Complex Neuromodulation Techniques

Abstract

Complex neuromodulation techniques are provided. Complex neuromodulation techniques for treating metabolic disorders are disclosed. Complex neuromodulation techniques may include dual-site neuromodulation or single-site neuromodulation. In embodiments, neuromodulation may be administered as a combination therapy with drug treatment.

Description

[Technical Field]

[0001] The subject matter disclosed herein relates to neuromodulation, and more particularly to techniques using complex neuromodulation therapies, including neuromodulation in combination with energy applied from an energy source and / or drug therapy. [Background technology]

[0002] Neuromodulation has been used to treat a variety of clinical conditions. For example, electrical stimulation at various locations along the spinal cord has been used to treat chronic low back pain. Such treatments are performed using implantable devices that periodically generate electrical energy and apply it to tissue, activating specific nerve fibers and resulting in reduced pain sensation. In spinal cord stimulation, stimulating electrodes are typically placed in the epidural space, but a pulse generator may be placed some distance from the electrode, such as in the abdomen or buttocks, and is connected to the electrode via electrical leads. Electrodes placed on or near specific target nerves perform neuromodulation by inducing action potentials in nerve fibers, resulting in neurotransmitter release at neural synapses and subsequent synaptic transmission. This propagation can result in physiological effects that are relatively large or more diffuse than desired, because implanted electrodes stimulate many nerves and axons at once. Because neural pathways are complex and interconnected, more targeted modulation effects may be clinically useful. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] US Patent Application Publication No. 2019 / 0388132 [Brief explanation of the drawings]

[0004] These and other features, aspects, and advantages of the present invention will be better understood from the following detailed description when read in conjunction with the accompanying drawings.

[0005] [Figure 1] FIG. 1 is a schematic diagram of the hypothalamic metabolic control center and its role in integrating hormonal / endocrine and neuronal / sensory information regarding glucose and nutrient availability, according to an embodiment of the present disclosure. [Figure 2] FIG. 1 is a schematic diagram of glucose-excitatory and glucose-inhibitory neuronal populations in the hypothalamus, according to embodiments of the present disclosure. [Figure 3A] FIG. 1 is a schematic diagram of autonomic input from the gut-liver-brain axis to the hypothalamus, according to an embodiment of the present disclosure. [Figure 3B] FIG. 1 is a schematic diagram of autonomic input from the gut-liver-brain axis to the hypothalamus, according to an embodiment of the present disclosure. [Figure 4] FIG. 1 is a schematic diagram of a hybrid neuromodulation technique combining non-invasive ultrasound neuromodulation of a portal vein sensor with additional stimulation to mimic fed-state signaling and induce glucose disposal according to an embodiment of the present disclosure. [Figure 5A] FIG. 1 shows glucose control in an animal model during daily hepatic portal plexus (single site) stimulation. [Figure 5B] The duration of effect in Figure 5A is shown to cease at 3 days with cessation of ultrasound after single-site treatment. [Figure 6] We demonstrate long-term remission in animal models after dual-site stimulation (i.e., hepatic portal plexus and superior mesenteric plexus), and demonstrate that dual-site stimulation with low-dose GLP agonist liraglutide further prolongs remission. [Figure 7] This study compared the duration of remission in an animal model between drug alone and combined neuromodulation, which combines single-site stimulation and drug treatment. [Figure 8A] 1 shows the effect on circulating glucose as a result of complex neuromodulation of dual-site stimulation in late-stage ZDF (Zucker diabetic fatty rats) animals. [Figure 8B]1 shows the effect on circulating glucose as a result of complex neuromodulation of two-site stimulation in late ZDF animals. [Figure 9] Dual-site versus single-site stimulation during metformin treatment in an animal model. [Figure 10A] 1 shows non-fasting blood glucose concentrations in animal models of combined neuromodulation therapy, single site stimulation, and control. [Figure 10B] Plasma GLP-1 concentrations in different single-site stimulation sites / target-alone animal models are shown. [Figure 10C] Circulating insulin concentrations in different single-site stimulation sites / target-alone animal models are shown. [Figure 10D] GABA concentrations in different single-site stimulation sites / target-alone animal models are shown. [Figure 10E] Circulating glucagon concentrations in different single-site stimulation sites / target-alone animal models are shown. [Figure 11] Circulating glucose concentrations during an acute oral glucose tolerance test in animal models of combined neuromodulation therapy, single-site stimulation, and control. [Figure 12] 1 shows non-fasting circulating glucose concentrations at various starting and stopping time points in neuromodulation therapy combination model and control animals. [Figure 13A] Circulating glucose concentrations during an acute oral glucose tolerance test are shown in single-site stimulation and control animal models with nutritional supplements. [Figure 13B] Circulating glucose concentrations during an acute oral glucose tolerance test are shown in single-site stimulation and control animal models with nutritional supplements. [Figure 14] FIG. 1 is a schematic diagram of a neuromodulation system using a pulse generator according to an embodiment of the present disclosure. [Figure 15] FIG. 1 is a block diagram of a neuromodulation system according to an embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0006] One or more specific embodiments are described below. In an effort to provide a concise description of these embodiments, not all features of an actual implementation are described herein. It should be understood that the development of such an actual implementation, like any engineering or design project, involves numerous implementation-specific decisions that will vary from implementation to implementation in order to achieve the developer's particular goals, including compliance with system-related and business-related constraints. Moreover, it should be understood that such a development effort may be complex and time-consuming, but would nevertheless be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill in the art having the benefit of this disclosure.

[0007] The examples or illustrations provided herein are not to be considered limiting, restrictive, or express definitions of any terms or terms with which they are used. Instead, these examples or illustrations are described with respect to various specific embodiments and are to be considered exemplary only. Those skilled in the art will understand that any term or term with which these examples or illustrations are used encompasses other embodiments, whether or not given therewith or elsewhere in this specification, and that all such embodiments are intended to be included within the scope of that term or term. Language designating such non-limiting examples and illustrations includes, but is not limited to, the following: "for example," "for instance," "such as," "including," "in certain embodiments," "in some embodiments," and "in one embodiment."

[0008] Provided herein are combined neuromodulation techniques that involve the application of neuromodulatory energy. The combined neuromodulation techniques produce targeted physiological outcomes (e.g., treatment, prevention, cure, or amelioration of a physiological condition) that are the result of the combined neuromodulation techniques. For example, combined neuromodulation techniques as provided herein can be used to enhance or increase the effectiveness of pharmaceutical or other neuromodulatory therapies and / or to make previously treatment-resistant or non-responsive patients more responsive. In embodiments, combined neuromodulation techniques include neuromodulation therapies in two or more different regions of interest to target different portions of a physiological control pathway. In embodiments, combined neuromodulation techniques include a neuromodulation therapy targeting at least one region of interest and at least one pharmaceutical treatment.

[0009] In embodiments of combined techniques, neuromodulation at a first site (e.g., the first portion of a combined neuromodulation technique) can be used to augment pharmaceutical treatment and / or augment neuromodulation at a second or different site on the patient. Thus, augmentation can allow for modification of pharmaceutical treatment or combined neuromodulation doses to adjust standard or recommended doses. In one example, a patient may be administered a smaller amount of pharmaceutical treatment composition to achieve the same clinical effect compared to the standard dose administered in the absence of neuromodulation. Therefore, lower dosages of the therapeutic composition may result in lower treatment costs. Furthermore, patients may be exposed to lower doses of certain drug regimens, resulting in a reduced risk of side effects and the ability to administer a particular drug regimen for a longer period of time. In another example, neuromodulation energy at one or more target sites may be adjusted (e.g., decreased) based on the augmentation of the combined treatment, such that less neuromodulation energy is applied to specific therapeutic targets within the region of interest to achieve a desired physiological outcome.

[0010] Augmentation may be bidirectional, such that the first part of the combined neuromodulation technique enhances the clinical effect of the second part of the combined neuromodulation technique, or vice versa. In one embodiment, neuromodulation is provided to a patient who is also receiving pharmaceutical treatment to enhance one or both of the clinical effect of the pharmaceutical treatment or the effect of the neuromodulation. For example, administration of pharmaceutical treatment can activate or alter the presence or level of molecules or cells in the patient that are associated with successful neuromodulation (e.g., successful application of neuromodulatory energy to a patient's region of interest to cause a physiological effect). Thus, pharmaceutical treatment may act to enhance neuromodulation or improve a patient's response to neuromodulation. Similarly, neuromodulation at one or both sites of a two-site treatment protocol may serve to enhance the clinical effect at the other site.

[0011] Ultrasound can stimulate peripheral nerve fields (both efferent and afferent nerve fields) in and around organs, including the spleen (which activates nerves that regulate immune cells and immune system function) and the liver (which activates nerves that regulate glucose / nutrient sensing and metabolic system function). Provided herein, in embodiments, is a technique for improving the antidiabetic effects of ultrasound therapy, with the unexpected result of providing long-term remission of type 2 diabetes, even in the genetic Fatty Diabetic Zucker (ZDF) rodent model. In embodiments, the technology involves dual stimulation treatment of the hypothalamic metabolic control center using both ultrasonic neuromodulation of the sensory field (i.e., hepatic / hepatoportal plexus neuromodulation of the ascending glucose sensor afferent pathway) and ultrasonic neuromodulation of the neuroendocrine field from the gastrointestinal tract (i.e., stimulation of glucagon-like peptide (GLP) secretion and related hormones and afferent pathways). In embodiments, the combined neuromodulation technique involves ultrasonic neuromodulation of the sensory field (i.e., hepatic / hepatoportal plexus neuromodulation of the ascending glucose sensor afferent pathway) and pharmaceutical activation of endocrine targets (i.e., liraglutide; GLP agonist). In embodiments, the complex neuromodulation technique includes targeting the superior mesenteric plexus, the inferior mesenteric plexus, and / or the fundus of the stomach as targets of neuromodulation energy.

[0012] The disclosed portions of the compound neuromodulation technique may be administered simultaneously, or different portions of the combination may be administered temporally separated. In one embodiment, different portions of the combination may be applied within 30 minutes of each other or within 60 minutes of each other. In one embodiment, different portions of the combination may be applied at least 5 minutes apart. Provided herein are specific time points between dual-site stimulation that provide temporally separated neuromodulation of the liver versus the gastrointestinal tract, enabling a synergistic two-site effect. The disclosed timing is designed to coincide with the afferent signaling that would occur during feeding to achieve the unique results shown (i.e., long-term diabetes remission). Thus, as provided herein, compound neuromodulation treatments can be tailored to the glucose kinetics induced by the initial energy application of compound neuromodulation. In one embodiment, the second or subsequent treatment is applied or administered within the first 40-60 minutes of the higher glucose period. In one embodiment, the second or subsequent treatment is applied or administered after the first 40-60 minutes of the lower glucose period. The different parts of the complex neuromodulation may be part of a treatment administered together in one patient visit.

[0013] It should be understood that a compound neuromodulation technique may be part of a patient treatment regimen that includes multiple compound neuromodulations administered on different days, weeks, or months. Compound neuromodulation as disclosed herein may be altered or adjusted over the course of a treatment regimen based on the patient's progression or clinical condition. Adjustments may include adjustments to energy application parameters and / or drug dosages.

[0014] Unexpected benefits of combining neuromodulation techniques include long-term remission in diabetic animal models using the two dual-modulation techniques described above. Furthermore, ultrasound activation of hepatic regions (i.e., the hepatoportal nerve plexus) has been demonstrated using lower ultrasound powers than previously demonstrated. This low-power activation is achieved by stimulating hepatic regions for a longer duration / stimulation time than previously described high-power activation / neuromodulation.

[0015] Embodiments of the disclosed complex neuromodulation techniques include pharmaceutical therapy and neuromodulation techniques for treating patients with metabolic disorders. Particular embodiments of the present disclosure are discussed in the context of blood glucose regulation. Figure 1 is a schematic diagram of the hypothalamic metabolic control center, illustrating its role in integrating hormonal / endocrine and neuronal / afferent sensory information regarding glucose and nutrient availability. Specific nuclei within the hypothalamus function in glucose homeostasis and may mediate a systemic glucose "set point" (i.e., target blood glucose concentration) based on sensory information in conjunction with peripheral effector tissues (e.g., liver, intestine, muscle, pancreas, etc.). Furthermore, the importance of this region of the brain in integrating sensory information from both hormonal / endocrine and nerve / autonomic inputs is supported by the presence of cellular / neuronal populations in this region that contain hormone and neurotransmitter receptors and monitor both the hormonal and autonomic environment through integration with the circulatory blood (at the median eminence of the hypothalamus and circumventricular organs) and direct and indirect connections to afferent peripheral sensory nerves.

[0016] Figure 2 shows a schematic diagram of glucose-excited and glucose-inhibited neuronal populations in the hypothalamus. Populations of sensory neurons that increase or decrease their firing rates based on current glucose concentrations are components of the glucoregulatory system. Cell populations within the arcuate nucleus (ARC) of the hypothalamus (i.e., a region located near the median eminence, or blood access) have incoming and outgoing sensory neuronal innervation (direct and indirect from other nuclei) and efferent outputs to major metabolic control centers, such as the paraventricular nucleus (PVN). POMC (pro-opiomelanocortin) / CART (cocaine- and amphetamine-regulated transcript) neurons are associated with generalized appetite reduction (and appetite suppression / satiety), whereas NPY (neuropeptide Y) / AgRP (agouti-related peptide) neurons are associated with appetite stimulation (and appetite enhancement). This neuronal population has acute effects on glucose disposal versus glucose uptake, as shown in Figure 2. Furthermore, these neurons are also associated with inhibitory GABA (gamma aminobutyric acid) neurons, which function as inhibitory inputs to the NPY / AgRP neurons.

[0017] Figures 3A-3B show autonomic input from the gut-liver-brain axis to the hypothalamus. Figure 3A is a schematic diagram of intestine / liver portal-mesenteric nutrient sensing. The hepatoportal plexus contains a set of afferent neurons that communicate with the hypothalamus. These neurons are associated with the portal vein and hepatic artery and change their firing rate based on the glucose gradient between the portal vein and peripheral blood supply to the liver (e.g., a meal is associated with an increase in glucose from the intestine or an increase in glucose concentrations in the portal vein and hepatic artery). Stimulating this site and activating these neurons has profound effects in animal models of diabetes, e.g., under daily stimulation conditions.

[0018] However, as shown in Figure 3B, the function of this hepatic portal plexus depends on more than the direct detection of "food" (i.e., glucose) in the portal vein. One interaction is with the gut incretins and the autonomic sensory system. In addition to altering the firing rate of hepatic glucose sensors, the ingestion of food and the presence of nutrients in the gastrointestinal tract trigger the secretion of incretins (and other hormones) from the cells lining the gastrointestinal tract wall. These hormones serve multiple functions: 1) alter gastric motility and emptying (potentially altering the kinetics / duration of portal glucose sensor firing); 2) modulate the firing rate / signaling of gut hormone-dependent sensory neurons in the gut (which are also regulated by nutrient receptors and are known to project to the hypothalamus); and 3) spread to the circulatory system, altering hypothalamic function via the median eminence / circumventricular organs via the blood rather than via direct neural pathways.

[0019] Figure 4 is a schematic diagram of a compound neuromodulation technique involving ultrasound neuromodulation of portal sensors and additional stimulation (which could be another ultrasound neuromodulation or drug therapy at a different target). Dual-site or dual-stimulation neuromodulation results in diabetic "remission" or long-term glycemic control even after stimulation is stopped, an unexpected result of embodiments of the present disclosure. There are multiple pathways that are activated during feeding and cause metabolic changes (and are necessary for healthy glucose regulation). As a non-invasive technique, the ultrasound neuromodulation technique as part of compound neuromodulation can be used to stimulate or induce multiple pathways. This can be done by stimulating more than one pathway with ultrasound (i.e., stimulating both the portahepatic and a gastrointestinal site, as described above) or by combining ultrasound stimulation of one pathway (i.e., neurotherapy) with a drug (i.e., a hormone or receptor pathway). Furthermore, such "dual stimulation" can be timed and spaced apart to mimic "natural" signaling during feeding and eating.

[0020] Example The following sections provide experimental parameters and results for several aspects of the examples provided herein.

[0021] Materials and Methods 1.1MHz Single Element Focused Ultrasound System The 1.1 MHz single-element focused ultrasound system included a signal 5 generator (model 33120A, Agilent Technologies Inc., Santa Clara, CA), an RF power amplifier (model 350L, Electronics & Innovation Ltd., Rochester, NY), and a 1.1 MHz focused single-element ultrasound transducer (model H102, Sonic Concepts Inc.). The transducer was coupled to the animal via a matching network (Model H102, Sonic Concepts Inc.). The transducer element was 64 mm in diameter and had a 63.2 mm radius of curvature, with a 20 mm diameter hole in the center into which a miniature imaging transducer could be inserted for image guidance. The transducer was acoustically coupled to the animal via a 6 cm tall plastic standoff cone filled with degassed water.

[0022] The nominal settings for the waveform are shown below. Carrier frequency -1.1MHz 135mVpk-pk signal generator amplitude 150 microsecond pulse length 5Hz pulse repetition frequency Maximum negative pressure = 0.78 MPa Stimulation time = 3 min total (per anatomical site) Pulse average intensity (Isppa) = 23 W / cm 2 Time average intensity (Ispta)=6,263mW / cm 2 MI=0.6 TI=0.7

[0023] The acoustic performance of the systems was characterized in an ISO / IEC 17025:2017 accredited laboratory (Acertara Acoustic Labs, Longmont, CO). Under the above setup, three separate 20 systems were characterized, resulting in an average mechanical index (MI) of 1.79 (±0.10 standard deviation), a reduced peak negative pressure (PR) of 1.87 (±0.11) MPa, and a power rating of 125.7 (±15.0) W / cm. 2 The spatial peak pulse average intensity Isppa.3 was 94.3 (±11.2) mW / cm 2 A spatial-peak temporal-averaged intensity (Ispta) of 3 was obtained. Good linearity (0.9947 R2) of the MI was observed over the amplitude range of 100 mV pk-to-pk to 200 mV pk-to-pk, so the acoustic power can be reasonably linearly extrapolated to other amplitudes, pulse durations, and pulse repetition frequencies. The simulated pressure profile had a full-width half-maximum amplitude of 1.8 mm laterally and 6 mm in depth using Field II. The peak's -6 dB diameter was measured in a certified laboratory to be 1.45 (±0.02) mm in the X-scan axis and 1.46 (±0.02) mm in the Y-scan axis.

[0024] Pre-neuromodulation ultrasound scans were performed using a Vivid E9 ultrasound system (GE Healthcare™) or an 11L probe (GE Healthcare™). The imaging beam of the probe was aligned with the ultrasound stimulation beam, allowing for confirmation of the ultrasound beam hitting the area of ​​interest using images of the targeted organ / lymph node (visualized on the Vivid E9).

[0025] The following parameters are for an alternative handheld ultrasound system used for low-power (e.g., trickle) neuromodulation experiments. GE V-scan handheld ultrasound system (GE Healthcare trademark). Setting #117 - Abdominal / Deep probe preset, unit set to color mode (MI: 1.0, Tis: 1.2). The depth of focus at this setting was estimated to be 4.1 cm. Compared to the prototype stimulators described above, these settings provide lower power stimuli. Carrier frequency -1.8MHz PRF=68Hz Duty cycle = 0.0048 Isppa (peak sound intensity) = 200 W / cm 2 Ispta (average sound intensity) = 10mW / cm 2 Stimulation time - 20 minutes (per anatomical site)

[0026] The following describes the diabetic animal model and the ultrasonic neuromodulation method used. Type 2 diabetes animal model Zucker diabetic fatty (ZDF) rat model Adult male ZDF rats (Charles River, Kingston, NY, USA) were ordered to arrive before 8 weeks of age. All animals were fed a high-calorie rodent diet (Purina 5008) and had free access to water. All rats were housed at 25°C with a 12-hour light-dark cycle and were acclimated to handling for at least 1 week prior to conducting experiments to minimize potential confounding of glucose measurements by stress responses. All procedures were performed in accordance with National Institutes of Health (NIH) guidelines under protocols approved by the GE Global Research™ Institutional Animal Care and Use Committee (IACUC). ZDF animals were acclimated to handling daily to prevent stress-induced changes in circulating glucose. After 8 weeks, ZDF rodents began to show rapid development of the diabetic phenotype and were then divided into sham-CTRL (control) or pFUS (peripheral focused ultrasound) treatment groups and subjected to acute or chronic ultrasound stimulation.

[0027] All rats were anesthetized with 1-4% isoflurane at 1 L / min O2. The rats were then placed on a water-circulating heating pad, and body temperature was maintained with a rectal thermometer probe. The abdomen was completely shaved, and the hair was completely removed with a nail to allow for subsequent stimulation of nerve plexuses in the hepatic or digestive system. The locations of the hepatic hilum, fundus, superior mesenteric nerve, and inferior mesenteric nerve were identified using a custom-made ultrasound imaging device (Vivid E9; GE Healthcare: trademark). These locations were then marked with a permanent marker, and the ultrasound stimulation probe was applied to the target areas.

[0028] Single-site stimulation conditions: After identifying targets in the liver / portal vein, fundus, superior mesenteric plexus, or inferior mesenteric plexus, one of the following designated ultrasound treatments was performed using either the pFUS prototype device or a handheld ultrasound device: The prototype pFUS device then delivered 3 min of stimulation (1.1 MHz, 150 burst cycles, 500 μs burst period). Stimulation was performed for 20 minutes using a portable ultrasound device at low power settings (1.8 MHz, pulse repetition frequency 68 Hz, duty cycle 0.0048%).

[0029] Blood glucose levels were monitored daily in the chronic study and at 5-minute intervals in the acute and OGTT studies. In the chronic study, additional blood samples were taken weekly for analysis of circulating markers. A final blood sample was taken at the time of euthanasia and used to assess circulating insulin levels.

[0030] Dual-site stimulation condition: After single-site stimulation testing, combinations of lead target sites identified in the single-site stimulation testing (portal region, superior mesenteric nerve plexus, etc.) were tested in combination during a single session.

[0031] With the prototype pFUS device, stimulation (1.1 MHz, 200 mV per pulse, 150 burst cycles, 500 μ burst period) was applied to each target site for 3 minutes. For example, pFUS was first applied to the hepatic portal vein region for 3 minutes. Immediately after pFUS stimulation to the hepatic portal vein region, the probe was moved to the superior mesenteric nerve region and a second pFUS was applied. The application of pFUS to the liver and superior mesenteric regions was varied daily, alternating the initial stimulation site (e.g., day 1: liver pFUS -> superior mesenteric pFUS, day 2: superior mesenteric pFUS -> liver pFUS, etc.). There was a delay period of approximately 5 minutes between stimulation of the first and second, or second and third, anatomical target sites.

[0032] The handheld ultrasound device applied the above-mentioned low-intensity stimulation to each region for 20 minutes per target. As with the prototype pFUS device, the handheld ultrasound device was applied first to one target, then to the second target, with targets alternated daily (e.g., Day 1: liver pFUS -> superior mesenteric pFUS, Day 2: superior mesenteric pFUS -> liver pFUS, etc.). However, with the handheld device, a 20-minute rest period was used between target stimulations to somewhat reduce heating of the handheld device, which could confound results.

[0033] Blood samples were collected from the tail vein using a Freestyle Freedom Lite (Abbott Diabetes Inc.). Because the Freestyle Freedom Lite meter uses a small volume of blood (0.3 μL), no additional transfusion was required to restore total volume after blood collection.

[0034] Rats were anesthetized, had a tail vein catheter placed, and were fasted overnight (12–16 h) before baseline blood samples were collected. Immediately after baseline collection, a single pFUS stimulation was performed, and the animals were allowed to recover from anesthesia. After recovery, a single 2 g / kg glucose solution was administered orally via oral syringe. After glucose administration, blood samples were collected every 5 minutes via catheterization from the tail vein. All procedures were performed in accordance with the GE Global Research™ Institutional Animal Care and Use Committee (IACUC). AUC-OGTT (oral glucose tolerance test) was calculated by considering the area under the curve (AUC) determined from the glucose concentrations at baseline and 120 minutes after glucose overload.

[0035] The disclosed neuromodulation techniques include the administration of a pharmaceutical treatment to a patient. The pharmaceutical treatment can be any compound, agent, drug, treatment, or other therapeutic dose, regimen, or protocol that has a desired therapeutic or clinical effect when combined with the neuromodulation techniques. The pharmaceutical treatment can include, by way of example, one or more of oral, parenteral, intravenous, topical, inhaled, or mucosally administered drugs.

[0036] The combination of pFUS therapy with pharmaceutical intervention was tested to determine the effect of dual therapy on treatment efficacy, as well as duration of efficacy. Using the human dose-response relationships for metformin and liraglutide, the least effective dose (less than or equal to efficacious dose affecting, ED20) was selected. This dose (ED20) represents the dose that produces a quantitative effect (all or nothing) in 20% of the population treated, representing a typical low-dose option.

[0037] Metformin (33-100 mg) was administered orally twice daily, 12 hours apart, in sterile water to avoid taste aversion. This administration regimen (timing) is identical to that used in humans, and was administered before breakfast and dinner meals.

[0038] Liraglutide (20 μg-50 μg) was formulated for parenteral injection with 1.42 mg disodium phosphate dihydrate, 14 mg propylene glycol, 5.5 mg phenol, and water and administered subcutaneously once daily in the morning in all cases, taking into account the long plasma half-life of liraglutide (approximately 15 hours).

[0039] Figure 5A shows glucose control in an animal model during daily hepatic portal plexus (single-site) stimulation. Figure 5B shows the duration of the effect of Figure 5A following cessation of ultrasound at 3 days. Daily stimulation is adequate to prevent or reverse hyperglycemia in the ZDF model, as shown in Figure 5A. However, as shown in Figure 5B, upon cessation of ultrasound treatment, hyperglycemia returns in only 3 days. Data related to stimulation of a naive / non-diabetic cohort and sham control (i.e., ultrasound probe placement but no stimulation / ultrasound energy delivery) also demonstrate the gradual rise in blood glucose (i.e., diabetes progression) expected in an untreated ZDF model. Thus, the ultrasound-mediated control of circulating glucose demonstrated in Figure 5A returns to an uncontrolled state after cessation of the ultrasound stimulation dose, as shown in Figure 5B, while a separate group using focused ultrasound demonstrates a decrease in circulating glucose.

[0040] Figure 6 shows longer-term remission in this animal model after dual-site stimulation (i.e., hepatic portal plexus and superior mesenteric plexus, results shown in Data 100) and even longer-term remission when dual-site stimulation was additionally used with the low-dose GLP agonist liraglutide (results shown in Data 102). The results demonstrate that dual-site stimulation (i.e., stimulation of both the hepatic portal plexus and superior mesenteric plexus) further reduces blood glucose (after 7 days of daily stimulation). Furthermore, after 7 days of treatment, ultrasound stimulation was stopped for the remainder of the experiment (day 8), and mean blood glucose levels remained below 200 mg / dL for 36 days (without additional treatment) after ultrasound cessation. This is one of the longest remission periods reported in the ZDF model.

[0041] Furthermore, when dual-site stimulation was performed in addition to low-dose treatment with the GLP agonist liraglutide (i.e., a drug that targets effector tissues related to the superior mesenteric plexus stimulus location; 102), the duration that ZDF animals maintained blood glucose levels below 200 mg / dL was further extended (again, after ultrasound was stopped on day 8; blood glucose levels remained below 200 mg / dL for 56 days post-ultrasound).

[0042] Figure 7 compares the duration of remission in an animal model of neuromodulation using drug monotherapy and single-site stimulation combined with drug therapy. In contrast to the dual therapy results shown in Figure 6, Figure 7 demonstrates that drug monotherapy (i.e., low-dose liraglutide; data shown in 200) or drug therapy plus a single hepatic portal plexus stimulation treatment (i.e., portal site-only ultrasound for 7 days, followed by drug alone from day 8 onward; data shown in 202) did not result in the same long-term remission. Even with continued liraglutide treatment (data 200), blood glucose rose to above 200 mg / dL within 13 days (after ultrasound). Single-site stimulation (i.e., 7 days of hepatic portal plexus stimulation alone) improved the duration below 200 mg / dL to 21 days, but did not result in long-term remission (i.e., potentially longer than 4 weeks of remission in the ZDF model). It should be understood that, in one embodiment, the disclosed therapeutic effects may be achieved by administering two or more drugs.

[0043] Figures 8A-8B show the effects on circulating glucose as a result of combined neuromodulation of dual-site stimulation in late-stage ZDF animals. In a separate experiment, dual-site ultrasound stimulation treatment (i.e., hepatic portal plexus and superior mesenteric plexus stimulation) was performed in a late-stage ZDF cohort (in which mean blood glucose had already reached >400 mg / dL). The few treatments that previously demonstrated long-term remission in the ZDF model (i.e., 4-week remission after FGF1 injection into the intracerebroventricular space or the ventricle below the arcuate nucleus) were ineffective in ZDF animals already well into the disease progression (i.e., mean glucose >300 mg / dL). However, as shown in Figure 8A, 7 of 11 animals treated with ultrasound stimulation responded with a 30-day remission (glucose levels below 200 mg / dL) after 5 days of daily ultrasound stimulation (ultrasound treatment was subsequently discontinued on day 6). Interestingly, 4 of the animals in this late-stage cohort, shown in Figure 8B, showed elevated blood glucose levels by day 8 after ultrasound treatment was discontinued and continued to progress to hyperglycemia despite receiving additional 1-day "bolus" dual-site stimulation on days 17, 27, and 34 (even though each stimulation resulted in a transient decrease in blood glucose levels). This data suggests that long-term remission with combined neuromodulation treatment may be associated with intact insulin signaling.

[0044] Figure 9 shows dual-site versus single-site stimulation during metformin treatment in an animal model. In an additional experimental cohort, single-site and dual-site stimulation treatments (again, ultrasound stimulation was administered for 7 days, then discontinued for the remainder of the experiment) were performed with or without the administration of low-dose antidiabetic drug metformin (i.e., an alternative, non-GLP pathway diabetes medication). Data 300 from the dual-site stimulation study again demonstrates the achievement of long-term remission (i.e., greater than 4 weeks) after ultrasound cessation in the dual ultrasound stimulation (no drug) cohort. Dual-site stimulation consisted of stimulation of the liver (hepatoportal plexus) and gastrointestinal tract (superior mesenteric plexus). However, adding low doses of metformin to this cohort did not result in prolonged remission (similar to the use of GLP agonists in Figure 6; data 302). The metformin-alone cohort, shown in data 304, temporarily reduced blood glucose levels to below 200 mg / dL, but was not as effective as the liraglutide-alone cohort (in terms of absolute reduction in blood glucose or length of time below 200 mg / dL). Similarly, the metformin + single-site (hepatic portal plexus) stimulation group, shown in data 306, was not as effective as the single-site + liraglutide cohort in terms of time to blood glucose reduction, as shown in Figure 6.

[0045] Figures 10A–E show data from dual-site, multi-site, and single-site ultrasound stimulations. In Figure 10A, blood glucose was monitored over 14 days following the dual-site treatment described above (i.e., hepatic portal plexus stimulation and superior mesenteric plexus stimulation; shown as data 400), multiple single-site stimulations (i.e., hepatic portal plexus / liver (shown as data 402) alone, or superior mesenteric plexus / liver (shown as data 403) alone), or triple stimulation (i.e., hepatic portal plexus / liver, superior mesenteric plexus / GI, and direct stimulation of the pancreas). Previous reports have shown that direct stimulation of the pancreas increases insulin secretion (without affecting glucagon).

[0046] Stimulation of the liver and GI sites rapidly reduced blood glucose in the ZDF model cohorts to values ​​below 200 mg / dL (shown as data 404). The addition of a third site (pancreas; shown as data 406) did not result in any additional benefit or reduction in blood glucose levels. Both single-site stimulation treatments (i.e., liver or GI stimulation; 402, 404) resulted in a slower reduction in blood glucose (compared to dual stimulation treatments) that did not achieve the same reduction in glucose on day 14. ZDFs receiving a sham control / probe placement without ultrasound did not show any reduction in glucose levels. Figure 10B shows plasma GLP-1 concentrations in an animal model with single-site stimulation alone. Figure 10C shows circulating insulin concentrations in an animal model with single-site stimulation alone. Figure 10D shows GABA concentrations in an animal model with single-site stimulation alone. Figure 10E shows circulating glucagon levels in an animal model with single-site stimulation alone. As shown in Figures 10B-E, pancreatic stimulation increased blood insulin levels (Figure 10C), and GI stimulation increased GLP levels (Figure 10B) and GABA levels (Figure 10D).

[0047] Figure 11 shows circulating glucose levels during an acute oral glucose tolerance test in an animal model combining neuromodulation therapy, single-site stimulation, and control. In the ZDF model, liver stimulation alone (shown in data 500; portal region stimulation) resulted in improved glucose regulation compared to sham control (shown in data 502). That is, glucose levels decreased after 40 minutes during the oral glucose tolerance test. Surprisingly, none of the gastrointestinal (GI) sites showed improvement during these short-term / acute OGTT tests, and each different GI site exhibited a different effect on the OGTT glucose curve. That is, single-site stimulation of the superior mesenteric plexus, inferior mesenteric plexus (shown in data 503), or gastric fundus elevated glucose levels during the first 40 minutes of the OGTT compared to sham control. Furthermore, the two mesenteric sites had higher glucose levels throughout the test (up to 200 minutes). Interestingly, with dual stimulation of the superior mesenteric plexus and portal vein sites, OGTT curve kinetics were similar to those of a single GI site for the first 40 minutes, but similar to those of a single liver site between 40 and 200 minutes. While the long-term benefits of dual-site stimulation in the above figures are clear and profound (i.e., long-term remission in the ZDF model), the additional benefits of GI stimulation are less readily apparent in acute OGTT tests. A further surprising result is the similar kinetics between dual-site stimulation (shown in data 504) and gastric fundus stimulation (shown in data 506), suggesting that a single autonomic nerve stimulation site may produce results similar to a "mixed" or dual-site stimulation treatment. It should be understood that, in embodiments, the disclosed remission results can be achieved with single-site stimulation. In one embodiment, single-site stimulation of gastrointestinal tissue can induce remission of metabolic disorders. In one embodiment, single-site stimulation can be the combined duration of dual-site stimulation as disclosed herein.

[0048] Figure 12 shows non-fasted circulating glucose concentrations in animal models with neuromodulation therapy and controls at various start and stop time points. The data in Figures 5-11 are from a relatively high power level (i.e., 6,263 mW / cm). 2 ), which is far below the levels associated with cavitation and / or tissue damage. Figure 12 shows the power dissipation achieved using a handheld / battery-powered ultrasound system (i.e., ~10 mW / cm). 2 We have shown that ultrasound stimulation at much lower power levels, using the GEVscan™ (described above), can also produce neuromodulatory results. However, in this cohort (low-power stimulation), each anatomical site was treated / stimulated for 20 minutes (i.e., 40 or 20 minutes per site for dual-site treatments, and 60 minutes for triple-site treatments, 20 minutes per site). In all previous high-power stimulation studies, stimulation time per site was only 3 minutes. Also, in this cohort, single-site stimulation was initiated on day 0, GI sites were added for dual-stimulation treatment on day 11, and pancreatic sites were added on day 25. As shown above, the addition of multi-site stimulation resulted in further reductions in glucose compared to single-site stimulation alone. All ultrasound treatments were discontinued on day 32. In this cohort, blood levels were very high at baseline (even higher than in Figure 8), and remission after cessation of ultrasound treatment lasted approximately 15 days.

[0049] Figure 13A shows circulating glucose concentrations during an acute oral glucose tolerance test in animal models with single-site stimulation with nutrient support and controls and controls. Figure 13B shows circulating glucose concentrations during an acute oral glucose tolerance test in animal models with single-site stimulation with nutrient support and controls and controls. As shown in Figure 13A, liver-only stimulation (shown as data 600; portal vein stimulation) resulted in improved glucose regulation compared to sham controls (shown as data 602). That is, glucose levels decreased after 40 minutes during the oral glucose tolerance test. While neither gastrointestinal (GI) site showed improvement during these short-term / acute OGTTs when administered alone, the combination of GI stimulation with a single oral dose of omega-3 fat solution produced OGTT curve kinetics similar to those of a single GI site during the first 40 minutes, but similar to those of a single liver site during the period between 40 and 200 minutes. Furthermore, the combination of omega-3 fat solution with single-site GI stimulation produced an OGTT curve (Figure 13B) identical to dual-site stimulation (liver and GI), suggesting that this single autonomic stimulation site combined with nutritional feeding may produce similar results as a "mixed" or dual-site stimulation procedure. Neuromodulation in Complex Neuromodulation Methods

[0050] The neuromodulation of one or more regions of interest (e.g., single-site or multi-site) provided herein allows for the local and nonablative application of energy only to the targeted region or region of interest (e.g., stimulation site or sites) and not outside the region or region of interest. Energy application may induce downstream effects outside the targeted region of interest, for example, in the same organ, tissue, or structure containing the region of interest, or in other organs and structures that do not contain the targeted region of interest. In some embodiments, downstream effects may be induced in the hypothalamus, for example. Energy application may also induce effects along the target nerve upstream from the energy application site. In some embodiments, effects outside one or more regions of interest can be achieved without applying energy directly to a region outside the one or more regions of interest where the downstream or upstream effect is induced. Thus, local energy application can be used to achieve or achieve systemic effects, which may include local, downstream, and / or upstream effects. The targeted region or region of interest may be any tissue or structure in the body with axon terminals that form synapses with non-neuronal cells or body fluids. In one example, the region of interest may be a subregion of an organ or structure such as the spleen, liver, pancreas, gastrointestinal tissue, etc. In another example, the region of interest may be in lymphatic tissue.

[0051] Neuromodulation of a targeted region may alter physiological processes, interrupting, decreasing, or augmenting one or more physiological pathways in a subject to produce a desired physiological outcome. Furthermore, because localized energy application can produce systemic changes, different physiological pathways may be altered in different ways and at different locations within the body, resulting in an overall characteristic profile of physiological changes in the subject that is induced by and characteristic of targeted neuromodulation for a particular subject. While these changes are complex, the neuromodulation techniques of the present invention provide the treated subject with one or more measurable targeted physiological outcomes that are the result of neuromodulation and may not be achievable without the application of energy to the targeted region or other interventions. Furthermore, other types of interventions (e.g., drug treatments as part of complex neuromodulation) may facilitate or enhance the physiological changes induced by neuromodulation.

[0052] The complex neuromodulation techniques discussed herein can be used to cause the physiological outcome of a change in the concentration (eg, increase, decrease) of a molecule of interest and / or a change in the properties of the molecule of interest. That is, selective modulation of one or more molecules of interest (e.g., a first molecule of interest, a second molecule of interest, etc.) controls the concentration (circulation, tissue) or properties (covalent modification) of the molecules as a result of application of energy to one or more regions of interest (e.g., a first region of interest, a second region of interest, etc.) in one or more tissues (e.g., a first tissue, a second tissue, etc.). Modulation of a molecule of interest can include changes in molecular properties, such as expression, secretion, translocation of proteins, and direct activity changes based on ion channel effects, either resulting from energy application itself or as a result of molecules directly affecting ion channels. Modulation of a molecule of interest can also refer to maintaining a desired concentration of a molecule without the concentration changes or fluctuations that would be expected as a result of neuromodulation. Modulation of a molecule of interest can also refer to enzyme-mediated covalent modifications (e.g., phosphorylation, acetylation, ribosylation, etc.). It can also refer to making a change in a molecular property, such as a change in the activity of a molecule (e.g., a change in the activity of a molecule). That is, it should be understood that selective modulation of a molecule of interest can refer to molecular concentration and / or molecular property. The molecule of interest can be a biological molecule, such as one or more of a carbohydrate (monosaccharide, polysaccharide), lipid, nucleic acid (DNA, RNA), or protein. In certain embodiments, the molecule of interest can be a signaling molecule, such as a hormone (an amine hormone, a peptide hormone, or a steroid hormone).

[0053] Certain embodiments described herein provide complex neuromodulation techniques that induce targeted physiological outcomes for the treatment of glucose metabolism and related disorders. Glucose regulation is complex and involves distinct local and systemic metabolic pathways. Application of energy to a target region / region of interest induces characteristic changes in these metabolic pathways to improve glucose regulation. In some embodiments, modulation in one or more regions of interest can be used to treat disorders including, but not limited to, diabetes (i.e., type 1 or type 2 diabetes), hyperglycemia, sepsis, trauma, infection, physiologic stress, diabetes-associated dementia, obesity, or other eating or metabolic disorders. In some embodiments, neuromodulation can be used to promote weight loss, control appetite, treat cachexia, or increase appetite. In one example, physiological stress is medically defined and can include various acute medical conditions (infection, severe injury / trauma, heart attack, bypass) as well as surgical cases that present with hyperglycemia. For example, direct stimulation of the pancreas increases appetite, while direct stimulation of the liver decreases NPY and promotes satiety signals. The desired physiological outcome can include regulating a subject's circulating (i.e., blood) glucose concentration to within a desired range related to normal glucose levels and avoiding hyperglycemia or hypoglycemia. In this manner, selective modulation of a molecule of interest can be achieved.This regulation is thought to be the result of inducing changes in glucoregulatory hormones in blood or tissues via targeted neuromodulation to produce desired glucose concentrations (i.e., desired glucose endpoints). Furthermore, glucose regulation is beneficial for healthy patients who have not been diagnosed with disease but who are prediabetic or wish to maintain a healthy weight.

[0054] To that end, the disclosed neuromodulation techniques can be used in conjunction with a neuromodulation system. FIG. 14 is a schematic diagram of a system 1010 for neuromodulation to achieve neurotransmitter release and / or activation of synaptic components (e.g., presynaptic cells, postsynaptic cells) in response to the application of energy. The depicted system includes a pulse generator 1014 coupled to an energy application device 1012 (e.g., an ultrasound transducer). The energy application device 1012 is configured to receive energy pulses, e.g., via a lead or wireless connection, which, during use, are directed to a region of interest in a subject's internal tissue or organ, resulting in a targeted physiological outcome. In certain embodiments, the pulse generator 1014 and / or energy application device 1012 can be implanted in a biocompatible site (e.g., the abdomen), with leads or leads internally coupling the energy application device 1012 and pulse generator 1014. For example, the energy applicator 1012 may be a MEMS transducer, such as a capacitive micromachined ultrasound transducer.

[0055] In certain embodiments, the energy application device 1012 and / or the pulse generator 1014 may communicate wirelessly with a controller 1016, which may, for example, in turn provide instructions to the pulse generator 1014. In other embodiments, the pulse generator 1014 may be an extracorporeal device, e.g., operable to apply energy transdermally or non-invasively from a location outside the subject's body, and in certain embodiments may be integrated within the controller 1016. In embodiments in which the pulse generator 1014 is extracorporeal, the energy application device 1012 may be operated by a caregiver and positioned at or above the subject's skin to deliver energy pulses transdermally to desired internal tissues. Once positioned to apply energy pulses to the desired site, the system 10 can initiate neuromodulation to achieve a targeted physiological result or clinical effect.

[0056] In certain embodiments, system 10 may include an evaluation device 1020 coupled to controller 1016 that evaluates a characteristic indicative of whether a target physiological outcome of the modulation has been achieved. In one embodiment, the target physiological outcome may be local. For example, modulation may result in a local tissue or functional change, such as a change in tissue structure, a local change in the concentration of a particular molecule, tissue displacement, increased fluid movement, etc.

[0057] The modulation may result in systemic or non-local changes, and the desired physiological outcome may be related to a change in the concentration of a circulating molecule or a change in the properties of tissues that do not include the region of interest to which energy is directly applied. In one example, displacement may be a surrogate measure of the desired modulation, and a displacement measurement below an expected displacement value may result in a modulation of the modulation parameters until the expected displacement value is induced. Accordingly, the evaluation device 1020 may be configured to evaluate concentration changes in some embodiments. In some embodiments, the evaluation device 1020 may be an imaging device configured to evaluate changes in organ size and / or position. While the depicted elements of the system 10 are shown separately, it should be understood that some or all of the elements may be combined with each other. Additionally, some or all of the elements may be in wired or wireless communication with each other.

[0058] Based on the evaluation, modulation parameters of the controller 1016 may be altered. For example, if the desired modulation relates to a change in concentration (circulating or tissue concentration of one or more molecules) within a defined time window (e.g., 5 minutes, 30 minutes after treatment initiation of energy application) or relative to a baseline at the start of treatment, a change in modulation parameters, such as pulse frequency or other parameters, may be desired and, as a result, may be provided by an operator or via an automatic feedback loop to the controller 1016 to define or adjust energy application or modulation parameters of the pulse generator 1014.

[0059] The system 1000 provided herein can provide energy pulses according to various modulation parameters. For example, modulation parameters can include various stimulation time patterns, ranging from continuous to intermittent. In intermittent stimulation, energy is delivered at a certain frequency for a fixed duration during a signal-on time. The signal-on time is followed by a period during which no energy is delivered, called a signal-off time. Modulation parameters also include the frequency and duration of stimulation application. The application frequency can be continuous or can be delivered over various time periods, e.g., within a day or a week. Treatment duration can last for various durations, including, but not limited to, from several minutes to several hours. In certain embodiments, treatment duration with a particular stimulation pattern can last for one hour, repeated at 72-hour intervals, for example. In certain embodiments, treatment can be administered more frequently, e.g., every three hours, for a shorter duration, e.g., 30 minutes. The application of energy according to modulation parameters, such as treatment duration and frequency, can be adjustably controlled to achieve desired results.

[0060] 15 is a block diagram of certain components of a system 1010. As provided herein, the system 1010 for neuromodulation can include a pulse generator 1014 adapted to generate a plurality of energy pulses for application to tissue of a subject. The pulse generator 1014 can be separate or integrated into an external device, such as a controller 1016. The controller 1016 includes a processor 1030 for controlling the device. Software code or instructions are stored in a memory 1032 of the controller 1016 for execution by the processor 1030 to control various components of the device. The controller 1016 and / or the pulse generator 1014 can be connected to the energy application device 1012 via one or more leads 1033 or wirelessly.

[0061] The controller 1016 also includes a user interface having input / output circuitry 1034 and a display 1036 adapted to allow a clinician to provide selection inputs or modulation parameters to the modulation programs. Each modulation program can include one or more sets of modulation parameters, including pulse amplitude, pulse width, pulse frequency, etc. The pulse generator 1014 modifies its internal parameters in response to control signals from the controller device 1016 to vary the stimulation characteristics of the energy pulses delivered to the subject via leads 1033 by the energy application device 1012. Any suitable type of pulse generation circuit can be employed, including, but not limited to, constant current, constant voltage, multiple independent current or voltage sources, etc. The applied energy is a function of current amplitude and pulse width duration. The control device 1016 enables adjustable control of energy by modifying the modulation parameters and / or by initiating or canceling / reducing energy application at certain times. In one embodiment, adjustable control of the energy application device is based on information regarding the concentration of one or more molecules (e.g., circulating molecules) within the subject. If the information is from the evaluation device 1020, a feedback loop can drive the adjustable control. For example, if the circulating glucose concentration measured by the evaluation device 1020 exceeds a predetermined threshold or range, the controller 1016 may initiate energy application to a region of interest (e.g., the liver) and with modulation parameters associated with reducing circulating glucose. The initiation of energy application may be triggered by the glucose concentration drifting above a predetermined (e.g., desired) threshold or outside a predetermined range. In another embodiment, the adjustable control may be in the form of altering modulation parameters if the initial application of energy does not result in an expected change in a targeted physiological outcome (e.g., concentration of a molecule of interest) within a predetermined time frame (e.g., 1 hour, 2 hours, 4 hours, 1 day).

[0062] In one embodiment, the memory 1032 stores different operational modes selectable by an operator. For example, the stored operational modes may include instructions for executing a set of modulation parameters associated with a particular treatment region, such as regions of interest in the liver, pancreas, gastrointestinal tract, or spleen. Different regions may have different associated modulation parameters. Rather than having the operator manually enter the mode, the controller 1016 may be configured to execute the appropriate instructions based on the selection. In another embodiment, the memory 1032 stores operational modes for different types of treatment. For example, activation may be associated with different stimulation pressures or frequency ranges versus those associated with inhibiting or blocking tissue function. In a specific example, if the energy application device is an ultrasound transducer, the time-averaged power (time-averaged intensity) and peak positive pressure may be greater than 1 mW / cm. 2 ~30,000mW / cm 2 (time-averaged intensity) and range from 0.1 MPa to 7 MPa (peak pressure). In one example, the time-averaged intensity is 35 W / cm in the region of interest to avoid levels associated with thermal damage and ablation / cavitation. 2 In another embodiment, when the energy application device is a mechanical actuator, the amplitude of the vibration is in the range of 0.1 to 10 mm. The selected frequency may depend on the mode of energy application, e.g., ultrasonic or mechanical actuator.

[0063] In another embodiment, the memory 1032 stores a calibration or setup mode that allows for adjustment or modification of modulation parameters to achieve a desired result. In one example, stimulation starts at a low energy parameter and gradually increases, either automatically or upon receiving operator input. In this way, the operator can achieve adjustment of induced effects while modulation parameters are changed.

[0064] The system 1000 may also include an imaging device to facilitate focusing of the energy application device 1012. In one embodiment, the imaging device is integrated with or may be the same device as the energy application device 1012, such that different ultrasound parameters (e.g., frequency, numerical aperture, or energy) are applied to select (e.g., spatially select) a region of interest and focus energy on the selected region of interest for targeting and subsequent neuromodulation. In another embodiment, the memory 1032 stores one or more targeting or focusing modes used to spatially select a region of interest within an organ or tissue structure. Spatial selection may include selecting a subregion of an organ to identify a volume of the organ corresponding to the region of interest. Spatial selection may depend on image data as provided herein. Based on the spatial selection, the energy application device 1012 can focus on the selected volume corresponding to the region of interest. For example, the energy application device 1012 may be configured to initially operate in a targeting mode to apply targeting mode energy used to capture image data used to identify a region of interest. The targeting mode energy is applied at a level and / or modulation parameters appropriate for preferential activation. However, once the region of interest has been identified, the controller 1016 may then operate in a treatment mode according to the modulation parameters associated with preferential activation.

[0065] The controller 1016 may also be configured to receive input related to a targeted physiological outcome as input to the selection of modulation parameters. For example, if an imaging modality is used to assess tissue properties, the controller 1016 may be configured to receive a calculated index or parameter of the property. The modulation parameters may be altered based on whether the index or parameter is above or below a predefined threshold. In one embodiment, the parameter may be a measure of tissue displacement of the affected tissue or a measure of the depth of the affected tissue. Other parameters may include assessing the concentration of one or more molecules of interest (e.g., assessing one or more of: a change in concentration relative to a threshold or baseline / control; a rate of change; or a determination of whether the concentration is within a desired range). Additionally, the energy application device 1012 (e.g., an ultrasound transducer) may operate under the control of the controller 1016 to a) acquire tissue image data that can be used to spatially select a region of interest within the target tissue, b) apply modulated energy to the region of interest, and c) acquire images (e.g., via displacement measurements) to determine that a targeted physiological outcome has occurred. In such an embodiment, the imaging device, evaluation device 1020, and energy application device 1012 may be the same device.

[0066] In another embodiment, the desired modulation parameter set may also be stored by the controller 1016. In this manner, subject-specific parameters may be determined. Furthermore, the effectiveness of such parameters may be evaluated over time. If the effectiveness of a particular parameter set decreases over time, the subject may be experiencing insensitivity to the activated pathway. If the system 10 includes an evaluation device 1020, the evaluation device 1020 may provide feedback to the controller 1016. In certain embodiments, the feedback may be received from a user or the evaluation device 1020 indicating characteristics of the target physiological outcome. The controller 1016 may be configured to cause the energy application device to apply energy according to the modulation parameters and dynamically adjust the modulation parameters based on the feedback. For example, based on the feedback, the processor 1016 may automatically modify the modulation parameters (e.g., frequency, amplitude, or pulse width of the ultrasound beam or mechanical vibration) in real time in response to feedback from the evaluation device 1020.

[0067] As an example, the technology of the present invention can be used to treat subjects with metabolic disorders. The technology can also be used to regulate blood glucose levels in subjects with glucose regulation disorders. Thus, the technology can be used to promote homeostasis of a target molecule or to promote a desired circulating concentration or concentration range of one or more target molecules (e.g., glucose, insulin, glucagon, or a combination thereof). In one embodiment, the technology can be used to control circulating (i.e., blood) glucose levels. In one embodiment, the following thresholds can be used to maintain blood glucose levels in a dynamic equilibrium within the normal range: Fasted: Less than 50 mg / dL (2.8 mmol / L): Insulin shock 50~70mg / dL (2.8~3.9mmol / L): Hypoglycemia / hypoglycemia 70-110 mg / dL (3.9-6.1 mmol / L): Normal value 110-125mg / dL (6.1-6.9mmol / L): High / impaired (prediabetes) 125(7mmol / L): Diabetic patients Non-fasting (approximately 2 hours after a meal): 70-140mg / dL: Normal 140-199 mg / dL (8-11 mmol / L): High or "borderline" / Prediabetic More than 200mg / dL: (11mmol / L): Diabetes For example, the technology can be used to maintain circulating glucose concentrations below about 200 mg / dL and / or above about 70 mg / dL. The technology can be used to maintain glucose in the range of about 4-8 mmol / L or about 70-150 mg / dL. The technology can also be used to maintain a subject's (e.g., patient's) normal blood glucose range, where the normal blood glucose range may be individualized based on the patient's individual factors, such as weight, age, and clinical history. Thus, the application of energy to one or more regions of interest can be adjusted in real time based on the desired final concentration of the molecule of interest or in a feedback loop based on input from the evaluation device 1020. For example, if the evaluation device 1020 is a circulating glucose monitor or blood glucose monitor, real-time glucose measurements can be used as input to the controller 16.

[0068] The energy application device 1012 may include an ultrasound transducer (e.g., a non-invasive ultrasound transducer or a handheld ultrasound transducer) capable of applying energy to a target, shown as a liver by way of non-limiting example. The energy application device 1012 may include control circuitry for controlling the ultrasound transducer. The control circuitry of the processor 1030 may be integral with the energy application device 1012 (e.g., via an integrated controller 1016) or may be a separate component. The ultrasound transducer may also be configured to acquire image data to assist in spatially selecting a desired or target region of interest and focusing the applied energy to the region of interest of the target tissue or structure based on the acquired image data.

[0069] The desired target within the region of interest may be an internal tissue or organ containing axon terminals and synapses with non-neuronal cells. Synapses can be stimulated by applying energy directly to the axon terminals within the focal region of an ultrasound transducer focused on the target region of interest, causing the release of molecules into the synaptic space. For example, axon terminals synapse with hepatocytes, and neurotransmitter release and / or changes in ion channel activity cause downstream effects, such as the activation of glucose metabolism. In one embodiment, liver stimulation or modulation may refer to modulation of the hepatic hilum or a region of interest adjacent to the hepatic hilum.

[0070] The energy may be concentrated or substantially concentrated in a region of interest, concentrating only on a portion of an internal tissue or organ, e.g., less than about 50%, 25%, 10%, or 5% of the total tissue volume. In one embodiment, energy may be applied to two or more regions of interest within the target tissue, and the combined volume of the two or more regions of interest may be less than about 90%, 50%, 25%, 10%, or 5% of the total tissue volume. In certain embodiments, energy is applied to only about 1%-50% of the total tissue volume, only about 1%-25% of the total tissue volume, only about 1%-10% of the total tissue volume, or only about 1%-5% of the total tissue volume. In certain embodiments, only axon terminals within the region of interest in the target tissue directly receive the applied energy and release neurotransmitters, while unstimulated axon terminals outside the region of interest do not receive substantial energy and therefore are not similarly activated / stimulated. In some embodiments, axon terminals in the portion of the tissue directly receiving the energy will induce altered neurotransmitter release. In this aspect, tissue subregions may be targeted for neuromodulation in a granular manner, e.g., one or more subregions may be selected. In some embodiments, energy application parameters may be selected to induce preferential activation of either neural or non-neural components within the tissue directly receiving the energy to induce a desired composite physiological effect. In certain embodiments, energy is applied over a range of approximately 25 mm 3 The energy can be focused or concentrated within a volume of about 0.5 mm. 3 -50mm 3 The focal volume and depth of focus for focusing or concentrating energy within the region of interest may be affected by the size / configuration of the energy application device 1012. The focal volume of the energy application may be defined by the field of focus of the energy application device 1012.

[0071] As provided herein, energy may be applied substantially only to a region or areas of interest, and not substantially in a general or non-specific manner throughout the tissue, to preferentially activate synapses in a targeted manner to achieve a targeted physiological outcome.

[0072] Technical effects of the disclosed embodiments include using the disclosed complex neuromodulation techniques to induce improvements in ultrasound stimulation therapy. In one embodiment, ultrasound stimulation administered to a diabetic patient already undergoing medication can: 1) enable a reduction in the dosage of currently administered medications (and subsequent side effects), 2) transition the patient from multiple medications to a single medication (potentially saving money), or 3) eliminate medication altogether by using multi-site ultrasound stimulation instead of medication. Furthermore, multi-site ultrasound testing can be initiated in pre-diabetic patients (e.g., subjects with insulin resistance) in addition to dietary and exercise recommendations to prevent progression to diabetes. Ultrasound can also be administered on an irregular basis (e.g., monthly or quarterly) to reinforce dietary and exercise therapy.

[0073] Additionally, monostimulus ultrasound therapy can be used in combination with a monotherapy, such that the two therapies act on overlapping and / or synergistic molecular and / or neural pathways. Liver ultrasound and GLP agonist therapy compared to dual-site ultrasound therapy as disclosed herein is an exemplary case.

[0074] Examples are used herein as part of the disclosure and will enable any person skilled in the art to practice the disclosed embodiments, including making and using any devices or systems, and performing any methods incorporated therein. The patentable scope is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements that do not differ substantially from the literal language of the claims. [Explanation of symbols]

[0075] 1010: System 1012: Energy application device 1014: Pulse generator 1016: Controller 1020: Evaluation device 1030: Processor 1032: Memory 1033: Lead wire 1034: Input / output circuit 1036: Display

Claims

1. 1. A complex neuromodulation system comprising: an energy application controller; The energy application controller applying a first ultrasound energy to a first region of interest in a subject having a metabolic disorder; applying second ultrasound energy to a second region of interest of the subject at a different time than the first ultrasound energy; is executed, configured to control an energy application device to repeat applying the first ultrasound energy to the first region of interest and applying the second ultrasound energy to the second region of interest less than once per month; the first region of interest is in a subregion of the liver; the second region of interest is in a gastrointestinal tissue of the subject; the energy application controller is configured to repeatedly apply the first ultrasound energy to the first region of interest and the second ultrasound energy to the second region of interest to treat the metabolic disorder, whereby an afferent pathway is stimulated and a remission of the metabolic disorder is achieved for greater than four weeks; the first ultrasound energy and the second ultrasound energy are applied to the subject at a time interval of less than one hour; The application of the first and second ultrasonic energy results in a further decrease in circulating glucose concentration in the subject compared to application of the first ultrasonic energy alone.

2. 1. A complex neuromodulation system comprising: an energy application controller; The energy application controller applying a first ultrasound energy to a first region of interest in a subject having a metabolic disorder; applying second ultrasound energy to a second region of interest of the subject at a different time than the first ultrasound energy; is executed, configured to control an energy application device to repeat applying the first ultrasound energy to the first region of interest and applying the second ultrasound energy to the second region of interest less than once per month; the first region of interest is in the liver or the hepatic portal plexus; the second region of interest is in a gastrointestinal tissue of the subject; the energy application controller is configured to repeatedly apply the first ultrasound energy to the first region of interest and the second ultrasound energy to the second region of interest to treat the metabolic disorder, wherein an afferent pathway is stimulated and remission of the metabolic disorder is achieved for greater than four weeks.

3. The system of claim 1 , wherein the metabolic disorder is type II diabetes.

4. The system of claim 1 , wherein the metabolic disorder is obesity or insulin resistance.

5. 10. The system of claim 1, further comprising an evaluation device that provides feedback to the energy application controller indicative of circulating glucose concentrations after the application of multiple ultrasound waves.

6. 6. The system of claim 5, wherein the evaluation device comprises receiving a pre-treatment circulating glucose concentration of the subject, and wherein the circulating glucose concentration after the multiple ultrasound applications is lower than the pre-treatment circulating glucose concentration.

7. 7. The system of claim 6, wherein the application of the first ultrasonic energy and the application of the second ultrasonic energy are automatically triggered by a circulating glucose concentration being above a predetermined threshold or outside a predetermined range.

8. The system of claim 1 , wherein the second region of interest includes the superior mesenteric plexus or the posterior gastric nerve.

9. 2. The system of claim 1, wherein the energy application controller is configured to repeat applying the first ultrasound energy to the first region of interest and the second ultrasound energy to the second region of interest once per day for multiple days to treat the metabolic disorder.

10. The system of claim 1 , wherein the metabolic disorder is prediabetes or insulin resistance.

11. 1. A complex neuromodulation system comprising: an energy application controller; The energy application controller applying first ultrasound energy to a first region of interest in a subject having a metabolic disorder to neuromodulate a glucose sensor afferent pathway; applying second ultrasound energy to a second region of interest of the subject at a different time than the first ultrasound energy to neuromodulate a neuroendocrine cortex; is executed, configured to control an energy application device to repeat applying the first ultrasound energy to the first region of interest and applying the second ultrasound energy to the second region of interest less than once per month; the first region of interest is in a subregion of the liver; the second region of interest is in a gastrointestinal tissue of the subject; the energy application controller is configured to repeatedly apply the first ultrasound energy to the first region of interest and the second ultrasound energy to the second region of interest to treat the metabolic disorder, whereby an afferent pathway is stimulated and a remission of the metabolic disorder is achieved for greater than four weeks; the first ultrasound energy and the second ultrasound energy are applied to the subject at a time interval of less than one hour; The application of the first and second ultrasonic energy results in a further decrease in circulating glucose concentration in the subject compared to application of the first ultrasonic energy alone.

12. 1. A complex neuromodulation system comprising: an energy application controller; The energy application controller applying first ultrasound energy to a first region of interest in a subject having a metabolic disorder to neuromodulate a glucose sensor afferent pathway; applying second ultrasound energy to a second region of interest of the subject at a different time than the first ultrasound energy to neuromodulate a neuroendocrine cortex; is executed, configured to control an energy application device to repeat applying the first ultrasound energy to the first region of interest and applying the second ultrasound energy to the second region of interest less than once per month; the first region of interest is in the liver or the hepatic portal plexus; the second region of interest is in a gastrointestinal tissue of the subject; the energy application controller is configured to repeatedly apply the first ultrasound energy to the first region of interest and the second ultrasound energy to the second region of interest to treat the metabolic disorder, wherein an afferent pathway is stimulated and remission of the metabolic disorder is achieved for greater than four weeks.

13. The system of claim 1 or 12, wherein the depth of focus of the energy application device is preset to a predetermined value.

14. The system of claim 13 , wherein the energy applicator comprises a handheld ultrasound probe.

15. The system of claim 12 , wherein the region of interest includes a fundus.

16. 1. A complex neuromodulation system comprising: an energy application controller; The energy application controller focusing a first ultrasound energy on a first region of interest in a subject having a metabolic disorder, the region of interest being a subregion of the liver; applying the first ultrasound energy to the first region of interest at a first power level for a first time; the first region of interest is in the liver or the hepatic portal plexus; focusing second ultrasound energy on a second region of interest in the subject, the second region of interest being in the superior mesenteric plexus, the inferior mesenteric plexus, and / or the gastric fundus; configured to drive the application of the second ultrasound energy to the second region of interest at a second power level and for a second duration; configured to repeat applying the first ultrasound energy to the first region of interest and applying the second ultrasound energy to the second region of interest less than once per month; the second power level is higher than the first power level; the second time period is shorter than the first time period; the energy application controller is configured to repeatedly apply the first ultrasound energy to the first region of interest and the second ultrasound energy to the second region of interest to treat the metabolic disorder, wherein an afferent pathway is stimulated and remission of the metabolic disorder is achieved for greater than four weeks.

17. 17. The complex neuromodulation system of claim 16, comprising a first energy application device configured to apply the first ultrasound energy to the first region of interest and a second energy application device configured to apply the second ultrasound energy to the second region of interest.

18. 17. The complex neuromodulation system of claim 16, comprising an energy application device configured to apply the first ultrasound energy to the first region of interest and the second ultrasound energy to the second region of interest.

19. 20. The complex neuromodulation system of claim 18, wherein a focal depth of the energy application device is preset to a predetermined value for application of the first ultrasonic energy and the second ultrasonic energy.

20. 20. The complex neuromodulation system of claim 19, wherein the second ultrasound energy is applied at least 5 minutes after the first ultrasound energy is applied.

21. 17. The complex neuromodulation system of claim 16, wherein the second region of interest is a subregion of gastrointestinal tissue.

22. The first power level may be configured to provide the first ultrasonic energy at about 200 W / cm ２ and a peak acoustic intensity of approximately 10 mW / cm ２ 17. The complex neuromodulation system of claim 16, wherein the system is applied at an average acoustic intensity of:

23. 17. The system of claim 1, 12 or 16, wherein the afferent pathway carries signals from sensory sites in the abdomen to the hypothalamus of the brain.

24. 17. The complex neuromodulation system of claim 16, wherein the first period of time is at least 15 minutes and the second period of time is less than 5 minutes.

25. 17. The complex neuromodulation system of claim 16, wherein the first and second ultrasound energies are sufficient to elicit an autonomic signal response in the subject.

26. an evaluation device; 26. The complex neuromodulation system of claim 25, wherein the autonomic signal response is detected by the evaluation device via changes in hypothalamic activity or function.

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