Compositions containing octadecaneuropeptides (ODNs) and their synthetic derivatives, and methods of use for the regulation of food intake, obesity, body weight, nausea, and vomiting

Abstract

Compositions and methods for the management and treatment of obesity, metabolic disorders, nausea and vomiting using ODN peptides and their synthetic derivatives are disclosed.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 384,306, filed November 18, 2022, the entire contents of which are incorporated herein by reference as if set forth in full.

[0002] Incorporation by Reference of Materials Submitted in Electronic Format The contents of the electronic sequence listing (UPNK-114PCT.xml; size: 35,858 bytes; and creation date: November 20, 2023) are incorporated herein by reference in their entirety.

[0003] The present invention relates to the field of neuroactive compounds and their methods of use for the management and treatment of food intake, obesity, nausea and vomiting. More specifically, certain endozepines, such as octadecaneuropeptides (ODNs) and their precursor diazepam-binding inhibitor (DBI) peptides, and their derivatives, are provided, which act in the central nervous system to not only control food intake but also protect neurons and astrocytes from programmed cell death by reducing inflammation, apoptosis, and oxidative stress. [Background technology]

[0004] Several publications and patent documents are cited throughout this specification in order to describe the state of the art to which this invention pertains, all of which are incorporated herein by reference.

[0005] Obesity affects more than 40% of the US population. The excess fat in obesity was originally thought to be harmless (benign). However, evidence suggests that excess fat can cause chemical changes in the blood, increasing the risk of heart disease. When fat cells enlarge, they secrete hormones that trigger chronic inflammation. Inflammation interferes with the efficient use of insulin, leading to insulin resistance. Insulin resistance can lead to poor blood sugar regulation, resulting in metabolic syndrome. This condition is associated with several risk factors that increase the likelihood of developing heart disease: high blood lipids (high LDL cholesterol, high total cholesterol, high triglycerides), high blood pressure (hypertension), high blood sugar (hyperglycemia), and low HDL cholesterol. Obesity increases other heart disease risk factors, such as sleep disorders and type 2 diabetes.

[0006] Overweight and obesity are defined by the World Health Organization (WHO) as abnormal or excessive fat accumulation that poses a risk to health. Obesity is measured by the body mass index (BMI), which is weight in kilograms divided by height in meters squared. People with a BMI of 30 or higher are generally considered obese. People with a BMI of 25 or higher are considered overweight. Losing 5% to 10% of your body weight can reduce risk factors for heart disease, and small lifestyle changes can improve metabolic syndrome and reduce your risk of heart disease.

[0007] Lifestyle interventions related to diet and physical activity are the first line of treatment for managing obesity and overweight, but their effectiveness is limited, and weight regain is common. Bariatric surgery is highly effective for weight loss in severely obese and high-risk patients, but its use is limited by its invasive nature, cost, and risk of perioperative adverse events, including perioperative mortality. While several medications have proven effective in weight loss, pharmacotherapy for obesity treatment is limited by barriers to treatment, including the slow weight loss induced by most medications, the development of dependence, the side effect profile of some medications, contraindications, poor compliance, and underprescription. Summary of the Invention [Problem to be solved by the invention]

[0008] Clearly, improved, non-toxic medications for the treatment of obesity are needed to enable sustainable and manageable food intake and reduce the adverse health effects associated with such medications, such as nausea and vomiting. [Means for solving the problem]

[0009] In accordance with the present invention, there is provided an isolated and / or purified octadecaneuropeptide (ODN) or a synthetic derivative thereof, comprising at least one amino acid sequence selected from SEQ ID NOs: 1 to 21, or a functional fragment thereof, or a sequence having at least 95% identity thereto, which effectively regulates food intake and body weight. In a preferred embodiment, the octadecaneuropeptide suppresses vomiting and nausea.

[0010] In other embodiments, the octadecaneuropeptide comprises one or more modified amino acids selected from those listed in Table 2 and / or one or more of Gln (alkyne), Ala (alkyne), Gly (alkyne), and Lys (N3). Certain modified amino acids are suitable for bioconjugation of desired moieties, such as lipids, cell-penetrating peptides, cell-targeting peptides, e.g., transferrin, Fc peptides, reporter molecules, e.g., fluorescent tags, etc. In a preferred embodiment, the octadecaneuropeptide is SEQ ID NO: 3, or a synthetic derivative of the TDN of SEQ ID NO: 3, or a functionally modified variant thereof.

[0011] In another aspect, a composition is provided comprising any one of the above octadecaneuropeptides in a pharmaceutically acceptable carrier. In another aspect, an isolated nucleic acid encoding the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 21 is also disclosed. In another aspect, the isolated nucleic acid is present in a vector for robust expression and production in an organism of interest.

[0012] In yet another embodiment, a nucleic acid sequence encoding any one of the octadecaneuropeptides having amino acid sequences SEQ ID NO:1 to SEQ ID NO:21 is disclosed. In another embodiment, the nucleic acid encodes the polypeptide of SEQ ID NO:3. The nucleic acids of the present invention may be contained in a vector and / or encapsulated in a liposome or extracellular vesicle, or attached to a nanoparticle or lipid nanoparticle to facilitate in vivo delivery. Examples of vectors include, but are not limited to, plasmid vectors, lentiviral vectors, AAV vectors, AAV9, and AAV8 viral vectors. Also disclosed are compositions comprising a nucleic acid encoding the above-described octadecaneuropeptides in a pharmaceutically acceptable carrier or buffer.

[0013] In a preferred embodiment, a method for treating a metabolic disease or disorder in a subject in need thereof is provided, comprising administering an effective amount of a composition comprising any one of the above-described octadecaneuropeptides in a pharmaceutically acceptable carrier. In one approach, the composition is effective in inhibiting the relaxin-3 receptor, which causes anorexia. In another approach, the composition functions to regulate glucose tolerance, counterregulatory responses, and one or more of 5-TG-induced hyperglycemia, insulin sensitivity, and weight loss. In another approach, the composition is capable of crossing the blood-brain barrier and penetrating the brain. Routes of administration can include systemic administration, parenteral administration, transdermal patch administration, intramuscular administration, subcutaneous administration, intracerebral administration, oral administration, buccal administration, and the like. A skilled clinician will recognize the many possible routes of administration. Metabolic diseases or disorders include, but are not limited to, obesity, diabetes, dyslipidemia, insulin resistance, hepatic steatosis, hypercholesterolemia, and nonalcoholic fatty liver disease. In certain embodiments, the method may also include administering a second therapeutic agent that treats or inhibits the progression of at least one disorder selected from obesity, diabetes, dyslipidemia, insulin resistance, hepatic steatosis, hypercholesterolemia, and non-alcoholic fatty liver disease.In a preferred embodiment, the patient's weight is reduced after administering the peptide.The method may also include evaluating the patient for the alleviation of symptoms associated with at least one disorder selected from obesity, diabetes, dyslipidemia, insulin resistance, hepatic steatosis, hypercholesterolemia, and non-alcoholic fatty liver disease. [Brief explanation of the drawings]

[0014] [Figure 1] Centrally administered ODN dose-dependently suppressed food intake in standard chow and HFD-fed rats. The effects of fourth ventricle ODN administration (0.2, 2, 20 μg / 2 μL in aCSF) on 24-hour food intake (grams; Figure 1A, Figure 1C) and body weight change (Figure 1B, Figure 1D) in standard chow-fed and HFD-fed rats. All data are mean ± SEM. [Figure 2]Eating pattern data after central ODN administration in standard-diet rats. Effects of fourth ventricle ODN administration (0.2, 2, or 20 μg / 2 μL in aCSF) on food intake (grams; Figures 2A-2D), meal number (Figures 2E-2H), time spent eating (seconds; Figures 2I-2L), meal length (seconds / meal; Figures 2M-2P), and meal size (grams / meal; Figures 2Q-2T) at time intervals of 1, 6, 6-12, and 12-24 hours after administration. All data are means ± SEM. [Figure 3] Heatmap representation of feeding behavior (bout) and feeding parameters 3 hours after central ODN administration in standard-fed rats. Effects of fourth ventricle ODN (0.2, 2, or 20 μg / 2 μL in aCSF) on food intake (grams; Figure 3A), number of feeding attempts (Figure 3B), time spent eating (seconds; Figure 3C), number of meals (Figure 3D), and time spent eating (Figure 3E). *Indicates difference from Veh (P=0.05). All data are mean ± SEM. [Figure 4] Eating pattern data after central ODN administration in HFD-fed rats. Effects of fourth ventricle ODN administration (0.2, 2, or 20 μg / 2 μL in aCSF) on food intake (grams; Figures 4A-4D), meal number (Figures 4E-4H), time spent eating (seconds; Figures 4I-4L), meal length (seconds / meal; Figures 4M-4P), and meal size (grams / meal; Figures 4Q-4T) at time intervals of 1, 6, 6-12, and 12-24 hours post-administration. All data are means ± SEM. [Figure 5] Heatmap representation of feeding behavior and dietary parameters in HFD-fed rats 24 hours after central ODN administration. Effects of fourth ventricle ODN (0.2, 2, or 20 μg / 2 μL in aCSF) on food intake (grams; Figure 5A), number of feeding behaviors (Figure 5B), time spent eating behaviors (seconds; Figure 5C), number of meals (Figure 5D), and time spent eating (Figure 5E). *Indicates difference from Veh (P=0.05). All data are mean ± SEM. [Figure 6]First feeding event data after central ODN administration in standard diet- and HFD-fed rats. Effects of fourth ventricle ODN (0.2, 2, 20 μg / 2 μL in aCSF) on latency to first meal (seconds; Figure 6A, Figure 6E), first meal size (grams; Figure 6B, Figure 6F), latency to first eating behavior (seconds; Figure 6C, Figure 6G), and first eating behavior size (grams; Figure 6D, Figure 6H) in standard diet- and HFD-fed rats. All data are mean ± SEM. [Figure 7] Effects of recombinant DBI protein administered into the fourth ventricle of the hindbrain on the dietary pattern and food intake of standard diet and HFD rats. Centrally administered recombinant DBI protein suppresses food intake in rats fed a standard diet. Effects of recombinant DBI protein (20 μg / 2 μL) administered into the fourth ventricle on 24-hour food intake in rats fed a standard diet (Figure 7A) and HFD (Figure 7B) and on body weight change in standard diet and HFD rats (Figure 7C). All data are mean ± SEM. [Figure 8] Heatmap representation of feeding behavior and dietary parameters 24 hours after central administration of recombinant DBI protein in standard chow-fed rats. Effects of fourth ventricle recDBI (20 μg / 2 μL in aCSF) on food intake (grams; Figure 8A), number of feeding behaviors (Figure 8B), time spent eating behaviors (seconds; Figure 8C), number of meals (Figure 8D), and time spent eating (Figure 8E). *Indicates difference from Veh (P=0.05). All data are mean ± SEM. [Figure 9] Pretreatment with antibodies against DBI suppressed centrally administered exendin-4-induced anorexia in standard diet-fed rats. Rats were pretreated in the fourth ventricle with DBI antibody (AB 3 μg / 3 μL) or vehicle, followed by treatment in the fourth ventricle with ODN (20 μg / 2 μL), Ex-4 (0.3 μg / 2 μL), or vehicle. Figures 9A and 9B show 24-h food intake in standard diet (Figure 9A) and HFD (Figure 9B)-fed rats, kaolin intake in standard diet (Figure 9C) and HFD (Figure 9D)-fed rats, and post-treatment body weight change in standard diet (Figure 9E) and HFD (Figure 9F)-fed rats. # indicates difference from Veh / Veh (P=0.05). All data are mean ± SEM. [Figure 10]Pretreatment with an ODN antagonist suppressed the anorexia induced by centrally administered exendin-4. Rats were pretreated in the fourth ventricle with an ODN antagonist (AntOP 200 μg / 2 μL) or vehicle, followed by treatment in the fourth ventricle with ODN (20 μg / 2 μL), Ex-4 (0.3 μg / 2 μL), or vehicle. Figures 10A and 10B show 24-hour food intake in rats fed a standard diet (Figure 10A) and an HFD (Figure 10B), kaolin intake in rats fed a standard diet (Figure 10C) and an HFD (Figure 10D), and body weight change in rats fed a standard diet (Figure 10E) and an HFD (Figure 10F) after treatment. # indicates difference from Veh / Veh (P=0.05). All data are mean ± SEM. [Figure 11] Pretreatment with an ODN antagonist attenuates peripheral liraglutide-induced appetite suppression. Rats were pretreated in the lateral ventricle with an ODN antagonist (AntOP 100 μg / 2 μL) or vehicle, followed by intraperitoneal administration of liraglutide (50 μg / kg) or vehicle. 48-hour food intake in standard chow (Figure 11A) and HFD (Figure 11B)-fed rats, kaolin intake in standard chow (Figure 11C) and HFD (Figure 11D)-fed rats, and body weight change in standard chow (Figure 11E) and HFD (Figure 11F)-fed rats after treatment. All data are mean ± SEM. [Figure 12] Coadministration of ODN for 5 days potentiates peripheral liraglutide-induced anorexia. Rats were administered ODN (100 μg / 2 μL) or vehicle into the fourth ventricle, followed by intraperitoneal administration of liraglutide (25 μg / kg) or vehicle for 5 days. Daily (FIG. 12A) and cumulative (FIG. 12B) food intake, daily (FIG. 12C) and cumulative (FIG. 12D) kaolin intake, and daily body weight (FIG. 12E) in HFD-fed rats after treatment. Food intake on day 1 (FIG. 12F). All data are mean ± SEM. [Figure 13]Central administration of tridecaneuropeptide (TDN), a cleavage product of ODN, suppresses 24-hour food intake in HFD-fed rats. Effects of TDN (20 μg / 2 μL) administered into the fourth ventricle on 24-hour food intake in standard chow (Figure 13A) and HFD-fed (Figure 13B)-fed rats, kaolin intake in standard chow (Figure 13C) and HFD-fed (Figure 13D)-fed rats, and body weight change in standard chow (Figure 13E) and HFD-fed (Figure 13F)-fed rats after treatment. All data are mean ± SEM. [Figure 14] Centrally administered TDN suppresses 24-hour food intake in rats. Effects of lateral ventricle TDN (20 μg / 2 μL or 200 μg / 2 μL) on 24-hour food intake in standard diet (FIG. 14A) and HFD (FIG. 14B)-fed rats, kaolin intake in standard diet (FIG. 14C) and HFD (FIG. 14D)-fed rats, and body weight change in standard diet (FIG. 14E) and HFD (FIG. 14F)-fed rats after treatment. All data are mean ± SEM. [Figure 15] Peripheral ODN suppresses 24-hour food intake in shrews without vomiting. Effects of intraperitoneal administration of ODN (500 μg / kg or 5000 μg / kg) on ​​24-hour food intake (Figure 15A), body weight change (Figure 15B), and vomiting episodes (Figure 15C). All data are means ± SEM. [Figure 16] Centrally administered ODN-based drugs suppress 24-hour food intake in HFD-fed rats. Effects on 24-hour food intake (Figure 16A) and body weight change (Figure 16B) after treatment with fourth ventricle ODN, TDN, SUODN04, and SUODN05 (20 μg / kg). All data are mean ± SEM. [Figure 17]Nutritional status and GLP-1R agonism regulate NTS / AP DBI protein expression. Rats were either fed ad libitum or fasted for 24 h and then injected with vehicle exendin-4 (0.3 μg / 2 μL) into the fourth ventricle 90 min before sacrifice. Brains were harvested and stained with DBI antibody to detect DBI protein expression, and its fluorescence was quantified at low, moderate, and strong levels using HALO-AI software. DBI protein expression was measured in the NTS at the pre-AP level in standard diet (Figure 17A) and HFD (Figure 17B)-fed rats, in the NTS at the AP level in standard diet (Figure 17C) and HFD (Figure 17D)-fed rats, in the NTS at the fourth ventricle level in standard diet (Figure 17E) and HFD (Figure 17F)-fed rats, and in the AP in standard diet (Figure 17G) and HFD (Figure 17H)-fed rats. [Figure 18] Centrally administered ODN suppresses food intake more strongly in females than in males fed a standard diet. Effects of lateral ventricle ODN (20, 100, and 200 μg / 2 μL) on 24-hour food intake (FIG. 18A) and body weight (FIG. 18B) in female standard-diet rats and on 24-hour food intake (FIG. 18C) and body weight (FIG. 18D) in male standard-diet rats. All data are mean ± SEM. [Figure 19] Centrally administered ODN suppresses food intake more potently in overnight-fasted females than in males fed a standard diet. Effects of lateral ventricle ODN (20 and 100 μg / 2 μL) on 24-hour food intake (FIG. 19A) and body weight (FIG. 19B) in overnight-fasted female rats fed a standard diet, and on 24-hour food intake (FIG. 19C) and body weight (FIG. 19D) in overnight-fasted male rats fed a standard diet. All data are mean ± SEM. [Figure 20] Centrally administered ODN suppressed food intake more strongly in HFD-fed females than in males. Effects of lateral ventricle ODN (20, 100, and 200 μg / 2 μL) on 24-hour food intake (Figure 20A) and body weight (Figure 20B) in female HFD-fed rats, and on 24-hour food intake (Figure 20C) and body weight (Figure 20D) after treatment in male HFD-fed rats. All data are mean ± SEM. [Figure 21]Centrally administered ODN weakly suppressed food intake in overnight-fasted HFD-fed males. Effects of lateral ventricle ODN (20 and 100 μg / 2 μL) on 24-hour food intake (FIG. 21A) and body weight (FIG. 21B) in overnight-fasted HFD-fed female rats, and on 24-hour food intake (FIG. 21C) and body weight (FIG. 21D) in overnight-fasted HFD-fed male rats. All data are mean ± SEM. [Figure 22] Endogenous DBI protein expression in the hindbrain was higher in females. Brains from male and female rats fed a standard diet were harvested and stained with DBI antibody to detect DBI protein expression. The fluorescence was quantified at low, medium, and high levels using HALO-AI software. DBI protein expression was measured in the AP (Figure 22A), the area postrema border (Figure 22B), the NTS at the AP level (Figure 22C), the border of the fourth ventricle (Figure 22D), and the NTS at the post-AP level (Figure 22E). [Figure 23] Female rats exhibited higher expression of endogenous relaxin-3 protein in nerve fibers. Brains from male and female rats fed a standard diet were collected and stained with relaxin-3 (Rln3) antibodies to detect Rln3 protein expression. The fluorescence was quantified using HALO-AI software at low, medium, and high levels. Rln3 protein expression was measured in the central intercalated nucleus (Figure 23A) and lateral intercalated nucleus (Figure 23B), which are rich in Rln3-positive neuronal cell bodies; the more posterior central intercalated nucleus (Figure 23C) and lateral intercalated nucleus (Figure 23D), which are rich in Rln3-positive nerve fibers; and the NTS at the AP level (Figure 23E) and the fourth ventricle level (Figure 23F). [Figure 24] ODN pretreatment suppressed the appetite stimulation induced by centrally administered relaxin-3. After treatment, the effects of lateral ventricle ODN (20 μg / 2 μL) on acute 3-hour food intake (FIG. 24A) and 24-hour body weight (FIG. 24B) in male HFD-fed rats. All data are mean ± SEM. [Figure 25]Centrally optimized ODN and TDN suppress 24-h food intake in rats. ODN and TDN were optimized using acetate precipitation rather than TFA salt precipitation to maintain a neutral pH > 4.5 in HEPES buffer. Effects of fourth ventricle ODN (200 μg / 2 μL) on 24-h food intake (Figure 25A) and body weight (Figure 25B) in male standard diet-fed rats, and effects of lateral ventricle TDN (200 μg / 2 μL) on 24-h food intake (Figure 25C) and body weight (Figure 25D) in female HFD-fed rats. All data are mean ± SEM. [Figure 26] Peripheral TDN suppresses 24-h food intake in overnight-fasted mice. Effects of IP TDN (5 mg / kg) on ​​24-h food intake (Figure 26A) and body weight (Figure 26B) in overnight-fasted male HFD-fed mice. All data are means ± SEM. [Figure 27] Chronic peripheral TDN suppresses food intake in mice. Effects of daily IP TDN (5 mg / kg) on ​​daily 24-hour body weight change (Figure 27A) and food intake (Figure 27B) and cumulative body weight change (Figure 27C) and food intake (Figure 27D) over 9 days in male HFD-fed mice. All data are mean ± SEM. [Figure 28] Hindbrain ODN improves glucose tolerance in standard-diet rats. The effects of fourth ventricle ODN (20 and 200 μg / 2 μL) on glucose tolerance (FIG. 28A), area under the glucose tolerance curve (FIG. 28B), and oral glucose-stimulated plasma insulin (FIG. 28C) in standard-diet fed rats after treatment. All data are mean ± SEM. [Figure 29] ODN signaling in the hindbrain controls counterregulatory responses. One-hour blood glucose levels (Figure 29A), 30-minute plasma corticosterone levels (Figure 29B), and plasma glucagon levels (Figure 29C) were measured in standard diet-fed rats pretreated with ODN (20 μg / 2 μL) or vehicle in the fourth ventricle, followed by treatment with 5-TG (210 μg / 2 μL) or vehicle in the fourth ventricle. All data are mean ± SEM. [Figure 30]Hindbrain ODN improves insulin resistance in standard-diet rats. Effects of fourth ventricle ODN (20 and 200 μg / 2 μL) on insulin resistance (Figure 30A), glucose tolerance test area under the curve (Figure 30B), and oral glucose-stimulated plasma insulin (Figure 30C) in standard-diet fed rats after administration. All data are mean ± SEM. [Figure 31] Pretreatment with relaxin-3 and ODN antagonists inhibited the improvement of insulin resistance by hindbrain ODN in standard-fed rats. Insulin tolerance test in pretreated standard-fed rats. The fourth ventricle was treated with relaxin-3 (Rln3 0.5 μg / 2 μL), AntOP (100 μg / 2 μL), or vehicle, followed by ODN (200 μg / 2 μL) or vehicle. All data are mean ± SEM. [Figure 32] (Figure 32A) DLS (Zetasizer Ultra, Malvern Panalytical) scan of TDN at concentrations of 10 nM to 10 mM in 50 mM HEPES buffer, pH 7.4. (Figure 32B) Agonism of the relaxin-3 receptor by the agonist relaxin-3-B chain peptide (RLN3b). [Figure 33]Coronal sections of 89Zr-labeled TDN PET scans (n ​​= 3) in athymic nude mice (n = 3) demonstrate significant brain uptake. Insulin-resistant TDN-DFO (0.19 mg, 80 nmol) was reconstituted in saline (pH 7) and reacted with 89Zr (930 mCi, 34.4 MBq) at a 1:5 (peptide:activity) ratio for 45 min at RT. Radiochemical purity was assessed by instant thin-layer silica gel chromatography (iTLC-SG; Agilent Technologies) using 50 mM EDTA as the mobile phase. The reaction was quenched by adding EDTA (5 mL, 50 mM) to sequester unreacted activity. Unbound radiometal was removed by centrifugation through a spin column (MWCO = 10 kDa) in sterile saline. Athymic nude mice (n = 3) were injected intravenously via the lateral tail vein with [89Zr]-DFO-TDN (4.8-5.6 MBq, 130-150 Ci, 60-70 nmol) in sterile saline. Small-animal PET was performed 48 hours after injection using a Bruker Albira Si PET / CT system while the mice were anesthetized with 2% isoflurane. Images were reconstructed by maximum likelihood expectation maximization with 12 iterations and a voxel resolution of 0.75 mm and analyzed using PMOD version 4.3 software. Volumes of interest were manually measured by drawing the target site across various planar sections and expressed as percent injected dose per tissue volume (%ID / mL). DETAILED DESCRIPTION OF THE INVENTION

[0015] (Detailed explanation) Endozepines are a family of astroglia-secreted proteins, including diazepam-binding inhibitors (DBIs) and their processing products. They were originally isolated and characterized as endogenous ligands of benzodiazepine receptors. The octadecaneuropeptide ODN, acting via central benzodiazepine receptors or orphan metabotropic receptors, has now been shown to exert important functions, including conflict-promoting behavior, anxiety induction, suppression of pentobarbital-induced sleep, and reduced water and food intake. To mediate their effects, ODNs regulate both glial and neuronal activity by affecting neurosteroid biosynthesis and neuropeptide expression. Furthermore, ODNs stimulate astrocyte proliferation and protect both neurons and astrocytes from oxidative stress-induced cell death. The anti-apoptotic effect of ODN on neurons is mediated by activation of ODN metabotropic receptors, which positively couple to the PKA, PKC, and MAPK / ERK signaling pathways, ultimately reducing the pro-apoptotic gene Bax and stimulating Bcl-2 expression, thereby suppressing the accumulation of intracellular reactive oxygen species. An imbalance favoring Bcl-2 promotes mitochondrial function and inhibits caspase activation, while ODN activates the endogenous antioxidant system, namely, glutathione biosynthesis and the expression and activity of antioxidant enzymes. In cultured astrocytes, moderate oxidative stress increases DBI expression and increases authentic ODN production, suggesting that ODN may act as a paracrine factor protecting neighboring neurons.

[0016] Obesity alters various aspects of hindbrain ODN signaling, including a prolonged time course of ODN hypophagocytosis, potential differences in the cleavage of diazepam-binding inhibitor (DBI) to ODNs (recombinant DBI protein induces hypophagocytosis in high-fat-fed (HFD) rats but not in standard-fed rats), and the ODN antagonist AntOP induces hypophagocytosis in HFD rats but not in standard-fed rats, potentially altering novel sites of action and / or target interactions.

[0017] ODN signaling is downstream of glucagon-like peptide-1 receptor (GLP-1R) signaling and may partially mediate its effects, thus partially mediating all GLP-1-based drug therapies currently used to treat diabetes and obesity. Nevertheless, signaling from ODNs and novel peptide derivatives (e.g., TDNs) does not appear to be maximized by GLP-1R agonism, as coadministration of ODNs and GLP-1R agonists exhibits additive anorectic effects.

[0018] Antagonizing ODN signaling with either DBI-targeting antibodies or ODN receptor peptide antagonists (AntOPs) attenuates the appetite-suppressing and weight-reducing effects of central and peripheral GLP-1R agonists. GLP-1R signaling may contribute to this effect, as nutritional status controls hindbrain DBI protein expression. ODNs may facilitate transport of GLP-1R agonists across the blood-brain barrier, particularly at the tanycyte interface in both the fourth ventricle and the subparenchymal border, thereby modulating the brain permeability of GLP-1R agonists. Multiple cleavage products of ODNs are physiologically active and suppress food intake, allowing the creation of novel, non-naturally occurring, optimized peptide forms.

[0019] In this study, we demonstrated that ODN receptors are atypical. ODNs are endogenous antagonists of the relaxin-3 receptor (RXFP3), and we discovered that novel peptide derivatives of ODN (e.g., TDN; tridecaneuropeptide; SEQ ID NO: 3) also antagonize the relaxin-3 receptor. Relaxin-3 is an orexigenic neuropeptide whose expression promotes weight gain and is upregulated in obesity to protect against weight loss. Therefore, antagonizing this signal to maintain low body weight may be a powerful tool to overcome the weight loss plateau commonly observed with current anti-obesity drug therapies. definition

[0020] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings that are commonly understood by those of ordinary skill in the art. In addition to the definitions contained in this subsection, additional term definitions are scattered throughout the text.

[0021] As used herein, "a," "or," and "an" can mean "at least one," "one or more," etc., unless the context clearly indicates otherwise. The term "or" means "and / or" unless otherwise specified. However, in the case of multiple dependent claims, the use of the term "or" refers back to multiple preceding claims in the alternative only.

[0022] Furthermore, a compound "selected from the group consisting of" refers to one or more of the compounds in the following list, including mixtures (i.e., combinations) of two or more compounds. According to the present invention, an isolated or biologically pure molecule is a compound that has been removed from its natural environment. As such, "isolated" or "biologically pure" does not necessarily reflect the extent to which a compound has been purified. An isolated compound of the present invention can be obtained from its natural source, can be produced using laboratory synthesis techniques, or can be produced by such chemical synthetic routes.

[0023] The terms "agent" and "test compound" refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal (especially mammalian) cells or tissues. Biological macromolecules include peptides, peptide / DNA complexes, siRNAs, shRNAs, antisense oligonucleotides, and nucleic acid-based molecules encoding proteins described herein.

[0024] Additionally, the term "compound" or "compounds" refers to the compounds discussed herein and is intended to include precursors and derivatives of the compounds, as well as pharmaceutically acceptable salts of the compounds, precursors and derivatives.

[0025] The phrase "consisting essentially of" when referring to a particular nucleotide or amino acid means a sequence having the characteristics of a given SEQ ID NO. For example, when used in reference to an amino acid sequence, the phrase includes the sequence itself and molecular modifications that do not affect the functional and novel characteristics of the sequence.

[0026] A "derivative" of a polypeptide, polynucleotide, or fragment thereof refers to a sequence that has been altered by, for example, altering the sequence of the construct, either by manipulation of the nucleic acid encoding the protein or by altering the protein itself. A "derivative" of a polypeptide sequence refers to an isolated amino acid molecule that has significant sequence similarity to the peptide sequence or a portion thereof. Furthermore, "derivative" includes such isolated nucleic acids containing modified nucleotides or mimetics of naturally occurring nucleotides that encode the derivative peptide.

[0027] As used herein, the term "functional" means that the nucleic acid or amino acid sequence is functional for a stated assay or purpose.

[0028] For purposes of the present invention, the terms "nucleic acid," "nucleotide sequence," or "nucleic acid molecule" as used herein refer to any DNA or RNA molecule, either single-stranded or double-stranded, and, if single-stranded, to the molecule of complementary sequence, either linear or circular. In discussing nucleic acid molecules, the sequence or structure of a particular nucleic acid molecule may be described herein according to the usual convention of providing the sequence in the 5' to 3' direction. The term "isolated nucleic acid" is sometimes used with respect to the nucleic acids of the present invention. When applied to DNA, this term refers to a DNA molecule separated from sequences with which it is immediately adjacent in the naturally occurring genome of the organism from which it originates. For example, an "isolated nucleic acid" can include a DNA molecule inserted into a vector, such as a plasmid or viral vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism. Alternatively, the term can refer to DNA that is sufficiently separated (e.g., substantially free) from other cellular components with which it would naturally be associated. "Isolated" does not imply artificial or synthetic mixture with other compounds or materials, or the presence of impurities that do not interfere with basic activity or that may be present, for example, due to incomplete purification. When applied to RNA, the term "isolated nucleic acid" primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term can refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it is associated in its natural state (i.e., in cells or tissues). Isolated nucleic acid (either DNA or RNA) can also refer to a molecule produced directly by biological or synthetic means and separated from other components present during its production.

[0029] A "specific binding pair" comprises a specific binding member (sbm) and a binding partner (bp) that have a particular specificity for each other and that, under normal conditions, bind preferentially to one another over other molecules. Examples of specific binding pairs include antigens and antibodies, biotin and streptavidin, ligands and receptors, and complementary nucleotide sequences. Those skilled in the art will recognize many other examples. Furthermore, the term "specific binding pair" also applies when either or both of the specific binding member and binding partner form part of a larger molecule. In embodiments where the specific binding pair comprises nucleic acid sequences, they will be of a length sufficient to hybridize to each other under assay conditions, preferably at least 10 nucleotides in length, more preferably at least 15 or 20 nucleotides in length.

[0030] According to the present invention, an isolated or biologically pure molecule or cell is a compound that has been removed from its natural environment. Therefore, "isolated" or "biologically pure" does not necessarily reflect the extent to which the compound has been purified. An isolated compound of the present invention can be obtained from its natural source, can be produced using laboratory synthesis techniques, or can be produced by such a chemical synthesis route.

[0031] As used herein, the term "delivery" refers to the introduction of a foreign molecule (i.e., a miRNA encoding a polypeptide of interest) into a cell. As used herein, the term "administration" refers to the introduction of a foreign molecule into the body or a cell. This term is intended to be synonymous with the term "delivery." peptide

[0032] The peptides of the present invention inhibit or regulate ODN activity. The term "inhibition" or "suppression" refers to the reduction or cessation of some event (such as protein-ligand binding), or the reduction or cessation of some phenotypic characteristic, or the reduction or cessation of the incidence, degree, or likelihood of that characteristic. "Reduce" or "inhibit" refers to the decrease, reduction, or cessation of an activity, function, and / or amount compared to a baseline. Inhibition or reduction need not be complete. For example, in certain embodiments, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 20% or more. In other embodiments, "reduce" or "suppress" refers to the ability to cause an overall reduction of 50% or more. In yet other embodiments, "reduce" or "suppress" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95%, or more.

[0033] As used herein, the term "modulate" refers to the ability of a compound to measurably alter an activity relative to an appropriate control. The presence of a compound in an assay may result in an increase or decrease in activity relative to a control in which the compound is absent. Preferably, an increase in activity is at least 25%, more preferably at least 50%, and most preferably at least 100% compared to the activity level in the absence of the compound. Similarly, a decrease in activity is preferably at least 25%, more preferably at least 50%, and most preferably at least 100% compared to the activity level in the absence of the compound. A compound that increases a known activity is an "agonist." An agent that decreases or blocks a known activity is an "antagonist."

[0034] Throughout this document, values ​​expressed in range format should be interpreted flexibly to include not only the numerical values ​​expressly stated as the limits of the range, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were expressly stated. For example, the range "about 0.1% to about 5%" or "about 0.1% to about 5%" should be interpreted to include not only about 0.1% to about 5%, but also individual values ​​(e.g., 1%, 2%, 3%, 4%) and subranges within the stated range (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%).

[0035] The term "inhibitor" refers to an agent that slows or blocks a particular chemical reaction, signaling pathway, or other process, or reduces the activity of a particular reactant, compound, or enzyme.

[0036] "Octadecaneuropeptides" or "ODNs" and their precursor, diazepam-binding inhibitors (DBIs), are peptides belonging to the endozepine family. Endozepines are produced exclusively by astroglial cells in the mammalian central nervous system, and their release is regulated by stress signals and neuroactive compounds. There is now compelling evidence that ODNs protect cultured neurons and astrocytes from apoptotic cell death induced by various neurotoxins.

[0037] " ODN antagonist " refers to a group of drugs that inhibit the action of ODN. " ODN agonist " refers to a group of drugs that enhance or enhance ODN activity. The peptides of interest herein include, in particular, naturally occurring ODN and its derivatives, as shown in Table 1.

[0038] (Table 1) ODN and ODN-based peptide sequences. The peptides ODN, AntOP, TDN, OP, and SUODN-03, -04, and -05 were synthesized and assayed. Gln (alkyne), Ala (alkyne), Gly (alkyne), and Lys (N3) are modified amino acids for bioconjugation, including lipidation and fluorescent tags.* ODN:Gln-Ala-Thr-Val-Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys AntOP:Arg-Pro-Gly-Leu-(D-Leu)-Asp-Leu-Lys TDN (SUODN-01):Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys OP (SUODN-02):Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys SUODN-03:Arg-Pro-Gly-Leu-Leu SUODN-04:Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly SUODN-05:Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu SUODN-06:Gln-Ala-Thr-Val-Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly SUODN-07:Gln-Ala-Thr-Val-Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu SUODN-08:Gln(Alkyne)-Ala-Thr-Val-Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys(N3) SUODN-09:Gln(Alkyne)-Ala-Thr-Val-Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Lys(N3)-Leu-Asp-Leu-Lys SUODN-10:Gln(アルキン)-Ala-Thr-Val-Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Lys(N3)-Asp-Leu-Lys SUODN-11:Gln(アルキン)-Ala-Thr-Val-Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Lys(N3)-Lys SUODN-12:Gln-Ala(アルキン)-Thr-Val-Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys(N3) SUODN-13:Gln-Ala(アルキン)-Thr-Val-Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Lys(N3)-Leu-Asp-Leu-Lys SUODN-14:Gln-Ala(アルキン)-Thr-Val-Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Lys(N3)-Asp-Leu-Lys SUODN-15:Gln-Ala(アルキン)-Thr-Val-Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Lys(N3)-Lys SUODN-16:Gln-Ala-Thr-Val-Gly(アルキン)-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys(N3) SUODN-17:Gln-Ala-Thr-Val-Gly(アルキン)-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly- Lys(N3)-Leu-Asp-Leu-Lys SUODN-18:Gln-Ala-Thr-Val-Gly(アルキン)-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Lys(N3)-Asp-Leu-Lys SUODN-19:Gln-Ala-Thr-Val-Gly(Alkyne)-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Lys(N3)-Lys *SEQ ID NOs: 1-21 are listed in descending order.

[0039] In certain embodiments, the residues of a protein or peptide are contiguous, without any non-genetically encoded amino acids or synthetic amino acids interrupting the sequence of amino acid residues. The peptide can be modified as described above. In other embodiments, the sequence can include one or more non-genetically encoded or synthetic amino acid moieties. In certain embodiments, the sequence of residues of a peptide can be interrupted by one or more non-genetically encoded or synthetic amino acid moieties, including, but not limited to, those shown in Table 2. [Table 2]

[0040] [Table 3]

[0041] Such improvements result in analogs with long half-lives that can be administered, for example, once a week.

[0042] In certain embodiments, the present invention includes peptides having at least 80% sequence identity to any of the peptides described herein. In certain embodiments, peptides of the present invention have a sequence identity of at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity.

[0043] Preferably, the peptides of the above sequences and their functional equivalents are administered to regulate obesity. As used herein, the term "functional equivalent" is intended to include amino acid sequence variants with amino acid substitutions in part or all of the protein, or amino acid additions or deletions in part of the protein. The amino acid substitutions are preferably conservative substitutions. Examples of conservative substitutions of naturally occurring amino acids include: aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn), and sulfur-containing amino acids (Cys, Met). The amino acid deletions are preferably located in regions that are not directly involved in the activity of the peptide.

[0044] As used herein, the term "variant" refers to a nucleic acid sequence or polypeptide that comprises a sequence that differs from a wild-type nucleic acid or polypeptide sequence at one or more nucleic acid or amino acid positions (by deletion, insertion, and / or substitution of a nucleic acid or amino acid, an L or D stereoisomer of an amino acid, or a non-naturally occurring amino acid).

[0045] As used herein, the term "linker" refers to the connection between two protein-coding sequences or their protein products. A linker comprises a contiguous nucleic acid or amino acid sequence and retains at least one cleavage site that allows the genes or their products to be separated by cleavage of the linker. Preferably, the linker comprises a cleavage site at its 5'-terminus and a cleavage site at its 3'-terminus, or a cleavage site at its N-terminus and a cleavage site at its C-terminus.

[0046] Peptides can be fused with biotin, polylysine, lysozyme, green fluorescent protein (and its derivatives), SUMO, or other desired proteinaceous tags for attachment to electrodes, nanotubes, or desired surfaces (e.g., electrically conductive, mineral), as well as any protein interaction partners desired to be investigated. Production of the desired peptide sequences can be carried out in E. coli using existing techniques, for example, with protein fusion tags that can be removed or left as desired. In certain embodiments, the peptide of interest may be fused via a linker.

[0047] The peptide can be fused with one or more cell penetrating peptides (CPPs) useful for facilitating delivery of the peptide to target cells. CPPs are known to those skilled in the art and include, but are not limited to, penetratin (RQIKIWFQNRRMKWKK) (SEQ ID NO: 22), VP22 peptide (DAATATRGRSAASRPTER PRAPARSASRPRRVD) (SEQ ID NO: 23), MAP (KLALKLALKALKAALKLA-amide) (SEQ ID NO: 24), transpectin (GWTLNSAGYLLGKINLKALAALAKKIL-amide) (SEQ ID NO: 25), R7 (RRRRRRR) (SEQ ID NO: 26), MPG (GALFLGWLGAAGSTMGAPKKRKV) (SEQ ID NO: 27), and Pep-1 (KETWWETWWTEWSQPKKKRKV) (SEQ ID NO: 28) and tat (YGRKKRRQRRR) (SEQ ID NO: 29).

[0048] The peptides can be expressed as fusions with larger proteins, facilitating large-scale expression, ease of purification, and ensuring product quality. Expression systems can also be utilized to generate large sequence libraries, enabling directed evolution for targeted properties. Peptides can also be incorporated into other proteins via fusion, making them useful for treating obesity and other metabolic disorders.

[0049] As mentioned above, the present invention also includes polynucleotides encoding the peptides described herein or fusion proteins containing the peptides. Those skilled in the art understand the degeneracy of the genetic code and understand that various polynucleotides can encode the same polypeptide. In some embodiments, the polynucleotide (i.e., the polynucleotide encoding the fusion polypeptide) can be codon-optimized for expression in a particular cell, including, but not limited to, a plant cell, a bacterial cell, or an algal cell. Any polynucleotide sequence encoding the desired form of the polypeptide described herein can be used. The polynucleotide sequences encoding the polypeptides of the present invention represent sequences that do not occur in nature. Computer programs for generating degenerate coding sequences are available and can be used for this purpose. Degenerate coding sequences can be generated using pencil, paper, the genetic code, and even by hand.

[0050] In the present context, the term "codon optimization" refers to changing the codons of a nucleotide sequence to favor expression in a particular species, without changing the amino acid sequence it encodes. Codon optimization is used to increase the abundance of the peptide or protein that a nucleotide sequence encodes, as "rare" codons are removed and replaced with abundant codons.

[0051] With respect to the fusion polypeptides disclosed herein, the terms "% sequence identity," "percent identity," or "% identity" refer to the percentage of residue matches between at least two amino acid sequences aligned using a standardized algorithm. Methods of amino acid sequence alignment are well known. Some alignment techniques consider conservative amino acid substitutions. Such conservative substitutions generally preserve the charge and hydrophobicity at the substitution site, preserving the structure (and therefore function) of the polypeptide. Percent amino acid sequence identity can be determined as understood in the art. Structural similarity is typically at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity.

[0052] Polypeptide sequence identity may be measured over the length of the entire defined polypeptide sequence, e.g., as defined by a particular SEQ ID NO:, or over a shorter length, such as the length of a fragment taken from a larger, defined polypeptide sequence, e.g., a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 contiguous residues. It is understood that such lengths are exemplary, and that any fragment length supported by the sequences set forth herein can be used to describe the length over which percent identity may be measured.

[0053] In vitro peptide synthesis Peptides can be chemically synthesized in solution or on solid phase. This process involves the directed and selective formation of an amide bond between an N-protected amino acid and an amino acid bearing a free amino group and a protected carboxylic acid. In solid phase synthesis, the carboxyl protecting group is attached to a polymer support. After bond formation, the amino protecting group of the dipeptide is removed, and the next N-protected amino acid is attached.

[0054] Solid-phase peptide synthesis (SSPS) is the most frequently used method for peptide synthesis due to its efficiency, simplicity, speed, and ease of parallelization. SPPS involves sequential addition of amino acid residues and side-chain protected amino acid residues to an amino acid or peptide bound to an insoluble polymer support. N-α-protection is achieved using the acid-labile Boc group (Boc SPPS) or the base-labile Fmoc group (Fmoc SPPS). After removal of this protecting group, the next protected amino acid is added using a coupling reagent or a pre-activated protected amino acid derivative. The C-terminal amino acid is anchored to the resin via a linker, the nature of which determines the conditions required to release the peptide from the support after chain elongation. Side-chain protecting groups are often selected so that they are cleaved simultaneously with the peptide's release from the resin.

[0055] While peptides of 50 amino acids can be routinely prepared, the synthesis of proteins of more than 100 amino acids is commonly reported. Longer proteins can be made by native chemical ligation of fully deprotected peptides in solution. This method allows the synthesis of natural peptides that are difficult to express in bacteria, the incorporation of unnatural amino acids and D-amino acids, and the generation of circular, branched, labeled, and post-translationally modified peptides.

[0056] Solution-phase peptide synthesis, which typically utilizes Boc or Z-amino protection, has largely been superseded by solid-phase peptide synthesis, except as an existing process for large-scale industrial peptide synthesis. Desired sequences can be developed by commercial organizations such as Sigma-Aldrich and Avivasysbio, which offer this service for a fee.

[0057] Vectors and production Transgenic cells expressing the polynucleotides also form an aspect of the present invention. Transgenic cells can be obtained by introducing a recombinant nucleic acid molecule encoding a protein of the present disclosure. As used herein, the term "recombinant nucleic acid" refers to a polynucleotide that has been manipulated by human intervention. A recombinant nucleic acid molecule can contain two or more nucleotide sequences linked in such a way that the resulting product is not found in cells in nature. In particular, two or more nucleotide sequences can be operably linked, e.g., to encode a fusion polypeptide. Recombinant nucleic acid molecules can also be based on naturally occurring polynucleotides but engineered to be different from them. For example, a polynucleotide can contain one or more nucleotide changes that bias the first codon normally present in the polynucleotide toward chloroplast codon usage. Alternatively, a sequence of interest, such as a restriction endonuclease recognition site, a splice site, a promoter, or an origin of DNA replication, can be introduced into the polynucleotide.

[0058] Any suitable technique for introducing recombinant nucleic acid molecules into cells can be used. Techniques for nuclear and chloroplast transformation are known and include, but are not limited to, electroporation, biological transformation (also called microprojectile / particle bombardment), agitation in the presence of glass beads, and Agrobacterium-based transformation.

[0059] As used herein, the term "construct" refers to a recombinant polynucleotide, including, but not limited to, DNA and RNA, which may be single-stranded or double-stranded and may represent a sense or antisense strand. A recombinant polynucleotide is a polynucleotide formed by laboratory methods that includes polynucleotide sequences derived from at least two different natural sources, and may be synthetic. Thus, a construct may include, for example, a new modification of an endogenous gene introduced by genome editing technology. A construct also includes, for example, a recombinant polynucleotide produced using recombinant DNA methods.

[0060] A "vector" is a vector capable of transferring a gene sequence into a target cell. Generally, the terms "vector construct," "expression vector," and "gene transfer vector" refer to any nucleic acid construct capable of inducing expression of a gene of interest and transferring a gene sequence into a target cell. Thus, the term includes cloning vectors, expression vectors, and integrating vectors.

[0061] The constructs and vectors provided herein can be prepared by methods available to those skilled in the art.In particular, each of the claimed constructs or expression cassettes is a recombinant molecule and does not exist in nature.In general, the nomenclature used herein and the experimental procedures utilized in the present invention include molecular biological, biochemical and recombinant DNA techniques that are well known and commonly employed in the art.Standard techniques available to those skilled in the art can be used for cloning, DNA and RNA isolation, amplification and purification.Such techniques are fully described in the literature.

[0062] The constructs and expression cassettes provided herein can include a promoter operably linked to any one of the polynucleotides described herein, but do not necessarily have a promoter and can be used for homologous recombination into a cell. Alternatively, the constructs can include a promoter, which can be a heterologous promoter or an endogenous promoter associated with the polypeptide.

[0063] As used herein, the terms "heterologous promoter," "promoter," "promoter region," or "promoter sequence" generally refer to the transcriptional regulatory region of a gene, which may be found 5' or 3' to a polynucleotide described herein, within the coding region of the polynucleotide, or within an intron of the polynucleotide. Typically, a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3') coding sequence. A typical 5' promoter sequence is surrounded at its 3' end by a transcription start site and extends upstream (5') to include the minimum number of bases or elements required to initiate transcription detectable at levels above background. Within the promoter sequence is a transcription start site (easily defined by mapping with nuclease S1) and a protein binding domain (consensus sequence) responsible for binding RNA polymerase.

[0064] In some embodiments, the disclosed polynucleotide is operably linked to a promoter.As used herein, a polynucleotide is "operably linked" or "operably linked" when it is placed in a functional relationship with a second polynucleotide sequence.For example, a promoter is operably linked to a polynucleotide when the promoter is linked to the polynucleotide so as to affect the transcription of the polynucleotide.In various embodiments, a polynucleotide can be operably linked to at least one, at least two, at least three, at least four, at least five, or at least ten promoters.

[0065] Heterologous promoters useful in practicing the present invention include, but are not limited to, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and tissue-specific promoters. Heterologous promoters can be plant, animal, bacterial, fungal, or synthetic promoters.

[0066] Treatment and administration In the methods described herein, acts may be performed in any order unless a temporal or operational order is expressly recited. Furthermore, certain acts may be performed simultaneously unless express claim language clearly recites them performed separately. For example, a claimed act of performing X and a claimed act of performing Y may be performed simultaneously in a single operation, and the resulting process falls within the literal scope of the claimed process.

[0067] As used herein, the terms "alleviate" or "inhibit" refer to the administration of a compound prior to or during the onset of clinical symptoms of a disease or condition, so as to reduce the physical manifestations of abnormalities associated with the disease or condition.

[0068] As used herein, a "condition in need of treatment" refers to a determination by a caregiver (e.g., in the case of a human, a physician, nurse, nurse practitioner, or individual; in the case of an animal, including a non-human mammal, a veterinarian) that a subject is in need of or will benefit from treatment. This determination is within the expertise of the caregiver, but is made based on a variety of factors, including knowledge that the subject is ill or will become ill as a result of a condition treatable by the disclosed compounds.

[0069] As used herein, a "subject" includes, but is not limited to, animals, plants, bacteria, viruses, parasites, and other organisms or entities. A subject may be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig, or rodent), fish, bird, reptile, or amphibian. A subject may be an invertebrate, more specifically an arthropod (e.g., an insect or crustacean). The term does not denote a particular age or sex; thus, adults, newborns, and fetuses of either sex are included. A patient refers to a subject suffering from a disease or disorder. The term "patient" includes human and veterinary subjects.

[0070] As used herein, the terms "treat," "treat," and "treatment" refer to eliciting a desired biological response, such as a therapeutic or prophylactic effect, respectively. In some embodiments, the therapeutic effect includes one or more of the following after administration of the agent or a composition comprising the agent: reduction / reduction in obesity; reduction / reduction in the severity of obesity (e.g., reduction or inhibition of growth or obesity); reduction / reduction in symptoms and obesity-related effects; delaying the onset of symptoms and obesity-related effects; reducing the severity of obesity-related symptoms; reducing the severity of acute episodes; reducing the number of symptoms and obesity-related effects; shortening or reducing the latency of symptoms and obesity-related effects; ameliorating symptoms and obesity-related effects; reducing secondary symptoms; reducing secondary infections; preventing recurrence of obesity; reducing the number or frequency of recurrent episodes; increasing the time between symptom episodes; increasing the time to sustained progression; promoting remission; inducing remission; enhancing remission; hastening recovery; increasing the effectiveness of alternative therapeutic agents or reducing resistance; and / or increasing the survival time of the affected host animal. A prophylactic effect can consist of completely or partially avoiding / inhibiting or delaying (e.g., completely or partially avoiding / inhibiting or delaying) the onset / progression of obesity or prolonging the survival of an affected host animal following administration of a treatment protocol. Treating obesity includes treating a subject already diagnosed with obesity, regardless of clinical stage or condition, delaying the onset or progression or worsening or progression of symptoms or signs of obesity, and / or preventing and / or reducing the severity of obesity.

[0071] In another aspect, provided herein is a method for treating a patient, comprising administering a peptide of interest.In any of the embodiments described herein, the subject may have obesity or excess weight, elevated BMI, elevated body fat mass, percentage, or volume, and / or excessive food intake.In any of the embodiments described herein, the subject may be obese.In any of the embodiments described herein, the subject may have excess weight.In any of the embodiments described herein, the subject may have elevated BMI.In any of the embodiments described herein, the subject may have elevated body fat mass, percentage, or volume.In any of the embodiments described herein, the subject may have excessive food intake.

[0072] Symptoms of obesity include, but are not limited to, excess body fat (especially around the waist), shortness of breath, increased sweating, snoring, inability to tolerate sudden exercise, daily fatigue, back and joint pain, and skin problems (due to fluid accumulation in skin folds).

[0073] Provided is a method for treating the subject with obesity, comprising administering ODN peptide or its functional derivative to the subject.Also disclosed is a method for treating overweight subject, comprising administering ODN peptide to the subject.This disclosure also provides a method for treating the subject with elevated BMI, comprising administering an effective amount of ODN peptide to the subject.Also described is a method for treating the subject with elevated body fat mass, body fat percentage or body fat mass, comprising administering ODN peptide to the subject.Finally, also disclosed is a method for treating the subject with excessive food intake, comprising administering ODN peptide.

[0074] The present disclosure also provides a method of treating a subject to prevent weight gain or maintain weight loss, the method comprising administering to the subject an effective amount of an ODN peptide.

[0075] In certain embodiments, methods are provided for treating a metabolic disease or disorder in a subject, comprising administering to the subject an effective amount of an ODN peptide.

[0076] As used herein, the term "metabolic disease" or "metabolic abnormality" refers to a series of abnormal conditions, also known as metabolic syndrome, that increase the risk of cerebrovascular disease and diabetes, such as increased body fat, elevated blood pressure, elevated blood glucose levels, and abnormal blood lipids. Metabolic diseases are not single diseases but are comprehensive diseases caused by genetic predisposition and environmental factors. In the present invention, metabolic diseases may be selected from the group consisting of obesity, diabetes, dyslipidemia, insulin resistance, hepatic steatosis, hypercholesterolemia, and non-alcoholic fatty liver disease, and are more preferably obesity or diabetes, but are not limited thereto.

[0077] As used herein, the term "diabetes" refers to a metabolic disorder characterized by hyperglycemia (high blood glucose levels), which results from insufficient or improper insulin secretion and the release of glucose in the urine, causing various symptoms and signs of hyperglycemia. Diabetes includes type 1 diabetes, which is caused primarily by destruction of pancreatic beta cells, resulting in insulin secretion failure, and type 2 diabetes, which is caused by insufficient insulin secretion in the body or insulin resistance, which prevents cells from responding to insulin. In the present invention, diabetes includes both type 1 diabetes and type 2 diabetes. In certain embodiments, the method further comprises administering a second therapeutic agent for treating or suppressing obesity. Non-limiting examples of therapeutic agents that treat or inhibit obesity and / or BMI increase include, but are not limited to, GLP1R agonists, melanocortin 4 receptor (MC4R) agonists, sibutramine, orlistat, phentermine, lorcaserin, naltrexone, liraglutide, diethylpropion, bupropion, metformin, pramlintide, topiramate, and zonisamide, or combinations thereof.

[0078] The administration of the therapeutic agent and / or ODN peptide for treating or suppressing obesity can be repeated, for example, after 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months or 3 months.Repeated administration can be the same dose or different doses.Administration can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times.For example, according to certain administration regimen, subject can be treated for a long period of time, for example, 6 months, 1 year or more.

[0079] The therapeutic agent and / or ODN peptide for treating or inhibiting obesity can be administered by any suitable route, including, but not limited to, parenteral, intravenous, oral, buccal, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Pharmaceutical compositions for administration are preferably sterile and substantially isotonic and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., single dose). Pharmaceutical compositions can be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. Formulations vary depending on the selected route of administration. The term "pharmaceutically acceptable" means that the carrier, diluent, excipient, or adjuvant is compatible with the other ingredients of the formulation and is not substantially harmful to the recipient thereof.

[0080] A non-limiting example of an effective dose range for the therapeutic compounds described herein is about 0.1 to 5,000 mg / kg body weight / day. One of ordinary skill in the art would be able to examine the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0081] The compounds may be combined with one or more pharmaceutically acceptable carriers and / or excipients that are believed to be safe and effective and can be administered to an individual without causing unwanted biological side effects or undesirable interactions. A carrier refers to all ingredients present in a pharmaceutical formulation other than the active ingredient. See, for example, Remington's Pharmaceutical Sciences, latest edition, by EW Martin Mack Pub. Co., Easton, PA. This publication discloses typical carriers that can be used in conjunction with the preparation of formulations of the compounds described herein and conventional methods for preparing pharmaceutical compositions, and is incorporated herein by reference. The most common are standard carriers for administering compositions to humans. In one embodiment, these include solutions such as sterile water, saline, and buffered solutions at physiological pH. Other compounds are administered according to standard procedures used by those skilled in the art.

[0082] These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.

[0083] In some embodiments, the therapeutic agent and / or ODN peptide (any of the peptide ligands disclosed herein) for treating or inhibiting obesity is administered intrathecally (i.e., into the subarachnoid space of the spinal cord or into the spinal canal, or into an anatomical or potential space within the sheaths, including, but not limited to, the arachnoid membrane of the brain or spinal cord, so that the therapeutic agent can reach the subject's cerebrospinal fluid). In some embodiments, intrathecal administration results in the therapeutic agent acting on, but not limited to, the cerebral cortex, cerebellum, striatum, cervical vertebrae, lumbar vertebrae, or thoracic vertebrae. An intrathecally administered therapeutic agent may ultimately affect targets throughout the central nervous system. In some embodiments, intrathecal administration is performed in the (retrocerebellomedullary cisterna magna) or lumbar region or area. In some embodiments, intrathecal administration to the lumbar region or area delivers the therapeutic agent to the distal spinal canal.

[0084] Exemplary methods of intrathecal administration are described, for example, in Lazorthes et al., Advances in Drug Delivery Systems and Applications in Neurosurgery, 143-192. In some embodiments, intrathecal administration is via injection, bolus injection, catheter, or pump. In some embodiments, intrathecal administration is via lumbar puncture. In some embodiments, the pump is an osmotic pump. In some embodiments, the pump is implanted in the subarachnoid space of the spinal canal, under the skin in the abdomen, or behind the chest wall. In some embodiments, intrathecal administration is via an intrathecal delivery system for the therapeutic agent, including a reservoir containing a volume of the therapeutic agent and a pump configured to deliver at least a portion of the therapeutic agent contained in the reservoir. In some embodiments, intrathecal administration is via intermittent or continuous access to an implanted intrathecal drug delivery device (IDDD). In some embodiments, the therapeutic agent is an inhibitory nucleic acid molecule. In some embodiments, the amount of nucleic acid molecule administered intrathecally ranges from about 10 μg to about 2 mg, about 50 μg to about 1500 μg, or about 100 μg to about 1000 μg. In some embodiments, the therapeutic agent is disposed in a pharmaceutical composition. In some embodiments, the pharmaceutical composition does not contain a preservative.

[0085] Parenteral formulations The peptides described herein can be formulated for parenteral administration, including intravenous, intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intravitreal, intratumoral, intramuscular, subcutaneous, subconjunctival, intravesical, intrapericardial, and intraumbilical administration by injection and infusion.

[0086] Parenteral preparations can be prepared as aqueous compositions by using the techniques known in the art.Typically, these compositions can be prepared as injectable preparations, for example, solution or suspension; the solid form that is suitable for being used to prepare solution or suspension by adding reconstitution medium before injection; emulsion, such as water-in-oil (w / o) emulsion, oil-in-water (o / w) emulsion and their microemulsions, liposome or emulsome.

[0087] For intravenous administration, the composition is prepared in a sterile isotonic aqueous buffer solution. If necessary, the composition can also contain a solubilizing agent. The components of the composition are supplied in unit dosage form, for example, as a dry lyophilized powder or concentrated solution, separately or mixed together in a sealed container such as an ampoule or sachet indicating the amount of active ingredient. When the composition is administered by infusion, it can be administered using an infusion bottle containing sterile pharmaceutical water or saline. When the composition is administered by injection, an ampoule of sterile water or saline can be provided so that the components can be mixed before injection.

[0088] The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), oil, for example, vegetable oil (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and / or by using surfactants. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride.

[0089] Solutions and dispersions of the active compounds as free acids or bases or pharmacologically acceptable salts thereof can be prepared in water or other solvent or dispersion medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersing agents, emulsifying agents, pH adjusters, viscosity adjusters, and combinations thereof.

[0090] Suitable surfactants include anionic, cationic, amphoteric, or nonionic surfactants. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate, and sulfate ions. Examples of anionic surfactants include long-chain alkylsulfonates, such as sodium, potassium, and ammonium, and alkylarylsulfonates, such as sodium dodecylbenzenesulfonate; dialkylsodium sulfosuccinates, such as sodium dodecylbenzenesulfonate; dialkylsodium sulfosuccinates, such as sodium bis(2-ethylthio)sulfosuccinate; and alkylsulfates, such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds, such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyldimethylbenzylammonium chloride, polyoxyethylene, and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl 4-oleate, sorbitan acylate, sucrose acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbate, polyoxyethylene octylphenyl ether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-β-alanine, sodium N-lauryl-β-iminodipropionate, myristoamphoacetate, lauryl betaine, and lauryl sulfobetaine.

[0091] The formulation may contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. The formulation may also contain an antioxidant to prevent the decomposition of the active agent.

[0092] Upon reconstitution, formulations are typically buffered for parenteral administration at a pH of 3-8. Suitable buffers include, but are not limited to, phosphate, acetate, and citrate buffers.

[0093] Water-soluble polymers are commonly used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, polyethylene glycol, etc.

[0094] Sterile injectable solution can be prepared by mixing the required amount of active compound in suitable solvent or dispersion medium, and optionally with one or more of the above-mentioned additives, and then filter sterilization.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains basic dispersion medium and other necessary ingredients from the above-mentioned.For the sterile powder to prepare sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technology, and obtains powder of active ingredient and any additional desired ingredients from the solution that has been previously sterilized by filtering.Powder can be prepared in a way that makes particles porous, which can increase the solubility of particles.The method of producing porous particles is well known in the art.

[0095] The parenteral formulations described herein can be formulated for controlled release, including immediate release, delayed release, sustained release, pulsatile release, and combinations thereof.

[0096] Nanoparticles and microparticles For parenteral administration, one or more compounds, and optionally one or more additional active agents, can be incorporated into microparticles, nanoparticles, or combinations thereof that provide controlled release of the compounds and / or one or more additional active agents. In forms in which the formulation includes two or more peptides, the peptides can be formulated for the same type of controlled release (e.g., delayed, extended, immediate, or pulsatile), or the peptides can be independently formulated for different types of release (e.g., immediate and delayed, immediate and extended, delayed and extended, delayed and pulsatile, etc.).

[0097] For example, the compound and / or one or more additional active agents can be incorporated into polymeric microparticles that provide controlled release of the peptide. Release of the peptide is controlled by diffusion of the protein from the microparticle and / or degradation of the polymeric microparticle by hydrolysis and / or enzymatic degradation. Suitable polymers include ethylcellulose and other natural or synthetic cellulose derivatives.

[0098] Polymers that dissolve slowly and form gels in aqueous environments, such as hydroxypropylmethylcellulose and polyethylene oxide, are also suitable materials for protein-containing microparticles. Other polymers include, but are not limited to, polyanhydrides, poly(ester anhydrides), polyhydroxy acids, such as polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly-3-hydroxybutyric acid (PHB) and copolymers thereof, poly-4-hydroxybutyric acid (P4HB) and copolymers thereof, polycaprolactone and copolymers thereof, and combinations thereof.

[0099] Alternatively, proteins can be incorporated into microparticles prepared from materials that are insoluble in aqueous solutions or slowly soluble in aqueous solutions but decompose in the digestive tract by means including enzymatic degradation, the surfactant action of bile acids, and / or mechanical erosion. As used herein, the term "slowly soluble in water" refers to a material that does not dissolve in water within 30 minutes. Preferred examples include fats, fatty substances, waxes, wax-like substances, and mixtures thereof. Suitable fats and fatty substances include fatty alcohols (such as lauryl, myristylstearyl, cetyl, or cetostearyl alcohol), fatty acids and their derivatives, including, but not limited to, fatty acid esters, fatty acid glycerides (mono-, di-, and triglycerides), and hydrogenated fats and oils. Specific examples include, but are not limited to, hydrogenated vegetable oil, hydrogenated cottonseed oil, hydrogenated castor oil, hydrogenated oils available under the trade name Sterotex®, stearic acid, cocoa butter, stearyl alcohol, and the like. Suitable waxes and wax-like substances include natural or synthetic waxes, hydrocarbons, and conventional waxes. Specific examples of waxes include beeswax, glycowax, castor oil wax, carnauba wax, paraffin, candelilla wax, etc. As used herein, wax-like substances are defined as any substance that is normally solid at room temperature and has a melting point of about 30-300°C.

[0100] In some cases, it may be desirable to change the rate at which water penetrates the microparticles. For this purpose, a rate-controlling agent (wicking agent) can be added along with the oils and waxes mentioned above. Examples of rate-controlling materials include certain starch derivatives (e.g., waxy maltodextrin and drum-dried cornstarch), cellulose derivatives (e.g., hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose, and carboxymethylcellulose), alginic acid, lactose, and talc. Furthermore, a pharmaceutically acceptable surfactant (e.g., lecithin) may be added to promote the disintegration of such microparticles.

[0101] Water-insoluble proteins, such as zein, can also be used as materials for forming protein-containing microparticles. Furthermore, water-soluble proteins, polysaccharides, and combinations thereof can be formulated into microparticles with peptides and then crosslinked to form an insoluble network. For example, cyclodextrins can be complexed with individual drug molecules and then crosslinked.

[0102] Nano- and micro-particle manufacturing method Encapsulation or incorporation of a drug into a carrier material to produce drug-containing microparticles can be achieved by known pharmaceutical formulation techniques. When formulated with oils, waxes, or wax-like substances, the carrier material is generally heated above its melting point, and the drug is added to form a mixture containing drug particles suspended in the carrier material, drug dissolved in the carrier material, or a mixture thereof. Microparticles can be formulated by several methods, including, but not limited to, solidification, extrusion, spray cooling, and aqueous dispersion. In a preferred process, wax is heated above its melting temperature, the drug is added, and the molten wax-drug mixture is allowed to solidify under constant agitation while cooling. Alternatively, the molten wax-drug mixture can be extruded and spheronized to form pellets or beads. These processes are known in the art.

[0103] Depending on the carrier material, it may be desirable to prepare drug-containing microparticles using a solvent evaporation method, in which the drug and carrier material are co-dissolved in a mutual solvent, after which the microparticles can be produced by several techniques, including, but not limited to, forming an emulsion in water or other suitable medium, spray drying, or evaporating the solvent from a bulk solution and milling the resulting material.

[0104] In some embodiments, the particulate drug is uniformly dispersed in a water-insoluble or slowly water-soluble substance. To minimize the size of the drug particles in the composition, the drug powder itself may be milled to produce fine particles before formulation. For this purpose, a jet milling process known in the pharmaceutical arts may be used. In other embodiments, the particulate drug is uniformly dispersed in the wax or wax-like substance by heating the wax or wax-like substance above its melting point and adding the drug particles while stirring the mixture. In this case, a pharmaceutically acceptable surfactant may be added to the mixture to promote dispersion of the drug particles.

[0105] Particles can also be coated with one or more modified-release coatings. Solid esters of fatty acids hydrolyzed by lipases can be spray-coated onto microparticles or drug particles. Zein is an example of a naturally occurring, water-insoluble protein. It can be coated onto drug-loaded microparticles or drug particles using spray coating or wet granulation techniques. In addition to naturally water-insoluble substances, digestive enzyme substrates also include those that form water-insoluble networks through crosslinking. Many methods for crosslinking proteins have been reported, initiated by both chemical and physical means. One of the most common methods for achieving crosslinking is the use of chemical crosslinkers. Examples of chemical crosslinkers include aldehydes (glutaraldehyde, formaldehyde), epoxy compounds, carbodiimides, and genipin. In addition to these crosslinkers, oxidized and native sugars have been used to crosslink gelatin. Crosslinking can also be achieved by enzymatic methods. For example, transglutaminase has been approved as a GRAS substance for crosslinking in seafood. Finally, crosslinking can be initiated by physical means such as heat treatment, UV irradiation, or gamma irradiation.

[0106] To prepare a coating layer of crosslinked protein surrounding drug-containing microparticles or drug particles, a water-soluble protein can be spray-coated onto the microparticles and then crosslinked by one of the methods described above. Alternatively, drug-containing microparticles can be microencapsulated in a protein by coacervation-phase separation (e.g., by adding salt) and then crosslinked. Suitable proteins for this purpose include gelatin, albumin, casein, and gluten.

[0107] Polysaccharides can also be cross-linked to form water-insoluble networks. In many polysaccharides, the main polymer chains can be cross-linked by reaction with calcium salts or polyvalent cations. Pectin, alginate, dextran, amylose, and guar gum cross-link in the presence of polyvalent cations. Complexes can also be formed between polysaccharides with opposite charges; for example, pectin and chitosan can complex through electrostatic interactions.

[0108] Injectable / implantable preparations The compounds described herein can be incorporated into injectable / implantable solid or semi-solid implants, such as polymeric implants. In some embodiments, the compounds are incorporated into polymers that are liquid or pasty at room temperature but increase in viscosity and form semi-solids or solids upon contact with aqueous media, such as physiological fluids. Exemplary polymers include, but are not limited to, hydroxyalkanoic acid polyesters, derived from the copolymerization of at least one unsaturated hydroxy fatty acid copolymerized with a hydroxyalkanoic acid. The polymer can be melted, mixed with an active agent, and cast or injection molded into a device. Such melt processing requires a polymer with a melting point below the temperature at which the polymer decomposes or reacts with the feed material. Devices can also be prepared by solvent casting, in which a polymer is dissolved in a solvent, the drug is dissolved or dispersed in the polymer solution, and the solvent is then evaporated. Solvent processing requires that the polymer be soluble in an organic solvent. Another method is compression molding of a mixed powder of polymer and drug, or polymer particles loaded with an active agent.

[0109] Alternatively, the compounds can be incorporated into a polymer matrix and molded, compressed, or extruded into a solid device at room temperature. For example, the compounds can be incorporated into biodegradable polymers such as polyanhydrides, polyhydroalkanoates (PHAs), PLA, PGA, PLGA, polycaprolactones, polyesters, polyamides, polyorthoesters, polyphosphazenes, proteins, and polysaccharides such as collagen, hyaluronic acid, albumin, gelatin, and combinations thereof, and compressed into a solid device such as a disk or extruded into a device such as a rod.

[0110] The release of one or more compounds from the implant can be varied by the choice of polymer, the molecular weight of the polymer, and / or modifying the polymer to facilitate degradation (such as by forming pores and / or incorporating hydrolyzable linkages). Methods for altering the properties of biodegradable polymers to alter the release profile of compounds from the implant are well known in the art.

[0111] Enteral / Oral / Lingual Formulations Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, sodium saccharin, starch, magnesium stearate, cellulose, magnesium carbonate, etc. Such compositions will contain a therapeutically effective amount of the compound and / or antibiotic with an appropriate amount of carrier to provide an appropriate form for the patient based on the mode of administration being used.

[0112] Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, lozenges, etc. Tablets can be made using compression or molding techniques well known in the art. Gelatin or non-gelatin capsules can be prepared using techniques well known in the art as hard or soft capsule shells that can enclose liquid, solid, or semi-solid fills.

[0113] The formulation can be prepared using a pharmaceutically acceptable carrier.The term "carrier" as generally used herein includes but is not limited to diluents, preservatives, binders, lubricants, disintegrants, swelling agents, fillers, stabilizers, and combinations thereof.

[0114] The carrier also includes all components of the coating composition, including plasticizers, pigments, colorants, stabilizers, lubricants, and the like.

[0115] Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, methacrylic resins commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, polysaccharides.

[0116] Additionally, the coating agent may include conventional carriers such as plasticizers, pigments, colorants, lubricants, stabilizers, pore formers, surfactants, and the like.

[0117] "Diluents," also known as "fillers," are typically required to increase the bulk of a solid dosage form to provide a practical size for tablet compression or bead or granule formation. Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starch, pregelatinized starch, silicon dioxide, titanium oxide, magnesium aluminum silicate, powdered sugar, and the like.

[0118] "Binders" are used to impart cohesion to solid dosage forms, preventing tablets, beads, or granules from crumbling after formulation. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose, and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia and tragacanth, celluloses such as sodium alginate, hydroxypropylmethylcellulose, hydroxypropylcellulose, and ethylcellulose, and synthetic polymers such as veegum, acrylic and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylic acid copolymers, aminoalkyl methacrylic acid copolymers, polyacrylic acid / polymethacrylic acid, and polyvinylpyrrolidone.

[0119] "Lubricants" are used to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, mineral oil, and the like.

[0120] "Disintegrants" are used to facilitate the breakup or "break-up" of a dosage form after administration and typically include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clay, cellulose, arginine, gums, or cross-linked polymers such as cross-linked PVP (Polyplasdone® XL from GAF Chemical Corp).

[0121] "Stabilizers" are used to inhibit or slow down decomposition reactions of pharmaceuticals, such as oxidation. Suitable stabilizers include, but are not limited to, antioxidants, butylated hydroxytoluene (BHT), ascorbic acid, its salts and esters, vitamin E, tocopherol and its salts, sulfites such as sodium metabisulfite, cysteine ​​and its derivatives, citric acid, propyl gallate, and butylated hydroxyanisole (BHA).

[0122] Oral dosage forms such as capsules, tablets, solutions, and suspensions can be formulated for controlled release. For example, one or more compounds and optionally one or more additional active agents can be formulated into nanoparticles, microparticles, and combinations thereof, and then encapsulated in soft or hard gelatin or non-gelatin capsules, or dispersed in a dispersion medium to form oral suspensions or syrups. Particles can be formed with a drug and a release-controlling polymer or matrix. Alternatively, drug particles can be coated with one or more release-controlling coatings before being incorporated into a finished dosage form.

[0123] In another embodiment, the compound(s) and optionally the additional active agent(s) are in dosage form, wherein the compound(s) and optionally the additional active agent(s) are formulated into a marketed oral dosage form such as a tablet or capsule, and the solid dosage form is coated with one or more controlled-release coatings, such as a delayed-release coating or a sustained-release coating. The coating may also include the compound(s) and / or the additional active agent(s).

[0124] The following materials and methods are provided to facilitate the practice of the present invention.

[0125] animal Adult male Sprague-Dawley rats (Charles River) were housed individually in a temperature- and humidity-controlled satellite vivarium under a 12-hour light:12-hour dark cycle and provided with ad libitum access to water and a standard diet (5001, LabDiet) or a 60% high-fat diet (HFD; D12492, Research Diets). Kaolin pellets (K50001, Research Diets) were also available ad libitum as needed. Rats were exposed to kaolin for at least 5 days before measuring kaolin intake in the pica test. For all feeding tests, except those performed at BioDAQ, rats were housed in hanging wire cages to allow accurate measurement of food spillage.

[0126] Male shrews (Suncus murinus) weighing ~50–80 g were bred and maintained in the de Jonge Laboratory at the University of Pennsylvania. These animals are descendants of a colony previously maintained at the University of Pittsburgh Cancer Institute (Dr. Charles Horn); the Taiwanese strain was originally derived from stock provided by the Chinese University of Hong Kong. They were housed singly in plastic cages (37.3 x 23.4 x 14 cm, Innovive) under a 12-hour light:12-hour dark cycle in a temperature- and humidity-controlled environment. They were fed a diet consisting of a mixture of cat food (75% Laboratory Feline Diet 5003, Lab Diet) and mink food (25% High Density Ferret Diet 5LI4, Lab Diet) ad libitum and had tap water ad libitum.

[0127] Experiments were performed in accordance with the National Institutes for Health Guide for the Care and Use of Laboratory Animals, and all procedures were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.

[0128] surgery For cannula implantation, rats were anesthetized with an intraperitoneal injection of a mixture containing ketamine (90 mg / kg, Butler Animal Health Supply), xylazine (2.7 mg / kg, Anased), and acepromazine (0.64 mg / kg, Butler Animal Health Supply) (KAX) and placed in a stereotaxic apparatus. Each rat received a 26-gauge guide cannula (Plastics One) stereotaxically implanted into the fourth ventricle (guide cannula coordinates: midline, 2.5 mm anterior to the occipital suture, 5.2 mm ventral to the skull; internal cannula coordinates: 7.2 mm ventral to the skull) or lateral ventricle (guide cannula coordinates: 1.5 mm lateral to the midline, 0.9 mm posterior to the sternum, 1.8 mm ventral to the skull; internal cannula coordinates: 3.8 mm ventral to the skull). For all cannulae, a dummy cannula (not protruding beyond the guide) was inserted into the guide cannula and left in place until fluid infusion was administered. In all surgeries, rats received postoperative temperature support and received analgesics (2 mg / kg meloxicam) immediately after surgery and for 2 days postoperatively.

[0129] Studies on food and kaolin intake In all studies measuring food intake after drug administration, central injections were administered in a volume of 2 μL using a Hamilton syringe terminating in a syringe tip extending 2.0 mm outward from the guide cannula. Intraperitoneal injections were administered based on body weight (0.1 mL / 100 g body weight). On acute treatment days, rats were food-deprived for 2 h before the dark cycle, and injections were administered immediately before the onset of the dark cycle. Food intake and kaolin intake were measured 1, 3, 6, and 24 h after the end of injection, and food crumbs were weighed and accounted for between time points. Body weight was measured during and 24 h after injection. Injection treatments were administered in a balanced, within-subject design, with at least 72 h between treatments. In chronic feeding studies, drug injections were administered once daily, and body weight, food intake, and kaolin intake were recorded every 24 h immediately before the onset of the dark cycle. Similar injections were also administered to rats housed in a Biodaq system (Research Diets, Inc.) for dietary pattern analysis. The BioDaq system records in 1-second increments, allowing for the measurement of episodic food intake. Individual bouts (bouts) were initiated by the animal at the onset of feeding and terminated at the end of feeding. Bouts were separated by a 5-second inter-meal interval (IBI). A meal was defined as at least one meal of a minimum 0.02g volume separated by a 5-minute uninterrupted inter-meal interval (IMI). Cumulative food intake, meal count, meal duration, mean meal size (g / meal), and mean meal duration (sec / meal) were calculated for 0-1 h, 0-6 h, 6-12 h, and 12-24 h after drug injection. Additionally, cumulative food intake (g), meal count, meal duration, meal count, and meal duration were calculated at 20-minute intervals for 3 hours after injection in standard chow-fed rats and at 1-hour intervals for 24 hours after injection in HFD-fed rats.

[0130] Pharmaceuticals Most of the drugs used in these studies (ODN, TDN, OP, AntOP, SUODN04, and SUODN05) were synthesized by the Doyle laboratory at Syracuse University. Commercially available drugs include: rat recombinant diazepam-binding inhibitor (DBI) protein (LS-G136996, Lifespan Biosciences) and exendin-4 (11096, Caymen Chemical) dissolved in artificial cerebrospinal fluid (aCSF, Harvard Apparatus); liraglutide (24727, Caymen Chemical) dissolved in 40 mM Tris-HCl buffer (pH 8) with 0.02% Tween-80; and rabbit anti-DBI primary antibody (ab231910, Abcam) used to neutralize endogenous DBI, undiluted; the appropriate vehicle was a 1:1 solution of glycerol and aCSF.

[0131] Immunohistochemistry and quantification of hindbrain DBI Rats fed a standard diet or HFD were allowed to eat ad libitum or fasted for 24 hours. All rats were injected with aCSF or exendin-4 (0.3 μg / 2 μL) into the fourth ventricle 90 minutes before sacrifice. Rats were deeply anesthetized with KAX and transcardially perfused with 0.1 M phosphate-buffered saline (PBS; pH 7.4), followed by 4% paraformaldehyde in 0.1 M PBS on ice. Brains were removed from the skull, postfixed in 4% paraformaldehyde for 24 hours, and stored at 4°C in 20% sucrose in 0.1 M PBS until submerged. Coronal dorsal vagal complex (DVC) sections (30 μm) were sliced ​​and directly mounted on slides (12-550-15; Superfrost Plus, Fisher Scientific) using a cryostat and stored at -80°C until immunohistochemistry (IHC) was initiated. Briefly, tissues were washed three times for 8 min with 0.1 M PBS and incubated in blocking solution [5% normal donkey serum (Jackson Immunoresearch) in PBST; PBS plus 0.3% Triton X], followed by incubation overnight at 4°C with rabbit anti-DBI primary antibody (1:500; ab231910, Abcam) and chicken anti-vimentin primary antibody (1:2000; ab24525, Abcam). The next morning, slides were washed three times for 8 min with PBST and incubated for 3 h at room temperature with donkey anti-rabbit fluorescent secondary antibody (1:500; AlexaFluor 647, Jackson Immunoresearch) and donkey anti-chicken fluorescent secondary antibody (1:500; AlexaFluor 488, Jackson Immunoresearch). Finally, the slides were washed three times for 5 min with PBST and once with PBS, and then coverslipped with antifade mounting medium containing DAPI (H-1200, Vector Laboratories, Inc.). Slides were visualized under a fluorescence microscope (BZ-X810, Keyence).Image analysis was performed using the HALO® FISH-IF and Area Quantification FL modules to quantify the fluorescence intensity of DBI protein staining in the nucleus tractus solitarius (NTS) and posterior region (AP), as well as the colocalization rate of DBI and vimentin staining in the AP, area postrema, and fourth ventricle border.

[0132] Quantitative real-time (qPCR) testing Rats fed a standard diet were injected with aCSF or exendin-4 (0.3 μg / 2 μL) into the fourth ventricle 90 min before sacrifice. Brains were rapidly removed, flash-frozen in isopentane at -70°C, and stored at -80°C until processing. Micropunch tissue samples were taken from the DVC of each brain. Total RNA was extracted from each tissue site using TRIzol (Invitrogen) and the RNeasy kit (Qiagen). cDNA was synthesized from 200 ng of total RNA using the Advantage RT-for-PCR kit (Clontech). Relative mRNA levels of DBI were quantified using quantitative real-time PCR. Rat GapDH (VIC-MGB) was used as an internal control. PCR reactions were performed using TaqMan Gene Expression Kits (DBI: Rn00821402_g1 and GapDH: Rn01775763_g1), and PCR reagents were from Applied Biosystems. Samples were analyzed on a QuantStudio 6 Pro system (Applied Biosciences). Calculation of relative mRNA expression was completed using the comparative threshold cycle method.

[0133] Hindbrain glucose sensing studies Standard-diet rats were deprived of food 2 h before the dark cycle and then injected into the fourth ventricle immediately before the onset of the dark cycle. Baseline glucose levels were collected from tail vein blood using a glucometer (Concur) before injection and 30 and 60 min after injection. In one study, rats were pretreated with vehicle (aCSF) or OP (20 μg / 2 μL) and then administered vehicle or 5-thio-d-glucose (5-TG; 210 μg / 2 μL). In another study, rats were pretreated with vehicle or AntOP (20 μg / 2 μL) and then administered vehicle or D-glucose (5.5 M in 3 μL). After the final blood glucose measurement, food was restored 1 h after injection, and food intake was recorded 2, 4, 6, and 24 h after injection. Body weight was measured during and 24 h after injection.

[0134] Peptide synthesis and purification Solid-phase peptide synthesis was performed on ProTide Rink amide resin (CEM Corporation cat # R002) using a microwave-assisted CEM Liberty Blue peptide synthesizer (Matthews, NC). Fmoc-protected amino acids were coupled to the resin using 0.25 M Oxyma Pure (CEM Corporation cat # S001) and 0.125 M N,N'-diisopropylcarbodiimide (Sigma-Aldrich cat # D125407) as the activator and activated base, respectively. Fmoc was removed during coupling with 20% piperidine (Sigma-Aldrich cat # 8.22299.0500). Global deprotection and cleavage of the peptide from the solid support resin was achieved using a CEM Razor instrument by incubation for 40 min at 40 °C in a mixture of 95% TFA (Sigma-Aldrich cat # 8.08260.2501), 2.5% TIPS (Sigma-Aldrich cat # 233781), and 2.5% water. The peptide was purified on an Agilent 1200 Series high-performance liquid chromatography (HPLC) instrument (10-75% HPLC-grade acetonitrile (VWR cat # BDH83639.400)) using an Agilent Zorbax C18 column (5 μm, 9.4 x 250 mm) at a flow rate of 2 mL / min for 20 min, monitoring at 220 nm.

[0135] Centrally administered ODN antagonizes the relaxin-3 method Rats were stereotactically implanted with bilateral guide cannulae (26-gauge, Plastics One) aimed at the intercalary nucleus (guide cannula coordinates: ±0.5 lateral to the midline, 9.5 mm anterior to the sternum, 5.8 mm ventral to the skull; internal cannula coordinates: 7.8 mm ventral to the skull). Recombinant human relaxin-3 was purchased (130-10, PreproTech Inc.), and ODN was synthesized in the Doyle laboratory. Injection treatments [pretreatment with ODN (10 μg) or vehicle, treatment with relaxin-3 (32.4 nmol) or vehicle] were arranged in a logarithmically balanced, within-subject design, with a ≥72-h interval. Rats were deprived of food 2 h before the dark cycle, and injections were administered immediately before the onset of the dark cycle. All injections were performed in aCSF at a rate of 20 nL / s with a volume of 100 nL using a micropump-depressor (PHD 2000, Harvard Apparatus) equipped with a Hamilton syringe terminated in an injector tip extending 2.0 mm distally from bilateral guide cannulae implanted at the insertion coordinates described above. Food intake over 24 hours was recorded using a Biodaq system (Research Diets, Inc.). Body weight was measured during and 24 hours after injection.

[0136] DVC tissue extraction from Sprague Dawley male rats Male Sprague Dawley rats were anesthetized with isoflurane, rapidly decapitated, and their brains were removed and snap-frozen in isopentane at -70°C and stored at -80°C. Using a cryostat, 1 mm3 micropunches were taken from the DVC of the rat brainstem (consisting of the occipital cortex, nucleus tractus solitarius, and dorsal motor nucleus of the vagus nerve) at the level of the AP. These micropunches were pooled in cryovials and refrigerated at -80°C for the following protein / GPCR tissue extraction procedure.

[0137] ODN-binding protein / GPCR extraction procedure from rat DVC tissue Membrane proteins were extracted from rat brain tissue using the GPCR Extraction and Stabilization Reagent (GESR) (ThermoFisher Scientific, Rockford, IL) tissue protocol. Briefly, tissue samples were suspended in 1 mL of cold (4°C) PBS and washed repeatedly. The PBS was decanted, and 1 mL of cold (4°C) GESR was added to the tissue sample. The tissue sample was homogenized by pipetting 15–20 times until a uniform suspension was obtained. The homogenate was transferred to a new tube and incubated at 4°C for 30 minutes with end-to-end agitation. The sample was centrifuged at 16,000 x g for 20 minutes at 4°C. The supernatant containing the stabilized protein receptor was saved and stored at 4°C until analysis.

[0138] Binding assay Binding analysis was performed on a Nicoya open surface plasmon resonance instrument using a Nicoya streptavidin sensor chip. The coupling procedure followed the streptavidin sensor chip protocol, including surface conditioning and activation steps. For ligand immobilization, ODN-biotin (20 μg / mL in PBST pH 7 running buffer) was injected onto channel 2, with an interaction time of 5 minutes. This process was repeated several times to optimize immobilization. GPCR extract supernatant was injected into channels 1 and 2 of the chip, and a background-corrected binding curve was obtained. The chip was then immersed in 5 mL of MeOH for 16 hours at 4 °C. Electronic absorption spectra of the MeOH used to immerse the chip were obtained using a Nanodrop One. The remaining MeOH was mixed with water, lyophilized, and sent for MS / MS sequencing.

[0139] The following examples are provided to illustrate certain embodiments of the present invention, but are not intended to limit the invention in any way. [Example]

[0140] Example 1 Synthesis and testing of ODN peptides and derivatives Central administration of ODN into the fourth ventricle at 0.2, 2, and 20 μg / 2 μL dose-dependently reduced food intake 1 h after injection in standard chow-fed rats (Figure 1A) without affecting 24-h body weight change (Figure 1B). Furthermore, in diet-induced obese (DIO) rats maintained on a 60% high-fat diet (HFD), ODN dose-dependently reduced food intake at 12 and 24 h after injection (Figure 1C), while non-significantly reduced 24-h body weight change (Figure 1D). These data support the dose-dependent suppression of standard chow and HFD intake by intracerebroventricular administration of ODN, although the time course of the appetite suppression differed. ODN acts acutely in lean standard chow-fed rats, but its suppression is longer-lasting and more effective in obese rats.

[0141] Meal pattern data were obtained from standard diet-fed rats at time intervals of 0–1 h, 0–6 h, 6–12 h, and 12–24 h after ODN injection, examining food intake (grams), meal frequency, meal duration (seconds), meal length (seconds / meal), and meal size (grams / meal). ODN dose-dependently reduced food intake during the first hour (Figure 2A), but not during other time periods (Figures 2B–2D). ODN did not affect meal frequency (Figures 2E–2H) or time spent eating (Figures 2I–2L) at any time point. Meal length was reduced with 0.2 μg ODN during the first hour, but not during other time periods (Figures 2M–2P). ODN dose-dependently reduced meal size during the first hour (Figure 2Q), but not during other time periods (Figures 2R–2T). These data support the finding that ODN suppresses food intake during the first hour after injection in standard diet-fed rats by reducing meal size.

[0142] Heatmap representation of food intake (Figure 3A, grams), number of eating behaviors (Figure 3B), time spent eating behaviors (Figure 3C, seconds), number of meals (Figure 3D), and time spent eating (Figure 3E, seconds) 3 hours after injection in rats fed a standard diet. Compared to vehicle, 2 μg and 20 μg ODN reduced food intake at 60 min after injection, and 20 μg ODN also reduced food intake at 80 and 100 min (Figure 3A). Number of eating behaviors, time spent eating behaviors, and number of meals were unaffected by ODN (Figures 3B-3D), but 0.2 μg ODN reduced eating time at 80 min after injection compared to vehicle (Figure 3E).

[0143] Food intake (grams), meal frequency, meal duration (seconds), meal length (seconds / meal), and meal size (grams / meal) were measured in HFD-maintained rats at the following time intervals after ODN injection: 0-1 h, 0-6 h, 6-12 h, and 12-24 h. 20 μg ODN reduced food intake between 6 and 12 h after injection, but not at other times (Figures 4A-4D). ODN did not affect food intake patterns at any time frame (Figures 4E-4T).

[0144] Heatmaps of food intake (Figure 5A, grams), number of eating behaviors (Figure 5B), time spent eating (Figure 5C, seconds), number of meals (Figure 5D), and time spent eating (Figure 5E, seconds) in HFD-fed rats 24 hours after injection were displayed. ODN at 0.2, 2, and 20 μg reduced food intake at 12 and 15 hours after injection, at 18 hours at 0.2 μg and 20 μg, and even at 24 hours at 20 μg (Figure 5A). ODN did not significantly alter number of eating behaviors, time spent eating, number of meals, or meal duration (Figures 5B-5E).

[0145] The feeding pattern data for first eating behavior and meal intake after ODN injection in standard diet- and HFD-fed rats are shown in Figures 6A-6H. Consistent with the acute effects of ODN in standard diet-fed rats, ODN did not alter the latency to the first meal (Figure 6A), but 20 μg of ODN decreased the size of the first meal (Figure 6B). 2 μg of ODN increased the latency to the first meal (Figure 6C), but did not alter the size of the first meal (Figure 6D). Consistent with the delayed effects of ODN in obese rats, ODN did not affect the latency to the first meal or the size of the first meal (Figures 6E-6H).

[0146] ODN is a cleavage product of DBI. To understand how the parent peptide DBI regulates food intake in standard diet-fed and HFD-fed rats, we injected recombinant DBI protein into the fourth ventricle. In standard diet-fed rats, DBI significantly reduced food intake at 4, 5, 7, and 24 h postinjection (Figure 7A). Surprisingly, DBI did not reduce food intake in HFD-fed rats (Figure 7B). Body weight change at 24 h was not significantly reduced in either diet group (Figure 7C). These results indicate that exogenous DBI does not suppress food intake, while ODN is more effective in obese rats. These results suggest that there may be impaired cleavage of DBI to ODN in obesity, which could be targeted to improve endogenous ODN signaling.

[0147] Heatmaps of food intake (Figure 8A, grams), number of feeding behaviors (Figure 8B), time spent feeding behaviors (Figure 8C, seconds), number of meals (Figure 8D), and time spent eating (Figure 8E, seconds) 24 hours after injection of recombinant DBI in rats fed a standard diet are shown. DBI reduced food intake at 4, 5, and 7-24 hours post-injection (Figure 8A), and reduced locomotion at 8 and 21 hours post-injection (Figure 8B), but did not alter time spent eating behaviors (Figure 8C). DBI reduced meal frequency at 8-10 and 12-21 hours post-injection (Figure 8D), and time spent eating at 8-24 hours post-injection (Figure 8E). These data support the strong suppression of food intake by restricting meal duration and meal frequency in rats fed a standard diet.

[0148] ODN expression and release have been shown to be regulated by nutritional status, decreasing with fasting and increasing with refeeding and glucose uptake. We hypothesized that postprandially induced glucagon-like peptide-1 (GLP-1) might also regulate ODN signaling. To investigate the contribution of ODN signaling to the appetite-suppressing effect of GLP-1, rats fed a standard diet or HFD were pretreated with a DBI-targeting antibody (AB) to neutralize endogenous DBI protein or vehicle into the fourth ventricle. Subsequently, ODN, the GLP-1 receptor agonist exendin-4 (Ex-4), or vehicle were administered into the fourth ventricle. AB pretreatment reduced the appetite-suppressing effect of Ex-4 24 h postinjection (Figure 9A), but did not affect HFD intake (Figure 9B). Kaolin intake was measured, and pica behavior was assessed. As expected, Ex-4 increased kaolin consumption in standard diet-fed rats compared with controls, which was not significantly reduced numerically by AB pretreatment (Fig. 9C). Kaolin intake in HFD-fed rats did not differ among any of the treatment groups (Fig. 9D). Ex-4 reduced 24-hour body weight change in both standard diet-fed and HFD-fed rats relative to the control group, regardless of pretreatment (Fig. 9E-9F).

[0149] We repeated this study using an ODN antagonist (AntOP) administered into the fourth ventricle instead of the DBI-targeting antibody. AntOP pretreatment again demonstrated that the anorectic effect of Ex-4 was reduced in standard-diet and HFD-fed rats 24 h postinjection (Figures 10A-10B). Furthermore, AntOP pretreatment alone reduced 24-h HFD intake (Figure 10B). Ex-4 increased kaolin intake in standard-diet and HFD-fed rats compared with controls, but AntOP pretreatment of Ex-4 with AntOP did not significantly increase kaolin intake in standard-diet rats compared with controls (Figures 10C-10D). In standard-diet rats, AntOP pretreatment attenuated the robust weight loss induced by Ex-4 (Figure 10E), and this trend was also seen in HFD-fed rats (Figure 10F). AntOP pretreatment alone also reduced 24-h body weight change in HFD rats (Figure 10F).

[0150] Next, we wanted to assess whether antagonizing ODN signaling attenuates the anorectic response to peripheral GLP-1 receptor agonists. AntOP or vehicle was administered via the lateral ventricle, followed by intraperitoneal injections of liraglutide or vehicle. Liraglutide reduced food intake in standard diet-fed rats regardless of pretreatment (Figure 11A). Liraglutide reduced HFD intake, which was attenuated by AntOP pretreatment 1 and 3 h postinjection (Figure 11B). Kaolin intake was increased and body weight was reduced by liraglutide, but not by AntOP pretreatment in standard diet-fed or HFD-fed rats (Figures 11C-11F). The results in Figures 9-11 indicate that the anorectic response to central and peripheral GLP-1 receptor agonists is mediated, in part, by central ODN signaling.

[0151] Next, we investigated whether ODN and a GLP-1 receptor agonist could cooperate to reduce food intake by injecting ODN or vehicle into the fourth ventricle and liraglutide or vehicle intraperitoneally for 5 days. Liraglutide reduced daily food intake on days 1 and 2 relative to vehicle and cumulative food intake on days 1–5, and ODN potentiated the liraglutide-induced food intake suppression on day 1 (Figures 12A–12B). Daily and cumulative intake of kaolin and daily body weight did not differ between treatment groups (Figures 12C–12E). When we isolated food intake on day 1, ODN alone did not alter food intake, but significantly potentiated the appetite-suppressing effect of liraglutide (Figure 12F). These data suggest that, as we propose, the action of ODN may be downstream of GLP-1 signaling, and therefore, GLP-1 receptor agonists do not maximally stimulate ODN signaling. This finding suggests that the combination of an ODN agonist with a GLP-1 agonist can more effectively control appetite.

[0152] Tridecaneuropeptide (TDN), a predicted cleavage product of ODN, did not suppress standard chow intake when administered into the fourth ventricle (20 μg), but suppressed HFD intake 24 hours after injection (Figures 13A-13B). TDN did not alter kaolin intake in standard chow-fed rats, but mildly increased kaolin intake in HFD-fed rats (Figures 13C-13D). 24-hour body weight change tended to be reduced by TDN in HFD-fed rats (Figure 13E).

[0153] Next, we administered 20 and 200 μg of TDN into the lateral ventricle of rats fed a standard diet and an HFD. We observed that 200 μg of TDN reduced 24-hour standard diet intake and 1-hour and 24-hour HFD intake (Figures 14A-14B). TDN did not affect kaolin intake in either dietary group, and no significant changes were observed in body weight at 24 hours (Figures 14C-14F). Figures 13 and 14 show that centrally administered TDN also has an appetite-suppressing effect.

[0154] To investigate whether intraperitoneal administration of ODN suppresses appetite and to evaluate the emetic response to peripheral ODN in a vomiting model, shrews were intraperitoneally injected with 500 μg and 5000 μg of ODN. Both doses of ODN suppressed food intake for 24 hours without significantly changing body weight over the 24 hours. Only one of nine shrews experienced a vomiting episode in response to the low dose of ODN, whereas no vomiting episodes were observed with the high dose of ODN (Figures 15A-15C).

[0155] Several novel ODN-derived peptides (ODN 20 μg, TDN 20 μg, SUODN04 20 μg, SUODN05 20 μg) were administered into the fourth ventricle to test for appetite suppression. All ODN-based peptides suppressed HFD intake for 24 hours compared to vehicle, without significant changes in 24-hour body weight (Figures 16A-16B).

[0156] DBI is synthesized by glial cell populations in the dorsal vagal complex (DVC) of the hindbrain, including the area postrema (AP) and nucleus tractus solitarius (NTS). To investigate where DVC DBI protein expression is regulated by nutritional status and GLP-1 receptor signaling, we quantified the amount of fluorescently labeled DBI protein in the AP and NTS in both standard-diet and HFD-fed rats fed ad libitum, fasted for 24 hours, or fasted for 24 hours followed by an intravenous injection of the GLP-1 receptor agonist exendin-4 (Ex-4). At the pre-AP level, DBI expression in the NTS was unchanged by treatment in either standard-diet or HFD-fed rats (Figures 17A-17B). DBI expression at the AP level in the NTS was reduced by Ex-4 treatment in standard-diet-fed rats compared with both fed and fasted groups, but did not differ by treatment in HFD-maintained rats (Figures 17C-17D). NTS DBI expression at the level of the fourth ventricle was decreased by Ex-4 treatment in standard diet-fed rats compared with fasted rats, but there was no difference between treatments in HFD-maintained rats (Figures S17E-S17F). In the AP, DBI expression was increased by fasting and decreased by Ex-4 treatment in standard diet-fed rats, but there was no difference between treatments in HFD-fed rats (Figures S17G-S17H). These data suggest that DBI protein expression in the NTS and AP is regulated by fasting and is inversely regulated by Ex-4 treatment in the fasted state in standard diet-fed rats but not in HFD-fed rats.

[0157] Further studies showed that females were more sensitive to the anorectic effects of exogenous lateral ventricular ODN than males fed a standard diet or HFD (Figures 15-21). The effects of increasing doses of lateral ventricular ODN (20, 100, and 200 μg / 2 μL) on 24-h food intake were compared for body weight and intake in male and female rats. The results are shown in Figures 18A-18D. The data indicate that exogenous lateral ventricular ODN has a weaker anorectic effect after overnight fasting in male and female rats fed a standard diet or HFD. See also Figures 19A-19D. Figure 20 shows that centrally administered ODN suppresses food intake more strongly in HFD-fed females than in males. The effects of lateral ventricular ODN (20, 100, and 200 μg / 2 μL) on 24-h food intake and body weight in male and female rats. See Figures 20A-20D. FIG. 21 shows that centrally administered ODN weakly suppresses food intake in overnight fasted HFD-fed males.

[0158] As shown in Figure 22, females exhibited higher expression of endogenous hindbrain DBI protein. Brains from male and female rats fed a standard diet were harvested and stained with DBI antibody to detect DBI protein expression. The fluorescence was quantified at low, moderate, and strong levels using HALO-AI software. DBI protein expression was measured in the AP (Figure 22A), the border of the area postrema (Figure 22B), the NTS at the AP level (Figure 22C), the border of the fourth ventricle (Figure 22D), and the NTS at the post-AP level (Figure 22E). We also examined the degree of DBI and vimentin coexpression in the dorsal vagal complex (DVC) region, where tanycytes are concentrated. In the AP, 87.9% of vimentin-positive cells coexpressed DBI, and 16.9% of DBI-positive cells coexpressed vimentin. At the border of the AP and NTS, 74.8% of vimentin-positive cells co-expressed DBI, and 47.4% of DBI-positive cells co-expressed vimentin. At the border of the fourth ventricle, 58.9% of vimentin-positive cells co-expressed DBI, and 81.5% of DBI-positive cells co-expressed vimentin.

[0159] There is also data suggesting that endogenous relaxin-3 protein expression in nerve fibers is higher in females (see Figure 23). Brains from male and female rats fed a standard diet were harvested and stained with relaxin-3 (Rln3) antibodies to detect Rln3 protein expression. The fluorescence was quantified at low, medium, and high levels using HALO-AI software. Rln3 protein expression was measured in the central intercalated nucleus (Figure 23A) and lateral intercalated nucleus (Figure 23B), which are rich in Rln3-positive neuronal cell bodies; the more posterior central intercalated nucleus (Figure 23C) and lateral intercalated nucleus (Figure 23D), which are rich in Rln3-positive nerve fibers; and the NTS at the AP level (Figure 23E) and the fourth ventricle level (Figure 23F). Furthermore, as shown in Figure 24, pretreatment with ODN suppressed the increased feeding induced by centrally administered relaxin-3.

[0160] After chemical synthesis, the purity of selected peptides was measured by MALDI-ToF MS and RP-HPLC, as shown in Figures 21A-14E. The results are as follows: ODN [1911.13 g / mol; 11.521 min TR]; 100% purity; TDN [1454.63 g / mol; 11.587 min TR]; 100% purity; OP [909.95 g / mol; 5.017 min TR]; 96.4% purity; AntOP [909.95 g / mol; 14.941 min TR]; 96% purity; SUODN-03 [553.68 g / mol; 11.372 min TR]; 95% purity. TR = retention time on a Zorbax analytical C18 column.

[0161] Centrally optimized ODN and TDN suppress 24-h food intake in rats. ODN and TDN were optimized using acetate precipitation rather than TFA salt precipitation to maintain a neutral pH > 4.5 in HEPES buffer. The effects of fourth ventricle ODN (200 μg / 2 μL) on 24-h food intake (Figure 25A) and body weight (Figure 25B) in male standard diet-fed rats and the effects of lateral ventricle TDN (200 μg / 2 μL) on 24-h post-treatment food intake (Figure 25C) and body weight (Figure 25D) in female HFD-fed rats are shown. Peripheral TDN suppresses fasting-induced refeeding in DIO mice. See Figure 26. This data also demonstrates that chronic peripheral TDN suppresses food intake in mice. FIG. 27 shows the effects of daily IP TDN (5 mg / kg) treatment on 24-hour body weight change and food intake, 9-day cumulative body weight change, and food intake in male HFD-fed mice.

[0162] Previous studies have shown that central ODN signaling mediates glucose sensing in the hindbrain. We pretreated the fourth ventricle with an ODN agonist (OP 20 μg / 2 μL) or vehicle, followed by 5-TG (210 μg / 2 μL) or vehicle. We measured 24-hour food intake, 1-hour blood glucose levels, and body weight changes in standard-diet rats and compared these results with 24-hour food intake. We also pretreated the fourth ventricle with an ODN antagonist (AntOP 100 μg / 2 μL) or vehicle, followed by glucose (5.5 mM, 3 μL) or vehicle.

[0163] Figures 28A-28C show that hindbrain ODN improves glucose tolerance in standard-diet rats. Hindbrain ODN signaling controls counterregulatory responses. Figure 29 is a graph showing the effects of hyperglycemia (210 μg / 2 μL) or vehicle on 1-hour blood glucose levels, and 30 minutes later, plasma corticosterone and plasma glucagon in standard-diet-fed rats after administration of ODN (20 μg / 2 μL) or vehicle into the fourth ventricle followed by administration of 5-TG into the fourth ventricle.

[0164] Further study has observed that hindbrain ODN improves insulin resistance in rats fed with standard diet.See Figure 30.Furthermore, as shown in Figure 31, pretreatment with relaxin-3 and ODN antagonist blocks the improvement of insulin resistance in rats fed with standard diet by hindbrain ODN.

[0165] Dynamic light scattering data were also generated, demonstrating that TDNs have various hydrodynamic radii (d. nm) and form complex structures (monomers, dimers, tetramers, etc.) in solution at different concentrations. These structures can exhibit activity and can favorably interact with or modulate different functions of target receptors. Finally, the structures in Figure 33 89 Zirconium-labeled TDN PET scans showed significant uptake in the brain in coronal images of athymic nude mice (n = 3). Insulin-resistant TDN-DFO (0.19 mg, 80 nmol) was reconstituted in saline (pH 7) and reacted with 89Zr (930 mCi, 34.4 MBq) at a 1:5 (peptide:activity) ratio for 45 min at RT. Radiochemical purity was assessed by instant thin-layer silica gel chromatography (iTLC-SG; Agilent Technologies) using 50 mM EDTA as the mobile phase. The reaction was quenched with EDTA (5 mL, 50 mM) to encapsulate unreacted activity, and unbound radiometal was removed by centrifugation through a spin column (MWCO = 10 kDa) in sterile saline. Athymic nude mice (n = 3) were injected intravenously via the lateral tail vein with [89Zr]-DFO-TDN (4.8-5.6 MBq, 130-150 Ci, 60-70 nmol) in sterile saline. Small-animal PET was performed 48 hours after injection using a Bruker Albira Si PET / CT system while the mice were anesthetized with 2% isoflurane. Images were reconstructed by maximum likelihood expectation maximization with 12 iterations and a voxel resolution of 0.75 mm and analyzed using PMOD version 4.3 software. Volumes of interest were manually measured by drawing the target site across various planar sections and expressed as percent injected dose per tissue volume (%ID / mL).

[0166] Collectively, these results indicate that centrally administered ODNs and novel ODN-based peptides (e.g., TDN) reduce food intake in standard-chow and HFD-fed rats and mice, that hindbrain DBI protein expression is regulated by nutritional status, that GLP-1 agonism in standard-chow rats is blunted in HFD-fed rats, that centrally administered GLP-1 agonism upregulates DBI mRNA expression in standard-chow-fed rats, that blocking ODN signaling with DBI-targeting antibodies or ODN antagonists attenuates the appetite-suppressing effects of central and peripheral GLP-1 analogs, that ODN and GLP-1 signaling cooperate to suppress food intake, and that ODN is involved in the hindbrain glucose-sensing response.

[0167] Example 2 Drug target screening using the gpcrMAX and orphanMAX assays The gpcrMAX and orphanMAX panels are intended to provide a cost-effective means for identifying potential interactions with some of the known GPCR or orphan GPCR targets. Compounds are typically tested at a single concentration, resulting in a semi-quantitative determination of efficacy. Potential interactions can then be confirmed with dose-response follow-up studies.

[0168] gpcrMAX - agonist mode Activation of GPCRs by compounds acting as agonists increases the recruitment of β-arrestin to the target GPCR. Results tables (Tables 4 and 5) show the mean, standard deviation (SD), and %CV of control values ​​for both baseline (Control 1) and maximum control ligand response (Control 2 - Max). Compound RLU (raw measurements) are provided, along with the mean RLU, standard deviation, and %CV for the test compound. Compound % activity is calculated as the % activity relative to baseline (0% activity) and maximum (100% activity).

[0169] gpcrMAX - antagonist mode Inhibition of GPCR activation by compounds that act as antagonists of ligand binding results in reduced recruitment of β-arrestin to the target GPCR. Results tables show the mean, standard deviation (SD), and %CV of control values ​​for both the EC80 (Control 1) and basal ligand response (Control 2 - Basal). Compound RLU (raw measurements) are provided along with the mean RLU, SD, and %CV of the test compound. Percent compound inhibition is calculated as percent inhibition relative to the EC80 (0% inhibition) and basal values ​​(100% inhibition). These assays provide a means to determine whether compound activity observed in the panel is potentially significant and worthy of follow-up. Different approaches are recommended for different panel types and assay modes.

[0170] orphanMAX - agonist mode Activation of an orphan GPCR by a compound acting as an agonist increases the recruitment of β-arrestin to the target orphan GPCR. Results tables show the mean, standard deviation (SD), and %CV of the observed baseline activity control values. Because different GPCRs exhibit varying expression levels and constitutive arrestin recruitment, baseline mean RLU values ​​vary for each target. Compound RLU (raw measurements) are provided along with the mean RLU, standard deviation, and %CV for the test compound. Because known ligands are typically not available for orphan GPCR assays, activity is calculated differently for the OrphanMAX GPCR Panel compared to the gpcrMAX Panel. Activity is calculated based on the baseline response only. Therefore, a 2-fold increase in mean RLU for a compound over baseline results in a % activity value of 100%. Similarly, a 3-fold increase corresponds to 200% activity.

[0171] Each assay can be performed on a GPCR with a known or unknown ligand.

[0172] The neuropeptide relaxin-3 (rxfp3) is synthesized in the intercalary nucleus of the hindbrain and has orexigenic properties that increase food intake and promote weight gain. Eurofins' proprietary GPCR screening identified the glial synthetic saturating factor (ODN) as an endogenous antagonist of the relaxin-3 receptor. To investigate whether ODN plays a functional role in antagonizing the effects of relaxin-3 in vivo, we injected ODN into the intercalary nucleus, a nucleus that not only synthesizes relaxin-3 but also expresses high levels of the relaxin-3 receptor, prior to relaxin-3 injection into rats. As expected, relaxin-3 increased 24-h food intake and body weight, but pretreatment with ODN significantly reversed these effects (Figure 31). These data support the idea that ODN is an endogenous antagonist of the relaxin-3 receptor rxfp3 and provide a mechanism for the appetite suppression mediated by centrally administered ODN signaling.

[0173] Table 4: Control dose-response curves for selected GPCR biosensor assays [Table 4] JPEG2025540675000005.jpg214170JPEG2025540675000006.jpg217170JPEG2025540675000007.jpg21817 0JPEG2025540675000008.jpg216170JPEG2025540675000009.jpg215170JPEG2025540675000010.jpg65170

[0174] *The assay mode for each compound was agonist. **The assay name for each compound is Arrestin.

[0175] Table 5: Agonist and antagonist activities of compounds measured by GPCR biosensor assay [Table 5] JPEG2025540675000012.jpg220170JPEG2025540675000013.jpg220170JPEG2025540675000014.jpg22017 0JPEG2025540675000015.jpg220170JPEG2025540675000016.jpg220170JPEG2025540675000017.jpg84170

[0176] While certain features of the invention have been described herein, many modifications, substitutions, changes, and equivalents will occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

1. An isolated or purified octadecaneuropeptide (ODN) or a synthetic derivative thereof, comprising an amino acid sequence selected from one or more of SEQ ID NOs: 1 to 21, or a functional fragment thereof, or a sequence having at least 95% identity thereto, wherein the octadecaneuropeptide regulates food intake and body weight.

2. The octadecaneuropeptide of claim 1 , wherein the octadecaneuropeptide suppresses vomiting and nausea.

3. 3. The octadecaneuropeptide of claim 1 or claim 2, comprising one or more of the following: i) a modified amino acid listed in Table 2, and / or ii) Gln(alkyne), Ala(alkyne), Gly(alkyne) and Lys(N3).

4. The octadecaneuropeptide of claim 3, wherein the modified amino acids facilitate bioconjugation of one or more agents selected from N- and / or C-terminal protection moieties, lipids, recombinant FC-peptides, and cell-penetrating peptides, enhancing one or more of the function, stability, and bioavailability of the peptide.

5. An octadecaneuropeptide according to any one of the preceding claims, which is a synthetic derivative of SEQ ID NO: 3 or a functionally modified variant thereof.

6. A composition comprising an octadecaneuropeptide according to any one of the preceding claims in a pharmaceutically acceptable carrier or buffer.

7. A nucleic acid sequence encoding any one of the octadecaneuropeptides having amino acids of SEQ ID NOs: 1 to 21 according to claim 1.

8. 8. The nucleic acid of claim 7, which encodes the polypeptide of SEQ ID NO:

3.

9. 9. The nucleic acid of claim 7 or 8, encapsulated in a liposome or extracellular vesicle, or attached to a nanoparticle or lipid nanoparticle.

10. A vector comprising the nucleic acid of claim 7 or 8.

11. The vector according to claim 10, which is a plasmid vector, a lentiviral vector, an AAV vector, AAV9, or AAV8.

12. 12. The vector of claim 10 or 11, which is encapsulated in a liposome or extracellular vesicle, or attached to a nanoparticle or lipid nanoparticle.

13. A composition comprising a nucleic acid encoding an octadecaneuropeptide described in any one of the preceding claims in a pharmaceutically acceptable carrier or buffer.

14. 14. A method of treating a metabolic disease or disorder in a patient in need thereof, comprising administering an effective amount of the composition of claim 6 or the composition of claim 13.

15. 15. The method of claim 14, wherein the metabolic disease or disorder is selected from obesity, diabetes, dyslipidemia, insulin resistance, hepatic steatosis, hypercholesterolemia, and non-alcoholic fatty liver disease.

16. 16. The method of claim 15, wherein the diabetes is selected from type 1 diabetes or type 2 diabetes.

17. 17. The method of any one of claims 14 to 16, comprising administering a second therapeutic agent that treats or inhibits progression of at least one disorder selected from obesity, diabetes, dyslipidemia, insulin resistance, hepatic steatosis, hypercholesterolemia, and non-alcoholic fatty liver disease.

18. 18. The method of any one of claims 14 to 17, further comprising providing the subject with a lifestyle intervention relating to diet and / or physical activity.

19. 18. The method of any one of claims 14 to 17, wherein the patient has reduced food intake 1, 3, 6, and / or 24 hours after administration of the peptide or nucleic acid encoding the peptide compared to untreated controls.

20. The method of any one of claims 14 to 17, wherein the patient loses weight after administration of the peptide.

21. 21. The method of any one of claims 14-20, further comprising evaluating the patient for a reduction in symptoms associated with at least one disorder selected from obesity, diabetes, dyslipidemia, insulin resistance, hepatic steatosis, hypercholesterolemia, and non-alcoholic fatty liver disease.

22. 21. The method of any one of claims 14 to 20, wherein the composition or carrier is formulated to cross the blood-brain barrier and administration is via an intracranial, intraparenchymal, intraventricular, or transmeningeal route.

23. 21. The method of any one of claims 14 to 20, wherein administration of the peptide is via a route selected from systemic, parenteral, transdermal patch, intramuscular, subcutaneous, buccal and oral.

24. 24. The method of any one of claims 14 to 23, wherein the composition is effective to inhibit relaxin-3 receptors, resulting in an appetite suppressant effect.

25. 23. The method of any one of claims 13 to 22, wherein the composition functions to modulate one or more of the following: i) improve glucose tolerance; ii) modulating the activity of the relaxin-3 receptor; iii) control counterregulatory responses and suppress 5-tg-induced hyperglycemia; iv) improving insulin sensitivity; v) Promotes weight loss vi) Penetrates the brain.

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