Neuromodulation to target glucose transporter and / or incretin pathways

Abstract

To provide methods for neuromodulation that include applying energy (e.g., ultrasound energy) to tissue so as to cause a change in a glucose transporter pathway molecule and / or an incretin pathway molecule.SOLUTION: A method comprises: applying ultrasound energy to an internal tissue of a subject having a metabolic disorder so as to cause a change in a glucose transporter pathway molecule and / or an incretin pathway molecule in the subject to treat the metabolic disorder, where the ultrasound energy is applied by an extracorporeal energy application device; measuring the change or an indication of the change in the subject; and determining, based on the measurement, that the ultrasound energy has caused the change in the glucose transporter pathway molecule and / or the incretin pathway molecule to treat the subject.SELECTED DRAWING: Figure 9A

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 817,373, entitled "NEUROMODULATION TO TARGET GLUCOSE TRANSPORTER AND / OR INCRETIN PATHWAYS," filed March 12, 2019, and U.S. Provisional Patent Application No. 62 / 826,517, entitled "NEUROMODULATION TO TARGET GLUCOSE TRANSPORTER AND / OR INCRETIN PATHWAYS," filed March 29, 2019, the contents of which are incorporated by reference in their entirety for all purposes. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under Grant No. HR0011-18-C-0040 awarded by the Defense Advanced Research Projects Agency (DARPA). The Government has certain rights in this invention. [Technical field]

[0003] The subject matter disclosed herein relates to neuromodulation or regulation of secretory cells by stimulating components of peripheral nerves or secretory cells within target tissues, and more specifically to techniques for targeting pathways such as the glucose transporter and / or incretin pathways by neuromodulation. [Background technology]

[0004] Neuromodulation or neurostimulation involves using stimulation devices to target specific neural pathways for clinical utility. For example, stimulation of central nervous system structures may be used to treat pain. Certain neurostimulation strategies use permanently implanted electrodes, transcutaneous electromagnetic fields, or adapted brain stimulation techniques to stimulate large nerves that can be accessed by implanted devices or nerves close to the skin surface. However, the smaller nerves of the peripheral nervous system that reside in or terminate in organs are more difficult to target than the larger central nervous system structures. The anatomy of the peripheral nervous system (PNS) presents a difficult challenge. Within peripheral nerves, individual axons are tightly bundled together in groups (bundles) and wrapped in protective tissue. This makes it difficult to selectively stimulate a subset of axons that terminate in a specific organ and uniquely regulate the function of transmitter cells within the organ. Novel neurostimulation methods are needed to non-invasively stimulate specific targets and correlate organ-specific neural activity with function for broad clinical interpretation and treatment of clinical symptoms. [Brief description of the drawings]

[0005] These and other features, aspects, and advantages of the present invention will be better understood from the following detailed description taken in conjunction with the accompanying drawings, in which like reference numerals represent like parts throughout.

[0006] [Figure 1] Shown are protein concentration levels measured by ELISA for sodium glucose cotransporter protein (SGLT1 in the intestine or SGLT2 in the kidney), glucose transporter 2 (GLUT-2), and glucagon-like peptide-1 (GLP-1) in liver, kidney and intestinal tissues collected from both hepatically stimulated and sham control obese Zucker animals such that intestinal tissue samples were collected using a luminal stripping technique to allow examination of both glucose transporter protein concentration changes or translocation changes across the luminal barrier tissue. [Diagram 2]Shown are mRNA, protein, and glucose-6-phosphate concentration levels from liver, muscle, intestine, and kidney tissues collected from both hepatically stimulated and sham-control obese Zucker animals. [Diagram 3] Shown are mRNA, protein, and glucose-6-phosphate concentration levels from hypothalamic tissue collected from both hepatically stimulated and sham-control obese Zucker animals. [Figure 4] Figure 1 shows protein concentration levels measured by ELISA from intestinal or kidney tissue collected from both hepatically stimulated and sham control obese Zucker animals, showing elevated GLUT2 in intestinal and kidney tissue and elevated GLP1 in intestinal tissue. [Diagram 5] 1 shows mRNA concentration levels in various tissues collected from both hepatically stimulated and sham control obese Zucker animals, indicating concurrent and interconnected changes mediated by various glucose utilization, insulin utilization and / or metabolism-related proteins. [Figure 6] 5'-activated protein kinase concentration levels as a result of stimulation versus control in hepatic, pancreatic or gastrointestinal tissues, and glutamate levels as a result of hepatic stimulation versus control in animals of an LPS-induced hyperglycemia model, showing that pancreatic stimulation is itself caused by insulin release and direct insulin signaling in the hypothalamus. [Figure 7A] We present an experimental platform for peripherally focused ultrasound (pFUS) neural activation in in vitro three-dimensional (3D) neural cultures. [Figure 7B] FIG. 7A shows the experimental timeline and imaged peripheral nerve network, showing calcium transients within a region of interest of cells in in vitro culture. [Figure 7C] FIG. 7B shows the fluorescence intensity after pulsed ultrasound stimulation of the cells in the in vitro culture of FIG. 7A. [Figure 7D] FIG. 7A shows the relationship between calcium transients and applied ultrasound pressure in in vitro cultures. [Figure 8A] FIG. 1 is a schematic diagram of a peripheral focused ultrasound target that focuses ultrasound stimulation at multiple locations containing cell body junctions and / or synaptic junctions along the vagus and parasympathetic nervous systems. [Figure 8B] FIG. 1 shows a timeline of ultrasound stimulation and blood sampling performed in animals in the LPC-induced inflammation and hyperglycemia model. [Figure 8C] Shown are TNF (cytokine) and glucose concentrations following pFUS targeted to different anatomical sites within the spleen and intestinal cholinergic anti-inflammatory pathways (i.e., spleen, sacral ganglion, and ganglion without treatment) compared to LPS alone (i.e., no ultrasound) controls. [Figure 8D] Measures of plasma / blood concentrations of neurotransmitters (i.e., epinephrine, norepinephrine, dopamine, and acetylcholine) following pFUS at three different anatomical target sites compared to LPS control (LPS CTRL). [Figure 9A] Schematic of pFUS-based high-precision organ-based neuromodulation in which innervation points of known axon populations are targeted for stimulation using focused pulsed ultrasound. [Figure 9B] FIG. 13 is a timeline of pFUS stimulation and blood sampling performed within the LPS-induced hyperglycemia model. [Figure 9C] Circulating blood glucose levels are shown at 0, 5, 15, 30, and 60 minutes for LPS control (no pFUS, yellow circles), liver pFUS (blue circles), pancreatic pFUS (purple circles), and GI pFUS (orange circles) groups. [Figure 10A] Hypothalamic markers insulin receptor substrate 1 (IRS1), phosphorylated protein kinase B (phos-Akt), glucose transporter 4 (GLUT4), and glucose-6-phosphate (G-6-phos) are shown. [Figure 10B] Circulating hormones and markers from collected blood samples including insulin, glucagon, leptin, and GLP1 concentrations. [Figure 11A]Shown are blood levels of epinephrine after pFUS and the no LPS / pFUS control (LPS CTRL) at each target stimulation site. [Figure 11B] Shown are blood concentrations of norepinephrine after pFUS and the LPS / no pFUS control (LPS CTRL) at each target stimulation site. [Figure 11C] Shown are blood levels of dopamine after pFUS and the LPS / no pFUS control (LPS CTRL) at each target stimulation site. [Figure 12] FIG. 1 is a schematic diagram of a neuromodulation system using a pulse generator, according to an embodiment of the present disclosure. [Figure 13] FIG. 1 is a block diagram of a neuromodulation system according to an embodiment of the present disclosure. [Figure 14] FIG. 1 is a flow diagram of a neuromodulation technique, according to an embodiment of the present disclosure. [Figure 15] FIG. 1 is a flow diagram of a neuromodulation technique, according to an embodiment of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] One or more specific embodiments are described below. In order to provide a concise description of these embodiments, not all features of an actual implementation are described herein. It will be appreciated that the development of an actual implementation, such as an engineering or design project, requires numerous implementation-specific decisions to be made to achieve the developer's particular goals, such as addressing system-related and business-related constraints, which may vary from implementation to implementation. Moreover, it will be appreciated 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.

[0008] Any examples or illustrations provided herein should not be considered in any way as representing a restriction, limitation, or definition to any one or more of the terms in which they are used. Instead, these examples or illustrations should be considered as being described in terms of various specific embodiments and should be considered as illustrative only. Those skilled in the art will understand that any one or more of the terms in which these examples or illustrations are utilized encompass other embodiments that may or may not be provided therewith or elsewhere in this specification, and all such embodiments are intended to be included within the scope of that one or more terms. Language designating such non-limiting examples and illustrations includes, but is not limited to, "for example," "for instance," "such as," "eg," "including," and "in one embodiment."

[0009] Metabolic pathways are controlled by complex feedback. For example, during food scarcity, the neuroendocrine system may be downregulated, slowing metabolism and increasing reabsorption of excess glucose that is being removed by the kidney. In contrast, an oversupply of glucose may result in abnormal activation and abnormal regulation of the endocrine system, peripheral nervous system, and / or central nervous system due to physiological functions of these systems that contribute to, and even play a causative role in, the development of type 2 diabetes mellitus (T2DM), obesity, or other metabolic diseases.

[0010] Certain drug applications or pharmaceutical treatments stimulate or inhibit the molecular pathways related to glucose transporters responsible for reabsorption or glucose uptake in both the kidney (SGLT2) and intestine (GLUT2) as well as the incretin / hormonal pathway that enhances insulin-stimulated glucose utilization. In the kidney, SGLT2 inhibitors (a class of T2DM drugs) are used to directly block glucose reabsorption (at the S1 segment of the proximal tubule or at the location of the SGLT2 protein). This subsequently increases glucose excretion in the urine, resulting in lower blood glucose levels. In the intestine, SGLT1 activity is thought to be involved in initiating the translocation of GLUT2 from the basolateral to the apical side of intestinal absorptive epithelial cells, as changes in the sodium (Na) cotransporter into the tissue cause depolarization, activation of L-type calcium channels, and Ca-dependent reorganization of cytoskeletal structures involved in GLUT2 transport. Activating this translocation can increase glucose absorption from the apical side / lumen into intestinal absorptive epithelial cells (and ultimately into the blood) after a meal. SGLT1 inhibitors (a second class of T2DM drugs) block this enhanced glucose absorption mechanism, retaining even more glucose inside the intestine for secretion.

[0011] Furthermore, for refined flour / sugar and low fiber based diets, the ability for rapid absorption in the early segments of the small intestine, either due to GLUT translocation or standard SGLT1 activity, may prevent food / sugar from reaching the distal part of the ileum. This is problematic in that this lower part of the small intestine (i.e., the ileum) contains glands that secrete incretins such as GLP1, a class of hormones involved in metabolism. Signaling from these hormones can signal other peripheral tissues to regulate their response to insulin (i.e., insulin sensitivity). Another class of T2DM drugs, GLP agonists, SGLT1 and SGLT2 act to mimic incretins and their functions in activating and sensitizing insulin secretion. The activity of SGLT1 and SGLT2 may be influenced by adrenergic signaling by binding to adrenergic receptors via adenylyl cyclase.

[0012] Provided herein are techniques for neuromodulation and secretory cell regulation targeting specific metabolic pathways and molecules. Such metabolic pathways or molecules may include glucose absorption vs. excretion in the intestine (SGLT1), glucose absorption vs. excretion in the kidney (SGLT2), and GLP / incretin signaling via neural pathways (altered intestinal glucose concentration via SGLT1 / GLUT2 activity and transport). Thus, in one embodiment, ultrasound energy may be applied to focus on a region of interest in a metabolic tissue (liver, gastrointestinal tract, pancreas, kidney). One or more regions of interest may be subjected to a treatment that includes one or more doses of ultrasound energy applied once or in individual amounts administered over a period of time as part of a treatment regimen. As a result of the treatment, the activity, concentration, and / or location of the specific molecule of interest is altered. The change may be in the tissue in which the one or more regions of interest are located, or in other tissues that are responsive to the neural signaling resulting from the application of energy directly to the region of interest. In one embodiment, the treatment is used to treat a subject with a metabolic disease or clinical condition. In one embodiment, a patient with a metabolic disease is treated by applying ultrasound energy to an internal tissue of the patient, the ultrasound energy being applied by an external energy application device, and applying the ultrasound energy causes changes to glucose transporter pathway molecules and / or incretin pathway molecules in the patient. In another embodiment, a system for treating a metabolic disease may include an energy application device configured to apply energy to an internal tissue of a subject, and a controller adapted to control the energy application device to apply energy and cause changes to glucose transporter pathway molecules and / or incretin pathway molecules in the subject. In one embodiment, treatment is assessed by measuring the change directly or by measuring a characteristic or indicator of the change.

[0013] Experimental results show that daily stimulation of metabolic tissues at levels consistent with activation of metabolic components of the autonomic nervous system acts to modify the concentration, location and / or activity of glucose transporter pathway molecules and / or incretin pathway molecules, altering glucose uptake versus excretion levels in a therapeutic manner. Moreover, this is accomplished in a localized manner and does not affect other pathways associated with side effects. Thus, neuromodulation techniques as disclosed may have fewer side effects compared to drug treatments to achieve similar physiological changes / therapeutic outcomes and may serve as an alternative or complement to existing drug therapies for subjects with metabolic diseases.

[0014] FIG. 1 shows protein concentrations measured by enzyme-linked immunosorbent assay (ELISA) in kidney, intestine and liver tissues in obese Zucker rats in a diabetic model in a liver stimulation group compared to a sham control. In this stimulation group, animals were stimulated with daily ultrasonic stimulation of the liver with sufficient power to allow activation of neural pathways and sensory or secretory cells and provide measured changes in glucose transport and incretin activity, concentration or location in downstream target tissues including the intestine and kidney. Sham controls were performed with an ultrasound transducer placed on the target organ but without applying ultrasonic stimulation. After daily treatments over several days, tissue samples were collected after the animals were sacrificed. Protein levels in the intestine (vs. other tissues and / or mRNA data from the intestine) are from samples taken by scraping from the lining of the intestine (thus collecting only the apical or luminal side of the epithelial lining).

[0015] Neuromodulation of the liver results in changes in metabolic pathways and signaling molecules in both blood markers and other tissues. A reduction in circulating blood glucose was measured in the ultrasound stimulation group, which is associated with altered absorption / secretion pathways. Daily energy application to the liver also results in changes in GLUT2 in the kidney and intestine, and GLP-1 in the intestine, compared to sham controls.

[0016] The measured protein changes were accompanied by changes in RNA expression measured by mRNA sequencing. Figure 2 shows protein, mRNA and assay data for various molecules of interest in liver, muscle, intestine and kidney tissues from both hepatically stimulated and sham-controlled obese Zucker animals treated as described in Figure 1. Figure 3 shows protein, mRNA and assay data for various molecules of interest in hypothalamic tissues from both stimulated and sham-controlled obese Zucker animals treated as described in Figure 1. SGLT2 is a key protein for glucose reabsorption, while renal GLUT2 is the primary means of glucose elimination or excretion in urine. Daily ultrasound stimulation in a rat model resulted in upregulation of both SGLT1 and GLUT2 mRNA in the intestine, but downregulation of both SGLT2 and GLUT2 mRNA in the kidney. As described above, expression and / or translocation of glucose transporters results in altered glucose reabsorption versus excretion. The stimulation and resulting effects also modulate the concentrations of GLP / incretins in the metabolic system. Daily ultrasound stimulation in a rat model led to upregulation of both GLUT2 mRNAs in the liver.

[0017] FIG. 4 shows protein concentration levels measured by ELISA from intestinal or kidney tissue collected from both hepatically stimulated and sham-control obese Zucker animals, showing an increase in GLUT2 in intestinal and kidney tissues and an increase in GLP1 in intestinal tissue.

[0018] Figure 5 shows mRNA concentration levels in various tissues collected from both hepatically stimulated and sham-controlled obese Zucker animals, indicating concurrent and interconnected changes mediated by cell membrane proteins. For example, glucose regulation in the liver is mediated by GLUT2, which is elevated as a result of daily hepatic ultrasound stimulation in a rat model. Skeletal muscle transport of glucose is mediated by GLUT4. In the kidney, multiple concurrent changes were associated with daily hepatic ultrasound stimulation in a rat model. Downregulation of protein kinase C (PKC), SGLT2, GLUT2, and Irs2 may be associated with impaired glucose excretion in urine and reabsorption in blood.

[0019] 6 shows 5'-activated protein kinase concentration levels after stimulation vs. control in hepatic, pancreatic or gastrointestinal tissues, and glutamate levels after hepatic stimulation vs. control in animals of the LPS-induced hyperglycemia model. 5'AMP is a known measure of insulin signaling directly to the hypothalamus and activation of insulin-dependent pathways.

[0020] Experimental results are also provided herein confirming direct peripheral focused ultrasound (pFUS) neural activation by applying ultrasound stimulation to in vitro three-dimensional (3D) neural cultures. The disclosed novel 3D culture system (Figure 7A) provides an in vitro experimental platform for peripheral nerve examination compared to traditional excised nerve bundle preparations. The formation of complex neural networks was observed within the culture platform, and the in vitro system was used to couple ultrasound transducers and observation fluorescence microscopes to neurons. The use of calcium indicator dyes allowed direct observation of neuronal activity. Ultrasound pressure associated with in vivo neuromodulation was confirmed to cause direct neural activation in the culture system. The results demonstrated that ultrasound stimulation can activate peripheral neurons.

[0021] FIG. 7A is a schematic diagram of a 3D in vitro peripheral neuron culture system. Isolated peripheral neurons (i.e., dorsal root ganglion cells, DRG) were mixed with hydrogel microparticles and co-injected into culture plates. After injection, the microparticle hydrogel was annealed, leaving a heterogeneous scaffold containing neurons that provided both a mechanically rigid growth surface for neural cell attachment and interconnected micropores for neurite outgrowth. DRG neurons were cultured in 200 μL of NbActiv4 containing 25 ng / ml NGF in an incubator at 37° C. and 5% CO2 concentration, and 50% of the medium was replaced every 3 days. DRG neurons were incubated with Fluo-4 Direct Calcium Assay Kit containing 250-fold of 10-3 M probenecid stock solution for Ca imaging. Briefly, 5 mL of calcium assay buffer was mixed and vortexed with 100 μL of probenecid stock solution to make a 2x loading dye solution, then the dye solution was added to the cells in a 1:1 ratio with the medium and incubated for 1 h before imaging, allowing for sufficient diffusion through the hydrogel.

[0022] FIG. 7B shows the experimental timeline shown with the corresponding right field and fluorescence images of DRG neuronal cells in the 3D hydrogel scaffold. The diameter of the hydrogel was about 100 μm, and the pore size of the hydrogel particles was about 1 μm. DRG neuronal cells proliferated three-dimensionally between the pores of the hydrogel particles. The fluorescence images show the time course of calcium imaging during pFUS stimulation. pFUS stimulation was performed 10 seconds after the start of observation and stopped again 120 seconds after the start. Ultrasound was then stopped for 2 minutes before stimulation was resumed (allowing for taking images after ultrasound or at the time of stopping). The calcium concentration of the DRG neuronal cells increased after ultrasound firing, as indicated by the increase in fluorescence inside the imaged neurons. After pFUS simulation, the calcium concentration of the cells returned to the same level as before stimulation.

[0023] Figure 7C shows the average ΔF / F0 and maximum ΔF / F0 values ​​at each time point for imaged neurons. ΔF / F0 does not change without pFUS stimulation (left), whereas ΔF / F0 increases during pFUS stimulation (right). Figure 7D shows the relationship between different amplitudes of pFUS utilized in replicate experiments. 200 mVpp (or 0.83 MPa peak positive pressure) showed a large change in fluorescence inside similar DRG cultures (N>5 for each group). This corresponds to the effective ultrasound pressure demonstrated for neuromodulation.

[0024] DRG responded well to the 3D culture environment, attaching to the hydrogel microparticle surface and projecting neurites into the micropores. Both cell body and synaptic neural features were evident in the cultures in addition to axonal projections (Figure 7B). Axonal extension led to neuronal network formation across the micropore gaps formed between the microparticles. Calcium transients in regions of interest within the cultures were imaged by imaging peripheral neuronal networks in Ca2+ indicator-filled medium (Fluo-4 Direct Calcium Assay Kit) (Figure 7B). To measure ultrasound-induced changes in calcium signaling (i.e., ultrasound-induced increases in neuronal activity), the ultrasound transducer (and focus) was aligned to the center of the optical field of view (FOV). Figure 7B demonstrates that the fluorescence intensity (and therefore the average calcium concentration within the neurons) in the FOV increased after pulsed ultrasound stimulation (using a 1.1 MHz, 136.36 μs pulse length, 0.5 ms pulse repetition period US pulse). The appearance and amplitude of these calcium transients were ultrasound pressure dependent, and were observed at maximal neuronal activation (Figure 7D, 0.83 MPa or 23 mW / cm 2 The applied pressure achieving this (match) corresponded to the effective ultrasound pressure for neuromodulation in the animal model.

[0025] Further provided herein is pFUS stimulation of alternative sites known to contain peripheral nerve cell bodies and synapses, including sensory ganglia (i.e., inferior or no-dose ganglia of the vagus nerve) and peripheral ganglia with mixed (sensory and efferent) innervation (i.e., sacral ganglion). Activation of cholinergic anti-inflammatory pathways (i.e., modulation of LPS-induced blood cytokine concentrations) was observed following ultrasound stimulation at each site (i.e., end organ / spleen, no-dose, and sacral ganglion), and both the magnitude of cytokine reduction and the presence of other off-target effects (i.e., concomitant changes in blood glucose levels) were shown to be stimulation site dependent. Finally, intervention in specific pathological conditions (i.e., reduction of LPS-induced hyperglycemia) was achieved by stimulation of multiple strategic anatomical locations associated with metabolic control. These exploratory stimulation sites included liver sites containing peripheral glucose sensors, pancreas associated with insulin-secreting beta cells, and intestinal sites containing incretin-secreting enteroendocrine cells. Ultrasound-induced attenuation of hyperglycemia was achieved by stimulating the respective anatomical targets. However, the magnitude of blood glucose reduction depends on the stimulation site, and the effect may be driven by different molecular mechanisms at each site. Together, these data demonstrate that ultrasound stimulation can directly activate peripheral nerves (and other neuroendocrine cells) and that pFUS can be used to efficiently evaluate the efficacy of alternative therapeutic bioelectronic drug stimulation sites.

[0026] 8A-8C show the results of pFUS activating the cholinergic anti-inflammatory pathway (CAP) at multiple locations within the brain-splenic nerve pathway. Figure 8A shows an activation schematic where pFUS stimulation was focused at multiple locations containing cell body junctions and / or synaptic junctions along the vagus and parasympathetic nervous systems. These include the untreated ganglion (i.e., the location of the cell bodies of multiple vagus afferents that project to the brain), the sacral ganglion (i.e., the location of cell body junctions and synaptic junctions of the mixed sympathetic and parasympathetic nervous systems), and the spleen (i.e., the location of CAP-specific neurons).

[0027] Figure 8B shows a timeline of the in vivo neuromodulation study conducted herein, in which LPS was first introduced (as generally disclosed herein) to create an inflammatory and hyperglycemic state. Ultrasound was then applied, and the effects of ultrasound neuromodulation were measured by comparing inflammatory or metabolic markers in the LPS control (i.e., no ultrasound stimulation) vs. the LPS + ultrasound stimulation cohort.

[0028] FIG. 8C shows that splenic neuromodulation and activation of the CAP are sufficient to attenuate the LPS response. Furthermore, local activation of the CAP at the splenic site limits additional (non-CAP or non-targeted) effects typically observed using implant-based VNS CAP activation, such as suppression of LPS-induced hyperglycemia. pFUS stimulation at both the untreated and sacral ganglia also results in attenuation of the LPS response. Furthermore, untreated ultrasound neuromodulation attenuated the effects of LPS to a greater extent than stimulation at either the sacral or splenic sites. This confirms the importance of vagal afferents in triggering activation of the CAP (i.e., neurons that enter the CNS through the untreated ganglion). However, this also expands the efferent arch of the pathway, which additionally includes vagal and sympathetic neurons.

[0029] However, stimulation at the ganglion without dosing also resulted in suppression of LPS-induced hyperglycemia, despite increased levels of cytokine suppression (Figure 8D). Targeted or precise ultrasound neuromodulation at the splenic site (reportedly not containing vagal afferent neurons) results in separable activation of CAP versus other non-target pathways (such as glucose-sensing cells in the liver). Herein, separable modulation of inflammatory versus metabolic pathways is also possible at the sacral stimulation site, but stimulation of vagal afferents at the ganglion without dosing results in activation of multiple pathways (similar to conventional cervical VNS).

[0030] Furthermore, analysis of changes in blood neurotransmitters after stimulation in different sites also fits the updated overall picture of the CAP pathway. Figure 8D shows the extent of blood concentrations / blood levels of neurotransmitters (i.e., epinephrine, norepinephrine, dopamine, and acetylcholine) after pFUS at three different anatomical target sites compared to LPS control (LPS CTRL). Asterisks characterize statistical significance (with a p-value threshold of less than 0.05) using a two-tailed t-test versus LPS-only control, and double crosses characterize significance versus both LPS control and alternative stimulation site data. N=5 for each experimental condition. Blood neurotransmitter profiles are dominated by acetylcholine (Ach) after targeted stimulation of the splenic site, consistent with the activation of abundant choline acetyltransferase (ChaT)-positive T cells present. This is accompanied by a modest rise in catecholamines at the splenic site (compared to stimulation without administration) for ChaT T cell activation. In contrast, stimulation without dosing produced a more modest increase in ACh but a larger increase in blood catecholamines (consistent with system-wide activation of multiple efferent CAP arms, i.e., splenic and intestinal CAP pathways). Finally, sacral ganglion stimulation produced modest increases in both ACh and catecholamines (compared to the splenic stimulation site or stimulation without dosing, respectively), but stimulation at this site produced the highest increase in blood dopamine. This suggests activation of additional vagus-mediated anti-inflammatory pathways mapped to the adrenal gland. Taken together, these results demonstrate that image / anatomically targeted pFUS can activate neurons at multiple locations within a mapped reflex and can be used to assess the level of target activation relative to non-targeted neural pathways within the PNS.

[0031] 9A-9C show organ-based peripheral focused ultrasound neuromodulation (pFUS) across the metabolic system. FIG. 9A is a schematic of pFUS-based high-precision organ-based neuromodulation in which innervation points of known axon populations are targeted for stimulation using focused pulsed ultrasound. Targets investigated herein include innervation points and sensory endings within the liver, pancreatic tail, and jejunal region of the small intestine (methods used to locate and target these sites are described in the Methods section). FIG. 9B is a timeline of pFUS stimulation and blood sampling performed within the LPS-induced hyperglycemia model. FIG. 9C shows circulating blood glucose levels are shown at 0, 5, 15, 30, and 60 min for LPS control (no pFUS, yellow circle), liver pFUS (blue circle), pancreatic pFUS (purple circle), and GI pFUS (orange circle) groups, n=6 for each group.

[0032] 10A-10B show a comparison of the effects of alternative pFUS stimulation sites on metabolic markers. FIG. 10A shows hypothalamic markers in animals stimulated using pFUS. ELISA optical intensity and concentrations of molecules related to insulin signaling and glucose utilization including insulin receptor substrate 1 (IRS1), phosphorylated protein kinase B (phos-Akt), glucose transporter 4 (GLUT4), and glucose-6-phosphate (G-6-phos). Data is shown for LPS control (no pFUS, LPS CTRL), liver pFUS (liver), pancreatic pFUS (pancreas), and gastrointestinal pFUS (GI). FIG. 8B shows blood markers in animals stimulated using pFUS. ELISA-based concentrations of blood hormones and markers from collected blood samples include insulin concentration, glucagon concentration, leptin concentration, and GLP1 concentration. Asterisks mark statistical significance using two-tailed t-tests versus LPS-only controls (with p-value threshold <0.05). n-6 in each experimental condition.

[0033] Gastrointestinal (GI) pFUS had significantly different effects on hypothalamic and blood metabolic markers (FIGS. 10A and 10B) compared to liver stimulation results. Like pancreatic stimulation, GI stimulation had no effect on blood insulin, blood glucagon, or blood leptin. However, GI stimulation also had no effect on IRS activity or GLUT4 and glucose-6-phosphate levels in the hypothalamus. Thus, unlike pancreatic or liver stimulation, the GI results suggest that non-insulin signaling pathways may be responsible for the GI stimulation effect in LPS-induced hyperglycemia. Furthermore, the increase in pAkt activity in GI stimulated hypothalamic samples suggests the hypothalamus may be involved in this other pathway, either directly or as an indirect effect. GI ultrasound stimulation may result in the regulation of incretin and glucagon-like peptide 1 (GLP-1) secretion in the small intestine in certain embodiments of the present technology.

[0034] FIG. 11A shows the blood concentration of epinephrine after pFUS and the control without LPS / pFUS (LPS CTRL) at each target stimulation site. FIG. 11B shows the blood concentration of norepinephrine after pFUS and the control without LPS / pFUS (LPS CTRL) at each target stimulation site. FIG. 11C shows the blood concentration of dopamine after pFUS and the control without LPS / pFUS (LPS CTRL) at each target stimulation site. Asterisks characterize statistical significance using a two-tailed t-test versus the LPS only control (with a p-value threshold of less than 0.05). There were n=6 in each experimental condition. As provided herein, hepatic pFUS caused an increase in blood norepinephrine. Meanwhile, pancreatic stimulation caused no obvious change in norepinephrine concentration, and intestinal stimulation caused a specific change in dopamine in the neurotransmitter profile (FIGS. 11A-11C). Sympathetic nerves are co-located with glucose- and fatty acid-sensing afferent fibers at the hepatic hilum. These nerves may provide a mechanism for hepatic vasoconstriction and blood pressure regulation. Dopaminergic neurons are present in the gut (but to a lesser extent in the liver and pancreas). These nerves are co-localized with neuroendocrine cells that produce GLP and act as an additional regulatory component in metabolic control and homeostasis. The data herein demonstrate that pFUS can be applied to rapidly screen potential therapeutic stimulation sites (both neural and neuroendocrine cell origins) to stimulate specific anatomical targets locally with high precision, providing novel techniques of regulation and further understanding of physiological systems.

[0035] pFUS experiments as disclosed herein for both in vivo and in vitro stimulation were performed as follows. The system used for pFUS stimulation consisted of a 1.1 MHz single element transducer (Sonic Concepts H106), a matching network (Sonic Concepts), an RF power amplifier (ENI 350L) and a function generator (Agilent 333120A). The 70 mm diameter transducer had a spherical surface with a radius of curvature of 65 mm. The transducer had a 20 mm diameter hole for inserting the imaging transducer centrally between the transducer alignment and the anatomical target. The numerically simulated pressure profile had a full width at half maximum (FWHM) amplitude of 1.8 mm laterally and 12 mm in depth. Acoustic coupling to the animal was achieved using a 6 cm cone filled with degassed water. A function generator was used to generate a pulsed sinusoidal waveform that was amplified by an RF amplifier and sent to an impedance matching network. The experiments in this manuscript were performed with a pulse repetition period of 0.5 ms (corresponding to a pulse repetition frequency of 2000 Hz) and a pulse width of 23 W / cm 2 A pulse amplitude of 1.0 s (burst average) and a burst duration of 136 μs (corresponding to a duty cycle of 0.27) were used. A voltage-pressure calibration of the transducer was performed prior to use with a needle-type hydrophone (ONDA HNA-0400) and is reported.

[0036] For the in vivo experiments as provided herein, a Vivid E9 or 11L ultrasound system and probe (GE Healthcare) was used to image the anatomical targets prior to pFUS neuromodulation. The pFUS transducer was then positioned in the target area based on this initial imaging. A second ultrasound scan was performed using a smaller imaging probe (3S, GE Healthcare). This was placed at the pFUS transducer and the opening of the coupling cone. The imaging beam of the 3S probe was aligned with the pFUS transducer to allow for confirmation of the organ and anatomical target of interest. An ultrasound standoff was used if the focal depth of the pFUS transducer needed to be adjusted. Anatomical markers were used to position and align the ultrasound stimulus at each target stimulation site. Anatomical markers were used to position the untreated ganglion contained within the internal carotid artery (as the vagus nerve runs along it towards the skull) and the posterior foramen lacunae, where ganglion is known to exist as an enlargement of the vagus nerve cell body at the base of the skull. For the sacral ganglion, the four vertebrae of the sacrum were positioned and the ultrasound transducer was aligned in the direction of the sacral foramen. The anatomical marker used for the liver stimulation experiments is the hepatic hilum (the so-called portal vein's entrance into the liver). For the pancreas, the splenic vein (imaged via the lateral left lateral side through the spleen to avoid non-specific liver stimulation) was used as the anatomical marker of the pancreatic tail. The pancreatic tail was chosen because it is known to contain dense islet cells and reside distal to the duodenum. Imaging of the upper left quadrant of the abdominal cavity was used to identify the jejunal region of the small intestine, which appears under diagnostic imaging with Doppler ultrasound as a highly vascularized folded structure with numerous folds. Figures 8A and 9A provide a schematic diagram and description of the location of the anatomical ultrasound targets of the ganglion and end organs.

[0037] Adult male Sprague-Dawley rats (250–300 g, Charles River Laboratories), aged 8–12 weeks, were housed on a 12-h light / dark cycle and allowed to acclimate for 1 week before experiments were performed. Housing was maintained at 25°C, and regular rodent chow was available ad libitum. Lipopolysaccharide (LPS) from Escherichia coli (0111:B4, Sigma Aldrich) was used to induce metabolic disturbance (i.e., hyperglycemia). 10 mg / kg LPS was administered by intraperitoneal injection, and blood was collected from the tail vein at 0, 5, 15, 30, and 60 min after LPS administration. The selected time points correspond to the onset and progression of hyperinsulinemia / hyperglycemia within the LPS-induced cytokine storm and insulin resistance model. Blood glucose levels were measured by a OneTouch Elite glucometer (LifeScan, Johnson and Johnson). After the 60 min time point, brain tissue (hypothalamus) samples were also collected terminally and homogenized in a solution of phosphate buffered saline (PBS) containing phosphatase (0.2 mM phenylmethylsulfonyl fluoride, 5 ug / mL aprotinin, 1 mM PC benzamidine, 1 mM sodium orthovanadate, and 2 uM cantharidin) and protease inhibitors (1 uL to 20 mg of tissue, as per Roche diagnostics instructions). Additional tissue samples (e.g., ganglia, spleen, liver, and intestinal tissue) were obtained at the 60 min time point and quickly frozen in liquid nitrogen after analysis. Blood samples collected throughout the study were stored with disodium (ethylenedinitrilo)tetraacetate (EDTA) anticoagulant. All samples were stored at -80°C prior to analysis.Tissues and terminal blood samples were analyzed by enzyme-linked immunosorbent assay (ELISA) for metabolic hormones or signaling molecules including TNF (tumor necrosis factor, Invitrogen), insulin (Crystal Chem), glucagon (Aviva Systems Biology), leptin (Aviva Systems Biology), IRS1 (insulin receptor substrate 1, Santa Cruz), phos-Akt (phosphorylated protein kinase B, MyBioSource), GLUT4 (glucose transporter 4, Lifespan Biosciences), and glucose-6-phosphate (G-6-phos, Lifespan Biosciences) according to the manufacturer's instructions. Catecholamine concentrations were measured from tissue samples using an HPLC protocol, and serum samples were injected directly into the machine without pretreatment within a short time. Tissue homogenates were first homogenized with 0.1 M perchloric acid and centrifuged for 15 min. After the supernatant was separated, the samples were injected into the HPLC. Catecholamines, norepinephrine and epinephrine were analyzed by HPLC equipped with an in-line UV detector. The test column used for this analysis was a Supelco Discovery C18 (15 cm x 4.6 mm ID, 5 μm particle size). The biphasic mobile phase consisted of [A] acetonitrile: [B] 50 mM KH2PO4 and was set to pH 3 (using phosphoric acid). The solution was then buffered using 100 mg EDTA and 200 mg / L 1-octane-sulfonic acid. The final concentration of the mobile phase mixture was set to A:B = 5:95. A flow rate of 1 mL / min was used to minimize column pressure compression resulting from the viscosity of the mobile phase utilized, improving overall peak resolution while keeping the column consistently at 20°C. The UV detector was maintained at 254 nm wavelength, which is known to capture the absorption of catecholamines, including norepinephrine, epinephrine and dopamine.

[0038] For stimulation, animals were anesthetized using 2-4% isoflurane and placed on a water-circulating heating pad to prevent hyperthermia during the procedure. Prior to neuromodulation, the area above the anatomical region of interest was shaved with a disposable razor and animal clippers. After targeting (as described above), ultrasound stimulation was applied for 1 min duration. LPS was then administered immediately after the first ultrasound stimulation (as described above). A 1 min ultrasound stimulation was then applied after LPS administration for a total duration of 2 min. Animals were then incubated under anesthesia and blood samples were taken as described above. After incubation, animals were euthanized and tissue and blood samples were obtained as described above.

[0039] The disclosed neuromodulation technology caused different changes to glucose transporter pathway molecules and / or incretin pathway molecules in individual tissues such as the intestine, the gastrointestinal tract, and the kidney. In one embodiment, the reduction in SGLT2 in the kidney occurred simultaneously with the increase in GLP1, SGLT1, GLUT2, and GLUT5 in the intestine. As a result of the overall complex changes, the patient's blood glucose rise rate is increased because the neuromodulation changes in the kidney act to impede glucose reabsorption into the blood, and the neuromodulation changes in the intestine act to increase gastric emptying. The changes can be mediated by the hypothalamus through signaling after hepatic neuromodulation.

[0040] In certain embodiments, the neuromodulation provided herein may be used as a treatment for subjects with metabolic diseases. Techniques that may be applied to treat glucose metabolism and related diseases and techniques that may alter disease progression are also provided herein. In one embodiment, liver modulation in one or more regions of interest may be used to treat diabetes (i.e., type 1 or type 2 diabetes), hyperglycemia, sepsis, trauma, infection, diabetes-related dementia, obesity, or eating disorders or metabolic diseases. In one embodiment, patients diagnosed with a disease may participate in neuromodulation treatment. After treatment, the patient may have achieved clinical benchmarks related to healthy patients. For example, a patient suffering from diabetes may exhibit blood glucose and / or insulin levels above the normal range. After treatment, the patient may have blood glucose and / or insulin levels within the normal range. Routine measurements of abnormal insulin and glucose utilization include fasting blood glucose tests, oral glucose tolerance tests, continuous glucose monitoring, HOMA score or HOMA index, glucose and insulin clamps, and other related techniques.

[0041] As provided herein, the neuromodulation therapy provided herein may involve the application of energy to an area of ​​interest as part of a treatment regimen. The treatment regimen may include one dose or multiple doses applied within a defined treatment time. For example, neuromodulation may be once a day in an area of ​​interest (e.g., the hepatic hilum), whereby the once a day treatment may be for a period of two or more consecutive days according to preset regulation parameters.

[0042] In certain embodiments, energy is applied to target tissues that are internal tissues or internal organs that are difficult to access using electrical stimulation techniques. Potential tissue targets include gastrointestinal (GI) tissues (stomach, intestines), muscle tissues (cardiac muscle, smooth muscle, and skeletal muscle), epithelial tissues (epithelial layers, lining of organs / GI), connective tissues, glandular tissues (exocrine / endocrine glands), and the like. In one example, focused application of energy to a neuromuscular junction promotes neurotransmitter release at the neuromuscular junction without an upstream action potential. In another example, focused application of energy at a terminal axon end provides cumulative activity that triggers action potentials within sensory neurons. In yet another example, focused application of energy at secretory cells, with or without adjacent nerve cells, causes those cells to activate and secrete. Potential regulatory targets may include the portion of the pancreas responsible for controlling insulin release, the portion of the liver responsible for glucose regulation, or the portion of the kidney responsible for glucose reabsorption and excretion.

[0043] Neuromodulation to a targeted region of interest can effect changes in a physiological manner to disrupt, reduce or enhance one or more physiological pathways in a subject to obtain a desired physiological outcome. Furthermore, because localized energy application produces systemic changes, different physiological pathways may be altered in different ways and at different locations in the body, resulting in an overall characteristic profile of physiological changes in the subject caused by the targeted neuromodulation properties for a particular subject. While these changes are complex, current neuromodulation techniques provide one or more measurable targeted physiological results for the treated subject that are the result of neuromodulation and would not be obtainable without the application of energy or other intervention to one or more targeted regions of interest. Furthermore, other types of interventions (e.g., drug therapy) can obtain a portion of the physiological changes caused by neuromodulation. In certain embodiments, the profile of physiological changes induced as a result of neuromodulation can be specific to the neuromodulation (and its associated modulation parameters) in one or more targeted regions of interest and can vary from patient to patient.

[0044] Neuromodulation techniques discussed herein can be used to induce physiological outcomes such as changes in the concentration (e.g., increase, decrease) and / or properties of a molecule of interest, such as glucose transporter pathway molecules and / or incretin pathway molecules. As provided herein, glucose transporter pathway molecules are a group of membrane proteins that facilitate glucose transport across cell membranes. Glucose transporter pathway molecules can include one or more of the GLUT or SLC2A family proteins, such as GLUT-1, GLUT-2, GLUT-3, GLUT-4, GLUT-5, GLUT-6, GLUT-7, GLUT-8, GLUT-9, GLUT-10, GLUT-11, GLUT-12, or GLUT-13. As provided herein, incretin pathway molecules can include glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), also known as incretins and based on their ability to enhance glucose-stimulated insulin secretion. Incretins are secreted by endocrine cells in the small intestine. Other approaches include stimulating tissues to target changes in molecules upstream of incretins and their secretion, including enzymes involved in their production and release, including DPP-4.

[0045] Causing a change via neuromodulation of a molecule of interest, i.e., one or more glucose transporter pathway molecules and / or incretin pathway molecules, can refer to modulating or affecting the concentration (blood, tissue) or properties (covalent modifications) of a molecule 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 glucose transporter pathway molecules and / or incretin pathway molecules can include changes in molecular properties such as protein expression, secretion, translocation, and changes in the properties of the molecule that are induced or influenced by the application of energy itself. It may include direct activity changes based on ion channel effects, either as a result of a molecule directly affecting an ion channel. Modulation of a molecule of interest may also refer to maintaining a desired concentration of the molecule, such that no predicted changes or fluctuations in concentration occur as a result of neuromodulation. Modulation of a molecule of interest may refer to causing a change in molecular properties, such as enzyme-mediated covalent modifications (phosphorylation, acetylation, ribosylation, etc.). That is, it will be understood that selective modulation of a molecule of interest may refer to molecular concentration and / or molecular properties. The molecule of interest may be a biological molecule, such as one or more of carbohydrates (monosaccharides, polysaccharides), lipids, nucleic acids (DNA, RNA), or proteins. In certain embodiments, the molecule of interest may be a signaling molecule, such as a hormone (amine hormone, peptide hormone, or steroid hormone).

[0046] The disclosed neuromodulation and cell regulation techniques may be used in combination with a neuromodulation system. FIG. 9 is a schematic diagram of a system 10 for neuromodulation to achieve neurotransmitter release and / or activate components of a synapse (e.g., presynaptic cells, postsynaptic cells) in response to the application of energy. The illustrated system includes a pulse generator 14 coupled to an energy application device 12 (e.g., an ultrasound transducer). The energy application device 12 is configured to be used directed, for example, via a lead or wirelessly connected, to a region of interest in an internal tissue or organ of a subject and subsequently receive an energy pulse that produces changes in glucose transporter pathway molecules and / or incretin pathway molecules. In certain embodiments, the pulse generator 14 and / or the energy application device 12 may be implanted in a biocompatible site (e.g., the abdomen), with one or more leads coupled to the energy application device 12 and the interior of the pulse generator 14. For example, the energy application device 12 may be a MEMS transducer, such as a capacitive micromachined ultrasound transducer.

[0047] In certain embodiments, the energy application device 12 and / or the pulse generator 14 may be in wireless communication with the controller 16, which may then provide instructions to the pulse generator 14, for example. In other embodiments, the pulse generator 14 may be an external device, applying energy in a non-invasive manner, for example, percutaneously or from a location outside the subject's body. In certain embodiments, it may be integrated within the controller 16. In embodiments where the pulse generator 14 is external, the energy application device 12 may be operated by a caregiver and placed at or on a point on the subject's skin such that the energy pulse is delivered percutaneously to the desired internal tissue. Once positioned to apply the energy pulse to the desired site, the system 10 may initiate neuromodulation to achieve a clinical effect, such as treating a metabolic disease.

[0048] In certain embodiments, the system 10 includes an evaluation device 20 coupled to the controller 16 for evaluating a characteristic indicative of whether a change in glucose transporter pathway molecules and / or incretin pathway molecules as a result of neuromodulation is obtained. In one embodiment, the characteristic may be local. For example, modulation may result in a change in local tissue or function, such as a change in tissue structure, a local change in a particular molecule concentration, tissue displacement, increased fluid movement, etc.

[0049] The modulation may result in systemic or non-localized changes, and the target physiological outcome may be related to a concentration change of a molecule in blood, or a change in a property of a tissue that does not include the region of interest where the energy is directly applied. In one example, displacement may be a surrogate measurement for the desired modulation, and a displacement measurement below a predicted displacement value may result in a modification of the modulation parameters until the predicted displacement value is induced. Thus, the evaluation device 20 may be configured to measure a concentration change in some embodiments. In some embodiments, the evaluation device 20 may be an imaging device configured to evaluate changes in organ size and / or position. While the illustrated elements of the system 10 are shown individually, it will be understood that some or all of the elements may be combined with each other. Furthermore, some or all of the components may communicate with each other in a wired or wireless manner.

[0050] Based on the evaluation, adjustment parameters of the controller 16 may be altered. For example, a change in adjustment parameters such as pulse frequency or other parameters may be desired if the desired adjustment relates to a change in concentration (blood or tissue concentration of one or more molecules) within a defined time window (e.g., 5 minutes, 30 minutes after the energy application procedure has begun) or compared to a baseline measurement at or before the start of the procedure. This may then be provided to the controller 16 either by the operator to define or adjust the energy application or adjustment parameters of the pulse generator 14, or via an automatic feedback loop.

[0051] The system 10 provided herein may provide energy pulses according to various adjustment parameters. For example, the adjustment parameters may include various stimulation time patterns ranging from continuous to intermittent. Energy may be delivered for a period of time at a specific frequency during the signal light time while stimulating intermittently. The signal light time is followed by a period of no energy delivery called the signal off time. The adjustment parameters may also include the frequency and duration of stimulation application. The application frequency may be continuous, for example, or may be delivered for various periods of time, such as within a day or within a week. The treatment period may last for various periods of time, including, but not limited to, minutes to hours. In certain embodiments, the treatment period with a specific stimulation pattern may last for one hour and may be repeated at intervals of, for example, 72 hours. In certain embodiments, treatment may be delivered more frequently, such as every three hours, or for shorter durations, such as 30 minutes. The application of energy according to adjustment parameters, such as treatment period and frequency, may be adjustably controlled to obtain a desired result.

[0052] 10 is a block diagram of certain components of the system 10. As provided herein, the system 10 for neuromodulation may include a pulse generator 14 adapted to generate a plurality of energy pulses for application to tissue of a target. The pulse generator 14 may be separate or integrated into an external device such as a controller 16. The controller 16 includes a processor 30 for controlling the device. Software codes and instructions for execution by the processor 30 to control the various components of the device are stored in a memory 32 of the controller 16. The controller 16 and / or the pulse generator 14 may be connected to the energy application device 12 via one or more leads 33 or may be connected wirelessly.

[0053] The controller 16 also includes a user interface and display 36 with input / output circuitry 34 adapted to allow the physician to select or provide adjustment parameters to the adjustment program. Each adjustment program may include one or more sets of adjustment parameters, including pulse amplitude, pulse width, pulse frequency, etc. The pulse generator 14 responds to control signals from the controller device 16 to modify its internal parameters to change the stimulation characteristics of the energy pulses transmitted via the leads 33 to the subject to which the energy application device 12 is applied. Any suitable type of pulse generating circuit may be used, including, but not limited to, a constant current source, a low voltage source, multiple independent current or voltage sources, etc. The applied energy is a function of the current amplitude and pulse width duration. The controller 16 allows the energy to be adjustably controlled by modifying the adjustment parameters and / or initiating or canceling / reducing the energy application at specific times. In one embodiment, the adjustable control of the energy application device is based on information regarding the concentration of one or more molecules (e.g., blood molecules) in the subject. If the information is from the evaluation device 20, a feedback loop can activate the adjustable control. For example, if blood or urine glucose concentration is above a predefined threshold or range as measured by the evaluation device 20, the controller 16 may initiate energy application to a region of interest (e.g., liver) having a regulation parameter associated with a reduction in blood glucose. Initiation of energy application may be triggered by the glucose concentration moving above a predefined (e.g., desired) threshold or outside a defined range. In another embodiment, the adjustable control may be in the form of altering the regulation parameter when an initial application of energy fails to produce a predicted change in a target physiological outcome (e.g., concentration of a molecule of interest) within a defined time frame (e.g., 1 hour, 2 hours, 4 hours, 1 day).

[0054] In one embodiment, the memory 32 stores different operation modes selectable by the operator. For example, the stored operation modes may include instructions for executing a set of adjustment parameters associated with a particular treatment site, such as an area of ​​interest in the liver, pancreas, gastrointestinal tract, or spleen. Different sites may have different associated adjustment parameters. Rather than having the operator manually enter the mode, the controller 16 may be configured to execute the appropriate instructions based on the selection. In another embodiment, the memory 32 stores operation modes for different types of treatments. For example, activation may be associated with different stimulation pressures or frequency ranges versus those associated with inhibiting or blocking tissue function. In one specific example, when the energy application device is an ultrasound transducer, the time-averaged power (time-averaged intensity) and peak positive pressure are greater than 1 mW / cm. 2 ~30,000mW / cm 2 (time-averaged intensity) and in the range of 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 vibration is in the range of 0.1 to 10 mm. The selected frequency may vary depending on the mode of energy application, for example, ultrasonic actuator or mechanical actuator.

[0055] In another embodiment, the memory 32 stores a calibration or setup mode that allows adjustment or modification of tuning parameters to obtain a desired result. In one example, stimulation begins at low energy parameters and gradually increases either automatically or upon receiving operator input. In this manner, the operator can obtain adjustment of the induced effect as tuning parameters are changed.

[0056] The system may also include an imaging device to facilitate focusing of the energy application device 12. In one embodiment, the imaging device may be integrated with the energy application device 12 or may be a device similar to the energy application device 12 such that different ultrasound parameters (frequency, aperture, or energy) are applied to select (e.g., spatially select) a region of interest and to focus energy to the selected region of interest for targeting and subsequent neuromodulation. In another embodiment, the memory 32 may store one or more targeting or focusing modes used to spatially select a region of interest within an organ or tissue structure. The spatial selection may include selecting a sub-region of the organ to identify a volume of the organ corresponding to the region of interest. The spatial selection may be based on image data provided herein. Based on the spatial selection, the energy application device 12 may be focused to the selected volume corresponding to the region of interest. For example, the energy application device 12 may be configured to be initially operated in a targeting mode and to apply energy in the targeting mode used to capture image data used to identify the region of interest. The energy in the targeting mode is applied at a level and / or with adjustment parameters other than those suitable for the preferred activation, however, once a region of interest is identified, the controller 16 may then be operated in the treatment mode according to the adjustment parameters associated with the preferred activation.

[0057] The controller 16 may also be configured to receive inputs related to the target physiological outcome as inputs for the selection of the modulation parameters. For example, when using imaging modalities to assess tissue characteristics, the controller 16 may be configured to receive a calculated index or parameter of the characteristic. Based on whether the index or parameter is above or below a defined threshold, the modulation parameters may be altered. In one embodiment, the parameter may be a measurement of tissue displacement of the affected tissue or a measurement of the depth of the affected tissue. Other parameters may include evaluating the concentration of one or more molecules of interest (e.g., evaluating changes in one or more concentrations compared to a threshold or baseline / control, percentage of change, determining whether the concentration is within a desired range). Furthermore, the energy application device 12 (e.g., an ultrasound transducer) may operate under the control of the controller 16 to a) acquire tissue image data that can be used to spatially select a region of interest within the target tissue, b) apply modulation energy to the region of interest, and c) acquire images to determine that a target physiological outcome associated with a change in glucose transporter pathway molecules and / or incretin pathway molecules occurs (e.g., displacement measurements). In one such embodiment, the imaging device, the evaluation device 20 and the energy application device 12 may be the same device.

[0058] In another embodiment, the desired adjustment parameter set may also be stored by the controller 16. In this manner, subject-specific parameters may be determined. Furthermore, the effectiveness of such parameters may be evaluated over time. If a particular set of parameters is less effective over time, the subject may develop insensitivity to the activated pathway. If the system 10 includes an evaluation device 20, the evaluation device 20 may provide feedback to the controller 16. In certain embodiments, feedback may be received from a user or from the evaluation device 20 that represents a characteristic of the target physiological outcome. The controller 16 may be configured to cause the energy application device to apply energy according to the adjustment parameters, and dynamically adjust the adjustment parameters based on the feedback. For example, based on the feedback, the processor 16 may automatically change the adjustment parameters (e.g., frequency, amplitude, or pulse width of the ultrasound beam or mechanical vibration) in real time and in response to feedback from the evaluation device 20.

[0059] In one example, the technology may be used to treat a subject with a metabolic disease. The technology may be used to regulate blood glucose levels in a subject with a glucose regulation disease. Thus, the technology may be used to promote homeostasis of a molecule of interest or promote a desired blood concentration range or concentration range of one or more molecules of interest (e.g., glucose, insulin, glucagon, or a combination thereof). In one embodiment, the technology may be used to control blood (i.e., blood) glucose levels. In one embodiment, the following thresholds may be used to maintain blood glucose levels in dynamic equilibrium in the normal range: Fasting: Less than 50 mg / dL (2.8 mmol / L): Insulin shock 50~70mg / dL (2.8~3.9mmol / L): Hypoglycemia / hypoglycemia 70~110mg / dL (3.9~6.1mmol / L): Normal 110-125mg / dL (6.1-6.9mmol / L): Elevated / impaired (prediabetic) 125(7mmol / L): Diabetic Non-fasting (approximately 2 hours after a meal): 70~140mg / dL: Normal 140-199 mg / dL (8-11 mmol / L): Elevated or "borderline" / prediabetic Over 200 mg / dL (11 mmol / L): Diabetes. For example, the technique can be used to maintain blood glucose concentrations below about 200 mg / dL and / or above about 70 mg / dL. The technique can be used to maintain glucose in the range of about 4-8 mmol / L or about 70-150 mg / dL. The technique can be used to maintain a subject's (e.g., patient's) euglycemic range, which may be an individual range based on the patient's individual factors such as weight, body mass index, age, sex, genetics, clinical history, etc. Thus, the application of energy (e.g., ultrasound energy) to one or more regions of interest may be adjusted in real time based on the desired target concentration of the molecule of interest, or in a feedback loop based on input from the evaluation device 20. For example, if the evaluation device 20 is a glucose monitor (e.g., blood glucose, excreted glucose), the real time glucose measurement may be used as an input to the controller 16.

[0060] In another embodiment, the technology may be used to induce a characteristic profile of physiological changes. For example, the characteristic profile may include a group of molecules of interest that increase in concentration in tissues and / or blood as a result of energy application, and another group of molecules of interest that decrease in concentration in tissues and / or blood as a result of energy application. The characteristic profile may also include a group of molecules that do not change as a result of energy application. The characteristic profile may define concurrent changes associated with a desired physiological outcome. For example, the profile may include a rise in blood glucose excreted in urine along with a rise in glucose excreted in urine. It may also include a reduction in mid-range glucose.

[0061] The desired target tissue may be an internal tissue or organ that contains a synapse between an axon terminal of a peripheral nerve and a non-neuronal cell. This synapse may be stimulated by applying energy directly to the axon terminal within the focal region of an ultrasound transducer focused on a region of interest in the target tissue or organ, causing the release of molecules into the synaptic cleft. For example, neurotransmitter release and / or changes in ion channel activity subsequently cause downstream effects such as activation of glucose metabolism. The region of interest may be selected to include a particular type of axon terminal, such as axon terminals of a particular neuron type, and / or axon terminals that form synapses with a particular type of non-neuronal cell. Thus, the region of interest may be selected to correspond to a portion of the target tissue that has the desired axon terminal (and associated non-neuronal cell). The energy application may be selected to preferentially cause the release of one or more molecules, such as neurotransmitters, from the nerve within the synapse, or to directly activate the non-neuronal cell itself by direct energy transfer (i.e., mechanical signaling or voltage-activated proteins within the non-neuronal cell), or to cause activation within both neuronal and non-neuronal cells that induce the desired physiological effect. The region of interest may be selected as a site of neural entry into the organ. In one embodiment, liver stimulation or modulation may refer to modulation of a region of interest at or adjacent to the hilum of the liver.

[0062] Energy may be focused or substantially concentrated at the region of interest and to only a portion (i.e., a small area) of the internal tissue or organ. For example, this may be less than about 50%, 25%, 10%, or 5% of the total volume of the tissue or organ. In one embodiment, energy may be applied to two or more regions of interest in the target tissue, and the total volume of the two or more regions of interest may be less than about 90%, 50%, 25%, 10%, or 5% of the total volume of the tissue. In one embodiment, energy is applied to only about 1%-50% of the total volume of the tissue, only about 1%-25% of the total volume of the tissue, only about 1%-10% of the total volume of the tissue, or only about 1%-5% of the total volume of the tissue. In certain embodiments, only axonal terminals 46 in the region of interest of the target tissue directly receive the applied energy and release neurotransmitters, while unstimulated axonal terminals outside the region of interest do not receive substantial energy and are therefore not activated / stimulated in the same manner. In some embodiments, axon endings 46 in the portion of the tissue that directly receives this energy may induce alterations in neurotransmitter release. In this manner, tissue subregions may be targeted in a granular manner for neuromodulation. For example, 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 that directly receives the energy to induce a desired combined physiological effect. In certain embodiments, the energy is delivered to a site about 25 mm apart. 3 In certain embodiments, the energy may be focused or concentrated within a volume of less than about 0.5 mm 3 ~50mm 3 The focal volume and focal depth for focusing or concentrating energy within a region of interest may be affected by the size / configuration of the energy application device 12. The focal volume of the energy application may be defined by the focal region of the energy application device 12.

[0063] As provided herein, energy may be applied substantially only to one or more regions of interest to preferentially activate synapses in a targeted manner to obtain a targeted physiological outcome, and not substantially in a general or non-specific manner throughout the tissue. Thus, only small regions of axon terminals of different types in the tissue are exposed to the direct energy application. Imaging data of the tissue or organ may serve to identify a region of interest in the target organ such that energy can be focused to the region of interest and not to the surrounding tissue. For example, a region of interest within an organ containing either blood vessels, nerves, or other anatomical markers may be spatially selected and used to identify a region with specific axon terminals or synapses. The disclosed selection of a region of interest using imaging information may be used in combination with organ or tissue structures (e.g., liver, pancreas, gastrointestinal tissue).

[0064] The disclosed techniques may be used to evaluate neuromodulation or cell modulation effects, which may then be used as input or feedback to select or modify neuromodulation parameters. The disclosed techniques may use direct assessment of tissue status or function as the target physiological outcome. Assessment may be performed before neuromodulation (i.e., baseline assessment), during neuromodulation, and / or after neuromodulation.

[0065] The evaluation techniques may include at least one of magnetic resonance functional imaging, diffusion tensor magnetic resonance imaging, positron emission tomography, or acoustic monitoring, thermal monitoring. The evaluation techniques may also include protein and / or marker concentration evaluation. Images from the evaluation techniques may be received by a system for automatic or manual evaluation. Based on the image data, the modulation parameters may also be changed. For example, changes or displacements in organ size may be used as a marker of local neurotransmitter concentration, may be used as a surrogate marker for exposing local cells to phenotype-modulating neurotransmitters, and may be effectively used as a marker of predicted effects on glucose metabolic pathways. Local concentration may refer to the concentration within the focal region of energy application.

[0066] Additionally or alternatively, the system may assess the presence or concentration of one or more molecules in tissue or blood. Concentrations in tissue may be referred to as local or resident concentrations. Tissue may be obtained by fine needle aspiration, and assessment of the presence or levels of a molecule of interest (e.g., metabolic molecule, marker of metabolic pathway, peptide signaling molecule, catecholamine) may be performed by any suitable technique known to those of skill in the art, such as enzyme-linked immunosorbent assay, mRNA sequencing, etc.

[0067] In other embodiments, the target physiological outcome may include, but is not limited to, tissue displacement, changes in tissue size, changes in concentration of one or more molecules (local, non-local, or blood concentration), changes in gene expression or marker expression, afferent activation, and cell migration. For example, tissue displacement (e.g., liver displacement) may occur as a result of the application of energy to the tissue. By evaluating the tissue displacement (e.g., via imaging), other effects may be inferred. For example, a particular displacement may be specific to a particular change in molecule concentration. In one example, a liver displacement of 5% may represent or be associated with a desired reduction in blood glucose concentration based on empirical data. In another example, tissue displacement may be evaluated by comparing reference image data (tissue image before application of energy to the tissue) with post-treatment image data (tissue image acquired after application of energy to the tissue) to determine a parameter of the displacement. The parameter may be a maximum or average displacement value of the tissue. If the displacement parameter is greater than a displacement threshold, the application of energy may be evaluated as tending to cause the desired target physiological outcome.

[0068] FIG. 11 is a flow diagram of a method 50 of treating a patient (i.e., a subject). In the method 50, a subject is identified 52. The subject may be a patient with a metabolic disease as provided herein. Identification may also be via a suitable diagnostic technique. Once identified, the subject is treated for the metabolic disease by applying energy to a target tissue of the subject 54. In one embodiment, an energy application device (ultrasonic energy, mechanical energy) is positioned such that an energy pulse is focused at a desired region of interest in an internal tissue (e.g., liver, GI, kidney, pancreas) at step 54, and a pulse generator applies one or more energy pulses to the region of interest of the target tissue to preferentially activate a subset of synapses in the target tissue. For example, this is positioned to stimulate axon terminals and alter glucose transporter pathway molecules and / or incretin pathway molecules as provided herein. In certain embodiments, the method 50 may include a step of evaluating the effect of the energy application. For example, one or more direct or indirect evaluations may be used. Based on the evaluation, adjustment parameters of one or more energy pulses may be altered (eg, dynamically or adjustably controlled) to achieve treatment of the subject.

[0069] In one embodiment, evaluation may be performed before and after application of the energy pulse to assess the change in glucose concentration as a result of the modulation. If the glucose concentration is above or below a threshold, appropriate changes can be made to the modulation parameters. For example, if the glucose concentration has a desired physiological outcome, the energy applied during neuromodulation may be backed off to a minimum level that supports the desired outcome. If the change in the characteristic relative to the threshold is associated with an insufficient change in glucose concentration, certain modulation parameters may be altered, such as, but not limited to, modulation amplitude or frequency, pulse shape, stimulation pattern and / or stimulation location. FIG. 12 is a flow diagram of a method 60 that, once identified (block 62), the subject is treated by applying non-invasive ultrasound energy to a region of interest in the liver (block 64). Application of non-invasive ultrasound energy alters glucose transporter pathway molecules and / or incretin pathway molecules in one or more tissues throughout the body as a result of neuromodulation of the liver. These changes in turn result in changes in blood glucose (e.g., a decrease) and changes in glucose excreted in the urine (e.g., an increase) that are indicative of successful treatment or the target physiological outcome. It will be appreciated that neuromodulation can result in many physiological effects related to glucose transporter pathway molecules and / or incretin pathway molecules, and one or more of these changes can be assessed.

[0070] The evaluated characteristic or condition may be a value or index, such as, for example, flow rate, concentration, cell population, or any combination thereof, which may then be analyzed by suitable techniques. For example, a relative change above a threshold may be used to determine whether a regulatory parameter has been altered. The desired modulation may be evaluated by a measured clinical outcome, such as the presence or absence of an increase in the size of a tissue structure (e.g., lymph node size), a change in the concentration of one or more released molecules (e.g., change relative to baseline concentration before neuromodulation). In one embodiment, the desired modulation may involve a concentration increase above a threshold, such as, for example, an increase in concentration of about 50%, 100%, 200%, 400%, 1000% relative to baseline. For blocking treatments, the evaluation may involve tracking a decrease in the molecule concentration over time, such as, for example, at least 10%, 20%, 30%, 50%, or 75% for the molecule of interest. Furthermore, for a particular subject, the desired blocking treatment may involve maintaining a relatively stable concentration of the molecule of interest in the context of other clinical signs that may have a tendency to increase the molecule concentration. That is, the desired block may block the potential rise. The rise or fall or other induced and measurable effect may be measured within a particular time window from the start of the treatment. For example, this may be within about 5 minutes, within about 30 minutes. In certain embodiments, when determining whether neuromodulation is desired, the change in neuromodulation is an instruction to stop the application of energy pulses. In another embodiment, one or more neuromodulation parameters are changed if neuromodulation is not desired. For example, the change in the modulation parameter may be an increase in pulse repetition frequency, such as a stepwise increase in frequency from 1 to 10,000 Hz, and evaluation of the desired characteristics until the desired neuromodulation is achieved. In another embodiment, the pulse width may be changed. In other embodiments, two or more parameters may be changed together, either in parallel or in sequence. After changing multiple parameters, if neuromodulation is not desired, the focus (so-called site) of energy application may be changed.

[0071] The technical effects of the present invention include inducing targeted changes in metabolic pathways and / or metabolic molecules through neuromodulation to treat a subject.

[0072] This specification uses examples to disclose the invention, including the best mode, and to enable those skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention 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 in substantial way from the literal language of the claims.

[0073] 10. System 12 Energy application devices 14 Pulse Generator 16 Controller 20 Evaluation Devices 30 processors 32 Memory 34 Input / Output Circuit 36 Display 50, 60 ways

Claims

1. 1. A method of treating a subject having a metabolic disease, comprising: applying ultrasonic energy to an internal tissue of the subject having the metabolic disorder to induce changes in glucose transporter pathway molecules and / or incretin pathway molecules in the subject; treating the metabolic disease, wherein the ultrasound energy is applied by an external energy application device; measuring said alteration or an indicator of said alteration in said subject; and determining based on the measurement that the ultrasound energy caused the change to the glucose transporter pathway molecule and / or the incretin pathway molecule to treat the subject.

2. The method of claim 1 , wherein the internal tissue is the liver.

3. The method of claim 2 , wherein said applying comprises applying said ultrasound energy to only a small region of the liver.

4. The method of claim 3, wherein the subregion is the hilum of the liver.

5. The method of claim 1 , wherein the internal tissue is a gastrointestinal tissue.

6. The method of claim 1 , comprising imaging the internal tissue to identify a region of interest in the internal tissue, and applying the ultrasound energy to the region of interest.

7. 2. The method of claim 1, wherein the metabolic disease is type 2 diabetes.

8. 2. The method of claim 1, wherein the metabolic disease is hyperglycemia.

9. 2. The method of claim 1, wherein the metabolic disease is insulin resistance.

10. The method of claim 1 , wherein the alteration comprises a change in concentration, activity, and / or location of the glucose transporter pathway molecule and / or the incretin pathway molecule.

11. 2. The method of claim 1, wherein the change comprises a change in concentration of the glucose transporter pathway molecule and / or the incretin pathway molecule in an internal organ that is not the internal tissue.

12. 2. The method of claim 1, wherein the glucose transporter pathway molecule and / or the incretin pathway molecule is one or more of sodium glucose cotransporter 2 protein (SGLT2), glucose transporter 2 (GLUT-2), glucose transporter 4 (GLUT-4), or glucagon-like peptide-1 (GLP-1).

13. The method of claim 1 , wherein measuring the indicator of the change comprises measuring the rise in glucose excreted in urine relative to a baseline measurement taken before the application.

14. 1. A system for treating a metabolic disease in a subject, the system comprising: an energy application device configured to apply energy to an internal tissue of the subject; a controller adapted to control the energy application device to apply the energy and cause changes to glucose transporter pathway molecules and / or incretin pathway molecules in the subject; and an evaluation device configured to receive an input representative of the change.

15. The system of claim 14 , wherein the evaluation device is a glucose monitor.

16. The system of claim 14 , wherein the energy application device comprises an ultrasonic transducer or a mechanical actuator.

17. The system of claim 14 , wherein the controller is configured to modify one or more parameters controlling the application of energy based on the input.

18. 1. A method of treating a subject having a metabolic disease, comprising: selecting a region of interest in a liver of the subject; applying ultrasound energy to the region of interest in the liver to induce changes to glucose transporter pathway molecules and / or incretin pathway molecules in the subject; Measuring blood glucose level, excreted glucose concentration, or both in said subject; and determining based on the blood glucose level, the excreted glucose concentration, or both that the ultrasound energy has caused the changes to the glucose transporter pathway molecules and / or the incretin pathway molecules to treat the subject.

19. 20. The method of claim 18, wherein determining that the ultrasound energy causes the change comprises determining that the blood glucose level has decreased relative to a baseline or threshold and that the excreted glucose concentration has increased relative to a baseline or threshold.

20. 20. The method of claim 18, wherein the subject is not taking medication for the metabolic disorder.

21. 1. A method of treating a subject having a metabolic disease, comprising: selecting a region of interest in a gastrointestinal tissue of the subject; applying ultrasonic energy to a region of interest in the gastrointestinal tissue to induce changes to glucose transporter pathway molecules and / or incretin pathway molecules; Measuring the subject's blood glucose level, the blood glucagon-like peptide 1 concentration, or both; and determining based on the blood glucose level, the blood glucagon-like peptide 1 concentration, or both, that the ultrasound energy caused the change to the glucose transporter pathway molecule and / or the incretin pathway molecule and treated the subject.

22. 22. The method of claim 21, wherein the region of interest is in the jejunum region of the small intestine.

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