[0045] Furthermore, drug-resistant hypertension precludes all existing pharmaceutical strategies. DBS is being explored as an alternative. In 2005, a 61-year-old man with drug-resistant hypertension was admitted to the hospital with chronic neuropathic pain and underwent deep brain stimulation of the periaqueductal gray area (PAG) (Green et al., 2007). In this first-in-human study, electrical stimulation of the PAG reduced blood pressure from 157.4 / 87.6 mmHg to 132.4 / 79.2 mmHg, which was unresponsive to antihypertensive medication. However, 20 s after stimulation was turned off, blood pressure returned to baseline. Two further clinical studies investigated the relationship between blood pressure reduction and analgesia, as well as potential long-term antihypertensive effects (Pereira et al., 2010; Patel et al., 2011). However, DBS is an invasive procedure with potential infection and bleeding risks (Fenoy and Simpson, 2014). Therefore, new strategies for treating hypertension are needed that are non-invasive, long-lasting, and do not require antihypertensive drugs.

[0046] One long-term focused ultrasound treatment for hypertension is renal denervation. After ultrasound ablation of the renal nerve, a recent multicenter clinical study demonstrated a reduction in blood pressure for at least two months (Azizi et al., 2021). Ultrasound renal denervation is approved in Europe to treat hypertension. However, renal anatomical restrictions (including the requirement that the diameter and length of the main renal artery must be within specified ranges) limit the availability of this treatment. A 2021 study excluded 108 patients based on these anatomical criteria. Furthermore, 17% of patients experienced persistent pain (>2 days) after the procedure.

[0047] This disclosure demonstrates that transcranial focused ultrasound in conjunction with barbiturate-loaded nanodroplets provides a noninvasive method for directly stimulating the PAG and a minimally invasive method for triggering the delivery of anesthetic agents. Both approaches have been shown to reduce high blood pressure (180 / 140 mmHg) to within a healthy range (140 / 100 mmHg). The use of PBND to locally deliver anesthetic agents was found to extend the effects of FUS from 2 to 5 days post-treatment.

[0048] As shown in the example below, focused ultrasound alone did not affect blood pressure in hypertensive rats (Figure 7). Comparing sonication of the frontal cortex with that of the vlPAG indicates that the decrease in blood pressure was dependent on brain anatomy. The decrease in blood pressure 2 h after PBND+FUS may be related to nonspecific drug release in the vlPAG and related inhibitory pathways upstream of the control region (Figures 8D and 8E). Using an acoustic controller to define the required acoustic pressure, combined with local delivery of pentobarbital, increased the reliability of the treatment. Four of four hypertensive rats responded to PBND+FUS (vlPAG), whereas one of four rats treated with FUS alone did not.

[0049] Immunohistochemical analysis was also used to explore the pathway of the antihypertensive effect. c-Fos is an immediate early gene (IEG) associated with neuronal firing. In contrast to our previous study in which pentobarbital was delivered to the motor cortex, where c-Fos suppression was mapped, our experiments show increased c-Fos expression within the vlPAG region. Regional differences in c-Fos expression in response to pentobarbital have been investigated by microinjection of the anesthetic into specific anatomical brain regions. Pentobarbital, a GABAA receptor agonist, administered by microinjection into the tuberomammillary nucleus of rats, was found to increase c-FOS expression in the ventrolateral preoptic area (VLPO), but suppress expression in the tuberomammillary nucleus, leaving expression in the locus coeruleus unchanged (Nelson et al., 2002). These anatomical differences are related to the distribution of GABAergic receptors, neurons, and interneurons. Furthermore, by injecting GABA directly into the PAG region of rats, the majority of GABAergic receptors present within this region have been mapped, signaling a significant GABAergic network responsible for widespread inhibitory pathways ( Behbehani et al., 1990 ).

[0050] In an exemplary method of the present invention, plasma hormone assays were performed to investigate the mechanism by which blood pressure reduction was promoted. Two hours after sonication of the vlPAG, a weak negative correlation was found between noradrenaline content in venous plasma samples and blood pressure, but no significant difference was found for adrenaline. Release of noradrenaline is typically associated with increases in metabolic function, heart rate, and blood pressure. However, the relationship between GABAergic receptors in the PAG region and norepinephrine has been investigated in the context of pain suppression. Norepinephrine injection has been shown to inhibit pain-modulating neurons (Basbaum and Fields, 1984), and increased noradrenaline release as a result of PAG stimulation has been reported with DBS, which has been shown to inhibit spinal dorsal horn neurons, resulting in analgesia (Cui et al., 1999).

[0051] An additional consideration is the clearance of noradrenaline; an increase in circulating noradrenaline in venous plasma may signal a decrease in the clearance rate (Esler et al., 1984). It has been shown that noradrenaline clearance is slower in normotensive individuals compared with hypertensive individuals (Grimm et al., 1980). Therefore, the increase in circulating noradrenaline may be the combined result of increased PAG-stimulated noradrenaline release and a decrease in clearance rate. Without intending to be limited by theory, we hypothesize that increased PAG c-Fos expression and increased plasma noradrenaline levels indicate PAG stimulation, which is responsible for an inhibitory response triggered by the GABAergic network, resulting in a decrease in blood pressure.

[0052] The methods, examples, and results disclosed herein demonstrate that FUS-mediated neuromodulation therapy for reducing hypertension is promising and can have clinical impact. While hypertension remains a fatal condition and its prevalence continues to increase in low-income countries, FUS offers a minimally invasive approach that has the potential to be optimized as a cost-effective, sustained treatment for hypertension. Indeed, the examples provided below demonstrate that continuous sonication combined with anesthetic-loaded nanodroplets reduces hypertension to within healthy limits for up to five days.

[0053] 2 provides a block diagram illustrating an exemplary embodiment of a system for performing a diagnostic or therapeutic transcranial procedure. Control and processing hardware 300 is operably connected to transcranial headset 100, optionally by transducer driver electronics / circuitry 380.

[0054] The control and processing hardware 300 includes one or more processors 310 (e.g., CPU / microprocessors), a bus 305, memory 315 which may include random access memory (RAM) and / or read-only memory (ROM), a data acquisition interface 320, a display 325, external storage 330, one or more communication interfaces 335, a power source 340, and one or more input / output devices and / or interfaces 345 (e.g., a speaker, user input devices such as a keyboard, keypad, mouse, position tracking stylus, position tracking probe, foot switch, and / or microphone for capturing voice commands).

[0055] The volumetric image data 370 and transducer registration data 375 may be stored in an external database or in the memory 315 or storage 330 of the control and processing hardware 300 .

[0056] A tracking system 365 may optionally be used to track the patient's position and orientation by detecting one or more alignment markers 460 attached to the transcranial headset 400 and, optionally, one or more medical instruments or devices that also have alignment markers attached thereto. For example, passive or active signals emitted from the alignment markers may be detected by a stereographic tracking system that employs two tracking cameras. The transducer drive electronics / circuitry 380 may include, for example, but is not limited to, a Tx / Rx switch, a transmit and / or receive beamformer.

[0057] The control and processing hardware 300 may be programmed with programs, subroutines, applications, or modules 350 that include executable instructions that, when executed by one or more processors 310, cause the system to perform one or more methods described in this disclosure. Such instructions may be stored, for example, in memory 315 and / or other storage.

[0058] In the illustrated exemplary embodiment, the transducer control module 355 includes executable instructions for controlling the transducers of the transcranial headset 400 to deliver energy to the PAG region based on registration of the transducer's position and orientation with the volumetric image data in accordance with the transducer registration data 375. For example, the transcranial headset 400 may support multiple phased array transducers, and the transducer control module 355 may control beamforming applied (on transmit and / or receive) to deliver one or more focused energy beams to a region of interest within the far-field region of the transcranial ultrasound transducer array elements based on the known position and orientation of the phased array transducer with respect to the volumetric image data. The region of interest may be specified by a user during surgery (e.g., via a user interface controlled by the control and processing hardware 300) or according to a pre-established surgical plan.

[0059] Registration module 360 may optionally be used to register volumetric image data 370 to an intraoperative frame of reference associated with tracking system 365. Optional guidance user interface module 362 includes executable instructions for displaying a user interface showing spatially registered volumetric images for image-guided procedures. Registration module 360 may also receive spatial correction information during surgery based on a detected spatial offset between the transcranial frame and the patient's head (which may be provided by a subset of distance-sensing transducers), and may use this spatial correction information to dynamically adjust (e.g., correct) the registration between the transducers and the volumetric image data.

[0060] 2 shows only one of each component, any number of each component can be included in the control and processing hardware 300. For example, a computer typically includes several different data storage media. Furthermore, while the bus 305 is shown as a single connection between all components, it will be understood that the bus 305 can represent one or more circuits, devices, or communication channels linking two or more of the components. For example, in a personal computer, the bus 305 often includes or is a motherboard. The control and processing hardware 300 can include more or fewer components than those shown.

[0061] The control and processing hardware 300 may be implemented as one or more physical devices coupled to the processor 310 via one of several communication channels or interfaces. For example, the control and processing hardware 300 may be implemented using an application specific integrated circuit (ASIC). Alternatively, the control and processing hardware 300 may be implemented as a combination of hardware and software, where the software is loaded into the processor from memory or via a network connection.

[0062] Some aspects of the present disclosure may be embodied at least in software that, when executed on a computing system, transforms the computing system into a special-purpose computing system capable of performing the methods disclosed herein. That is, the techniques may be implemented in a computer system or other data processing system in response to its processor, such as a microprocessor, executing sequences of instructions contained in a memory, such as a ROM, volatile RAM, non-volatile memory, cache, magnetic and optical disk, or remote storage device. Furthermore, the instructions may be downloaded to a computing device over a data network in the form of a compiled and linked version. Alternatively, logic for performing processes such as those discussed above may be implemented in additional computer- and / or machine-readable media, such as large-scale integrated circuits (LSIs), application-specific integrated circuits (ASICs), or discrete hardware components such as firmware, e.g., electrically erasable programmable read-only memories (EEPROMs) and field-programmable gate arrays (FPGAs).

[0063] Computer-readable media can be used to store software and data that, when executed by a data processing system, causes the system to perform various methods. Executable software and data can be stored in various locations, including, for example, ROM, volatile RAM, non-volatile memory, and / or cache. Portions of this software and / or data can be stored in any one of these storage devices. In general, machine-readable media includes any mechanism that provides (i.e., stores and / or transmits) information in a form accessible by a machine (e.g., a computer, a network device, a personal digital assistant, a manufacturing tool, any device with a set of one or more processors, etc.).

[0064] Examples of computer-readable media include, but are not limited to, recordable and non-recordable type media, such as volatile and non-volatile memory devices, read-only memory (ROM), random access memory (RAM), flash memory devices, floppy and other removable disks, magnetic disk storage media, optical storage media (e.g., compact discs (CDs), digital versatile discs (DVDs), etc.), etc. The instructions may be embodied in electrical, optical, acoustic, or other forms of propagated signals, such as digital and analog communication links for carrier waves, infrared signals, digital signals, etc. As used herein, the terms "computer-readable material" and "computer-readable storage medium" refer to all computer-readable media, excluding the transitory propagated signals themselves.

[0065] Referring now to Figure 3, a non-limiting exemplary patient-specific headset 400 for performing transcranial diagnostic or therapeutic procedures is shown worn on a patient's head 50. The patient-specific headset 400, which includes a patient-specific frame (support structure) 410 supporting multiple transducers 420, conforms to the anatomical contours of at least a portion of the patient's head. The patient-specific frame 410, shown in cross section in Figure 3, mechanically supports the transducers 420 in preselected positions and orientations. The transducers 420 may be used to transmit and / or receive energy for diagnostic or therapeutic purposes in the brain or for localization on the skull surface.

[0066] The patient-dedicated frame 410 includes a plurality of attachment interfaces for receiving and supporting the transducers 420. In the exemplary embodiment shown in FIG. 3, the attachment interfaces are provided as apertures (recesses) into which the transducers 420 are placed. The transducers 420 may be affixed to the patient-dedicated frame 410 by a variety of different means, including, but not limited to, by an attachment mechanism (e.g., via fasteners extending into optionally pre-formed holes in the patient-dedicated frame 410) or by an adhesive such as glue. In the exemplary implementation shown in FIG. 3, the transducers 420 are remotely interfaced with electronics via wires or via a flexible printed circuit board 440. The transducers 420 may be removably attachable to the patient-dedicated frame 410.

[0067] The exemplary patient-specific headset shown in FIG. 3 may also include a coupling layer 430 disposed adjacent to the inner surface of the patient-specific frame. The outer surface of the coupling layer 430 contacts the distal surface of the transducer 420, and the inner surface of the coupling layer contacts the patient's head 50, thereby facilitating coupling of energy between the transducer within the patient-specific frame and the patient's head. The inclusion of the coupling layer 430, as well as the configuration and / or geometry of the coupling layer, may depend on the type of transducer 420. For example, if the transducer 420 is an ultrasound transducer, the coupling layer 430 may be an acoustic coupling layer that facilitates propagation of acoustic waves and reduces reflections at the interface. In one exemplary implementation, the coupling layer 430 includes an elastic membrane that maintains a liquid layer between the transducer surface and the elastic membrane to achieve coupling to the skin. [Example]

[0068] The following examples are presented to enable those skilled in the art to understand and practice embodiments of the present disclosure, and should not be considered as limitations on the scope of the disclosure, but merely as being illustrative and representative thereof.

[0069] In the following examples, two experiments were performed: first, healthy normotensive Wistar rats were used to evaluate the duration of blood pressure reduction after a single 10-min sonication of the vlPAG region. The effects of general anesthetics, FUS alone, FUS in nanodroplets, and FUS in pentobarbital-loaded nanodroplets (PBND) targeted to the vlPAG were then compared with those given as a single treatment or in a series of daily treatments. In a second experiment, a model of hypertension (spontaneously hypertensive rats (SHRs)) was used to compare five daily treatments of FUS alone and FUS in PBND targeted to the vlPAG or the frontal cortex (as a control). Noninvasive blood pressure measurements, plasma hormone assessments, and immunohistochemical examination of neuronal activity were performed in conscious rats to explore the underlying mechanisms.

[0070] Example 1: Methods Barbiturate-encapsulated nanodroplets Ultrasound-responsive nanodroplets with a diameter of 200 nm were synthesized according to a modified condensation protocol (Sheeran et al., 2011, 2017; Matsunaga et al., 2012) as previously described (Lea-Banks et al., 2021). Definity lipid solution (Lantheus Medical Imaging, USA) was combined with pentobarbital (Sigma Aldrich Millipore Sigma, USA) by tip sonication, degassed, and filled with decafluorobutane (CF4F10). Precursor microbubbles were formed by stirring (VialMix, Lantheus Medical Imaging, USA), condensed at -10°C, washed by centrifugation (300 g, 8 min, 4°C), and filtered through a 0.8 μm filter (Minisart Syringe Filter, Sartorius, Germany). Nanodroplets were stored on ice prior to use and used within 2 h of preparation. Sham nanodroplets (Sham ND) were similarly prepared without the addition of pentobarbital.

[0071] animal Two cohorts of rats were used to evaluate ultrasound-induced changes in blood pressure. Normotensive, healthy Wistar rats (23 males, 4 females) were purchased from Charles River Laboratories (Wilmington, MA, USA) and weighed 319 ± 12 g (males) and 218 ± 3 g (females) on the first treatment day. Spontaneously hypertensive rats (SHR) (5 males, 5 females) were purchased from Envigo (Indianapolis, IN, USA) and weighed 256 ± 12 g (males) and 172 ± 8 g (females) on the first treatment day. Animals were housed under a reversed light cycle at the Sunnybrook Research Institute Animal Facility (Toronto, ON, Canada) and had free access to food and water. All animal procedures were approved by the Sunnybrook Research Institute Animal Care Committee and adhered to the guidelines of the Canadian Council on Animal Care and ARRIVE.

[0072] Animal preparation To induce general anesthesia, 5% isoflurane (ISO) in medical air was used, decreasing to 2% for the duration of the ultrasound procedure. To allow for coupling between the transducer and scalp, hair was removed with an electric razor and depilatory cream, and a thin layer of ultrasound gel was applied to the scalp (Wavelength CL, ON, Canada). The ultrasound gel was then spun down in a centrifuge tube and used to remove air bubbles (1000G, 10 min). A 22-gauge tail vein catheter was inserted. After blood collection, 2 mL of saline was administered subcutaneously; all other injections were administered intravenously. The rat was positioned in the stereotaxic frame of the FUS system and breathed into a nose cone connected to the ISO machine, scavenged with an F / AIR charcoal filter. A warmed saline bag was placed over the animal to maintain body temperature. PBND or sham ND was administered as a slow bolus via the tail vein catheter at a dose of 1.0 mL / kg (containing approximately 25 μg / mL pentobarbital).

[0073] Transcranial FUS System Using a preclinical prototype system (RK50, FUS Instruments Inc., ON, Canada) with atlas-based targeting, focused ultrasound (FUS) at 540 kHz was delivered to the ventrolateral periaqueductal gray matter (vlPAG) region (D / V 5.8 mm, A / P -7.6 mm, L / M -0.6 mm relative to bregma) or the frontal cortex (D / V 4.0 mm, A / P 3.0 mm, L / M -0.6 mm relative to bregma). The skull geometry was co-registered with the focal point of a single-element transducer (center frequency 540 kHz, element diameter 35 mm, focal length 25 mm), which was circularly cut to accommodate a narrowband PZT (lead zirconate titanate) hydrophone (center frequency 270 kHz, element diameter 5 mm, unfocused) (Figure 5A). Sonication was performed with a pulse length of 10 ms, a pulse repetition frequency of 1 Hz, and a duration of 10 min (Fig. 5B).

[0074] To assess the unique ultrasound pressure required for droplet evaporation in each rat, a 3-minute ultrasound pressure gradient was performed following intravenous administration of nanodroplets. The ultrasound pressure was increased in 8 kPa increments from 0.45 MPa until subharmonic emissions exceeded 3.5 times the baseline value. This pressure was set as the evaporation threshold, and sonication was fixed at this pressure for the next 10 minutes of treatment. In the ultrasound-only group, a fixed pressure of 1.1 MPa was used, based on the average pressure required for evaporation.

[0075] Blood pressure measurements in conscious rats Blood pressure and heart rate were measured noninvasively in conscious rats using a tail cuff system (CODA system, Kent Scientific, Torrington, CT, USA). Rats were placed under a blanket on a heated platform (CODA system) inside a cylindrical holder and allowed to acclimate for 10 min before measurements were initiated. Fifteen cycles were performed, and mean systolic and diastolic blood pressures and heart rates were recorded. Recordings containing tail movements or insufficient blood volume were automatically excluded by the CODA system software. Rats were acclimatized by daily blood pressure measurements for 5 consecutive days before the first treatment.

[0076] Tail temperatures of hypertensive rats were assessed using an infrared thermometer (TW2, Thermoworks, Salt Lake City, UT, USA) by measuring the base of the tail at three time points: (1) immediately after placement in the holder, (2) after 10 min of acclimation, and (3) immediately after measurement. All rats were weighed daily.

[0077] Assessment of plasma epinephrine and norepinephrine To quantify epinephrine and norepinephrine plasma levels (hormones related to metabolic function and blood pressure) in SHRs, venous blood was collected before and 24 h after treatment. 0.6 mL of blood was collected via a tail vein catheter and placed in a heparin-lined Eppendorf tube. Samples were stored on ice for 30 min and then centrifuged at 1000×G for 10 min. Plasma was separated and stored at −80°C until analysis. Epinephrine and norepinephrine were assessed in triplicate using ELISA (Abnova, KA1877, Taipei, Taiwan) according to the manufacturer's specifications.

[0078] Immunohistochemical examination of spontaneously hypertensive rats Ninety minutes after treatment, rats were intracardially perfused with ice-cold saline and 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer. Brains were removed and fixed overnight in 4% PFA, then equilibrated in 30% sucrose in 0.1 M phosphate buffer. Samples were then embedded in OCT, horizontally cryosectioned into 40 μm-thick free-floating sections, and stored in cryoprotectant at -20°C.

[0079] For c-Fos expression analysis, one SHR was used from each group: naive, FUS only (vlPAG), and PBND + FUS (vlPAG). Horizontal sections 5.8 mm deep from bregma were selected for immunofluorescence staining based on the target coordinates of sonication. Sections were rinsed in 0.3% Triton X-100 in 0.1 M PBS and then treated with 10% donkey serum blocking solution at room temperature for 1 hour. Sections were incubated overnight at 4°C in primary antibodies, anti-cfos (ab190289, 1:1000, Abcam Inc., Cambridge, MA, USA) and anti-NeuN (ab104224, 1:1000, Abcam Inc.). Sections were then washed and incubated with fluorescently labeled secondary antibodies, donkey Alexa Fluor® 488 anti-mouse (A21202, 1:1000, Invitrogen, Eugene, OR, USA) and donkey Alexa Fluor® 568 anti-rabbit (A10042, 1:1000, Invitrogen), diluted in blocking buffer for 2 hours at room temperature. Sections were washed with PBS and mounted on X-tra glass slides (Leica Microsystems, Wetzlar, Germany) using aqueous mounting medium containing DAPI (Fluoroshield™ with DAPI, Sigma-Aldrich Corporation, St. Louis, MO, USA). Slides were stored in the dark at 4°C until imaging.

[0080] Sections were imaged at 20x magnification (512 × 512 pixel field of view) and quantification of c-Fos and colocalization analysis of NeuN were performed using 1 mm sections as previously described ( Lunde and Glover, 2020 ).2 The analysis was performed within a region of interest (ROI). Two custom binary images were created based on the raw data of the multichannel input image using the Colocalization Image Creator plugin (ImageJ, National Institutes of Health, Bethesda, Maryland, USA). Background signal intensity was reduced by global threshold binarization for each channel to generate the binarized images. The binarized images were Boolean ANDed and then macro-transformed to remove intracellular holes and filtered to remove cells smaller than 5 μm in size. The first binarized image contained the DAPI and c-Fos channels and represented the total number of c-Fos-labeled cells. The second binarized image contained the DAPI, NeuN, and c-Fos channels and represented the signal overlap across the three input channels, indicating colocalization between NeuN and c-Fos. The number of c-Fos-positive cells and the number of NeuN-positive cells colocalized with c-Fos were then automatically counted using the Colocalization Object Counter plugin (ImageJ). The total number of c-Fos-positive cells and the total number of c-Fos-NeuN colocalized cells detected within the sonicated and unsonicated PAG regions are reported. The percentage of c-Fos-positive cells that also showed NeuN expression was also calculated.

[0081] To investigate treatment safety, erythrocyte extravasation and cell apoptosis were assessed by hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (DeadEnd™ Colorimetric TUNEL System, Promega, Madison, WI, USA). All rats were sacrificed 7 days after the final treatment and perfused intracardially with saline and buffered formalin. Brains were excised and immersed in 10% neutral buffered formalin for 24 h, then transferred to 70% ethanol and embedded in paraffin 48 h later. Five-micrometer-thick longitudinal sections were taken at 500-micrometer intervals and stained.

[0082] statistical analysis Blood pressure and heart rate data are presented as individual data points or bars showing the mean + / - standard deviation with error bars. Significance was assessed using paired and unpaired t-tests, with the significance level set at p<0.05. Correlations were evaluated using the Pearson coefficient (R 2 ) is evaluated.

[0083] Example 2: Results Habituation to blood pressure measurement Five days of acclimation were required for the rats to tolerate the noninvasive blood pressure measurement procedure (Figure 9). After acclimation, tail movement during measurements significantly decreased, and the consistency of blood pressure and heart rate measurements improved (Figure 9). After acclimation, baseline systolic and diastolic blood pressures in normotensive rats were 133±16 mmHg and 103±14 mmHg, respectively, and heart rates were 258±40 bpm. Baseline systolic and diastolic blood pressures in hypertensive rats (SHR) were 183±18 mmHg and 142±23 mmHg, respectively, and heart rates were 263±42 bpm.

[0084] Transcranial focused ultrasound transiently decreases blood pressure in normotensive rats After a single 10-min sonication of the vlPAG, 6 of 6 healthy Wistar rats showed significant reductions in systolic blood pressure (-37 ± 7 mmHg) and diastolic blood pressure (-28 ± 9 mmHg), which remained significantly reduced from baseline 6 h after treatment (p < 0.05, Fig. 4E, Fig. 4F). Heart rate did not change significantly (Fig. 4G).

[0085] Transcranial focused ultrasound with barbiturate nanodroplets maintains blood pressure reduction in normotensive rats General anesthesia (ISO alone) and anesthesia combined with injection of pentobarbital-loaded nanodroplets (ISO+PBND) did not alter blood pressure when measured 2 to 48 hours after treatment (Figure 5). After a single insonification of the vlPAG region (Figures 5A-5D), FUS-treated rats (ISO+FUS) showed a decrease in systolic blood pressure (-26 + / - 7 mmHg) and diastolic blood pressure (-22 + / - 7 mmHg) 2 hours after treatment. Sham nanodroplets (non-loaded, pentobarbital-free) combined with FUS (ISO+FUS+ShamND) similarly temporarily decreased blood pressure (-19 + / - 14 mmHg systolic, -15 + / - 18 mmHg diastolic). Combining FUS with pentobarbital-loaded nanodroplets (ISO+FUS+PBND) proved effective, with blood pressure remaining lower for 24 hours after a single treatment (systolic -25 + / - 5 mmHg and diastolic -21 + / - 8 mmHg at 2 hours; systolic -17 + / - 6 mmHg and diastolic -18 + / - 10 mmHg at 24 hours).

[0086] The following week, two treatments were administered on consecutive days (Figure 5E). Again, ISO alone and ISO + PBND showed no change in blood pressure through 48 hours after either treatment. After sequential insonification of the vlPAG region, ISO + FUS reduced systolic and diastolic blood pressure 2 hours after both treatments (systolic -28 + / - 5 mmHg, diastolic -27 + / - 3 mmHg), as did sham nanodroplets and FUS (systolic -29 + / - 13 mmHg, diastolic -29 + / - 10 mmHg), with both treatment groups returning to baseline after 24 hours. Sequential treatment with ISO+FUS+PBND maintained its effect, with blood pressure remaining lower for 48 hours after two consecutive treatments (systolic -26 + / - 7 mmHg, diastolic -12 + / - 10 mmHg at 2 hours; systolic -14 + / - 10 mmHg, diastolic -15 + / - 13 mmHg at 24 hours; systolic -11 + / - 10 mmHg, diastolic -9 + / - 11 mmHg at 48 hours). Changes in body weight and heart rate were comparable to control rats (Figure 10).

[0087] Transcranial focused ultrasound reduces blood pressure in hypertensive rats, maintained with barbiturate nanodroplets We used male and female spontaneously hypertensive rats (SHRs) to investigate the effects of FUS on blood pressure reduction, the efficacy of PBND, and the role of brain regions in the treatment of hypertension. A crossover study was conducted in which five treatment groups of SHRs received five consecutive treatments separated by 24 hours, followed by 6 days of monitoring before crossover (Figure 7A). FUS was applied to the left frontal cortex (a region not involved in blood pressure regulation, serving as a positive control) (FUS(positive control)) or the vlPAG region (FUS(vlPAG)) (Figure 6G).

[0088] Figure 6 shows the change in systolic blood pressure for each SHR treated for five consecutive days and monitored for six days thereafter. Naive SHR showed no change in blood pressure throughout the study (Figure 6A). Three of four hypertensive rats showed a decrease in blood pressure after vlPAG insomnia (Figure 6I). FUS (vlPAG) showed a greater decrease in blood pressure (systolic -43 + / - 29 mmHg, diastolic -39 + / - 31 mmHg at 2 hr; systolic -32 + / - 28 mmHg, diastolic -38 + / - 34 mmHg at 24 hr) than FUS (active control) (systolic -18 + / - 29 mmHg, diastolic -11 + / - 27 mmHg at 2 hr; systolic 1 + / - 14 mmHg, diastolic 6 + / - 13 mmHg at 24 hr) (Figure 6C, Figure 6I). FUS(vlPAG) reduced blood pressure for 48 hours after 5 ultrasound treatments, returning to baseline after 3 days.

[0089] Four of four hypertensive rats showed a decrease in blood pressure after vlPAG sonication with PBND. PBND+FUS (vlPAG) showed the largest and most consistent decrease in blood pressure (-57 + / - 17 mmHg systolic, -48 + / - 17 mmHg diastolic at 2 hr) and remained significantly decreased (p<0.05) for 4 (systolic) or 5 (diastolic) days after the final sonication. PBND+FUS (active control) showed a transient decrease in blood pressure 2 hr after sonication (-37 + / - 16 mmHg systolic, -16 + / - 5 mmHg diastolic at 2 hr), returning to baseline 24 hr after the final sonication. Animal weight and tail base temperature were comparable to naive rats (Figure 10).

[0090] At sufficient ultrasonic pressure, known as the vaporization threshold, the nanodroplets transitioned from a liquid to a gaseous state, releasing the therapeutic agent and emitting a unique acoustic signal. Using linear regression analysis, the occurrence of subharmonic and second harmonic waves was found to correlate with a decrease in blood pressure (R 2 = 0.88 and 0.75, Pearson coefficients), acoustic emissions detected from the active control region or by FUS alone showed no correlation with changes in blood pressure (R 2 <0.4, Pearson coefficient).

[0091] Plasma hormone content in hypertensive rats correlates with blood pressure reduction and acoustic response of nanodroplets Plasma levels of adrenaline and noradrenaline were quantified from plasma samples collected before treatment, 24 hours after treatment, and 1 week after treatment. Mean noradrenaline levels were higher in rats sonicated in the vlPAG (Figures 6J-6L) compared with those after sonication in the control region (Figures 6D-6F). 24 hours after sonication in the vlPAG region, noradrenaline levels were significantly correlated with systolic blood pressure (R 2 =0.56) (Fig. 7K) and diastolic blood pressure (R 2 = 0.69) (Figure 6L), but no such relationship existed in the active control group (Figures 6E, 6F) or adrenaline levels (Figure 11). Furthermore, the noradrenaline levels of hypertensive rats in the PBND+FUS (vlPAG) group were mapped to subharmonic release (R 2 = 0.84) (Figure 6J). In contrast, hypertensive rats in the PBND+FUS (active control) group showed no significant relationship between hormone levels and blood pressure or acoustic emission (Figures 6D-F).

[0092] Immunohistochemical assessment maps neuronal stimulation of FUS Ninety minutes after treatment, three animals were perfused to assess c-Fos expression and NeuN antibody (Figure 8). FUS and FUS combined with pentobarbital nanodroplets (PBND) demonstrated a higher number of c-Fos-positive cells within the sonicated area of the VLPAG compared to the contralateral side (Figure 8A). Seventy-nine percent of c-Fos-positive cells were also found to express NeuN after FUS alone, and 61% of c-Fos-positive cells were found to express NeuN after PBND + FUS (Figure 8G). H&E and TUNEL staining performed 7 days after the final treatment showed no signs of hemorrhage or edema and no increase in the number of apoptotic cells (Figure 12).

[0093] Example 3: Experimental demonstration that ultrasound-induced blood pressure reduction is dose-dependent Many treatments for hypertension are ineffective in certain patient populations due to drug resistance and pregnancy. Previously, daily continuous focused ultrasound (FUS) stimulation of the ventrolateral periaqueductal gray (vlPAG) in spontaneously hypertensive rats reduced blood pressure (BP) for up to 24 hours. Here, we investigated the effect of ultrasound amplitude on the resulting reduction in BP.

[0094] Normotensive Wistar rats (18 males and 18 females) were used to evaluate BP reduction after FUS stimulation. BP was measured in awake, acclimated rats using a noninvasive tail cuff system (CODA, Kent Scientific) (Figure 14A). A single-element focused transducer (540 kHz, 34 mm element diameter, 3 mm beamwidth) with a stereotactic guided positioning system (FUS Instruments) was used to target the vlPAG. Sonication (10 min duration, 10 ms burst length, 1 Hz pulse repetition frequency) was performed in anesthetized rats (isoflurane and medical air) at six sound pressure amplitudes (0.000, 0.275, 0.550, 1.100, 1.650, and 2.100 MPa) (Figure 14B). Immunohistochemistry was performed 90 min after FUS stimulation to map neuronal firing and neuronal subtypes, and histology was performed 24 h after FUS stimulation to assess safety.

[0095] Two hours after FUS, we observed a pressure-dependent decrease in BP, ranging from the lowest pressure (0.275 MPa, -19.6 ± 17.8 mmHg systolic, -19.3 ± 17.6 mmHg diastolic) to the highest pressure (2.100 MPa, -36.6 ± 33.4 mmHg systolic, -34.1 ± 31.8 mmHg diastolic) (Figures 14C and 14D). 24 hours after FUS, BP returned to baseline for all ultrasound pressure amplitudes. There was a significant correlation between ultrasound pressure and the decrease in systolic BP (p = 0.02), with a slope of -11.1 mmHg / MPa. At the highest pressure, the change in systolic BP was linearly correlated with baseline systolic BP (R = 0.65, p = 0.05), suggesting that ultrasound-induced BP reduction is most effective in subjects with elevated resting BP, similar to the association with hypertension. There was no histological evidence of hemorrhage, edema, or tissue damage after exposure. The data indicate that ultrasound-induced BP reduction is dose-dependent.

[0096] While the specific embodiments described above are shown by way of example, it should be understood that these embodiments are susceptible to various modifications and alternative forms. It should also be understood that the claims are not intended to limit the scope of the invention to the particular forms disclosed, but rather to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure. References Airan, RDet al. (2017) "Noninvasive Targeted Transcranial Neuromodulation via Focused Ultrasound Gated Drug Release from Nanoemulsions", Nano letters.2017 / 01 / 17.American Chemical Society,17(2),pp.652-659.doi:10.1021 / acs.nanolett.6b03517. Azizi,M.et al.(2021)「Ultrasound renal denervation for hypertension resistant to a triple medication pill(RADIANCE-HTN TRIO):a randomised,multicentre,single-blind,sham-controlled trial」,The Lancet.Elsevier,397(10293),pp.2476-2486. Basbaum,A.I.and Fields,H.L.(1984)「Endogenous pain control systems:brainstem spinal pathways and endorphin circuitry」,Annual review of neuroscience.Annual Reviews 4139 El Camino Way,PO Box 10139,Palo Alto,CA 94303-0139,USA,7(1),pp.309-338. Behbehani,M.M.et al.(1990)「The effect of GABA and its antagonists on midbrain periaqueductal gray neurons in the rat」,Pain.Elsevier,40(2),pp.195-204. Cui,M.et al.(1999)「Periaqueductal gray stimulation-induced inhibition of nociceptive dorsal horn neurons in rats is associated with the release of norepinephrine,serotonin,and amino acids」,Journal of Pharmacology and Experimental Therapeutics.ASPET,289(2),pp.868-876. Davidson,B.et al.(2020)「Magnetic resonance-guided focused ultrasound capsulotomy for refractory obsessive compulsive disorder and major depressive disorder:clinical and imaging results from two phase I trials」,Molecular psychiatry.Nature Publishing Group,25(9),pp.1946-1957. Elias,W.J.et al.(2016)「A randomized trial of focused ultrasound thalamotomy for essential tremor」,New England Journal of Medicine.Mass Medical Soc,375(8),pp.730-739. Esler,M.et al.(1984)「Plasma noradrenaline kinetics in humans」,Journal of the autonomic nervous system.Elsevier,11(2),pp.125-144. Fenoy,A.J.and Simpson,R..(2014)「Risks of common complications in deep brain stimulation surgery:management and avoidance」,Journal of Neurosurgery JNS.American Association of Neurological Surgeons,120(1),pp.132-139.doi:10.3171 / 2013.10.JNS131225. Fomenko, A. et al. (2018) "Low-intensity ultrasound neuromodulation: An overview of mechanisms and emerging human applications", Brain Stimulation, 11(6), pp. 1209-1217. doi: https: / / doi.org / 10.1016 / j.brs.2018.08.013. FUSfoundation (2021) State of the field report. Available at: https: / / www.fusfoundation.org / images / pdf / FUSF_State_of_the_Field_2021_Final_Web.pdf. Green, A. L. et al. (2007) "Deep brain stimulation: a new treatment for hypertension?", Journal of clinical neuroscience. Elsevier, 14(6), pp. 592-595. Grimm, M. et al. (1980) "Norepinephrine clearance and pressor effect in normal and hypertensive man", Klinische Wochenschrift. Springer, 58(21), pp. 1175-1181. Hakimova, H. et al. (2015) "Ultrasound stimulation inhibits recurrent seizures and improves behavioral outcome in an experimental model of mesial temporal lobe epilepsy", Epilepsy & Behavior. Elsevier, 49, pp. 26-32. Laurent,S.(2017)「Antihypertensive drugs」,Pharmacological research.Elsevier,124,pp.116-125. Lea-Banks,H.et al.(2020)「Localized anesthesia of a specific brain region using ultrasound-responsive barbiturate nanodroplets」,Theranostics,10(6),pp.2849-2858. Lea-Banks,H.et al.(2021)「Ultrasound-sensitive nanodroplets achieve targeted neuromodulation」,Journal of Controlled Release.Elsevier B.V.,332,pp.30-39.doi:10.1016 / j.jconrel.2021.02.010. Li,D.et al.(2020)「Low-intensity focused ultrasound stimulation treatment decreases blood pressure in spontaneously hypertensive rats」,IEEE Transactions on Biomedical Engineering.IEEE,67(11),pp.3048-3056. Lunde,A.and Glover,.C.(2020)「A versatile toolbox for semi-automatic cell-by-cell object-based colocalization analysis」,Scientific reports.Nature Publishing Group,10(1),pp.1-26. Martinez-Fernandez,R.et al.(2018)「Focused ultrasound subthalamotomy in patients with asymmetric Parkinson’s disease:a pilot study」,The Lancet Neurology.Elsevier,17(1),pp.54-63. Matsunaga,T.O.et al.(2012)「Phase-change nanoparticles using highly volatile perfluorocarbons:toward a platform for extravascular ultrasound imaging」,Theranostics.2012 / 12 / 23.Ivyspring International Publisher,2(12),pp.1185-1198.doi:10.7150 / thno.4846. Meng,Y.et al.(2021)「Focused ultrasound neuromodulation」,International Review of Neurobiology.Elsevier,159,pp.221-240. Mooney,S.J.et al.(2016)「Focused ultrasound-induced neurogenesis requires an increase in blood-brain barrier permeability」,PloS one.Public Library of Science San Francisco,CA USA,11(7),p.e0159892. Nelson,L.E.et al.(2002)「The sedative component of anesthesia is mediated by GA BAA receptors in an endogenous sleep pathway」,Nature neuroscience.Nature Publishing Group,5(10),pp.979-984. Ozdas,M.S.et al.(2020)「Non-invasive molecularly-specific millimeter-resolution manipulation of brain circuits by ultrasound-mediated aggregation and uncaging of drug carriers」,Nature communications.Nature Publishing Group,11(1),pp.1-15. Patel,N.K.et al.(2011)「Deep brain stimulation relieves refractory hypertension」,Neurology.AAN Enterprises,76(4),pp.405-407. Pereira,E.A.C.et al.(2010)「Sustained reduction of hypertension by deep brain stimulation」,Journal of Clinical Neuroscience.Elsevier,17(1),pp.124-127. Podymow,T.and August,P.(2011)「Antihypertensive drugs in pregnancy」,in Seminars in nephrology.Elsevier,pp.70-85. Scarcelli,T.et al.(2014)「Stimulation of hippocampal neurogenesis by transcranial focused ultrasound and microbubbles in adult mice」,Brain stimulation.Elsevier,7(2),pp.304-307. Sharabi,S.et al.(201)「Non-thermal focused ultrasound induced reversible reduction of essential tremor in a rat model」,Brain stimulation.Elsevier,12(1),pp.1-8. Sheeran,P.S.et al.(2011)「Formulation and Acoustic Studies of a New Phase-Shift Agent for Diagnostic and Therapeutic Ultrasound」,Langmuir.American Chemical Society,27(17),pp.10412-10420.doi:10.1021 / la2013705. Sheeran,P.S.et al.(201)「More Than Bubbles:Creating Phase-Shift Droplets from Commercially Available Ultrasound Contrast Agents」,Ultrasound in Medicine and Biology.Elsevier,43(2),pp.531-540.doi: 10.1016 / j.ultrasmedbio.2016.09.003. Stern,J.M.et al.(2021)「Safety of focused ultrasound neuromodulation in humans with temporal lobe epilepsy」,Brain Stimulation.Elsevier. Wang,J.B.et al.(2018)「Noninvasive Ultrasonic Drug Uncaging Maps Whole-Brain Functional Networks」,Neuron.Cell Press,100(3),pp.728-738.e7.doi:10.1016 / J.NEURON.2018.10.042. WHO(2022)World health statistics 2022:monitoring health for the SDGs,sustainable development goals.Available below: https: / / cdn.who.int / media / docs / default-source / gho-documents / world-health-statistic-reports / worldhealthstatistics_2022.pdf?sfvrsn=6fbb4d17_3. Zhang,M.,Li,B.,Liu,Y.,et al.(2021) "Different Modes of Low-Frequency Focused Ultrasound-Mediated Attenuation of Epilepsy Based on the Topological Theory", Micromachines. Multidisciplinary Digital Publishing Institute, 12(8), p. 1001. Zhang,M.,Li,B.,Lv,X.,et al.(2022) "Low-Intensity Focused Ultrasound-Mediated Attenuation of Acute Seizure Activity Based on EEG Brain Functional Connectivity", Brain Sciences. Multidisciplinary Digital Publishing Institute, 11(6), p. 711. Zhang,T.et al.(2022) "Transcranial focused ultrasound stimulation of periaqueductal gray for analgesia", IEEE Transactions on Biomedical Engineering. IEEE.

Claims

1. 1. A method for treating hypertension using focused ultrasound, comprising delivering focused ultrasound to norepinephrine-producing regions of the brain following intravenous injection of nanodroplets containing a therapeutic agent, wherein the focused ultrasound is configured to promote evaporation of the nanodroplets and release of the therapeutic agent.

2. The method of claim 1 , wherein the therapeutic agent comprises a barbiturate.

3. The method of claim 1 , wherein the therapeutic agent comprises a lipid-soluble anesthetic.

4. 4. The method of claim 3, wherein the lipid-soluble anesthetic has a molecular weight of less than 400 Da and a logarithm of the partition coefficient approximately equal to 2.

5. The method of any one of claims 1 to 4, wherein the norepinephrine-producing region comprises the periaqueductal gray region.

6. The method according to any one of claims 1 to 4, wherein the norepinephrine-producing region comprises the ventrolateral periaqueductal gray region.

7. 5. The method of claim 1, wherein the norepinephrine-producing region comprises at least one of the locus coeruleus, the thalamus, the hypothalamus, the neocortex, and the cerebellum.

8. 1. A method for treating high blood pressure using focused ultrasound, comprising delivering focused ultrasound to norepinephrine-producing regions of the brain, the focused ultrasound being configured to induce repetitive mechanical stress on the tissue by creating two or more closely spaced ultrasound acoustic pressure nodes, the adjacent nodes being in different acoustic phases such that forces are generated between the nodes to stimulate or inhibit neuronal activity.

9. 10. The method of claim 8, wherein the ultrasonic field is generated by one of a phased array, a lens, a reflector, a waveguide, and multiple overlapping ultrasonic fields.

10. 10. The method of claim 8 or 9, wherein the norepinephrine-producing region comprises the periaqueductal gray region.

11. 9. The method of claim 8, wherein the norepinephrine-producing region comprises at least one of the locus coeruleus, the thalamus, the hypothalamus, the neocortex, and the cerebellum.

12. 9. The method of claim 8, wherein the norepinephrine-producing region comprises the ventrolateral periaqueductal gray region.

13. 10. The method of claim 8, wherein the focused ultrasound delivery is performed after intravenous injection of nanodroplets containing a therapeutic agent to promote evaporation of the nanodroplets and release of the therapeutic agent.

14. 10. The method of claim 8, wherein the focused ultrasound delivery is combined with a systemically delivered drug, such as an anesthetic, that enhances the ultrasound impact on brain tissue.

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