Intraluminal delivery of bitter tasting oligopeptides for the treatment of type 2 diabetes

Abstract

Described herein are methods and compositions for treating diabetes and / or obesity. Bitter taste oligopeptide molecules formulated for enteral delivery modulate signals involving receptors facing the lumen of the gastrointestinal tract that involve hormones involved in gastric emptying and inhibition of appetite, such as glucagon-like peptide-1 (GLP-1) and peptide tyrosine-tyrosine (PYY). As a novel way of treating diabetes with limited side effects, the described invention uses the body's own endocrine system to treat diabetes, which is an advantage over existing therapies that can only provide disease management without a cure or require more aggressive approaches such as surgical intervention.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201680034226.0, filed on April 22, 2016, entitled "Intestinal-delivered bitter oligopeptide for the treatment of type 2 diabetes". Technical Field

[0002] This invention relates to compositions and methods for treating diabetes. Background Technology

[0003] All disclosures herein are incorporated by reference as if each individual disclosure or patent application had been expressly and individually indicated to be incorporated by reference. The following description includes information useful for understanding the invention. It is not acknowledged that any information provided herein is prior art or related to the currently claimed invention, nor is it acknowledged that any disclosure expressly or implicitly referenced is prior art.

[0004] On the surface of the gastrointestinal epithelium, molecular sensing mechanisms are endowed for detecting dietary components and intestinal microbial metabolites. Many types of enteroendocrine cells have been identified, and they are primarily classified by the specific contents of their endocrine neurotransmitters. Some key examples include enteroendocrine I cells containing cholecystokinin (CCK); and L cells containing glucagon-like peptide-1 (GLP-1) and peptide tyrosine-tyrosine (PYY). The release of these agents into the bloodstream is generated by the interaction of receptors on the cellular luminal surface with nutritional or environmental factors in the intestinal contents. Each has a specific and essential function in gastrointestinal responses, including local and systemic metabolism.

[0005] GLP-1 is derived from the transcription product of the proglucagon gene. The biologically active forms of GLP-1 are GLP-1-(7-37) and GLP-1-(7-36)NH2. Once in circulation, GLP-1 has a half-life of less than 2 minutes due to rapid degradation by the enzyme dipeptidyl peptidase-4 (DPP4). It is a potent antiglycemic hormone, thereby inducing glucose-dependent stimulation of insulin secretion while inhibiting glucagon secretion. This glucose-dependent effect is particularly attractive because GLP-1 no longer stimulates insulin to induce hypoglycemia when plasma glucose concentrations are within the normal fasting range. GLP-1 restores the glucose sensitivity of pancreatic β-cells through mechanisms involving increased expression of GLUT2 and glucokinase. GLP-1 also inhibits pancreatic β-cell apoptosis, stimulates the proliferation and differentiation of insulin-secreting β-cells, and inhibits gastric secretion and motility. This delays gastric emptying, which promotes satiety and weight loss. In fact, GLP-1 analogs and inhibitors of endogenous GLP-1 degradation have been developed and demonstrated efficacy in treating obesity-related types of type II diabetes. These analogs have not only been shown to significantly improve insulin secretion and glucose control, but they have also been found to reduce gastric emptying and increase satiety, resulting in weight loss benefits. L-cells also release two circulating forms of PYY: PYY1-36 and PYY3-36. The latter form is believed to be the dominant form during both fasting and feeding states and is produced by the peptidase DPP4 cleaving the N-terminal Tyr-Pro residues from PYY1-36. PYY inhibits food uptake via the PYY-2 receptor expressed in neurons of the arcuate nucleus of the hypothalamus. Other effects of PYY include slowing gastric emptying and slowing small intestinal motility.

[0006] Numerous studies using animal and human models have been conducted to probe the fundamental mechanisms of nutrient-sensing receptors in the gut. These studies have identified taste receptors (sweet, umami, and bitter) and fatty acid receptors (activated by a wide range of chain lengths in fatty acids) in various enteroendocrine cells. Sweet and umami receptors are most likely to sense or taste energy nutrients and amino acids in food, while bitter receptors potentially sense or taste any harmful and toxic components in food. Once these food components are sensed, several metabolic pathways are activated. In the case of bitter-sensing components, pathways that slow gastric emptying and food absorption may be activated. Many of these associations are not well characterized, especially in the context of human physiology, and have been found to be slow. Several studies have shown that the release of GLP-1 or other hormones such as PYY, CCK, and ghrelin can be achieved by activating one of these receptors. These associations are crucial for understanding the detailed role of these gut taste receptors in food digestion.

[0007] The physiological role of peptide hormones released from endocrine cells lining the gastrointestinal lumen has been known for some time, but the mechanisms by which these hormones are "sensed" and secreted through intestinal contents were previously unknown. It has now been discovered that taste receptors, previously thought to be confined to the lingual epithelium, are also present in the stomach, small intestine, and colon. Several enteroendocrine cell types express TAS2R family bitter taste receptors and T1R2 / 3 sweet taste receptors. In fact, enteroendocrine cells respond to bitter ligands such as phenylthiourea (PTC) by secreting GLP-1, peptide YY (PYY), or CCK, which are specific activators of bitter taste receptors TAS2R38 and denatonium, activating the bitter taste receptor TAS2R47.

[0008] To understand the nutrient-mediated signaling of L cells in the context of peptide hormone release, the inventors focused on the bitter taste receptor TAS2R38 to investigate its mechanistic role in the release of GLP-1 hormone from L cells. The inventors have identified four bitter oligopeptides (BPx1, BPx2, BPx3, and BPx4) derived from whey protein that can be used as therapeutic agents for patients with type 2 diabetes. Summary of the Invention

[0009] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, compositions and methods intended to be exemplary and illustrative rather than limiting.

[0010] This document provides a method for treating, inhibiting, reducing the severity of diabetes and / or obesity in a subject of need, slowing the progression of diabetes and / or obesity, and / or promoting the prevention of diabetes and / or obesity. The method includes providing an agent that increases the release of intestinal hormones and administering an effective amount of the composition to a subject of need to treat, inhibit, reduce the severity of diabetes and / or obesity in the subject, slow the progression of diabetes and / or obesity, and / or promote the prevention of diabetes and / or obesity. In one embodiment, the diabetes is type 2 diabetes.

[0011] In some embodiments, the intestinal hormone is any one or more of glucagon peptide-1 (GLP-1), peptide tyrosine-tyrosine (PYY), or combinations thereof. In some embodiments, GLP-1 is any one or more of GLP-1-(7-37), GLP-1-(7-36)NH2, or combinations thereof.

[0012] In some embodiments of the method, the agent is one or more bitter oligopeptides. In exemplary embodiments, the bitter oligopeptides include any one or more of YGLF, YPFPGPIPN, IPAVF, LLF, combinations thereof, or analogs thereof, pharmaceutical equivalents, or peptide mimics, substantially composed of or composed of such agents.

[0013] In one embodiment, the bitter oligopeptide is conjugated with a pharmaceutical agent to increase intestinal retention. In some exemplary embodiments, the pharmaceutical agent for increasing intestinal retention is any one or more of cellulose, fatty acids, polyethylene glycol (PEG), or combinations thereof.

[0014] In some embodiments, the methods described herein further include providing food-derived fatty acids or plant molecules that increase the release of intestinal hormones. In exemplary embodiments, food-derived fatty acids or plant molecules include, but are not limited to, any one or more of the following: urolithin A, ellagic acid, ursolic acid, oleanolic acid, 6-m-propyl-2-thiouracil, propionic acid, butyrate, palmitic acid, or combinations thereof.

[0015] In various embodiments of the methods described herein, the increase in intestinal hormone release is mediated by G protein-coupled receptors (GPCRs), wherein the GPCRs are expressed on the surface of endocrine L-cells. In some embodiments, the GPCR is any one or more of SSTR2, TAS2R38, TAS2R39, TAS2R46, TAS2R47, FFAR1, FFAR2, FFAR4, or FFAR4.

[0016] In various embodiments of the method, the composition is administered orally, enterally into the small intestine, or via rectal suppository or enema into the colon. In one embodiment, the subject is a human.

[0017] In various embodiments, the methods described herein also include administration of any one or more of an SGLT2 inhibitor, insulin, inhaled insulin, sulfonylureas, metformin, acarbose, thiazolidinediones, or combinations thereof.

[0018] In some embodiments, the effective amount of the agent is about 0.1 mg / kg / day to 0.5 mg / kg / day, 0.5 mg / kg / day to 5 mg / kg / day, 5 mg / kg / day to 10 mg / kg / day, 10 mg / kg / day to 20 mg / kg / day, 20 mg / kg / day to 50 mg / kg / day, 50 mg / kg / day to 100 mg / kg / day, 100 mg / kg / day to 200 mg / kg / day, or 200 mg / kg / day. The dosage ranges from 300 mg / kg / day to 400 mg / kg / day, 400 mg / kg / day to 500 mg / kg / day, 500 mg / kg / day to 600 mg / kg / day, 600 mg / kg / day to 700 mg / kg / day, 700 mg / kg / day to 800 mg / kg / day, 800 mg / kg / day to 900 mg / kg / day, or 900 mg / kg / day to 1000 mg / kg / day. The composition may be administered to the subject before, during, or after the development of diabetes and / or obesity. In some embodiments, the composition is administered to the subject 1-3 times daily or 1-7 times weekly. In some embodiments, the composition is administered to the subject for 1-5 days, 1-5 weeks, 1-5 months, or 1-5 years.

[0019] In each implementation scheme, agents that increase the release of intestinal hormones and fatty acids are administered sequentially or simultaneously.

[0020] This document also provides pharmaceutical compositions comprising one or more bitter oligopeptides and a pharmaceutically acceptable carrier. In some embodiments, the bitter oligopeptide stimulates the release of glucagon-like peptide-1 (GLP-1), peptide tyrosine tyrosine (PYY), or a combination thereof. In some embodiments, the release of GLP-1, PYY, or a combination thereof is mediated by G protein-coupled receptor (GPCR) signaling. In some embodiments, the GPCR comprises one or more of TAS2R38, TAS2R39, TAS2R46, TAS2R47, FFAR1, FFAR2, FFAR4, or FFAR4, SSTR2. In some embodiments, the bitter oligopeptide comprises one or more of YGLF, YPFPGPIPN, IPAVF, LLF, combinations thereof, or analogs thereof, pharmaceutical equivalents, or peptide mimics. In some embodiments, the bitter oligopeptide is conjugated to any one or more of cellulose, fatty acids, polyethylene glycol (PEG), or combinations thereof.

[0021] This document also provides a method for screening peptides for the treatment of diabetes and / or obesity. The method includes providing one or more candidate peptides, contacting the peptides with cells that secrete GLP-1, and determining whether the contact causes an increase in GLP-1 secretion, the increase in GLP-1 secretion indicating that the candidate peptide may be used to treat diabetes. The screening method includes contacting each of a plurality of candidate peptides to be tested, respectively. In some embodiments, the plurality of candidate peptides includes more than about 10 4 Multiple samples. In some embodiments, the multiple samples include more than about 5 x 10 samples. 4 One sample. In some embodiments, the candidate peptide is a bitter oligopeptide. In some embodiments, the cells are Hu-Tu80 cells. Attached Figure Description

[0022] Exemplary embodiments are illustrated in the referenced figures. The various embodiments and figures disclosed herein are to be regarded as illustrative rather than restrictive.

[0023] Figure 1 This section describes various embodiments of the invention, including known receptors and their potential functions in L-cells, including an overview of receptor-mediated control of GLP-1 and PYY secretion from enteroendocrine cells. Question marks indicate receptors that have not yet been identified.

[0024] Figure 2 The binding pocket of α-lactamase tetrapeptide (cyan) in the bitter taste receptor TAS2R38 is depicted according to various embodiments of the present invention.

[0025] Figure 3 Dose-response data are depicted for GLP-1 release from HuTu-80 cells for bitter oligopeptides (BPx1 = YGLF, BPx2 = YPFPGPIPN, BPx3 = IPAVF, BPx4 = LLF), fatty acid ligands, and metformin, according to various embodiments of the present invention.

[0026] Figure 4A and Figure 4B The release of GLP-1 in mice treated with PTU-cellulose, PTU, or cellulose is depicted according to various embodiments of the present invention. Figure 4A Synthesis of PTU-cellulose. Figure 4BThe mean GLP-1 release in seven mice treated with PTU-cellulose, PTU, or cellulose was measured. In male C57Bl / 6 mice, the PTU-cellulose dose was 5 g / kg body weight; the PTU dose was 200 mg / kg body weight; and the cellulose dose was 5 g / kg body weight. Blood was drawn at indicated times and serum GLP-1 levels from said blood were measured using an EGLP-35K glucagon-like peptide-1 (active) ELISA kit (Millipore, MA).

[0027] Figure 5 According to various embodiments of the invention, GLP-1 release is stimulated in cultured HuTu-80 cells by the known TAS2R38 ligand PTU, said cells either untreated or treated with the medium and the indicated concentration for 30 minutes. Conditioned culture medium is collected and frozen until GLP-1 is measured. Values ​​are mean ± SE, N = 2 (PTU). GLP-1 is measured using a Luminex assay.

[0028] Figure 6 The fold change of TAS2R38 mRNA in HuTu-80 cells, siRNA control, and receptor siRNA is depicted according to various embodiments of the present invention.

[0029] Figure 7A and Figure 7BAccording to various embodiments of the invention, TAS2R38 is expressed in most HuTu-80 cells. Human duodenal epithelial cancer cells HuTu-80 (catalog number HTB-40, ATCC, Manassas, VA) were seeded in 4-well slides (Nunc, Thermo Fisher Scientific, Rochester, NY) coated with poly-L-lysine (Sigma, St. Louis, MO). The confluent cells were fixed with 4% paraformaldehyde (Sigma) for 30 min, washed three times with PBS (Sigma), permeated with 0.25% Triton X-100 in PBS for 10 min, washed three times with PBS, and blocked with 5% BSA in PBS for 2 h. The fixed cells were incubated overnight with rabbit TAS2R38 antibody (ab65509, Abcam, Cambridge, MA) at a concentration of (A) 1:150 or (B) 1:250. Cells were washed three times with PBS and incubated overnight with 1:400 Alexa Fluor 488 goat anti-rabbit IgG antibody. They were then washed three times with PBS and counterstained with mounting medium containing DAPI (Vector Labs, Burlingame, CA). Fluorescent green (TAS38R) and blue (DAPI) were visualized on a Nikon Eclipse Ti-U microscope using proprietary Elements software (Nikon, Melville, NY).

[0030] Figure 8According to various embodiments of the invention, siTAS2R38 is knocked down in most HuTu-80 cells. Human duodenal epithelial cancer cells HuTu-80 (catalog number HTB-40, ATCC, Manassas, VA) were transfected via nuclear perforation (Lonza, Walkersville, MD) with either control siRNA or TAS2R38 siRNA (Qiagen, Valencia, CA). These transfected HuTu-80 cells were seeded in four-well slides coated with poly-L-lysine (Sigma, St. Louis, MO) (Nunc, Thermo Fisher Scientific, Rochester, NY). The confluent cells were fixed with 4% paraformaldehyde (Sigma) for 30 min, washed three times with PBS (Sigma), permeated with 0.25% Triton X-100 in PBS for 10 min, washed three times with PBS, and blocked with 5% BSA in PBS for 2 h. Fixed cells were incubated overnight with a 1:250 concentration of rabbit TAS2R38 antibody (ab65509, Abcam, Cambridge, MA). Cells were washed three times with PBS and incubated overnight with a 1:400 concentration of second Alexa Fluor 488 goat anti-rabbit IgG antibody, washed three times with PBS, and counterstained with mounting medium containing DAPI (VectorLabs, Burlingame, CA). Fluorescent green (TAS38R) and blue (DAPI) were visualized and captured using proprietary Elements software on a Nikon Eclipse Ti-U microscope.

[0031] Figure 9A and Figure 9B Various embodiments of the invention are described, illustrating the stimulation of GLP-1 release in cultured HuTu-80 cells by known bitter ligands, phytochemicals, and fatty acid ligands. Figure 9A Stimulated with pomegranate tannin metabolites and loquat extract TP components; Figure 9B FFAR was activated by short-chain and long-chain free fatty acids. HuTu-80 cells were treated untreated (zero time) or with indicated concentrations of the medium and various phytochemicals for 30 minutes. Conditioned medium was collected and frozen until GLP-1 was measured as described in the methods below. Values ​​are mean ± SE, N = 3, *P < 0.05, **P < 0.01

[0032] Figure 10 The invention describes the in vivo GLP-1 release in healthy mice following administration of BPx1 bitter oligopeptide at two different concentrations according to various embodiments.

[0033] Figure 11A and Figure 11B According to various embodiments of the invention, when TAS2R expression is inhibited, the increase in GLP-1 release is reduced in the BPx1 case, thereby indicating that GLP-1 release is mediated by TAS2R38 activation mediated by BPx1. Detailed Implementation

[0034] All references cited herein are incorporated herein by reference in their entirety as if fully explained. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. Allen et al., Remington: The Science and Practice of Pharmacy, 22nd ed., Pharmaceutical Press (September 15, 2012); Hornyak et al., Introduction to Nanoscience and Nanotechnology, CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 3rd ed., revised edition, J. Wiley & Sons (New York, NY 2006); Smith, March's Advanced Organic Chemistry Reactions, Mechanisms and Structure, 7th ed., J. Wiley & Sons (New York, NY 2013); Singleton, Dictionary of DNA and Genome Technology, 3rd ed., Wiley-Blackwell (November 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY). (2012), providing general guidance to those skilled in the art on many of the terms used in this application. For references on how to prepare antibodies, see Greenfield, Antibodies: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press (Cold Spring Harbor NY, 2013); And Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. July 1976, 6(7):511-9; Queen and Selick, Humanized immunoglobulins, US Patent No. 5,585,089 (December 1996); and Riechmann et al., Reshaping human antibodies for therapy, Nature March 24, 1988, 332(6162):323-7.

[0035] For references on pediatrics, see Schwartz et al., The 5-Minute Pediatric Consult, 4th edition, Lippincott Williams & Wilkins, (June 16, 2005); Robertson et al., The Harriet Lane Handbook: A Manual for Pediatric House Officers, 17th edition, Mosby, (June 24, 2005); and Hay et al., Current Diagnosis and Treatment in Pediatrics (Current Pediatrics Diagnosis & Treatment), 18th edition, McGraw-Hill Medical, (September 25, 2006).

[0036] Those skilled in the art will recognize that many methods and materials similar to or equivalent to those described herein can be used to practice this invention. In fact, this invention is by no means limited to the methods described herein. For the purposes of this invention, the following terms are defined below.

[0037] As used herein, the terms “comprising” or “comprises” are used with respect to compositions, methods, and their respective components that can be used in embodiments, and remain open-ended terms that include unspecified elements, whether or not said elements are required. Those skilled in the art will understand that, in general, the terms used herein are typically intended to be “open-ended” terms (e.g., the term “comprising” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “comprising” should be interpreted as “including but not limited to,” etc.).

[0038] Unless otherwise indicated, the terms “a / an” and “the” and similar references used in the context of describing particular embodiments of this application (particularly in the context of the claims) are to be understood to cover both singular and plural. The numerical ranges listed herein are merely intended as a way of abbreviating each individual value falling within the range. Each individual value is incorporated into this specification as if it were individually listed herein, unless otherwise indicated herein or the context clearly contradicts it. All methods described herein may be performed in any suitable order. The use of any and all instances or exemplary language (e.g., “as”) provided relative to certain embodiments herein is merely intended to better elucidate the application and not to impose limitations on the scope of the application as otherwise claimed. The abbreviation “eg” derives from the Latin word for example (exempli gratia) and is used herein to indicate non-limiting instances. Therefore, the abbreviation “eg” is synonymous with the term “for example.” The language in the specification should not be construed as indicating any unclaimed element necessary for practicing this application.

[0039] "Beneficial outcomes" may include, but are by no means limited to, mitigating or reducing the severity of the disease state, preventing the disease state from worsening, preventing the disease state from progressing, reducing the patient's likelihood of developing the disease state, and extending the patient's life or life expectancy. Beneficial or desired clinical outcomes include, but are not limited to, relief of one or more symptoms, reduction in the degree of impairment, stabilization (i.e., non-worsening) of diabetes progression, delay or slowing of diabetes, and improvement or mitigation of diabetes-related symptoms. Treatment also includes a reduction in mortality or an increase in lifespan compared to untreated subjects.

[0040] As used herein, “administering” and / or “administer” refers to any route of delivery of the pharmaceutical composition to a patient. In one embodiment, the composition described herein is administered enterically into the small intestine. Routes of delivery may include non-invasive oral (oral), topical (skin), transmucosal (nasal, buccal / sublingual, vaginal, ocular, and rectal) and inhalation routes, as well as parenteral routes and other methods known in the art. “Parenteral” refers to routes of delivery typically associated with injection, including intraorbital, infusion, intra-arterial, intracarotid, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intravertebral, intrasternal, intrasheath, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal routes. Through parenteral routes, the composition may be in the form of a solution or suspension for infusion or injection, or in the form of a lyophilized powder.

[0041] As used herein, the term "effective amount" refers to an amount in which a pharmaceutical composition comprising one or more peptides or mutants, variants, analogs, or derivatives thereof disclosed herein alleviates at least one or more symptoms of a disease or condition, and involves an adequate amount of the pharmaceutical composition to provide the desired effect. The phrase "therapeutic effective amount" as used herein refers to an adequate amount of a composition that treats a condition in a reasonable benefit / risk ratio applicable to any medical treatment.

[0042] A significant therapeutic or preventative reduction in symptoms is defined as a measurement of, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, or more, compared to the condition of a control or untreated subject or the subject prior to administration of the oligopeptides described herein. Measurable or detectable parameters include clinically detectable markers of the disease, such as elevated or decreased levels of biomarkers, and parameters associated with clinically accepted scales for the symptoms or markers of diabetes. However, it should be understood that the total daily dosage of the compositions and formulations disclosed herein will be determined by the attending physician within the bounds of reasonable medical judgment. The exact amount required will vary depending on factors such as the type of disease being treated, the subject's sex, age, and weight.

[0043] As used herein, “subject” means a person or animal. Animals are typically vertebrates, such as primates, rodents, livestock, or game animals. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, species of felines such as domestic cats, and species of canines such as dogs, foxes, and wolves. The terms “patient,” “individual,” and “subject” are used interchangeably herein. In one embodiment, a subject is a mammal. Mammals can be humans, non-human primates, mice, rats, dogs, cats, horses, or cattle, but are not limited to these examples. Furthermore, the methods described herein can be used to treat domesticated animals and / or pets. The terms do not indicate a specific age or sex. Therefore, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this terminology.

[0044] As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatment aimed at reversing, alleviating, improving, suppressing, slowing, or stopping the progression or severity of symptoms associated with a disease or condition. The term “treatment” includes reducing or alleviating at least one side effect or symptom of a symptom, disease, or condition such as diabetes (e.g., type 2 diabetes). Treatment is generally “effective” if one or more symptoms or clinical markers decrease. Alternatively, treatment is “effective” if the progression of the disease is slowed or stopped. That is, “treatment” includes not only improvement of symptoms or markers but also the cessation or at least slowing of the progression or worsening of symptoms without treatment. Beneficial or desired clinical outcomes include, but are not limited to, reduction of one or more symptoms, reduction of disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. The term “treatment” for a disease also includes providing relief of the symptoms or side effects of the disease (including palliative care).

[0045] As used in this article, “modulation” or “modulates” or “modulating” refers to the upregulation (i.e., activation or stimulation) or downregulation (i.e., inhibition or suppression) of a response, or both, in combination or separately.

[0046] As used herein, "pharmaceuticalally acceptable carrier" refers to a conventional pharmaceutically acceptable carrier applicable to this invention.

[0047] As used in this article, “promote” and / or “promoting” refers to an increase in a specific behavior of a cell or organism.

[0048] As used herein, "therapeutic agent" refers to a medicine used to treat, inhibit, prevent, alleviate, reduce the severity of, or decrease the likelihood of developing a disease, slow its progression, and / or cure it. Diseases targeted by therapeutic agents include, but are not limited to, diabetes, such as type 2 diabetes.

[0049] As used in this article, “peptide mimics” are small protein-like chains designed to mimic protein functions. They can be modifications of existing peptides or novel designs to mimic known peptides. They can be, for example, peptide-like substances and / or β-peptides and / or D-peptides.

[0050] "Recombinant virus" refers to a virus that has been genetically modified (e.g., by adding or inserting a heterologous nucleic acid construct into the particle).

[0051] A "gene," or "coding sequence," or sequence that "encodes" a specific protein or peptide is a nucleic acid molecule that, when placed under the control of appropriate regulatory sequences, is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo. The boundaries of a gene are determined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxyl) end. Genes can include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. The transcription termination sequence is typically located at the 3' end of the gene sequence.

[0052] The term "control element" collectively refers to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, etc., which together enable the replication, transcription, and translation of coding sequences in recipient cells. Not all of these control elements need to be present, provided that the selected coding sequence can replicate, transcribe, and translate in the appropriate host cell.

[0053] The term “promoter region” is used in its common sense in this document to refer to a nucleotide region that includes a DNA regulatory sequence derived from a gene capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence.

[0054] "Operationally linked" refers to an arrangement of elements in which the components described therein are configured to perform their normal functions. Thus, control elements operably linked to a coding sequence enable the expression of that coding sequence. The control element need not be adjacent to the coding sequence, as long as it functions to guide the expression of the coding sequence. Therefore, for example, an untranslated but transcribed sequence can be present between a promoter sequence and a coding sequence, and the promoter sequence can still be considered "operationally linked" to the coding sequence.

[0055] “Gene transfer” or “gene delivery” refers to a method or system for reliably inserting foreign DNA into a host cell. Such methods can produce transient expression of non-integrating transferred DNA, expression of extrachromosomal replication and transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of a host cell. Gene transfer provides a unique approach to treating acquired and hereditary diseases. Many systems have been developed for transferring genes into mammalian cells. See, for example, U.S. Patent No. 5,399,346. Examples of well-known vectors used for gene transfer include adenoviruses and recombinant adenoviruses (RAd), adeno-associated virus (AAV), herpes simplex virus type 1 (HSV-1), and lentiviruses (LV).

[0056] As used herein, "genetically modified cell," "genetically engineered cell," or "modified cell" refers to a cell expressing a polynucleotide sequence having any one or more of the sequences of BPx1, BPx2, BPx3, BPx4, or combinations thereof, or variants, derivatives, pharmaceutical equivalents, peptide mimics, or analogs thereof. BPx1 consists of the sequence YGLF (SEQ ID NO:1). BPx2 consists of the sequence YPFPGPIPN (SEQ ID NO:2). BPx3 consists of the amino acid sequence IPAVF (SEQ ID NO:3). BPx4 consists of the sequence LLF. In some embodiments, BPx1, BPx2, BPx3, BPx4, or variants, derivatives, pharmaceutical equivalents, peptide mimics, or analogs thereof are conjugated with agents that increase intestinal retention, such as cellulose, fatty acids, polyethylene glycol (PEG), or combinations thereof.

[0057] As used herein, “naked DNA” refers to DNA encoding a polypeptide having any one or more sequences of BPx1, BPx2, BPx3, BPx4, or combinations thereof, or variants, derivatives thereof, pharmaceutical equivalents, peptide mimics, or the like, cloned in a suitable expression vector for expression. Viral vectors that may be used include, but are not limited to, SIN lentiviral vectors, retroviral vectors, foam virus vectors, adeno-associated virus (AAV) vectors, hybrid vectors, and / or plasmid transposons (e.g., the Sleeping Beauty transposon system) or integrase-based vector systems. Other vectors that may be used with alternative embodiments of the present invention will be apparent to those skilled in the art.

[0058] As used herein, “polynucleotide” includes, but is not limited to, DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (short nucleolar RNA), miRNA (microRNA), genomic DNA, synthetic DNA, synthetic RNA, and / or tRNA.

[0059] The term “transfection” is used herein to refer to the uptake of foreign DNA by a cell. A cell is “transfected” when foreign DNA is introduced into the cell membrane. Many transfection techniques are generally known in the art. See, for example, Graham et al., Virology, 52:456 (1973); Sambrook et al., Molecular Cloning, a laboratory manual, ColdSpring Harbor Laboratories, New York (1989); Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene 13:197 (1981). Such techniques can be used to introduce one or more portions of foreign DNA, such as plasmid vectors and other nucleic acid molecules, into a suitable host cell. The term refers to both stable and transient uptake of genetic material.

[0060] As used in this article, "vector," "cloning vector," and "expression vector" refer to media that can introduce polynucleotide sequences (e.g., foreign genes) into host cells to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequences. Vectors include plasmids, bacteriophages, viruses, etc.

[0061] As used in this article, "intestine" refers to the gut. L cells containing GLP-1 and PYY are mainly located in the distal small intestine (ileum) and colon.

[0062] As used in this article, "bitter peptide" refers to a peptide molecule that tastes bitter. BP refers to bitter peptide.

[0063] As mentioned above, elevated postprandial glucagon-like peptide-1 (GLP-1) levels have been observed after bariatric surgery. Because increased GLP-1 has a significant glucose-regulating effect, the potential molecular mechanisms underlying the effects of surgery on GLP-1 secretion may play an important role in diabetes remission. Bariatric surgery (especially those that deliver ingested nutrients directly to the small and mid-intestinal tract) alters food digestion, shifting the digestion and absorption of nutrients, which normally occur in the upper small intestine, to the lower small intestine, where enteroendocrine L-cells responsible for GLP-1 release are located. Therefore, the luminal contents of the distal small intestine will contain partially digested dietary components post-surgery, and the molecular components of undigested food may activate receptors on these cells to trigger GLP-1 release. Some nutrient receptors, such as taste receptors and fatty acid receptors, or their effectors, have been shown to exist in these L-cells in human and animal tissues. Several food-based bitter oligopeptides derived from hydrolysates of whey proteins (derived from β-lactoglobulin, α-lactalbumin, albumin, and β-casein) have been identified, which can individually induce GLP-1 release in an in vitro L-cell model. These peptides can be delivered intestinally to the small intestine where the target receptors are located. The interaction of the oligopeptides will cause GLP-1 to be released into the bloodstream, thereby inducing diabetes regression. By mimicking the underlying mechanisms behind diabetes regression after obesity surgery, this treatment shows great promise for regressing type 2 diabetes.

[0064] In addition to weight loss in patients undergoing bariatric surgery, the procedure has also provided highly favorable outcomes in the regression of type 2 diabetes mellitus (T2DM). Diabetes regression typically precedes significant weight loss and occurs within days or weeks of surgery. This indicates a fundamental shift in metabolic events responding to food intake following these surgeries. The long-term efficacy of these surgeries for weight loss and diabetes prevention has been supported by several longitudinal studies tracking metabolic biomarkers in surgical and control patients for just weeks and up to 10 years. Several studies have shown that increased β-cell glucose sensitivity is not associated with weight loss. The interaction between glucose sensitivity and GLP-1 response is complex. Studies have shown that an excessive GLP-1 response leads to improved β-cell function and therefore plays a crucial role in improved glucose tolerance within the first day post-surgery. After this period, improved hepatic insulin sensitivity is thought to play a role in glucose tolerance. One study demonstrated the specific role of GLP-1 in normalized glucose levels in patients after bariatric surgery using Exendin 9-39 (a GLP-1 receptor antagonist). This study found that antagonizing GLP-1R led to a return of β-cell glucose sensitivity to preoperative levels.

[0065] Because bariatric surgery (especially Roux-en-Y gastric bypass (RYGB) bypassing the proximal intestine) places the digestion of dietary macronutrients in the mid-small intestine, there are higher concentrations of intermediate digestive products (including oligopeptides) that reach the ileum and colon and are typically fully digested and absorbed in the proximal small intestine. Profiling the digestive products of the luminal contents is extremely challenging due to the impractical and risky nature of sampling the luminal contents during meals. The inventors propose that oligopeptides presented on the luminal surface may be at least partially responsible for the excessive GLP-1 response to test diets following bypass surgery. The inventors have also found bitter taste receptors (especially TAS2R38) on L cells in the small intestine, and therefore propose that bitter oligopeptides may interact with TAS2R38 and possibly other bitter taste receptors on L cells to induce GLP-1 release.

[0066] The pepsin / trypsin / chymotrypsin digests of milk proteins, such as β-lactoglobulin, α-lactalbumin, albumin, and β-casein, produce bitter oligopeptide products (Liu et al., J. Agric. Food Chem. 2014, 62, 5719-5725). The inventors synthesized four of these oligopeptides and found that they potently enhance GLP-1 release. The inventors' data suggest that low concentrations of food-derived bitter peptides delivered to the ileum or colon can induce a metabolic response beneficial to diabetes control by releasing GLP-1 (and peptide tyrosine tyrosine (PYY)) from L cells. The inventors hypothesize that the regression of T2DM following bariatric surgery (particularly RYGB) can be mimicked by delivering such oligopeptides to the distal small intestine and envision that the oligopeptides stimulate GPCRs on L cells, thereby generating an enhanced GLP-1 response and subsequent T2DM regression.

[0067] The bitter taste receptor TAS2R38 is a G protein-coupled receptor (GPCR) that has been found in many extraoral sites such as the gastrointestinal (GI) system, respiratory system, and brain, but its function in these sites is only beginning to be understood. To probe the receptor's potential metabolic role, immunohistochemical analysis of human ileal tissue showed that the receptor co-localizes with glucagon-like peptide-1 (GLP-1) in L cells.

[0068] The present invention described herein proposes a treatment for diabetes. Because the mechanism proposed by the invention involves receptors facing the lumen of the gastrointestinal tract, it is suggested that these oligopeptide molecules (e.g., BPx1, BPx2, BPx3, BPx4, or combinations thereof) would be effective if formulated for intestinal delivery. Treatments exist that use GLP-1 analogs and methods to prolong the lifespan of secreted GLP-1 by inhibiting its breakdown. However, a solution as described herein does not exist. This is advantageous because the present invention uses the body's own endocrine system to treat diabetes, as current therapies have adverse effects associated with the agents used. Examples include GLP-1 mimicry agents that have been consistently associated with significant adverse effects such as pancreatitis and pancreatic cancer.

[0069] This document provides pharmaceutical compositions comprising, and substantially comprising, an agent that increases the release of intestinal hormones (such as GLP-1, PYY, or combinations thereof), and an acceptable carrier / excipient. In some embodiments, the agent is a bitter oligopeptide, indicating that it interacts with bitter taste receptors on the tongue, which are also present in the ileum and colon of the gastrointestinal tract. In some embodiments, the bitter oligopeptide is any one or more of BPx1, BPx2, BPx3, BPx4, or combinations thereof, or analogs thereof, pharmaceutical equivalents, and / or peptide mimics as described herein. In certain aspects of all embodiments of the invention, the oligopeptide further comprises a fusion protein. Specifically, the fusion protein comprises an oligopeptide described herein fused with any one or more of an epitope tag, a half-life extender, or a combination thereof. In some embodiments, the one or more oligopeptides cause a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% increase in the release of intestinal hormones (such as GLP-1, PYY, or combinations thereof). When administered therapeutically, the oligopeptide composition typically also comprises a pharmaceutically acceptable solution or carrier. In some aspects, the polypeptide or protein (e.g., BPx1, BPx2, BPx3, BPx4, or combinations thereof) is a “modified polypeptide” containing non-naturally occurring amino acids. In some aspects, the polypeptide contains a combination of naturally occurring and non-naturally occurring amino acids, and in some embodiments, the peptide contains only non-naturally occurring amino acids.

[0070] In one embodiment, the agent causing an increase in intestinal hormones (such as GLP-1, PYY, or combinations thereof) comprises, or is substantially composed of, the amino acid sequence YGLF (BPx1; SEQ ID NO:1) or an analogue, pharmaceutical equivalent, or peptide mimic. In some embodiments, BPx1 or variants, derivatives, pharmaceutical equivalents, peptide mimics, or analogues thereof are conjugated to an agent that increases intestinal retention. Examples of agents that increase intestinal retention include, but are not limited to, cellulose, fatty acids, polyethylene glycol (PEG), or combinations thereof.

[0071] In another embodiment, the agent causing an increase in intestinal hormones (such as GLP-1, PYY, or combinations thereof) comprises, or is substantially composed of, the amino acid sequence YPFPGPIPN (BPx2; SEQ ID NO:2) or an analogue, pharmaceutical equivalent, or peptide mimic. In some embodiments, BPx2 or variants, derivatives, pharmaceutical equivalents, peptide mimics, or analogues thereof are conjugated to an agent that increases intestinal retention. Examples of agents that increase intestinal retention include, but are not limited to, cellulose, fatty acids, polyethylene glycol (PEG), or combinations thereof.

[0072] In another embodiment, the agent causing an increase in intestinal hormones (such as GLP-1, PYY, or combinations thereof) comprises, or is substantially composed of, the amino acid sequence IPAVF (BPx3; SEQ ID NO:3) or an analogue, pharmaceutical equivalent, or peptide mimic. In some embodiments, BPx3 or variants, derivatives, pharmaceutical equivalents, peptide mimics, or analogues thereof are conjugated to an agent that increases intestinal retention. Examples of agents that increase intestinal retention include, but are not limited to, cellulose, fatty acids, polyethylene glycol (PEG), or combinations thereof.

[0073] In another embodiment, the agent causing an increase in intestinal hormones (such as GLP-1, PYY, or combinations thereof) comprises, or is substantially composed of, the amino acid sequence LLF (BPx4) or an analogue thereof, a pharmaceutical equivalent, or a peptide mimic. In some embodiments, BPx4 or variants, derivatives, pharmaceutical equivalents, peptide mimics, or analogues thereof are conjugated to an agent that increases intestinal retention. Examples of agents that increase intestinal retention include, but are not limited to, cellulose, fatty acids, polyethylene glycol (PEG), or combinations thereof.

[0074] In some embodiments, the BPx1, BPx2, BPx3, BPx4 peptides or combinations thereof or their analogues, pharmaceutical equivalents, and / or peptide mimics are modified peptides. "Modified peptides" may include the incorporation of lactam bridges, head-tail cyclization, non-natural amino acids into the peptides of the present invention, including synthetic non-natural amino acids, substituted amino acids, or one or more D-amino acids into the peptide (or other components of the composition, excluding protease recognition sequences), which is desirable in certain circumstances. Peptides containing D-amino acids exhibit increased stability in vitro or in vivo compared to L-amino acid-containing forms. Therefore, the construction of peptides incorporating D-amino acids can be particularly useful when greater in vivo or intracellular stability is required or demanded. More specifically, D-peptides are resistant to endogenous peptidases and proteases, thus providing such properties when better oral transepithelial and transdermal delivery of linked drugs and conjugates, improved bioavailability of membrane-permanent complexes (see below for further discussion), and prolonged intravascular and interstitial lifespan are desired. The use of D-isomer peptides can also enhance the transdermal and oral epithelial delivery of linked drugs and other cargo molecules. Furthermore, D-peptides are not efficiently processed for major histocompatibility complex II-restricted presentation to T helper cells and are therefore unlikely to induce humoral immune responses throughout the organism. Therefore, peptide conjugates can be constructed using, for example, D-isomer forms of cell-penetrating peptide sequences, L-isomer forms of cleavage sites, and D-isomer forms of therapeutic peptides. Thus, in some embodiments, the disclosed peptide comprises L amino acids and D amino acids, containing no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 D-amino acids. In some aspects, the peptide comprises more than 10 D-amino acids, and in some aspects, all amino acids of the peptide are D-amino acids.

[0075] In some embodiments, the agents that cause increased release of intestinal hormones (such as GLP-1, PYY, or combinations thereof) are inverted-reverse peptides of BPx1 peptide, BPx2 peptide, BPx3 peptide, BPx4 peptide, or combinations thereof, or analogs thereof, pharmaceutical equivalents, and / or peptide mimics. An "inverted-reverse peptide" is a peptide with the orientation of its peptide bonds reversed at at least one position, i.e., the amino and carboxyl ends of the side chains relative to the amino acids. Therefore, inverted-reverse analogs have reversed ends and reversed peptide bond orientations while substantially maintaining the topology of the side chains as in the native peptide sequence. Inverted-reverse peptides may contain L-amino acids or D-amino acids, or a mixture of L-amino acids and D-amino acids, up to all amino acids being D-isomers. Partially inverted-reverse peptide analogs are polypeptides in which only a portion of the sequence is reversed and replaced by enantiomeric amino acid residues. Because the reverse-inverted portion of this analogue has reversed amino and carboxyl terms, the amino acid residues flanking the reverse-inverted portion are replaced by side-chain-similar α-substituted geminitroaminomethane and malonic acid esters, respectively. It has been found that the reverse-inverted form of the cell-penetrating peptide functions as efficiently as the native form in transmembrane translocation. The synthesis of reverse-inverted peptide analogues is described in Bonelli, F. et al., Int J Pept Protein Res. 24(6):553-6 (1984); Verdini, A and Viscomi, GC, J. Chem. Soc. Perkin Trans. 1:697-701 (1985); and U.S. Patent No. 6,261,569, all of which are incorporated herein by reference in their entirety. A method for the solid-phase synthesis of partially reverse-inverted peptide analogues (EP 97994-B) has also been described and is incorporated herein by reference in its entirety.

[0076] Other variants of the peptides described herein (e.g., BPx1, BPx2, BPx3, and BPx4) may contain conserved substituted sequences, meaning that one or more amino acid residues of the original peptide are replaced by different residues, and the conservedly substituted peptides retain the desired biological activity, namely, the ability to increase the release of intestinal hormones (such as GLP-1, PYY, or combinations thereof) substantially equivalent to that of the original peptide. Examples of conserved substitutions include substitutions of amino acids that do not alter the secondary and / or tertiary structure of BPx1, BPx2, BPx3, and / or BPx4; substitutions that do not alter the overall or local hydrophobic properties; substitutions that do not alter the overall or local charge; substitutions by residues of equivalent side chain size; or substitutions by side chains with similar reactive groups.

[0077] Other examples involve the substitution of non-evolutionarily conserved amino acids in parental sequences between species. Advantageously, in some embodiments, when conserved substitutions are produced, these conserved amino acids and structures are not altered.

[0078] A given amino acid can be substituted with residues having similar physiological and chemical characteristics, such as replacing one aliphatic residue with another (e.g., Ile, Val, Leu, or Ala replacing each other), or replacing one polar residue with another (e.g., between Lys and Arg; between Glu and Asp; or between Gln and Asn). Other such conserved substitutions, such as substitution of an entire region with similar hydrophobic characteristics or substitution of residues with similar side chain volumes, are well known. Isolated peptides containing conserved amino acid substitutions can be tested in any of the assays described herein to confirm that the desired activity, such as the retention of increased release of intestinal hormones (e.g., GLP-1, PYY, or combinations thereof), is as determined by assays described elsewhere herein.

[0079] Amino acids can be grouped according to the similarity of the properties of their side chains (in Allhninger, in Biochemistry, 2nd edition, pp. 73-75, Worth Publishers, New York (1975)): (1) Nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) Uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) Acidic: Asp (D), Glu (E); (4) Basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be grouped based on their common side-chain properties: (1) hydrophobic: ortholeucine, Met, Ala, Val, Leu, Ile, Phe, Trp; (2) neutral-hydrophilic: Cys, Ser, Thr, Asn, Gln, Ala, Tyr, His, Pro, Gly; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe, Pro, His, or hydroxyproline. Non-conservative substitution would require replacing members of one of these categories with members of another.

[0080] Particularly preferred conservative substitutions for the variants described herein are as follows: Ala substituted with Gly or Ser; Arg substituted with Lys; Asn substituted with Gln or His; Asp substituted with Glu or Asn; Cys substituted with Ser; Gln substituted with Asn; Glu substituted with Asp; Gly substituted with Ala or Pro; His substituted with Asn or Gln; Ile substituted with Leu or Val; Leu substituted with Ile or Val; Lys substituted with Arg, Gln, or Glu; Met substituted with Leu, Tyr, or Ile; Phe substituted with Met, Leu, or Tyr; Ser substituted with Thr; Thr substituted with Ser; Trp substituted with Tyr or Phe; Tyr substituted with Phe or Trp; and / or Phe substituted with Val, Tyr, Ile, or Leu. Generally, conservative substitutions encompass residue exchanges with residues having similar physicochemical properties (i.e., replacing another hydrophobic amino acid with a hydrophobic residue).

[0081] Any cysteine ​​residues that do not participate in maintaining the proper conformation of the isolated peptide as described herein may be substituted (usually with serine) to improve the oxidative stability of the molecule and prevent undesirable crosslinking. Conversely, one or more cysteine ​​bonds may be added to the isolated peptide as described herein to improve its stability or promote polymerization.

[0082] As used herein, a “functional fragment” is a segment or region of a peptide that contains at least three, four, or five amino acids and can increase the release of intestinal hormones (such as GLP-1, PYY, or combinations thereof) according to the assays described herein. Functional fragments may contain conserved substitutions of the sequences disclosed herein, provided they retain the function of increasing the release of intestinal hormones (such as GLP-1, PYY, or combinations thereof). This can be tested by detecting an increase in the release of at least 30%, at least 40%, or at least 50% of the parental (e.g., original) form of the peptide.

[0083] To enhance the stability, bioavailability, and / or delivery of peptides into cells, peptides may be modified. For example, in some embodiments, the isolated peptides described herein may contain at least one peptide bond substitution. A single peptide bond or multiple peptide bonds, such as 2, 3, 4, 5, or 6 or more bonds, or all peptide bonds, may be substituted. The isolated peptides described herein may contain one type or multiple types of peptide bond substitution, such as 2, 3, 4, 5, or more types of peptide bond substitution. Non-limiting examples of peptide bond substitutions include urea, thiourea, carbamates, sulfonylureas, trifluoroethylamine, o-(aminoalkyl)-phenylacetic acid, p-(aminoalkyl)-phenylacetic acid, m-(aminoalkyl)-phenylacetic acid, thioamides, tetrazolium, borate esters, olefinic groups, and derivatives thereof. In some embodiments, BPx1, BPx2, BPx3, BPx4, or variants, derivatives, pharmaceutical equivalents, peptide mimics, or analogs thereof are conjugated to an agent that increases intestinal retention. Examples of agents that increase intestinal retention include, but are not limited to, cellulose, fatty acids, polyethylene glycol (PEG), or combinations thereof.

[0084] In some embodiments, the isolated peptides as described herein may comprise naturally occurring amino acids commonly found in polypeptides and / or proteins produced by living organisms, such as Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M), Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q), Asp (D), Glu (E), Lys (K), Arg (R), and His (H). In some embodiments, the isolated peptides as described herein may comprise substituted amino acids. Non-limiting examples of alternative amino acids include D-amino acids, β-amino acids, homocysteine, phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, γ-carboxyglutamic acid, hippuric acid, octahydroindole-2-carboxylic acid, statins, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine (3-mercapto-D-valine), ornithine, citrulline, α-methyl-alanine, p-benzoylphenylalanine, p-aminophenylalanine, p-fluorophenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine, diaminobutyric acid, 7-hydroxy-tetrahydroisoquinoline carboxylic acid, naphthylalanine, and biphenylalanine. Amino acids, cyclohexylalanine, amino-isobutyric acid, valine, leucine, tert-leucine, tetrahydroisoquinoline carboxylic acid, pipercoic acid, phenylglycine, homophenylalanine, cyclohexylglycine, dehydroleucine, 2,2-diethylglycine, 1-amino-1-cyclopentane carboxylic acid, 1-amino-1-cyclohexane carboxylic acid, amino-benzoic acid, amino-naphthoic acid, γ-aminobutyric acid, difluorophenylalanine, piperidinecarboxylic acid, α-aminobutyric acid, thienyl-alanine, tert-butylglycine, trifluorovaline, hexafluoroleucine, fluorinated analogs, azide-modified amino acids, alkyne-modified amino acids, cyano-modified amino acids and their derivatives.

[0085] In some embodiments, the isolated peptide may be modified, for example, by adding a portion to one or more amino acids constituting the peptide. In some embodiments, the isolated peptide as described herein may comprise one or more moieties, such as one or more moieties per peptide, two or more moieties per peptide, five or more moieties per peptide, ten or more moieties per peptide, or more moieties per peptide. In some embodiments, the isolated peptide as described herein may comprise one or more types of modifications and / or moieties, such as one type of modification, two types of modifications, three types of modifications, or more types of modifications. Non-limiting examples of modifications and / or moieties include PEGylation; glycosylation; HES-ization; ELP-ization; esterification; acetylation; amidation; end-capping modification; cyanoation; phosphorylation; and cyclization. In some embodiments, end-capping modification may include N-terminal acetylation, N-terminal acylation, and N-terminal formylation. In some embodiments, end-capping modification may include C-terminal amidation, introduction of C-terminal alcohol, aldehyde, ester, and thioester moieties.

[0086] The isolated peptides described herein may be coupled and / or linked to a second functional molecule, peptide, and / or polypeptide. In some embodiments, the isolated peptides described herein are coupled to a target molecule. In some embodiments, the isolated peptides described herein are coupled to the target molecule by expressing the peptide and the target molecule as a fusion peptide, optionally with a peptide linker sequence inserted therebetween. As used herein, a “target molecule” can be any molecule, such as a peptide, antibody or fragment thereof, antigen, targeting liposome, or small molecule that can bind to or be bound to a specific cell or tissue type. As a non-limiting example, if it is desired to target the isolated peptides described herein to the intestine (e.g., to treat, inhibit, reduce the severity of, and / or slow the progression of diabetes such as type 2 diabetes), an isolated peptide comprising the amino acid sequence of any of BPx1, BPx2, BPx3, BPx4 or variants, derivatives, pharmaceutical equivalents, peptide mimics, or analogs thereof may be coupled to an antibody or fragment thereof that is specific to the small intestine and colon (e.g., an antibody or antibody fragment as described in U.S. Patent Publication 2005 / 0287066). As a non-limiting example, if it is desired to target the isolated peptides described herein to the intestine to increase the release of intestinal hormones such as GLP-1 and / or PYY to treat, inhibit, reduce the severity of, and / or slow the progression of diabetes, then the isolated peptides containing the amino acid sequence of any of BPx1, BPx2, BPx3, BPx4 or variants, derivatives, pharmaceutical equivalents, peptide mimics or analogs thereof may be conjugated to an antibody or fragment thereof that is specific to the intestine (e.g., an IgA antibody).

[0087] In some embodiments, the isolated peptide, as described herein, may be a fusion peptide or a polypeptide. A fusion polypeptide may comprise a peptide linker domain inserted between a first domain of a peptide comprising an amino acid sequence LLF derivative, variant, functional fragment, prodrug, or analogue thereof, as described herein, or a peptide comprising an amino acid sequence SEQ ID NO:1-3 or a derivative, variant, functional fragment, prodrug, or analogue thereof, and at least a second domain of the fusion peptide. Where the partner domain is formed upon complementation of the constituent fragments, the first peptide domain may be an N-terminal domain, a C-terminal domain, or an internal sequence. Methods for synthesizing or producing fusion proteins are well known to those skilled in the art. As used herein, the term "fusion protein" refers to a recombinant protein of two or more proteins. Fusion proteins may be produced, for example, by linking a nucleic acid sequence encoding one protein to a nucleic acid sequence encoding another protein such that they form a single open reading frame, which can be translated in a cell into a single polypeptide containing all the intended proteins. The protein sequence may vary. Fusion proteins may contain epitope tags or half-life extenders. Epitope tags include biotin, FLAG tags, c-myc, hemagglutinin, His6, digoxin, FITC, Cy3, Cy5, green fluorescent protein, V5 epitope tag, GST, β-galactosidase, AU1, AU5, and avidin. Half-life extenders include Fc domains and serum albumin.

[0088] In some embodiments, the isolated peptides described herein may be pharmaceutically acceptable prodrugs. As used herein, a "prodrug" refers to a compound that can be converted into a therapeutic agent by some chemical or physiological process, such as enzymatic processes and metabolic hydrolysis. Therefore, the term "prodrug" also refers to a precursor of a pharmaceutically acceptable biologically active compound. When administered to a subject, a prodrug may be inactive (i.e., an ester) but is converted into an active compound in vivo, for example, by hydrolysis to a free carboxylic acid or a free hydroxyl group. Prodrug compounds typically offer advantages such as solubility, tissue compatibility, or delayed release in the body. The term "prodrug" is also intended to include any covalently bonded carrier that releases an active compound in vivo when the prodrug is administered to a subject. Prodrugs of active compounds can be prepared by modifying functional groups present in said active compound in a manner that, in a conventional procedure or in vivo, cleaves the modifier into a parent active compound. Prodrugs include compounds in which a hydroxyl, amino, or thiol group is bonded to any group that, when administered to a subject as a prodrug of said active compound, is cleaved to form a free hydroxyl, free amino, or free thiol group, respectively. Examples of prodrugs include (but are not limited to) acetate, formate, and benzoate derivatives of alcohols in the active compound, or acetamide, formamide, and benzamide derivatives of amine functional groups in the active compound. See Harper, “Drug Latentiation” in Jucker, ed., Progress in Drug Research 4:221-294 (1962); Morozowich et al., “Application of Physical Organic Principles to Prodrug Design” in E.B. Roche, ed., Design of Biopharmaceutical Properties through Prodrugs and Analogs, APHA Acad. Pharm. Sci. 40 (1977); Bioreversible Carriers in Drug in Drug Design, Theory and Application, E.B. Roche, ed., APHA Acad. Pharm. Sci. (1987); Design of Prodrugs, H. Bundgaard, Elsevier (1985); Wang et al., “Prodrug approaches to the improved delivery of peptide drug” in Curr. Pharm. Design.In 5(4):265-287(1999); Pauletti et al. (1997) Improvement in peptide bioavailability: Peptidomimetics and Prodrug Strategies, Adv. Drug. Delivery Rev. 27:235-256; Mizen et al. (1998) “The Use of Esters as Prodrugs for Oral Delivery of (3-Lactam antibiotics," Pharm. Biotech. ll,:345-365; Gaignault et al. (1996) “Designing Prodrugs and Bioprecursors I. Carrier Prodrugs," Pract. Med. Chem. 671-696; Asgharnejad, “Improving Oral Drug Transport", in Transport Processes in Pharmaceutical Systems, G.L. Amidon, P.I. Lee and E.M. Topp, eds., Marcell Dekker, pp. 185-218 (2000); Balant et al., “Prodrugs for the improvement of drug absorption via different routes of administration", Eur. J. Drug Metab. Pharmacokinet., 15(2):143-53(1990); Balimane and Sinko, “Involvement of multiple transporters in the oral absorption of nucleoside analogues", Adv. Drug Delivery Rev., 39(1-3):183-209(1999); Browne, “Fosphenytoin (Cerebyx)", Clin. Neuropharmacol. 20(1):1-12(1997); Bundgaard, “Bioreversible derivatization of drugs—principle and applicability to improve the therapeutic effects of drugs", Arch. Pharm.Chemi 86(1):1-39(1979); Bundgaard H. "Improved drug delivery by the prodrug approach", Controlled Drug Delivery 17:179-96(1987); Bundgaard H. "Prodrugs as a means to improve the delivery of peptide drugs", Arfv. Drug Delivery Rev. 8(1):1-38(1992); Fleisher et al. "Improved oral drug delivery: solubility limitations overcome by the use of prodrugs", Arfv. Drug Delivery Rev. 19(2):115-130(1996); Fleisher et al. "Design of prodrugs for improved gastrointestinal absorption by intestinal enzyme targeting", Methods Enzymol. 112(Drug Enzyme Targeting, Pt. A):360-81,(1985); Farquhar D, et al., "Biologically Reversible Phosphate-Protective Groups", Pharm. Sci., 72(3):324-325(1983); Freeman S, et al., "Bioreversible Protection for the Phospho Group: Chemical Stability and Bioactivation of Di(4-acetoxy-benzyl)Methylphosphonate with Carboxyesterase," Chem. Soc., Chem. Commun., 875 - 877 (1991); Friis and Bundgaard, "Prodrugs of phosphates and phosphonates: Novel lipophilic alphaacyloxyalkylester derivatives of phosphate - or phosphonate containing drugs masking the negative charges of these groups", Eur. J. Pharm. Sci. 4: 49 - 59 (1996); Gangwar et al., "Pro - drug, molecular structure and percutaneous delivery", Des. Biopharm. Prop. Prodrugs Analogs, [Symp.] Meeting Date 1976, 409 - 21. (1977); Nathwani and Wood, "Penicillins: a current review of their clinical pharmacology and therapeutic use", Drugs 45(6): 866 - 94 (1993); Sinhababu and Thakker, "Prodrugs of anticancer agents", Adv. Drug Delivery Rev. 19(2): 241 - 273 (1996); Stella et al., "Prodrugs. Do they have advantages in clinical practice?", Drugs 29(5): 455 - 73 (1985); Tan et al "Development and optimization of anti - HIV nucleoside analogs and prodrugs: A review of their cellular pharmacology, structure - activity relationships and pharmacokinetics", Adv. Drug Delivery Rev. 39(1 - 3): 117 - 151 (1999); Taylor, "Improved passive oral drug delivery via prodrugs", Adv. Drug Delivery Rev.,19(2):131-148(1996); Valentino and Borchardt, “Prodrug strategies to enhance the intestinal absorption of peptides”, Drug Discovery Today 2(4):148-155(1997); Wiebe and Knaus, “Concepts for the design of anti-HIV nucleoside prodrugs for treating cephalic HIV infection”, Adv. Drug Delivery Rev.:39(1-3):63-80(1999); Waller et al., “Prodrugs”, Br. J. Clin. Pharmac. 28:497-507(1989), which are incorporated herein by reference in their entirety.

[0089] In some embodiments, the isolated peptide as described herein may be a pharmaceutically acceptable solvate. The term "solvate" refers to the isolated peptide as described herein in a solid state, wherein molecules of a suitable solvent are incorporated into the crystal lattice. A suitable solvent for therapeutic administration is physiologically tolerable at the administered dose. Examples of suitable solvents for therapeutic administration are ethanol and water. When water is used as the solvent, the solvate is referred to as a hydrate. Typically, a solvate is formed by dissolving the compound in a suitable solvent and separating the solvate by cooling or using an antisolvent. Solvates are typically dried or azeotropically under ambient conditions.

[0090] In some implementations, the isolated peptides, as described herein, may be non-crystalline, i.e., in an amorphous solid form.

[0091] On one hand, this document describes a vector comprising a nucleic acid encoding a peptide as described herein. As used herein, the term "vector" refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term "vector" encompasses any genetic element capable of replicating and transferring a gene sequence into a cell when associated with appropriate control elements. Vectors can include, but are not limited to, cloning vectors, expression vectors, plasmids, bacteriophages, transposons, granules, chromosomes, viruses, viral particles, etc. Many vectors suitable for transferring exogenous genes into target mammalian cells are available. The vector can be an adjunct type, such as a plasmid, a viral vector such as cytomegalovirus, adenovirus, etc., or can be integrated into the target cell genome via homologous recombination or random integration, such as retroviral vectors such as MMLV, HIV-1, ALV, etc. Many viral vectors are known in the art and can be used as vectors for the delivery of nucleic acid regulatory compounds into cells. For example, constructs containing nucleic acids encoding polypeptides can be integrated and packaged into the genomes of non-replicating defective viruses such as adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV), or others (including retroviral and lentiviral vectors) for infection or transduction into cells. Alternatively, the constructs can be incorporated into vectors capable of aposomeric replication (e.g., EPV and EBV vectors). The nucleic acids incorporated into the vector can be operatively linked to expression control sequences such that the expression control sequences control and regulate the transcription and translation of the polynucleotide sequence.

[0092] As used herein, the term "expression vector" refers to a vector from which a sequence linked to a transcriptional regulatory sequence directs the expression of RNA or polypeptide. The expressed sequence is typically, but not necessarily, heterologous to the cell. Expression vectors may contain additional elements; for example, they may have two replication systems, thus allowing them to be maintained in two organisms, such as for expression in human cells and for cloning and amplification in a prokaryotic host.

[0093] As used herein, the term "transfection" refers to methods for introducing exogenous nucleic acids (such as nucleic acid sequences encoding peptides as described herein) into cells, such as chemical methods. As used herein, the term transfection does not cover virus-based methods for introducing exogenous nucleic acids into cells. Transfection methods include physical therapy (electroporation, nanoparticles, magnetic transfection) and chemical-based transfection methods. Chemical-based transfection methods include, but are not limited to, methods using cyclodextrins, polymers, liposomes, nanoparticles, cationic lipids or mixtures thereof (e.g., DOPA, Lipofectamine, and UptiFectin) and cationic polymers such as DEAE-glucan or polyethyleneimine.

[0094] As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin and has the ability to be packaged into viral vector particles. Viral vectors may contain nucleic acids encoding peptides as described herein, in place of non-essential viral genes. Vectors and / or particles can be used to transfer any nucleic acid into cells in vitro or in vivo. Many forms of viral vectors are known in the art. When used to refer to a viral vector, the term "non-replicating" means that the viral vector cannot further replicate and package its genome. For example, when a subject's cells are infected with non-replicating recombinant adeno-associated virus (rAAV) viral particles, a heterologous (also called a transgene) gene is expressed in the patient's cells, but the rAAV is replication-deficient (e.g., lacking a helper gene encoding an essential protein for packaging the virus), and viral particles cannot form in the patient's cells. The term "transduction," as used herein, refers to the use of viral particles or viruses to introduce foreign nucleic acids into cells.

[0095] Retroviruses, such as lentiviruses, provide a convenient platform for delivering nucleic acid sequences encoding target drugs. Selected nucleic acid sequences can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells, e.g., in vitro or ex vivo. Retroviral systems are well known in the art and are described, for example, in U.S. Patent No. 5,219,740; Kurth and Bannert (2010) "Retroviruses: Molecular Biology, Genomics and Pathogenesis" Calster Academic Press (ISBN: 978-1-90455-55-4); and Hu and Pathak Pharmacological Reviews 2000 52:493-512, all of which are incorporated herein by reference in their entirety.

[0096] In some implementations, the target nucleotide sequence is inserted into an adenovirus-based expression vector. Unlike retroviruses that integrate into the host genome, adenoviruses persist extrachromosomally, thereby minimizing the risk associated with insertional mutagenesis (Haj-Ahmad and Graham (1986) J. Virol. 57:267-74; Bett et al. (1993) J. Virol. 67:5911-21; Mittereder et al. (1994) Human Gene Therapy 5:717-29; Seth et al. (1994) J. Virol. 68:933-40; Barr et al. (1994) Gene Therapy 1:51-58; Berkner, KL (1988) BioTechniques 6:616-29; and Rich et al. (1993) Human Gene Therapy 4:461-76). Adenovirus vectors offer several advantages in gene therapy. They infect a wide variety of cells, have a broad host range, exhibit high infection efficiency, guide high levels of heterologous sequence expression, and enable long-term expression of these sequences in vivo. The virus is fully infectious as a cell-free viral particle and therefore does not require injection of production cell lines. Regarding safety, adenoviruses are not associated with severe human pathology, and recombinant vectors derived from the virus can be made replication-deficient by deletion in early region 1 (“E1”) of the viral genome. Adenoviruses can also be produced in large quantities relatively easily. For all these reasons, vectors derived from human adenoviruses with at least the E1 region deleted and replaced by the target gene have been widely used in preclinical and clinical gene therapy experiments. Adenoviral vectors used in conjunction with the compositions and methods described herein can be derived from any of a variety of adenovirus serotypes, including but not limited to any of more than 40 adenovirus serotype strains, such as serotypes 2, 5, 12, 40, and 41. The adenoviral vectors used in the methods described herein are typically replication-deficient and contain the target sequence under the control of a suitable promoter. For example, U.S. Patent No. 6,048,551 (which is incorporated herein by reference in its entirety) describes a replication-defective adenovirus vector containing a human genome controlled by a Rous sarcoma virus (RSV) promoter. Other recombinant adenoviruses with various serotypes and containing different promoter systems can be created by those skilled in the art. See, for example, U.S. Patent No. 6,306,652, which is incorporated herein by reference in its entirety.Other useful adenovirus-based vectors for delivering nucleic acid sequences include, but are not limited to: “minimal” adenovirus vectors as described in U.S. Patent No. 6,306,652, which retain at least a portion of the viral genome required for capsidation (capsidation signal) and at least one copy of at least one functional portion or derivative of the ITR; and “gutless” (helper virus dependent) adenoviruses in which the vast majority of the viral genome has been removed and which produce virtually no viral proteins, such vectors allow gene expression to persist for more than a year after a single administration (Wu et al. (2001) Anesthes. 94:1119-32; Parks (2000) Clin. Genet. 58:1-11; Tsai et al. (2000) Curr. Opin. Mol. Ther. 2:515-23).

[0097] In some implementations, a nucleotide sequence encoding a peptide as described herein is inserted into an adeno-associated virus (AAV)-based expression vector. AAV is a parvovirus belonging to the genus AAV and possesses several characteristics not found in other viruses. AAV can infect a wide range of host cells, including non-dividing cells. AAV can infect cells from different species. AAV is not associated with any human or animal disease and does not appear to alter the biological characteristics of the host cell upon integration. In fact, it is estimated that 80%–85% of the population has been exposed to the virus. Finally, AAV is stable under a wide range of physical and chemical conditions, thus facilitating its production, storage, and transport. AAV is a helper virus-dependent virus; that is, it requires co-infection with a helper virus (e.g., adenovirus, herpesvirus, or vaccinia) to form AAV viral particles in the wild. In the absence of co-infection with a helper virus, AAV establishes a latent state in which the viral genome is inserted into the host cell chromosome but does not produce infectious viral particles. Subsequent helper virus infection rescues the integrated genome, allowing it to replicate and package its genome into infectious AAV viral particles. Although AAV can infect cells from different species, the helper virus must be of the same species as the host cell. Therefore, for example, human AAV will replicate in canine cells co-infected with canine adenovirus. Adeno-associated virus (AAV) has been successfully used in gene therapy. AAV has been engineered to deliver target genes by deleting internal non-repetitive portions of the AAV genome (i.e., the rep and cap genes) and inserting a heterologous sequence (in this case, the sequence encoding the drug) between the ITRs. The heterologous sequence is typically functionally linked to a heterologous promoter (constitutive, cell-specific, or inducible) capable of driving expression in the patient's target cells under appropriate conditions. Recombinant AAV viral particles containing a nucleic acid sequence encoding the target drug can be produced using a variety of techniques recognized in the art, such as those described in U.S. Patent Nos. 5,139,941; 5,622,856; 5,139,941; 6,001,650; and 6,004,797, the contents of which are incorporated herein by reference in their entirety. The vectors and cell lines required for preparing rAAV raw materials without helper-free viruses are commercially available as the AAV Helper-Free System (Catalogue No. 240071) (Agilent Technologies, Santa Clara, Calif.).

[0098] Other viral vectors suitable for delivering nucleic acid molecules encoding peptides as described herein include those derived from the poxvirus family, including vaccinia virus and fowlpox virus. Alternatively, fowlpox viruses, such as chickenpox virus and canarypox virus, can be used to deliver genes. The use of fowlpox virus vectors in the cells of humans and other mammalian species is advantageous in terms of safety because members of the fowlpox virus genus can only replicate effectively in susceptible avian species. Methods for generating recombinant fowlpox viruses are known in the art and employ genetic recombination, see, for example, WO 91 / 12882; WO 89 / 03429; and WO92 / 03545.

[0099] Molecular conjugate vectors, such as adenovirus chimeric vectors, can also be used to deliver sequences encoding peptides as described herein (Michael et al. (1993) J. Biol. Chem. 268:6866-69 and Wagner et al. (1992) Proc. Natl. Acad. Sci. USA 89:6099-6103). Members of the alphavirus genus, such as Sindbis virus and Semliki Forest viruses, can also be used as viral vectors for delivering nucleic acid sequences (see, for example, Dubensky et al. (1996) J. Virol. 70:508-19; WO 95 / 07995; WO 96 / 17072).

[0100] In some embodiments, the vector also comprises a signal peptide operatively linked to the peptide. A signal peptide is a terminally (typically N-terminally) positioned peptide sequence that provides a pathway for protein entry into or across a membrane. Different signal peptides can be used in different applications. For example, with respect to cellular systems used to produce isolated peptides as described herein, secreted signal peptides can allow for increased yield and ease of purification. As another example, with respect to cells that produce peptides as described herein and administer them to a subject for therapeutic purposes, multiple signal peptides, such as peptide signaling for secretion from a first cell, peptide signaling for internalization through a second cell, and a final peptide signaling for nuclear localization, can increase the amount of peptide reaching the target environment. As yet another example, with respect to applications such as gene therapy, peptide signaling for nuclear localization can increase the amount of peptide reaching the target environment. Signal peptides are known in the art. Non-limiting examples of nuclear localization signaling (NLS) peptides for use in mammalian cells include: SV40 large T antigen NLS (PKKKRKV) (SEQ ID NO:4); nucleoplasmic protein NLS (KR[PAATKKAGQA]KKKK) (SEQ ID NO:5); KK / RXK / R (SEQ ID NO:6); KKXR (SEQ ID NO:7); KKXK (SEQ ID NO:8); KRXK (SEQ ID NO:9); KRXR (SEQ ID NO:10); and PY-NLS (see, for example, Dingwall et al. J Cell Biol 188 107:841-9 and Makkerh et al. Curr Biol. 1996 6:1025-7; both of which are incorporated herein by reference in their entirety for further discussion). Non-limiting examples of secretory signaling peptides for use in mammalian cells include human albumin signal peptide (MKWVTFISLLFLFSSAYS) (SEQ ID NO:4). (SEQ ID NO:11); human chymotrypsin signal peptide (MAFLWLLSCWALLGTTGF) (SEQ ID NO:12); human interleukin-2 signal peptide (MQLLSCIALILALV) (SEQ ID NO:13); human trypsinogen-2 signal peptide (MNLLLILTFVAAAVA) (SEQ ID NO:14); and sequences including coding regions for signals used for precursor cleavage by signal peptidase, furin protease or other pro-hormone convertases (e.g., PC3).For example, signal (peptide) sequences cleaved by furin protease (also known as PACE, see U.S. Patent No. 5,460,950), other subtilisin proteases (including PC2, PC1 / PC3, PACE4, PC4, PC5 / PC6, LPC / PC7, IPC8 / SPC7, and SKI-I; Nakayama, Biochem. J., 327:625-635 (1997)), enterokinase (see U.S. Patent No. 5,270,181), or chymotrypsin may be incorporated into the signal (peptide) sequences defined herein. Other signal peptides are known in the art, and the selection of a signal peptide may be influenced by cell type, growth conditions, and the desired purpose of the peptide.

[0101] On one hand, this document describes a cell expressing a vector containing a nucleic acid encoding a peptide as described herein. In some embodiments, the cell expressing the vector as described herein is a cell suitable for producing polypeptides. Cells suitable for producing polypeptides can be prokaryotic or eukaryotic cells, such as bacteria, viruses, yeast, fungi, mammalian cells, insect cells, plant cells, etc. As a non-limiting example, cells used for producing proteins are commercially available, such as bacterial cells (BL21-derived cells – catalog number 60401-1, Lucigen; Middleton, WI) and mammalian cells (293F cells – catalog number 11625-019, Invitrogen; Grand Island, NY).

[0102] Recombinant molecules, such as the vectors described herein, can be transported via transformation, particularly transduction, conjugation, lipid transfection, protoplast fusion, mobilization, particle bombardment, electroporation (Neumann et al., “Gene Transfer into Mouse Lyoma Cells by Electroporation in High Electric Fields,” EMBO J. 1(7): 841–845 (1982); Wong et al., “Electric Field Mediated Gene Transfer,” Biochem Biophys Res Commun 107(2): 584–587 (1982); Potter et al., “Enhancer-dependent Expression of Human Kappa Immunoglobulin Genes Introduced into Mouse pre-B Lymphocytes by Electroporation,” Proc. Natl. Acad. Sci. USA 81(22): 7161–7165 (1984), which are hereby incorporated herein by reference in their entirety), polyethylene glycol-mediated DNA uptake (Joseph Sambrook & David...). W. Russell, Molecular Cloning: A Laboratory Manual cp.16 (2nd edition, 1989, which is hereby incorporated herein by reference in its entirety), or the fusion of protoplasts with other entities (e.g., microcells, cells, lysosomes, or other readily fused lipid surfaces containing chimeric genes) (Fraley et al., “Liposome-mediated Delivery of Tobacco Mosaic Virus RNA into Tobacco Protoplasts: A Sensitive Assay for Monitoring Liposome-protoplast Interactions,” Proc. Natl. Acad. Sci. USA, 79(6):1859–1863 (1982), which is hereby incorporated herein by reference in its entirety) is introduced into cells. The host cells are then cultured in a suitable medium under conditions conducive to the expression of the target protein or peptide. After culture, the cells are destroyed by physical or chemical methods, and the protein or peptide is purified from the resulting crude extract. Alternatively, the culture may include conditions that allow the protein or peptide to be secreted into the growth medium of the recombinant host cells, and the protein or peptide to be isolated from said growth medium. Alternative methods may be used where appropriate.

[0103] Peptides can also be linked to adjuvants. The term "adjuvant" refers to a compound or mixture that enhances the immune response and / or promotes an appropriate rate of absorption after vaccination, and as used herein, encompasses any uptake promoter. Non-limiting examples of adjuvants include chemokines (e.g., defensins, HCC-1, HCC4, MCP-1, MCP-3, MCP4, MIP-1α, MIP-1β, MIP-1δ, MIP-3α, MIP-2, RANTES); other ligands of chemokine receptors (e.g., CCR1, CCR-2, CCR-5, CCR6, CXCR-1); and cytokines (e.g., IL-1β, IL-2, IL-1β ... 4. IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17 (AF), IL-18; IFNα, IFN-γ; TNF-α; GM-CSF; TGF-β; FLT-3 ligand; CD40 ligand; other ligands of the receptors of those cytokines; Th1 cytokines, including but not limited to IFN-γ, IL-2, IL-12, IL-18, and TNF; Th2 cytokines, including but not limited to IL-4, IL-5, IL-10, and IL-13; and Th17 cytokines, including but not limited to IL-17 (A to F), IL-23, TGF-β, and IL-6; immunostimulatory CpG motifs in bacterial DNA or oligonucleotides; derivatives of lipopolysaccharides such as monophospholipid A (MPL); muramyl dipeptide (MDP) and its derivatives (e.g., moradil ester, threonyl-MDP). Muraminoyl tripeptide, N-acetyl-muramino-L-threonyl-D-isoglutamine (thr-MDP); N-acetyl-nor-muramino-L-alanyl-D-isoglutamine (CGP11637, also known as nor-MDP); N-acetyl-muramino-L-alanyl-D-glutamine acyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycerol-3-hydroxyphosphoryloxy)-ethylamine (CGP) 19835A, referred to as MTP-PE); MF59 (see International Publication No. WO 90 / 14837); Poly[di(carboxyphenoxy)phosphazene] (PCPP polymer; Virus Research Institute, USA); RIBI (GSK), which contains three components extracted from bacteria in a 2% squalene / Tween 80 emulsion: monophospholipid A, trehalose dimethicone ester, and cell wall skeleton (MPL+TDM+CWS); OM-174 (a lipid A-associated glucosamine disaccharide; OM Pharma SA, Meyrin, Switzerland); heat shock proteins and their derivatives; Leishmania homologs of elF4a and its derivatives;Bacterial ADP-ribosylated exotoxins and their derivatives (e.g., genetic mutants, fragments containing A and / or B subunits, chemically toxin-like forms); chemical conjugates or genetic recombinants containing bacterial ADP-ribosylated exotoxins or their derivatives; C3d tandem arrays; lipid A and its derivatives (e.g., monophosphoryl or diphosphoryl lipid A, lipid A analogs, AGP, ASO2, ASO4, DC-Chol, Detox, OM-174); ISCMS and saponins (e.g., Quil A, QS-21); (Cambridge Bioscience, Worcester, MA); squalene; superantigens; or salts (e.g., aluminum hydroxide or aluminum phosphate, calcium phosphate). For other useful adjuvants, see also Nohria et al., Biotherapy, 7:261-269, 1994; Richards et al., in Vaccine Design, edited by Powell et al., Plenum Press, 1995; and Pashine et al., Nature Medicine, 11:S63-S68, 4 / 2005). Other examples of adjuvants may include the RIBI adjuvant system (Ribi Inc., Hamilton, MT.), alum, mineral gels such as aluminum hydroxide gel, oil-in-water emulsions, water-in-oil emulsions, such as Freund's complete and incomplete adjuvants, block copolymers (CytRx, Atlanta GA), QS-21 (Cambridge Biotech Inc., Cambridge MA), and SAF-M (Chiron, Emeryville CA). Adjuvants, saponins, Quil A or other saponin fractions, monophospholipid A and avridin lipid-amine adjuvants, and Other suitable adjuvants may include, for example, surfactants such as lysophosphatidylcholine, complex polyols, polyanionic peptides, oil or hydrocarbon emulsions, key foraminifera hemocyanin, and dinitrophenol.

[0104] In some embodiments, cells may be genetically engineered to express the peptides described herein, and the genetically engineered cells may be used in cell therapy. Examples of cells that may be used include, but are not limited to, dendritic cells, T lymphocytes (T cells), and naive T cells (T cells). N ), memory T cells (e.g., central memory T cells (T cells) CM ), effector memory cells (T) EMNatural killer cells, hematopoietic stem cells, and / or pluripotent embryonic / induced stem cells capable of producing treatment-related progeny. In one embodiment, the genetically engineered cells are autologous cells. As an example, the individual T cells of the present invention can be CD4+ / CD8-, CD4- / CD8+, CD4- / CD8-, or CD4+ / CD8+. T cells can be a mixed population of CD4+ / CD8- cells and CD4- / CD8+ cells or a population of single clones. When co-cultured in vitro with cells expressing the peptides (e.g., CD20+ and / or CD19+ tumor cells), CD4+ T cells can produce IL-2, IFNα, TNFα, and other T cell effector cytokines. When co-cultured in vitro with target cells, CD8+ T cells can produce IL-2, IFNα, TNFα, and other T cell effector cytokines. + T cells can lyse antigen-specific target cells. In some implementations, the T cells may be CD45RA. + CD62L + Initial cells, CD45RO + CD62L + Central memory cells, CD62L - Effector memory cells or any one or more of them (Berger et al., Adoptive transfer of virus-specific and tumor-specific T cell immunity. Curr Opin Immunol 2009 21(2)224-232).

[0105] In some embodiments, tolerant antigen-presenting cells can be used in cell therapy. Examples include B cells, dendritic cells, macrophages, etc. The cells can be of any origin, including human. The peptides described herein can be used to induce tolerance in the cells. In some embodiments, tolerance is induced in the presence of cytokines.

[0106] In some implementations, cells that produce peptides as described herein may be administered to a subject, for example, to treat or suppress diabetes (such as type 2 diabetes), reduce its severity, and / or slow its progression.

[0107] In some embodiments, nanoparticles comprising peptides as described herein may be administered to a subject. In some embodiments, the nanoparticles used in conjunction with the peptides described herein may be as described in Levine et al., Polymersomes: A new multi-functional tool for cancer diagnosis and therapy. Methods 2008, Vol. 46, pp. 25–32, or as described in S Jain et al., Gold nanoparticles as novel agents for cancer therapy. Br J Radiol. 2012 Feb; 85(1010):101–113.

[0108] In some implementations, the cells expressing a vector encoding a peptide as described herein may be cells of a subject, such as a subject receiving gene therapy for treating, suppressing diabetes (e.g., type 2 diabetes), reducing its severity, and / or slowing its progression. Vectors used for gene therapy may include viral or non-viral vectors as described elsewhere herein.

[0109] How to use

[0110] This document provides a method for treating, inhibiting, reducing the severity of, slowing the progression of, and / or promoting the prevention of a disease state in a subject of need. The method includes providing a composition comprising an agent that increases the release of intestinal hormones (such as GLP-1 and / or PYY), and administering an effective amount of the composition to the subject to treat, inhibit, reduce the severity of, and / or promote the prevention of the disease state. In some embodiments, GLP-1 is any one or more of GLP-1-(7-37), GLP-1-(7-36)NH2, or combinations thereof. In various embodiments, the disease state is diabetes. In one embodiment, the diabetes is type 2 diabetes. In another embodiment, the disease state is obesity. In some embodiments, the agent is an agonist of any one or more of SSTR2, TAS2R38, TAS2R39, TAS2R46, TAS2R47, FFAR1, FFAR2, FFAR4, or the FFAR4 receptor. In another embodiment, the agent is an antagonist of somatostatin. In one embodiment, the agent comprises a bitter oligopeptide. In some embodiments, the agent that increases intestinal hormone release is any one or more of BPx1, BPx2, BPx3, BPx4 or combinations thereof, or analogs thereof, pharmaceutical equivalents, or peptide mimics. In some embodiments, the bitter oligopeptide is conjugated to an agent that increases intestinal (e.g., distal small intestine) retention. In exemplary embodiments, examples of agents that increase intestinal retention include, but are not limited to, cellulose, fatty acids, polyethylene glycol (PEG), or combinations thereof. In some embodiments, treating, inhibiting, reducing the severity of, and / or promoting the prevention of the disease state further includes the administration of fatty acids and / or plant molecules found in food. In exemplary embodiments, fatty acids or plant molecules found in food include, but are not limited to, any one or more of the following: urolithin A, ellagic acid, ursolic acid, oleanolic acid, 6-m-propyl-2-thiouracil, propionic acid, butyrate, palmitic acid, or combinations thereof. In some embodiments, treating, suppressing, reducing the severity of, and / or promoting the prevention of the disease state further includes administering a cellulose-conjugated PTU (PTU-cellulose). In various embodiments, a combination of an agent that increases intestinal hormone release and fatty acids, plant molecules, and / or PTU-cellulose found in food is administered sequentially or simultaneously. In various embodiments, the combination of the agent that increases intestinal hormone release and fatty acids and / or PTU-cellulose is administered orally, enterally to the small intestine, or via rectal suppositories or enemas to the colon. In one embodiment, the subject is a human. In various embodiments, the compositions described herein are administered to the subject before, during, or after the subject has the disease state.In some embodiments, the composition is administered to the subject 1-3 times daily or 1-7 times weekly. In some embodiments, the composition is administered to the subject for 1-5 days, 1-5 weeks, 1-5 months, or 1-5 years.

[0111] This document also provides methods for treating, inhibiting, reducing the severity of diabetes, slowing the progression of diabetes, and / or promoting the prevention of diabetes in a subject of need. The methods include providing a composition comprising an agent that increases the release of intestinal hormones (such as GLP-1 and / or PYY), and administering an effective amount of the composition to the subject to treat, inhibit, reduce the severity of diabetes, and / or promote the prevention of diabetes. In some embodiments, GLP-1 is any one or more of GLP-1-(7-37), GLP-1-(7-36)NH2, or combinations thereof. In one embodiment, the diabetes is type 2 diabetes. In some embodiments, the agent is an agonist of any one or more of SSTR2, TAS2R38, TAS2R39, TAS2R46, TAS2R47, FFAR1, FFAR2, FFAR4, or FFAR4 receptors. In another embodiment, the agent is an antagonist of somatostatin. In one embodiment, the agent comprises a bitter oligopeptide. In some embodiments, the agent that increases intestinal hormone release is any one or more of BPx1, BPx2, BPx3, BPx4 or combinations thereof, or analogues thereof, pharmaceutical equivalents, or peptide mimics. In some embodiments, the bitter oligopeptide is conjugated with an agent that increases intestinal (e.g., distal small intestine) retention. In exemplary embodiments, agents that increase cell retention include, but are not limited to, cellulose, fatty acids, polyethylene glycol (PEG), or combinations thereof. In some embodiments, treating, inhibiting, reducing the severity of diabetes, and / or promoting the prevention of diabetes further includes the administration of fatty acids and / or plant molecules found in food that increase GLP-1 release. In exemplary embodiments, fatty acids or plant molecules found in food include, but are not limited to, any one or more of the following: urolithin A, ellagic acid, ursolic acid, oleanolic acid, 6-m-propyl-2-thiouracil, propionic acid, butyrate, palmitic acid, or combinations thereof. In some embodiments, treating, inhibiting, reducing the severity of diabetes, and / or promoting the prevention of diabetes further includes the administration of PTU conjugated with cellulose (PTU-cellulose). In various embodiments, the combination of the agent that increases the release of intestinal hormones with fatty acids and / or PTU-cellulose is administered sequentially or simultaneously. In various embodiments, the combination of the agent that increases the release of intestinal hormones with fatty acids and / or PTU-cellulose is administered orally, enterally into the small intestine, or via rectal suppositories or enemas into the colon. In one embodiment, the subject is a human. In various embodiments, the composition described herein is administered to the subject before, during, or after the subject has the disease state. In some embodiments, the composition is administered to the subject 1-3 times daily or 1-7 times weekly.In some embodiments, the composition is administered to a subject for 1-5 days, 1-5 weeks, 1-5 months, or 1-5 years.

[0112] This document further provides a method for treating, inhibiting, reducing the severity of obesity in a subject, and / or promoting the prevention of obesity in the subject. The method includes providing a composition comprising an agent that increases the release of intestinal hormones (such as GLP-1 and / or PYY), and administering an effective amount of the composition to the subject to treat, inhibit, reduce the severity of obesity in the subject, and / or promote the prevention of obesity in the subject. In some embodiments, GLP-1 is any one or more of GLP-1-(7-37), GLP-1-(7-36)NH2, or combinations thereof. In some embodiments, the agent is an agonist of any one or more of SSTR2, TAS2R38, TAS2R39, TAS2R46, TAS2R47, FFAR1, FFAR2, FFAR4, or FFAR4 receptors. In another embodiment, the agent is an antagonist of somatostatin. In one embodiment, the agent comprises a bitter oligopeptide. In some embodiments, the agent that increases intestinal hormone release is any one or more of BPx1, BPx2, BPx3, BPx4 or combinations thereof, or analogues thereof, pharmaceutical equivalents, or peptide mimics. In some embodiments, the bitter oligopeptide is conjugated with an agent that increases intestinal (e.g., distal small intestine) retention. In exemplary embodiments, agents that increase cell retention include, but are not limited to, cellulose, fatty acids, polyethylene glycol (PEG), or combinations thereof. In some embodiments, treating, inhibiting, reducing the severity of obesity, and / or promoting the prevention of obesity further includes the administration of fatty acids and / or plant molecules found in food that increase GLP-1 release. In exemplary embodiments, fatty acids or plant molecules found in food include, but are not limited to, any one or more of the following: urolithin A, ellagic acid, ursolic acid, oleanolic acid, 6-m-propyl-2-thiouracil, propionic acid, butyrate, palmitic acid, or combinations thereof. In some embodiments, treating, inhibiting, reducing the severity of obesity, and / or promoting the prevention of obesity further includes the administration of PTU conjugated with cellulose (PTU-cellulose). In various embodiments, the combination of the agent that increases the release of intestinal hormones with fatty acids and / or PTU-cellulose is administered sequentially or simultaneously. In various embodiments, the combination of the agent that increases the release of intestinal hormones with fatty acids and / or PTU-cellulose is administered orally, enterally into the small intestine, or via rectal suppositories or enemas into the colon. In one embodiment, the subject is a human. In various embodiments, the composition described herein is administered to the subject before, during, or after the subject has the disease state. In some embodiments, the composition is administered to the subject 1-3 times daily or 1-7 times weekly. In some embodiments, the composition is administered to the subject for 1-5 days, 1-5 weeks, 1-5 months, or 1-5 years.

[0113] In some embodiments, BPx1, BPx2, BPx3, BPx4 and / or combinations thereof, or analogues thereof, pharmaceutical equivalents or peptide mimics, as described herein for the treatment of diabetes, may be administered co-administered with existing treatments for diabetes. Oligopeptides BPx1, BPx2, BPx3, BPx4 and / or combinations thereof, or analogues thereof, pharmaceutical equivalents or peptide mimics, may be administered sequentially or simultaneously with existing treatments for diabetes. In some embodiments, existing treatments that may be used with the methods described herein include, but are not limited to, SGLT2 inhibitors (such as canagliflozin (Invokana), dapagliflozin (Farxiga), epaligliflozin (Jardiance)), insulin, inhaled insulin, sulfonylureas, metformin, acarbose, thiazolidinediones or combinations thereof.

[0114] In various embodiments, the effective amount of the agent that increases the release of intestinal hormones (such as GLP-1, PYY, or combinations thereof) (e.g., any one or more of BPx1, BPx2, BPx3, BPx4, and / or combinations thereof, or analogues thereof, pharmaceutical equivalents, or peptide mimics) is about 0.01 to 0.05 μg / kg / day, 0.05-0.1 μg / kg / day, 0.1 to 0.5 μg / kg / day, 0.5 to 5 μg / kg / day, 5 to 10 μg / kg / day, etc. 10 to 20 μg / kg / day, 20 to 50 μg / kg / day, 50 to 100 μg / kg / day, 100 to 150 μg / kg / day, 150 to 200 μg / kg / day, 200 to 250 μg / kg / day, 250 to 300 μg / kg / day, 300 to 350 μg / kg / day, 350 to 400 μg / kg / day, 400 to 500 μg / kg / day, 500 to 600 μg / kg / day, 600 to 700 μg / kg / day g / kg / day, 700 to 800 μg / kg / day, 800 to 900 μg / kg / day, 900 to 1000 μg / kg / day, 0.01 to 0.05 mg / kg / day, 0.05-0.1 mg / kg / day, 0.1 to 0.5 mg / kg / day, 0.5 to 1 mg / kg / day, 1 to 5 mg / kg / day, 5 to 10 mg / kg / day, 10 to 15 mg / kg / day, 15 to 20 mg / kg / day, 20 to 50 mg / kg / day Any one or more of the following doses: g / kg / day, 50 to 100 mg / kg / day, 100 to 200 mg / kg / day, 200 to 300 mg / kg / day, 300 to 400 mg / kg / day, 400 to 500 mg / kg / day, 500 to 600 mg / kg / day, 600 to 700 mg / kg / day, 700 to 800 mg / kg / day, 800 to 900 mg / kg / day, 900 to 1000 mg / kg / day, or combinations thereof. Typical doses of agents that increase the release of intestinal hormones (such as GLP-1, PYY, or combinations thereof) (e.g., any one or more of BPx1, BPx2, BPx3, BPx4 and / or combinations thereof, or analogues thereof, pharmaceutical equivalents, or peptide mimics) may be within the range recommended by the manufacturer for the use of known therapeutic compounds and may also be indicated to those skilled in the art by in vitro reactions or reactions in animal models. Such doses can typically be reduced in concentration or amount by up to about an order of magnitude without loss of the associated biological activity. The actual dose may depend on the physician's judgment, the patient's condition, and the effectiveness of the treatment method, which is based on, for example, the in vitro reactivity of the relevant cultured cells or tissue samples (such as biopsied malignant tumors) or the response observed in appropriate animal models.In various embodiments, the composition of the present invention comprising an agent that increases the release of intestinal hormones (such as GLP-1, PYY, or combinations thereof) (e.g., BPx1, BPx2, BPx3, BPx4 and / or combinations thereof, or analogues thereof, pharmaceutical equivalents, or peptide mimics) may be administered once daily (SID / QD), twice daily (BID), three times daily (TID), four times daily (QID), or more times daily to administer an effective amount to the subject, wherein the effective amount is any one or more of the doses described herein.

[0115] In each implementation scheme, the subjects are selected from the group consisting of: humans, non-human primates, monkeys, apes, dogs, cats, cows, horses, rabbits, mice, and rats.

[0116] This document also provides a method for screening peptides for the treatment of diabetes. The method includes providing one or more candidate peptides, fatty acids, or plant molecules found in food, contacting said candidate peptides, fatty acids, or plant molecules with GLP-1-secreting cells, and determining whether said contact results in an increase in GLP-1 secretion. An increase in GLP-1 secretion indicates that the candidate peptides, fatty acids, or plant molecules found in food may be used to treat diabetes. In one embodiment, the diabetes is type 2 diabetes. In some embodiments, the candidate peptide is a bitter oligopeptide. In some embodiments, the method includes contacting each of a plurality of candidate peptides to be tested separately. In some embodiments, the plurality of candidate peptides includes more than about 10 4 Multiple samples. In some embodiments, the multiple samples include more than about 5 x 10^6 samples. 4 One sample. In some embodiments, the cells secreting GLP-1 are Hu-Tu80 cells. In an exemplary embodiment, the increase in secretion is relative to a reference value. The reference value may be a negative control (e.g., the absence of the candidate peptide) or the GLP-1 secretion level in the presence of PTU or the GLP-1 secretion level in the presence of the fatty acids described herein, or a combination thereof. Other systems that test the ability of the ligand to interact with receptors known on L cells and responsible for regulating GLP-1 release may be used as an alternative to HuTu80 cells.

[0117] Pharmaceutical Composition

[0118] This document provides pharmaceutical compositions comprising, or substantially comprising, a therapeutic agent that increases the release of intestinal hormones and an acceptable carrier / excipient. In some embodiments, the therapeutic agent increases the release of any one or more of GLP-1, PYY, or combinations thereof to treat diabetes in a subject of need. In one embodiment, the diabetes is type 2 diabetes mellitus (T2DM). In one embodiment, the therapeutic agent is an oligopeptide. In another embodiment, the therapeutic agent is a small molecule. In some exemplary embodiments, the oligopeptide comprises the amino acid sequence YGLF (BPx1, SEQ ID NO:1) or its analogues, pharmaceutical equivalents, or peptide mimics, YPFPGPIPN (BPx2, SEQ ID NO:2) or its analogues, pharmaceutical equivalents, or peptide mimics, IPAVF (BPx3, SEQ ID NO:3) or its analogues, pharmaceutical equivalents, or peptide mimics, LLF or its analogues, pharmaceutical equivalents, or peptide mimics, or combinations thereof, comprising or substantially comprising thereof.

[0119] In various embodiments, the pharmaceutical compositions according to the invention can be formulated for delivery via any route of administration. "Route of administration" can refer to any route of administration known in the art, including but not limited to aerosol, nasal, oral, mucosal, percutaneous, parenteral, or enteral. In some embodiments, the pharmaceutical composition can be administered enterically to the small intestine. In some embodiments, the pharmaceutical composition can be administered orally. In some embodiments, the pharmaceutical composition can be administered to the colon via a rectal suppository or enema. "Parenteral" refers to routes of administration typically associated with injection, including intraorbital, infusion, intra-arterial, intra-bursal, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intravertebral, intrasternal, intrasheath, intrauterine, intravenous, subarachnoid, subbursal, subcutaneous, transmucosal, or transtracheal routes. via the parenteral route, the composition can be in the form of a solution or suspension for infusion or injection, or in the form of a lyophilized powder. via the parenteral route, the composition can be in the form of a solution or suspension for infusion or injection. The pharmaceutical composition may be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres, lipid vesicles, or polymer vesicles via the enteral route.

[0120] As used herein, the phrases “parenteral administration” and “extragastric administration” refer to modes of administration other than intravenous and local administration, typically by injection. As used herein, the phrases “systemic administration,” “systemic administration,” “peripheral administration,” and “peripheral administration” refer to the administration of a therapeutic agent that increases the release of intestinal hormones beyond direct access to the target site, tissue, or organ, so that the therapeutic agent enters the subject’s circulatory system and is thus metabolized and undergoes other similar processes.

[0121] "Pharmaceutically acceptable excipients" means excipients that are generally safe, non-toxic, and suitable for the preparation of pharmaceutical compositions, and includes excipients acceptable for veterinary and human pharmaceutical use. Such excipients may be solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous.

[0122] The pharmaceutical compositions according to the invention may also contain any pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable substance, composition, or medium relating to carrying or delivering a target compound from one tissue, organ, or part of the body to another tissue, organ, or part of the body. For example, a carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating substance, or a combination thereof. Each component of the carrier must be "pharmaceutical acceptable" because it must be compatible with the other components of the formulation. It must also be suitable for contact with any tissue or organ to which it may come into contact, meaning it must not carry the risk of toxicity, irritation, allergic reactions, immunogenicity, or any other complications that outweigh its therapeutic benefit.

[0123] The pharmaceutical compositions according to the invention can also be encapsulated, tableted, or prepared in the form of emulsions or syrups for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate its preparation. Liquid carriers include syrups, peanut oil, olive oil, glycerin, saline, alcohol, and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, clay, magnesium stearate or stearic acid, talc, pectin, gum arabic, agar, or gelatin. The carrier may also include sustained-release substances, such as glyceryl monostearate or glyceryl distearate alone or in combination with waxes.

[0124] Pharmaceutical formulations are prepared following conventional pharmaceutical techniques, including grinding, mixing, granulation, and, where necessary, compression (for tablets); or grinding, mixing, and filling (for hard gelatin capsules). When a liquid carrier is used, the formulation will be in the form of a syrup, elixir, emulsion, or aqueous or non-aqueous suspension. Such liquid formulations can be administered directly orally or filled into soft gelatin capsules.

[0125] The pharmaceutical compositions according to the invention can deliver a therapeutically effective amount. The precise therapeutically effective amount is the amount at which the composition will produce the most effective result in terms of therapeutic efficacy in a given subject. This amount will vary depending on a number of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dose, and type of drug), the nature of one or more pharmaceutically acceptable carriers in the formulation, and the route of administration. Those skilled in the art of clinical and pharmacology will be able to determine the therapeutically effective amount through routine experiments, such as by monitoring the subject's response to the administered compound and adjusting the dose accordingly. For further guidance, see Remington: The Science and Practice of Pharmacy (Gennaro, ed., 20th edition, Williams & Wilkins PA, USA) (2000).

[0126] The therapeutic agents described herein can be administered to a patient in a single dose or in multiple doses. When multiple doses are administered, the doses may be spaced apart from each other, for example, 1 hour, 3 hours, 6 hours, 8 hours, 1 day, 2 days, 1 week, 2 weeks, or 1 month. For example, the therapeutic agent may be administered for, for example, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or more weeks. In various embodiments, the composition is administered to the subject 1-3 times daily or 1-7 times weekly. In various embodiments, the composition is administered to the subject for 1-5 days, 1-5 weeks, 1-5 months, or 1-5 years. It should be understood that for any particular subject, the specific dosing regimen should be adjusted over time according to the individual's needs and the professional judgment of the person administering or supervising the administration of the composition. For example, if a lower dose does not provide sufficient therapeutic activity, the dose of the therapeutic agent may be increased. Although the attending physician will ultimately determine the appropriate dosage and regimen, therapeutically effective doses of one or more peptides, or mutants, variants, analogs, or derivatives thereof, as disclosed herein, may be provided at doses of 0.0001, 0.01, 0.01, 0.1, 1, 5, 10, 25, 50, 100, 500, or 1,000 mg / kg or μg / kg. Effective doses can be extrapolated from dose-response curves obtained from in vitro or animal model bioassays or systems.

[0127] As used herein, an effective quantity will also include quantities sufficient to delay the development of disease symptoms, alter the course of disease symptoms (e.g., but not limited to slowing the progression of disease symptoms), or reverse disease symptoms. Therefore, it is impossible to specify a precise “effective quantity.” However, for any given situation, an appropriate “effective quantity” can be determined by one of ordinary skill in the art using only conventional experimental methods.

[0128] Effective doses, toxicity, and therapeutic efficacy can be determined in cell cultures or laboratory animals using standard pharmaceutical procedures, such as those used to determine LD50 (the dose that is lethal to 50% of the population) and ED50 (the dose that is therapeutically effective in 50% of the population). The dose can vary depending on the dosage form and route of administration used. The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD50 / ED50. Compositions and methods exhibiting a large therapeutic index are preferred. The therapeutically effective dose can initially be evaluated from cell culture assays. Additionally, doses can be formulated in animal models to achieve circulating plasma concentration ranges including the IC50 (i.e., concentrations of therapeutic agents that increase intestinal hormone release, achieving half-maximal inhibition of symptoms), as determined in cell cultures or suitable animal models. Plasma levels can be measured, for example, by high-performance liquid chromatography. The effect of any particular dose can be monitored by appropriate bioassays. The dose can be determined by a physician and adjusted as necessary to suit the observed therapeutic effect.

[0129] The phrase "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within reasonable medical judgment, are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and are commensurate with a reasonable benefit / risk ratio. As used herein, the phrase "pharmaceutically acceptable carrier" or "pharmaceuticalally acceptable excipient" refers to pharmaceutically acceptable materials, compositions, or mediators that participate in maintaining the stability, solubility, or activity of therapeutic agents that increase the release of intestinal hormones, such as liquid or solid fillers, diluents, excipients, solvents, mediators, encapsulating materials, manufacturing aids (e.g., lubricants, magnesium talc, calcium stearate, zinc stearate, or stearic acid), or solvent encapsulating materials. Each carrier must be "acceptable" in the sense of compatibility with other components of the formulation and harmlessness to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth gum; (5) malt; (6) gelatin; (7) excipients, such as cocoa butter and suppository waxes; (8) oils, such as peanut oil and cottonseed oil. Oils, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (9) glycols, such as propylene glycol; (10) polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol (PEG); (11) esters, such as ethyl oleate and ethyl laurate; (12) agar; (13) buffers, such as magnesium hydroxide and aluminum hydroxide; (14) alginic acid; (15) pyrogen-free water; (16) isotonic saline; (17) Ringer's solution; (18) pH buffer solutions; (19) polyesters, polycarbonates, and / or polyanhydrides; (20) fillers, such as peptides and amino acids; (21) serum components, such as serum albumin, HDL, and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic and compatible substances used in pharmaceutical preparations. Release agents, coating agents, preservatives, and antioxidants may also be present in the formulation. Terms such as “excipients,” “carriers,” and “pharmaceutically acceptable carriers” are used interchangeably in this document.

[0130] Therapeutic agents that increase the release of intestinal hormones described herein may be specifically formulated for administration to a subject in solid, liquid, or gel form, including those suitable for: (1) parenteral administration, e.g., as a sterile solution or suspension or sustained-release formulation via subcutaneous, intramuscular, intravenous, or epidural injection; (2) local application, e.g., as a cream, ointment, controlled-release patch, or spray applied to the skin; (3) intravaginal or rectal application, e.g., as a pessary, cream, or foam; (4) ocular application; (5) percutaneous application; (6) transmucosal application; (7) intraenteral application; or (8) nasal application. Furthermore, therapeutic agents that increase the release of intestinal hormones described herein may be implanted in the patient or injected using a drug delivery system. See, for example, Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24:199-236 (1984); Lewis, ed., "Controlled Release of Pesticides and Pharmaceuticals" (Plenum Press, New York, 1981); U.S. Patent No. 3,773,919; and U.S. Patent No. 3,270,960.

[0131] The following description can be used for other implementation schemes of formulations and administration methods of therapeutic agents that increase the release of intestinal hormones in the methods described herein.

[0132] Parenteral formulations. Parenteral formulations of therapeutic agents that increase the release of intestinal hormones can also be administered to subjects via various routes, including but not limited to subcutaneous, intravenous (including bolus), intramuscular, and intra-arterial administration. Since administration of parenteral formulations typically bypasses the patient's natural defenses against contaminants, parenteral formulations are preferably sterile or capable of being sterilized prior to administration to the patient. Examples of parenteral formulations include, but are not limited to, solutions prepared for injection, anhydrous products prepared for dissolution or suspension in pharmaceutically acceptable injectable media, suspensions prepared for injection, controlled-release parenteral formulations, and emulsions.

[0133] Suitable media for providing the parenteral dosage forms of this disclosure are well known to those skilled in the art. Examples include, but are not limited to: sterile water; water for injection (USP); saline solution; glucose solution; aqueous media, such as, but not limited to, sodium chloride injection, Ringer's solution, glucose injection, glucose and sodium chloride injection, and lactated Ringer's solution; water-miscible media, such as, but not limited to, ethanol, polyethylene glycol, and propylene glycol; and non-aqueous media, such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0134] Aerosol formulations. Therapeutic agents that increase enterosteroid release can be packaged in pressurized aerosol containers with suitable propellants (such as hydrocarbon propellants like propane, butane, or isobutane) and conventional adjuvants. Therapeutic agents that increase enterosteroid release can also be administered in non-pressurized forms, such as in nebulizers or sprayers. Therapeutic agents that increase enterosteroid release can also be administered directly to the airways in dry powder form, for example, using an inhaler.

[0135] As an illustration, suitable powder compositions include powder formulations of therapeutic agents that increase the release of intestinal hormones and are well mixed with lactose, or other inert powders acceptable for intrabronchial administration. The powder composition may be administered via an aerosol dispenser or encapsulated in a breakable capsule that can be inserted into a device by a subject, the device being able to puncture the capsule and expel the powder in a stable flow suitable for inhalation. The composition may contain a propellant, a surfactant, and a co-solvent, and may be filled into a conventional aerosol container sealed by a suitable metering valve.

[0136] Aerosols for delivery to the respiratory tract are known in the art. See, for example, Adjei, A. and Garren, J. Pharm. Res., 1:565-569 (1990); Zanen, P. and Lamm, J.-WJ Int. J. Pharm., 114:111-115 (1995); Gonda, I. "Aerosols for delivery of therapeutic an diagnostic agents to the respiratory tract," in Critical Reviews in Therapeutic Drug Carrier Systems, 6:273-313 (1990); Anderson et al., Am. Rev. Respir. Dis., 140:1317-1324 (1989)) and also have the potential for systemic delivery of peptides and proteins (Patton and Platz, Advanced Drug Delivery). Reviews, 8:179-196 (1992)); Timsina et al., Int. J. Pharm., 101:1-13 (1995); and Tansey, IP, Spray Technol. Market, 4:26-29 (1994); French, DL, Edwards, DA and Niven, RW, Aerosol Sci., 27:769-783 (1996); Visser, J., Powder Technology 58:1-10 (1989)); Rudt, S. and RHMuller, J. Controlled Release, 22: 263-272 (1992); Tabata, Y, and Y. Ikada, Biomed. Mater. Res., 22: 837-858 (1988); Wall, DA, Drug Delivery, 2: 10 1-201995); Patton, J. and Platz, R., Adv. Drug Del. Rev., 8:179-196 (1992); Bryon, P., Adv. Drug Del. Rev., 5:107-132 (1990); Patton, JS, et al., Controlled Release, 28:1579-85 (1994); Damms, B. and Bains, W., Nature Biotechnology (1996); Niven, RW, et al., Pharm. Res.,12(9); 1343-1349(1995); and Kobayashi, S., et al., Pharm. Res., 13(1):80-83(1996), the entire contents of which are incorporated herein by reference.

[0137] The formulations of therapeutic agents that increase the release of intestinal hormones described herein also encompass anhydrous pharmaceutical compositions and dosage forms containing the disclosed compounds as active ingredients, as water can promote the degradation of some compounds. For example, the addition of water (e.g., 5%) is widely accepted in the pharmaceutical industry as a method to simulate long-term storage in order to determine properties such as shelf life or stability of the formulation over time. See, for example, Jens T. Carstensen, Drug Stability: Principles & Practice, 379-80 (2nd edition, Marcel Dekker, NY, NY: 1995). The anhydrous pharmaceutical compositions and dosage forms of this disclosure can be prepared using anhydrous or low-moisture components and under low-moisture or low-humidity conditions. Pharmaceutical compositions and dosage forms containing lactose and at least one active ingredient, including a primary or secondary amine, are preferably anhydrous if substantial contact with moisture and / or humidity during manufacturing, packaging, and / or storage is desired. It is preferred to package the anhydrous compositions using materials known to prevent exposure to water so that they can be included in a suitable formulary kit. Examples of suitable packaging include, but are not limited to, hermetically sealed foil, plastic, unit-dose containers (e.g., vials) with or without desiccant, blister packs, and strip packs.

[0138] Controlled and delayed release formulations. In some embodiments of the methods described herein, therapeutic agents that increase the release of intestinal hormones may be administered to subjects via controlled or delayed release. Ideally, the use of optimally designed controlled-release formulations in medical treatment is characterized by the use of minimal drug substance to cure or control symptoms in the shortest possible time. Advantages of controlled-release formulations include: 1) prolonged drug activity; 2) reduced dose frequency; 3) improved patient compliance; 4) less total drug use; 5) reduced local or systemic side effects; 6) minimized drug accumulation; 7) reduced fluctuations in blood levels; 8) improved therapeutic efficacy; 9) reduced enhancement or loss of drug activity; and 10) increased speed of disease or symptom control (Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000)). Controlled-release formulations can be used to control the onset, duration of action, plasma levels within the therapeutic window, and peak blood levels of the compound. Specifically, controlled or extended-release formulations or formulations can be used to ensure maximum effectiveness of the compound of formula (I) while minimizing potential adverse effects and safety issues that may occur due to insufficient drug dosage (i.e., below the minimum therapeutic level) and exceeding the toxicity level of the drug.

[0139] A variety of known controlled or prolonged release dosage forms, formulations, and devices are suitable for use with therapeutic agents that increase the release of enterosteroids described herein. Examples include, but are not limited to, those described in U.S. Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185B1, each of which is incorporated herein by reference in its entirety. These dosage forms can be used with, for example, different proportions of hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, permeable systems (such as... (Alza Corporation, Mountain View, Calif.USA)), multilayer coatings, microparticles, liposomes or microspheres, or combinations thereof, can be used to provide the desired release characteristics for the slow or controlled release of one or more active ingredients. Furthermore, ion exchange materials can be used to prepare fixed, adsorbed salt forms of the disclosed compounds, thereby achieving controlled drug delivery. Specific examples of anion exchangers include, but are not limited to, A568 and AP143 (Rohm & Haas, Spring House, Pa. USA).

[0140] In some embodiments, the therapeutic agent for increasing enterosteroid release used in the methods described herein is administered to the subject via continuous release or pulsed administration. Pulsed therapy is not a form of discontinuous administration of the same amount of composition over time, but rather comprises administration of the same dose of composition at a reduced frequency or a reduced dose. Continuous release or pulsed administration is particularly preferred when symptoms persist in the subject, such as when the subject has persistent or chronic symptoms of a viral infection. The dose per pulse can be reduced, and the total amount of the therapeutic agent for increasing enterosteroid release administered to the patient during treatment can be minimized.

[0141] If necessary, the interval between pulses can be determined by those skilled in the art. Typically, the time interval between pulses can be calculated by administering another dose of the composition when the composition or its active component is no longer detectable in the subject before the next pulse is delivered. The time interval can also be calculated from the in vivo half-life of the composition. The time interval can be calculated to be greater than the in vivo half-life, or 2, 3, 4, 5, or even 10 times the half-life of the composition. Various methods and apparatus for delivering pulsed compositions to patients by infusion or other means are disclosed in U.S. Patent Nos. 4,747,825; 4,723,958; 4,948,592; 4,965,251, and 5,403,590.

[0142] The reagent kit of the present invention

[0143] The present invention also provides a kit for treating, inhibiting, and / or reducing the severity of diabetes in a subject of need. The kit comprises a composition containing a therapeutic agent that increases the release of intestinal hormones, and instructions for using the composition to treat, inhibit, and / or reduce the severity of diabetes in a subject of need. In some embodiments, the therapeutic agent that increases the release of intestinal hormones is an oligopeptide, wherein the oligopeptide comprises the amino acid sequence YGLF (SEQ ID NO:1) or an analogue thereof, a pharmaceutical equivalent, or a peptide mimic, YPFPGPIPN (SEQ ID NO:2) or an analogue thereof, a pharmaceutical equivalent, or a peptide mimic, IPAVF (SEQ ID NO:3) or an analogue thereof, a pharmaceutical equivalent, or a peptide mimic, LLF or an analogue thereof, a pharmaceutical equivalent, or a peptide mimic, or a combination thereof, constitutes or substantially constitutes thereof.

[0144] The kit is a collection of materials or components comprising at least one composition described herein. Therefore, in some embodiments, the kit contains a composition comprising an oligopeptide, wherein the oligopeptide comprises the amino acid sequence YGLF (SEQ ID NO:1) or an analogue, pharmaceutical equivalent, or peptide mimic, YPFPGPIPN (SEQ ID NO:2) or an analogue, pharmaceutical equivalent, or peptide mimic, IPAVF (SEQ ID NO:3) or an analogue, pharmaceutical equivalent, or peptide mimic, LLF or an analogue, pharmaceutical equivalent, or peptide mimic, or a combination thereof, or any one or more combinations thereof, comprising or substantially comprising thereof.

[0145] The accuracy of the components configured in the kit of the present invention depends on the intended purpose of the kit. In one embodiment, the kit is specifically configured for human subjects. In other embodiments, the kit is configured for veterinary applications treating subjects, such as, but not limited to, livestock, domestic animals, and laboratory animals.

[0146] Instructions for use may be included in the kit. These instructions typically include a clear description of the techniques used to achieve the desired results, such as treating or suppressing diabetes in the subject, or reducing the severity of said diabetes. Optionally, the kit may also contain other suitable components, such as measuring instruments, diluents, buffers, pharmaceutically acceptable carriers, syringes, or other suitable accessories that will be readily recognized by those skilled in the art.

[0147] Materials or components configured in the kit to maintain the operability and utility of materials or components can be stored in any convenient and suitable manner to provide practitioners with the necessary storage. For example, components may be in dissolved, dehydrated, or lyophilized form; they may be provided at room temperature, refrigerated temperature, or frozen temperature. Components are typically contained in suitable packaging materials. As used herein, the phrase "packaging material" refers to one or more physical structures for containing the contents of the kit (such as the compositions of the present invention). Packaging materials are constructed by well-known methods and preferably provide a sterile, contaminant-free environment. As used herein, the term "packaging" refers to a suitable solid matrix or material, such as glass, plastic, paper, foil, etc., capable of containing individual kit components. Thus, for example, packaging may be a bottle for containing an appropriate amount of the compositions of the present invention containing pharmaceutical compositions as described herein. The packaging material typically has an external label indicating the contents and / or purpose of the kit and / or its components.

[0148] Example

[0149] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. The specific materials mentioned are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can develop equivalent means or reactants without exercising the capabilities of the invention and without departing from its scope.

[0150] Example 1

[0151] Experimental methods

[0152] Human ileal tissue from Cedars-Sinai Biobank was analyzed using IRB protocol 34332 to observe the presence of the receptor in L cells by immunohistochemistry targeting the colocalization of TAS2R38 and GLP-1. In a parallel study, the previously determined structure of the taster haplotype PAV of TAS2R38 [J. Tan, et al., 3D Structure Prediction of TAS2R38 Bitter Receptors Bound to Agonists Phenylthiocarbamide (PTC) and 6-n-Propylthiouracil (PROP), J Chem Inf Model 52 (2012) 1875-1885] was used for structure-based virtual ligand screening of commercially available compounds from the ZINC database [JJ Irwin, T, et al., ZINC: a free tool to discover chemistry for biology, J Chem Inf Model 52 (2012) 1757-1768]. Three of the popular molecules were purchased and their potential for GLP-1 release was tested in in vitro and in vivo assays using HuTu-80 cells. One of the molecules was also tested using TAS2R38 knockout cells. The methods and materials used in these studies are described below.

[0153] Immunohistochemistry

[0154] Experiments were designed to determine whether the TAS2R38 receptor is expressed on human enteroendocrine L cells. To identify whether it co-localizes with GLP-1 on native L cells, IHC was performed on human GI tissue using double immunochromatography with previously validated GPCRs and GLP-1 antibodies. The antibodies used were: TAS2R38 [rabbit polyclonal (H:ab65509, Abcam)] and GLP-1 [goat polyclonal (sc-26637, Santa Cruz Biotechnology)]. The number of cells stained with GLP-1 or TAS2R38, or both, was visually counted.

[0155] Virtual ligand screening

[0156] The DOCK Blaster server

[38] was used in virtual ligand screening (VLS) studies using previously predicted PTU-binding PAV conformations

[36] , which has access to several compound libraries, including a library from the ZINC database with approximately 2 million commercially available compounds [JJIrwin, et al., ZINC: A Free Tool to Discover Chemistry for Biology, J Chem Inf Model (2012)]. For each ligand molecule, the server hooks multiple ligand conformations corresponding to its internal free torsion to a user-provided putative binding site and sorts the molecules using a scoring function. The top 500 most popular molecules were obtained from this server and prioritized using a more accurate scoring function based on the all-atom Dreiding force field [SLMayo, BDOlafson, WAGoddard, Dreiding—a Generic Force-Field for Molecular Simulations, Journal of Physical Chemistry 94(1990)8897-8909] to select the top 200 different small molecules corresponding to different chemical scaffolds. The top 15 molecules were selected from this list, and three of them were purchased for further research.

[0157] PTU - Cellulose Synthesis

[0158] The synthesis reaction is summarized in Figure 4A In this study, PTUs with substituted methyl groups were synthesized by first performing a Claisen condensation of 2,2-dimethyl-1,3-dioxane-4,6-dione with glutaric anhydride to produce 7-ethoxy-5,7-dioxoheptanoic acid, which was then condensed with thiourea to produce carboxyl-substituted PTUs. These carboxyl-substituted PTUs were then conjugated with the hydroxyl groups of cellulose in DMSO using N,N'-diisopropylcarbodiimide (DIC) and 4-dimethylaminopyridine (DMAP). The structure of the product was confirmed by IR spectroscopy.

[0159] In vitro studies

[0160] a. GLP-1 release.HuTu-80 cells (HTB-40, ATCC) were proliferated in Dulbecco's modified Eagle's medium (DMEM) supplemented with a mixture of 10% FBS and PSG antibiotics, containing 4 mM L-glutamine, 1.5 g / L sodium bicarbonate, and 4.5 g / L glucose, and maintained at 10% CO2 and 37°C. For experiments, these cells were seeded in 6-well plates or 100-mm... 2 In tissue culture dishes, cells were proliferated to confluence (5–7 days) and incubated overnight in serum-free medium before treatment. Prior to experiments, discarded medium was replaced with OPTI-MEM (Gibco, Life Technologies, Grand Island, NY) containing a mixture of Halt protease and phosphatase inhibitors (Thermo Fisher Scientific, Rockford, IL), and administered to each well for 30 minutes. For experiments, dose-dependent GLP-1 release from HuTu-80 cells into the medium was determined. Medium from the cells was collected and frozen for subsequent peptide hormone release measurements using the Luminex MAGPIX Metabolic Multiplex Assay Kit (HMHMAG-34K) according to the manufacturer's instructions.

[0161] b. Inhibition of GPCR expression. To determine the specific GPCR-mediated response to a particular ligand, in vitro RNA silencing of TAS2R38 was performed. RNA silencing was performed using a commercial kit (FlexiTube siRNA, Qiagen) consisting of four pre-designed oligonucleotides specifically targeting each gene transcript. Each kit also included a concomitant control and quantification assay for detecting gene knockdown. Cultured HuTu-80 cells were transfected with single and mixed oligonucleotide siRNAs of TAS2R38 using HiPerFect transfection reagent (Qiagen) or Lipofectamine 2000 (Life Tech) according to the manufacturer's recommended procedure. Cells were maintained in basal medium for up to 48 hours. Gene knockdown was assessed by RT-qPCR and Western blotting using appropriate antibodies to ensure inhibition. In parallel groups of transfected HuTu-80 cells, GLP-1 release was stimulated using an effective dose of the specific ligand. Dosage-response curves for the ligand with and without siRNA treatment were generated.

[0162] In vivo studies

[0163] The tested ligands were administered via gavage to pathogen-free male BALB / c mice aged 6 to 8 weeks: the predicted ligand, a PTU-cellulose conjugate; PTU calculated as the same amount in PTU-cellulose; or cellulose alone. Retroorbital blood was obtained, and GLP-1 was measured at regular time intervals.

[0164] Example 2

[0165] The presence of GLP-1 / PYY-releasing GPCR receptors on the luminal surface of regulatory L cells in human intestinal tissue from bariatric surgery patients was determined using immunohistochemistry (IHC) and / or in situ hybridization (ISH).

[0166] Members of the TAS2R and FFAR GPCR subfamilies are functionally expressed in human L cell lines (HuTu-80 and H716 cells) and detectable in intestinal tissue by RT-PCR analysis. Numerous L cells from human GI tissues of the ileum and colon were identified using immunohistochemistry based on staining with antibodies against GLP-1 using Cedars-Sinai Medical Center Biobank and its Microscopy Core. Several of these L cells showed colocalization of GLP-1 with the bitter taste receptor TAS2R38, the fatty acid receptor GPR40 (FFAR1)4, or the somatostatin receptor SSTR2, as determined by antibodies against the corresponding receptors. These receptors are primarily located on the cell surface, and GLP-1 was also found in cytoplasmic granules within these cells. These colocalization data suggest a role for these receptors in regulating GLP-1 release from L cells.

[0167] Somatostatin receptor subtype 5 (SSTR5) has been shown in animal models to be used by GLP-1 to regulate itself (via paracrine signaling) by inducing the release of somatostatin from adjacent D cells, which inhibits incretin release from L cells. Models demonstrating this include... Figure 1The invention illustrates the use of somatostatin antagonists to enhance GLP-1 release. While the inventors did not find SSTR5 receptors in human ileal L cells, they detected another subtype, SSTR2, on the apical surface of L cells. This indicates the presence of ligands generated within the intestinal lumen that regulate the GLP-1 secretory response in L cells. These ligands could be digestive products of food or products of action of bacteria present in the intestinal lumen. Others have identified GLP-1 receptors in the human gastrointestinal mucosa and enteric neurons, which could potentially mediate this paracrine signaling on D cells. As described herein, SSTR2 ligands can be used to regulate GLP-1 release from L cells. Specifically, SSTR2 agonists, alone and in combination with bitter oligopeptides, can be used to modulate the level of GLP-1 release. Furthermore, agents acting as SSTR2 antagonists, alone and in combination with bitter oligopeptides, fatty acids, and / or plant molecules from food, can be used to enhance GLP-1 release from L cells.

[0168] Identified molecular structures of L cell Gpcrs bound to oligopeptides to guide in vitro and in vivo studies

[0169] The inventors have used a validated computational method to predict the structure of the bitter taste receptor TAS2R38, which binds to flavor enhancers, to identify small molecules from a macromolecular library that subsequently induced GLP-1 release in in vitro and in vivo studies. Combining these methods with experiments to predict ligands / peptides provides the most efficient way to explore mechanisms.

[0170] Atomic-level structural prediction of the bitter taste receptor TAS2R38 requires consideration of known haplotypes of the receptor (tasting and non-tasting individuals, as described below). Phenylethiourea (PTC) and 6-n-propylthiouracil (PTU) are two potent and specific agonists of TAS2R38. PTCs are very bitter to some individuals but essentially tasteless to others, a phenomenon traced back to two common forms of the TAS2R38 gene exhibiting single nucleotide polymorphisms (SNPs) at three sites: a) amino acid (AA) position 49, encoding Pro / Ala; b) AA position 262, encoding Ala / Val; and c) AA position 296, encoding Val / Ile. These variations result in two frequent haplotypes, hTAS2R38PAV and hTAS2R38AVI, with hTAS2R38PAV showing a strong response to PTC at μM concentrations, while hTAS2R38AVI is unresponsive to PTC. Mutation analysis has shown that amino acid positions 49 and 262 are crucial for cellular responses. To address the molecular differences between bitter taster and non-tasting populations based on the hTAS2R38 receptor and the corresponding activation mechanisms of its agonists PTC and PTU, the inventors used their own computational methods to predict the atomic-level structures of a range of bitter receptor haplotypes: from taster hTAS2R38PAV to non-tasting hTAS2R38AVI (non-tasting).

[0171] The inventors then used the GenDock method to predict the binding sites and binding affinity of PTC and PTU for both the taster and non-tasterant receptor forms. Both PTC and PTU formed a stable H bond with residue 262 of the taster (hTAS2R38PAV) receptor form, but not with the residue of the non-tasterant (hTAS2R38AVI) receptor form. Therefore, this residue appears to be crucial for the experimentally consistent PTC / TAS2R38 taster difference. Recently, the inventors docked one of the bitter oligopeptides (α-lactamase) to TAS2R38. The binding pocket is located in... Figure 2 As shown in the image.

[0172] Studies using L-cell in vitro models and mouse in vivo models were conducted to validate the role of oligopeptides mediated by receptor-based mechanisms.

[0173] These studies were conducted using HuTu-80 cells, an excellent model of enteroendocrine L cells. Previous studies have demonstrated that at least 17 members of the TAS2R family are expressed in human colon tissue, and 11 of them were also detected in HuTu-80 cells. Furthermore, these cells respond to bitter ligands such as PTC ligands that release GLP-1. To confirm the presence of receptors for this substance, RT-PCR analysis was performed. HuTu-80 cell cDNA was used as a template to detect the presence of TAS2R and FFAR transcripts by RT-PCR. Transcripts of TAS2R, FFAR, and L cell markers, glucagon (GCG) / GLP-1, PYY, and chromogranin A (CgA) are readily detectable at basal levels in these cells, making them suitable for in vitro studies in this protocol.

[0174] GLP-1 release was measured using HuTu-80 cells exposed to bitter oligopeptides derived from milk protein and fatty acid ligands. Figure 3 The dose-response effect of each of the ligands is illustrated, thus demonstrating for the first time that bitter oligopeptides derived from the digestion of dietary proteins can activate GLP-1 release. GLP-1 secretion responses in response to propionate and butyrate fatty acids known to activate FFAR3, and palmitate known to activate FFAR1, were also observed. Finally, the results indicate that metformin can activate GLP-1 release, consistent with recent reports, and suggest that the action of metformin is partially mediated by receptors on L cells. These results suggest that HuTu80 cells provide a large platform for receptor-associated GLP-1 secretion.

[0175] To determine whether a TAS2R38 receptor-specific agonist (PTU) could activate L-cell secretion of GLP-1 in animals, PTU or a PTU-cellulose conjugate (calculated to obtain the same number of PTU molecules) were administered. The conjugate was intended to retain PTU in the intestinal lumen to demonstrate that the activity of PTU in inducing GLP-1 release was due to interaction with the luminal surface of the TAS2R38 receptor. The molecules were administered via gavage to pathogen-free 6- to 8-week-old male BALB / c mice. Retroorbital blood was obtained, and GLP-1 levels were measured at regular time intervals. Figure 4BThe results showed that both PTU and its conjugates induced GLP-1 release into the bloodstream, with a greater increase and longer duration of release in the PTU conjugate case compared to PTU alone. Cellulose alone had no effect. These results indicate the release of GLP-1 via administration of a TAS2R38 agonist via gastric tube. Using structural analysis, novel molecules that interact with TAS2R38 to induce GLP-1 release in HuTu80 cells were identified. These molecules were administered to mice via gastric tube, and they induced GLP-1 release in mice. This result further validates the inventors' modeling approach for ligand association of specific receptors.

[0176] The most potent and efficient bitter peptides were synthesized in a GMP facility, combined with an FDA-approved formulation designed for colonic / ileal targeted delivery, and encapsulated for administration to patients with prediabetes.

[0177] For the in vitro testing of bitter oligopeptides, the inventors outsourced the synthesis of 10 mg of each of four peptides (BPx1 to BPx4). Based on in vivo studies in mice ( Figure 3 The most efficient oligopeptides BPx1 and BPx2 will be synthesized in moderate quantities (<100 gms) in a Good Manufacturing Practice (GMP) facility.

[0178] Example 3

[0179] Immunohistochemistry was used to identify numerous cells colocalizing with TAS2R38 and GLP-1 in human GI tissues from the ileum and colon using the Cedars-Sinai Medical Center Biobank and its Microscopy Core. Cells stained with an antibody against GLP-1 were considered to be L cells. GLP-1 colocalizes with TAS2R38, which is primarily observed on the cell surface and at a higher level facing the lumen. Additionally, visual counting of stained cells covering ileal and colonic tissues showed that approximately 8% of ileal L cells and approximately 12% of colonic L cells were stained against TAS2R38. Detailed figures are shown in Table 1. These figures should be considered qualitative, as quantitative counting of colocalization is challenging due to the small surface area of ​​the pyramidal L cells' apex and the lumen-facing surface.

[0180] Table 1. Number of cells stained only with TAS2R38 and GLP-1, and for ileal tissue (first four rows) and colon co-localization.

[0181]

[0182] A dose-response study was conducted on the effects of PTU and Z7 on GLP-1 release in wild-type and TAS2R38 knockout HuTu-80 cells. Figure 5 As shown, PTU induces the release of GLP-1, which shifts to the right in cells with TAS2R38 knockdown. Figure 6 , Figure 7A , Figure 7B and Figure 8 The results show receptor expression and knockdown in HuTu-80 cells using TAS2R38 receptor siRNA. These results strongly indicate that the PTU ligand interacts with this receptor. The fact that larger concentrations of the ligand can induce GLP-1 release in the presence of reduced receptor expression suggests that these ligands also interact with other receptors to induce GLP-1 release, or that only a small fraction of the receptor is required to provide a complete response. These preliminary data also indicate that GLP-1 release in HuTu-80 cells is sensitive and specific to some bitter ligands, making these cells an ideal model for rapid screening of new compounds.

[0183] The PTU-cellulose conjugate was tested to observe whether GLP-1 release could be maintained by slowing the absorption of PTU from the gastrointestinal tract. Because the target receptor can enter through the lumen, the potential drug molecule does not need to be distributed systemically according to its pharmacological action. Figure 4B The results showed that both PTU and PTU-cellulose induced GLP-1 release into the bloodstream, with a greater magnitude and longer duration of increase in the PTU-cellulose case compared to PTU alone. Cellulose alone had no effect. The PTU-cellulose data suggest that functionalizing PTU to reduce absorption enhances GLP-1 release, indicating that intestinal restriction of potential drugs can be used to control GLP-1 release in addition to minimizing side effects caused by systemic circulation.

[0184] These data indicate that activation of TAS2R38 induces the release of the intestinal peptide hormone GLP-1, making TAS2R38 a novel diabetes target as an agent that increases GLP-1 levels, and demonstrating the effectiveness of GLP-1 analogs in diabetes management. These data suggest that a complex signaling cascade can regulate the release of intestinal peptides and potentially induce metabolic effects. This also makes nutrient receptors highly attractive therapeutic targets for metabolic diseases with aberrant intestinal peptide signaling. In the context of TAS2R38 described in this study, bitter (but safe) components of foods that can activate this receptor could be promising therapeutic candidates. The results presented in this study regarding the TAS2R38 receptor reveal the possibility of screening and identifying novel “bitter” molecules capable of activating this receptor and inducing desired metabolic effects, particularly for diabetes remission.

[0185] Example 4

[0186] GLP-1 is released through plant bitter compounds and fatty acid ligands.

[0187] GLP-1 release was also measured in HuTu-80 cells exposed to plant bitter compounds, fatty acid ligands, PTC, and compounds predicted by computer modeling to interact with TAS2R38.

[0188] The ability of plant signals to promote GLP-1 release was tested. Figure 9A The results showed that pomegranate-derived ellagitannin metabolites urolithin A and ellagic acid, or loquat triterpenoid components ursolic acid and oleanolic acid, caused a significant increase in GLP-1 relative to basal release within 30 minutes.

[0189] also, Figure 9B The results showed that both short-chain fatty acid propionates and butyrates, or long-chain palmitic acid, stimulated GLP-1 release comparable to that of TAS2R38 ligands PTC and PTU (10 mM). For these experiments, concentrations with measurable effects were selected.

[0190] Figure 10 The left figure shows the GLP-1 response in healthy mice after administration of glucose and different doses of PTU molecules as ligands for the TAS2R38 receptor. Figure 10 The right figure shows the GLP-1 response in healthy mice after administration of two different doses of the bitter peptide BPx1.

[0191] Figure 11A and Figure 11B The image shows the effect of bitter peptide BPx1 in cultured HuTu-80 cells treated with untreated (si control) or with TAS2R38 siRNA medium (siTAS2R38) and the indicated concentration for 30 minutes. Figure 11A ) and disordered polyalanine peptide Ala-Ala-Ala-Ala ( Figure 11B GLP-1 release was stimulated. Conditioned culture media were collected and frozen until GLP-1 was measured. Values ​​are mean ± SD, N = 3. GLP-1 was measured using a Luminex assay. Data indicate that the effect of bitter peptides on GLP-1 release is primarily mediated through TAS2R38. Furthermore, out-of-order peptides such as polyalanine are effective in inducing GLP-1 release. These data suggest that GLP-1 release is specific to BPx1.

[0192] The various methods and techniques described above provide numerous ways to implement the invention. It should be understood, of course, that not all of the stated objectives or advantages can be achieved according to any particular embodiment described herein. Therefore, for example, those skilled in the art will recognize that the methods can be carried out in a manner that achieves or optimizes one or more advantages as taught herein, without necessarily requiring the achievement of other objectives or advantages taught or indicated herein. Various advantageous and disadvantageous alternatives are mentioned herein. It should be understood that some preferred embodiments specifically include one, another, or several advantageous features, while other embodiments specifically exclude one, another, or several disadvantageous features, and still other embodiments specifically weaken currently disadvantageous features by including one, another, or several advantageous features.

[0193] Furthermore, those skilled in the art will recognize the applicability of various features from different implementations. Similarly, those skilled in the art can combine and match the various elements, features, and steps described above, as well as other known equivalents of such elements, features, or steps, to perform the method according to the principles described herein. In different implementations, some of the elements, features, and steps will be specifically included while others will be specifically excluded.

[0194] Although the invention has been disclosed in the context of certain embodiments and examples, those skilled in the art will understand that embodiments of the invention extend the specifically disclosed embodiments to other alternative embodiments and / or uses, as well as modifications and equivalents thereof.

[0195] Numerous variations and alternatives have been disclosed in embodiments of the invention. Further variations and alternatives will be apparent to those skilled in the art. Among these variations (but not limited to) are oligopeptide design, receptors regulated by such oligopeptides, methods of administering such compositions, and related therapies, including specific uses of products resulting from the teachings of the invention. Various embodiments of the invention may specifically include or exclude any of these variations or elements.

[0196] In some embodiments, the numerical values ​​representing quantities of components, properties (such as concentration, reaction conditions, etc.) used to describe and claim certain embodiments of the invention should, in some cases, be understood to be modified by the term "about". Therefore, in some embodiments, the numerical parameters set forth in the written description and appended claims are approximations that can vary with the desired properties sought to be obtained through a particular embodiment. In some embodiments, numerical parameters should be interpreted according to the number of significant figures reported and by applying general rounding techniques. While the numerical ranges and parameters describing a broad range of embodiments of the invention are approximations, the values ​​set forth in particular instances are reported as precisely as possible. The numerical values ​​provided in some embodiments of the invention may contain some errors necessarily arising from the standard deviations seen in their respective experimental measurements.

[0197] In some embodiments, the terms “a / an” and “the” and similar references used in the context of describing particular embodiments of the invention (particularly in the context of certain following claims) are to be understood to cover both singular and plural. The numerical ranges listed herein are intended merely as a way of abbreviating each individual value falling within the range. Unless otherwise specified herein, each individual value is incorporated into this specification as if each individual value were listed separately herein. All methods described herein may be performed in any suitable order unless otherwise specified herein or otherwise obviously contradicted by the context. The use of any and all instances or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is solely intended to better illustrate the invention without imposing a limitation on the scope of the invention as otherwise claimed. No language in the specification should be construed as indicating that any unclaimed element is necessary for carrying out the invention.

[0198] The grouping of alternative elements or embodiments of the invention disclosed herein should not be construed as limiting. Each member of a group may be mentioned and claimed individually, or in any combination with other members of that group or other elements appearing herein. For convenience and / or patentability reasons, one or more members of a group may be included in or removed from one group. When any such inclusion or deletion occurs, this specification shall be deemed to include the modified group herein, thus satisfying all written descriptions of the Markush groups as used in the appended claims.

[0199] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments will become apparent to those skilled in the art upon reading the foregoing description. It is expected that those skilled in the art can appropriately use these variations and can practice the invention in other ways specifically described herein. Therefore, many embodiments of the invention include modifications and equivalents to all the subject matter recited in the appended claims, as permitted by applicable law. Furthermore, unless otherwise indicated herein or clearly contradicted by the context, any combination of the foregoing elements in all possible variations is covered within the scope of the invention.

[0200] Furthermore, this specification makes numerous references to patents and printed publications. Each of the references and printed publications cited above is incorporated herein by way of individual citation.

[0201] It should be understood that the embodiments of the invention disclosed herein are illustrative examples of the principles of the invention. Other modifications may be used within the scope of the invention. Therefore, alternative configurations of the invention may be used in accordance with the teachings of this document, for example, but not limited to. Therefore, embodiments of the invention are not limited to those precisely shown and described. sequence list <110> Cedars-Sinai Medical Center <120> Intestinal-delivered bitter oligopeptides for the treatment of type 2 diabetes <150> 62 / 151,306 <151> 2015-04-22 <160> 15 <170> SIPOSequenceListing 1.0 <210> 1 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptides <400> 1 Tyr Gly Leu Phe 1 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptides <400> 2 Tyr Pro Phe Pro Gly Pro Ile Pro Asn 1 5 <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptides <400> 3 Ile Pro Ala Val Phe 1 5 <210> 4 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> peptides <400> 4 Leu Leu Phe 1 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> peptides <400> 5 Pro Lys Lys Lys Arg Lys Val 1 5 <210> 6 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> peptides <400> 6 Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 1 5 10 15 <210> 7 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> peptides <220> <221> UNSURE <222> (4)..(4) <223> Xaa can be any naturally occurring amino acid. <400> 7 Lys Lys Arg Xaa Lys Arg 1 5 <210> 8 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptides <220> <221> UNSURE <222> (3)..(3) <223> Xaa can be any naturally occurring amino acid. <400> 8 Lys Lys Xaa Arg 1 <210> 9 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptides <220> <221> UNSURE <222> (3)..(3) <223> Xaa can be any naturally occurring amino acid. <400> 9 Lys Lys Xaa Lys 1 <210> 10 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptides <220> <221> UNSURE <222> (3)..(3) <223> Xaa can be any naturally occurring amino acid. <400> 10 Lys Arg Xaa Lys 1 <210> 11 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptides <220> <221> UNSURE <222> (3)..(3) <223> Xaa can be any naturally occurring amino acid. <400> 11 Lys Arg Xaa Arg 1 <210> 12 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> peptides <400> 12 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser <210> 13 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> peptides <400> 13 Met Ala Phe Leu Trp Leu Leu Ser Cys Trp Ala Leu Leu Gly Thr Thr 1 5 10 15 Gly Phe <210> 14 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> peptides <400> 14 Met Gln Leu Leu Ser Cys Ile Ala Leu Ile Leu Ala Leu Val 1 5 10 <210> 15 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> peptides <400> 15 Met Asn Leu Leu Leu Ile Leu Thr Phe Val Ala Ala Ala Val Ala 1 5 10 15

Claims

1. Use of one or more bitter oligopeptides in the preparation of a medicament for treating or inhibiting type 2 diabetes in a subject in need, reducing the severity of type 2 diabetes in a subject in need, or slowing the progression of type 2 diabetes in a subject in need. The one or more bitter oligopeptides increase the release of intestinal hormones; wherein the bitter oligopeptide is an oligopeptide with the amino acid sequence LLF, the plurality of bitter oligopeptides include oligopeptides with the amino acid sequence LLF, the plurality of bitter oligopeptides also include oligopeptides with the amino acid sequence YGLF, oligopeptides with the amino acid sequence YPFPGPIPN, oligopeptides with the amino acid sequence IPAVF, or combinations thereof.

2. The use as claimed in claim 1, wherein at least one of the bitter oligopeptides is conjugated with a pharmaceutical agent to increase intestinal retention; wherein the pharmaceutical agent for increasing intestinal retention includes cellulose, fatty acids, or polyethylene glycol (PEG).

3. The use as described in claim 1, wherein the medicament further comprises providing a fatty acid or plant molecule derived from food; wherein the fatty acid or plant molecule derived from food is any one or more of the following: urolithin A, ellagic acid, ursolic acid, oleanolic acid, 6-m-propyl-2-thiouracil, propionic acid, butyrate, palmitic acid.

4. The use as described in claim 1, wherein the drug is administered orally, enterically into the small intestine, or via rectal suppository or enema into the colon.

5. The use as described in claim 1, wherein the drug further comprises an SGLT2 inhibitor.

6. The use as described in claim 1, wherein the drug further comprises any one or more of insulin, sulfonylureas, metformin, acarbose, and thiazolidinediones.

7. Use of one or more bitter oligopeptides in the preparation of a medicament for treating or alleviating obesity in a subject of need. The one or more bitter oligopeptides increase the release of intestinal hormones; wherein the bitter oligopeptide is an oligopeptide with the amino acid sequence LLF, the plurality of bitter oligopeptides include oligopeptides with the amino acid sequence LLF, the plurality of bitter oligopeptides also include oligopeptides with the amino acid sequence YGLF, oligopeptides with the amino acid sequence YPFPGPIPN, oligopeptides with the amino acid sequence IPAVF, or combinations thereof.

8. The use as described in claim 7, wherein at least one of the one or more bitter oligopeptides is conjugated with a pharmaceutical agent to increase intestinal retention; The agents mentioned above that increase intestinal retention include cellulose, fatty acids, or polyethylene glycol (PEG).

9. The use as described in claim 1 or 7, wherein the medicament comprises one or more of the bitter oligopeptides and a pharmaceutically acceptable excipient.

10. The use as claimed in claim 9, wherein the pharmaceutically acceptable excipient comprises any one or more of cellulose and PEG.

11. The use as described in claim 1 or 7, wherein at least one of the bitter oligopeptides is a D-isomer peptide containing one or more D-enantiomeric amino acids.

12. Use of bitter oligopeptides in the preparation of medicaments for the treatment or inhibition of type 2 diabetes or obesity in subjects in need, for reducing the severity of type 2 diabetes or obesity in subjects in need, for slowing the progression of type 2 diabetes or obesity in subjects in need. The amino acid sequence of the bitter oligopeptide is YGLF, wherein, The oligopeptide is conjugated with cellulose.

13. Use of bitter oligopeptides in the preparation of medicaments for treating or inhibiting type 2 diabetes or obesity in subjects in need, reducing the severity of type 2 diabetes or obesity in subjects in need, or slowing the progression of type 2 diabetes or obesity in subjects in need. The bitter oligopeptide is conjugated with cellulose, and the amino acid sequence of the bitter oligopeptide is YPFPGPIPN.

14. Use of bitter oligopeptides in the preparation of medicaments for the treatment or inhibition of type 2 diabetes or obesity in subjects in need, for reducing the severity of type 2 diabetes or obesity in subjects in need, for slowing the progression of type 2 diabetes or obesity in subjects in need. The bitter oligopeptide is conjugated with cellulose, and the amino acid sequence of the bitter oligopeptide is IPAVF.

15. The use as described in any one of claims 12-14, wherein at least one of the bitter oligopeptides is a D-isomer peptide containing one or more D-enantiomeric amino acids.

16. The use as described in any one of claims 12-14, wherein the drug further comprises a fatty acid or plant molecule derived from food; wherein the fatty acid or plant molecule derived from food is any one or more of the following: urolithin A, ellagic acid, ursolic acid, oleanolic acid, 6-m-propyl-2-thiouracil, propionic acid, butyrate, palmitic acid.

17. The use as described in any one of claims 12-14, wherein the drug further comprises any one or more of SGLT2 inhibitors, insulin, sulfonylureas, metformin, acarbose, and thiazolidinediones.

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