Olfactory delivery of therapeutic peptides

Abstract

The present disclosure describes methods and devices for olfactory delivery of therapeutic peptides such as insulin. Devices and methods of the present disclosure may provide convenient and effective ways to accomplish olfactory delivery of therapeutic peptides.

Description

OLFACTORY DELIVERY OF THERAPEUTIC PEPTIDESCROSS REFERENCE

[0001] This PCT application claims the benefit to U.S. Provisional Application No. 63 / 606,059, filed December 4, 2023, the contents of which are incorporated herein by reference.FIELD

[0002] The present disclosure relates generally to delivery of small peptide therapeutics, and more specifically, delivery via the olfactory.BACKGROUND

[0003] Certain peptides are known to provide therapeutic effects to subjects. Insulin, and similar small-peptide therapeutics have been found to treat a wide variety of disorders in a variety of physiological systems, including the central nervous system. While therapeutic effects of peptides on the central nervous system (or a region, tissue, or organ th er co I') are of interest, delivery is known to present challenges. Olfactory delivery is a possibility for therapeutic peptides.

[0004] The therapeutic potential of drugs targeting the brain through olfactory neural pathways via intranasal application is significant, including decreased systemic absorption, smaller doses, and reduced risk of complications from over-medication. Nasal mist devices are a standard method to deliver drugs to the nasal cavity. Recent research has established the inefficiency of these methods compared to methods designed to deliver drugs more locally into the nasal cavity where drug action is desired. Focal delivery promises to deposit the medication bolus directly to the olfactory cleft immediately below the cribriform plate where the olfactory nerves pass into the brain, whereas standard nasal misters deposit drug dosage throughout the nasal cavity and sinuses with potential significant dilution prior to reaching the olfactory cleft, due to dispersion over a large surface area. Moreover, spray devices typically atomize or vaporize the drug compound, creating small droplets that are degraded by high velocity airflow through the nasal valve. Shear forces can destroy some drug molecules and biologies as they are atomized into droplets. With few exceptions, existing devices typically deliver only 5-8% of a dose to the olfactory cleft. There remains a need for formulations, methods, and devices for targeted delivery of therapeutic peptides to subregions of the nasal cavity, such as the olfactory cleft.SUMMARY

[0005] It is appreciated by the inventors that challenges related to delivery of therapeutic peptides to certain regions of the central nervous system include the fragility of peptides, and difficulty in crossing the blood-brain barrier. For example, the blood brain barrier and / or the blood-cerebrospinal fluid barrier may limit or prevent peptides from having therapeutic effects when they are delivered intravenously. Intranasal delivery efficiencies have typically been low, even for non-protein, small-molecule therapeutics. Challenges associated with olfactory delivery of peptides include difficulties in accommodating for variations in the nasal channel, difficulties in targeting deposition of the peptide at the correct location in the nasal channel, and difficulties providing a device which can reliably and repeatably deliver a composition to locations within the nasal channel with a high degree of precision across a population of subjects, without damaging the peptides. Methods and devices of the present invention provide for targeted delivery of therapeutic peptides to the olfactory region such that the therapeutic peptides are not denatured by damaging shear forces or delivered off- target, thereby improving delivery efficiency and therapeutic efficacy. Additional advantages of the present invention may include the avoidance of needles (particularly relevant to subjects with needle-phobia), the possibility of self-administration, and enablement of the delivery of formulations with viscosity too high for intravenous injection.

[0006] It is appreciated by the inventors that difficulties in nasal delivery include anatomical, cognitive, and dexterity related challenges related to providing a reliable and effective nasal delivery device. For example, the internal nasal valve is a flow-limiting segment of the nasal channel bounded medially by the dorsal septum, laterally by the caudal portion of the upper lateral cartilage and inferiorly by the head of the inferior turbinate that together present a physical barrier between the nasal vestibule and the rest of the nasal channel, including the respiratory region and the olfactory cleft. The internal nasal valve is bounded by nasal tissue that can swell or block the path to the nasal channel, which varies based on time of day, environmental factors, and genetically among individuals. Devices may need to be inserted at particular angles or depths in order to target a specific area, and users may struggle with correct placement and actuation of a device. Users with motor skill impairment (e.g., persons with Parkinson’s disease, arthritis) or cognitive impairments (e.g., Alzheimer’s disease) may especially struggle to correctly articulate, position, and actuate an intranasal delivery device.

[0007] To counter such difficulties, this disclosure provides formulations, methods, and devices for olfactory delivery of therapeutic peptides such as insulin. This disclosure provides an introducer device for targeted delivery of a peptide-containing composition to a target region of a nasal cavity of a subject, which can readily be actuated and positioned by users of various patient populations, and reliably deliver the composition to a target subregion of the nasal cavity, to enable precision delivery of the peptides. As a non-limiting example, the olfactory cleft can be a target sub-region of the nasal cavity, such that nonspray, targeted delivery of a therapeutic to the olfactory cleft produces a desired effect. Additional target sub-regions of the nasal cavity may be identified.

[0008] In one aspect is a method of olfactory delivery of therapeutic peptides, the method comprising: selectively delivering a formulation comprising a therapeutically effective amount of the therapeutic peptide to an olfactory region of the subject, wherein at least a portion of a formulation is delivered to one or more of the group consisting of cerebrospinal fluid, the central nervous system, or a region, tissue, or organ thereof. In some embodiments, the therapeutic peptide may be delivered to a target or organ system other than the CNS. Non-limiting examples include the lymphatic system, Nasal Associated Lymphatic Tissue (NALT), and Cribriform Lymphatic / Glymphatic, Systemic, etc. Therapeutic peptides may also elicit an immune response in the CNS.

[0009] In some embodiments, the formulation is ejected from a dispensing element, wherein at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the formulation passes through a circular opening having a diameter of between about 5 mm and about 6 mm at a distance of about 25 mm from where the formulation is ejected from the dispensing element. In some embodiments, the formulation has a viscosity of between about 0.5 cP and about 100 cP. In some embodiments, the formulation has a viscosity of between about 0.5 cP and about 50 cP. In some embodiments, the formulation has a viscosity of between about 0.5 cP and about 10 cP. In some embodiments, the formulation has a viscosity of between about 45 cP and about 55 cP. In some embodiments, the formulation is ejected from the dispensing element at a velocity of between about 1 m / s and about 30 m / s. In some embodiments, the formulation is ejected from the dispensing element at a velocity of between about 2 m / s and about 4 m / s. In some embodiments, the formulation is ejected from the dispensing element at a velocity of between about 25 m / s and about 30 m / s. In some embodiments, the formulation is ejectedfrom a dispensing element, wherein at least 75% of the formulation passes through a circular opening having a diameter of between about 5 mm and about 6 mm at a distance of about 25 mm from where the formulation is ejected from the dispensing element, wherein the formulation has a viscosity of between about 0.5 cP and about 50 cP, and wherein the formulation is ejected from the dispensing element at a velocity of between about 1 m / s and about 30 m / s.

[0010] In some embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the formulation is delivered to the olfactory region of the subject. In some embodiments, delivery of the therapeutic peptide to the olfactory region of the subject results in a plasma concentration of the therapeutic peptide about 120 min after delivery that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% of a plasma concentration of the therapeutic peptide when about the same dose is delivered intravenously.

[0011] In some embodiments, the formulation is delivered as a laminar flow. In some embodiments, the formulation is delivered as a liquid jet. In some embodiments, the formulation is delivered as a flow having a Reynold’s number of 2300 or less. In some embodiments, the delivering of the formulation as a laminar flow, a liquid jet, or a flow having a Reynold’s number of 2300 or less: a) increases on target delivery of the composition to the target region, b) decreases off target delivery of the composition to the nasal cavity, or c) both, compared to delivering the formulation with a spray ejection profile.

[0012] In some embodiments, selectively delivering the formulation to the olfactory region of the subject comprises ejecting the formulation from an ejection zone in a nasal cavity of the subject, wherein the ejection zone is: (a) 0mm to 30mm superior to a horizontal line that intersects the anterior aspect of the internal nasal valve, and (b) 0mm to 20mm anterior to an inclined line that intersects the anterior aspect of the middle turbinate and the posterior aspect of the vestibule. In some embodiments, the ejection zone is further: i. 0mm to 40mm inferior to a horizontal line that is parallel to the inferior aspect of the olfactory cleft, ii. 0mm to 20mm posterior to the internal nasal dorsum, iii. 10mm to 50mm superior to a horizontal line that intersects the inferior aspect of the columella, iv. 0mm to 30mm superior to a horizontal line that intersects the superior aspect of the inferior turbinate, v. 0mm to 3mm from the septum, or vi. any combination thereof. In some embodiments, delivering the formulation from the ejection zone: a) increases on target delivery of the composition to thetarget region, b) decreases off target delivery of the composition to the nasal cavity, or c) both, compared to dispensing the formulation outside the ejection zone.

[0013] In some embodiments, the formulation is delivered by a device comprising the dispensing element, wherein the device is inserted into a nasal cavity of the subject at an angle of between about 30 degrees and about 40 degrees from a vertical line, wherein the vertical line is perpendicular to a horizontal line that is parallel to an inferior aspect of the olfactory cleft of the subject.

[0014] In some embodiments, the formulation delivered to the olfactory region remains at the olfactory region for at least about 10 seconds, at least about 30 seconds, at least about 1 min, at least about 2 min, at least about 3 min, at least about 4 min, at least about 5 min, at least about 6 min, at least about 7 min, at least about 8 min, at least about 9 min, at least about 10 min, at least about 11 min, at least about 12 min, at least about 13 min, at least about 14 min, or at least about 15 min.

[0015] In some embodiments, the therapeutic peptide is insulin, oxytocin, glutathione, a neurotrophic factor, a glial cell-derived neurotrophic factor (GDNF), a nerve growth factor (NGF), glucagon, a glucagon like peptide 1 (GLP-1), exendin-4, dulaglutide, a GLP-1 agonist, an incretin mimetic, or a galanin-like peptide (GALP). In some embodiments, the therapeutic peptide is an analog of one of the group consisting of: insulin, oxytocin, glutathione, a neurotrophic factor, a glial cell-derived neurotrophic factor (GDNF), a nerve growth factor (NGF), glucagon, a glucagon like peptide 1 (GLP-1), exendin-4, dulaglutide, a GLP-1 agonist, an incretin mimetic, a galanin-like peptide (GALP), erythropoietin (EPO), a glycoprotein hormone, salmon calcitonin, buserelin acetate, desmopressin acetate (peptide drug), Gonadorelin, Nafarelin acetate, Protirelin, and cyanocobalamine.

[0016] In some embodiments, the therapeutic peptide is insulin, or a variant or analog thereof. In some embodiments, delivery of the therapeutic peptide results in reduction in brain activity in the subject. In some embodiments, delivery of the therapeutic peptide results in reduction in brain activity in the subject for at least about 15 min, at least about 30 min, at least about 45 min, or at least about 60 min after delivery. In some embodiments, delivery of the therapeutic peptide results in reduction in brain activity in the subject for a longer period of time than when about the same dose of the therapeutic peptide is delivered by a nasal spray. In some embodiments, the therapeutic peptide is exendin-4, or a variant or analog thereof.

[0017] In some embodiments, the therapeutic peptide has a length of less than 50 amino acids. In some embodiments, the therapeutic peptide is conjugated to a therapeutic agent. In some embodiments, the therapeutic agent is a drug or a radioisotope.

[0018] In some embodiments, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% of the therapeutic peptide in the formulation is delivered to one or more of the group consisting of cerebrospinal fluid, the central nervous system, and a region, tissue, or organ thereof.

[0019] In some embodiments, the method provides delivery of a greater amount of the therapeutic peptide to one or more of the group consisting of cerebrospinal fluid, the central nervous system, and a region, tissue, or organ thereof compared to intravenous delivery of an identical dose of the therapeutic peptide. In some embodiments, the therapeutic peptide provides a therapeutic effect upon delivery to the cerebrospinal fluid, the central nervous system or region, tissue, or organ thereof. In some embodiments, the subject has been diagnosed with a disease or disorder related to the central nervous system. In some embodiments, delivery of the therapeutic peptide provides effective treatment for Alzheimer's disease, Parkinson's disease, a cancer of the brain, a cancer of the CNS, a non- CNS disorder, an immunological disorder, an oncological disorder, a hematological disorder, an opthalmological disorder, a neurological disorder, a respiratory disorder, a cardiovascular disorder, an infectious disease, vitamin deficiency, multiple sclerosis, improvement of uterine contractions, postmenopausal osteoporosis, cryptorchism, endometriosis, central precocious puberty (CPP), Primary nocturnal enuresis, central cranial diabetes insipidus, nocturia, cranial diabetes insipidus or nocturia associated with multiple sclerosis, stroke, epilepsy, central nervous system trauma, a metabolic disorder, an infectious agent (i.e. Naegleria fowleri) or protein (i.e. Creutzfeldt Jacobs disease), a malignant tumor, a benign tumor, glioblastoma, anosmia, or an endocrine disorder. In some embodiments, delivery of the therapeutic peptide provides effective treatment for depression, dementia, or Alzheimer’s disease.

[0020] In some embodiments, the formulation is delivered without exposing the therapeutic peptide to shear forces sufficient to damage a significant portion of the therapeutic peptide,without aerosolizing the formulation, or by passing the formulation through a shear disintegrating tip. In some embodiments, the therapeutic peptide bypasses the blood-brain barrier, the blood-cerebrospinal fluid barrier, or the arachnoid membrane. In some embodiments, the non-CNS-target is blood.

[0021] In some embodiments, the formulation is delivered via a device comprising: a. a housing defining first and second insertable portions, each for insertion into a nasal channel of the subject, wherein, upon insertion of at least one of the insertable portions into the nasal channel of the subject, the at least one insertable portion engages tissue within the nasal channel to open or expand an internal nasal valve of the subject thereby positioning the at least one insertable portion for delivery of the formulation to the olfactory region of the subject; and b. an actuator which delivers the formulation from at least one of the insertable portions when the device is actuated.

[0022] In another aspect is a device for olfactory delivery of therapeutic peptides, the device comprising: a housing comprising an insertable portion comprising a distal end, and a proximal end; and a subject-engaging portion which engages a columella region of the subject to seat the distal end of the insertable portion within an ejection zone of a nasal channel of the subject; wherein the device is configured to deliver a formulation comprising a therapeutically effective amount of the peptide to the olfactory region of the subject.

[0023] In some embodiments, the device dispenses the formulation as a laminar jet. In some embodiments, the device comprises a compliant dispensing tip comprising a compliant and flexible soft nib. In some embodiments, application of pressure by the subject-engaging portion to the columella region of the subject enables and / or causes delivery of the formulation to the subject from the insertable portion. In some embodiments, the insertable portion comprises a dispensing element for delivery of the formulation to the olfactory region of the subject.

[0024] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.INCORPORATION BY REFERENCE

[0025] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description and accompanying drawings, that set forth illustrative embodiments in which the principles of the present disclosure are utilized.

[0027] FIG. 1A depicts a bottom view of an exemplary embodiment of a subject’s nose.

[0028] FIG. IB depicts a side view of an exemplary embodiment of a subject’s nose.

[0029] FIG. 1C depicts a side view of a representative subject’s nasal channel.

[0030] FIG. ID depicts a side view of an exemplary embodiment of a representative subject’s nasal channels from the vestibule to the olfactory cleft based on a posteriorly oriented plane.

[0031] FIG. IE depicts a side view of an exemplary embodiment of a representative subject’s nasal channels from the vestibule to the olfactory cleft based on an anteriorly oriented plane.

[0032] FIG. IF depicts a side view of an exemplary embodiment of a representative target ejection zone, according to some embodiments.

[0033] FIG. 1G depicts a front, base and perspective view of an exemplary embodiment of a subject’s nose and the columella region.

[0034] FIG. 1H depicts a side view of an exemplary embodiment of a representative subject’s nasal cavity, including internal anatomical features.

[0035] FIG. II depicts a side view of an exemplary embodiment of a representative subject’s sinus, according to some embodiments.

[0036] FIG. 1J depicts a side view of a subject’s nasal cavity.

[0037] FIG. 2A depicts an exemplary embodiment of an Exemplary Device in a second configuration, according to some embodiments.

[0038] FIG. 2B depicts an exemplary embodiment top view of an Exemplary Device in the second configuration, according to some embodiments.

[0039] FIG. 2C depicts an exemplary embodiment trigger button side view of an Exemplary Device in the second configuration, according to some embodiments.

[0040] FIG. 2D depicts an exemplary embodiment chassis side view of an Exemplary Device in the second configuration, according to some embodiments.

[0041] FIG. 2E depicts an exemplary embodiment bottom view of an Exemplary Device in the second configuration, according to some embodiments.

[0042] FIG. 2F depicts an exemplary embodiment perspective view of an Exemplary Device in the first configuration, according to some embodiments.

[0043] FIG. 2G depicts an exemplary embodiment front view of another Exemplary Device in the first configuration, according to some embodiments.

[0044] FIG. 2H depicts an exemplary embodiment front view of an Exemplary Device in the second configuration, according to some embodiment.

[0045] FIG. 21 depicts an exemplary embodiment front view of another Exemplary Device in the first configuration, according to some embodiments.

[0046] FIG. 2J depicts an exemplary embodiment front view of an Exemplary Device in the second configuration, according to some embodiment.

[0047] FIG. 3A depicts an exemplary embodiment of an Exemplary Device in the second configuration with one dispensing element revealed and a side view of an exemplary embodiment of a subject’s nose, according to some embodiments.

[0048] FIG. 3B depicts an exemplary embodiment of an Exemplary Device in the second configuration with one dispensing element revealing along the passageway and a side view of an exemplary embodiment of a representative subject’s nasal channels from the vestibule to the olfactory cleft based on an anteriorly oriented plane, according to some embodiments

[0049] FIG. 4A depicts an exemplary embodiment of a Cartesian reference plane of (left to right) a front, a top, and a side view of an Exemplary Device, according to some embodiments.

[0050] FIG. 4B depicts an exemplary embodiment of the subject plane, according to some embodiments.

[0051] FIG. 4C depicts an exemplary embodiment of (left to right) a front Coronal Plane, a top Transverse Plane, and a back Sagittal Plane view, according to some embodiments.

[0052] FIG. 4D depicts an exemplary embodiment of an Exemplary Device Sagittal Angle positioning in the subject, according to some embodiments.

[0053] FIG. 4E depicts an exemplary embodiment of an Exemplary Device Coronal- Medial Angle positioning in the subject, along a front view of the Coronal Plane, and a top view along a transverse plane, according to some embodiments.

[0054] FIG. 4F depicts an exemplary embodiment of an Exemplary Device Depth positioning along a front view of the Coronal Plane, and along a side view of the Sagittal Plane in the subject, according to some embodiments.

[0055] FIG. 4G depicts an exemplary embodiment of an Exemplary Device along a side view of the sagittal plane of delivery to the target region in the subject, according to some embodiments.

[0056] FIG. 4H depicts an exemplary embodiment of an Exemplary Device along a side view of the sagittal plane aiming from the respiratory region in the subject, according to some embodiments.

[0057] FIG. 41 depicts a side view of a target ejection zone, according to some embodiments.

[0058] FIG. 4J depicts a side view of a target ejection zone with respect to other nasal cavity anatomy, according to some embodiments.

[0059] FIGS. 5A-C depict images of a transparent model nasal cavity with a colored solution demonstrating delivery to an olfactory region of the model.

[0060] FIG. 6A depicts a mouse in an olfactory delivery study with a catheter inserted into the nostril to deliver a formulation to the olfactory region.

[0061] FIG. 6B depicts a diagram of the targeted olfactory delivery shown in FIG. 6A.

[0062] FIG. 7A depicts a mouse in an intranasal delivery study with a pipet tip inserted into the nostril to deliver a formulation to the nasal cavity.

[0063] FIG. 7B depicts a diagram of the non-targeted intranasal delivery shown in FIG. 7A.

[0064] FIG. 8 depicts amplitude of low frequency fluctuation (ALFF) maps showing changes in brain activation compared to baseline at 15 and 60 minutes post intranasal delivery of insulin for subjects 1-4.

[0065] FIG. 9 depicts amplitude of low frequency fluctuation (ALFF) maps showing changes in brain activation compared to baseline at 15 and 60 minutes post intranasal delivery of insulin with MucoLox for subjects 5-8.

[0066] FIG. 10 depicts amplitude of low frequency fluctuation (ALFF) maps showing changes in brain activation compared to baseline at 15 and 60 minutes post intranasal delivery of insulin for Block 1 participants who received insulin without MucoLox and Block 2 participants who received insulin with 15% MucoLox.

[0067] FIG. 11 depicts amplitude of low frequency fluctuation (ALFF) maps showing a comparison of spray delivery between subject groups 1 and 2.

[0068] FIGs. 12A-12B depict region of interest analysis for two representative brain regions.

[0069] FIG. 13 shows images of flow time series for olfactory delivery of technetium-99.

[0070] FIG. 14A is a graph showing exendin-4 plasma concentration after olfactory delivery (OD) or intravenous (IV) delivery in rats.

[0071] FIG. 14B is a graph showing exendin-4 concentrations in brain and olfactory after olfactory delivery (OD) or intravenous (IV) delivery in rats.DETAILED DESCRIPTION

[0072] This disclosure provides formulations, methods, and devices for olfactory delivery of therapeutic peptides. As used herein, the term “peptide” refers to a molecule including two or more amino acids.

[0073] This disclosure provides an introducer device for targeted delivery of a composition to a target region of a nasal cavity of a subject, which can be readily actuated and positioned by users of various patient populations, and reliably deliver the composition to the target region of the nasal cavity. Exemplary devices utilize a dual nostril inserter with a columella engaging portion positioned therebetween in order to quickly and reliably seat the insertable portions of the device within a target ejection zone of a subject’s nasal cavity. The exemplary devices disclosed herein can permit for quick, easy, and reliable positioning of a dispensing element within the nasal channel so as to permit for accurate and targeted deposition of compositions to target regions of the nasal cavity. Further, exemplary devices disclosed herein can enable quick, easy, and reliable positioning and targeted deposition across diverse patient populations (e.g., users that are elderly, cognitively impaired, dexterity impaired, or have variations in nasal anatomy), by users of varying skill (e.g., untrained bystanders), and under high stress circumstances (e.g., a medical emergency), which may otherwise prevent proper use of intranasal delivery devices.

[0074] The devices of the present disclosure may comprise housing comprising one or a combination of the following: a subject engaging portion, one or more insertable portions, one or more dispensing elements, and a trigger.

[0075] FIG. 1A depicts a bottom view of an exemplary embodiment of a subject’s nose. FIG. IB depicts a side view of an exemplary embodiment of a subject’s nose. In some embodiment’s the nose 1 has a columella region 10 between the entrance to two nasal channels 20. In some embodiments, the nose 1 has an external nasal valve 12 coupled to the nasal channel 20. In some embodiments, the nose 1 has an internal nasal valve 13 (INV) coupled to the nasal channel 20.

[0076] FIG. 1C depicts a side view of an exemplary embodiment of a subject’s nasal cavity showing an inferior turbinate 16, a middle turbinate 15, and a superior turbinate 14. FIG. ID depicts an angled side view of an exemplary embodiment of a representation subject’s nasal channels 20 from the vestibules 21 to the olfactory clefts 23 based on a posteriorly oriented plane 17 showing the middle turbinates 15. FIG. IE depicts an angled side view of an exemplary embodiment of a representation subject’s nasal channels 20 from the vestibules to the olfactory clefts based on an anteriorly oriented plane 18. FIG. IF depicts a side view of an exemplary embodiment of a representation subject’s target ejection area 19. FIG. 1G depicts a front, base and perspective view of an exemplary embodiment of a representation subject’s nose exposing the columella region 10. The respiratory regions comprise turbinates that present physical obstacles to delivery to the upper reaches of a nasal channels 20, e.g., the olfactory clefts 23. Each respiratory region comprises at least one superior turbinate 14. Each respiratory region comprises at least one middle turbinate 15. Each respiratory region comprises at least one inferior turbinate 16. Each respiratory region comprises at least one posterior pathway 17 that involves at least one middle turbinate 15. Each respiratory region comprises at least one anterior pathway 18 that does not involve at least one middle turbinate 15.

[0077] In some cases, the middle turbinate 15 comprises a physical obstruction for composition delivery to an olfactory cleft. In some cases, the middle turbinate 15 comprises a most anterior aspect about aligned with the cheek bone. In some embodiments, the middle turbinate 15 comprises a most anterior aspect not aligned with the cheek bone. In some cases, the nasal channels 20 simplify anteriorly, and comprise angled pathways without one or more turbinates presenting physical obstacles to delivering a composition 111 to theupper nasal channels, including the olfactory clefts 23, or directing compositions 111 down one or more meatuses, e.g., the middle meatus 30, to the nasopharynx. In some cases, the nasal channel 20 comprises one pathway from the vestibule 21 to the olfactory cleft 23 based on an anteriorly oriented plane 18 with a target ejection point 19. In some embodiments, the nose comprises the nasal septum 24, upper lateral cartilage 25, and lower lateral cartilage 26.

[0078] FIG. 1H depicts a side view of an exemplary embodiment of a representation subject’s nasal cavity. In some cases, the subject’s nasal cavity 11 comprises the nasal vestibule 21, inferior turbinate 16, middle turbinate 15, superior turbinate 14, cribriform plate 31 or a combination thereof.

[0079] FIG. 11 depicts a side view of an exemplary embodiment of a representation subject’s sinus. In some cases, the subject’s sinus comprises the inferior turbinate 16, middle turbinate 15, superior turbinate 14, cribriform plate 31, middle meatus 30, or a combination thereof.

[0080] FIG. 1J depicts a side view of an exemplary embodiment of a representation subject’s nasal cavity. In some embodiments, the nasal cavity 11 comprises the nasal bone 33, septal-lateral cartilage junction 27, lower lateral cartilage 26, anterior nasal spine 32, or a combination thereof.

[0081] The “columella” is the firm tissue bridge that separates the nostrils at the base of the nose. The “columella” is the most anteroinferior portion of the nasal septum. The term “columella” or “columella region” is the subnasale, or an anterior nasale spine, or a combination thereof. The columella region may comprise a subnasale, or a combination thereof. The columella shape may be defined by an anterior nasal spine located posteriorly to the columella.

[0082] The “introduction pathway” is, in sequence, the vestibule, the anterior aspect of the internal nasal valve, and the anterior aspect of the respiratory region - anterior of the turbinates.

[0083] The “internal nasal valve” (INV) is a space bounded medially by the dorsal septum 24 (or just septum), laterally by the caudal portion of the upper lateral cartilage, and inferiorly by the head of the inferior turbinate.

[0084] The term “nasal cavity” includes two nasal channels, each comprises a vestibule, respiratory region and olfactory cleft, and a nasopharynx.

[0085] The term “turbinates” refers to superior turbinate, middle turbinate, or inferior turbinate, or a combination thereof.

[0086] Nasal Cavity: This is the large, air-filled space behind the nose, where air passes on its way to the throat during inhalation.

[0087] Internal Nasal Valve: This is the narrowest part of the nasal airway, located just beyond the nostril. It's formed by the edge of the nasal septum, the upper lateral cartilage, and the floor of the nose. The internal nasal valve plays a critical role in regulating airflow through the nose. The area of interest is superior (above) to this structure.

[0088] Nasal Septum: This is the thin wall of bone and cartilage that separates the right and left nostrils. It forms the medial (towards the middle) boundary of the region of interest.

[0089] Lateral Nasal Wall: This is the side wall of the nasal cavity, which is opposite to the nasal septum. It's a complex structure that includes the turbinates (long, curled bones that protrude into the nasal cavity) and the meatuses (grooves or channels between the turbinates). The lateral nasal wall forms the lateral (towards the side) boundary of the region of interest.

[0090] Middle and Superior Meatuses: These are the spaces within the nasal cavity located between the turbinates. The middle meatus is located beneath the middle turbinate and above the inferior turbinate, and the superior meatus is located beneath the superior turbinate. The region of interest encompasses parts of these spaces.

[0091] Nostrils (External Nares): These are the two openings of the nose where air enters.

[0092] Nasal Vestibule: The nasal vestibule is the most anterior part of the nasal cavity, just inside the nostrils. It's the area of the nose that protrudes outside the face predominantly. This area is lined with skin and contains hair follicles, and it acts as the initial filtering and warming area for inhaled air before it moves deeper into the nasal cavity. The nasal vestibule extends posteriorly to the nasal valve, which is the narrowest part of the nasal airway and located just beyond the nostril.

[0093] Nasal Septum: This is a thin wall made of bone and cartilage that separates the left and right sides of the nasal cavity.

[0094] Turbinates (Nasal Conchae): These are three pairs of bony projections (inferior, middle, and superior) covered in mucous membrane that protrude into the nasal cavity from the lateral walls. They increase the surface area of the nasal cavity, aiding in the warming, humidification, and filtration of inhaled air.

[0095] Meatuses: These are the spaces located between the turbinates. Each turbinate has a corresponding meatus underneath it (i.e., inferior, middle, and superior meatus).

[0096] Olfactory Region: This is a small area located at the top of the nasal cavity, where the sense of smell is located.I. Method

[0097] In one aspect, provided herein is a method of delivering a therapeutic peptide to a subject in need thereof, the method comprising: a. selectively delivering a formulation comprising a therapeutically effective amount of the therapeutic peptide to an olfactory region of the subject; wherein at least a portion of formulation is delivered to one or more of the group consisting of cerebrospinal fluid, the central nervous system or a region, tissue, or organ thereof, and a non-CNS target. In one aspect, provided herein is a method of delivering a therapeutic peptide to a subject in need thereof. The method may comprise delivering a formulation comprising a therapeutically effective amount of the therapeutic peptide to an olfactory region of the subject. At least a portion of the formulation may be delivered to the cerebrospinal fluid. At least a portion of the formulation may be delivered to the central nervous system. At least a portion of the formulation may be delivered to a region, tissue, or organ of the central nervous system. At least a portion of the formulation may be delivered to a non-CNS target. At least a portion of the formulation may be delivered to one or more of the group consisting of cerebrospinal fluid, the central nervous system, or a region, tissue, or organ thereof, and a non-CNS target.

[0098] The method can deliver at least 50% of the formulation to the olfactory region.

[0099] In some embodiments, at least about 50% of the formulation is delivered to the olfactory region of the subject. In some embodiments, at least about 55% of the formulation is delivered to the olfactory region of the subject. In some embodiments, at least about 60% of the formulation is delivered to the olfactory region of the subject. In some embodiments, at least about 65% of the formulation is delivered to the olfactory region of the subject. In some embodiments, at least about 70% of the formulation is delivered to the olfactory region of the subject. In some embodiments, at least about 75% of the formulation is delivered to the olfactory region of the subject. In some embodiments, at least about 80% of the formulation is delivered to the olfactory region of the subject. In some embodiments, at least about 85% of the formulation is delivered to the olfactory region of the subject. In some embodiments, at least about 90% of the formulation is delivered to the olfactory region ofthe subject. In some embodiments, at least about 95% of the formulation is delivered to the olfactory region of the subject.

[0100] The formulation can be ejected from a dispensing element. In some embodiments, at least about 70% of the formulation can pass through a circular opening having a diameter of between about 5 mm and 6 mm placed about 25 mm from where the formulation is ejected from the dispensing element. In some embodiments, at least about 75% of the formulation can pass through a circular opening having a diameter of between about 5 mm and 6 mm placed about 25 mm from where the formulation is ejected from the dispensing element. In some embodiments, at least about 80% of the formulation can pass through a circular opening having a diameter of between about 5 mm and 6 mm placed about 25 mm from where the formulation is ejected from the dispensing element. In some embodiments, at least about 85% of the formulation can pass through a circular opening having a diameter of between about 5 mm and 6 mm placed about 25 mm from where the formulation is ejected from the dispensing element. In some embodiments, at least about 90% of the formulation can pass through a circular opening having a diameter of between about 5 mm and 6 mm placed about 25 mm from where the formulation is ejected from the dispensing element. In some embodiments, at least about 95% of the formulation can pass through a circular opening having a diameter of between about 5 mm and 6 mm placed about 25 mm from where the formulation is ejected from the dispensing element.

[0101] In some embodiments, the formulation has a viscosity of between about 0.5 cP and about 100 cP. In some embodiments, the formulation has a viscosity of between about 0.5 cP and about 90 cP. In some embodiments, the formulation has a viscosity of between about 0.5 cP and about 80 cP. In some embodiments, the formulation has a viscosity of between about 0.5 cP and about 70 cP. In some embodiments, the formulation has a viscosity of between about 0.5 cP and about 60 cP. In some embodiments, the formulation has a viscosity of between about 0.5 cP and about 50 cP. In some embodiments, the formulation has a viscosity of between about 0.5 cP and about 40 cP. In some embodiments, the formulation has a viscosity of between about 0.5 cP and about 30 cP. In some embodiments, the formulation has a viscosity of between about 0.5 cP and about 20 cP. In some embodiments, the formulation has a viscosity of between about 0.5 cP and about 10 cP. In some embodiments, the formulation has a viscosity of between about 0.5 cP and about 5 cP. In some embodiments, the formulation has a viscosity of between about 5 cP and about 10 cP.In some embodiments, the formulation has a viscosity of between about 10 cP and about 15 cP. In some embodiments, the formulation has a viscosity of between about 15 cP and about 20 cP. In some embodiments, the formulation has a viscosity of between about 20 cP and about 25 cP. In some embodiments, the formulation has a viscosity of between about 25 cP and about 30 cP. In some embodiments, the formulation has a viscosity of between about 30 cP and about 35 cP. In some embodiments, the formulation has a viscosity of between about 35 cP and about 40 cP. In some embodiments, the formulation has a viscosity of between about 40 cP and about 45 cP. In some embodiments, the formulation has a viscosity of between about 45 cP and about 50 cP. In some embodiments, the formulation has a viscosity of between about 50 cP and about 55 cP. In some embodiments, the formulation has a viscosity of between about 55 cP and about 60 cP. In some embodiments, the formulation has a viscosity of between about 60 cP and about 65 cP. In some embodiments, the formulation has a viscosity of between about 65 cP and about 70 cP. In some embodiments, the formulation has a viscosity of between about 70 cP and about 75 cP. In some embodiments, the formulation has a viscosity of between about 75 cP and about 80 cP. In some embodiments, the formulation has a viscosity of between about 85 cP and about 90 cP. In some embodiments, the formulation has a viscosity of between about 95 cP and about 100 cP. In some embodiments, the formulation has a viscosity of higher than 100 cP. In some embodiments, the formulation has a viscosity of between about 45 cP and about 55 cP. In some embodiments, the formulation has a viscosity of between about 48 cP and about 52 cP. In some embodiments, the formulation has a viscosity of about 1 cP. In some embodiments, the formulation has a viscosity of about 50 cP.

[0102] In some embodiments, the formulation is ejected from the dispensing element at a velocity of between about 1 m / s and about 30 m / s. In some embodiments, the formulation is ejected from the dispensing element at a velocity of between about 2 m / s and about 4 m / s. In some embodiments, the formulation is ejected from the dispensing element at a velocity of between about 25 m / s and about 30 m / s. In some embodiments, the formulation is ejected from the dispensing element at a velocity. In some embodiments, the velocity is about 1 m / s. In some embodiments, the velocity is about 2 m / s. In some embodiments, the velocity is about 3 m / s. In some embodiments, the velocity is about 4 m / s. In some embodiments, the velocity is about 5 m / s. In some embodiments, the velocity is about 6 m / s. In some embodiments, the velocity is about 7 m / s. In some embodiments, the velocity is about 8 m / s.In some embodiments, the velocity is about 9 m / s. In some embodiments, the velocity is about 10 m / s. In some embodiments, the velocity is about 11 m / s. In some embodiments, the velocity is about 12 m / s. In some embodiments, the velocity is about 13 m / s. In some embodiments, the velocity is about 14 m / s. In some embodiments, the velocity is about 15 m / s. In some embodiments, the velocity is about 16 m / s. In some embodiments, the velocity is about 17 m / s. In some embodiments, the velocity is about 18 m / s. In some embodiments, the velocity is about 19 m / s. In some embodiments, the velocity is about 20 m / s. In some embodiments, the velocity is about 21 m / s. In some embodiments, the velocity is about 22 m / s. In some embodiments, the velocity is about 23 m / s. In some embodiments, the velocity is about 24 m / s. In some embodiments, the velocity is about 25 m / s. In some embodiments, the velocity is about 26 m / s. In some embodiments, the velocity is about 27 m / s. In some embodiments, the velocity is about 28 m / s. In some embodiments, the velocity is about 29 m / s. In some embodiments, the velocity is about 30 m / s. In some embodiments, the velocity is between about 1 m / s and about 2 m / s. In some embodiments, the velocity is between about 4 m / s and about 6 m / s. In some embodiments, the velocity is between about 6 m / s and about 8 m / s. In some embodiments, the velocity is between about 8 m / s and about 10 m / s. In some embodiments, the velocity is between about 10 m / s and about 12 m / s. In some embodiments, the velocity is between about 12 m / s and about 14 m / s. In some embodiments, the velocity is between about 14 m / s and about 16 m / s. In some embodiments, the velocity is between about 16 m / s and about 18 m / s. In some embodiments, the velocity is between about 18 m / s and about 20 m / s. In some embodiments, the velocity is between about 20 m / s and about 22 m / s. In some embodiments, the velocity is between about 22 m / s and about 24 m / s. In some embodiments, the velocity is between about 24 m / s and about 26 m / s. In some embodiments, the velocity is between about 26 m / s and about 28 m / s. In some embodiments, the velocity is between about 28 m / s and about 30 m / s.

[0103] In some embodiments, the formulation is ejected from a dispensing element, wherein at least about 75% of the formulation passes through a circular opening having a diameter of between about 5 mm and about 6 mm placed about 25 mm from where the formulation is ejected from the dispensing element. In some embodiments, the formulation has a viscosity of about 1 cP and is ejected from the dispensing element at a velocity of between about 2 m / s and about 4 m / s. In some embodiments, the formulation has a viscosity of about 50 cP and is ejected from the dispensing element at a velocity of between about 25m / s and about 30 m / s. In some embodiments, the formulation has a viscosity of between about 1 cP and about 50 cP. In some embodiments, the formulation is ejected from the dispensing element at a velocity of between about 1 m / s and about 30 m / s.

[0104] The formulation can be delivered as a liquid jet or laminar flow. The formulation can be delivered as a flow having a Reynold’s number of 2300 or less.

[0105] Laminar flow can refer to a smooth, regular movement of fluids where adjacent layers of particles experience relatively little mixing.

[0106] In some embodiments, the formulation is delivered as a liquid jet.

[0107] In some embodiments, the formulation is delivered as a laminar flow.

[0108] In some embodiments, delivering the formulation as a laminar flow increases on target delivery of the composition to the target region compared to delivering the formulation with a spray ejection profile. In some embodiments, delivering the formulation as a laminar flow decreases off target delivery of the composition to the nasal cavity compared to delivering the formulation with a spray ejection profile. In some embodiments, delivering the formulation as a laminar flow increases on target delivery of the composition to the target region and decreases off target delivery of the composition to the nasal cavity compared to delivering the formulation with a spray ejection profile.

[0109] In some embodiments, the formulation is delivered as a liquid jet.

[0110] In some embodiments, delivering the formulation as a liquid jet increases on target delivery of the composition to the target region compared to delivering the formulation with a spray ejection profile. In some embodiments, delivering the formulation as a liquid jet decreases off target delivery of the composition to the nasal cavity compared to delivering the formulation with a spray ejection profile. In some embodiments, delivering the formulation as a liquid jet increases on target delivery of the composition to the target region and decreases off target delivery of the composition to the nasal cavity compared to delivering the formulation with a spray ejection profile.

[0111] In some embodiments, the formulation is delivered as a flow having a Reynold’s number of 2300 or less. In some embodiments, the formulation is delivered as a flow having a Reynold’s number of 2200 or less. In some embodiments, the formulation is delivered as a flow having a Reynold’s number of 2100 or less. In some embodiments, the formulation is delivered as a flow having a Reynold’s number of 2000 or less. In some embodiments, the formulation is delivered as a flow having a Reynold’s number of 1900 or less. In someembodiments, the formulation is delivered as a flow having a Reynold’s number of 1800 or less. In some embodiments, the formulation is delivered as a flow having a Reynold’s number of 1700 or less. In some embodiments, the formulation is delivered as a flow having a Reynold’s number of 1600 or less. In some embodiments, the formulation is delivered as a flow having a Reynold’s number of 1500 or less. In some embodiments, the formulation is delivered as a flow having a Reynold’s number of 1400 or less. In some embodiments, the formulation is delivered as a flow having a Reynold’s number of 1300 or less. In some embodiments, the formulation is delivered as a flow having a Reynold’s number of 1200 or less. In some embodiments, the formulation is delivered as a flow having a Reynold’s number of 1100 or less. In some embodiments, the formulation is delivered as a flow having a Reynold’s number of 1000 or less. In some embodiments, the formulation is delivered as a flow having a Reynold’s number of 900 or less. In some embodiments, the formulation is delivered as a flow having a Reynold’s number of 800 or less. In some embodiments, the formulation is delivered as a flow having a Reynold’s number of 700 or less. In some embodiments, the formulation is delivered as a flow having a Reynold’s number of 600 or less. In some embodiments, the formulation is delivered as a flow having a Reynold’s number of 500 or less. In some embodiments, the formulation is delivered as a flow having a Reynold’s number of 400 or less. In some embodiments, the formulation is delivered as a flow having a Reynold’s number of 300 or less.

[0112] In some embodiments, delivering the formulation as a flow having a Reynold’s number of 2300 or less increases on target delivery of the composition to the target region compared to delivering the formulation with a spray ejection profile. In some embodiments, delivering the formulation as a flow having a Reynold’s number of 2300 or less decreases off target delivery of the composition to the nasal cavity compared to delivering the formulation with a spray ejection profile. In some embodiments, delivering the formulation as a flow having a Reynold’s number of 2300 or less increases on target delivery of the composition to the target region and decreases off target delivery of the composition to the nasal cavity compared to delivering the formulation with a spray ejection profile.

[0113] As shown herein, delivery of the therapeutic peptide by the method disclosed herein can result in a plasma concentration of the therapeutic peptide for a period of time after delivery. In some embodiments, the period of time is 5 min after delivery. In some embodiments, the period of time is 15 min after delivery. In some embodiments, the periodof time is 30 min after delivery. In some embodiments, the period of time is 60 min after delivery. In some embodiments, the period of time is 90 min after delivery. In some embodiments, the period of time is 120 min after delivery. In some embodiments, the period of time is 150 min after delivery. In some embodiments, the period of time is 180 min after delivery. In some embodiments, the period of time is 200 min after delivery. In some embodiments, the period of time is 250 min after delivery.

[0114] In some embodiments, delivery of the therapeutic peptide to the olfactory region of the subject results in a plasma concentration of the therapeutic peptide about 60 min after delivery that is at least about 5% of a plasma concentration of the therapeutic peptide when about the same dose is delivered intravenously. In some embodiments, delivery of the therapeutic peptide to the olfactory region of the subject results in a plasma concentration of the therapeutic peptide about 60 min after delivery that is at least about 6% of a plasma concentration of the therapeutic peptide when about the same dose is delivered intravenously. In some embodiments, delivery of the therapeutic peptide to the olfactory region of the subject results in a plasma concentration of the therapeutic peptide about 60 min after delivery that is at least about 7% of a plasma concentration of the therapeutic peptide when about the same dose is delivered intravenously. In some embodiments, delivery of the therapeutic peptide to the olfactory region of the subject results in a plasma concentration of the therapeutic peptide about 60 min after delivery that is at least about 8% of a plasma concentration of the therapeutic peptide when about the same dose is delivered intravenously. In some embodiments, delivery of the therapeutic peptide to the olfactory region of the subject results in a plasma concentration of the therapeutic peptide about 60 min after delivery that is at least about 9% of a plasma concentration of the therapeutic peptide when about the same dose is delivered intravenously. In some embodiments, delivery of the therapeutic peptide to the olfactory region of the subject results in a plasma concentration of the therapeutic peptide about 60 min after delivery that is at least about 10% of a plasma concentration of the therapeutic peptide when about the same dose is delivered intravenously. In some embodiments, delivery of the therapeutic peptide to the olfactory region of the subject results in a plasma concentration of the therapeutic peptide about 120 min after delivery that is at least about 10% of a plasma concentration of the therapeutic peptide when about the same dose is delivered intravenously. In some embodiments, delivery of the therapeutic peptide to the olfactory region of the subjectresults in a plasma concentration of the therapeutic peptide about 120 min after delivery that is at least about 20% of a plasma concentration of the therapeutic peptide when about the same dose is delivered intravenously. In some embodiments, delivery of the therapeutic peptide to the olfactory region of the subject results in a plasma concentration of the therapeutic peptide about 120 min after delivery that is at least about 30% of a plasma concentration of the therapeutic peptide when about the same dose is delivered intravenously. In some embodiments, delivery of the therapeutic peptide to the olfactory region of the subject results in a plasma concentration of the therapeutic peptide about 120 min after delivery that is at least about 40% of a plasma concentration of the therapeutic peptide when about the same dose is delivered intravenously. In some embodiments, delivery of the therapeutic peptide to the olfactory region of the subject results in a plasma concentration of the therapeutic peptide about 120 min after delivery that is at least about 50% of a plasma concentration of the therapeutic peptide when about the same dose is delivered intravenously. In some embodiments, delivery of the therapeutic peptide to the olfactory region of the subject results in a plasma concentration of the therapeutic peptide about 200 min after delivery that is at least about 50% of a plasma concentration of the therapeutic peptide when about the same dose is delivered intravenously. In some embodiments, delivery of the therapeutic peptide to the olfactory region of the subject results in a plasma concentration of the therapeutic peptide about 200 min after delivery that is at least about 60% of a plasma concentration of the therapeutic peptide when about the same dose is delivered intravenously. In some embodiments, delivery of the therapeutic peptide to the olfactory region of the subject results in a plasma concentration of the therapeutic peptide about 200 min after delivery that is at least about 70% of a plasma concentration of the therapeutic peptide when about the same dose is delivered intravenously. In some embodiments, delivery of the therapeutic peptide to the olfactory region of the subject results in a plasma concentration of the therapeutic peptide about 200 min after delivery that is at least about 80% of a plasma concentration of the therapeutic peptide when about the same dose is delivered intravenously. In some embodiments, delivery of the therapeutic peptide to the olfactory region of the subject results in a plasma concentration of the therapeutic peptide about 200 min after delivery that is at least about 90% of a plasma concentration of the therapeutic peptide when about the same dose is delivered intravenously.

[0115] In some embodiments, selectively delivering the formulation to the olfactory region of the subject comprises inserting a dispensing element into an ejection zone of a nasal cavity of the subject.

[0116] In some embodiments, the method further comprises inserting a dispensing element into a nasal cavity of the subject.

[0117] In some embodiments, the dispensing element is positioned from about 0.1 mm to about 30 mm from the olfactory region or an anterior entry to the olfactory region. In some embodiments, the tip is positioned from about 0.1 mm to about 25 mm from the olfactory region or an anterior entry to the olfactory region. In some embodiments, the tip is positioned from about 0.1 mm to about 3 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 9 mm, about 0.1 mm to about 12 mm, about 0.1 mm to about 15 mm, about 0.1 mm to about 18 mm, about 0.1 mm to about 20 mm, about 0.1 mm to about 25 mm, about 0.1 mm to about 30 mm, about 3 mm to about 5 mm, about 3 mm to about 9 mm, about 3 mm to about 12 mm, about 3 mm to about 15 mm, about 3 mm to about 18 mm, about 3 mm to about 20 mm, about 3 mm to about 25 mm, about 3 mm to about 30 mm, about 5 mm to about 9 mm, about 5 mm to about 12 mm, about 5 mm to about 15 mm, about 5 mm to about 18 mm, about 5 mm to about 20 mm, about 5 mm to about 25 mm, about 5 mm to about 30 mm, about 9 mm to about 12 mm, about 9 mm to about 15 mm, about 9 mm to about 18 mm, about 9 mm to about 20 mm, about 9 mm to about 25 mm, about 9 mm to about 30 mm, about 12 mm to about 15 mm, about 12 mm to about 18 mm, about 12 mm to about 20 mm, about 12 mm to about 25 mm, about 12 mm to about 30 mm, about 15 mm to about 18 mm, about 15 mm to about 20 mm, about 15 mm to about 25 mm, about 15 mm to about 30 mm, about 18 mm to about 20 mm, about 18 mm to about 25 mm, about 18 mm to about 30 mm, about 20 mm to about 25 mm, about 20 mm to about 30 mm, or about 25 mm to about 30 mm, including increments therein. In some embodiments, the tip is positioned from about 0.1 mm, about 3 mm, about 5 mm, about 9 mm, about 12 mm, about 15 mm, about 18 mm, about 20 mm, about 25 mm, or about 30 mm from the olfactory region or an anterior entry to the olfactory region. In some embodiments, the tip is positioned from at least about 0.1 mm, about 3 mm, about 5 mm, about 9 mm, about 12 mm, about 15 mm, about 18 mm, about 20 mm, or about 25 mm from the olfactory region or an anterior entry to the olfactory region. In some embodiments, the dispensing element is positioned from at most about 3 mm, about 5 mm, about 9 mm, about 12 mm, about 15 mm, about 18 mm, about 20 mm,about 25 mm, or about 30 mm from the olfactory region or an anterior entry to the olfactory region.

[0118] In some embodiments, the method further comprises ejecting the formulation from an ejection zone of the nasal cavity.

[0119] In some embodiments, dispensing the formulation from the ejection zone increases on target delivery of the composition to the target region compared to dispensing the formulation outside the ejection zone. In some embodiments, dispensing the formulation from the ejection zone decreases off target delivery of the composition to the nasal cavity compared to dispensing the formulation outside the ejection zone. In some embodiments, dispensing the formulation from the ejection zone increases on target delivery of the composition to the target region and decreases off target delivery of the composition to the nasal cavity compared to dispensing the formulation outside the ejection zone.

[0120] Referring to FIGs. 4A-4J. In some embodiments, the ejection zone 29 is: 0mm to 30mm superior to a horizontal line 627 that intersects the anterior aspect of the internal nasal valve 13, and 0mm to 20mm anterior to an inclined line 610 that intersects the anterior aspect of the middle turbinate 15 and the posterior aspect of the vestibule 21.

[0121] In some embodiments, the ejection zone 29 is further 0 mm to 40mm inferior to a horizontal line 621 that is parallel to the inferior aspect of the olfactory cleft 23, 0mm to 20mm posterior to the internal nasal dorsum 622, 10mm to 50mm superior to a horizontal line 617 that intersects the inferior aspect of the columella 10, 0mm to 30mm superior to a horizontal line 624 that intersects the superior aspect of the inferior turbinate 16, 0mm to 3mm from the septum 24 , or any combination thereof.

[0122] In some embodiments, the ejection zone 29 is a trapezium or irregular quadrilateral comprising (i) an inferior side 29A being a 10-25mm line extending posteriorly and horizontally from the anterior aspect of the internal nasal valve 13, (ii) an anterior side 29B being a 10-35mm line extending superiorly and parallel to the internal nasal dorsum 622 from the anterior aspect of the internal nasal valve 13, (iii) a superior side 29C being a 10- 25mm line extending posteriorly and horizontally from a point on the internal nasal dorsum 622 that is 0-10mm inferior to the inferior aspect of the olfactory cleft 23, and (iv) a posterior line 29D being a 10-35mm line extending vertically along a plane that intersects the anterior aspect of the middle turbinate 15.

[0123] In some embodiments, dispensing the formulation from the ejection zone 29 increases on target deposition of the formulation to an olfactory cleft 23.

[0124] In some embodiments, the subject engaging portion 106 engages a columella region 10 of the subject to seat a distal end 128 of the insertable portion 107 within an ejection zone 29 of a nasal cavity 11 of the subject, wherein the ejection zone 29 is: 0mm to 30mm superior to a horizontal line 627 that intersects the anterior aspect of the internal nasal valve 13, and 0mm to 20mm anterior to an inclined line 610 that intersects the anterior aspect of the middle turbinate 15 and the posterior aspect of the vestibule 21, and one or more of the following: 0mm to 40mm inferior to a horizontal line 621 that is parallel to the inferior aspect of the olfactory cleft 23, 0mm to 20mm posterior to the internal nasal dorsum 622, 10mm to 50mm superior to a horizontal line 623 that intersects the inferior aspect of the columella 10, 0mm to 30mm superior to a horizontal line 624 that intersects the superior aspect of the inferior turbinate 16, 0mm to 3mm from the septum 24 or any combination thereof.

[0125] In some embodiments, the ejection zone is superior to a horizontal line that intersects the anterior aspect of the internal nasal valve by about 1 mm to about 30 mm. In some embodiments, the ejection zone is superior to a horizontal line that intersects the anterior aspect of the internal nasal valve by about 1 mm to about 2 mm, about 1 mm to about 4 mm, about 1 mm to about 6 mm, about 1 mm to about 8 mm, about 1 mm to about 10 mm, about 1 mm to about 14 mm, about 1 mm to about 18 mm, about 1 mm to about 22 mm, about 1 mm to about 26 mm, about 1 mm to about 30 mm, about 2 mm to about 4 mm, about 2 mm to about 6 mm, about 2 mm to about 8 mm, about 2 mm to about 10 mm, about 2 mm to about 14 mm, about 2 mm to about 18 mm, about 2 mm to about 22 mm, about 2 mm to about 26 mm, about 2 mm to about 30 mm, about 4 mm to about 6 mm, about 4 mm to about 8 mm, about 4 mm to about 10 mm, about 4 mm to about 14 mm, about 4 mm to about 18 mm, about 4 mm to about 22 mm, about 4 mm to about 26 mm, about 4 mm to about 30 mm, about 6 mm to about 8 mm, about 6 mm to about 10 mm, about 6 mm to about 14 mm, about 6 mm to about 18 mm, about 6 mm to about 22 mm, about 6 mm to about 26 mm, about 6 mm to about 30 mm, about 8 mm to about 10 mm, about 8 mm to about 14 mm, about 8 mm to about 18 mm, about 8 mm to about 22 mm, about 8 mm to about 26 mm, about 8 mm to about 30 mm, about 10 mm to about 14 mm, about 10 mm to about 18 mm, about 10 mm to about 22 mm, about 10 mm to about 26 mm, about 10 mm to about 30mm, about 14 mm to about 18 mm, about 14 mm to about 22 mm, about 14 mm to about 26 mm, about 14 mm to about 30 mm, about 18 mm to about 22 mm, about 18 mm to about 26 mm, about 18 mm to about 30 mm, about 22 mm to about 26 mm, about 22 mm to about 30 mm, or about 26 mm to about 30 mm, including increments therein. In some embodiments, the ejection zone is superior to a horizontal line that intersects the anterior aspect of the internal nasal valve by about 1 mm, about 2 mm, about 4 mm, about 6 mm, about 8 mm, about 10 mm, about 14 mm, about 18 mm, about 22 mm, about 26 mm, or about 30 mm. In some embodiments, the ejection zone is superior to a horizontal line that intersects the anterior aspect of the internal nasal valve by at least about 1 mm, about 2 mm, about 4 mm, about 6 mm, about 8 mm, about 10 mm, about 14 mm, about 18 mm, about 22 mm, or about 26 mm. In some embodiments, the ejection zone is superior to a horizontal line that intersects the anterior aspect of the internal nasal valve by at most about 2 mm, about 4 mm, about 6 mm, about 8 mm, about 10 mm, about 14 mm, about 18 mm, about 22 mm, about 26 mm, or about 30 mm. In some embodiments, the ejection zone is anterior to an inclined line that intersects the anterior aspect of the middle turbinate and the posterior aspect of the vestibule by about 1 mm to about 20 mm. In some embodiments, the ejection zone is anterior to an inclined line that intersects the anterior aspect of the middle turbinate and the posterior aspect of the vestibule by about 1 mm to about 2 mm, about 1 mm to about 4 mm, about 1 mm to about 6 mm, about 1 mm to about 8 mm, about 1 mm to about 10 mm, about 1 mm to about 12 mm, about 1 mm to about 14 mm, about 1 mm to about 16 mm, about 1 mm to about 18 mm, about 1 mm to about 20 mm, about 2 mm to about 4 mm, about 2 mm to about 6 mm, about 2 mm to about 8 mm, about 2 mm to about 10 mm, about 2 mm to about 12 mm, about 2 mm to about 14 mm, about 2 mm to about 16 mm, about 2 mm to about 18 mm, about 2 mm to about 20 mm, about 4 mm to about 6 mm, about 4 mm to about 8 mm, about 4 mm to about 10 mm, about 4 mm to about 12 mm, about 4 mm to about 14 mm, about 4 mm to about 16 mm, about 4 mm to about 18 mm, about 4 mm to about 20 mm, about 6 mm to about 8 mm, about 6 mm to about 10 mm, about 6 mm to about 12 mm, about 6 mm to about 14 mm, about 6 mm to about 16 mm, about 6 mm to about 18 mm, about 6 mm to about 20 mm, about 8 mm to about 10 mm, about 8 mm to about 12 mm, about 8 mm to about 14 mm, about 8 mm to about 16 mm, about 8 mm to about 18 mm, about 8 mm to about 20 mm, about 10 mm to about 12 mm, about 10 mm to about 14 mm, about 10 mm to about 16 mm, about 10 mm to about 18 mm, about 10 mm to about 20 mm, about 12mm to about 14 mm, about 12 mm to about 16 mm, about 12 mm to about 18 mm, about 12 mm to about 20 mm, about 14 mm to about 16 mm, about 14 mm to about 18 mm, about 14 mm to about 20 mm, about 16 mm to about 18 mm, about 16 mm to about 20 mm, or about 18 mm to about 20 mm, including increments therein. In some embodiments, the ejection zone is anterior to an inclined line that intersects the anterior aspect of the middle turbinate and the posterior aspect of the vestibule by about 1 mm, about 2 mm, about 4 mm, about 6 mm, about 8 mm, about 10 mm, about 12 mm, about 14 mm, about 16 mm, about 18 mm, or about 20 mm. In some embodiments, the ejection zone is anterior to an inclined line that intersects the anterior aspect of the middle turbinate and the posterior aspect of the vestibule by at least about 1 mm, about 2 mm, about 4 mm, about 6 mm, about 8 mm, about 10 mm, about 12 mm, about 14 mm, about 16 mm, or about 18 mm. In some embodiments, the ejection zone is anterior to an inclined line that intersects the anterior aspect of the middle turbinate and the posterior aspect of the vestibule by at most about 2 mm, about 4 mm, about 6 mm, about 8 mm, about 10 mm, about 12 mm, about 14 mm, about 16 mm, about 18 mm, or about 20 mm. In some embodiments, the ejection zone is inferior to a horizontal line that is parallel to the inferior aspect of the olfactory cleft by about 1 mm to about 35 mm. In some embodiments, the ejection zone is inferior to a horizontal line that is parallel to the inferior aspect of the olfactory cleft by about 1 mm to about 2 mm, about 1 mm to about 3 mm, about 1 mm to about 4 mm, about 1 mm to about 5 mm, about 1 mm to about 8 mm, about 1 mm to about 12 mm, about 1 mm to about 15 mm, about 1 mm to about 18 mm, about 1 mm to about 21 mm, about 1 mm to about 25 mm, about 1 mm to about 30 mm, about 1 mm to about 35 mm, about 2 mm to about 3 mm, about 2 mm to about 4 mm, about 2 mm to about 5 mm, about 2 mm to about 8 mm, about 2 mm to about 12 mm, about 2 mm to about 15 mm, about 2 mm to about 18 mm, about 2 mm to about 21 mm, about 2 mm to about 25 mm, about 2 mm to about 30 mm, about 2 mm to about 35 mm, about 3 mm to about 4 mm, about 3 mm to about 5 mm, about 3 mm to about 8 mm, about 3 mm to about 12 mm, about 3 mm to about 15 mm, about 3 mm to about 18 mm, about 3 mm to about 21 mm, about 3 mm to about 25 mm, about 3 mm to about 30 mm, about 3 mm to about 35 mm, about 4 mm to about 5 mm, about 4 mm to about 8 mm, about 4 mm to about 12 mm, about 4 mm to about 15 mm, about 4 mm to about 18 mm, about 4 mm to about 21 mm, about 4 mm to about 25 mm, about 4 mm to about 30 mm, about 4 mm to about 35 mm, about 5 mm to about 8 mm, about 5 mm to about 12 mm, about 5 mm to about 15 mm, about5 mm to about 18 mm, about 5 mm to about 21 mm, about 5 mm to about 25 mm, about 5 mm to about 30 mm, about 5 mm to about 35 mm, about 8 mm to about 15 mm, about 8 mm to about 18 mm, about 8 mm to about 21 mm, about 8 mm to about 25 mm, about 8 mm to about 30 mm, about 8 mm to about 35 mm, about 12 mm to about 15 mm, about 12 mm to about 18 mm, about 12 mm to about 21 mm, about 12 mm to about 25 mm, about 12 mm to about 30 mm, about 12 mm to about 35 mm, about 15 mm to about 18 mm, about 15 mm to about 21 mm, about 15 mm to about 25 mm, about 15 mm to about 30 mm, about 15 mm to about 35 mm, about 18 mm to about 21 mm, about 18 mm to about 25 mm, about 18 mm to about 30 mm, about 18 mm to about 35 mm, about 21 mm to about 25 mm, about 21 mm to about 30 mm, about 21 mm to about 35 mm, about 25 mm to about 30 mm, about 25 mm to about 35 mm, or about 30 mm to about 35 mm, including increments therein. In some embodiments, the ejection zone is inferior to a horizontal line that is parallel to the inferior aspect of the olfactory cleft by about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 8 mm, about 12 mm, about 15 mm, about 18 mm, about 21 mm, about 25 mm, about 30 mm, or about 35 mm. In some embodiments, the ejection zone is inferior to a horizontal line that is parallel to the inferior aspect of the olfactory cleft by at least about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 8 mm, about 12 mm, about 15 mm, about 18 mm, about 21 mm, about 25 mm, or about 30 mm. In some embodiments, the ejection zone is inferior to a horizontal line that is parallel to the inferior aspect of the olfactory cleft by at most about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 8 mm, about 12 mm, about 15 mm, about 18 mm, about 21 mm, about 25 mm, about 30 mm, or about 35 mm. In some embodiments, the ejection zone is posterior to the internal nasal dorsum by about 1 mm to about 20 mm. In some embodiments, the ejection zone is posterior to the internal nasal dorsum by about 1 mm to about 2 mm, about 1 mm to about 3 mm, about 1 mm to about 4 mm, about 1 mm to about 6 mm, about 1 mm to about 8 mm, about 1 mm to about 10 mm, about 1 mm to about 12 mm, about 1 mm to about 14 mm, about 1 mm to about 16 mm, about 1 mm to about 18 mm, about 1 mm to about 20 mm, about 2 mm to about 3 mm, about 2 mm to about 4 mm, about 2 mm to about 6 mm, about 2 mm to about 8 mm, about 2 mm to about 10 mm, about 2 mm to about 12 mm, about 2 mm to about 14 mm, about 2 mm to about 16 mm, about 2 mm to about 18 mm, about 2 mm to about 20 mm, about 3 mm to about 4 mm, about 3 mm to about 6 mm, about 3 mm to about 8 mm, about 3 mm to about 10 mm, about 3 mm to about 12 mm, about 3 mm to about 14 mm,about 3 mm to about 16 mm, about 3 mm to about 18 mm, about 3 mm to about 20 mm, about 4 mm to about 6 mm, about 4 mm to about 8 mm, about 4 mm to about 10 mm, about 4 mm to about 12 mm, about 4 mm to about 14 mm, about 4 mm to about 16 mm, about 4 mm to about 18 mm, about 4 mm to about 20 mm, about 6 mm to about 8 mm, about 6 mm to about 10 mm, about 6 mm to about 12 mm, about 6 mm to about 14 mm, about 6 mm to about 16 mm, about 6 mm to about 18 mm, about 6 mm to about 20 mm, about 8 mm to about 10 mm, about 8 mm to about 12 mm, about 8 mm to about 14 mm, about 8 mm to about 16 mm, about 8 mm to about 18 mm, about 8 mm to about 20 mm, about 10 mm to about 12 mm, about 10 mm to about 14 mm, about 10 mm to about 16 mm, about 10 mm to about 18 mm, about 10 mm to about 20 mm, about 12 mm to about 14 mm, about 12 mm to about 16 mm, about 12 mm to about 18 mm, about 12 mm to about 20 mm, about 14 mm to about 16 mm, about 14 mm to about 18 mm, about 14 mm to about 20 mm, about 16 mm to about 18 mm, about 16 mm to about 20 mm, or about 18 mm to about 20 mm, including increments therein. In some embodiments, the ejection zone is posterior to the internal nasal dorsum by about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 6 mm, about 8 mm, about 10 mm, about 12 mm, about 14 mm, about 16 mm, about 18 mm, or about 20 mm. In some embodiments, the ejection zone is posterior to the internal nasal dorsum by at least about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 6 mm, about 8 mm, about 10 mm, about 12 mm, about 14 mm, about 16 mm, or about 18 mm. In some embodiments, the ejection zone is posterior to the internal nasal dorsum by at most about 2 mm, about 3 mm, about 4 mm, about 6 mm, about 8 mm, about 10 mm, about 12 mm, about 14 mm, about 16 mm, about 18 mm, or about 20 mm. In some embodiments, the ejection zone is superior to a horizontal line that intersects the inferior aspect of the columella by about 10 mm to about 50 mm. In some embodiments, the ejection zone is superior to a horizontal line that intersects the inferior aspect of the columella by about 10 mm to about 12 mm, about 10 mm to about 16 mm, about 10 mm to about 20 mm, about 10 mm to about 24 mm, about 10 mm to about 28 mm, about 10 mm to about 32 mm, about 10 mm to about 36 mm, about 10 mm to about 40 mm, about 10 mm to about 45 mm, about 10 mm to about 50 mm, about 12 mm to about 16 mm, about 12 mm to about 20 mm, about 12 mm to about 24 mm, about 12 mm to about 28 mm, about 12 mm to about 32 mm, about 12 mm to about 36 mm, about 12 mm to about 40 mm, about 12 mm to about 45 mm, about 12 mm to about 50 mm, about 16 mm to about 20 mm, about 16 mm to about 24 mm, about 16 mm to about 28 mm, about 16mm to about 32 mm, about 16 mm to about 36 mm, about 16 mm to about 40 mm, about 16 mm to about 45 mm, about 16 mm to about 50 mm, about 20 mm to about 24 mm, about 20 mm to about 28 mm, about 20 mm to about 32 mm, about 20 mm to about 36 mm, about 20 mm to about 40 mm, about 20 mm to about 45 mm, about 20 mm to about 50 mm, about 24 mm to about 28 mm, about 24 mm to about 32 mm, about 24 mm to about 36 mm, about 24 mm to about 40 mm, about 24 mm to about 45 mm, about 24 mm to about 50 mm, about 28 mm to about 32 mm, about 28 mm to about 36 mm, about 28 mm to about 40 mm, about 28 mm to about 45 mm, about 28 mm to about 50 mm, about 32 mm to about 36 mm, about 32 mm to about 40 mm, about 32 mm to about 45 mm, about 32 mm to about 50 mm, about 36 mm to about 40 mm, about 36 mm to about 45 mm, about 36 mm to about 50 mm, about 40 mm to about 45 mm, about 40 mm to about 50 mm, or about 45 mm to about 50 mm, including increments therein. In some embodiments, the ejection zone is superior to a horizontal line that intersects the inferior aspect of the columella by about 10 mm, about 12 mm, about 16 mm, about 20 mm, about 24 mm, about 28 mm, about 32 mm, about 36 mm, about 40 mm, about 45 mm, or about 50 mm. In some embodiments, the ejection zone is superior to a horizontal line that intersects the inferior aspect of the columella by at least about 10 mm, about 12 mm, about 16 mm, about 20 mm, about 24 mm, about 28 mm, about 32 mm, about 36 mm, about 40 mm, or about 45 mm. In some embodiments, the ejection zone is superior to a horizontal line that intersects the inferior aspect of the columella by at most about 12 mm, about 16 mm, about 20 mm, about 24 mm, about 28 mm, about 32 mm, about 36 mm, about 40 mm, about 45 mm, or about 50 mm. In some embodiments, the ejection zone is superior to a horizontal line that intersects the superior aspect of the inferior turbinate by about 1 mm to about 30 mm. In some embodiments, the ejection zone is superior to a horizontal line that intersects the superior aspect of the inferior turbinate by about 1 mm to about 2 mm, about 1 mm to about 4 mm, about 1 mm to about 6 mm, about 1 mm to about 8 mm, about 1 mm to about 10 mm, about 1 mm to about 12 mm, about 1 mm to about 15 mm, about 1 mm to about 18 mm, about 1 mm to about 21 mm, about 1 mm to about 25 mm, about 1 mm to about 30 mm, about 2 mm to about 4 mm, about 2 mm to about 6 mm, about 2 mm to about 8 mm, about 2 mm to about 10 mm, about 2 mm to about 12 mm, about 2 mm to about 15 mm, about 2 mm to about 18 mm, about 2 mm to about 21 mm, about 2 mm to about 25 mm, about 2 mm to about 30 mm, about 4 mm to about 6 mm, about 4 mm to about 8 mm, about 4 mm to about 10 mm, about 4 mm to about12 mm, about 4 mm to about 15 mm, about 4 mm to about 18 mm, about 4 mm to about 21 mm, about 4 mm to about 25 mm, about 4 mm to about 30 mm, about 6 mm to about 8 mm, about 6 mm to about 10 mm, about 6 mm to about 12 mm, about 6 mm to about 15 mm, about 6 mm to about 18 mm, about 6 mm to about 21 mm, about 6 mm to about 25 mm, about 6 mm to about 30 mm, about 8 mm to about 10 mm, about 8 mm to about 12 mm, about 8 mm to about 15 mm, about 8 mm to about 18 mm, about 8 mm to about 21 mm, about 8 mm to about 25 mm, about 8 mm to about 30 mm, about 10 mm to about 12 mm, about 10 mm to about 15 mm, about 10 mm to about 18 mm, about 10 mm to about 21 mm, about 10 mm to about 25 mm, about 10 mm to about 30 mm, about 12 mm to about 15 mm, about 12 mm to about 18 mm, about 12 mm to about 21 mm, about 12 mm to about 25 mm, about 12 mm to about 30 mm, about 15 mm to about 18 mm, about 15 mm to about 21 mm, about 15 mm to about 25 mm, about 15 mm to about 30 mm, about 18 mm to about 21 mm, about 18 mm to about 25 mm, about 18 mm to about 30 mm, about 21 mm to about 25 mm, about 21 mm to about 30 mm, or about 25 mm to about 30 mm, including increments therein. In some embodiments, the ejection zone is superior to a horizontal line that intersects the superior aspect of the inferior turbinate by about 1 mm, about 2 mm, about 4 mm, about 6 mm, about 8 mm, about 10 mm, about 12 mm, about 15 mm, about 18 mm, about 21 mm, about 25 mm, or about 30 mm. In some embodiments, the ejection zone is superior to a horizontal line that intersects the superior aspect of the inferior turbinate by at least about 1 mm, about 2 mm, about 4 mm, about 6 mm, about 8 mm, about 10 mm, about 12 mm, about 15 mm, about 18 mm, about 21 mm, or about 25 mm. In some embodiments, the ejection zone is superior to a horizontal line that intersects the superior aspect of the inferior turbinate by at most about 2 mm, about 4 mm, about 6 mm, about 8 mm, about 10 mm, about 12 mm, about 15 mm, about 18 mm, about 21 mm, about 25 mm, or about 30 mm. In some embodiments, the ejection zone is away from the septum by about 0.1 mm to about 3 mm. In some embodiments, the ejection zone is away from the septum by about 0.1 mm to about 0.2 mm, about 0.1 mm to about 0.4 mm, about 0.1 mm to about 0.6 mm, about 0.1 mm to about 0.8 mm, about 0.1 mm to about 1 mm, about 0.1 mm to about 1.25 mm, about 0.1 mm to about 1.5 mm, about 0.1 mm to about 1.75 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 2.5 mm, about 0.1 mm to about 3 mm, about 0.2 mm to about 0.4 mm, about 0.2 mm to about 0.6 mm, about 0.2 mm to about 0.8 mm, about 0.2 mm to about 1 mm, about 0.2 mm to about 1.25 mm, about 0.2 mm to about 1.5 mm, about 0.2 mm to about1.75 mm, about 0.2 mm to about 2 mm, about 0.2 mm to about 2.5 mm, about 0.2 mm to about 3 mm, about 0.4 mm to about 0.6 mm, about 0.4 mm to about 0.8 mm, about 0.4 mm to about 1 mm, about 0.4 mm to about 1.25 mm, about 0.4 mm to about 1.5 mm, about 0.4 mm to about 1.75 mm, about 0.4 mm to about 2 mm, about 0.4 mm to about 2.5 mm, about 0.4 mm to about 3 mm, about 0.6 mm to about 0.8 mm, about 0.6 mm to about 1 mm, about 0.6 mm to about 1.25 mm, about 0.6 mm to about 1.5 mm, about 0.6 mm to about 1.75 mm, about 0.6 mm to about 2 mm, about 0.6 mm to about 2.5 mm, about 0.6 mm to about 3 mm, about 0.8 mm to about 1 mm, about 0.8 mm to about 1.25 mm, about 0.8 mm to about 1.5 mm, about 0.8 mm to about 1.75 mm, about 0.8 mm to about 2 mm, about 0.8 mm to about2.5 mm, about 0.8 mm to about 3 mm, about 1 mm to about 1.25 mm, about 1 mm to about1.5 mm, about 1 mm to about 1.75 mm, about 1 mm to about 2 mm, about 1 mm to about2.5 mm, about 1 mm to about 3 mm, about 1.25 mm to about 1.5 mm, about 1.25 mm to about 1.75 mm, about 1.25 mm to about 2 mm, about 1.25 mm to about 2.5 mm, about 1.25 mm to about 3 mm, about 1.5 mm to about 1.75 mm, about 1.5 mm to about 2 mm, about1.5 mm to about 2.5 mm, about 1.5 mm to about 3 mm, about 1.75 mm to about 2 mm, about1.75 mm to about 2.5 mm, about 1.75 mm to about 3 mm, about 2 mm to about 2.5 mm, about 2 mm to about 3 mm, or about 2.5 mm to about 3 mm, including increments therein. In some embodiments, the ejection zone is away from the septum by about 0.1 mm, about 0.2 mm, about 0.4 mm, about 0.6 mm, about 0.8 mm, about 1 mm, about 1.25 mm, about1.5 mm, about 1.75 mm, about 2 mm, about 2.5 mm, or about 3 mm. In some embodiments, the ejection zone is away from the septum by at least about 0.1 mm, about 0.2 mm, about 0.4 mm, about 0.6 mm, about 0.8 mm, about 1 mm, about 1.25 mm, about 1.5 mm, about1.75 mm, about 2 mm, or about 2.5 mm. In some embodiments, the ejection zone is away from the septum by at most about 0.2 mm, about 0.4 mm, about 0.6 mm, about 0.8 mm, about 1 mm, about 1.25 mm, about 1.5 mm, about 1.75 mm, about 2 mm, about 2.5 mm, or about 3 mm.

[0126] The dispensing element can be a dispensing element of a device for olfactory delivery of the therapeutic peptide. The device can be inserted into the nasal cavity of the subject for delivery. The device can be inserted at an angle from a vertical line. In some embodiments, the vertical line is a line perpendicular to a horizontal line that is parallel to the inferior aspect of the olfactory cleft, as depicted as line c-c of FIG. 4J.

[0127] In some embodiments, the device is inserted into the nasal cavity of the subject at an angle of between about 30 degrees to about 40 degrees from a vertical line, the vertical line being perpendicular to a horizontal line that is parallel to an inferior aspect of the olfactory cleft of the subject. In some embodiments, the angle is about 30 degrees from the vertical line. In some embodiments, the angle is about 31 degrees from the vertical line. In some embodiments, the angle is about 32 degrees from the vertical line. In some embodiments, the angle is about 33 degrees from the vertical line. In some embodiments, the angle is about 34 degrees from the vertical line. In some embodiments, the angle is about 35 degrees from the vertical line. In some embodiments, the angle is about 36 degrees from the vertical line. In some embodiments, the angle is about 37 degrees from the vertical line. In some embodiments, the angle is about 38 degrees from the vertical line. In some embodiments, the angle is about 39 degrees from the vertical line. In some embodiments, the angle is about 40 degrees from the vertical line.

[0128] In some embodiments, the formulation has a viscosity of about 1 cP. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 1.0 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 1.1 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 1.2 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 1.3 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 1.4 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 1.5 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 1.6 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 1.7 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 1.8 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 1.9 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 2.0 m / s. In some embodiments, delivering theformulation comprises ejecting the formulation from the ejection zone at a velocity of about 2.1 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 2.2 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 2.3 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 2.4 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 2.5 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation n from the ejection zone at a velocity of about 2.6 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 2.7 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 2.8 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 2.9 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of between about 1.1 m / s and about 1.9 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of between about 1.0 m / s and about 2.2 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of between about 1.0 m / s and about 2.8 m / s.

[0129] In some embodiments, the formulation has a viscosity of about 50 cP. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 4 m / s. In some embodiments, the formulation has a viscosity of about 50 cP. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 4.7 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 5 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 6 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 7 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 8 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from theej ection zone at a velocity of about 9 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 10 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 11 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 12 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 13 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 14 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 15 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 16 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 17 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 18 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 18.6 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of about 19 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of between about 4.7 m / s and about 18.6 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of between about 13.9 m / s and about 15.8 m / s. In some embodiments, delivering the formulation comprises ejecting the formulation from the ejection zone at a velocity of between about 11.0 m / s and about 15.8 m / s.

[0130] As demonstrated herein, the formulation delivered by the method disclosed herein can remain at the olfactory cleft of the subject for a period of time longer than when the formulation is delivered by a nasal spray.

[0131] In some embodiments, the formulation delivered to the olfactory cleft of the subject remains at the olfactory cleft for at least about 10 seconds. In some embodiments, the formulation delivered to the olfactory cleft of the subject remains at the olfactory cleft for at least about 30 seconds. In some embodiments, the formulation delivered to the olfactory cleft of the subject remains at the olfactory cleft for at least about 1 min. In some embodiments, the formulation delivered to the olfactory cleft of the subject remains at theolfactory cleft for at least about 2 min. In some embodiments, the formulation delivered to the olfactory cleft of the subject remains at the olfactory cleft for at least about 3 min. In some embodiments, the formulation delivered to the olfactory cleft of the subject remains at the olfactory cleft for at least about 4 min. In some embodiments, the formulation delivered to the olfactory cleft of the subject remains at the olfactory cleft for at least about 5 min. In some embodiments, the formulation delivered to the olfactory cleft of the subject remains at the olfactory cleft for at least about 6 min. In some embodiments, the formulation delivered to the olfactory cleft of the subject remains at the olfactory cleft for at least about7 min. In some embodiments, the formulation delivered to the olfactory cleft of the subject remains at the olfactory cleft for at least about 8 min. In some embodiments, the formulation delivered to the olfactory cleft of the subject remains at the olfactory cleft for at least about 9 min. In some embodiments, the formulation delivered to the olfactory cleft of the subject remains at the olfactory cleft for at least about 10 min. In some embodiments, the formulation delivered to the olfactory cleft of the subject remains at the olfactory cleft for at least about 11 min. In some embodiments, the formulation delivered to the olfactory cleft of the subject remains at the olfactory cleft for at least about 12 min. In some embodiments, the formulation delivered to the olfactory cleft of the subject remains at the olfactory cleft for at least about 13 min. In some embodiments, the formulation delivered to the olfactory cleft of the subject remains at the olfactory cleft for at least about 14 min. In some embodiments, the formulation delivered to the olfactory cleft of the subject remains at the olfactory cleft for at least about 15 min. In some embodiments, the formulation delivered to the olfactory cleft of the subject remains at the olfactory cleft for longer than 15 min. In some embodiments, the formulation delivered to the olfactory cleft of the subject remains at the olfactory cleft for a period of time longer than if the formulation was delivered by nasal spray.

[0132] Any suitable therapeutic peptides may be used with the methods disclosed herein.

[0133] In some embodiments, the therapeutic peptide is a small peptide. In some embodiments, the therapeutic peptide has a length of less than 100 amino acids. In some embodiments, the therapeutic peptide has a length of less than 90 amino acids. In some embodiments, the therapeutic peptide has a length of less than 80 amino acids. In some embodiments, the therapeutic peptide has a length of less than 70 amino acids. In some embodiments, the therapeutic peptide has a length of less than 60 amino acids. In someembodiments, the therapeutic peptide has a length of less than 50 amino acids. In some embodiments, the therapeutic peptide has a length of less than 40 amino acids. In some embodiments, the therapeutic peptide has a length of less than 30 amino acids. In some embodiments, the therapeutic peptide has a length of less than 20 amino acids. In some embodiments, the therapeutic peptide has a length of less than 10 amino acids.

[0134] The therapeutic peptide can be a modified peptide. The therapeutic peptide can be conjugated to an agent. The agent can be a therapeutic agent, such as a drug or a radioisotope.

[0135] In some embodiments, the therapeutic peptide is conjugated to a therapeutic agent. In some embodiments, the therapeutic agent is a drug. In some embodiments, the therapeutic agent is a radioisotope.

[0136] Examples of therapeutic peptides include but are not limited to: insulin, exendin- 4, oxytocin, glutathione, a neurotrophic factor, a glial cell-derived neurotrophic factor (GDNF), a nerve growth factor (NGF), glucagon, a glucagon like peptide 1 (GLP-1), dulaglutide, a GLP-1 agonist, an incretin mimetic, a galanin-like peptide (GALP), erythropoietin (EPO), a glycoprotein hormone, salmon calcitonin, buserelin acetate, desmopressin acetate, gonadorelin, nafarelin acetate, protirelin, cyanocobalamine, and variants and analogs thereof.

[0137] In some embodiments, the therapeutic peptide is insulin, or a variant or analog thereof. As demonstrated herein, delivery of insulin using the method disclosed herein can result in reduction of brain activity in the subject for at least 60 minutes after delivery. The reduction in brain activity can last longer than where a similar dose is administered by spray.

[0138] In some embodiments, delivery of the therapeutic peptide results in reduction in prefrontal brain activity. In some embodiments, delivery of the therapeutic peptide results in reduction in brain activity for at least 5 min. In some embodiments, delivery of the therapeutic peptide results in reduction in brain activity for at least 15 min. In some embodiments, delivery of the therapeutic peptide results in reduction in brain activity for at least 30 min. In some embodiments, delivery of the therapeutic peptide results in reduction in brain activity for at least 45 min. In some embodiments, delivery of the therapeutic peptide results in reduction in brain activity for at least 60 min. In some embodiments, delivery of the therapeutic peptide results in longer lasting reduction in prefrontal brain activity as compared to delivery of about the same dose of the therapeutic peptide by nasalspray. In some embodiments, delivery of the therapeutic peptide results in at least 5 min longer reduction in prefrontal brain activity than delivery of about the same dose by nasal spray. In some embodiments, delivery of the therapeutic peptide results in at least 10 min longer reduction in prefrontal brain activity than delivery of about the same dose by nasal spray. In some embodiments, delivery of the therapeutic peptide results in at least 15 min longer reduction in prefrontal brain activity than delivery of about the same dose by nasal spray. In some embodiments, delivery of the therapeutic peptide results in at least 20 min longer reduction in prefrontal brain activity than delivery by nasal spray. In some embodiments, delivery of the therapeutic peptide results in at least 25 min longer reduction in prefrontal brain activity than delivery of about the same dose by nasal spray. In some embodiments, delivery of the therapeutic peptide results in at least 30 min longer reduction in prefrontal brain activity than delivery of about the same dose by nasal spray. In some embodiments, delivery of the therapeutic peptide results in at least 35 min longer reduction in prefrontal brain activity than delivery of about the same dose by nasal spray. In some embodiments, delivery of the therapeutic peptide results in at least 40 min longer reduction in prefrontal brain activity than delivery by nasal spray. In some embodiments, delivery of the therapeutic peptide results in at least 45 min longer reduction in prefrontal brain activity than delivery of about the same dose by nasal spray. In some embodiments, delivery of the therapeutic peptide results in at least 1 hour longer reduction in prefrontal brain activity than delivery of about the same dose by nasal spray.

[0139] In some embodiments, the therapeutic peptide is exendin-4, or a variant or analog thereof.

[0140] It is demonstrated herein that the method disclosed herein can result in delivery of the therapeutic peptide to a non-CNS target.

[0141] In some embodiments, the non-CNS target is blood.

[0142] The method disclosed herein is capable of delivering the therapeutic peptide to one or more of the group consisting of cerebrospinal fluid, the central nervous system, and a region, tissue, or organ thereof. In some embodiments, at least about 5% of the therapeutic peptide in the formulation is delivered to one or more of the group consisting of cerebrospinal fluid, the central nervous system, and a region, tissue, or organ thereof. In some embodiments, at least about 10% of the therapeutic peptide in the formulation is delivered to one or more of the group consisting of cerebrospinal fluid, the central nervoussystem, and a region, tissue, or organ thereof. In some embodiments, at least about 15% of the therapeutic peptide in the formulation is delivered to one or more of the group consisting of cerebrospinal fluid, the central nervous system, and a region, tissue, or organ thereof. In some embodiments, at least about 20% of the therapeutic peptide in the formulation is delivered to one or more of the group consisting of cerebrospinal fluid, the central nervous system, and a region, tissue, or organ thereof. In some embodiments, at least about 25% of the therapeutic peptide in the formulation is delivered to one or more of the group consisting of cerebrospinal fluid, the central nervous system, and a region, tissue, or organ thereof. In some embodiments, at least about 30% of the therapeutic peptide in the formulation is delivered to one or more of the group consisting of cerebrospinal fluid, the central nervous system, and a region, tissue, or organ thereof. In some embodiments, at least about 35% of the therapeutic peptide in the formulation is delivered to one or more of the group consisting of cerebrospinal fluid, the central nervous system, and a region, tissue, or organ thereof. In some embodiments, at least about 40% of the therapeutic peptide in the formulation is delivered to one or more of the group consisting of cerebrospinal fluid, the central nervous system, and a region, tissue, or organ thereof. In some embodiments, at least about 45% of the therapeutic peptide in the formulation is delivered to one or more of the group consisting of cerebrospinal fluid, the central nervous system, and a region, tissue, or organ thereof. In some embodiments, at least about 50% of the therapeutic peptide in the formulation is delivered to one or more of the group consisting of cerebrospinal fluid, the central nervous system, and a region, tissue, or organ thereof. In some embodiments, at least about 55% of the therapeutic peptide in the formulation is delivered to one or more of the group consisting of cerebrospinal fluid, the central nervous system, and a region, tissue, or organ thereof. In some embodiments, at least about 60% of the therapeutic peptide in the formulation is delivered to one or more of the group consisting of cerebrospinal fluid, the central nervous system, and a region, tissue, or organ thereof. In some embodiments, at least about 65% of the therapeutic peptide in the formulation is delivered to one or more of the group consisting of cerebrospinal fluid, the central nervous system, and a region, tissue, or organ thereof. In some embodiments, at least about 70% of the therapeutic peptide in the formulation is delivered to one or more of the group consisting of cerebrospinal fluid, the central nervous system, and a region, tissue, or organ thereof. In some embodiments, at least about 75% of the therapeutic peptide in the formulation is delivered to one or more of the group consistingof cerebrospinal fluid, the central nervous system, and a region, tissue, or organ thereof. In some embodiments, at least about 80% of the therapeutic peptide in the formulation is delivered to one or more of the group consisting of cerebrospinal fluid, the central nervous system, and a region, tissue, or organ thereof. In some embodiments, at least about 85% of the therapeutic peptide in the formulation is delivered to one or more of the group consisting of cerebrospinal fluid, the central nervous system, and a region, tissue, or organ thereof. In some embodiments, at least about 90% of the therapeutic peptide in the formulation is delivered to one or more of the group consisting of cerebrospinal fluid, the central nervous system, and a region, tissue, or organ thereof.

[0143] The method disclosed herein can provide delivery of a greater amount of the therapeutic peptide to one or more of the group consisting of cerebrospinal fluid, the central nervous system, and a region, tissue, or organ thereof compared to intravenous delivery of an identical dose of the therapeutic peptide.

[0144] The therapeutic peptide can provide a therapeutic effect upon delivery to the cerebrospinal fluid, the central nervous system, or region, tissue, or organ thereof via the method disclosed herein.

[0145] In some embodiments, the subject has been diagnosed with a disease or disorder related to the central nervous system.

[0146] In some embodiments, the delivery of the therapeutic peptide provides effective treatment for Alzheimer's disease, Parkinson's disease, a cancer of the brain, a cancer of the CNS, a non-CNS disorder, an immunological disorder, an oncological disorder, a hematological disorder, an opthalmological disorder, a neurological disorder, a respiratory disorder, a cardiovascular disorder, an infectious disease, vitamin deficiency, multiple sclerosis, improvement of uterine contractions, postmenopausal osteoporosis, cryptorchism, endometriosis, central precocious puberty (CPP), Primary nocturnal enuresis, central cranial diabetes insipidus, nocturia, cranial diabetes insipidus or nocturia associated with multiple sclerosis, stroke, epilepsy, central nervous system trauma, a metabolic disorder, an infectious agent (i.e. Naegleria fowleri) or protein (i.e. Creutzfeldt Jacobs disease), a malignant tumor, a benign tumor, glioblastoma, anosmia, or an endocrine disorder.

[0147] In some embodiments, the delivery of the therapeutic peptide provides effective treatment for depression, dementia, or Alzheimer’s disease.

[0148] The method disclosed herein can minimize shear forces exposed to the therapeutic peptide.

[0149] In some embodiments, the formulation is delivered without exposing the therapeutic peptide to shear forces sufficient to damage a significant portion of the therapeutic peptide, without aerosolizing the formulation, or by passing the formulation through a shear disintegrating tip.

[0150] In some embodiments, the therapeutic peptide bypasses the blood-brain barrier, the blood-cerebrospinal fluid barrier, or the arachnoid membrane.

[0151] In some embodiments, the subject is a mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a human.II. Device

[0152] In another aspect, provided herein is a device for olfactory delivery of a therapeutic peptide to an olfactory region of a subject, the device comprising: (a) a housing comprising an insertable portion comprising a distal end, and a proximal end; and (b) a subject-engaging portion which engages a columella region of the subject to seat the distal end of the insertable portion within an ejection zone of a nasal channel of the subject; wherein the device is configured to deliver a formulation comprising a therapeutically effective amount of the peptide to the olfactory region of the subject. In another aspect, provided herein is a device for olfactory delivery of a therapeutic peptide to an olfactory region of a subject. The device can comprise a housing comprising an insertable portion comprising a distal end and a proximal end. The device can comprise a subject-engaging portion which engages a columella region of the subject. The subject-engaging portion can seat the distal end of the insertable portion within an ejection zone of a nasal channel of the subject. The device can be configured to deliver a formulation comprising a therapeutically effective amount of the peptide to the olfactory region of the subject.

[0153] In some embodiments, the device dispenses the formulation as a laminar jet.

[0154] In some embodiments, application of pressure by the subject-engaging portion to the columella region of the subject enables and / or causes delivery of the formulation to the subject from the insertable portion.

[0155] In some embodiments, the insertable portion comprises a dispensing element for delivery of the formulation to the olfactory region of the subject.

[0156] The housing can comprise two insertable portions. In some embodiments, the two insertable portions comprise at least one dispensing element, each insertable portion for insertion into a nasal channel of the subject, wherein, upon insertion of the first insertable portion into a nasal channel of the subject, the first insertable portion engages tissue within the nasal channel to open or expand an internal nasal valve of the subject thereby positioning at least one of the dispensing elements for delivery of the formulation to the subject. In some embodiments, the subject engaging portion comprises a trigger, wherein upon application of pressure to the subject engaging portion, the trigger permits actuation of the device to deliver the formulation to the subject through the dispensing element.

[0157] The device can be used with the methods disclosed herein.III. Definitions

[0158] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0159] The term “about” or “approximately” when used in reference to a particular recited value, means the value may vary from the recited value by no more than 10%, 5%, 2% or 1%. Where a particular value is recited, it can be understood that the value is modified by the term “about” or “approximately”, unless indicated otherwise.

[0160] The term “comprise”, “comprising”, or the like means additional elements or components other than those recited may be present. Other terms such as “include”, “contain”, “have” and the like have similar meaning.

[0161] The term “consist of’, “consisting of’ or the like means no additional component is present.

[0162] As used herein, the singular forms “a,” “an,” and “the” include plural references, unless indicated otherwise. For example, a reference to “a molecule” can be a reference to more than one molecule.

[0163] The term “or” is used to mean “and / or”, unless it is indicated explicitly to refer to alternatives only. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the expression “A, B, and / or C” can mean A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.

[0164] As used herein, the term “peptide” refers to a molecule including two or more amino acids.

[0165] As used herein, a “therapeutically effective amount” means an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that elicits a desired biological response. In some embodiments, a therapeutically effective amount of a substance is an amount that is sufficient, when administered as part of a dosing regimen to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the effective amount of a provided compound in a formulation to treat a disease, disorder, and / or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and / or reduces incidence of one or more symptoms or features of the disease, disorder, and / or condition. In some embodiments, a “therapeutically effective amount” is at least a minimal amount of a provided compound, or composition containing a provided compound, which is sufficient for treating one or more symptoms of a disease or disorder.

[0166] As used herein, the term “variant” in the context of a peptide means a peptide that shares a certain percentage amino acid sequence identity with a reference upon alignment of the amino acid sequences.

[0167] The term “functional variant” in the context of a protein or polypeptide means a variant that is capable of having one or more activities of a reference protein or polypeptide. For example, a functional variant of insulin may refer to a variant of insulin that can retain the activity to reduce brain activity to the same extent or at least a substantially same extent as insulin.

[0168] The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the application. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.EXAMPLESExample 1 - Laminar Fluid Ejection Method

[0169] The primary goal of the study was to develop a method of ejecting fluid through a cannula for deposition at the olfactory cleft. In general, all devices consisted of a ImL polycarbonate syringe filled to 0.20mL of formulation. The syringe was capped with a cannula (inner diameter = 1.35mm) and mounted on a carbon-fiber reinforced mechanism. A coil spring housed within the mechanism provided the force to compress the syringe. The spring preload was adjustable with the use of an external threaded collar and shims. The spring surrounded an orifice-based damper that could be filled with different weight silicone oils and orifice plates with different sized holes and quantity of holes. The mechanism used a simple trigger that released a syringe plunger guided by aluminum rails. This bar pushed the plunger shaft of the syringe. The syringe was held accurately and firmly in place in front of the mechanism.

[0170] Aperture testing was conducted to evaluate the coherence of fluid post-ejection and to visually assess the laminar qualities of the ejected fluid. Fluids at two different viscosities (IcP and 50cP) were tested, each at a range of ejection velocities (2m / s - 27.7m / s). Device settings for a selection of velocities within this range were determined experimentally prior to conducting aperture testing. Velocity was determined using highspeed camera-footage of ejected fluid traveling a fixed distance from the tip of the canula. A small aperture (5.64mm in diameter) was placed 25mm away from the tip of the canula. The canula was supported by magnetic supports to reduce wobbling. A catching tray was placed on the opposite side of the aperture. The percent mass transferred from the syringe to the catching tray was calculated for each test-run (Mass in catching tray / Mass Ejected from syringe * 100). Tests of each combination of viscosity and speed were repeated three times. The mean percent mass transferred for each condition is presented in Table 1. (Note that the ejected mass is smaller than the total fill mass, as it does not include the residual left in the device. The residual left in the device did not meaningfully differ across conditions: 13.6% for IcP, 10.4% for 50cP).Table 1. Aperture TestingPercent mass transferred from the device through a small 5.64mm aperture and into a catching tray for low / high fluid viscosities and velocities.

[0171] In general, higher speeds were associated with reduced mass transfer, possibly due to increased cannula movement and splatter off the aperture edge. Qualitatively, it was observed that the device ejected fluid in a cohesive stream with a narrow diameter, especially compared to the plume emitted from a traditional spray device.

[0172] Next, a series of in-vitro deposition tests was conducted to determine the range of velocities that would optimally deposit fluid in the olfactory cleft of the human nasal passages. To test this, the mechanism was inserted into a transparent 3D-printed model of the nasal cavity. Optimal delivery was considered to be in the target zone, which represents the approximate location of the olfactory cleft in the model. If ejected too slowly, the fluid would deposit anterior to the front boundary of the target zone; too quickly and it would deposit beyond the back boundary. Two fluid viscosities (IcP and 50cP) were tested at each of three different insertion angles (30, 35, 40 degrees from the vertical). The cannula’s insertion depth was held constant at 37.5mm. The minimum velocity necessary to reach the Front Boundary (vf), the olfactory cleft (vopt), and the back boundary (vb), for each of the viscosities and deposition angles are presented in Table 2.Table 2. In-vitro Deposition TestingTable presents optimal velocities (Vopt) for fluids of low / high viscosity across three device angles. The Vf and Vb columns show the velocity limits at which the deposition was in front of or behind the target area, respectively.

[0173] For velocities within the optimal range, residence times were observed to exceed 10 minutes. Notably, angles of 35-40 degrees permitted a wider range of velocities, but using an angle of 30-degrees did not dramatically shift the optimal velocity. This suggested the device is adaptable to a wide range of angles. Optimal velocities (+ / -20%) were found to be 1.6m / s for IcP fluid and 14.0m / s for 50cP fluid. These optimal velocities may be associated with laminar flow.

[0174] A Reynold’s number (Re= p* V*L / |i, where p is the density of the fluid, V is the velocity of the flow, L is the diameter of the passage, and g is the dynamic viscosity of the fluid) can be computed to quantify whether a fluid exhibits laminar or turbulent flow. The Reynold’s number for water at a velocity of 1.6m / s and viscosity of I cP is approximately 2,160 with a 1.35 mm diameter (density of water is approximately 1000 kg / m3). Similarly for a fluid of 50 cP (with the same density as water), the Reynold’s number at 15m / s is approximately equal to 405 with a 1.35 mm diameter. Both Reynold’s numbers fall below 2,300 which is the commonly accepted threshold for laminar flow.Example 2 In-Vivo Evaluation of Laminar Fluid Ejection Method

[0175] The primary goal of these studies is to visualize delivery to the olfactory cleft (first with technetium-99, and then with methylene blue). The device was tuned to eject the fluid at a specific velocity. This velocity was chosen based on the viscosity of the fluid and the studies in Example 1 , to maximize the extent of laminar flow.

[0176] Visualizing Olfactory Cleft Delivery with Technetium-99

[0177] Participants'. Nine healthy participants (ages 19+) with no history of abnormal nasal or sinus symptoms or contraindications for nasal cannulation, Magnetic Resonance Imaging (MR1), or Single-Photon Emission Computed Tomography (SPECT). Participants completed one study visit undergoing magnetic resonance imaging (MR1) and single-photon emission computed tomography (SPECT) to visualize the deposition of a radiolabeled tracer (technetium-99) in their nasal passages. In total, nine participants were enrolled in the study, and eight completed all study procedures (Participant 1.02 withdrew before completing all imaging procedures). All study procedures were approved by the Horizon Health Network’s Human Research Protection Program. Prior to participating in the study, all participants were assessed by a licensed otolaryngologist physician to confirm their eligibility. Study Design: On Day 1 of the study, anatomical (T1 -weighted) magnetic resonance images were collected from participants at the Moncton MRI clinic (Moncton, NB, Canada) using aSiemens Skyra VD13 3T scanner. MRI data provided anatomical information regarding soft tissue structure in the nasal cavity and the location of each participant’s olfactory region.

[0178] On Day 2 of the study, a saline solution including the technetium-99 radiotracer (mean dose 4.6 mCi) was delivered to participants using the Laminar Fluid Ejection method described above. Day 2 study procedures were conducted at the Nuclear Imaging Department of Saint John Regional Hospital (Saint John, NB, Canada). At the time of delivery and for five minutes afterwards, 2D SPECT Flow images were acquired every three seconds using a gamma camera. Flow images were acquired in a single sagittal plane (128x128 with voxels 4.8mm diameter).

[0179] Image Analysis'. Image analysis was completed in Vivoquant (4.0). SPECT Flow data was resampled to match MRI resolution (320 x 320 with voxels 1.918mm isotropic) and manually registered to T1 images in x and y space with minimal rotation in the z-plane. This procedure enabled landmarking to nasal passage characteristics minimally visible in the SPECT data alone. Flow data was visually inspected to determine (1) the point in the time series when the bolus was dispensed, (2) the time of cannula removal, (3) the median time point in the series where the bolus initially lodged between points 2 and 4, (4) the time point at which the bolus began additional migration, and (5) the time point of the final image acquired during the five -minute SPECT series. All subjects’ SPECT Flow images are presented in FIG. 13.

[0180] Results: In seven out of eight participants imaging results demonstrated delivery of the radiotracer to the cribriform area. The radiotracer was detected in the cribriform area without any movement for at least 1.5 minutes in six of those seven participants. Additionally, clearance of the radiotracer was minor for the duration of the study for six out of those seven cases. In the two cases where sub-optimal outcomes were reached, it was the result of (i) bolus delivery anterior and superior to the cribriform (Participant 1.07), and (ii) bolus that cleared quickly after reaching the target, possibly due to the bolus remaining well intact as it directly impacted the cribriform (Participant 1.05). See Table 3 for a summary of the results in each participant.Table 3. Visualizing Olfactory Cleft Delivery with Technetium-99

[0181] Visualizing Olfactory Cleft Delivery with Methylene Blue

[0182] Participants'. Five healthy participants (ages 19+) with no history of sinonasal symptoms and no evidence of nasal inflammation were recruited for this study. Prior to recruitment of participants study procedures were approved by Providence Health Care research ethics board, (British Columbia, Canada) and conducted under Health Canada Investigational Testing Authorization #314993. All participants were assessed by a licensed otolaryngologist physician to confirm their eligibility. Participants completed two study visits during which a licensed otolaryngologist physician administered 0.1 mL of the visual dye tracer, methylene blue.

[0183] Study Design: During the first visit, the methylene blue was administered using the Laminar Fluid Ejection method (1.5mm cannula; velocity = 4.5m / s). During the second visit, methylene blue was administered using a conventional nasal spray device (Pharma systems Item #10272, UPC:063636802714). The Pharma systems spray device was used in these experiments because it was the device normally used by the compounding pharmacy for intranasal delivery. It was the device patients and clinicians were used to at this clinic.

[0184] Following delivery, the physician used a nasal endoscope to image the deposition of methylene blue to the olfactory cleft at up to five time points (1, 5, 8, 12, and 15 minutes following delivery). From each image, the physician judged whether at least 50% of the methylene blue fluid was delivered to the olfactory cleft. If <50% of the methylene blue was judged to have deposited in the olfactory cleft, imaging was stopped and that session’s trial was completed.

[0185] Results: Four of the five participants demonstrated successful delivery of >50% of the methylene blue to the olfactory cleft using the Laminar Fluid Ejection method. In eachof the five participants, the conventional nasal spray failed to deliver at least 50% of the dye to the olfactory cleft (Table 4).Table 4. Visualization of Delivery to the Olfactory Clef Using Methylene BlueResults from in-human tests of the Laminar Fluid Ejection (LFE) method using the dye methylene blue.Example 3: Insulin Delivery fMRI Analysis

[0186] In the in-human studies below, the device was tuned to eject the fluid at a specific velocity. This velocity was chosen based on the viscosity of the fluid and the studies described in Example 1, to maximize the extent of laminar flow.

[0187] Methods

[0188] Participants: Eight participants (male and female, ages 23 - 30) were recruited for this study from the New Brunswick area. Participants reported no history of abnormal nasal or sinus symptoms, insulin use, diabetes, metabolic disorder, or any complicating medical conditions, or contraindications for nasal cannulation, MR1 or venous blood sampling. Additionally, participants reported no previous diagnosis of SARS-CoV-2 with symptoms of anosmia. Study procedures were approved by Horizon Health Network’s Human Research Protection Program prior to recruitment of participants.

[0189] Study Design An open-label 4-arm crossover study design was used to understand the insulin brain effects between the focal deposition (cannula) and the standard nasal mister method. Undiluted solution of 801U (0.16 ml) prepared from U500 insulin was delivered to the olfactory region (condition A). Under this condition, 40 1U (0.08 ml) of human insulin was delivered directly by an ENT physician to each nostril. For condition B, undiluted solution containing 15% MucoLox (a thickening agent) and 801U (0.16 ml) insulin was delivered to the olfactory region. For condition C, a total of 1.6 ml insulin andsterile water mixture was prepared for N asal spray delivery. The nasal spray bottle contained 801U of U500 insulin mixed with a sterile water solution. The spray was delivered over 4 minutes with two puffs per nostril every minute. Condition sequences AC, CA, BC, and CB comprise the 4 arms of the study.

[0190] Participants were randomly assigned to one of two blocks. In Block 1 , participants were randomly assigned by alternating sequence to one of the A arms (AC or CA). Block 1 participants did not receive MucoLox in the cannula session. In Block 2, participants were randomly assigned by alternating sequence to one of the B arms (BC or CB). Block 2 participants did receive MucoLox in the cannula session. For a given participant, appointments for each condition were scheduled at a minimum of 14 days apart to ensure no carry-over effects from the previous condition.

[0191] MRI Acquisition'. Magnetic resonance imaging was completed at an imaging clinic in Moncton (Moncton MRI) using standard sequences on a 3T Siemens Skyra, VD13. Anatomical T1 -weighted MPRAGE images were acquired for each subject [TR = 1900ms, TE = 2.99ms, Flip Angle = 9 degrees, Voxel Size = 0.45 x 0.45 x 0.9mm, Dimensions = 448 x 512 x 160]. Resting state functional T2*-weighted BOLD images [TR = 2600ms, TE = 30ms, Flip Angle = 90 degrees, Voxel Size = 3 x 3 x 3mm, Dimensions = 80 x 80 x 45] were acquired for each subject at baseline and again 15 minutes and 60 minutes following intranasal delivery of insulin. Each resting state functional scan consisted of 180 volumes acquired over the course of 7 minutes and 48 seconds.

[0192] MRI Preprocessing'. MRI data was preprocessed using fMRIPrep. Preprocessing steps of the anatomical images included correction for intensity nonuniformity, skull stripping, segmentation of gray matter, white matter, and cerebrospinal fluid (CSF) masks, and normalization to MNI template space. Preprocessing steps for the functional images included slice time correction, head motion correction (calculation of framewise displacement and 6-degrees of motion parameters and their temporal derivatives), registration to the subjects Tlw image, normalization to MNI template space, and spatial smoothing (3mm FWHM Gaussian kernel).

[0193] fMRI Denoising'. Functional MRI data were denoised using a standard denoising pipeline implemented in the CONN Toolbox (v 22. a) and SPM (v 12.7). Denoising included the regression of potential confounding effects characterized by white matter time series (5 CompCor noise components), CSF time series (5 CompCor noise components), motionparameters and their first order derivatives (12 factors total), outlier scans (below 12 factors), session and task effects (linear detrending) and their first order derivatives (12 factors), and quadratic effects (3 factors) within each functional run. This was followed by bandpass frequency filtering of the BOLD timeseries between 0.008Hz and 0.09Hz. Compcor noise components within the white matter and CSF were estimated by computing the average BOLD signal as well as the largest principal components orthogonal to the BOLD average, motion parameters, and outlier scans within each subject’s eroded segmentation masks. From the number of noise terms included in this denoising strategy, the effective degrees of freedom of the BOLD signal after denoising were estimated to range from 353.9 to 365.9 (average 361.5) across all subjects.

[0194] fMRI ALFF Measurements'. Amplitude of low frequency fluctuations (ALFF) maps characterizing low-frequency BOLD signal variability at each voxel were estimated as the root mean square (RMS) of the BOLD signal after denoising and band-pass filtering between 0.008 Hz and 0.09 Hz. ALFF can be conceptualized as a measure of the spectral power (or amplitude) of BOLD signal within the filtered frequency band.

[0195] Group-level Analyses'. Two-sided t-tests were conducted at each voxel to determine differences in ALFF between conditions using AFNl’s 3dttest++ command. Un- thresholded maps are displayed in the top two rows of FIGS 8, 9, and 10 for the spray and cannula conditions. Warm colors in these maps highlight brain regions with higher ALFF signal (increased brain activity) in the 15 min. or 60 min. scans compared to baseline. Cool colors in these maps highlight brain regions with lower ALFF signal (decreased brain activity) in the 15 min. or 60 min. scans compared to baseline. Green-colored regions show little to no change between baseline and the 15 / 60 min. follow-up scans.

[0196] Thresholded maps are displayed when comparing cannula to spray (FIGS. 8 & 9), when comparing the cannula conditions with and without MucoLox (FIG. 10), and when comparing the spray condition across the two participant Blocks (FIG. 11). Opaque voxels outlined in black represent brain regions that show a statistically significant difference in ALFF between the two conditions being contrasted (liberal statistical threshold of p < 0.05, uncorrected for multiple comparisons). Because of the low sample size, non-significant voxels are also displayed with reduced opacity corresponding to larger pvalues (less significant difference between conditions).

[0197] Region of Interest Analysis: Mean ALFF signal was extracted from each of the 49 regions of the Harvard Oxford Subcortical / Cortical Atlases using the python package NiLearn. Plots tracking change in ALFF signal over time and across conditions were generated in R using the ggplot2 and ggpubr packages. FIG. 12 plots changes in mean ALFF within the anterior cingulate cortex and inferior temporal gyrus over time. Red lines represent cannula sessions and blue lines represent spray sessions. Sessions in which subjects received the MucoLox thickening agent are represented by triangles. A nonparametric LOESS regression was fit to each group’s data and is plotted as a thick line surrounded by a shaded gray area (95% confidence interval).

[0198] This example relates to assessment of whether the method used for intranasal delivery of insulin affected brain activity, as measured with functional magnetic resonance imaging (fMRI). Previous work in this field has demonstrated changes in brain signal associated with intranasal delivery of insulin via a traditional nasal mist (spray) device. In the current study researchers compared the traditional spray device to an olfactory delivery device. In two separate counterbalanced sessions, each participant received an intranasal dose of 80 IU insulin via both a traditional spray mister (1.6 mL) or an olfactory delivery device (0.16 mL). This difference in volume is an important difference between the two systems. The olfactory delivery device delivers a small volume in a very targeted profile. The spray device requires much more volume (about an order of magnitude) of the formulation to achieve the same concentration of drug. Additionally, during the olfactory delivery device delivery session four of the eight participants received a thickening agent (15% MucoLox) that was combined with the insulin. Resting state fMRI was acquired at baseline, and then again 15 and 60 minutes following intranasal delivery of insulin. Scans acquired after receiving insulin showed decreased prefrontal brain activity compared to baseline scans. The limited data from subjects in this pilot study suggest that the olfactory delivery device elicits a larger and longer-lasting decrease in prefrontal brain activity compared to the traditional spray mister. However, olfactory delivery of insulin mixed with the thickening agent MucoLox showed weaker and more transient prefrontal deactivation compared to olfactory delivery of insulin alone. Together, these results suggest that administration of insulin alone (without MucoLox) via a olfactory delivery device may provide targeted, longer lasting deactivation of prefrontal brain regions.

[0199] Key Findings:

[0200] For subjects in Block 1 (Insulin without Mucolox), there is a difference in brain activation elicited by delivery via the olfactory delivery device as compared to delivery via the spray device. Prefrontal deactivation was greater and more sustained when insulin was administered via the olfactory delivery device as compared to administration via the spray device.

[0201] For subjects in Block 2 (Insulin with MucoLox) there may be a difference in brain activation elicited by delivery via the olfactory delivery device as compared to delivery via the spray device. At 15 minutes, both the olfactory delivery with MucoLox and spray mister elicit a small decrease of prefrontal brain activity. However at 60 minutes, brain activity appears to have increased back to baseline levels in the olfactory device delivery condition with MucoLox, while it remained deactivated in the spray mister condition.

[0202] There is a difference in the brain activity patterns elicited by the olfactory delivery device conditions with and without MucoLox. When comparing olfactory device-delivery of insulin alone to olfactory device-delivery of insulin mixed with the thickening agent MucoLox, subjects who received insulin alone exhibited greater, longer lasting deactivation of prefrontal cortex.

[0203] Note on Interpreting fMRI Signal:

[0204] Insulin affects brain activity by inhibiting glucose metabolism in neurons. fMRI cannot measure glucose metabolism or neuronal action potentials directly. Instead, fMRI measures blood oxygenation level dependent (BOLD) signal, which is interpreted as a proxy for brain activity. Active neurons require oxygen as fuel which they receive from hemoglobin molecules in the bloodstream. As hemoglobin is deoxygenated (to provide oxygen to an active neuron), the hemoglobin’s magnetic properties change (from diamagnetic to paramagnetic). The BOLD signal measured by the MRI scanner is a ratio of oxygenated to deoxygenate hemoglobin levels measured at specific brain regions. BOLD signal can be thought of as a proxy for brain activity. Effective delivery of insulin to a brain region should result in inhibition of glucose metabolism, decreased neuronal firing, and a corresponding decrease (deactivation) in BOLD signal amplitude.

[0205] Results

[0206] Block 1 (Insulin without MucoLox): Four subjects were assigned to Block 1 of the study, receiving insulin via traditional spray mister and an olfactory delivery device.ALFF maps at 15 min. and 60 min. post insulin administration are shown in FIG. 8. Results demonstrate that delivery of insulin via both spray and olfactory delivery device are associated with a reduction in brain activity in prefrontal regions after 15 minutes. After 60 minutes, the reduction in prefrontal brain activity is even more intense in the olfactory delivery condition compared to the spray condition.

[0207] FIG. 8: ALFF maps show changes in brain activation compared to baseline at 15 and 60 minutes post intranasal delivery of insulin. Shown here are group maps from Block1 participants who received insulin via a standard spray mister and an olfactory delivery device a. The bottom row shows the group difference map of between cannula (middle row) and the spray (top row) conditions. Opaque voxels outlined in black represent brain regions that show a statistically significant difference in ALFF between the two conditions (liberal statistical threshold of p < 0.05, uncorrected for multiple comparisons). Because of the low sample size, non-significant voxels are also displayed with reduced opacity corresponding to larger p-values (less significant difference between conditions).

[0208] Block 2 (Insulin with MucoLox): Four subjects were assigned to Block 2 of the study, receiving insulin via traditional spray mister and a mixture of insulin and 15% MucoLox via an olfactory delivery device. ALFF maps at 15 min. and 60 min. post insulin administration are shown in FIG. 9. Results demonstrate that after 15 minutes, delivery of insulin via both spray and olfactory delivery device are associated with a similar reduction in brain activity in prefrontal regions. After 60 minutes, the reduction in prefrontal brain activity is more intense in the spray condition, and prefrontal brain activity appears to have returned to baseline levels in the olfactory delivery condition.

[0209] FIG 9: ALFF maps show changes in brain activation compared to baseline at 15 and 60 minutes post intranasal delivery of insulin. Shown here are group maps from Block2 participants who received insulin via a standard spray mister and a mixture of insulin and MucoLox via an olfactory delivery device. The bottom row shows the group difference map of between olfactory delivery device with MucoLox (middle row) and the spray (top row) conditions. Opaque voxels outlined in black represent brain regions that show a statistically significant difference in ALFF between the two conditions (liberal statistical threshold of p < 0.05, uncorrected for multiple comparisons). Because of the low sample size, nonsignificant voxels are also displayed with reduced opacity corresponding to larger p-values (less significant difference between conditions).

[0210] Effect of MucoLox: To investigate potential effects of adding 15% MucoLox to insulin we compared the ALFF maps from the olfactory delivery conditions in Block 1 (Insulin without MucoLox) and Block 2 (Insulin with MucoLox) participants. Four subjects were assigned to each Block. ALFF maps at 15 min. and 60 min. post insulin administration are shown in FIG. 10. Results demonstrate that after 15 minutes, delivery of insulin via olfactory delivery device is associated with a reduction in brain activity in prefrontal regions, but that this reduction is greater in the absence of MucoLox (Block 1). After 60 minutes, the reduction in prefrontal brain activity is more intense in Block 1 participants who did not receive MucoLox. At 60 minutes, Block 2 participants who received MucoLox show a return to baseline levels of prefrontal brain activity.

[0211] FIG 10: ALFF maps show changes in brain activation compared to baseline at 15 and 60 minutes post intranasal delivery of insulin via olfactory delivery device. Shown here are group maps from Block 1 participants who received insulin without MucoLox (top row) and Block 2 participants who received insulin with 15% MucoLox (middle row). The bottom row shows the group difference map. Opaque voxels outlined in black represent brain regions that show a statistically significant difference in ALFF between the two conditions (liberal statistical threshold of p < 0.05, uncorrected for multiple comparisons). Because of the low sample size, non-significant voxels are also displayed with reduced opacity corresponding to larger p-values (less significant difference between conditions).

[0212] Control Analysis (Comparing Spray Condition across Blocks 1 & 2): It is possible that changes in ALFF attributed to MucoLox in FIG. 10 could be caused by inherent group differences between subjects in Blocks 1 and 2. To account for this, we examined the difference in ALFF measures during the spray condition across Block 1 and Block 2 participants. In the spray condition, participants in both Blocks received only insulin without MucoLox. The expectation is that ALFF levels will not differ significantly across the two groups. After 15 minutes, subjects in Block 2 show increased brain activity in prefrontal cortex and reduced brain activity in posterior brain regions compared to Block 1 participants. At 60 minutes, Block 2 participants show decreased brain activity in select prefrontal and posterior regions compared to Block 1 participants.

[0213] This control analysis supports the interpretation in FIG. 10 that MucoLox is associated with reduced changes in brain activity. If it were simply the case that subjects in Block 2 showed uniformly increased prefrontal activation compared to Block 1, then theresults in FIG. 10 could be attributed to individual differences between the two groups. However, Block 2 participants actually showed greater prefrontal deactivation at the 60 minute scan. This suggests that reduced deactivation / retum to baseline of Block 2 participants in FIG. 10 is likely due to MucoLox.

[0214] FIG. 11 : As a control analysis, we compared ALFF measures between the two subject groups. Both groups received the same insulin dose via the traditional spray mister. If Group 2 had shown an overall increase in prefrontal brain activity, it would have suggested that individual differences were a major factor for the MucoLox related activation differences observed in FIG. 10. However, the results here suggest that individual differences alone likely do not account for the reduced deactivation seen in the MucoLox condition. Opaque voxels outlined in black represent brain regions that show a statistically significant difference in ALFF between the two conditions (liberal statistical threshold of p < 0.05, uncorrected for multiple comparisons). Because of the low sample size, nonsignificant voxels are also displayed with reduced opacity corresponding to larger p- values (less significant difference between conditions).

[0215] Region of Interest Analysis: A region of interest (ROI) analysis was conducted to examine how ALFF measures changed over time in specific brain regions (see FIG. 12). Regions were selected from the Harvard Oxford Cortical Atlas to be representative of the anterior decrease and posterior increase in ALFF that was observed in this study. The ROI analysis demonstrates that within the anterior division of the cingulate gyrus, ALFF decreased over time during both the olfactory delivery and spray conditions. Alternatively, within the inferior temporal gyrus, ALFF increased over time during both conditions. These plots demonstrate the significant heterogeneity between subjects in ALFF responses to insulin delivery.

[0216] FIG. 12: Region of interest analysis for two representative brain regions, highlighted in orange on axial slices from the MNI template brain. Plots show mean change in ALFF over time within these regions. Individual points and lines represent each of the 8 subjects. Triangles on the cannula plots indicate subjects who received 15% MucoLox with their insulin dose. Thick lines show a non-parametric LOESS fit of the data. Shaded gray regions represent a 95% confidence interval. (A) The anterior division of the cingulate gyrus shows a decrease in ALFF in both the olfactory delivery and spray conditions. (B) The temporooccipital part of the inferior temporal gyrus shows an increase in ALFF for bothconditions. (* p < 0.05 in a T-test comparing ALFF at various time-points, not corrected for multiple comparisons.)

[0217] This example demonstrates that intranasal delivery of insulin via the olfactory delivery device is associated with deactivation of the prefrontal cortex to a greater degree than traditional delivery via a spray mister.Example 4 Olfactory delivery of exendin-4 in rats

[0218] The primary goal of this study was to test whether olfactory delivery (OD) administration of small peptides performed comparably to IV administration in delivering drugs to the brain and plasma.

[0219] Exendin-4 was tested for each of two routes of administration (OD and IV). Five rats were used for each condition. Animals were anesthetized using Sutai / Xylazine. Blood samples were acquired at seven timepoints following compound administration: 5, 15, 30, 60, 90, 120, and 200 minutes. After 200 minutes, animals were euthanized via CO2. The brain, olfactory bulb, and liver were extracted from each animal and prepared for analysis via LC / MS.

[0220] Drug Administration and Sample Collection'. Intravenous (IV) administration was conducted by lOOpL tail vein injection. Olfactory delivery (OD) administration was conducted by a 0.699 mm diameter cannula inserted into the right nostril with a minimal angle of 20-degrees to the target of the correct meatus (FIG. 6A).

[0221] Sample Preparation: An aliquot of 30 pL plasma was spiked into a 1.5 mL tube, and 120 LIL of acetonitrile containing internal standard were added for protein precipitation. The mixture was vortexed, centrifuged at 14000 rpm for 10 min. The 50 pL supernatant was resolved with 150 LIL H2O, then injected for LC-MS / MS analysis. Brain or olfactory sample was homogenized with ice-cold phosphate buffer saline (pH 7.4) at a ratio of 4 (buffer): 1 (tissue) (v / w). An aliquot of 30 pL homogenate was spiked into 1.5 mL tube, and 120 pL of acetonitrile containing internal standard was added for protein precipitation. The mixture was vortexed, centrifuged at 14000 rpm for 10 min. Transfer 50 pL of supernatant was mixed with 150 pL of water and the final solution was injected for LC-MS / MS analysis.

[0222] Liquid Chromatography / Mass Spectrometry: An Ultra Performance Liquid Chromatography (UPLC) chromatographic system (Waters), equipped with an AB Sciex QTRAP 6500 mass spectrometer was used to analyze the samples. Chromatographic separation was achieved on the Waters HSS T3 column 50*2.1mm ID, 1.8um, and a gradientmobile phase changing from 80% H20 (0.1% formic acid) (A) and 90% acetonitrile (0.1% formic acid) (B). The flow rate was maintained at 0.6mL / min. Analyst 1.6 software packages (Applied Biosystems) were used to control the LC / MS / MS system, as well as for data acquisition and processing. A calibration curve was constructed in blank olfactory bulb homogenate.

[0223] Statistics: T-tests were conducted to compare tissue / plasma concentrations following each of the three routes of administration. Corresponding p-values were corrected for multiple comparisons using the Holm-Bonferroni method.

[0224] Results: Exendin-4 (5mpk) demonstrated non-inferior uptake in plasma (FIG. 14A, Table 5). In two animals, exendin-4 showed increased olfactory uptake, and in three animals exendin-4 showed increased brain uptake following OD compared to IV administration. However, across the entire group, these differences were not statistically significant (FIG. 14B, Table 6).Table 5. Plasma concentration of exendin-4 after olfactory delivery (OD) administration or intravenous (IV) administration _Plasma OD IVTime (min) ng / ml ng / ml5 52.27 12216.6615 47.59 4203.5730 55.85 2217.9860 47.73 873.8390 67.32 439.69120 86.07 212.81200 38.50 48.40Table 6. Tissue concentration of exendin-4 after olfactory delivery (OD) administration or intravenous (IV) administration _OD IVTime (min) Tissue ng / g ng / gBrain 19.7 BLOQ200 Olfactory Bulb BLOQ BLOQ*BLOQ: below 5 ng / gExample 5: Administration of insulin via olfactory delivery.

[0225] Objective: To compare olfactory delivery (OD) of insulin to intranasal (IN) and intravenous (IV) delivery.

[0226] Animals: SD male Rats (male, 150-250 g), n = 9

[0227] Model Development: OD, IN, and IV injection

[0228] Measurement: Plasma, olfactory bulb, rest brain PK

[0229] Group Information:

[0230] Group A: RSH cannula delivery (OD). To investigate OD via a 0.699 mm diameter cannula to simulate RSH device delivery. 25 pL single nostril.

[0231] Group B: Standard pipette delivery (IN). To investigate IN delivery via a standard pipette to simulate IN spray devices. 25 pL single nostril.

[0232] Group C: Intravenous administration (IV). Intravenous delivery to serve as uptake control. 25 pL tail vein.

[0233] Test Compound: insulin (0.5 - 5.0 mg / kg)

[0234] Fast: Animals will be fasted overnight (free access to water), minimum 6 hours pre-op.

[0235] Time point: Animals are euthanized at specified time points (15 minutes).

[0236] Sample Collection Method: At each time point, 3 rats from each group will be euthanized. Animal brain with olfactory bulb will be carefully collected without perfusion. The surface will be gently rinsed with saline and dried. Olfactory bulb and brain: Rats olfactory bulb and brain will be collected and snap-freeze in liquid nitrogen. The tissue will be stored at -80 C until analysis.

[0237] Plasma Sample Preparation: Full blood will be collected into EDTA-2K tube and centrifuge at 4C, 4000rpm centrifugation. The plasma will be stored at -80 C until analysis.

[0238] Sample analysis: Test compound in olfactory bulb, brain, and plasma samples will be analyzed by LC / MS / MS and / or ELISA. Calibration curve will be constructed in blank olfactory bulb homogenate.

[0239] Data analysis: Non-compartmental analysis.

[0240] Formulations: 5-50 mg / mL of insulin in an aqueous solution including ~40% propylene glycol, ~10% ethanol, ~5% sodium benzoate / benzoic acid, and ~1.5% benzyl alcohol.

[0241] Study Procedure:

[0242] Group A (OD): A 0.699 mm diameter cannula with lipophilic ointment spread on the catheter is carefully inserted into the right nostril with a minimal angle of 20° to the target of the correct meatus. During insertion the catheter is rotated gently to advance it through the tight nasal cavity. Once the catheter reaches the ethmoid turbinate it cannot be gently advanced anymore (~15 mm). 25 pL is slowly instilled in the olfactory region.

[0243] Group B (IN) A p200 pipet tip is placed into the nostril just up to the nasal valve and 25 pL is gently deposited into the lower nase.

[0244] Group C (IV): Tail vein injection of 100 pL.

[0245] Predicted results: At least a portion of the formulation comprising insulin is delivered to one or more of the group consisting of cerebrospinal fluid, the central nervous system, or a region, tissue, or organ thereof. For example, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% of the formulation comprising insulin is delivered to one or more of the group consisting of cerebrospinal fluid, the central nervous system, or a region, tissue, or organ thereof. The amount of insulin delivered to one or more of the group consisting of cerebrospinal fluid, the central nervous system, or a region, tissue, or organ thereof is sufficient to provide a therapeutic effect.Additional examples: Examples similar to Example 1 to demonstrate olfactory delivery of one or more peptides selected from the group comprising insulin, oxytocin, glutathione, a neurotrophic factor, a glial cell-derived neurotrophic factor (GDNF), a nerve growth factor (NGF), glucagon, a glucagon like peptide 1 (GLP-1), dulaglutide, a GLP-1 agonist, an incretin mimetic, a galanin-like peptide (GALP), erythropoietin (EPO), a glycoprotein hormone, salmon calcitonin, buserelin acetate (a luteinizing hormone-releasing hormone (LHRH) agonist), desmopressin acetate (peptide drug), Gonadorelin, LHRH (Gonadotropin-releasing hormone agonist), Nafarelin acetate, Protirelin (synthetic analogue of thyrotropin-releasing hormone (TRH)), Cyanocobalamine (synthetic form of vitamin Bl 2) or an analog of any of the proceeding.

[0246] While the present disclosure has been described with reference to examples, it is to be understood that the scope of the claims should not be limited by the embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

[0247] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present disclosure is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term.

[0248] The scope of the claims should not be limited by the embodiments and examples, but should be given the broadest interpretation consistent with the description as a whole.

Claims

CLAIMSWhat is claimed is:

1. A method of delivering a therapeutic peptide to a subject in need thereof, the method comprising: a. selectively delivering a formulation comprising a therapeutically effective amount of the therapeutic peptide to an olfactory region of the subject; wherein at least a portion of formulation is delivered to one or more of the group consisting of cerebrospinal fluid, the central nervous system or a region, tissue, or organ thereof, and a non-CNS target.

2. The method of claim 1, wherein the formulation is delivered as a liquid jet.

3. The method of any one of claims 1-2, wherein the formulation is delivered as a laminar flow.

4. The method of any one of claims 1-3, wherein the formulation is delivered as a flow having a Reynold’s number of 2300 or less.

5. The method of any one of claims 1-4, wherein the formulation is ejected from a dispensing element, wherein at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the formulation passes through a circular opening having a diameter of between about 5 mm and about 6 mm at a distance of about 25 mm from where the formulation is ejected from the dispensing element.

6. The method of any one of claims 1-5, wherein the formulation has a viscosity of between about 0.5 cP and about 100 cP.

7. The method of any one of claims 1-5, wherein the formulation has a viscosity of between about 0.5 cP and about 50 cP.

8. The method of any one of claims 1-5, wherein the formulation has a viscosity of between about 0.5 cP and about 10 cP.

9. The method of any one of claims 1-5, wherein the formulation has a viscosity of between about 45 cP and about 55 cP.

10. The method of any one of claims 1-9, wherein the formulation is ejected from the dispensing element at a velocity of between about 1 m / s and about 30 m / s.

11. The method of any one of claims 1-9, wherein the formulation is ejected from the dispensing element at a velocity of between about 2 m / s and about 4 m / s.

12. The method of any one of claims 1-9, wherein the formulation is ejected from the dispensing element at a velocity of between about 25 m / s and about 30 m / s.

13. The method of any one of claims 1-4, wherein the formulation is ejected from a dispensing element, wherein at least 75% of the formulation passes through a circular opening having a diameter of between about 5 mm and about 6 mm at a distance of about 25 mm from where the formulation is ejected from the dispensing element, wherein the formulation has a viscosity of between about 1 cP and about 50 cP, and wherein the formulation is ejected from the dispensing element at a velocity of between about 1 m / s and about 30 m / s.

14. The method of any one of claims 1-13, wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the formulation is delivered to the olfactory region of the subject.

15. The method of any one of claims 1-14, wherein delivery of the therapeutic peptide to the olfactory region of the subject results in a plasma concentration of the therapeutic peptide about 120 min after delivery that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% of a plasma concentration of the therapeutic peptide when about the same dose is delivered intravenously.

16. The method of any one of claims 2-4, wherein the delivering the formulation as a laminar flow, a liquid jet, or a flow having a Reynold’s number of 2300 or less: a) increases on target delivery of the composition to the target region, b) decreases off target delivery of the composition to the nasal cavity, or c) both, compared to delivering the formulation with a spray ejection profile.

17. The method of any one of claims 1-16, wherein selectively delivering the formulation to the olfactory region of the subject comprises ejecting the formulation from an ejection zone in a nasal cavity of the subject, wherein the ejection zone is:(a) 0mm to 30mm superior to a horizontal line that intersects the anterior aspect of the internal nasal valve, and(b) 0mm to 20mm anterior to an inclined line that intersects the anterior aspect of the middle turbinate and the posterior aspect of the vestibule.

18. The method of claim 17, wherein the ejection zone is further:i. Omm to 40mm inferior to a horizontal line that is parallel to the inferior aspect of the olfactory cleft, ii. Omm to 20mm posterior to the internal nasal dorsum, iii. 10mm to 50mm superior to a horizontal line that intersects the inferior aspect of the columella, iv. Omm to 30mm superior to a horizontal line that intersects the superior aspect of the inferior turbinate, v. Omm to 3mm from the septum, or vi. any combination thereof.

19. The method of claim 17 or 18, wherein delivering the formulation from the ejection zone: a) increases on target delivery of the composition to the target region, b) decreases off target delivery of the composition to the nasal cavity, or c) both, compared to dispensing the formulation outside the ejection zone.

20. The method of any one of claims 17-19, wherein the formulation is delivered by a device comprising the dispensing element, wherein the device is inserted into a nasal cavity of the subject at an angle of between about 30 degrees and about 40 degrees from a vertical line, wherein the vertical line is perpendicular to a horizontal line that is parallel to an inferior aspect of the olfactory cleft of the subject.

21. The method of any one of claims 1-20, wherein the formulation delivered to the olfactory region remains at the olfactory region for at least about 10 seconds, at least about 30 seconds, at least about 1 min, at least about 2 min, at least about 3 min, at least about 4 min, at least about 5 min, at least about 6 min, at least about 7 min, at least about 8 min, at least about 9 min, at least about 10 min, at least about 11 min, at least about 12 min, at least about 13 min, at least about 14 min, or at least about 15 min.

22. The method of any one of claims 1-21, wherein the therapeutic peptide is insulin, oxytocin, glutathione, a neurotrophic factor, a glial cell-derived neurotrophic factor (GDNF), a nerve growth factor (NGF), glucagon, a glucagon like peptide 1 (GLP- 1), exendin-4, dulaglutide, a GLP-1 agonist, an incretin mimetic, or a galanin-like peptide (GALP).

23. The method of any one of claims 1-22, wherein the therapeutic peptide is an analog of one of the group consisting of: insulin, oxytocin, glutathione, a neurotrophic factor, a glial cell-derived neurotrophic factor (GDNF), a nerve growth factor (NGF), glucagon, a glucagon like peptide 1 (GLP-1), exendin-4, dulaglutide, a GLP-1 agonist, an incretin mimetic, a galanin-like peptide (GALP), erythropoietin (EPO), a glycoprotein hormone, salmon calcitonin, buserelin acetate, desmopressin acetate (peptide drug), Gonadorelin, Nafarelin acetate, Protirelin, and cyanocobalamine.

24. The method of any one of claims 1-23, wherein the therapeutic peptide is insulin, or a variant or analog thereof.

25. The method of claim 24, wherein delivery of the therapeutic peptide results in reduction in brain activity in the subject.

26. The method of claim 24, wherein delivery of the therapeutic peptide results in reduction in brain activity in the subject for at least about 15 min, at least about 30 min, at least about 45 min, or at least about 60 min after delivery.

27. The method of claim 25 or 26, wherein delivery of the therapeutic peptide results in reduction in brain activity in the subject for a longer period of time than when about the same dose of the therapeutic peptide is delivered by a nasal spray.

28. The method of any one of claims 1-27, wherein the therapeutic peptide is exendin- 4, or a variant or analog thereof.

29. The method of any one of claims 1-28, wherein the therapeutic peptide has a length of less than 50 amino acids.

30. The method of any one of claims 1-29, wherein the therapeutic peptide is conjugated to a therapeutic agent.

31. The method of claim 30, wherein the therapeutic agent is a drug or a radioisotope.

32. The method of any one of claims 1-31, wherein at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, or at least about90% of the therapeutic peptide in the formulation is delivered to one or more of thegroup consisting of cerebrospinal fluid, the central nervous system, and a region, tissue, or organ thereof.

33. The method of any one of claims 1-32, wherein the method provides delivery of a greater amount of the therapeutic peptide to one or more of the group consisting of cerebrospinal fluid, the central nervous system, and a region, tissue, or organ thereof compared to intravenous delivery of an identical dose of the therapeutic peptide.

34. The method of any one of claims 1-33, wherein the therapeutic peptide provides a therapeutic effect upon delivery to the cerebrospinal fluid, the central nervous system or region, tissue, or organ thereof.

35. The method of any one of claims 1-34, wherein the subject has been diagnosed with a disease or disorder related to the central nervous system.

36. The method of any one of claims 1-35, wherein delivery of the therapeutic peptide provides effective treatment for Alzheimer's disease, Parkinson's disease, a cancer of the brain, a cancer of the CNS, a non-CNS disorder, an immunological disorder, an oncological disorder, a hematological disorder, an opthalmological disorder, a neurological disorder, a respiratory disorder, a cardiovascular disorder, an infectious disease, vitamin deficiency, multiple sclerosis, improvement of uterine contractions, postmenopausal osteoporosis, cryptorchism, endometriosis, central precocious puberty (CPP), Primary nocturnal enuresis, central cranial diabetes insipidus, nocturia, cranial diabetes insipidus or nocturia associated with multiple sclerosis, stroke, epilepsy, central nervous system trauma, a metabolic disorder, an infectious agent (i.e. Naegleria fowleri) or protein (i.e. Creutzfeldt Jacobs disease), a malignant tumor, a benign tumor, glioblastoma, anosmia, or an endocrine disorder.

37. The method of any one of claims 1-36, wherein delivery of the therapeutic peptide provides effective treatment for depression, dementia, or Alzheimer’s disease.

38. The method of any one of claims 1-37, wherein the formulation is delivered without exposing the therapeutic peptide to shear forces sufficient to damage a significant portion of the therapeutic peptide, without aerosolizing the formulation, or by passing the formulation through a shear disintegrating tip.

39. The method of any one of claims 1-38, wherein the therapeutic peptide bypasses the blood-brain barrier, the blood-cerebrospinal fluid barrier, or the arachnoid membrane.

40. The method of any one of claims 1-39, wherein the non-CNS -target is blood.

41. The method of any one of claims 1-40, wherein the formulation is delivered via a device comprising: a. a housing defining first and second insertable portions, each for insertion into a nasal channel of the subject, wherein, upon insertion of at least one of the insertable portions into the nasal channel of the subject, the at least one insertable portion engages tissue within the nasal channel to open or expand an internal nasal valve of the subject thereby positioning the at least one insertable portion for delivery of the formulation to the olfactory region of the subject; and b. an actuator which delivers the formulation from at least one of the insertable portions when the device is actuated.

42. A device for olfactory delivery of a therapeutic peptide to an olfactory region of a subject, the device comprising: a. a housing comprising an insertable portion comprising a distal end, and a proximal end; and b. a subject-engaging portion which engages a columella region of the subject to seat the distal end of the insertable portion within an ejection zone of a nasal channel of the subject; wherein the device is configured to deliver a formulation comprising a therapeutically effective amount of the peptide to the olfactory region of the subject.

43. The device of claim 42, wherein the device dispenses the formulation as a laminar jet.

44. The device of any one of claims 42-43, wherein the device comprises a compliant dispensing tip comprising a compliant and flexible soft nib.

45. The device of any one of claims 42-44, wherein application of pressure by the subject-engaging portion to the columella region of the subject enables and / or causes delivery of the formulation to the subject from the insertable portion.

6. The device of any one of claims 42-45, wherein the insertable portion comprises a dispensing element for delivery of the formulation to the olfactory region of the subject.

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