Non-invasive nerve imaging using ultrasound

Abstract

A method for non-invasive nerve imaging using ultrasound imaging in conjunction with administration of a myelin basic protein (MBP) nerve imaging contrast agent. A pharmaceutical formulation for the administration (e.g., intravenous administration) of the contrast agents for nerve identification in ultrasound imaging is also provided.

Description

NON-INVASIVE NERVE IMAGING USING ULTRASOUNDRELATED APPLICATIONS

[0001] This applications claims the priority benefit of U.S. Provisional Application No. 63 / 643,693, filed 7 May 2024.TECHNICAL FIELD

[0002] The present disclosure relates generally to relates to non-invasive nerve imaging using ultrasound techniques, and specifically to the field of intravenous (IV) administration of myelin basic protein (MBP) nerve imaging contrast agents. Also provided are pharmaceutical formulations for intravenous administration of the contrast agents to allow for nerve identification.BACKGROUND

[0003] The subject matter discussed in this section should not be assumed to be prior art merely as a result of its mention in this section. Similarly, a problem mentioned in this section or associated with the subject matter provided as background should not be assumed to have been previously recognized in the prior art. The subject matter in this section merely represents different approaches, which in and of themselves can also correspond to implementations of the claimed technology.

[0004] Unintended nerve injury is a leading cause of morbidity associated with many lifesaving surgical procedures. Complications arising from these injuries are dependent on the severity and location of the nerve injury and often result in symptoms that negatively impact the patient's quality of life, such as loss of function and / or sensation, muscle atrophy, paralysis, and chronic neuropathy. Unfortunately, many nerve-related diseases are not diagnosed in time. By the time a diagnosis is determined, patients typically undergo surgery due to nerve-related injury, which may lead to a longer path to recovery. Concomitantly, nerves within a neurovascular bundle are often difficult to visualize before and / or during surgery, even under magnification, due to their intricacy, size, and anatomic variations among individuals.

[0005] Accordingly, magnetic resonance imaging (MRI) is frequently employed to visualize nerves due to its ability to distinguish nerves. However, the use of MRI is often associated with barriers including high costs, lack of accessibility at the point of patient care, and scarcity. Although other imaging techniques, such as ultrasound, may be more accessible, they are often limited to certain types of nerves and / or visibility during imaging is limited. As such, there is currently a need for non-invasive imaging technique that allows for the detection and treatment of nerve-related diseases in a timely manner, is cost effective, and is a widely available tool.SUMMARY

[0006] Certain embodiments are summarized below. These embodiments are not intended to limit the scope of the disclosure. Indeed, the present disclosure may encompass a variety of forms that may be similar to or different from the embodiments set forth below. The embodiments herein include image enhancement methods that may be employed in analytical, diagnostic, or prognostic applications related to myelin basic protein detection. The applications may be useful for imaging labeled nerves that are labeled with an imaging agent (e.g., an ultrasound nerve labeling agent) using ultrasound imaging techniques. Such enhancement methods are described herein.

[0007] An embodiment of a method for imaging myelin basic protein in a non- invasive setting by administration of a nerve labeling agent includes the steps of administering an ultrasound nerve labeling agent to a subject. The method also includes positioning an ultrasound probe on the subject’s skin, wherein the ultrasound probe is configured to and may apply ultrasound energy to a region of interest of the subject. The method also includes acquiring ultrasound image data of the region of interest, wherein the ultrasound image data, when displayed, depicts the one or more nerves labeled with the nerve labeling agent.

[0008] One example of the present method has the ultrasound nerve labeling agent including a compound of Formula I or a salt thereof, wherein Formula I is:wherein R1is an alkyl group, R2is an electron donating group, and R3is an electron withdrawing group. In some cases, Formula I is:

[0009] In some instances, one or more nerves labeled with the ultrasound nerve labeling agent may have greater contrast than unlabeled nerves. In some cases, the ultrasound nerve labeling agent may be administered via an intravenous injection.

[0010] A further embodiment of the present invention provides an ultrasound imaging agent composition comprising a compound of the above Formula I, or a salt thereof, wherein Formula I is:and an aqueous pharmaceutical carrier. In some instances, R1may be a lower alkyl groups from 1 to 6 carbon atoms, or wherein R1may be a lower alkyl group from 1 to 4 carbon atoms. And in some instances, R2may be a primary amine, secondary amine,tertiary amine, or an alkoxy group, or R3may be -CHO, -COR, -COOR, -COOH, - CONH2, -CONHR, -CONR2, -CF3, -CN, C=C(CN)2-SO3H, -NH3+, -NR3+, -NO2, - SOR, -SO2R, -SO2NH2, -SO2NHR, or -SO2NR2.

[0011] In some cases, the ultrasound imaging agent may include at least two solvents selected from PEG-300, propylene glycol, polyvinyl pyrrolidone, and Laurocapram. In such instances, the aqueous pharmaceutical carrier may include 1- 30% PEG-300, 1-20% propylene glycol, 1-10% polyvinyl pyrrolidone, and 0-10% Laurocapram based on volume.

[0012] In some embodiments, the aqueous pharmaceutical carrier may be surfactants, lipids, cyclodextrins, phospholipids, detergents, buffer solutions, stabilizers, and preservatives. Alternatively, or in addition, the ultrasound imaging agent composition may bind to myelinated nerves.

[0013] Further, in some embodiments, the ultrasound imaging agent, R2may be - NR’R”, -NHR, -NH2, -NC(NH2)2, -OH, - OR, -SR, -NHCOR, -OCOR, -C6H5, or - CH=CR2.

[0014] Further, in some embodiments, the Formula I is a salt further including an anion or a polyatomic anion.

[0015] Yet another embodiment provides an ultrasound imaging agent as above, wherein Formula I is:where R1is a methyl group, R2is NH2, and R3is SCh CHa).

[0016] Still another embodiment of the invention provides a method of reading a nerve labeled ultrasound image that includes determining one or more nerves is labeled with an ultrasound nerve labeling agent, wherein the one or more labeled nerves has a contrast greater than unlabeled nerves; and generating a diagnosis of a nerve-related disorder for a patient based on the ultrasound image. In some cases, theultrasound nerve labeling agent binds to myelin basic protein. And in some cases, the nerve-related disorder includes multiple sclerosis, Guillain-Barre syndrome, leukodystrophies, metachromatic leukodystrophy, Refsum's disease, adrenoleukodystrophy, Krabbe's disease, phenylketonuria, Canavan disease, Pelizaeus-Merzbacher disease, Alexander's disease, diabetic neuropathy, chemotherapy induced neuropathy, Alzheimer's disease, vascular dementia, dementia with Lewy bodies, or any combination thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:

[0018] FIG. l is a block diagram of an ultrasound system, in accordance with aspects of the present disclosure;

[0019] FIG. 2 shows ultrasound images of a sciatic nerve in an obese rat pre- and post- administration of the optical nerve labeling agent, in accordance with an embodiment of the present disclosure; and

[0020] FIG. 3 shows a graph of a comparison of image intensity based on the mean grey value in the ultrasound images of the sciatic nerve pre- and post- administration of the optical nerve labeling agent, in accordance with an embodiment of the present disclosure.DETAILED DESCRIPTION

[0021] One or more specific embodiments of the present disclosure will be described below. In an effort to provide a concise description of these embodiments, all features of an actual implementation may not be described in the specification. It should be appreciated that in the development of any such actual implementation, as in any engineering or design project, numerous implementation-specific decisions must be made to achieve the developers’ specific goals, such as compliance with system-related and business-related constraints, which may vary from oneimplementation to another. Moreover, it should be appreciated that such a development effort might be complex and time consuming, but would nevertheless be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill having the benefit of this disclosure.

[0022] When introducing elements of various embodiments of the present disclosure, the articles “a,” “an,” “the,” and “said” are intended to mean that there are one or more of the elements. The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. Any examples of operating parameters and / or environmental conditions are not exclusive of other parameters / conditions of the disclosed embodiments.

[0023] “Myelin-associated neuropathy” generally refers to any condition in which the insulating material ensheathing portions of neuronal cells becomes damaged or dysfunctional as a component of a syndrome, disease, or other pathological condition, such as, but not limited to, multiple sclerosis, Guillain-Barre syndrome, leukodystrophies, metachromatic leukodystrophy, Refsum's disease, adrenoleukodystrophy, Krabbe's disease, phenylketonuria, Canavan disease, Pelizaeus-Merzbacher disease, Alexander's disease, diabetic neuropathy, chemotherapy induced neuropathy, Alzheimer's disease, vascular dementia, dementia with Lewy bodies, or any combination thereof.

[0024] “Agent” refers to a solution or carrier for introducing a compound into a subject in a manner to allow the compound to be administered at a desired concentration and efficacy. The agent may include, but is not limited to, solvents, stabilization aids, buffers, and fillers. A pharmaceutical agent refers to the agents having medicinal or other biological properties including, but not limited to, use in therapy or diagnostics. An imaging or contrast agent refers to the agents having image enhancement (e.g., contrast enhancement) properties that may improve the usability or quality of images obtained of a patient in which the contrast or imaging agent is present in the subject being imaged. Imaging or contrast agents may be imaging modality specific, such as ultrasound or X-ray specific.

[0025] An agent exhibits “specific binding” for myelin if it associates more frequently with, more rapidly with, for a longer duration with, and / or with greater affinity to, myelin than with tissues not containing myelin. “Non-specific binding” refers to binding of the agent to non-myelin containing tissue. For relative binding values, such as specific binding or non-specific binding, each sample should be measured under similar physical conditions (i.e., temperature, pH, formulation, and mode of administration). Generally, specific binding is characterized by a relatively high affinity of an agent to a target and a relatively low to moderate capacity. Typically, binding is considered specific when the affinity constant Kais at least 106M'1. A higher affinity constant indicates greater affinity, and thus typically greater specificity. For example, antibodies typically bind antigens with an affinity constant in the range of 106M-1to 109M'1or higher. “Non-specific” binding usually has a low affinity with a moderate to high capacity. Non-specific binding usually occurs when the affinity constant is below 106M'1. Controlling the time and method used to contact the agent with the tissues reduces non-specific binding.

[0026] “Washing” generally refers to any method, such as but not limited to, immersion in, or flushing by repeated application of, a non-labeling solution or other substance, such as but not limited to water, saline, buffered saline, or ethanol, so as to provide a medium for dissociation, dispersal, and removal of unbound or non- specifically bound labeling compound from non-myelinated components of the tissue or sample of tissue without eliminating specific binding to myelin.

[0027] “ Control sample representative of the tissue section” refers to a tissue sample of a similar size, morphology, or structure as the tissue sample to be analyzed, and with a level of myelin whereby the sample's level of myelin serves as a reference to which other samples' myelin levels may be compared.

[0028] “Pharmaceutical carrier” refers to a composition, which allows the application of the agent material to the site of the application, surrounding tissues, or prepared tissue section to allow the agent to have an effective residence time for specific binding to the target or to provide a convenient manner of release. Solubilization strategies may include but are not limited to pH adjustments, salt formation, formation of ionizable compounds, use of co-solvents, complexation, surfactants and micelles, emulsions and micro-emulsions. The pharmaceutical carriermay include, but is not limited to, a solubilizer, percutaneous enhancers, detergent, buffer solution, stabilizers, and preservatives. Examples of these include but are not limited to, HC1, citric acid, DMSO, propylene glycol, ethanol PEG 300, cyclodextrins, citrate, acetate, phosphate, carbonate or tris(hydroxymethyl)aminomethane. An example of a percutaneous enhancer is Laurocapram which is capable of also transporting or carrying a compound across a barrier such as a transdermal penetration.

[0029] “Demyelination model” refers to any experimentally-induced damage to, or dysfunction of, the insulating material ensheathing portions of neuronal cells, that may be utilized in the experimental study of neuropathic demyelination, including, but not limited to, experimental allergic encephalomyelitis.

[0030] “Remyelination” refers to the spontaneous, therapeutic, or experimentally induced repair, regeneration, or otherwise enhanced constitution or functionality of the insulating material ensheathing neuronal axons.

[0031] “Alkyl” is intended to include linear, branched, or cyclic hydrocarbon structures and combinations thereof, including lower alkyl and higher alkyl. Alkyl groups are those of C20 or below. “Lower alkyl” refers to alkyl groups of from 1 to 6 carbon atoms, preferably from 1 to 4 carbon atoms, and includes methyl, ethyl, n- propyl, isopropyl, and n-, s- and t- butyl. Higher alkyl refers to alkyl groups having seven or more carbon atoms, preferably 7-20 carbon atoms, and includes n-, s- and t- heptyl, octyl, and dodecyl. Cycloalkyl is a subset of alkyl and includes cyclic hydrocarbon groups of from 3 to 8 carbon atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and norbornyl. Alkenyl and alkynyl refer to alkyl groups wherein two or more hydrogen atoms are replaced by a double or triple bond, respectively.

[0032] “ Substituted” refers to residues, including, but not limited to, alkyl, alkylaryl, aryl, arylalkyl, and heteroaryl, wherein up to three H atoms of the residue are replaced with lower alkyl, substituted alkyl, aryl, substituted aryl, haloalkyl, alkoxy, carbonyl, carboxy, carboxalkoxy, carboxamido, acyloxy, amidino, nitro, halo, hydroxy, OCH(COOH)2, cyano, primary amino, secondary amino, acylamino,alkylthio, sulfoxide, sulfone, phenyl, benzyl, phenoxy, benzyloxy, heteroaryl, or heteroaryl oxy.

[0033] “Electron donating group” refers to chemical groups that add electron density to the conjugated TI system making it more nucleophilic. Electron donating groups may be recognized by lone pairs of electrons on an atom adjacent to the TI system. Examples of electron donating groups include, but are not limited to, - NR’R”, -NHR, -NH2, -NC(NH2)2, -OH, - OR, -SR, -NHCOR, -OCOR, -C6H5, and - CH=CR2.

[0034] “Electron withdrawing group” refers to chemical groups that remove electron density from the conjugated TI system rendering the structure less nucleophilic. Electron withdrawing groups may be recognized either by the atom adjacent to the TI system having several bonds to more electronegative atoms or, having a formal positive charge. Examples of electron withdrawing groups include, but are not limited to, -CHO, -COR, -COOR, -COOH, -CONH2, -CONHR, -CONR2, - CF3, -CN, C=C(CN)2-SO3H, -NH3+, -NR3+, -NO2, -SOR, -SO2R (e g., -SO2CH3), - SO2NH2, -SO2NHR, and -SO2NR2. It should be noted that the R groups within the electron withdrawing groups may be methyl, ethyl, propyl, etc.

[0035] An agent exhibits “specific uptake” for myelinated tissues if it associates more frequently with, more rapidly with, for a longer duration with, and / or with greater affinity to, and / or if it is absorbed more, or accumulates more in myelinated tissues than with non-myelinated tissues. Generally, specific uptake is characterized by a relatively high affinity of an agent to a target.

[0036] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention, by measurement error or variation, and / or by conventionally obtainable clinical tolerances. At the very least, each numericalparameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0037] Provided herein are image enhancement methods that may be applicable to analytical, diagnostic, or prognostic applications related to myelin basic protein detection. The applications may be particularly applicable in imaging labeled nerve fibers or cells, such as in contexts of peripheral nerve labeling, spinal imaging, brain tissue imaging, non-invasive in vivo measurement of myelination levels, and preclinical and basic neuroscience bench research aimed at the study of the function and process of myelination, and the dysfunction and repair of myelin. An imaging agent (e.g., an ultrasound nerve labeling agent, ultrasound imaging agent composition) may be used with ultrasound imaging as a diagnostic tool by a primary care provider. In this way, the primary care provider is enabled to view and identify nerve damage or other issues as part of an early detection method using non-invasive ultrasound imaging. In other embodiments, the imaging agent may be used with ultrasound imaging as a pre-surgical analysis to plan for surgery and / or during surgery (e.g., non-invasive, minimally invasive, open) to improve visualization and help surgeons avoid certain nerves if needed.

[0038] In one embodiment, an agent which binds specifically to myelin basic protein may be administered parenterally to a surgical subject, such as prior to surgery, such that the agent binds to myelin basic protein and may be cleared from tissues that do not contain myelin basic protein. The imaging agent binds to an outer layer of nerves that is composed of myelin basic protein. Accordingly, the embodiments described herein may be used for all types of nerves, including otherwise hard to visualize peripheral nerves. In one embodiment, the agent may be administered intravenously, wherein the nerves will be more easily viewed using ultrasound imaging after a certain time post-administration. Ultrasound imaging may be subsequently utilized to visualize and study the nerves.

[0039] Due to their specific binding to the imaging agent, nerves and other myelin containing tissue are distinguishable in ultrasound images from tissue not containing myelin basic protein. This enables the caregiver or clinician to view myelinated tissue in reconstructed ultrasound images and / or facilitates accurately administeringtreatment to the intended myelinated tissue. In certain embodiments the agent comprises the compound of Formula I.

[0040] An agent which specifically binds to myelin basic protein may be administered parenterally to a subject prior to a procedure or to treatments targeting a nerve or other myelin containing tissue, such as pharmaceutical or surgical nerve block. In certain embodiments the myelinated tissue may be peripheral nerve tissue and / or part of the spinal canal and intervertebral foramen. In other embodiments the myelinated tissue may be part of the brain. In certain embodiments the agent comprises a compound of Formula I or its salt.

[0041] In one embodiment the aforementioned agent, may be administered parenterally to a subject, prior to a procedure, to permit binding to myelin basic protein, and clearance from tissues that do not contain myelin basic protein without the elimination of specific myelin basic protein binding.

[0042] By inspection of the ultrasound images, the clinician may determine if, and where a nerve tissue (e.g., the spinal cord or associated nerve roots), is impinged, such as by the vertebral column or foreign matter. Additional scans may be conducted to provide additional information, such as the structure and relative positioning of elements of the vertebral column.

[0043] To determine whether myelination in the patient may be deficient, myelination levels may be compared to those exhibited by a subject or subjects believed or known not to be suffering from a myelin-associated neuropathy. In another embodiment, rates of demyelination or remyelination may be determined. Following treatment with a known or suggested therapeutic agent believed or anticipated to prevent or slow demyelination or to promote remyelination in patients suffering from myelin-associated neuropathies, myelination levels are evaluated by performing the imaging over time in the patients treated with the therapeutic agent. The imaging may be performed at different points of time and the level of myelination at one time point compared to that of another. As such level of myelination may be determined qualitatively or quantitatively.

[0044] A positive result suggestive of a myelin-associated neuropathy may be one in which the decrease of myelin basic protein of the subject, compared to a baseline measurement of myelin basic protein, in a control sample is statistically significant. The control sample may be from a similar sample free of a myelin-associated neuropathy or from the same subject with measurements taken over time.

[0045] Ultrasound imaging may then be used to assess the presence or quantity of an agent having specific binding to myelin basic protein in the tissue sample and may represent the presence or amount of myelin basic protein. In certain embodiments, the agent may comprise a compound of Formula I or its salt. The labeling with, and detection, visualization, or quantitation of an agent, may also be performed in conjunction with labeling with, and detection, visualization, or quantitation of at least one other compound that specifically binds a substance other than myelin basic protein.

[0046] Many of the compounds described herein may comprise one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)-. The chemical structure of the agent includes for example, without limitation, all such possible isomers, as well as, their racemic and optically pure forms. Optically active (R)- and (S)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also included.

[0047] In certain embodiments, a method for the qualitative or quantitative detection of myelin basic protein through application of an agent that specifically binds to myelin basic protein is provided. The specific binding to myelin basic protein may be by a compound of Formula I or its salt:I wherein R1is an alkyl group, R2is an electron donating group and R3is an electron withdrawing group.

[0048] In certain embodiments, the specific binding to myelin basic protein may be by a compound of Formula I or its salt, 1(a):1 (a)

[0049] Formula I (a) (e.g., GE3111) may be synthesized in a stepwise procedure. An example of such a method includes a tandem Heck coupling followed by a Horner-Wittig olefination using bromoaldehyde for the middle ring and readily available building blocks for the terminal rings. By way of example, preparation of the specific myelin binding protein Formula I (a) may be prepared as described in Molecular Imaging and Biology, “Intraoperative Fluorescence Imaging of Peripheral and Central Nerves Through a Myelin-Selective Contrast Agent” by Victoria E. Cotero, et al. DOI: 10.1007 / sl 1307-012-0555-1, which is incorporated by reference herein in its entirety and for all purposes.

[0050] In certain embodiments R1be a lower alkyl groups of from 1 to 6 carbon atoms, preferably from 1 to 4 carbon atoms, and includes methyl, ethyl, n-propyl, isopropyl, and n-, s- and t- butyl. The electron-donating group, R2, may include aprimary, secondary, or tertiary amine, or an alkoxy group. Preferably, R2may be an amine, and more preferably NH2.

[0051] In certain preferred embodiments, the specific binding to myelin basic protein may be by a compound of Formula I or its salt, 1(b):1 (b) wherein R4and R5may be used to improve aqueous solubility and reduce logP of the agent. R4and R5may be independently a hydrogen atom or an alkyl, preferably a lower alkyl group of from 1 to 6 carbon atoms. In other embodiments, R4and R5may independently be a substituted alkyl groups, such as, but not limited to an alkoxy or alcohol. In certain embodiments, the alkoxy group may contain ethylene glycol units or an ethylene glycol terminated alcohol; for example (CH2CH2O)nX or CH2CH2CH2 (OCH2CH2)nOX where n is an integer between 1 and 6 and X is hydrogen, methyl or ethyl. In still other embodiments, when R4and R5form an unsubstituted or substituted heterocyclic ring structure. The heterocyclic ring structure may be piperidine, piperazine, or morpholine or an alkyl or alkoxyl substituted piperidine, piperazine, or morpholine.

[0052] In each embodiment, R2and the sulfonamide group R4R5NSO2 are conjugated through the 71 double bond orbitals of the benzene rings and olefinic substituents, thereby providing a clear path for electrons to flow from the electrondonating group to the electron-withdrawing group.

[0053] In certain embodiments, the agent may be a salt of Formula I, wherein R4and R5may comprise an ammonium cation with an anion. The ammonium salt may be a tertiary ammonium salt wherein the anion may be a halide. In other embodiments, the anion may be polyatomic such as, but not limited to peroxide,nitrate, carbonate, sulfate, and phosphate. The polyatomic anion may also comprise a halide such as, but limited to, a chlorate, perchlorate, iodate, periodate, bromate, tetrafluoroborate, hexafluorophosphate, a fluoropolyphosphate, or a combination thereof. In still other examples, the anion may originate from carboxylic acids, such as, but not limited to, citrate, tartrate, maleate, malate, fumarate, itaconate, or ascorbate. For in vivo applications, those anions with low biological toxicity would be preferred.

[0054] Other non-limiting examples of Formula I, are shown in Table 1 (Formula I(c-j))-

[0055] Increase in solubility is obtained in comparison to similar materials such as structures d and e.

[0056] In certain embodiments, imaging agents as discussed herein, which have improved aqueous solubility compared to similar agents, may lessen nonspecific partitioning of the agents to the non-target tissue, such as adipose tissue. Also, improved aqueous solubility may enable the agents to be formulated in pharmaceutical carriers with less or no known toxic effects, thus making them more suitable for use in higher dosage and providing researchers and clinicians important diagnostic and treatment tools by facilitating non-invasive nerve imaging.

[0057] To determine whether myelination in the patient may be deficient, myelination levels may be compared to those exhibited by a subject or subjects believed or known not to be suffering from a myelin-associated neuropathy. In another embodiment, rates of demyelination or remyelination may be determined. Following treatment with a known or suggested therapeutic agent believed or anticipated to prevent or slow demyelination or to promote remyelination in patients suffering from myelin-associated neuropathies, myelination levels are evaluated by performing the non-invasive ultrasound nerve imaging over time in the patients treated with the imaging agent. The imaging may be performed at different points of time and the level of myelination at one time point compared to that of another. As such, level of myelination may be determined qualitatively or quantitatively.

[0058] After binding to myelin basic protein, the sample may be washed in a manner and medium suitable to remove any unbound and non-specifically bound label from the sample, without eliminating specific binding to myelin basic protein.

[0059] In certain embodiments, a pharmaceutical carrier may be used to enhance, at least one of, solubility, penetration or bioavailability of an imaging agent comprising a compound of Formula I or its salt. In certain embodiments the pharmaceutical carrier may be used to enhance the solubility of the compound in a solution as well as acting to carry or transport the compound across a barrier, for example to allow for percutaneous penetration.

[0060] In certain embodiments, the pharmaceutical carrier may comprise Carbopol, polyethylene glycol (such as PEG-300), propylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, or Laurocapram, for intraoperative local administration.

[0061] In certain embodiments, the pharmaceutical carrier is an aqueous solution comprising 1-30% PEG-300, 1-20% propylene glycol, 1-10% polyvinyl pyrrolidone, and 0-10% Laurocapram based on volume.

[0062] In certain embodiments, the pharmaceutical carrier is an aqueous solution comprises 20% PEG-300, 10% propylene glycol, 5% polyvinyl pyrrolidone, and 5% Laurocapram, based on volume.

[0063] Other pharmaceutical carriers may include, but are not limited to, surfactants including non-ionic surfactants, lipids including triglycerides, cyclodextrins, and phospholipids as well as other detergents, buffer solutions, stabilizers, and preservatives. In each case, the use of both water-soluble and waterinsoluble organic solvents may be used in combination with other pharmaceutical carriers to limit the occurrence of precipitation, pain, inflammation and homolysis upon administration.

[0064] Techniques to enhance solubility of the agent may include, pH adjustment, salt formation as described above co-solvents, complexation, emulsions, micelles, and liposomes. The pharmaceutical carrier may also include, but is not limited to, surfactants such as a detergent, buffer solutions, stabilizers, and preservatives. In certain embodiments, the carrier may also include a percutaneous enhancer which acts to carry a compound or drug across a barrier including transdermal delivery.

[0065] With the preceding in mind, and by way of providing useful context, FIG. 1 depicts a high-level view of components of an ultrasound system 10 that may be employed in accordance with the present approach. The illustrated ultrasound system 10 includes a transducer array 14 having transducer elements suitable for contact with a subject or patient 18 during an imaging procedure. The transducer array 14 may be configured as a two-way transducer capable of transmitting ultrasound waves into and receiving such energy from the subject or patient 18. In such an implementation, in the transmission mode the transducer array elements convert electrical energy into ultrasound waves and transmit it into the patient 18. In reception mode, the transducer array elements convert the ultrasound energy received from the patient 18 (backscattered waves) into electrical signals.

[0066] Each transducer element is associated with respective transducer circuitry, which may be provided as one or more application specific integrated circuits (ASICs) 20, which may be present in a probe or probe handle. That is, each transducer element in the array 14 is electrically connected to a respective pulser 22, transmit / receive switch 24, preamplifier 26, swept gain 34, and / or analog to digital (A / D) converter 28 provided as part of or on an ASIC 20. In other implementations, this arrangement may be simplified or otherwise changed. For example, components shown in the circuitry 20 may be provided upstream or downstream of the depictedarrangement, however, the basic functionality depicted will typically still be provided for each transducer element. In the depicted example, the referenced circuit functions are conceptualized as being implemented on a single ASIC 20 (denoted by dashed line), however it may be appreciated that some or all of these functions may be provided on the same or different integrated circuits.

[0067] Also depicted in FIG. 1, a variety of other imaging components are provided to enable image formation with the ultrasound system 10. Specifically, the depicted example of an ultrasound system 10 also includes a beam former 32, a control panel 36, a receiver 38, and a scan converter 40 that cooperate with the transducer circuitry to produce an image or series of images 42 that may be stored and / or displayed to an operator or otherwise processed as discussed herein. The transducer array 14 may communicate the ultrasound data to the beam-former via a wired connection or wireless connection (e.g., via a wireless communication unit that is part of the transducer array that communicates over a wi-fi network, utilizing Bluetooth® technique, or some other manner).

[0068] A processing component 44 (e.g., a microprocessor or processing circuitry) and a memory 46 of the system 10, such as may be present control panel 36, may be used to execute stored software code, instructions, or routines for processing the acquired ultrasound signals to generate meaningful images and / or motion frames, which may be displayed on a display 47 of the ultrasound system 10. The term "code" or "software code" used herein refers to any instructions or set of instructions that control the ultrasound system 10. The code or software code may exist in a computerexecutable form, such as machine code, which is the set of instructions and data directly executed by the processing component 44 of the control panel 36, human- understandable form, such as source code, which may be compiled in order to be executed by the processing component 44 of the control panel 36, or an intermediate form, such as object code, which is produced by a compiler. In some embodiments, the ultrasound system 10 may include a plurality of controllers.

[0069] As an example, the memory 46 may store processor-executable software code or instructions (e.g., firmware or software), which are tangibly stored on a non- transitory computer readable medium. Additionally or alternatively, the memory 46 may store data. As an example, the memory 46 may include a volatile memory, suchas random-access memory (RAM), and / or a nonvolatile memory, such as read-only memory (ROM), flash memory, a hard drive, or any other suitable optical, magnetic, or solid-state storage medium, or a combination thereof. Furthermore, processing component 44 may include multiple microprocessors, one or more “general -purpose” microprocessors, one or more special-purpose microprocessors, and / or one or more application specific integrated circuits (ASICS), or some combination thereof. For example, the processing component 44 may include one or more reduced instruction set (RISC) or complex instruction set (CISC) processors. The processing component 44 may include multiple processors, and / or the memory 46 may include multiple memory devices.

[0070] Ultrasound information may be processed by other or different mode- related modules (e.g., B-mode, Color Doppler, power Doppler, M-mode, spectral Doppler anatomical M-mode, strain, strain rate, and the like) to form 2D or 3D data sets of image frames and the like. For example, one or more modules may generate B- mode, color Doppler, power Doppler, M-mode, anatomical M-mode, strain, strain rate, spectral Doppler image frames and combinations thereof, and the like. The image frames are stored and timing information indicating a time at which the image frame was acquired in memory may be recorded with each image frame. The modules may include, for example, a scan conversion module to perform scan conversion operations to convert the image frames from Polar to Cartesian coordinates. A video processor module may be provided that reads the image frames from a memory and displays the image frames in real time while a procedure is being carried out on a patient. A video processor module may store the image frames in an image memory, from which the images are read and displayed. The ultrasound system 10 shown may include a console system, or a portable system, such as a hand-held or laptop-type system.

[0071] The ultrasound system 10 may be operable to continuously acquire ultrasound scan data at a frame rate that is suitable for the imaging situation in question. Typical frame rates may range from 20-120 but may be lower or higher. The acquired ultrasound scan data may be displayed on the display 47 at a display-rate that can be the same as the frame rate, or slower or faster. An image buffer may be included for storing processed frames of acquired ultrasound scan data that are notscheduled to be displayed immediately. Preferably, the image buffer is of sufficient capacity to store at least several minutes worth of frames of ultrasound scan data. The frames of ultrasound scan data are stored in a manner to facilitate retrieval thereof according to its order or time of acquisition. The image buffer may be embodied as any known data storage medium.

[0072] The display 47 may be any device capable of communicating visual information to a user. For example, the display 47 may include a liquid crystal display, a light emitting diode display, and / or any suitable display or displays. The display 47 can be operable to present ultrasound images and / or any suitable information.

[0073] Components of the ultrasound system 10 may be implemented in software, hardware, firmware, and / or the like. The various components of the ultrasound system 10 may be communicatively linked. Components of the ultrasound may be implemented separately and / or integrated in various forms.EXAMPLES

[0074] The following non-limiting examples are shown and describe various embodiments of the present invention. The examples include data acquired comparing administered ultrasound nerve labeling contrast agents. As shown it demonstrates that when properly formulated, nerve labeling contrast agent can selectively label nerves for non-invasive ultrasound imaging.

[0075] By way of example, FIG. 2 shows ultrasound images 50 of a sciatic nerve (shown via an arrow) in an obese rat pre- and post- administration of the ultrasound nerve labeling agent, in accordance with an embodiment of the present disclosure. The sciatic nerve of an obese diet induced obese (DIO) rat was located using an ultrasound imager GE Logic E. The leftmost image 52 was taken prior to intravenous administration (e.g., injection) of GE3111 (Formula 1(a)) (0.5 mg / mL). Anatomical landmarks were used to confirm proximity to the sciatic nerve. The leftmost image 52 shows a normal, unlabeled sciatic nerve, pointed to by a reference arrow. Following a 1-hour incubation time period with GE3111, the ultrasound imager was placed over the location again and subsequent images were collected. The rightmost image 54demonstrates an overall enhancement in the intensity of the sciatic nerve within the ultrasound image, as represented by the reference arrow.Table 1 shows a comparison of the of the mean grey image values pre-administration and post-administration of GE3111 taken at three different locations within the ultrasound images of FIG. 2.

[0076] Relatedly, Table 1 shows a comparison of mean grey image values acquired from the ultrasound images of FIG. 2. In general, the images of FIG. 2 were controlled for identical contrast and brightness for visual comparison. Mean image grey value was performed using Imaged. Table 1 illustrates that the mean grey image values acquired from the rightmost image 54 post-administration of GE3111 are greater relative to pre-administration of GE3111 (leftmost image 52).

[0077] With the preceding in mind, FIG. 3 shows a graph 60 of a comparison of image intensity based on the mean grey value in the ultrasound images of the sciatic nerve pre- and post- administration of the ultrasound nerve labeling agent, in accordance with an embodiment of the present disclosure. In general, the graph shows that the mean image grey value is greater for the rightmost image 54 taken postadministration of GE3111 relative to the leftmost image 52 taken pre-administration of GE3111. Accordingly, FIG. 3 demonstrates that the sciatic nerve of the obese DIOrat is easier to visualize via ultrasound 1-hour post-administration of GE3111 relative to the unlabeled nerve.

[0078] While only certain features have been illustrated and described herein, many modifications and changes will occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the disclosure.

Claims

AMENDED CLAIMS received by the International Bureau on 07 November 2025 (07.11.2025)What is claimed is:

1. A method for imaging myelin basic protein in a non-invasive setting by administration of an ultrasound nerve labeling agent comprising the steps of: administering the ultrasound nerve labeling agent to a subject, wherein the ultrasound nerve labeling agent comprises a compound of Formula I or a salt thereof, wherein Formula I is:wherein R1is an alkyl group, specifically a methyl group, R2is NH2, a primary amine acting as an electron donating group, and R3is -SO2(CH3), an electron withdrawing group, and the agent is formulated in an aqueous pharmaceutical carrier comprising 1-30% PEG-300, 1- 20% propylene glycol, 1-10% polyvinyl pyrrolidone, and 0-10% Laurocapram by volume; positioning an ultrasound probe on the subject’s skin, wherein the ultrasound probe is configured to apply ultrasound energy to a region of interest of the subject; and acquiring ultrasound image data of the region of interest, wherein, following administration of the agent, the ultrasound image data, when displayed, depicts the one or more nerves labeled with the ultrasound nerve labeling agent as exhibiting an increase in mean grayscale intensity of at least 164% relative to pre-administration baseline, measured 60 minutes post-administration under diagnostic ultrasound, and wherein the method is performed in the absence of gas-filled microbubbles or particulate ultrasound contrast agents.

2. The method of claim 1, wherein the ultrasound nerve labeling agent is administered via an intravenous injection.

3. The method of claim 1, wherein the ultrasound nerve labeling agent binds to myelinated nerves.

Images