Methods of treating hypertension by transcranial focused ultrasound

Abstract

A minimally invasive method of reducing hypertension using focused ultrasound is provided. Upon injection of ultrasound responsive nanodroplets loaded with a therapeutic agent, such as a barbital drug, focused ultrasound is delivered to the noradrenaline producing area, such as the area of grey matter around an aqueduct, thereby locally releasing the therapeutic agent payload from the nanodroplets and achieving a therapeutic reduction of blood pressure. For example, acoustic emissions from vaporized droplets may be employed to infer one or more treatment parameters from a pre-established correlation. For example, plasma hormone content and / or blood pressure changes may be inferred, thereby providing a method of real-time treatment monitoring. The example methods are useful for effecting blood pressure reduction in subjects exhibiting drug-resistant hypertension.

Description

[0001] Cross - reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 391,201, entitled "METHODS FOR THE TREATMENT OF HYPERTENSION VIA TRANSCRANIAL - FOCUSED - ULTRASOUND", filed on July 21, 2022, the entire content of which is incorporated herein by reference. Background of the Invention

[0003] The present disclosure relates to methods of neuromodulation using focused ultrasound.

[0004] Elevated blood pressure (i.e., hypertension) affects one - quarter of adults globally and the prevalence continues to rise (WHO, 2022). Current strategies for reducing hypertension typically use medications. Antihypertensive drugs have been clinically used for over 60 years and are classified into five major classes (beta - blockers, diuretics, angiotensin - converting enzyme inhibitors, angiotensin II receptor blockers, calcium channel blockers), which 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 resistant hypertension and during pregnancy (Laurent, 2017).

[0005] Focused ultrasound (FUS) is a sound wave with a frequency higher than 20 kHz and a maximum intensity focused on a single target, which can non - invasively modulate brain activity. This technique provides higher precision and deeper penetration than external electric and magnetic fields, enabling targeting of specific brain anatomical 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. Through precise MRI - guided thalamotomy (termed focused ultrasound thalamotomy), hand tremors can be significantly reduced and motor function enhanced (Elias et al., 2016), and it has currently been used to treat nearly 7,000 patients globally (InSightec). Subsequently, similar procedures have been used to treat Parkinson's disease (Martínez - Fernández et al., 2018), obsessive - compulsive disorder (OCD) and major depressive disorder (Davidson et al., 2020), and have been shown to be safe in the treatment of epilepsy (Stern et al., 2021).

[0006] At lower intensities, non-ablative ultrasound exposure can transiently modulate neuronal activity (Fomenko et al., 2018). This has been pre-clinically 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 an acute pain model, ultrasound treatment of the periaqueductal gray area (PAG) showed an analgesic effect (Zhang et al., 2022). In a hypertensive rat model, Li et al. showed that daily low-intensity ultrasound delivery to the ventrolateral periaqueductal gray (vlPAG) reduced systolic blood pressure, heart rate, and hypertension-related proteins in plasma (Li et al., 2020). The studies described above suggest that ultrasound can non-invasively achieve a blood pressure reduction effect similar to DBS. However, the duration of the blood pressure reduction after treatment has not been reported. Therefore, it is currently unclear whether seven consecutive daily treatments are optimal or necessary, and whether the effect persists within a clinically relevant time frame.

[0007] In addition to direct neuromodulation, FUS is also a non-invasive method for releasing anesthetics in the brain, which uses ultrasound-responsive drug carriers, such as droplets for delivering propofol (Airan et al., 2017) and liquids for delivering barbiturates (Lea-Banks et al., 2020), as well as microbubbles for releasing muscimol (Ozdas et al., 2020). Combining FUS with locally released anesthetics allows control of the duration of neuromodulation, thus prolonging the effect according to pharmacokinetics. Previously, the present inventors have demonstrated this off-line neuromodulation using nanodroplets loaded with pentobarbital (PBND), and the results showed that rats had persistent sensorimotor deficits 60 minutes (Lea-Banks et al., 2020) and 90 minutes (Lea-Banks et al., 2021) after the end of ultrasound treatment. In addition, encapsulation of the anesthetic can prevent off-target effects such as general sedation and improve the reliability of local area inhibition (Wang et al., 2018). Summary of the Invention

[0008] A minimally invasive method for reducing hypertension using focused ultrasound is provided. After injecting ultrasound-responsive nanodroplets loaded with a therapeutic agent (such as a barbiturate), the focused ultrasound is delivered to the norepinephrine-producing area, such as the periaqueductal gray area, so as to locally release the therapeutic agent payload from the nanodroplets and achieve a therapeutic reduction in blood pressure. For example, acoustic emissions from vaporized droplets can be used to infer one or more treatment parameters according to a pre-established correlation. For example, plasma hormone content and / or blood pressure changes can be inferred, thus providing a method for real-time treatment monitoring. This exemplary method can be used to achieve a reduction in blood pressure in subjects with drug-resistant hypertension.

[0009] Thus, in some aspects, there is provided a method for treating hypertension using focused ultrasound, the method comprising, after intravenous injection of nano-droplets loaded with a therapeutic agent:

[0010] delivering focused ultrasound to the norepinephrine-producing region of the brain, wherein the focused ultrasound is configured to promote vaporization of the nano-droplets and release of the therapeutic agent.

[0011] In some example embodiments of the method, the therapeutic agent comprises a barbiturate.

[0012] In some example embodiments of the method, the therapeutic agent comprises a lipophilic anesthetic. The lipophilic anesthetic may have a molecular weight of less than 400 Da and a logarithm of the partition coefficient of approximately 2.

[0013] In some example embodiments of the method, the norepinephrine-producing region comprises the periaqueductal gray region.

[0014] In some example embodiments of the method, the norepinephrine-producing region comprises the ventrolateral periaqueductal gray region.

[0015] In some example embodiments of the method, the norepinephrine-producing region comprises at least one of the locus coeruleus, thalamus, hypothalamus, neocortex, and cerebellum.

[0016] In another aspect, there is provided a method for treating hypertension using focused ultrasound, the method comprising:

[0017] delivering focused ultrasound to the norepinephrine-producing region of the brain, wherein the focused ultrasound is configured to induce repetitive mechanical stress on the tissue by forming two or more closely spaced ultrasonic pressure nodes, wherein adjacent nodes are in different acoustic phases, thereby generating a force that stimulates or inhibits neuronal activity between the nodes.

[0018] In some example embodiments of the method, the ultrasound field is generated by one of a phased array, lens, reflector, waveguide, and multiple overlapping ultrasound fields.

[0019] In some example embodiments of the method, the norepinephrine-producing region comprises the periaqueductal gray region.

[0020] In some example embodiments of the method, the norepinephrine-producing region comprises at least one of the locus coeruleus, thalamus, hypothalamus, neocortex, and cerebellum.

[0021] In some example embodiments of the method, the norepinephrine-producing region comprises the ventrolateral periaqueductal gray region.

[0022] In some example embodiments of the method, focused ultrasound delivery is performed after intravenous injection of nanodroplets loaded with a therapeutic agent to facilitate vaporization of the nanodroplets and release of the therapeutic agent.

[0023] In some example embodiments of the method, focused ultrasound delivery is combined with an agent such as systemic delivery of an anesthetic, which will enhance the effect of ultrasound on the brain tissue.

[0024] The functions and advantages of the present invention can be further understood by referring to the following detailed description and the drawings. Brief Description of the Drawings

[0025] Embodiments will now be described by way of example only with reference to the drawings, in which:

[0026] Figure 1 is a flowchart showing an example method of using transcranial focused ultrasound to deliver to the periaqueductal gray area (PAG) to reduce hypertension after injection of drug-loaded nanodroplets.

[0027] Figure 2 Shows an exemplary transcranial focused ultrasound system.

[0028] Figure 3 Shows an exemplary focused ultrasound headset.

[0029] Figure 4 Shows the persistence of blood pressure reduction in normotensive rats after a single ultrasound treatment. (A) Schematic of non-invasive blood pressure measurement in an awake rat in a cylindrical rodent holder, with an occluding cuff and a volume-pressure recording (VPR) cuff, showing representative pressure measurements before treatment. (B) Schematic of a focused ultrasound system targeting the ventrolateral periaqueductal gray (vlPAG), (C) ultrasound pulse protocol, (D) histological safety assessment showing no signs of bleeding or edema after ultrasound treatment (scale bar 50 μm). (E) Systolic blood pressure, (F) diastolic blood pressure, and (G) heart rate before and after ultrasound treatment. Lines show individual measurements of six healthy rats, and significance was assessed from baseline (*p < 0.05).

[0030] Figure 5 Shows the reduction of systolic and diastolic blood pressure in normotensive rats. Single treatment protocol, (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 show one standard deviation, N = 6, *p < 0.05.

[0031] Figure 6Show the effect of ultrasound-treated brain regions on reducing blood pressure in hypertensive rats. Show the change in systolic blood pressure for each rat, from baseline, during treatment (2 hours after treatment) to within 6 days after treatment, measured daily for (A) initial, (B) PBND+FUS (active control), (C) FUS (active control), (H) PBND+FUS (vlPAG), and (I) FUS (vlPAG). Graphs of the relationship between norepinephrine plasma content, acoustic emission at subharmonic frequencies, systolic and diastolic blood pressure 2 hours after ultrasound treatment of (D-F) active control regions and (J-L) vlPAG regions. Markers represent individual rats after treatment with FUS alone (circles) or with FUS and pentobarbital-loaded nanodroplets (PBND) (triangles). Linear correlation was evaluated using the Pearson coefficient (R 2 ).

[0032] Figure 7 Show the reduction in systolic and diastolic blood pressure in hypertensive rats. (A) Schematic of a crossover study design, including five consecutive treatments. (B) Acoustic emission of activated pentobarbital nanodroplets (PBND) mapped to the change in systolic blood pressure 2 hours after treatment. (C) Heart rate, (D) systolic blood pressure (BP), and (E) diastolic blood pressure (BP). Error bars show one standard deviation, N = 4, *p < 0.05.

[0033] Figure 8 Show the immunohistochemical analysis demonstrating increased neuronal activity after treatment. (A) Immunohistochemical sections of the VLPAG of 3 hypertensive rats (initial, FUS only, PBND+FUS), stained for C-Fos (red), NeuN (green), and DAPI (blue). Scale bar represents 100 μm. (B, C) C-Fos expression in the PAG region untreated (right) and treated with ultrasound (left), as shown in the schematic of the brain section in (D). (E, F) NeuN-positive cells in the PAG region untreated (right) and treated with ultrasound, and (G) the proportion of C-Fos-positive cells that are also NeuN-positive, N = 1.

[0034] Figure 9 Show the adaptation of blood pressure and heart rate measurements. (A1–A3) Normotensive rats and (B1–B3) SHR were trained for 5 days before treatment to improve tolerance, reduce tail movement, and reduce the variation (standard deviation, STD) in blood pressure and heart rate (beats per minute, bpm) measurements.

[0035] Figure 10Shows the body weight and body temperature assessments of (A) healthy normotensive Wistar rats (n = 6) and (B) spontaneously hypertensive rats (n = 4) during adaptation, treatment, and post-treatment follow-up. Error bars represent one standard deviation, and no significance (NS) was found between groups (p > 0.05).

[0036] Figure 11 Shows the plasma hormone adrenaline level assessment in spontaneously hypertensive rats, showing no correlation with systolic or diastolic blood pressure (BP), assessed with Pearson's coefficient (R 2 <0.1).

[0037] Figure 12 Shows representative images of histological safety assessment using (A) TUNEL and (B) H&E staining to identify bleeding or apoptosis. No damage was seen in the sonication areas of normotensive and hypertensive rats.

[0038] Figure 13 Is a table presenting the distribution of animals between experimental groups.

[0039] Figure 14A 、 14B 、14C and 14D provide experimental results demonstrating the dose-dependence of ultrasound-mediated blood pressure reduction. Detailed implementation

[0040] The various embodiments and aspects of the present disclosure will be 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 present disclosure. Many specific details are described to provide a thorough understanding of the various embodiments of the present disclosure. However, in some instances, well-known or conventional details are not described in order to provide a concise discussion of the embodiments of the present disclosure.

[0041] As used herein, the terms "comprises" and "comprising" should be interpreted as inclusive and open-ended, rather than exclusive. Specifically, when used in the specification and claims, the terms "comprises" and "comprising" and their variants mean including the specified features, steps, or components. These terms should not be construed as excluding the presence of other features, steps, or components.

[0042] As used herein, the term "exemplary" means "serving as an example, instance, or illustration" and should not be construed as more preferred or advantageous than other configurations disclosed herein.

[0043] As used herein, the terms "about" and "approximately" are intended to cover variations that may exist in the upper and lower limits of a range of values, such as variations in properties, parameters, and dimensions. Unless otherwise specified, the terms "about" and "approximately" each mean plus or minus 25% or less.

[0044] It is to be understood that, unless otherwise specified, any specified range or group is a shorthand way of separately referring to each member of the range or group and to every possible subrange or subgroup subsumed therein, and likewise for any subrange or subgroup thereof. Unless otherwise specified, this disclosure contemplates and expressly encompasses combinations of each specific member and subrange or subgroup.

[0045] As used herein, the term "about", when used in conjunction with a quantity or parameter, refers to a range spanning from about one-tenth to ten times the stated quantity or parameter.

[0046] Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise indicated (e.g., by context), as used herein, the following terms are intended to have the following meanings:

[0047] As described above, although previous studies involving the delivery of focused ultrasound to the PAG alone have successfully demonstrated a reduction in hypertension, the duration of this benefit has been unclear. As described in the examples provided below, the inventors have found that, in the case of a hypertensive rat model, the delivery of focused ultrasound to the PAG alone results in only a subset of the treated rats exhibiting a reduction in blood pressure after focused ultrasound treatment. Moreover, in the hypertensive rats that did exhibit a reduction in blood pressure, after five consecutive days of delivery of focused ultrasound to the PAG, the transient reduction lasted only 24 hours.

[0048] The inventors sought to improve the therapeutic efficacy and persistence of focused ultrasound on the brain to better reduce hypertension. The inventors have discovered a treatment modality involving the use of a combination of focused ultrasound and nanodroplets loaded with barbiturates, rather than simply sonicating the PAG region of the brain. It has been found that when nanodroplets loaded with barbiturates are injected into a subject, subsequent delivery of focused ultrasound to the PAG provides a dual-mechanism neuromodulatory therapeutic benefit through the combined action of focused ultrasound on the PAG region and through the effect of locally releasing the barbiturates from the nanodroplets within the PAG by focused ultrasound.

[0049] Accordingly, various exemplary embodiments of the present disclosure employ a combination of focused ultrasound (FUS) and nanodrops loaded with barbiturates as a minimally invasive method to reduce hypertension by modulating central brain activity. Figure 1An exemplary method of focused ultrasound neuromodulation for hypertension by triggering the release of payload-loaded nanodroplets of barbiturates is shown. In step 100, nanodroplets loaded with a therapeutic agent (e.g., pentobarbital) are injected into the bloodstream of a subject. Focused ultrasound is then delivered to the periaqueductal gray (PAG) region of the subject, as shown in step 110. The focused ultrasound is configured to facilitate the release of the focused ultrasound-triggered therapeutic agent from the nanodroplets.

[0050] As shown in optional step 120, acoustic emissions generated by the collapse of the nanodroplets can be monitored, for example, using one or more transducers for delivering 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 to control the delivery of the focused ultrasound, and / or as a means to verify or determine that an effective dose has been delivered by the vaporization of the nanodroplets, and / or as a means to determine or infer the blood pressure reduction caused by the treatment. For example, at a sufficient ultrasonic pressure (referred to as the vaporization threshold), the nanodroplets transition from a liquid state to a gaseous state, releasing the therapeutic agent and emitting a unique acoustic signal. The subharmonic frequency (half of the emission frequency) can be used as a marker for drug release (Lea-Banks et al. 2020, 2021). As described below, the inventors found a weak correlation between the acoustic emissions and the blood pressure reduction (R 2 = 0.46 and 0.36, Pearson coefficients) (see Figure 7 B), while the acoustic emissions detected from FUS alone had no correlation with the blood pressure changes (R 2 = 0.08 and 0.04, Pearson coefficients).

[0051] Referring again to Figure 1 , steps 100 and 110 (and optional step 120) can be repeated one or more times at appropriate time intervals to achieve a sustained reduction in blood pressure, as shown in 130. For example, the treatment can be repeated on two or more consecutive days. In some exemplary embodiments, the treatment is repeated for 3, 4, 5, 6, 7, 8, 9, or 10 consecutive days.

[0052] As shown in step 140, after one or more treatments including steps 100 and 110, an increase in blood pressure can be detected after a certain time interval (e.g., 5, 6, 7, 8, 9, or 10 days). Steps 100 - 130 can then be repeated to obtain another blood pressure reduction, as shown in 150.

[0053] Vaporization of nanodroplets can be driven by pressure changes and / or temperature changes. The underlying mechanisms are referred to as mechanical or thermal mechanisms (Lea-Banks et al., 2019, JCR). When using low ultrasound frequencies (<2 MHz), mechanical-driven vaporization predominates, which is typically used when performing ultrasound treatment through the skull. The vaporization threshold (the ultrasound pressure required to activate the nanodroplets) is directly related to the frequency, meaning that low-frequency exposure (50 kHz - 2 MHz) requires low ultrasound pressure (0.2 - 3.0 MPa), with burst lengths between 1 ms - 100 ms. The droplets can also be vaporized with shorter bursts, but may require higher pressure amplitudes. In a rodent model, it was found that nanodroplets loaded with pentobarbital could remain acoustically active in the circulation for up to 20 minutes after a single bolus injection (Lea-Banks et al. 2020, Theranostics). Therefore, an ultrasound treatment duration of 1 - 30 minutes after bolus administration can be effectively used, but if the nanodroplets are delivered by infusion or multiple bolus injections, the ultrasound treatment time can be extended.

[0054] PAG is a component in the pathway that controls cardiovascular function. The arcuate nucleus of the hypothalamus (ARC), the ventrolateral periaqueductal gray matter (vlPAG), and the rostral ventrolateral medulla (rVLM) are crucial in controlling the sympathetic excitatory cardiovascular reflex response. Without being bound by theory, the inventors believe that these alternative brain regions may be susceptible to the delivery of ultrasound-triggered therapeutic agents (such as barbiturates) and also affect blood pressure. Additionally, the examples provided below show that changes in norepinephrine excretion and clearance are associated with the antihypertensive effect. Therefore, the present exemplary embodiment can be used to target other centers of norepinephrine excretion in the entire norepinephrine system, such as the locus coeruleus, thalamus, hypothalamus, cerebral cortex, and cerebellum.

[0055] This exemplary embodiment employs a therapeutic agent (drug) loading strategy that is applicable to a wide range of therapeutic agents, including small molecule lipophiles. In the example of nano-droplets with a lipid shell, since the drug is incorporated into the lipid shell and then released and crosses the intact blood-brain barrier, the molecular weight of the drug should be less than 400 Da, and the LogP (logarithm of the partition coefficient) should be close to 2. Non-limiting examples of suitable barbiturates with a molecular weight less than 400 Da and a logarithm of the partition coefficient approximately equal to 2 include amobarbital, alphenal, butabarbital, butalbital, butethal, pentobarbital, phenobarbital, secobarbital, and thiopental. Since all barbiturates are agonists of the GABA A receptor, it is expected that the use of alternative barbiturates will trigger a similar inhibitory pathway, resulting in a decrease in blood pressure.

[0056] Although the various examples of the present disclosure relate to nano-droplets loaded with barbiturates, it should be understood that the nano-droplets can be loaded with other therapeutic agents, such as but not limited to small lipophilic anesthetics. Non-limiting examples of small lipophilic anesthetics include benzocaine, bupivacaine (Marcaine), ketamine, levobupivacaine, lidocaine, prilocaine, procaine, propofol, ropivacaine, tetracaine, which have a similar inhibitory effect on neuronal activity.

[0057] In some exemplary embodiments, the nano-droplets can be formed to have a core containing a low-boiling point perfluorocarbon (such as but not limited to decafluorobutane and octafluoropropane), and a shell containing one or more of phospholipids, proteins (such as albumin), and polymers (such as amphiphilic copolymers). Example methods of manufacturing nano-droplets are disclosed in U.S. Patent Application No. US20130336891, which is incorporated herein by reference in its entirety. Example methods of manufacturing nano-droplets loaded with a therapeutic agent are disclosed in U.S. Patent Application No. US20200368352, which is incorporated herein by reference in its entirety. In some exemplary embodiments, focused ultrasound delivered to the periaqueductal gray area induces repetitive mechanical stress on the tissue by forming two or more closely spaced ultrasound pressure nodes (or foci), where adjacent nodes are in different (opposite in the optimal configuration) acoustic phases, thereby generating a force that stimulates or inhibits neuronal activity between the nodes. Such an exemplary implementation can be performed after injecting nano-droplets loaded with a therapeutic agent, or in the case where nano-droplets loaded with a therapeutic agent are not injected.

[0058] The ultrasound field can be generated by a phased array, a lens, a reflector, a waveguide, and / or one of multiple overlapping ultrasound fields and / or any other method that allows control of the ultrasound field.

[0059] As shown in the examples provided below, after a single 10-minute ultrasound treatment, the blood pressure of healthy Wistar rats decreased within 6 hours. After 5 consecutive days of focused ultrasound (FUS) treatment with nano-droplets loaded with pentobarbital (PBND) every day, the diastolic blood pressure of hypertensive rats decreased for up to 5 days, while ultrasound treatment of the frontal cortex had no effect on blood pressure. vlPAG stimulation was confirmed by immunohistochemistry showing increased neuronal activity and enzyme-linked immunosorbent assay (ELISA) showing changes in plasma hormone content.

[0060] The results presented herein demonstrate the use of focused ultrasound as a minimally invasive treatment tool for hypertension with a clinically relevant time scale. Hypertension is the most common disorder during pregnancy. However, many antihypertensive drugs pose significant risks associated with fetal growth restriction, reduced placental blood flow, and fetal loss (Podymow and August, 2011). The mixed evidence regarding health risks has led to different regulatory approvals and clinical uses around the world; for example, in the UK, labetalol is the drug of choice for treating hypertension during pregnancy, while its use is avoided in North America (U.S. FDA Pregnancy Category C).

[0061] In addition, refractory hypertension rules out all existing pharmaceutical strategies. DBS has been investigated as an alternative. In 2005, a 61-year-old male with drug-resistant hypertension was admitted for chronic neuropathic pain and received deep brain stimulation of the periaqueductal gray (PAG) region (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 otherwise unresponsive to antihypertensive medications. However, blood pressure returned to baseline 20 seconds after stimulation was turned off. Two further clinical studies investigated the relationship between blood pressure reduction and analgesia and the potential long-term blood pressure-lowering effect (Pereira et al., 2010; Patel et al., 2011). However, DBS is an invasive procedure associated with potential risks of infection and bleeding (Fenoy and Simpson, 2014). Therefore, a new strategy for treating hypertension is needed that is non-invasive, persistent, and does not require antihypertensive medications.

[0062] A long-term focused ultrasound treatment for hypertension is achieved through renal denervation. In a recent multicenter clinical study, blood pressure was reduced for at least 2 months after ultrasound ablation of the renal nerves (Azizi et al., 2021). Ultrasound renal denervation has been approved in Europe for the treatment of hypertension. However, limitations in renal anatomy (including the main renal artery diameter and length must be within specified ranges) limit the accessibility of this treatment. In the 2021 study, 108 patients were excluded based on these anatomical criteria. In addition, 17% of patients experienced persistent pain (>2 days) after the procedure.

[0063] The present disclosure demonstrates that transcranial focused ultrasound in combination with nanodroplets loaded with barbiturates provides a non-invasive method for directly stimulating the PAG and a minimally invasive method for triggering anesthetic delivery. Both methods have been shown to reduce hypertension (180 / 140 mmHg) to the healthy range (140 / 100 mmHg). It was found that local delivery of anesthetic using PBND extended the effect of FUS from 2 days to 5 days after treatment.

[0064] As shown in the following example, ultrasound treatment of the frontal cortex using focused ultrasound alone had no effect on blood pressure in hypertensive rats ( Figure 7 ). Comparison of ultrasound treatment of the active control region and the vlPAG indicated that blood pressure reduction depends on brain anatomy. The blood pressure reduction 2 hours after PBND+FUS may be related to non-specific drug release in the active control region upstream of the vlPAG and related inhibitory pathways ( Figure 8D, 8E). The acoustic controller that defines the required pressure, in combination with local delivery of pentobarbital, improves the reliability of treatment. Four out of four hypertensive rats responded to PBND + FUS (vlPAG), while one out of four rats treated with FUS alone did not respond.

[0065] Immunohistochemical analysis was also employed to explore the pathway of the antihypertensive effect. C-Fos is an immediate early gene (IEG) associated with neuronal firing. Contrary to the previous work of the present inventors on delivering pentobarbital to the motor cortex (where inhibition of C-Fos was mapped), an increase in C-Fos expression was observed in the vIPAG region in this experiment. By microinjecting an anesthetic into specific anatomical regions of the brain, regional differences in C-Fos expression in response to pentobarbital were investigated. Pentobarbital is a GABAA receptor agonist, and by microinjecting it into the tuberomammillary nucleus of rats, it has been found to increase c-FOS expression in the ventrolateral preoptic nucleus (VLPO), but inhibit expression in the tuberomammillary nucleus and leave expression in the locus coeruleus unchanged (Nelson et al., 2002). These anatomical differences are related to the distribution of GABAergic receptors, neurons, and interneurons. In addition, by directly injecting GABA into the PAG region of rats, where most of the GABA receptors present have been mapped, it signals to an important GABA network responsible for profound inhibitory pathways (Behbehani et al., 1990).

[0066] In the present exemplary method, plasma hormone assays were performed to study the mechanism driving blood pressure reduction. After sonication of the vlPAG for 2 hours, a weak negative correlation was found between the norepinephrine content in venous plasma samples and blood pressure, but no significant difference was found for epinephrine. The release of norepinephrine is generally associated with an increase in metabolic function, heart rate, and blood pressure. However, the relationship between GABA receptors in the PAG region and norepinephrine has been explored in the context of pain inhibition. Norepinephrine injection has been shown to inhibit pain-modulating neurons (Basbaum and Fields, 1984), and it has been reported that PAG stimulation in DBS leads to an increase in norepinephrine release, which is shown to inhibit dorsal horn neurons, thereby producing an analgesic effect (Cui et al., 1999).

[0067] A further consideration is norepinephrine clearance, where an increase in circulating norepinephrine in venous plasma may indicate a decrease in clearance (Esler et al., 1984). It has been found that the clearance rate of norepinephrine is slower in normotensive humans compared to hypertensive patients (Grimm et al., 1980). Thus, an increase in circulating norepinephrine may be the combined result of increased norepinephrine release and decreased clearance through PAG stimulation. Without wishing to be bound by theory, the inventors hypothesize that increased PAG C-Fos expression and increased plasma norepinephrine levels indicate that PAG stimulation results in an inhibitory response triggered by the GABAergic network and leads to a decrease in blood pressure.

[0068] The methods, examples, and results disclosed herein demonstrate that FUS-mediated neuromodulation therapy for reducing hypertension is promising and may have clinical implications. Hypertension remains a deadly disease with a growing prevalence, but FUS provides a minimally invasive approach that has the potential to be optimized as a cost-effective long-term treatment for hypertension. Indeed, in the examples provided below, it is shown that continuous ultrasound treatment in combination with anesthetic-loaded nanodroplets can reduce hypertension to the healthy range for up to 5 days.

[0069] Figure 2 A block diagram is provided showing an example implementation of a system for performing diagnostic or therapeutic transcranial surgery. The control and processing hardware 300 is operably connected to the transcranial headset 100, optionally through the transducer drive electronics / circuitry 380.

[0070] The control and processing hardware 300 includes one or more processors 310 (e.g., CPU / microprocessor), a bus 305, a memory 315 (which may include random access memory (RAM) and / or read-only memory (ROM)), a data acquisition interface 320, a display 325, an external storage device 330, one or more communication interfaces 335, a power supply 340, and one or more input / output devices and / or interfaces 345 (e.g., speakers, user input devices such as keyboards, keypads, mice, position-tracking styli, position-tracking probes, foot switches, and / or microphones for capturing voice commands).

[0071] The volumetric image data 370 and the transducer registration data 375 may be stored in an external database or in the memory 315 or storage device 330 of the control and processing hardware 300.

[0072] The tracking system 365 can optionally be used to track the position and orientation of a patient by detecting one or more fiducial markers 460 attached to the transcranial headset 400 and optionally also one or more medical instruments or devices that also have fiducial markers attached thereto. For example, passive or active signals emitted from the fiducial markers can be detected by a stereotactic tracking system employing two tracking cameras. The transducer drive electronics / circuitry 380 can include, for example but not limited to, a Tx / Rx switch, a transmit and / or receive beamformer.

[0073] The control and processing hardware 300 can be programmed with a program, subroutine, application, or module 350 including executable instructions that, when executed by one or more processors 310, cause the system to perform one or more of the methods described in this disclosure. Such instructions can be stored, for example, in the memory 315 and / or other storage devices.

[0074] In the illustrated example embodiment, the transducer control module 355 includes executable instructions for controlling the transducer of the transcranial headset 400 to deliver energy to the PAG region based on the registration of the transducer position and orientation with volumetric image data according to the transducer registration data 375. For example, the transcranial headset 400 can support multiple phased array transducers, and the transducer control module 355 can control the applied beamforming (during transmission and / or reception) to deliver one or more focused energy beams to the region of interest in the far field of the transcranial ultrasound transducer array elements based on the known position and orientation of the phased array transducers relative to the volumetric image data. The region of interest can be specified by the user during the procedure (e.g., via a user interface controlled by the control and processing hardware 300) or according to a pre-established surgical plan.

[0075] Optionally, a registration module 360 can be employed to register the volumetric image data 370 to an intraoperative reference frame associated with the tracking system 365. The optional guidance user interface module 362 includes executable instructions for displaying a user interface that displays the volumetric image for spatial registration for image-guided surgery. The registration module 360 can also receive intraoperative spatial correction information based on a detected spatial offset between the transcranial frame and the patient's head (which can be provided by a subset of distance-sensing transducers) and employ this spatial correction information to dynamically adjust (e.g., correct) the registration between the transducer and the volumetric image data.

[0076] Although Figure 2Only one of each component is shown in the figure, but any number of each component may be included in the control and processing hardware 300. For example, computers typically contain many different data storage media. In addition, although the bus 305 is depicted as a single connection between all components, it should be understood that the bus 305 may represent one or more circuits, devices, or communication channels that link two or more components. For example, in a personal computer, the bus 305 typically includes or is the motherboard. The control and processing hardware 300 may include many more or fewer components than shown.

[0077] The control and processing hardware 300 may be implemented as one or more physical devices coupled to the processor 310 through one or more 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 a memory or through a network connection.

[0078] Some aspects of the present disclosure may be embodied at least in part in software that, when executed on a computing system, converts the computing system into a dedicated computing system capable of performing the methods disclosed herein. That is, these techniques may be performed in a computer system or other data processing system in response to its processor (e.g., a microprocessor) executing a sequence of instructions contained in a memory (e.g., a ROM, volatile RAM, non-volatile memory, cache, disk and optical disk, or remote storage device). In addition, the instructions may also be downloaded to a computing device over a data network in the form of a compiled and linked version. Alternatively, the logic for performing the above process may be implemented in an additional computer and / or machine-readable medium, such as a discrete hardware component such as a large-scale integrated circuit (LSI), an application-specific integrated circuit (ASIC), or firmware such as an electrically erasable programmable read-only memory (EEPROM) and a field programmable gate array (FPGA).

[0079] Computer-readable media can be used to store software and data that, when executed by a data processing system, cause the system to perform various methods. Executable software and data can be stored in various locations, such as ROM, volatile RAM, non-volatile memory, and / or cache. Portions of the software and / or data can be stored in any of these storage devices. In general, machine-readable media include any mechanism that provides (i.e., stores and / or transmits) information in a form accessible to 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.).

[0080] Examples of computer-readable media include, but are not limited to, recordable and non-recordable types of media such as volatile and non-volatile memory devices, read-only memory (ROM), random access memory (RAM), flash memory devices, floppy disks and other removable disks, magnetic disk storage media, optical storage media (e.g., compact discs (CDs), digital versatile discs (DVDs), etc.), and the like. The instructions can be embodied in digital and analog communication links for electrical, optical, acoustic, or other forms of propagated signals (such as carrier waves, infrared signals, digital signals, etc.). The phrases "computer-readable material" and "computer-readable storage medium" as used herein refer to all computer-readable media except the transient propagated signals themselves.

[0081] Now referring to Figure 3 , a non-limiting and 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 includes a patient-specific frame (support structure) 410 that supports a plurality of transducers 420 and that conforms to the anatomical profile of at least a portion of the patient's head. Figure 3 The patient-specific frame 410 shown in cross-section in [reference] mechanically supports the transducers 420 in a preselected position and orientation. The transducers 420 can be used to transmit and / or receive energy for brain diagnostic or therapeutic purposes or for localization on the surface of the skull.

[0082] The patient-specific frame 410 includes a plurality of attachment interfaces for receiving and supporting the transducers 420. In Figure 3 the example embodiment shown, the attachment interfaces are provided as holes (recesses) in which the transducers 420 are placed. The transducers 420 can be fixed to the patient-specific frame 410 in a variety of different ways, such as, but not limited to, using an attachment mechanism (e.g., by a fastener extending into the patient-specific frame 410, optionally into a pre-formed hole), or an adhesive, such as glue. In Figure 3 the exemplary embodiment shown, the transducers 420 are remotely connected to an electronic device via wires or a flexible printed circuit board 440. The transducers 420 can be detachably attached to the patient-specific frame 410.

[0083] Figure 3The exemplary patient-specific earphone shown 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 energy coupling between the transducer in the patient-specific frame and the patient's head. The inclusion of the coupling layer 430 and the composition and / or geometry of the coupling layer may depend on the type of transducer 420. For example, if the transducer 420 is an ultrasonic transducer, the coupling layer 430 may be an acoustic coupling layer that facilitates the propagation of sound waves and reduces reflection at the interface. In one exemplary embodiment, the coupling layer 430 includes an elastic membrane that retains a liquid layer between the transducer surface and the elastic membrane, thereby achieving coupling with the skin.

[0084] Example

[0085] The following examples are provided to enable those skilled in the art to understand and practice the embodiments of the present disclosure. They should not be construed as limiting the scope of the present disclosure, but are merely illustrative and representative.

[0086] In the following examples, two experiments were conducted: The first experiment used healthy normotensive Wistar rats to evaluate the persistence of blood pressure reduction after a single 10-minute ultrasound treatment of the vlPAG region. Subsequently, the effects of general anesthesia, FUS alone, FUS with nanodroplets, and FUS with pentobarbital-loaded nanodroplets (PBND) targeting the vlPAG administered in a single treatment or in a pair of treatments over consecutive days were compared. The second experiment used a hypertensive model - spontaneously hypertensive rats (SHR) - to compare FUS alone and FUS with PBND, targeting the vlPAG or the frontal cortex (as an active control), in 5 treatments over consecutive days. Non-invasive blood pressure measurements, plasma hormone assessments, and immunohistochemistry of neuronal activity were performed in awake rats to explore the underlying mechanisms.

[0087] Example 1: Method

[0088] Barbiturate-loaded nanodroplets

[0089] Ultrasound-responsive nanodroplets with a diameter of 200 m were synthesized according to the previously described method (Lea-Banks et al., 2021), following an improved condensation protocol (Sheeran et al., 2011, 2017; Matsunaga et al., 2012). Definity lipid solution (Lantheus Medical Imaging, USA) was mixed with pentobarbital (Sigma Aldrich MilliporeSigma, USA) by tip sonication, degassed, and filled with perfluorobutane (C4F10). Precursor microbubbles were formed by stirring (VialMix, Lantheus Medical Imaging, USA), concentrated at -10 °C, washed by centrifugation (300G, 8 min, 4 °C), and filtered at 0.8 μm (Minisart syringe filter, Sartorius, Germany). Nanodroplets were stored on ice before use and used within 2 hours after fabrication. Sham nanodroplets (sham ND) were fabricated in the same manner but without the addition of pentobarbital.

[0090] Animal

[0091] Two groups of rats were used to evaluate ultrasound-induced blood pressure changes. 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 day of treatment. 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 day of treatment. Animals were housed in the Sunnybrook Research Institute animal facility (Toronto, ON, Canada) on a reverse light cycle and had free access to food and water. All animal procedures were approved by the Sunnybrook Research Institute Animal Care Committee and were in accordance with the Canadian Council on Animal Care and ARRIVE guidelines.

[0092] Animal preparation

[0093] General anesthesia was induced with medical air containing 5% isoflurane (ISO) and its concentration was reduced to 2% during ultrasound surgery. To allow coupling between the transducer and the scalp, hair was removed using an electric shaver and depilatory cream, and a thin layer of ultrasound gel (Wavelength CL, ON, Canada) was applied to the scalp. The ultrasound gel was rotated in a centrifuge tube before use to remove air bubbles (1000G, 10 min). A 22-gauge tail vein catheter was inserted. After blood sampling, 2 mL of saline was injected subcutaneously, otherwise all injections were administered intravenously. The rats were placed in the stereotactic frame of the FUS system, breathed through a nose cone connected to the ISO machine, and scavenged with an F / AIR charcoal filter. A warm saline bag was placed on the animal to maintain body temperature. PBND or sham ND (containing approximately 25 μg / mL pentobarbital) was slowly injected via the tail vein catheter at a dose of 1.0 mL / kg.

[0094] Transcranial FUS system

[0095] Using a preclinical prototype system with atlas-based targeting (early prototype of RK50, FUS Instruments Inc., ON, Canada), focused ultrasound (FUS) at 540 kHz was targeted and delivered to the ventrolateral periaqueductal gray (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 geometry of the skull was co-registered with the focus of a single-element transducer (central frequency 540 kHz, element diameter 35 mm, focal length 25 mm), which had a central circular cutout to accommodate a narrowband PZT (lead zirconate titanate) hydrophone (central frequency 270 kHz, element diameter 5 mm, unfocused)( Figure 5 A). Sonication was performed at a pulse length of 10 ms and a pulse repetition frequency of 1 Hz for a duration of 10 minutes( Figure 5 B).

[0096] To evaluate the unique ultrasound pressure required for droplet vaporization in each rat, a 3-minute pressure ramp was performed after intravenous injection of the nanodroplets. The ultrasound pressure started at 0.45 MPa and increased in 8 kPa increments until the subharmonic emission exceeded 3.5 times the baseline value. This pressure was set as the vaporization threshold, and the subsequent 10-minute treatment ultrasound was fixed at this pressure. For the group using only ultrasound, a fixed pressure of 1.1 MPa was used based on the average pressure required for vaporization.

[0097] Measuring blood pressure in conscious rats

[0098] The blood pressure and heart rate of conscious rats were non-invasively measured using a tail-cuff system (CODA system, Kent Scientific, Torrington, CT, USA). The rats were placed in a cylindrical holder on a heating platform (CODA system), covered with a blanket, and allowed to acclimate for 10 minutes before starting the measurements. Fifteen cycles were performed, and the mean systolic and diastolic blood pressures and heart rate were recorded. The CODA system software automatically excluded recordings containing tail movement or insufficient blood volume. The rats were acclimated by performing blood pressure measurements daily for 5 consecutive days before the first treatment.

[0099] For hypertensive rats, tail temperature was evaluated by measuring the base of the tail at three time points using an infrared thermometer (TW2, Thermoworks, Salt Lake City, UT, USA): (1) immediately after placement in the holder, (2) after 10 minutes of acclimation, and (3) immediately after the measurement. All rats were weighed daily.

[0100] Assessment of plasma adrenaline and noradrenaline

[0101] Venous blood was collected before and 24 hours after treatment to determine the plasma levels of adrenaline and noradrenaline (hormones related to metabolic function and blood pressure) in SHR. 0.6 mL of blood was collected via a tail vein catheter and placed in an Eppendorf tube lined with heparin. The samples were kept on ice for 30 minutes and then centrifuged at 1000 G for 10 minutes. Plasma was separated and stored at -80 °C until analysis. Adrenaline and noradrenaline were evaluated in triplicate using ELISA (Abnova, KA1877, Taipei, China) according to the manufacturer's instructions.

[0102] Immunohistochemistry of spontaneously hypertensive rats

[0103] Ninety minutes after treatment, the rats were perfused intracardially with ice-cold saline and 0.1 M phosphate buffer containing 4% paraformaldehyde (PFA). The brains were harvested, post-fixed in 4% PFA overnight, and then equilibrated in 0.1 M phosphate buffer containing 30% sucrose. The samples were then embedded in OCT, cryosectioned horizontally at a thickness of 40 μm as free-floating sections, and stored in cryoprotectant at -20 °C.

[0104] One SHR per group was used for C-Fos expression analysis: naive, FUS(vlPAG) only, and PBND+FUS(vlPAG). According to the ultrasound treatment target coordinates, horizontal sections at a depth of 5.8 mm from the bregma were selected for immunofluorescence staining. The sections were rinsed with 0.1 M PBS containing 0.3% Triton X-100 and then treated in a blocking solution of 10% donkey serum for 1 hour at room temperature. The sections were incubated overnight at 4°C in primary antibodies anti-c-fos (ab190289, 1:1000, Abcam Inc., Cambridge, MA, USA) and anti-NeuN (ab104224, 1:1000, Abcam Inc.). The sections were then washed and incubated with fluorescently labeled secondary antibodies donkey Alexa 488 anti-mouse (A21202, 1:1000, Invitrogen, Eugene, OR, USA) and donkey Alexa 568 anti-rabbit (A10042, 1:1000, Invitrogen) for 2 hours at room temperature. The sections were washed with PBS and mounted onto X-tra glass slides (Leica Microsystems, Wetzlar, Germany) using an aqueous mounting medium containing DAPI (FluoroshieldTM with DAPI, Sigma-Aldrich Corporation, St. Louis, MO, USA). The slides were stored at 4°C in the dark until imaging.

[0105] Sections were imaged at 20x magnification (512×512 pixel field of view) to cover a 1 mm 2C-Fos quantification and NeuN colocalization analysis were performed in the region of interest as previously described (Lunde and Glover, 2020). Two custom binary images were created from the raw data of the multi-channel input image using the Colocalization Image Creator plugin (ImageJ, National Institutes of Health, Bethesda, Maryland, USA). Background signal intensity was reduced by global thresholding of each channel to generate a binary image. The binary images were combined with Boolean AND, macro transformation was performed to remove holes in the cells, and filtering was performed to remove cells with a size less than 5 μm. The first binary image included the DAPI and C-Fos channels and represented the total number of C-Fos-labeled cells. The second binary image included the DAPI, NeuN, and C-Fos channels and represented the signal overlap between the three input channels, indicating colocalization between NeuN and C-Fos. Next, the number of C-Fos-positive cells and the number of NeuN-positive cells colocalized with C-Fos were 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 in sonicated and non-sonicated PAG regions are reported. The proportion of C-Fos-positive cells showing NeuN expression was also calculated.

[0106] To investigate the safety of treatment, erythrocyte extravasation and apoptosis were assessed using hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (DeadEndTM colorimetric TUNEL system, Promega, Promega, Madison, WI, USA). All rats were sacrificed 7 days after the last treatment and intracardiac perfusion was performed with saline and buffered formalin. The brain was removed and immersed in 10% neutral buffered formalin for 24 hours, then transferred to 70% ethanol for 48 hours, and then embedded in paraffin. Axial sections of 5 μm thickness were taken at 500 μm intervals and stained.

[0107] Statistical analysis

[0108] Blood pressure and heart rate data are presented as individual data points or as bar graphs showing mean + / - standard deviation and error bars. Paired and unpaired t-tests were used to assess significance, with the significance level set at p < 0.05. Correlations were calculated using Pearson's coefficient (R 2 ) to evaluate.

[0109] Example 2: Results

[0110] Adaptation for blood pressure measurement

[0111] A 5-day acclimatization period is necessary for rats to become able to tolerate the NIBP procedure ( Figure 9 After adaptation, tail movement during measurement was significantly reduced, and the consistency of blood pressure and heart rate measurements was improved ( Figure 9 ). After adaptation, baseline systolic and diastolic blood pressures of normotensive rats were 133+ / -16mmHg and 103+ / -14mmHg, respectively, and heart rate was 258+ / 40bpm. Baseline systolic and diastolic blood pressures of hypertensive rats (SHR) were 183+ / -18mmHg and 142+ / -23mmHg, respectively, and heart rate was 263+ / -42bpm.

[0112] Transcranial focused ultrasound transiently reduces blood pressure in normotensive rats

[0113] After a single 10-minute ultrasound treatment of the vlPAG, systolic (-37 + / - 7 mmHg) and diastolic (-28 + / - 9 mmHg) blood pressures were significantly reduced in 6 of 6 healthy Wistar rats and remained significantly below baseline levels 6 hours after treatment (p < 0.05, Figure 4 E, 4F). Heart rate showed no significant changes ( Figure 4 G).

[0114] Transcranial focused ultrasound and barbiturate nanodroplets continuously reduce blood pressure in normotensive rats

[0115] General anesthesia (ISO only) and anesthesia combined with injection of pentobarbital nanodroplets (ISO+PBND) showed no changes in blood pressure when measured 2-48 hours after treatment

[0116] ( Figure 5 After a single ultrasound treatment of the vlPAG region ( Figure 5 A-5D), rats treated with FUS (ISO+FUS) had reduced systolic blood pressure (-26+ / -7mmHg) and reduced diastolic blood pressure (-22+ / -7mmHg) 2 hours after treatment. Pseudo-nanodroplets (unloaded, without pentobarbital) combined with FUS (ISO+FUS+pseudo-ND) also temporarily reduced blood pressure (systolic -19+ / -14mmHg, diastolic -15+ / -18mmHg). The study found that combining FUS with nanodroplets loaded with pentobarbital (ISO+FUS+PBND) maintained the effect, and blood pressure remained reduced for 24 hours after a single treatment (systolic -25+ / -5mmHg and diastolic -21+ / -8mmHg at 2 hours; systolic -17+ / -6mmHg and diastolic -18+ / -10mmHg at 24 hours).

[0117] The following week, two treatments were performed on consecutive days ( Figure 5E). Similarly, ISO alone and ISO+PBND showed no changes in blood pressure within 48 h after either treatment. After continuous ultrasound of the vlPAG region, ISO+FUS reduced both systolic and diastolic blood pressure 2 h after both treatments (-28 + / - 5 mmHg systolic and -27 + / - 3 mmHg diastolic), as did sham nanodroplets and FUS (-29 + / - 13 mmHg systolic and -29 + / - 10 mmHg diastolic), and both treatment groups returned to baseline after 24 h. Continuous treatment with ISO+FUS+PBND maintained the effect: blood pressure remained lowered for 48 hours after two consecutive treatments (2-hour systolic blood pressure -26+ / -7mmHg, diastolic blood pressure -12+ / -10mmHg; 24-hour systolic blood pressure -14+ / -10mmHg, diastolic blood pressure -15+ / -13mmHg; 48-hour systolic blood pressure -11+ / -10mmHg, diastolic blood pressure -9+ / -11mmHg). Changes in body weight and heart rate were comparable to those of control rats ( Figure 10 ).

[0118] Transcranial focused ultrasound reduces blood pressure in hypertensive rats and is maintained by barbiturate nanodroplets

[0119] Male and female spontaneously hypertensive rats (SHR) were used to investigate FUS-mediated blood pressure reduction, the efficacy of PBND, and the role of brain regions in the treatment of hypertension. A crossover study was performed in which five SHR treatment groups received five consecutive treatments separated by 24 h, followed by 6 days of monitoring, and then crossed over ( Figure 7 A). FUS was applied to the left frontal cortex (an area not involved in regulating blood pressure, serving as an active control) (FUS(active control)) or to the vlPAG region (FUS(vlPAG)) ( Figure 6 G).

[0120] Figure 6 The change in systolic blood pressure for each SHR treated for 5 consecutive days and then monitored for 6 days is shown. The initial SHR showed no change in blood pressure throughout the study ( Figure 6 A). After vlPAG ultrasound treatment, blood pressure decreased in 3 of 4 hypertensive rats ( Figure 6 I). FUS(vlPAG) showed greater blood pressure reduction (2 hours systolic -43+ / -29mmHg, diastolic -39+ / -31mmHg; 24 hours systolic -32+ / -28mmHg, diastolic -38+ / -34mmHg) compared to FUS(active control) (2 hours systolic -18+ / -29mmHg, diastolic -11+ / -27mmHg; 24 hours systolic 1+ / -14mmHg, diastolic 6+ / -13mmHg) ( Figure 6C, 6I). FUS (vlPAG) reduced blood pressure within 48 hours after 5 ultrasound treatments and returned to baseline after 3 days.

[0121] Following sonication of the vlPAG with PBND, blood pressure was reduced in 4 of 4 hypertensive rats. PBND+FUS(vlPAG) showed the greatest and most consistent reduction in blood pressure (-57+ / 17 mmHg systolic and -48+ / -17 mmHg diastolic at 2 hours), and the reduction remained significant (p<0.05) up to 4 days (systolic) or 5 days (diastolic) after the last sonication. PBND+FUS(active control) showed a transient reduction in blood pressure 2 hours after sonication (-37+ / -16 mmHg systolic and -16+ / -5 mmHg diastolic at 2 hours), which returned to baseline 24 hours after the final sonication. Animal body weights and tail-base temperatures were comparable to those of naive rats ( Figure 10 ).

[0122] At sufficient ultrasound pressure (known as the vaporization threshold), the nanodroplets transition from a liquid to a gaseous state, releasing the therapeutic agent and emitting a unique acoustic signature. Using linear regression analysis, subharmonic and second harmonic emissions were found to correlate with a reduction in blood pressure (R 2 =0.88 and 0.75, Pearson coefficients), while acoustic emissions detected from the active control area or using FUS alone showed no correlation with blood pressure changes (R 2 <0.4, Pearson coefficient).

[0123] Plasma hormone content in hypertensive rats is related to blood pressure reduction and acoustic response of nanodroplets

[0124] Plasma levels of epinephrine and norepinephrine were quantified from plasma samples collected before, 24 h after, and 1 week after treatment. Figure 6 Compared with rats treated with ultrasound at the vlPAG ( Figure 6 The mean norepinephrine level was higher in the vlPAG region (J-6L). Norepinephrine levels were correlated with systolic blood pressure (R) 24 h after ultrasound treatment in the vlPAG region. 2 =0.56)( Figure 7 K) and diastolic blood pressure (R 2 =0.69)( Figure 6 L) showed a weak negative correlation, but the active control group ( Figure 6 E, 6F) or adrenaline level ( Figure 11 ) did not have such a relationship. The norepinephrine level in the hypertensive rats in the PBND+FUS (vlPAG) group was also mapped to the subharmonic emission (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 emissions ( Figure 6 D-6F).

[0125] Immunohistochemical assessment maps neuronal stimulation by FUS

[0126] Three animals were perfused 90 minutes after treatment to assess C-Fos expression and NeuN antibody ( Figure 8 FUS and the combination of FUS and pentobarbital-loaded nanodroplets (PBND) showed that the number of C-Fos-positive cells in the VLPAG-sonicated area was higher compared with the contralateral side ( Figure 8 A). It was also found that after FUS alone, 79% of C-Fos positive cells expressed NeuN, while after PBND+FUS, 61% of C-Fos positive cells expressed NeuN ( Figure 8 G). H&E and TUNEL staining performed 7 days after the last treatment showed no signs of hemorrhage or edema, and the number of apoptotic cells did not increase ( Figure 12 ).

[0127] Example 3: Experimental evidence that ultrasound-mediated blood pressure reduction is dose-dependent

[0128] Many treatments for hypertension are ineffective in certain patient populations (e.g., 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 h. Here, the effect of ultrasound pressure amplitude on BP reduction was investigated.

[0129] Normotensive Wistar rats (18 males, 18 females) were used to assess the reduction in BP after FUS stimulation. BP ( Figure 14A vlPAG was localized using a single-element focused transducer (540 kHz, 34 mm element diameter, 3 mm beam width) and a stereotaxic localization system (FUSInstruments). Ultrasonication (duration 10 min, burst length 10 ms, pulse repetition frequency 1 Hz) was performed in anesthetized rats (isoflurane and medical air) at six pressure amplitudes (0.000, 0.275, 0.550, 1.100, 1.650, and 2.100 MPa) ( Figure 14B ). Immunohistochemical analysis was performed 90 min after FUS stimulation to map neuronal firing and neuronal subtypes, and histological analysis was performed 24 h after FUS stimulation to assess safety.

[0130] A pressure-dependent BP decrease was observed 2 h after FUS, from the lowest pressure (0.275 MPa, systolic -19.6 ± 17.8 mmHg, diastolic -19.3 ± 17.6 mmHg) to the highest pressure (2.100 MPa, systolic -36.6 ± 33.4 mmHg, diastolic -34.1 ± 31.8 mmHg) ( Figure 14C and 14D ). BP returned to baseline levels at all ultrasound pressure amplitudes 24 hours after FUS. There was a significant correlation between ultrasound pressure and systolic BP reduction (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 (R2=0.65, p=0.05), indicating that ultrasound-mediated BP reduction is most effective in subjects with high resting BP, as in the case of hypertension. After exposure, no histological signs of hemorrhage, edema, or tissue damage were seen. These data indicate that ultrasound-mediated BP reduction is dose-dependent.

[0131] The above specific embodiments are shown as examples only, and it should be understood that these embodiments can be easily modified and replaced. It should also be understood that the claims are not intended to be limited to the specific forms disclosed, but to cover all modifications, equivalents and alternatives that fall within the spirit and scope of the present disclosure.

[0132] References

[0133] Airan,RDeta / .(2017)'Noninvasive Targeted TranscranialNeuromodulation via Focused Ultrasound Gated Drug Release fromNanoemulsions',Nano letters.2017 / 01 / 17.American Chemical Society,17(2),pp.652-659.doi:

[0134] 10.1021 / acs.nanolett.6b03517.

[0135] Azizi,M.et al.(2021)‘Ultrasound renal denervation for hypertensionresistant to a triple medication pill(RADIANCE-HTN TRIO):a randomised,multicentre,single-blind,sham-controlled trial’,The Lancet.Elsevier,397(10293),pp.2476-2486.

[0136] Basbaum,A.I.and Fields,H.L.(1984)‘Endogenous pain control systems:brainstem spinal pathways and endorphin circuitry’,Annualreview ofneuroscience.Annual Reviews 4139 El Camino Way,PO Box 10139,Palo Alto,CA94303-0139,USA,7(1),pp.309-338.

[0137] Behbehani,M.M.et al.(1990)‘The effect of GABA and its antagonists onmidbrain periaqueductal gray neurons in the rat’,Pain.Elsevier,40(2),pp.195-204.

[0138] Cui,M.et al.(1999)‘Periaqueductal gray stimulation-induced inhibitionof nociceptive dorsal horn neurons in rats is associated with the release ofnorepinephrine,serotonin,and amino acids’,Journal of PharmacologyandExperimental Therapeutics.ASPET,289(2),pp.868-876.

[0139] Davidson,B.et al.(2020)‘Magnetic resonance-guided focused ultrasoundcapsulotomy for refractory obsessive compulsive disorder and major depressivedisorder:clinical and imaging results from two phase I trials’,Molecularpsychiatry.Nature Publishing Group,25(9),pp.1946-1957.

[0140] Elias,W.J.et al.(2016)‘A randomized trial of focused ultrasoundthalamotomy for essential tremor’,New England Journal of Medicine.MassMedical Soc,375(8),pp.730-739.

[0141] Esler,M.et al.(1984)‘Plasma noradrenaline kinetics in humans’,Journalof the autonomic nervous system.Elsevier,11(2),pp.125-144.

[0142] Fenoy,A.J.and Simpson,R..(2014)‘Risks of common complications in deepbrain stimulation surgery:management and avoidance’,Journal of NeurosurgeryJNS.American Association of Neurological Surgeons,120(1),pp.132-139.doi:10.3171 / 2013.10.JNS131225.

[0143] Fomenko,A.et al.(2018)‘Low-intensity ultrasound neuromodulation:Anoverview of mechanisms and emerging human applications’,Brain Stimulation,11(6),pp.1209-1217.doi: https: / / doi.org / 10.1016 / j.brs.2018.08.013.

[0144] FUSfoundation(2021)State of the field report.Available at:

[0145] https: / / www.fusfoundation.org / images / pdf / FUSF State of the Field 2021 Final Web.pdf.

[0146] Green,A.L.et al.(2007)‘Deep brain stimulation:a new treatment forhypertension?’,Journal of clinical neuroscience.Elsevier,14(6),pp.592-595.

[0147] Grimm,M.et al.(1980)‘Norepinephrine clearance and pressor effect innormal and hypertensive man’,Klinische Wochenschrift.Springer,58(21),pp.1175-1181.

[0148] Hakimova,H.et al.(2015)‘Ultrasound stimulation inhibits recurrentseizures and improves behavioral outcome in an experimental model of mesialtemporal lobe epilepsy’,Epilepsy&Behavior.Elsevier,49,pp.26-32.

[0149] Laurent,S.(2017)‘Antihypertensive drugs’,Pharmacologicalresearch.Elsevier,124,pp.116-125.

[0150] Lea-Banks,H.et al.(2020)‘Localized anesthesia of a specific brainregion using ultrasound-responsive barbiturate nanodroplets’,Theranostics,10(6),pp.2849-2858.

[0151] Lea-Banks,H.et al.(2021)‘Ultrasound-sensitive nanodroplets achievetargeted neuromodulation’,Journal of Controlled Release.Elsevier B.V.,332,pp.30-39.doi:10.1016 / j.jconrel.2021.02.010.

[0152] Li,D.et al.(2020)‘Low-intensity focused ultrasound stimulationtreatment decreases blood pressure in spontaneously hypertensive rats’,IEEETransactions on Biomedical Engineering.IEEE,67(11),pp.3048-3056.

[0153] Lunde,A.and Glover,.C.(2020)‘A versatile toolbox for semi-automaticcell-by-cell object-based colocalization analysis’,Scientific reports.NaturePublishing Group,10(1),pp.1-26.

[0154] Martinez-Fernández,R.et al.(2018)‘Focused ultrasound subthalamotomyin patients with asymmetric Parkinson’s disease:a pilot study’,The LancetNeurology.Elsevier,17(1),pp.54-63.

[0155] Matsunaga,T.O.et al.(2012)‘Phase-change nanoparticles using highlyvolatile perfluorocarbons:toward a platform for extravascular ultrasoundimaging’,Theranostics.2012 / 12 / 23.Ivyspring nternational Publisher,2(12),pp.1185-1198.doi:10.7150 / thno.4846.

[0156] Meng,Y.et al.(2021)‘Focused ultrasound neuromodulation’,InternationalReview of Neurobiology.Elsevier,159,pp.221-240.

[0157] Mooney,S.J.et al.(2016)‘Focused ultrasound-induced neurogenesisrequires an increase in blood-brain barrier permeability’,PoS one.PublicLibrary of Science San Francisco,CA USA,11(7),p.e0159892.

[0158] Nelson,L.E.et al.(2002)‘The sedative component of anesthesia ismediated by GA BAA receptors in an endogenous sleep pathway’,Natureneuroscience.Nature Publishing Group,5(10),pp.979-984.

[0159] Ozdas,M.S.et al.(2020)‘Non-invasive molecularly-specific millimeter-resolution manipulation of brain circuits by ultrasound-mediated aggregationand uncaging of drug carriers’,Nature communications.Nature Publishing Group,11(1),pp.1-15.

[0160] Patel,N.K.et al.(2011)‘Deep brain stimulation relieves refractoryhypertension’,Neurology.AAN Enterprises,76(4),pp.405-407.

[0161] Pereira,E.A.C.et al.(2010)‘Sustained reduction of hypertension bydeep brain stimulation’,Journal of Clinical Neuroscience.Elsevier,17(1),pp.124-127.

[0162] Podymow,T.and August,P.(2011)‘Antihypertensive drugs in pregnancy’,inSeminarsin nephrology.Elsevier,pp.70-85.

[0163] Scarcelli,T.et al.(2014)‘Stimulation of hippocampal neurogenesis bytranscranial focused ultrasound and microbubbles in adult mice’,Brainstimulation.Elsevier,7(2),pp.304-307.

[0164] Sharabi,S.et al.(201)‘Non-thermalfocused ultrasound inducedreversible reduction of essential tremor in a rat model’,Brainstimulation.Elsevier,12(1),pp.1-8.

[0165] Sheeran,P.S.et al.(2011)‘Formulation and Acoustic Studies of a NewPhase-Shift Agent for Diagnostic and Therapeutic Ultrasound’,Langmuir.American Chemical Society,27(17),pp.10412-10420.doi:10.1021 / la2013705.

[0166] Sheeran,P.S.et al.(201)‘More Than Bubbles:Creating Phase-ShiftDroplets from Commercially Available Ultrasound Contrast Agents’,UltrasoundinMedicine and Biology.Elsevier,43(2),pp.531-540.doi:

[0167] 10.1016 / j.ultrasmedbio.2016.09.003.

[0168] Stern,J.M.et al.(2021)‘Safety of focused ultrasound neuromodulationin humans with temporal lobe epilepsy’,Brain Stimulation.Elsevier.

[0169] Wang,J.B.et al.(2018)‘Noninvasive Ultrasonic Drug Uncaging MapsWhole-Brain Functional Networks’,Neuron.Cell Press,100(3),pp.728-738.e7.doi:10.1016 / J.NEURON.2018.10.042.

[0170] WHO(2022)World health statistics 2022:monitoring health for the SDGs,sustainable development goals.Available at:

[0171] https: / / cdn.who.int / media / docs / default-source / gho-documents / world- health-statistic-reports / worldhealthstatistics_2022.pdf?sfvrsn=6fbb4d17_3.

[0172] Zhang,M.,Li,B.,Liu,Y.,et al.(2021)‘Different Modes of Low-FrequencyFocused Ultrasound-Mediated Attenuation of Epilepsy Based on the TopologicalTheory’,Micromachines.Multidisciplinary Digital Publishing Institute,12(8),p.1001.

[0173] Zhang,M.,Li,B.,Lv,X.,et al.(202)‘Low-Intensity Focused Ultrasound-Mediated Attenuation of Acute Seizure Activity Based on EEG Brain FunctionalConnectivity’,Brain Sciences.Multidisciplinary Digital Publishing Institute,11(6),p.711.

[0174] Zhang,T.et al.(2022)‘Transcranial focused ultrasound stimulation ofperiaqueductalgray for analgesia’,IEEE Transactions on BiomedicalEngineering.IEEE.

Claims

1. A method for treating hypertension using focused ultrasound, the method comprising, after intravenous injection of nano-droplets loaded with a therapeutic agent: Delivering focused ultrasound to the norepinephrine-producing region of the brain, wherein the focused ultrasound is configured to promote vaporization of the nano-droplets and release of the therapeutic agent.

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

3. The method according to claim 1, wherein the therapeutic agent comprises a lipophilic anesthetic.

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

5. The method according to 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. The method according to any one of claims 1 to 4, wherein the norepinephrine-producing region comprises at least one of the locus coeruleus, thalamus, hypothalamus, neocortex, and cerebellum.

8. A method for treating hypertension using focused ultrasound, the method comprising: Delivering focused ultrasound to the norepinephrine-producing region of the brain, wherein the focused ultrasound is configured to induce repetitive mechanical stress on tissue by forming two or more closely spaced ultrasonic pressure nodes, wherein adjacent nodes are in different acoustic phases, thereby generating a force that stimulates or inhibits neuronal activity between the nodes.

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

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

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

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

13. The method according to claim 8, wherein the focused ultrasound delivery is performed after intravenous injection of nano-droplets loaded with a therapeutic agent to promote vaporization of the nano-droplets and release of the therapeutic agent.

14. The method according to claim 8, wherein the focused ultrasound delivery is combined with an agent such as systemic delivery of an anesthetic, the agent enhancing the effect of the ultrasound on brain tissue.

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