Intranasal delivery of tesamorelin for the treatment of obesity

Abstract

Methods for intranasal delivery of GLP-1 (glucagon-like peptide-1) drugs, such as tirzepatide (TZP), using drug formulations encapsulated in poly(lactic-co-glycolic acid) (PLGA) nanoparticles coated with chitosan (CS) and / or chitosan grafted with polyethylenimine (CS-PEI) under optimized conditions for surface charge alteration and particle size control. The resulting formulations successfully provided dose-dependent body fat reduction in mice, showing significant therapeutic effects.

Description

Related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application 63 / 589,289, filed October 10, 2023, by a trustee of Columbia University, entitled “Intranasal Delivery of Tirzepatide to Treat Obesity,” the entire disclosure of which is incorporated herein by reference. Technical Field

[0002] The subject matter disclosed in this article generally relates to the intranasal delivery of obesity drugs using pharmaceutical formulations encapsulated in coated nanoparticles. background

[0003] Numerous attempts have been made to treat the obesity pandemic, primarily including lifestyle modifications, surgery, and pharmacological treatments. However, a breakthrough only came with the approval of GLP-1 receptor agonists for the treatment of obesity. Among these, semaglutide (Wegovy®) and telpoxetine (Tzp, Zepbound®) have demonstrated significant clinical efficacy in controlling blood sugar levels and reducing weight. For example, a 72-week course of Tzp treatment resulted in a weight loss of up to 22.5% in obese adults. GLP-1 drugs primarily work by suppressing appetite via the central nervous system, increasing insulin secretion in the pancreas, reducing glucagon production in the liver, and slowing gastric emptying; however, the individual mechanisms of contribution remain to be differentiated. Despite their remarkable weight-loss efficacy, GLP-1 drugs face challenges related to adverse side effects, such as common to severe gastrointestinal adverse reactions, risk of thyroid tumors, acute kidney injury, acute pancreatitis, and gallbladder disease. GLP-1 drugs are currently primarily administered via subcutaneous injection, and high-dose, long-acting medications are a strategy that increases intervals to several weeks. Oral administration is expected to offer advantages in patient adherence, but is hampered by low bioavailability and strong gastrointestinal adverse reactions. However, painless, easy-to-self-administer, non-invasive drug delivery is needed to increase long-term patient adherence to GLP-1 drugs. More importantly, organ-specific drug delivery holds the greatest promise for avoiding the adverse side effects of systemic administration.

[0004] Telboride (TZP), a lipid-modified peptide drug developed by Eli Lilly and Company, was recently approved by the U.S. Food and Drug Administration (FDA) as an antidiabetic and anti-obesity drug. TZP is an agonist of both the GIP (glucose-dependent insulinotropic peptide) and GLP-1 (glucagon-like peptide-1) receptors, both of which are known to be involved in stabilizing blood glucose levels and regulating dietary preferences. Although newly marketed, TZP has already demonstrated significant clinical efficacy and commercial potential. Meanwhile, cases of rebound effects after discontinuation suggest that TZP may be an ideal option for long-term use. However, TZP is currently administered via subcutaneous injection, which is detrimental to long-term adherence. Therefore, less invasive routes such as intranasal delivery are more desirable. Nevertheless, to date, there is no precedent for intranasal alternatives to TZP for therapeutic purposes. A key challenge is that, due to the large molecular size and readily degradable nature of peptide drugs like TZP, they cannot be transported across mucosal barriers and enter the body spontaneously. Therefore, it is crucial to employ novel formulations of TZP to enhance its transport across the barrier and reliably maintain its therapeutic efficacy.

[0005] Any reference or identification of any document in this application is not an admission that the document is prior art to this invention. Overview

[0006] In one aspect, the present invention provides a method of delivering a GLP-1 drug to a patient, the method comprising intranasal delivery of a pharmaceutical composition containing a GLP-1 drug. In some embodiments, the GLP-1 drug is selected from the group consisting of telposide, smegglutide, and retaglutide. In some embodiments, the GLP-1 drug in the pharmaceutical composition is sufficient to treat obesity, obesity comorbidity, or any other condition causing hyperglycemia or fatty liver, or related conditions.

[0007] In some embodiments, intranasal delivery includes a nanoparticle delivery system. In some embodiments, the nanoparticle delivery system encapsulates a GLP-1 drug. In some embodiments, the nanoparticle delivery system comprises polymer nanoparticles comprising poly(lactic-co-glycolic acid) (PLGA) nanoparticles coated with at least one positively charged biocompatible polymer. In some embodiments, the positively charged biocompatible polymer is selected from chitosan (CS) and polyethyleneimine-grafted chitosan (CS-PEI). In some embodiments, the nanoparticle delivery system comprises polymer nanoparticles comprising poly(lactic-co-glycolic acid) (PLGA) nanoparticles coated with polyethyleneimine-grafted chitosan (CS-PEI). In some embodiments, the ratio of coating to PLGA is 0.25. In some embodiments, the coated nanoparticles have a polydispersity index (PdI) of 0.1 ± 0.02. In some embodiments, the coated nanoparticles have a zeta potential of 49.3 ± 1.3 mV.

[0008] In some implementations, the patient is being treated for obesity, obesity-related comorbidities, or any other condition causing hyperglycemia or fatty liver, or related conditions. In some implementations, the GLP-1 drug is delivered to the olfactory bulb. In some implementations, the GLP-1 drug is administered at approximately 50 nmol / kg. Apply by weight.

[0009] The foregoing and other aspects, features and advantages will be apparent from the description and drawings and the claims (if included). Brief description of the attached diagram

[0010] The patent or application document contains at least one drawing in color. A published copy of the patent or application with the color drawing will be provided by the patent office upon request and payment of the necessary fees.

[0011] The features and advantages of the invention will be described below in conjunction with the accompanying and / or included drawings, wherein like reference numerals denote like elements, and: Figure 1 – A schematic diagram illustrating how TZP-loaded nanoparticles alleviate obesity.

[0012] Figures 2A-2F – Optimization and characterization of coated PLGA nanoparticles. Figure 2A –2C) Based on size ( Figure 2A ), polydispersity index ( Figure 2B ) and ζ potential ( Figure 2C The coating polymer (CP:CS-PEI, CS: chitosan) and coating ratio for preparing coated PLGA nanoparticles were optimized; Figure 2D )Optimized hydrodynamic diameter distribution of coated PLGA nanoparticles; Figure 2E Representative TEM images of TZP-loaded PLGA nanoparticles; Figure 2F Sustained release curves of TZP-loaded PLGA nanoparticles under physiologically relevant in vitro conditions. Data are presented as mean ± SD.

[0013] Figures 3A-3C – Intranasal injection of PLGA-TZP resulted in weight loss and improved metabolic health. Mice treated with or without PLGA-TZP ( Figure 3A Body weight curve and ( Figure 3B Body weight changes. Mice treated with free TZP (free) and PLGA blank (Veh) were used as baseline controls. Figure 3C Changes in body composition 8 days post-injection. A two-way ANOVA was used to compare the changes in body composition between the carrier control (n=7) and the intranasal injection group of PLGA-TZ (PLGA-TZP group and carrier group n=7, free drug group n=8). p < 0.01 and **** p < 0.0001. Data are presented as mean ± SEM.

[0014] Figure 4 – Intranasal injection of PLGA-TZP resulted in weight loss and improved metabolic health. Weight changes in mice treated with and without PLGA-TZP are shown. Mice treated with free TZP (free) and PLGA blank (mediator) served as baseline controls.

[0015] Figures 5A-5K – Intranasal administration of GLP-1 RA nanoparticles has shown anti-obesity effects. Figure 5A A schematic diagram of the experimental design. Figures 5B-5C Characterization of Tzp-PLGA nanoparticles (Tzp-NP), including a histogram of their size distribution. Figure 5B ) and release curve in PBS ( Figure 5C ). ( Figure 5D-5F DIO C57BL / 6 male mice were administered Tzp, Tzp-NP, or a mediator control via intranasal administration once daily for 8 days at a dose of 100 nmol / kg bw. Figure 5D ) Schematic diagram. Figure 5E Weight changes. Figure 5F Changes in fat mass. Figure 5GDIO male mice were administered Tzp (Tzp, sc, 10 nmol / kg.bw) subcutaneously or Tzp-NP (Tzp-NP, na, 50 nmol / kg.bw) or a carrier substance once daily for 3 days. Figure 5H-5I After daily administration of subcutaneous semaglutide (Sema, sc, 10 nmol / kg.bw) or intranasal semaglutide nanoparticles (Sema-NP, 50 nmol / kg.bw) for 6 days, the body weight of mice ( Figure 5H ) and fat mass ( Figure 5I Changes in nasal fluid were observed. Water administered intranasally served as a control. Figure 5J-5K After daily administration of subcutaneous retaglutide (Reta, sc, 10 nmol / kg.bw) or intranasal retaglutide nanoparticles (Reta-NP, na, 50 nmol / kg.bw) for 7 days, the body weight of mice ( Figure 5J ) and fat mass ( Figure 5K Changes in body weight were observed. Intranasal water was used as a control. Data are presented as mean ± sem. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistical significance of changes in body weight was assessed using two-way ANOVA. Changes in fat mass were assessed using paired t-tests and compared to baseline body weight for each mouse on day 0. For (BC), n = 3. For (EF), n = 7, 7, 8 (control, Tzp-NP, and Tzp groups). For G, n = 7 in the control group and n = 6 in the treatment group. For (HI), n = 6, 7, 7 (control, Sema-NP, and Sema groups). For (JK), n = 8 in each group.

[0016] Figures 6A-6I Intranasal delivery concentrates GLP-1 drugs in the olfactory bulb and minimizes peripheral distribution. Figure 6A Brain signal distribution. Mice were administered 50 nmol / kg bw Cy5-labeled Tzp NP or 10 nmol / kg bw Cy5-labeled Tzp via intranasal (na) or subcutaneous (sc) routes. The control group received intranasal water. One hour later, in vivo imaging was performed using an IVIS optical imaging system to measure fluorescence signals. (The control group, na group, and sc group were n = 3, 4, and 3, respectively). Figures 6B-6C Diet-induced obese (DIO) male mice were fasted overnight. After treatment with Tzp NP (na), Tzp (sc), or water (medium control) for 1 hour, the mice were fed for another 1 hour, and food intake was measured. Figure 6B A schematic diagram of the experimental setup. Figure 6CQuantitative measurement of food intake. (The control group, na group, and sc group were n = 7, 7, and 6, respectively). Figure 6D Confocal microscopy analysis revealed the distribution of Tzp-NP and Tzp in the brain one hour after injection. Figure 6E Cy5 fluorescence signal detected in peripheral tissues 1 hour after injection. Figure 6F-6I Mice were administered 50 nmol / kg bw Cy7-labeled Tzp-NP or 10 nmol / kg bw Cy7-labeled Tzp via intranasal or subcutaneous routes. The control group received water intranasally. (The control group, na group, and sc group had n = 2, 3, and 3, respectively). Figure 6F Cy7 fluorescence signal in the brain 24 hours after injection. Figure 6G The plasma concentrations of Tzp at the indicated time points (n = 3, 6, and 5 for the control group, na group, and sc group, respectively). Figure 6H Cy7 fluorescence signals were detected in multiple organs 24 hours after injection. Figure 6I Quantification of signals from different organs in the H-map. Fluorescence scale unit is photons / s / cm. 2 / sr. Data is presented as mean ± sem.

[0017] Figures 7A-7L – The anti-obesity effect of long-term intranasal application of Tzp-NP. Figure 7A A schematic diagram of the experimental setup. Diet-induced obese (DIO) male mice were treated daily with Tzp-NP (na), Tzp (sc), or water (na) as a control for 4 weeks. Mice were sacrificed under ad libitum conditions. Food intake was measured for the first three days, and insulin tolerance tests (ITT) and glucose tolerance tests (GTT) were performed in the last two weeks. Figure 7B Weight changes during treatment. Figure 7C Average food intake during the three-day treatment period. Figure 7D Fat quality during 7E treatment ( Figure 7D ) and lean body mass ( Figure 7E The change of ). Figure 7F Representative images of mice after treatment. Figure 7G The percentage of body weight that is fat. Figure 7H The weight of the tissues at the time of execution. Figure 7I Images of iWAT and eWAT at the time of euthanasia after four weeks of processing. Figure 7J Histological analysis (H&E staining) of eWAT and iWAT. Figure 7K Distribution of adipocyte size in eWAT. Figure 7LGene expression in eWAT. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; significance was determined by two-way ANOVA. For (C), n = 7 for the vector group and n = 6 for the Tzp treatment group. For (AM) (excluding C), n = 8 for the vector group and na group and n = 7 for the sc group.

[0018] Figures 8A-8K – Intranasal delivery of Tzp-NP improves obesity-related metabolic disorders in a similar way to subcutaneous administration. Figures 8A-8H The experimental design is the same as described in Figure 3A. Figure 8A , 8B) Glucose tolerance tests (GTT) were performed after 2.5 weeks and 2 weeks of Tzp treatment, respectively. Figure 8A ) and insulin tolerance test (ITT) Figure 8B ). ( Figure 8C-8E Plasma insulin levels Figure 8C ), non-esterified fatty acids (NEFA) Figure 8D ) and triglycerides (TG) Figure 8E ). ( Figure 8F Histological analysis of liver tissue (H&E staining). Figure 8G Plasma alanine aminotransferase (ALT) levels. Figure 8H Gene expression analysis in the liver. Figure 8I Blood glucose and insulin levels in mice 1 hour after injection of Tzp-NP (na), Tzp (sc), or water as a control. Figure 8K Changes in glucose levels over time in mice receiving Tzp-NP or Tzp. Data are presented as mean ± sem. For (AH), n = 8 in the mediator and intranasal groups; n = 7 in the subcutaneous group. For (I, J), n = 7, 6, and 7 in the mediator, intranasal, and subcutaneous groups, respectively. For (K), n = 6 in each group. Statistical significance was determined using two-way ANOVA.

[0019] Figures 9A-9F – Characterization of TZP-PLGA nanoparticles includes ( Figure 9A Transmission electron microscopy (TEM) images, Figure 9B Fluid dynamics diameter, ( Figure 9C Polydispersity Index (PDI) and ( Figure 9D ζ potential. Figure 9E Weight changes in diet-induced obese (DIO) female mice following a single intranasal injection of Tzp-NP or water (control). Figure 9FBody weight changes in male DIO mice after receiving a single dose of freshly prepared Tzp-NP or after storing Tzp-NP at 4°C for 3 or 7 days. For (BD), n = 3 per group. For (EF), n = 5 per group. Statistical significance was determined using t-tests or two-way ANOVA.

[0020] The accompanying figures in this article are for illustrative purposes only and are not necessarily drawn to scale. Detailed Explanation

[0021] Detailed aspects and applications of this disclosure are described below in the accompanying drawings and detailed description of the technology. Unless otherwise specified, the words and phrases in the specification and claims are intended to have the common, conventional, and customary meanings as understood by one of ordinary skill in the art.

[0022] In the following description, and for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of various aspects of this disclosure. However, those skilled in the art will understand that embodiments of the technology disclosed herein can be implemented without these specific details. It should be noted that many different and alternative configurations, apparatuses, and techniques exist to which the disclosed technology can be applied. The full scope of the technology disclosed herein is not limited to the examples described below.

[0023] The singular forms “a,” “an,” and “the” include plural indicators unless the context clearly specifies otherwise. Thus, for example, referring to “a step” includes referring to one or more such steps.

[0024] The terms “exemplary,” “example,” or any of their various forms are used herein to mean anything used as an example, illustration, or description. Any aspect or design described herein as an “exemplary” or “example” is not necessarily to be construed as preferred or superior to other aspects or designs. Furthermore, examples are provided for clarity and understanding only and are not intended to limit or constrain the disclosed subject matter or any related parts of this disclosure in any way. It should be understood that numerous additional or alternative examples of various scopes could have been presented, but have been omitted for the sake of brevity.

[0025] When representing a range of values, another implementation includes from one specific value and / or to another specific value. Similarly, when a value is expressed as an approximation using the antecedent “about,” it will be understood that the specific value forms another implementation. All ranges include endpoints and can be combined with each other. Numerical ranges referred to by endpoints include all numbers and fractions contained within the corresponding range, as well as the endpoints referred to.

[0026] As used herein, the terms “about” or “approximately” when referring to measurable values ​​such as parameters, quantities, time intervals, etc., mean variations in the specified value and variations deviating from the specified value, such as variations of + / -10% or less, + / -5% or less, + / -1% or less, and + / -0.1% or less, and variations deviating from the specified value by + / -10% or less, + / -5% or less, + / -1% or less, and + / -0.1% or less, provided that such variations are suitable for implementation in the disclosed invention. It should be understood that the values ​​referred to by the modifier “about” or “approximately” are themselves specifically and preferably disclosed.

[0027] Throughout the description and claims of this specification, the words “comprising” and “including”, as well as variations of these words such as “containing” and “containing”, mean “including but not limited to” and are not intended to exclude (and do not exclude) other components.

[0028] Detailed embodiments of this disclosure are included herein as needed. It should be understood that the disclosed embodiments are merely examples of the invention, which can be implemented in various forms. Therefore, the specific structural and functional details disclosed herein should not be construed as limiting, but merely as a basis for teaching those skilled in the art to use the invention. The following specific examples will enable a better understanding of this disclosure. However, they are given only as guidance and do not imply any limitation.

[0029] This disclosure will be more readily understood by referring to the following detailed description, in conjunction with the accompanying drawings and embodiments, which form part of this disclosure. It should be understood that this disclosure is not limited to the specific materials, apparatus, methods, applications, conditions, or parameters described and / or shown herein, and the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to limit the claimed invention. As used herein, the term "more than one" means more than one. When indicating a range of values, another embodiment includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation using the antecedent "about," it will be understood that the particular value forms another embodiment. All ranges include end values ​​and can be combined with each other.

[0030] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology can be found in *Molecular Cloning: A Laboratory Manual*, 2008. ndedition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4 th edition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); Methods in Enzymology series (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2 nd edition 2013 (E.A. Greenfield ed.); Animal Cell Culture (1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al . (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley &Sons (New York, NY 1994), March, Advanced Organic Chemistry Reactions,Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2 nd edition (2011).

[0031] The terms “optional” or “optionally” mean that the event, situation, or substituent described below may or may not occur, and the description includes both the possibility that the event or situation occurs and the possibility that it does not occur.

[0032] As used herein, a “biological sample” may comprise intact cells and / or live cells and / or cell debris. A biological sample may comprise (or be derived from) “body fluids.” This invention includes embodiments in which body fluids are selected from: amniotic fluid, aqueous humor, vitreous humor, bile, serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudate, feces, female ejaculation, gastric acid, gastric juice, lymph, mucus (including nasal discharge and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, inflammatory secretions (rheum), saliva, semen (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vomitus, and mixtures of one or more thereof. Biological samples include cell cultures, body fluids, and cell cultures derived from body fluids. Body fluids can be obtained from mammalian organisms, for example, by puncture or other collection or sampling procedures.

[0033] The terms “subject,” “individual,” and “patient” are used interchangeably herein and refer to vertebrates, preferably mammals, and more preferably humans. Mammals include, but are not limited to, murines, apes, humans, farm animals, sporting animals, and pets. Tissues, cells, and their progeny from biological entities obtained in vivo or cultured in vitro are also included.

[0034] Various embodiments are described below. It should be noted that specific embodiments are not intended as an exhaustive description of the broader aspects discussed herein, nor as a limitation thereof. An aspect described in connection with a particular embodiment is not necessarily limited to that embodiment, but can be practiced through any other embodiment. Throughout the specification, references to “an embodiment,” “one embodiment,” or “an exemplary embodiment” mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Therefore, the phrases “in one embodiment,” “in one embodiment,” or “an exemplary embodiment,” appearing in various places throughout the specification, do not necessarily refer to the same embodiment, but may refer to the same embodiment. Furthermore, it will be apparent to those skilled in the art, based on this disclosure, that particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments. Moreover, while some embodiments described herein include some features included in other embodiments but exclude others, combinations of features from different embodiments are intended to be within the scope of the invention. For example, in the appended claims, any claimed embodiment can be used in any combination.

[0035] All publications, published patent documents and patent applications cited herein are incorporated herein by reference to the extent that each individual publication, published patent document or patent application is specifically and individually indicated to be incorporated by reference. Overview

[0036] GLP-1 inhibitors are blockbuster drugs for treating obesity. However, current subcutaneous injections present a range of problems, including reduced adherence and increased risks. Oral delivery either has extremely low bioavailability or is ineffective. This article reports intranasal delivery of TZP, offering a novel, more convenient, and potentially safer approach to maximizing its market potential.

[0037] Intranasal delivery offers unique advantages compared to conventional routes of administration. First, the nasal mucosal environment is less acidic, less digestible, and easier to administer than oral delivery. Second, due to the high permeability and large surface area of ​​the nasal vascular system, intranasal delivery provides superior bioavailability and faster absorption into the bloodstream, almost comparable to intravenous injection. Most notably, intranasal administration allows drugs to bypass the blood-brain barrier via the olfactory nerve epithelium, as demonstrated by the approval of esketamine (an antidepressant known to exert its therapeutic effects in the brain) and desmopressin for the treatment of central cranial diabetes insipidus. Indeed, due to these advantages, peptide drugs, including insulin, glucagon, and GLP-1 drugs, have been investigated for intranasal delivery, particularly in the case of type 2 diabetes. However, intranasal delivery of recombinant human GLP-1 successfully lowered blood glucose levels in patients with type 2 diabetes, but did not reduce weight, total food intake, or hunger (Ueno H, Mizuta M, Shiiya T, et al. Exploratory trial of intranasal administration of glucagon-like peptide-1 in Japanese patients with type 2 diabetes. Diabetes Care. 2014;37(7):2024-2027). Another study in obese adults, with intranasal administration of peptide YY3–36 (PYY3–36) for 12 weeks, also did not result in weight loss (Gantz I, Erondu N, Mallick M, et al. Efficacy and safety of intranasal peptide YY3-36 for weight reduction in obese adults. J Clin Endocrinol Metab. 2007;92(5):1754-1757). Therefore, there is a critical gap in unlocking the therapeutic potential of GLP-1 drugs in obesity management through this uniquely advantageous intranasal delivery route.

[0038] The applicant has developed an intranasal nanoparticle system for delivering GLP-1 drugs to the brain. By minimizing peripheral distribution through more confined brain delivery, the applicant has achieved generalized anti-obesity efficacy of GLP-1 drugs, including semaglutide, Tzp, and triple retaliglutide. This limited delivery reveals an interesting mechanism by which GLP-1 RA inhibits appetite evoked in the olfactory bulb, unlike conventional systemic administration. In addition to weight control, the metabolic benefits regarding improved insulin sensitivity and glucose and lipid metabolism are comparable to those of systemic subcutaneous administration. More importantly, by avoiding distribution to the hindbrain, intranasal delivery of retaliglutide reduced gastrointestinal adverse events in rats and minks. Therefore, intranasal delivery provides a promising approach to maximizing the therapeutic effects of GLP-1 drugs, with potentially increased adherence and safety.

[0039] The applicant first designed and developed a nanoparticle delivery system that can effectively encapsulate and deliver TZP via intranasal administration. It is a polymer nanoparticle primarily composed of two materials: PLGA and a chitosan derivative. PLGA, or poly(lactic-co-glycolic acid), is a family of biodegradable and biocompatible polymers approved by the FDA for therapeutic use (see, for example, Danhier F, Ansorena E, Silva JM, Coco R, Le Breton A, Préat V. PLGA-based nanoparticles: an overview of biomedical applications. J Control Release. 2012;161(2):505-522). Its fabrication and production are also highly flexible, enabling the formation of visible medical implants and nanoparticles encapsulating drugs. The degradability of PLGA can be tuned by adjusting the ratio between the two monomers, lactic acid and glycolic acid. It is a safe candidate for various drug delivery methods. To enhance transmucosal transport, the applicant developed a series of novel carriers by coating PLGA nanoparticles with chitosan (CS) and chitosan-derived polyethyleneimine-grafted chitosan (CS-PEI). Chitosan is a natural polysaccharide derived from chitin, a common component of the shells of crustaceans such as crabs and lobsters. It is highly biocompatible and biodegradable. Chitosan has been shown to facilitate transmucosal transport of nanoparticles because its amino groups carry a positive charge when protonated in water and interact with the negatively charged mucus layer. PEI grafting confers a higher positive charge density (which improves transport) and higher solubility in water (which contributes to the colloidal stability of the resulting nanoparticles) than chitosan. Experiments in obese mice showed that intranasal delivery with the aid of nanoparticles can produce promising TZP therapeutic effects comparable to subcutaneous injection, which cannot be achieved by intranasal administration of the free drug form alone.

[0040] Whether GLP-1 receptor agonists (RAs) exert their effects through the central and peripheral systems to control weight and improve metabolic function remains unclear, primarily limited by the lack of efficient delivery to the brain. The applicant demonstrates here that, despite minimal distribution in the circulation and peripheral tissues, intranasal delivery of telpolide to the brain mediated by nanomedicine achieves anti-obesity effects and metabolic improvements comparable to subcutaneous administration. In acute intranasal administration, telpolide was detected primarily in the olfactory bulb region, without reaching the hypothalamus; however, food intake and hyperglycemia were suppressed. Three-dimensional whole-brain c-Fos imaging revealed a significant overlap in neuronal activation regions between the intranasal and subcutaneous delivery pathways, but also some distinct regions. Notably, intranasal delivery of telpolide showed reduced gastrointestinal adverse events in rats and minks by avoiding distribution to the hindbrain (brainstem). These findings suggest that intranasal delivery of GLP-1 drugs potentially provides a safer and more patient-friendly treatment option for the management of obesity and diabetes.

[0041] Terms and Definitions The terms “therapeutic agent,” “therapeutic agent,” or “therapeutic medicine” are used interchangeably and refer to a molecule or compound that, when administered to a subject, imparts a beneficial effect. Beneficial effects include the ability to make a diagnostic decision; improvement of a disease, symptom, condition, or pathological condition; reduction or prevention of the onset of a disease, symptom, condition, or pathological condition; and generally resistance to a disease, symptom, condition, or pathological condition.

[0042] As used herein, the terms “treatment” or “treating,” or “relief” or “improvement” are used interchangeably. These terms refer to methods of obtaining beneficial or desired outcomes, including but not limited to therapeutic and / or preventative benefits. A therapeutic benefit is any treatment-related improvement or effect on one or more diseases, conditions, or symptoms during treatment. To obtain a preventative benefit, the composition may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject who reports one or more physiological symptoms of a disease, even if the disease, condition, or symptom may not yet have manifested. As used herein, “treatment” includes improving a disease, curing a disease, preventing a disease from worsening, slowing the rate of disease progression, or preventing a disease from recurring (i.e., preventing relapse).

[0043] The term "effective amount" or "therapeutic effective amount" refers to the amount of a drug agent sufficient to achieve a beneficial or desired outcome. Therapeutic effective amounts can vary depending on one or more of the following: the subject being treated and their disease condition, the subject's weight and age, the severity of the disease condition, the method of administration, etc., which can be readily determined by one of ordinary skill in the art. The term also applies to the dose that will provide an image for detection using any of the imaging methods described herein. The specific dose can vary depending on one or more of the following: the particular drug agent selected, the dosing regimen followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system carrying the drug.

[0044] "Pharmaceutical composition" means a composition that typically contains an excipient, such as a pharmaceutically acceptable carrier that is conventional in the art and suitable for administration to cells or subjects.

[0045] As used in this article, “biocompatibility” means compatibility with living tissues or systems in a non-toxic, harmless, physiologically non-reactive manner that does not cause immune rejection.

[0046] Treatment methods for obesity and obesity comorbidities In an exemplary embodiment, a subject is treated intranasally with a pharmaceutical composition or therapeutic agent comprising a GLP-1 drug. In an exemplary embodiment, the pharmaceutical composition is administered in the form of nanoparticles coated with a positively charged polymer.

[0047] Patients and diseases or conditions In exemplary embodiments, the compositions disclosed herein are used to treat subjects. Treatment may be preventative. In exemplary embodiments, the compositions disclosed herein may be used to treat subjects suffering from any obesity-related condition or disease, including but not limited to obesity comorbidities such as diabetes and hepatic steatosis, or any other condition causing hyperglycemia and / or fatty liver or associated with it. Non-limiting conditions or diseases include type 2 diabetes, atherosclerotic cardiovascular disease, cardiovascular disease, obesity, non-alcoholic fatty liver disease, metabolic liver disease, polycystic ovary syndrome, reward system disorders (e.g., addiction, bulimia, or substance use disorders), glucose intolerance, insulin resistance, and depression (e.g., GLP-1 agonists have shown antidepressant and neuroprotective effects). In exemplary embodiments, subjects at risk of obesity-related cancers (e.g., esophageal cancer, colorectal cancer, endometrial cancer, gallbladder cancer, kidney cancer, liver cancer, ovarian cancer, and pancreatic cancer, as well as meningioma and multiple myeloma). In exemplary embodiments, subjects suffering from any obesity category are treated. For example, treating patients with type I obesity-overweight (BMI 25.0-29.9 kg / m²) 2 Category II obesity - Obesity (BMI 30.0-39.9 kg / m²)2 Subjects with Class III obesity—extreme obesity (BMI > 40 kg / m2) are treated with the compositions described herein. In an exemplary embodiment, subjects with a BMI greater than 30 or a BMI greater than 27 and suffering from at least one weight-related comorbidity are treated with the compositions described herein. In an exemplary embodiment, the compositions disclosed herein are used for cosmetic weight loss.

[0048] In an exemplary implementation, the GLP-1 drug is administered at approximately 50 nmol / kg. Administered based on body weight. For example, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nmol / kg. Body weight, or an effective or therapeutically effective dose determined by a physician and the disease or condition being treated (e.g., an amount administered to a patient to reduce obesity). In an exemplary embodiment, humans are more sensitive to GLP-1 drugs than mice, resulting in lower doses for treating human patients compared to mice. In an exemplary embodiment, intranasal administration can be more frequent (e.g., before each meal, daily, every other day) rather than once per week. The advantage of more frequent administration compared to systemic injection is that the treatment can more closely mimic physiological fluctuations.

[0049] GLP-1 receptor agonists As used herein, “GLP-1 drugs” refers to glucagon-like peptide-1 (GLP-1) receptor agonists, also known as GLP-1 analogs, GLP-1DAs, or incretin mimics. GLP-1 receptor agonists also refer to a class of appetite-suppressing drugs that reduce blood glucose and energy intake by activating GLP-1 receptors. They mimic the action of the endogenous incretin hormone GLP-1 released from the gastrointestinal tract after eating. GLP-1 agonists were initially developed for type 2 diabetes. The 2022 American Diabetes Association Standards of Care recommend GLP-1 agonists as first-line treatment for type 2 diabetes, particularly in patients with atherosclerotic cardiovascular disease or obesity. It has also been noted that these drugs significantly reduce food intake and weight, and some are also approved for the treatment of obesity in the absence of diabetes. They have also been developed for other indications, such as non-alcoholic fatty liver disease, polycystic ovary syndrome, and reward system disorders, such as addiction. GLP-1 agonists work by activating GLP-1 receptors. They slow gastric emptying, inhibit glucagon release, and stimulate insulin production, thereby lowering high blood sugar in people with type 2 diabetes. They also reduce food intake, thus reducing weight, making them an effective therapy for obesity. Some metabolic effects of GLP-1 agonists in rodents are mediated via increased synthesis of fibroblast growth factor 21 (FGF21). Pharmaceutical companies have developed dual GLP-1 / FGF21 receptor agonists. In exemplary embodiments, the compositions disclosed herein comprise a GLP-1 / FGF21 receptor agonist.

[0050] Non-limiting GLP-1 drugs that can be incorporated into the nanoparticles described herein include exenatide (marketed as Byetta® and Bydureon), approved in 2005 / 2012. TM Liraglutide (manufactured by AstraZeneca), approved in 2010 (Victoza® for diabetes, Saxenda® for obesity, manufactured by Novo Nordisk), and abiglutide (Tanzeum) approved in 2014. TM (Manufactured by GSK), dulaglutide (Trulicity®, manufactured by Eli Lilly), approved in 2014, and liximabide (Lyxumia, Europe), approved in 2016. TM Adlyxin from the United States TMThe following drugs are approved in 2017: Smegglutide (manufactured by Sanofi), Ozempic® and Rybelsus® for diabetes, and Wegovy® for obesity (manufactured by Novo Nordisk); Telboride (TZP; a dual GLP-1 and GIP agonist; Mounjaro® for diabetes and Zepbound® for obesity, manufactured by Eli Lilly); dulaglutide (Trulicity®); exenatide (Byetta®); exenatide extended-release (Bydureon®); and Orforglipron. TM (a non-peptide oral GLP-1 receptor agonist), Efocipegtrutide TM (HM15211) (See, e.g., Abdelmalek MF, Suzuki A, Sanchez W, et al. A phase 2, adaptive randomized, double-blind, placebo-controlled, multicenter, 52-week study of HM15211 in patients with biopsy-confirmed non-alcoholic steatohepatitis - Study design and rationale of HM-TRIA-201 study. Contemp Clin Trials. 2023;130:107176) and retarglutide (RETA; LY3437943) (see, e.g., Coskun T, Urva S, Roell WC, et al. LY3437943, a noveltriple glucagon, GIP, and GLP-1 receptor agonist for glycemic control and weight loss: From discovery to clinical proof of concept. Cell Metab. 2022;34(9):1234-1247.e9; and Jastreboff AM, Kaplan LM, Frías JP, et al. Triple-Hormone-Receptor Agonist Retatrutide for Obesity - A Phase 2 Trial.N Engl J Med. 2023;389(6):514-526).

[0051] Nanoparticles In an exemplary embodiment, the GLP-1 drug is encapsulated in nanoparticles. In an exemplary embodiment, the nanoparticles are PLGA. In an exemplary embodiment, the PLGA nanoparticles are about 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, or 130 nm, preferably about 100 nm. In an exemplary embodiment, the PLGA nanoparticles are functionalized (i.e., coated) with one or more positively charged polymers. In an exemplary embodiment, PLGA nanoparticles are generated, and then a positively charged polymer is added, which coats the PLGA nanoparticles based on the electrostatic interaction between the negatively charged surface of the bare PLGA nanoparticles and the positively charged polymer. In a preferred embodiment, one or more positively charged polymers are selected from CS and CS-PEI coatings. In an exemplary embodiment, CS and / or CS-PEI coatings are used in different ratios to PLGA to obtain the desired nanoparticles. In an exemplary embodiment, a CS-PEI coating is used. In an exemplary embodiment, the ratio of coating to PLGA is 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, or 0.40, preferably 0.25. In an exemplary embodiment, the coated nanoparticles have a hydrodynamic diameter of about 110 nm, 115 nm, 120 nm, 125 nm, or 130 nm, preferably about 120.5 ± 1.2 nm. In an exemplary embodiment, the coated nanoparticles have a PdI of 0.1 ± 0.02 and a zeta potential of 49.3 ± 1.3 mV.

[0052] Generation of nanoparticles In exemplary embodiments, PLGA nanoparticles are generated by any method known in the art. In exemplary embodiments, PLGA nanoparticles are generated via an emulsion-evaporation method. This technique allows for the encapsulation of hydrophobic and hydrophilic drugs at the micron or nanoscale. In exemplary embodiments, PLGA is dissolved in an organic phase (oil), which is emulsified in an oil-immiscible phase (typically water) using a surfactant or stabilizer. The hydrophobic drug is added directly to the oil phase, while the hydrophilic drug can first be emulsified with a polymer solution before particle formation. High-intensity acoustic treatment pulses (bursts) can be used to promote the formation of small polymer droplets. The resulting emulsion is added to a larger aqueous phase and stirred for several hours to allow the solvent to evaporate. As the solvent is removed, the polymer precipitates, and the hardened nanoparticles are collected and washed by centrifugation, then lyophilized and stored long-term. In exemplary embodiments, PLGA nanoparticles are generated using an emulsion-based technique, followed by the addition of a positively charged polymer that coats the PLGA nanoparticles based on the electrostatic interaction between the negatively charged surface of the bare PLGA nanoparticles and the positively charged polymer.

[0053] In an exemplary embodiment, PLGA nanoparticles are generated via a nanoprecipitation method (see, for example, Salatin S, Barar J, Barzegar-Jalali M, Adibkia K, Kiafar F, Jelvehgari M. Development of a nanoprecipitation method for the entrapment of a very water-soluble drug into Eudragit RL nanoparticles. Res Pharm Sci. 2017; 12(1): 1–14). In this method, the polymer and drug are dissolved in an organic solvent (e.g., acetone or DMSO) and then added dropwise to water. The organic solvent is evaporated, and the particles are collected as a precipitate using centrifugation. In an exemplary embodiment, the nanoparticles are generated by rapid nanoprecipitation (FNP) (see, for example, Markwalter CE, Pagels RF, Wilson BK, Ristroph KD, Prud'homme RK. Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles. J Vis Exp. 2019;(143):10.3791 / 58757).

[0054] In an exemplary embodiment, PLGA nanoparticles are generated via microfluidic-assisted synthesis (see, for example, Hung LH, Lee AP. Microfluidic devices for the synthesis of nanoparticles and biomaterials. J Med Biol Eng. 2007; 27(1): 1–6; and Lababidi N, Sigal V, Koenneke A, Schwarzkopf K, Manz A, Schneider M. Microfluidics as tool to prepare size-tunable PLGA nanoparticles with high curcumin encapsulation for efficient mucus penetration). Beilstein J Nanotechnol . 2019; 10: 2280-2293).

[0055] Further embodiments are described in the examples below. These examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

[0056] Example Example 1 – TZP-loaded nanoparticles for intranasal delivery This invention reports TZP-loaded nanoparticles for intranasal delivery, exhibiting good anti-obesity effects. The applicant encapsulated TZP in PLGA nanoparticles and coated them with CS and CS-PEI under optimized conditions for surface charge alteration and particle size control. The resulting formulation was tested in obese mice. Consistent dose-dependent reduction in body fat was observed, demonstrating significant therapeutic effects. Figure 1 ).

[0057] The following is a brief description of the nanoparticle development. The applicant first generated PLGA nanoparticles using an emulsion-based technique. Then, to functionalize the nanoparticles with polymers, the applicant tested CS and CS-PEI coatings at different ratios to PLGA and characterized the self-assembly behavior of the resulting nanoparticles, including hydrodynamic size, polydispersity index (PdI), and zeta potential (Figure 2). Both coatings rely on the electrostatic interaction between the negatively charged surface of the bare PLGA nanoparticles and the positively charged polymer. Due to PEI grafting, CS-PEI has a higher positive charge density than CS. Therefore, the CS-PEI-coated PLGA nanoparticles not only have a higher surface charge but also form a denser coating layer, which explains why, at the same coating ratio, the CS-PEI-coated nanoparticles have a smaller size than the CS-coated nanoparticles. Considering that the size of the nanoparticles before coating was already 100 nm, minimal size increase after coating is desirable for efficient intranasal drug delivery. Therefore, the CS-PEI coating formulation with a coating-to-PLGA ratio of 0.25 was optimal. Furthermore, this formulation exhibited the lowest PdI value, indicating the most uniform and consistent nanoparticle size distribution. Thus, it was the final optimized formulation, with a hydrodynamic diameter of 120.5 ± 1.2 nm, a PdI of 0.1 ± 0.02, and a zeta potential of 49.3 ± 1.3 mV. Moreover, sustained release of TZP was demonstrated in vitro under physiologically relevant conditions at 37 °C in phosphate buffer. Release curves showed that only 50% of the loaded drug was released within the first 10 hours. At 48 hours, release reached only 60%, and then slowly climbed to 80% at 96 hours. These data demonstrate the good potential of intranasal nanocarriers for TZP, exhibiting attractive physical characteristics and controlled release behavior.

[0058] Next, the applicant investigated whether PLGA-TZP nanoparticles had an effect on weight loss. To induce obesity in C57BL / 6 mice, the applicant subjected them to a high-fat diet (HFD). After obesity induction, obese mice were administered PLGA-TZP intranasally daily at a dose of 100 nmol / kg body weight, and their weight changes were closely monitored. Over a week, PLGA-TZP resulted in a gradual decrease in body weight, approximately 2% per day. Figures 3A-3B After one week of treatment, the PLGA-TZP group showed approximately 10% weight loss compared to the mediator and TZP-free groups. Body composition analysis (MRI) showed that the weight loss was primarily due to a reduction in fat mass. Figure 3C ).

[0059] Following these findings, the applicant can investigate the metabolic benefits associated with PLGA-TZP. The applicant anticipates observing improvements in obesity-related glucose intolerance and insulin resistance with PLGA-TZP treatment.

[0060] The limitations of TZP in weight loss stem from its associated side effects. Pancreatitis, gastrointestinal adverse events, and injection site inflammation are common reports of TZP use. The controlled and constant release of PLGA-TZP is expected to alter the pharmacodynamics (PD) of TZP in vivo, thus avoiding the acute high concentrations currently observed with subcutaneous (SC) injections. To assess the potential reduction in TZP side effects by intranasal administration of PLGA-TZP compared to conventional subcutaneous (SC) injection, the applicant conducted a comparative analysis. This comparison may involve evaluating the side effects of TZP administered via intranasal versus SC injection, aiming to achieve equivalent levels of weight loss efficacy in both groups. The applicant anticipates that intranasal administration of PLGA-TZP will offer an advantage in avoiding side effects. The applicant may also assess the effects of TZP on bone and other organs.

[0061] In summary, this non-invasive intranasal administration of PLGA-TZP not only maintains the weight loss benefits and enhances metabolic health similar to SC TZP injections, but more importantly, it improves patient compliance and avoids side effects.

[0062] Example 2 – Development of GLP-1 RA Nanoparticles for Intranasal Delivery to Reduce Weight To investigate whether brain administration of GLP-1 drugs is suitable for weight control, the applicant administered Tzp to diet-induced obese (DIO) mice via daily intranasal injection, but this failed to reduce weight, indicating ineffectiveness in crossing the brain barrier to function. To overcome this limitation, the applicant encapsulated Tzp with a biodegradable and biocompatible polymer PLGA (poly(lactic-co-glycolic acid)) (an FDA-approved drug carrier), creating nanoparticles (Tzp-NP) to facilitate intranasal delivery. Figure 5A Tzp-NP was prepared using an emulsion-based technique, and the optimized formulation was characterized by a hydrodynamic diameter of 104.7 ± 2.1 nm, a polydispersity index (PdI) of 0.16 ± 0.01, and a zeta potential of -44.8 ± 0.6 mV. Figure 5B and 9A -9D). Tzp-NP also showed sustained release of Tzp, reaching 50% at 10 hours and slowly climbing to 80% at 96 hours ( Figure 5C ).

[0063] The applicant then administered 100 nmol / kg via daily intranasal injection. BW dose of Tzp-NP treatment in DIO mice ( Figure 5D These NP vectors enabled Tzp to effectively diffuse across the mucosal layer and the blood-brain barrier (BBB), resulting in a 12% weight loss over 8 days of treatment. This contrasts sharply with the diminished effects of free Tzp. Figure 5E Weight loss is primarily due to a reduction in fat mass. Figure 5F This was replicated in female DIO mice, showing a 3% weight loss within 24 hours. Figure 9E Furthermore, Tzp-NPs exhibited good stability, as they reduced the same weight after dissolving at 4 °C for up to 1 week. Figure 9F ).

[0064] Next, the applicant compared the anti-obesity efficacy of intranasal delivery versus conventional systemic delivery. In DIO mice, during a 3-day treatment, 50 nmol / kg Intranasal administration of bw doses of Tzp-NP achieved the same effect as subcutaneous injection of free Tzp (10 nmol / kg). bw) equivalent to 10% weight loss ( Figure 5G In addition, the applicant evaluated whether PLGA NP encapsulation is a universal method for intranasal delivery of GLP-1 RA. This is in contrast to subcutaneous injection of free smegglutinin (…). Figure 5H Compared to 10%, intranasal administration of sema NP for six days resulted in an 8% reduction in body weight in DIO mice, based on the same reduction in fat mass. Figure 5I Similarly, for the triple GLP-1 drug retaglutide, intranasal delivery of its NP resulted in 22% weight loss and 38.9% fat reduction within one week, with weaker efficacy than subcutaneous injection. Figure 5J-5K In summary, intranasal delivery of NP-encapsulated GLP-1 drugs has shown to be a viable therapy for treating obesity in a DIO mouse model.

[0065] Example 3 - Intranasal delivery concentrates GLP-1 drugs in the olfactory bulb and minimizes peripheral distribution. Intranasal delivery is known to primarily target the olfactory bulb, which expresses abundant GLP-1 receptors. However, it remains unclear whether GLP-1 plays a role in the olfactory bulb to influence appetite. Using fluorescently labeled Tzp, the applicant did detect a major Tzp-Cy5 signal in the olfactory bulb region within 1 hour after intranasal administration, in stark contrast to minimal brain signal observed after subcutaneous injection. Figure 6A Interestingly, using fasted mice, both delivery routes effectively suppressed appetite at 1 hour. Figures 6B-6CBrain slice imaging confirmed strong fluorescent signals in the olfactory region and cerebral cortex via intranasal delivery, while almost no signal was detected in the brains of mice that received subcutaneous injection. Figure 6D In peripheral tissues, 1 hour after administration, Tzp-Cy5 signaling was detected only in the liver via both delivery routes. Figure 6E This indicates the central mechanism by which Tzp reduces food intake.

[0066] Twenty-four hours after intranasal administration, Tzp fluorescence signals remained high in the olfactory bulb region while diffusing to other areas of the brain, whereas subcutaneous injection resulted in strong distribution in the brain, primarily the hypothalamus. Figure 6F Next, the applicant compared the pharmacokinetics of Tzp via different delivery routes. The subcutaneous route showed that Tzp rapidly entered circulation, reaching peak levels at 5 hours post-injection and remaining at high levels for 24 hours. Figure 6G This supports strong signals in peripheral organs. Figure 6H Notably, the plasma concentrations of intranasally delivered Tzp-Cy7 remained significantly low over the 24-hour period, reflecting its negligible peripheral tissue distribution. Figure 6H-6I The relatively strong signaling in the liver and kidneys after intranasal delivery is consistent with the rapid clearance characteristics of nanoparticles. Overall, intranasal administration is an effective route for delivering GLP-1 drugs to the brain and delivering appetite-suppressing functions.

[0067] Example 4 – Anti-obesity effect of long-term intranasal application of Tzp-NP The applicant will then continue to evaluate the long-term anti-obesity efficacy of intranasal delivery of Tzp-NP compared to subcutaneous administration by treating DIO mice daily for four consecutive weeks. Figure 7A Both treatments resulted in rapid weight loss during the initial two weeks, followed by near-stabilization over the next two weeks. Ultimately, intranasal delivery resulted in a 26.7% weight loss, while subcutaneous injection resulted in a 32.6% weight loss. Figure 7B The mild weight loss in the vector group was likely due to stress or solitary confinement during treatment. Recognizing that the primary mechanism of weight loss with GLP-1 drugs involves reduced food intake, the applicant continued to compare food consumption after administration of Tzp via intranasal or subcutaneous routes. Compared to the vector group, food intake was reduced by 70% and 80% in the intranasal and subcutaneous groups, respectively, and there was no significant difference between the two treatment groups. Figure 7C This forms the basis for their weight loss. While both treatments effectively reduce fat mass, subcutaneous injections are more effective at reducing both fat mass and lean body mass. Figures 7D-7E Four weeks later, the resulting lean phenotype was supported by a significant reduction in fat content. Figure 7F-7GIntranasal treatment resulted in a 50% reduction in visceral fat eWAT and subcutaneous iWAT indistinguishably, compared to a 60% reduction achieved with subcutaneous injection. Figure 7H Liver weight was also reduced in both Tzp treatment groups, while spleen size was unaffected. Tzp treatment reduced adipocyte hypertrophy in both eWAT and iWAT groups. Figure 7I-7L Adipocyte hypertrophy is often an indicator of harmful fat function in obesity. Consistent with the lean phenotype, the gene Dgat2, encoding a key triglyceride synthase, is downregulated in eWAT by Tzp treatment, while most adipocyte genes are normally expressed. Figure 7H Obesity is often associated with chronic inflammation in adipose tissue, and regardless of the delivery route, Tzp treatment downregulates inflammatory markers. Mcp1 and Il6 The expression of [unclear] indicates a reduction in fat inflammation. In summary, these compelling results suggest that intranasal administration is an effective alternative for Tzp delivery in obesity treatment.

[0068] Example 5 - Intranasal delivery of Tzp NP achieved considerable metabolic improvement In addition to weight control, GLP-1 drugs have also shown remarkable efficacy in treating diabetes and metabolic liver disease, both major comorbidities of obesity. Intranasal delivery of Tzp NP has shown improvements in glucose tolerance and insulin sensitivity as strong as subcutaneous injection. Figures 8A-8B Consistent with enhanced insulin sensitivity, long-term Tzp treatment decreased plasma insulin levels. Figure 8C Intranasal treatment only slightly reduces plasma triglyceride (TG) and non-esterified fatty acid (NEFA) levels. Figure 8D-8E Consistent with the reduction in liver weight (). Figure 7H Both delivery routes improved hepatic steatosis. Figure 8F Accompanied by a decreasing trend in plasma ALT levels (a marker of liver injury) Figure 8G ). and gluconeogenesis ( Foxo1 , G6pc and Fbp1 ) and fat production ( Srebf1 , Fasn , Scd1 , Elov6 and Dgat2 The overall downregulation of related genes is the basis for improved liver metabolic function. Figure 8H ).

[0069] GLP-1 RA effectively lowers blood glucose levels by stimulating insulin release through its action in the pancreas. The applicant then compared the acute effects of Tzp via different delivery routes. Due to the stimulation of insulin release ( Figure 8JIntranasal delivery of Tzp NP rapidly lowers blood glucose levels within 1 hour, which is the same as subcutaneous injection. Figure 8I Interestingly, although Tzp NP requires a higher dose to achieve a weight loss comparable to that achieved with subcutaneous injection (50 nmol / kg), bw comparison 10 nmol / kg bw), but Tzp NP is more effective in lowering blood sugar levels ( Figure 8K During this period, Tzp primarily reaches the olfactory bulb rather than the pancreas. Figure 6A and 6E This suggests that sympathetic pathways directed in the olfactory bulb stimulate insulin release.

[0070] Example 6 – Discussion Metabolic fluctuations are a fundamental characteristic of living organisms and a crucial pathway to achieving metabolic homeostasis. However, a single high-dose long-acting GLP-1 drug may flatten these fluctuations, despite its advantages in patient compliance. In contrast, intranasal delivery of short-term activated GLP-1 may better mimic the fluctuations in GLP-1 production and function under physiological conditions.

[0071] Intranasal drug delivery has emerged as a promising approach for treating a variety of conditions, particularly those targeting the central nervous system. In particular, for peptide drugs, oral administration is often impractical due to their rapid inactivation in the digestive system, leading to the common use of subcutaneous or intravenous administration. However, these routes present challenges such as inconvenience, pain, and the risk of infection, impacting patient adherence. In contrast, intranasal delivery offers a viable alternative. For example, it enables rapid absorption, as demonstrated by glucagon nasal sprays used for emergency hypoglycemia management. Furthermore, intranasal delivery allows direct access to the brain, bypassing the bloodstream, making it a widely used method for treating neurological disorders. Approved drugs such as esketamine and desmopressin exemplify this, as they target the brain via intranasal administration. In addition, intranasal delivery has been explored in trials involving GLP-1 drugs, including Exendin-4 and liraglutide, for the treatment of diabetes. However, the potential use of intranasal GLP-1 receptor agonists in the management of obesity remains uncertain. In this study, the applicant found that intranasal administration of GLP-1 drugs (including but not limited to smegglutide, telpogglutide, and retaglutide) showed similar efficacy to subcutaneous delivery in the treatment of obesity and related metabolic diseases, particularly fatty liver disease.

[0072] In this study, although intranasal and subcutaneous administration achieved considerable weight loss and food intake suppression (particularly evident during the initial treatment days), there were differences in distribution across different brain regions. This inconsistency not only indicates different pharmacokinetics of TZP via different pathways but also suggests the involvement of different neuronal circuits in the brain driven by GLP-1. The olfactory bulb is known to be involved in appetite regulation (Stark R. The olfactory bulb: A neuroendocrine spotlight on feeding and metabolism). J Neuroendocrinol . 2024;36(6):e13382) also includes proglucagon-1 cells that locally release GLP-1 (Thiebaud N, Gribble F, Reimann F, Trapp S, Fadool DA. A unique olfactory bulb microcircuit driven by neurons expressing the precursor to glucagon-like peptide 1. Sci Rep .2019;9(1):15542;Montaner M, Denom J, Jiang W, Magnan C, Trapp S, Gurden H. The local GLP-1 system in the olfactory bulb is required for odor-evoked cephalicphase of insulin release in mice. Mol Metab .2023;73:101738). Therefore, the high signal of TZP observed in the olfactory bulb after intranasal delivery can directly target the hypothalamus and brainstem via cerebrospinal fluid circulation (Born J, Lange T, Kern W, McGregor GP, Bickel U, Fehm HL. Sniffing neuropeptides: a transnasal approach to the human brain). Nat Neurosci2002;5(6):514-516). Whether there exists a cerebrospinal fluid-independent neuronal circuit from the olfactory bulb to the hypothalamus to regulate food intake remains unknown, but it is a very interesting possibility. The lower signal observed in the hypothalamus of the intranasal group not only suggests that the brain may not require high concentrations of GLP-1 to be effective, but also suggests the existence of other neuronal circuits controlling food intake. Subcutaneous injection of telposide results in persistently high plasma and organ levels, even up to 24 hours after injection. This dual nature of high plasma telposide levels presents both advantages and disadvantages. While high concentrations contribute to rapid and effective weight loss, prolonged exposure may lead to decreased sensitivity to GLP-1 drugs over time, requiring higher doses to achieve long-term efficacy in humans. Furthermore, the potential side effects of elevated telposide concentrations in non-brain organs must be considered. Direct delivery to the brain via intranasal administration is expected to avoid systemic side effects. This study showed that intranasal delivery resulted in better bone health and lower pancreatic concentrations 24 hours after injection compared to subcutaneous administration (which has been reported to cause acute pancreatitis in humans). However, it is important to note that mice may not be able to fully replicate the gastrointestinal side effects observed in human patients following GLP-1 drug administration; therefore, the applicant did not observe any differences in gastric histology or inflammatory marker gene expression (data not included). Further research is needed to evaluate the benefits of intranasal delivery in mitigating side effects, possibly using primate models for more accurate assessment.

[0073] While the nasal route, as discussed above, offers advantages, disadvantages such as nasal mucosal irritation and low bioavailability must also be considered. In this study, although the mediator group experienced some weight loss after prolonged PBS treatment, the nasal cavity exhibited normal morphology. This finding rules out the possibility that nasal irritation is a side effect of long-term use. In this study, the bioavailability of intranasal GLP1 drugs was approximately 20% compared to subcutaneous administration, consistent with previous reports (see, for example, patent EP1696960B1). However, this low bioavailability may increase translational costs in the clinical setting. Strategies to improve nasal absorption can mitigate this problem. For example, combining nanoparticles with absorption enhancers such as cell-penetrating peptides and tight junction modulators can improve bioavailability delivered intranasally. The presence of mucins in nasal mucus, containing high levels of sialic acid and sulfate, imparts a strong net negative charge to the mucus surface. The applicant found that coating PLGA-TZP with the positively charged material chitosan significantly improved delivery efficiency. Furthermore, further research is needed to explore methods to improve the efficiency of TZP's direct entry into the brain while minimizing systemic exposure to reduce side effects. Additionally, prolonging the release of nanoparticles into the brain could extend therapeutic efficacy, reduce treatment frequency, and improve patients' quality of life. These efforts provide a meaningful pathway for future research into optimizing intranasal drug delivery in obesity management.

[0074] In summary, the current research provides an alternative, painless, easy-to-self-administer, and non-invasive method for delivering GLP-1 drugs to address obesity and related metabolic diseases.

[0075] Example 7 – Method Preparation of GLP-1 drug nanomedicine. TZP was encapsulated in PLGA nanoparticles prepared via a double emulsion method, as described in previous literature (Saffarionpour S, One-step preparation of double emulsions stabilized with amphiphilic and stimuli-responsive block copolymers and nanoparticles for nutraceuticals and drug delivery. JCIS Open. 2021; Volume 3, 100020). TZP stock solutions were prepared by dissolving TZP (Peptide Sciences, CA) in ddH2O and stored on ice or at 4°C. The PLGA polymer (Resomer RG 504 H, Sigma-Aldrich, MA) was dissolved in chloroform, and each batch was prepared fresh for use. Sodium dodecyl sulfate (SDS, FisherBioReagents, PA) was used as a surfactant to stabilize the droplets during emulsification. The SDS solution was prepared in dd water. The double emulsion method was carried out in two steps. First, a Tzp stock solution (aqueous solution, 10 mg / ml) and a PLGA solution (organic solution, 10 mg / ml) were mixed at a volume ratio of 1:4 and sonicated using a 20 kHz ultrasonic processor to generate water droplets in the organic phase, which appeared opaque to the naked eye. This step is also known as the water-in-oil emulsion step. Immediately after the first step, an SDS solution was added to the aforementioned mixture at a volume four times that of the PLGA solution. The new mixture was then sonicated, also known as a water-in-oil-in-water emulsion. The organic solvent was removed by rotary evaporation. Subsequently, the nanoparticles were concentrated using an Amicon ultracentrifuge with a Mwco 10 kDa (MilliporeSigma, Darmstadt, Germany). SDS was removed by washing three times with dd water. The resulting nanoparticles were lyophilized and stored at -20 °C for later use.

[0076] To encapsulate the Tzp-loaded NPs with another polymer, a technique called rapid nanocomposite (FNC) was used (Hu H, Yang C, Li M, Shao D, Mao HQ, Leong KW. Flash Technology-Based Self-Assembly in Nanoformation: From Fabrication to Biomedical Applications). Mater Today (Kidlington) (2021;42:99-116). In short, because Tzp NPs have a negative surface charge, cationic polymers can coat these nanoparticles via electrostatic interactions. Nanoparticles and a polymer solution (aqueous solution) are injected through two separate channels via FNC, but are vigorously mixed together in a confined chamber. As a result, a homogeneous coating is generated. The ratio and concentration are variable and depend on the target surface charge.

[0077] Characterization of nanoparticles. The morphology of NPs was characterized using a FEI Talos transmission electron microscope (Thermo Fisher, MA). The hydrodynamic diameter and zeta potential of NPs in water or PBS were characterized using a Nano-ZS 90 Nanosizer (Malvern Instruments Ltd., Worcestershire, UK).

[0078] For drug release assays, lyophilized TZP-loaded nanoparticles were dispersed in PBS and gently shaken at 100 rpm at 37 °C. The amount of peptides released from the supernatant was measured at predetermined time points (0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 24, 48, 72, and 96 hours). The amount of peptides was quantified using a Pierce BCA protein assay kit (Thermo Scientific, MA).

[0079] Mice were administered the following treatments. Mice used in this study were bred from the C57BL / 6J strain and housed in the Columbia University animal facility. They were kept in a controlled environment with a temperature maintained at 23 ± 1 °C and subjected to a 12-hour light-dark cycle. Mice had free access to standard chow food (PicoLab Rodent 5053) and water. For experiments involving a high-fat diet (HFD), a diet consisting of 60% fat was obtained from Research Diets (D12492i). Obesity was induced in 25-week-old male mice by administering a high-fat diet (HFD) for six consecutive weeks. For short-term studies, diet-induced obese (DIO) mice were treated daily with subcutaneous injections of semaglutide, telpolide, and retaliglutide, or intranasal injections of their PLGA nanoparticle formulations for 6, 3, and 7 days, respectively. The control group received intranasal water. Food intake was measured during the first three days after telpolide treatment. For long-term telpolide studies, metabolic analyses, such as ITT and GTT measurements, were initiated two weeks after body weight stabilized. Body weight was tracked daily, and body composition was assessed weekly using EchoMRI. Four weeks after the initial injection, mice were euthanized under free-feeding conditions. For drug administration, mice were anesthetized with isoflurane and maintained at the surgical level of anesthesia. Following anesthesia induction, mice received subcutaneous or intranasal injections of the test drug. For intranasal administration, a 5 μL solution of the drug in water was delivered to each mouse. The subcutaneous dose was 10 nmol / kg, and the intranasal dose was 50 nmol / kg.

[0080] Metabolic Characterization. For the glucose tolerance test (GTT), mice were fasted for 16 hours in clean cages with fresh bedding, followed by an intraperitoneal (ip) injection of glucose at 2 g / kg body weight. Blood glucose levels were measured using a Breeze 2 (Bayer) blood glucose meter at 0, 15, 30, 60, 90, and 120 minutes post-injection, via tail vein sampling. For the insulin tolerance test (ITT), insulin was administered ip to mice fasted for 4 hours at a dose of 0.75 U / kg body weight. Blood glucose levels were measured at 0, 15, 30, 45, and 60 minutes post-injection. To measure food intake, mice were housed individually. Their high-fat diet was placed in stainless steel containers, and they were allowed unrestricted access to food and water. The food containers were weighed daily to track consumption.

[0081] Serum NEFA (Fujifilm Wako), TG (ThermoScientific), insulin (Mercodia Insulin ELISA), and alanine aminotransferase (ALT, TecoDiagnostics A526120) levels were quantified according to the respective manufacturers' instructions.

[0082] In vivo tissue distribution of dye-labeled telpolide. Telpolide was conjugated with Cy5 or Cy7 and encapsulated in PLGA nanoparticles. Mice received these labeled compounds subcutaneously or intranasally at doses of 10 nmol / kg bw or 50 nmol / kg bw, respectively. In vivo imaging was performed at 1, 2, 3, and 24 hours post-injection using the PerkinElmer IVIS system. After euthanasia, tissue signals were quantified at 3 and 24 hours post-injection using the same system. Blood was collected from the tail vein at 1, 2, 3, 5, 8, and 24 hours post-injection, and the Cy5 fluorescence signal in plasma was analyzed using a SpectraMax M2 plate reader (MolecularDevices, CA).

[0083] Bone preparation and analysis. Femurs and tibias were collected and fixed overnight at 4 °C in 10% neutral buffered formalin, followed by analysis of bone microstructure and lipid content. Microstructure was scanned using a Quantum FX μCT scanner. Bone mineral density (BMD) was measured at consistent 60-slice intervals, starting from the growth plate of the femur or tibia. For lipid analysis, bone was decalcified in 14% EDTA at 4 °C for at least 2 weeks, with the solution changed every 3 to 4 days. Bone was then stained with 1% osmium tetroxide and 2.5% potassium dichromate for 48 hours and further imaged by μCT. Bone marrow fat sections were identified at consistent 250-slice intervals, starting from the growth plate of the femur or tibia. Lipid volume was quantified using Analyze 12 software, while bone mineral density was determined using Analyze 14.

[0084] Gene expression. RNA was extracted from tissues using the Tri-Isolate RNA pure kit (IBIScientific) according to the manufacturer's instructions. Subsequently, 1,000 ng of RNA was reverse transcribed to produce cDNA using a high-capacity cDNA reverse transcription kit (AppliedBiosystems). Quantitative real-time PCR (qPCR) was then performed using the Bio-Rad CFX96 real-time PCR system and GoTaq qPCR Master Mix (Promega). Relative gene expression was assessed using the ΔΔCt method, where… Rpl23 or CpaUsed as a reference gene. Primer sequences are available upon request.

[0085] Histology. Following dissection, epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT), and liver were collected and immediately fixed in 10% formalin solution. The tissues were then embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) in preparation for subsequent microscopic examination. For brain immunohistochemistry, mice were first perfused with PBS, which was then converted to 10% buffered formalin for brain fixation. cFOS antibody was used. Following established protocols, gastric sections were immunohistochemically stained with primary antibodies against Atp4b (Abcam) and Ki67 (Cell Signaling Technology).

[0086] For histological analysis of the nasal cavity, the following procedure was used. After euthanasia, the mouse head was collected and carefully skinned. The mandible was then discarded. The remaining head tissue was fixed in 10% buffered formalin solution for 48 hours. After fixation, decalcification was performed using 10% EDTA solution for 20 consecutive days, with the solution changed every 3–4 days. The decalcified tissue was then processed for paraffin embedding, sectioning, and H&E staining for histological evaluation of the nasal cavity.

[0087] Statistical analysis. Two-way ANOVA was used to assess the significance of differences among the groups of mice. Paired t-tests were used to assess the significance of changes in body weight before and after treatment. A p-value less than 0.05 was considered statistically significant. Data in this study are presented as mean ± standard error of mean (sem). Prism 9.3.1 (GraphPad) was used for analysis.

[0088] Various modifications and alterations to the methods, pharmaceutical compositions, and kits described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in conjunction with specific embodiments, it should be understood that the invention can be further modified, and the claimed invention should not be unduly limited to these specific embodiments. In fact, various modifications to the described methods of carrying out the invention that are apparent to those skilled in the art are contemplated within the scope of the invention. This application is intended to cover any variations, uses, or adaptations of the invention that generally follow the principles of the invention, and includes such deviations from this disclosure that fall within known conventions in the field and may be applied to the essential features set forth herein.

Claims

1. A method of delivering a GLP-1 drug to a patient, the method comprising intranasal delivery of a pharmaceutical composition comprising the GLP-1 drug.

2. The method according to claim 1, wherein the GLP-1 drug is selected from telpolide, smegglutide, and retaloglutide.

3. The method according to any one of claims 1 and 2, wherein the GLP-1 drug in the pharmaceutical composition is sufficient to treat one or more of the following: obesity, obesity comorbidity, or any other condition causing hyperglycemia or fatty liver or related conditions.

4. The method according to any one of claims 1-3, wherein the intranasal delivery comprises a nanoparticle delivery system.

5. The method of claim 4, wherein the nanoparticle delivery system encapsulates the GLP-1 drug.

6. The method of claim 4 or 5, wherein the nanoparticle delivery system comprises polymer nanoparticles, the polymer nanoparticles comprising poly(lactic-co-glycolic acid) (PLGA) nanoparticles coated with at least one positively charged biocompatible polymer.

7. The method according to claim 6, wherein the positively charged biocompatible polymer is selected from chitosan (CS) and polyethyleneimine-grafted chitosan (CS-PEI).

8. The method of claim 6 or 7, wherein the nanoparticle delivery system comprises polymer nanoparticles, the polymer nanoparticles comprising poly(lactic-co-glycolic acid) nanoparticles coated with chitosan (CS-PEI) grafted with polyethyleneimine.

9. The method according to any one of the preceding claims, wherein the patient is receiving treatment for obesity, obesity comorbidity, or any other condition causing hyperglycemia or fatty liver or related conditions.

10. The method according to any one of the preceding claims, wherein the GLP-1 drug is delivered to the olfactory bulb.

11. The method according to any one of the preceding claims, wherein the GLP-1 drug is administered at about 50 nmol / kg body weight.

12. The method according to any one of claims 6-11, wherein the ratio of coating to PLGA is 0.

25.

13. The method according to any one of claims 6-12, wherein the coated nanoparticles have a polydispersity index (PdI) of 0.1 ± 0.

02.

14. The method according to any one of claims 6-13, wherein the coated nanoparticles have a ζ potential of 49.3 ± 1.3 mV.

Images