WO2014052611A1 - Génération de nouvelles cellules bêta du pancréas - Google Patents

Génération de nouvelles cellules bêta du pancréas Download PDF

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WO2014052611A1
WO2014052611A1 PCT/US2013/061947 US2013061947W WO2014052611A1 WO 2014052611 A1 WO2014052611 A1 WO 2014052611A1 US 2013061947 W US2013061947 W US 2013061947W WO 2014052611 A1 WO2014052611 A1 WO 2014052611A1
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seq
peptide
amino acid
antibody
cells
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PCT/US2013/061947
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English (en)
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Claresa LEVETAN
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Levetan Claresa
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Priority claimed from US13/662,209 external-priority patent/US8911776B2/en
Priority claimed from US13/662,245 external-priority patent/US9511110B2/en
Priority claimed from US13/662,253 external-priority patent/US9133440B2/en
Priority claimed from US13/662,232 external-priority patent/US20140120560A1/en
Application filed by Levetan Claresa filed Critical Levetan Claresa
Priority to EP13841793.6A priority Critical patent/EP2900691A4/fr
Priority to CA 2886442 priority patent/CA2886442A1/fr
Priority to JP2015534660A priority patent/JP2015533821A/ja
Priority to AU2013323462A priority patent/AU2013323462A1/en
Publication of WO2014052611A1 publication Critical patent/WO2014052611A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/474—Pancreatic thread protein; Reg protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel therapies derived from bioactive regions of the human Regla, Reglb, Reg3a and Reg4 gene protein for the generation of new pancreatic beta cells for treatment of new and existing type 1 and 2 diabetes, PreDiabetes, and diseases of insulin deficiency, beta cell deficiency, insulin resistance and impaired glucose metabolism.
  • Diabetes is one of the most serious health issues facing civilization with The World Health Organization reporting that approximately 346 million people worldwide have already been diagnosed with diabetes, making it a global challenge. Diabetes is a chronic disease that manifests when insulin production by the beta cells of the pancreas is insufficient. Among type 2 diabetes patients, there is a 50-80% reduction in beta cell mass by the time of diagnosis compared to a reduction in beta mass by 90% or more among type 1 patients, who commonly have an autoimmune component to their beta cell loss.
  • This invention identifies and confirms peptide sequences that are highly conserved and 100% identical within the human Reg la, human Reglb, human Reg3a and human Reg 4 proteins and identical Reg peptide sequences within four other mammalian species.
  • This inventor has previously described the role of a 14-amino acid Human Reg3a peptide in pancreatic islet development.
  • This present invention identifies peptide sequences contained within the human Regla, Reglb, Reg3a and Reg4 that are not contained within the previously described, prior art human 14-amino acid peptide Reg3a protein and the 15-amino acid hamster Reg3 gamma protein, resulting in beta cell regeneration.
  • the Reg peptides described within this invention bind directly to the Reg Receptor on pancreatic extra-islet ductal tissue.
  • 14-amino acid peptide which is shown to (Levetan CS et al, Endocr Pract. 2008;14(9):1075-1083) interact with the Reg receptor but does not directly bind to it.
  • This invention also specifically identifies a binding region within human Reg Receptor and the bio active peptides within the Reg gene protein that bind directly to the Reg Receptor that are not included in the prior art of the 15-amino acid hamster Reg3gamma sequence known as Islet Neogenesis Associated Protein/INGAP or the 14-amino acid human Reg3a peptide known as Human Proislet Peptide/HIP.
  • This invention demonstrates the utility of these peptides, derivatives, peptidomimetics and stimulatory antibodies that have been generated from the specific binding regions of the Reg Receptor for the generation of new human beta cells
  • REG genes encode proteins secreted by the exocrine pancreas, which has been associated with beta cell regeneration in rodents.
  • REG la, REGlb, REG3a and REG4 belong to the C-lectin family and are randomly clustered on 2pl2. They share structural and some functional properties and encode proteins that are members of the Reg family with sequence homology as described in this invention. Their products are secretory proteins of the C-type lectin superfamily that are involved in beta cell regeneration and proliferation.
  • this invention describes a novel approach for the reversal of the disease by administering an immune tolerance agent prior to the initiation of Reg peptides described in this invention.
  • an immune tolerance agent prior to the administration of the Reg peptide for beta regeneration in order to accelerate the regeneration process for successful reversal of diabetes.
  • This invention identifies peptide sequences that are within the human Regla, human Reglb, human Reg3a and human Reg4 proteins that have not been previously described for the usage of beta cell generation. This invention specifically demonstrates that homologous peptides within the human Regla, human Reglb, human Reg3a and Reg4 gene protein bind directly to the human Reg receptor, which results in the acceleration of the generation of new beta cells from pancreatic ductal tissue.
  • this invention is the first to demonstrate peptides not contained within the human 14-amino acid Reg3a, and contained within the human Regla, Reglb, Reg3a and Reg4 protein and bind directly to the Reg Receptor leading to new beta cell formation.
  • the Reg Receptor plays a key regulatory role in beta cell growth and generation from extra-islet exocrine tissue inclusive of ducts, acinar cells and progenitor cells contained within these cells. Further, the present invention identifies the binding region within human Regla, human Reglb, human Reg3a and human Reg4 and a binding domain on Reg Receptor, which are targets for the treatment of diabetes and other diseases for which there is need for new beta cells.
  • the present invention further demonstrates these peptides, which have not been described in the prior art, are unique Reg peptides for binding to the Reg Receptor on the surface pancreatic ductal tissue and are pivotal for new beta cell formation either via direct usage of the peptide, derivatives, optimized versions, peptidomimetics that bind to Reg Receptor or via stimulating antibodies generated from unique binding sites within the Reg Receptor that generate new beta cells.
  • This invention finds the Reg Receptor to be a pivotal receptor and a specific site within the Reg Receptor to be the site of Reg binding resulting in the translocation of the Reg Receptor through the cytoplasm to the nucleus of extra-islet exocrine tissue inclusive of ducts, acinar cells and progenitor cells contained within these cells, generating beta cell growth, acceleration and turnover.
  • Embodiments of the present invention provide for novel agents and methods for pancreatic beta cell regeneration agents that have not previously been described.
  • Agents include homologous human peptide sequences within the Regla, Reglb, Reg3a and human Reg4 protein, as well as, stimulatory antibodies generated from a unique 20-amino acid binding region within the 919-amino acid protein human Reg Receptor.
  • This invention includes formulations, derivatives, optimized forms of human peptides described, and also include peptidomimetics and stimulatory antibodies serving as peptidomimetics that are designed for the usage in the treatment of type 1 and 2 diabetes, and other conditions of insulin deficiency, beta cell deficiency, insulin resistance and abnormal glucose metabolism.
  • This invention includes methods for direct delivery of agents specified in this invention for generation of new beta cells from progenitor cells within the pancreas and provided to patients via oral, intravenous and subcutaneous delivery with and without organ specific targeting and may include direct delivery to the pancreas and also includes ex-vivo
  • transformation using the inventions herein into new beta cells from embryonic stem cells, human adult bone-marrow derived cells, induced pluripotent stem cells, mesenchymal stem cells, umbilical cord stem cells, or other stem cells and may include resident populations of endogenous stem cells that exist within the adult pancreas that are then administered to patients with new and existing type 1 and 2 diabetes, PreDiabetes and diseases of insulin deficiency, beta cell deficiency, insulin resistance and impaired glucose metabolism, with routes of delivery to include, but are not limited to oral, intravenous, subcutaneous delivery with and without organ specific targeting and may include direct administration to the pancreas or liver.
  • This invention includes methods for pancreatic beta cell generation and include both in vivo and ex-vivo beta cell generation and methods for treating new onset and previously existing type 1 and 2 diabetes, Latent Autoimmune Diabetes of Adulthood (LADA), those at risk for type 1 diabetes, including but not limited to those with positive autoimmune antibodies markers including who are Glutamic Acid Decarboxylase-65 antibody, those with PreDiabetes and diseases of hyperglycemia, glucose intolerance and beta cell impairment or deficiency, insulin resistance, associated conditions including, obesity, obesity prior to the development of diabetes, obesity in children leading to PreDiabetes, both type 1 and type 2 diabetes in childhood and adolescence and include, but are not limited to conditions such as polycystic ovarian syndrome, nonalcoholic steatohepatitis, hyperlipidemia and hypertriglyceridemia and other conditions related to the deficiency or lack of effective amounts of insulin.
  • LADA Latent Autoimmune Diabetes of Adulthood
  • the present invention provides for the discovery of specific regenerative bioactive peptide sequences within the human Regla protein that are referred to as embodiments of "Perle Peptides” including the discovery of the following bioactive sequence within human Regla and Reglb, NVWIGLHDP (SEQ ID NO: 1), found within the 166-amino acid human Regla protein (SEQ ID NO: 2) and within human Reglb (SEQ ID NO: 3) and SEQ. ID NO: 1 also has 100% homology to sequences found in seven other mammals (FIG: 17).
  • the 8-amino acid peptide VWIGLHDP (SEQ ID NO: 4) sequence within the human Regla protein (SEQ ID NO: 2), Regla (SEQ ID NO: 3) and within the human Reg3a protein (SEQ ID NO: 5), are demonstrated in this invention to bind directly to the human 919-amino acid Reg receptor (SEQ ID NO: 6). Sequence ID NO. 4 was also demonstrated to have 100% homology to 12 mammals including chimpanzee, rat, but not the mouse (FIG. 18).
  • This invention also identifies homologous peptide sequences within human Regla, Reglb, Reg3a and Reg4, WIGLHDP (SEQ ID NO: 7); and WIGLHDPT (SEQ ID NO: 8), and VWIGLHDPT (SEQ ID NO. 14), which are all contained within the human Reg3a (SEQ ID NO: 5).
  • SEQ ID NO. 8 has 100% homology to 12 other mammals (FIG: 19) including chimpanzee, rat, mouse, sheep and cow.
  • SEQ ID NO:7 has 100% homology to 14 mammals including chimpanzee, mouse and rat (FIG 20).
  • this discovery includes a 20-amino acid specific binding site (SEQ ID NO: 9) within the 919-amino acid Reg Receptor from which a stimulatory antibody to the Reg Receptor have been generated from this 20-amino acid.
  • this discovery also includes "Optimized Perle Peptides" including Perle peptidomimetics which refers to variations of Perle Peptides wherein the peptide has been modified to increase the stability, solubility, including formulations that have increased protease resistance, reduced immunogenicity, Tmax and bioavailability compared to the native peptide and/or provide greater ease in administration.
  • Optimized formulations include developed modifications rendering the peptide sequences less susceptible to protease cleavage in serum with those proteases that normally recognize free ends, thereby effectively increasing the Tmax and bioavailability of the peptides include, but are not limited to the 1) blocking with a c- terminal acetyl groups and an n-terminal amide groups, 2) derivatives thereof modified by adding a cysteine residue to the n-terminal, resulting in a compound which is capable of forming dimers in solution 3) modified by conventional and backbone cyclization to improve biological activity, increasing bioavailability and dosing efficacy.
  • stability refers to the peptide' s resistance to degradation by in-serum proteases which target and degrade Perle Peptides, Optimized Perle Peptides and Perle peptidomimetics.
  • bioavailability refers to the amount of peptide available for in vivo therapeutic use by the target cells, pathways and/or systemic mechanisms based on the Perle Peptides' ability to avoid degradation by proteases and other systemic pathways that degrade Perle Peptides and Optimized Perle Peptides that include blocking the peptide by the addition of an n-terminal amide group and a c-terminal acetyl group, pegylated, cyclization and other methods thereof.
  • generation of new pancreatic beta cells occurs from the administration of Perle Peptides and includes formulations, derivatives, optimized forms and also include peptidomimetics of the Perle Peptide, Optimized Perle Peptides and agents that bind to the human Reg Receptor and a specific binding region within the Reg Receptor are presented in this invention with the modality of delivery including, but is not limited to: oral, intravenous, subcutaneous with direct and indirect delivery to the pancreas and liver.
  • this inventions provides new therapy and specific methods for generation of new beta cells for usage in treatment of type 1 diabetes and autoimmune diabetes including 1) new onset and previously existing type 1, Latent Autoimmune Diabetes of Adulthood (LAD A), those at risk for type 1 diabetes who have autoimmune markers including those who are Glutamic Acid Decarboxylase-65 antibody positive via the usage of Perle Peptides, Optimized Perle Peptides, Perle Peptidomimetics and Stimulatory Antibodies to the Perle Peptide Receptor in combination with immune tolerance agents to prevent autoimmune destruction of the new beta cells formed from this invention.
  • LAD A Latent Autoimmune Diabetes of Adulthood
  • the fifth embodiment includes specific methodology and timing for administration of Perle Peptides, Optimized Perle Peptides, Perle Peptidomimetics and Stimulatory Antibodies to the Perle Peptide Receptor in parallel with an immune tolerance agent beginning on the day of the immune nadir of the specific immune agent, which varies vastly between the different agents utilized in man for immune tolerance among patients with type 1 diabetes.
  • this invention provides specific methods for monitoring and tapering exogenous insulin dosages as new beta cells are formed since there are numerous feedback mechanisms to prevent hypoglycemia that prevent beta cell regeneration as a patient approaches normal glucose levels as new beta cells are generated.
  • an innovative therapy for accelerated generation of the ex-vivo generation of beta cells by methodology for administration of Perle Peptides and/or
  • this invention provides for the use of Perle Peptides, including formulations, derivatives, optimized forms including peptidomimetics and stimulatory antibodies to the Reg Receptor for the accelerated generation of beta cells are given directly to patients via oral, intravenous, subcutaneous and may include targeted therapy to the pancreas or liver and may also include the direct delivery of beta cells to patients formed by the ex-vivo transformation to pancreatic beta cells from pluripotent stem cells including embryonic cells, adult somatic stem cells, human adult bone-marrow derived stem cells, umbilical cord stems cells, mesenchymal stem cells, human amniotic membrane-derived mesenchymal cells, pluripotent cells, mammalian stem cells, mammalian stem cells and ectodermal stem cells that are induced by this invention into beta cells for the treatment of new onset and previously existing type 2 diabetes, those at risk with PreDiabetes and diseases of hyperglycemia, glucose intolerance and beta cell impairment or deficiency, insulin resistance, metabolic syndrome and
  • a seventh embodiment of this invention includes the specific methods and treatment of new onset and previously existing type 2 diabetes, PreDiabetes, glucose intolerance, hyperglycemia, syndromes of insulin resistance and glucose impairment, diseases of hyperglycemia, glucose intolerance and beta cell impairment or deficiency, insulin resistance and associated conditions including: obesity, obesity prior to the development of diabetes, obesity in children leading to PreDiabetes, childhood diabetes (both type 1 and 2) and other conditions including but not limited to polycystic ovarian syndrome, nonalcoholic
  • Perle Peptide(s) including formulations, derivatives, optimized forms and peptidomimetics of the Perle Peptides that are administered alone or in conjunction of one or more diabetes agents, which may include all types of insulin, sulfonylureas, metformin, meglitinides, GLP-1 receptor analogs, DPP-4 inhibitors, thiazolidinediones, SGLT2 inhibitors, anti-inflammatory agents, pramlintide, Vitamin D and/or lifestyle modifications and other agents utilized to improve glucose in order to prevent and limit the destruction of the new beta cells formed by this invention.
  • diabetes agents which may include all types of insulin, sulfonylureas, metformin, meglitinides, GLP-1 receptor analogs, DPP-4 inhibitors, thiazolidinediones, SGLT2 inhibitors, anti-inflammatory agents, pramlintide, Vitamin D and/or lifestyle modifications and other agents utilized to improve glucose in order to prevent and limit the destruction of the new beta cells formed by this invention
  • the eighth embodiment of this invention includes the specific methods and treatment for tapering insulin and or diabetes medications patients as Perle Peptide(s), including formulations, derivatives, optimized forms and peptidomimetics of the Perle Peptides are administered in order to optimize efficacy. This includes providing specific methodology for optimizing the glycemic milieu before and during treatment with Perle Peptides and derivatives to optimize the formation of new beta cells.
  • the ninth embodiments of this invention include the specific methods and treatment for PreDiabetes, new onset and pre-existing type 2 diabetes utilizing Perle Peptide and includes formulations, derivatives, optimized forms and peptidomimetics of the Perle Peptide to accelerate the formation of new pancreatic beta cells that are administered to patients who diabetes-drug naive.
  • the present invention provide pharmaceutical formulations and unit dose forms of Perle Peptides alone or in combination with one or more other active pharmaceutical ingredients (APIs).
  • the API is an agent in soluble liposome preparations that allow Perle Peptides to be administered by a variety of routes, including subcutaneously, intramuscularly, intravenously, intra- arterially, and even orally, depending on the formulation selected.
  • the formulation is for general systemic administration, but in other embodiments, the formulation comprises a targeting agent for targeted administration to specific locations, receptors, cells, tissues, organs, or organ systems including targeting to the liver and/or pancreas.
  • methods are provided for treating a pathology associated specifically with impaired pancreatic function in a subject that may have diabetes or
  • PreDiabetes either type 1 or 2 and in need of such treatment.
  • the method comprises one or more of the steps of (1) intensifying glycemic control (2) administering oral vitamin D to maintain 25 -hydro xyvitamin levels above 40 mg/ml; (3) administering one or more immune therapies for protecting new islet cell formation, including administration of immunosuppressive agents; (4) administering Perle Peptide formulations, derivatives, optimized forms and peptidomimetics in combination with insulin or other diabetes agents, (5) reducing, or tapering off of other diabetes therapies as new beta cell populations are restored (6) administering repeat dosages of immunotherapy for the protection of beta cells formed by Perle Peptide formulations, derivatives, optimized forms and peptidomimetics on routine basis depending on the selected immune therapy and (7) administering Perle Peptide formulations, derivatives, optimized forms and peptidomimetics at intervals to maintain a minimum number of beta cells for normal glucose metabolism.
  • Embodiments of the invention also provide kits for treating a patient having type 1 or type 2 diabetes or other condition in which there are aberrant glucose and insulin levels, perturbation in glucose metabolism or insulin resistance, acute and comprising a therapeutically effective dose of Perle Peptides, Optimized Perle Peptides or Perle Peptide peptidomimetics and other Perle formulations.
  • kits for measuring Perle Peptides, Optimized Perle Peptides and Perle peptidomimetics levels in a sample comprising a Perle Peptides, Optimized Perle Peptides and Perle peptidomimetics-specific antibody and optionally Perle Peptides, Optimized Perle Peptides and Perle peptidomimetics and optionally a labeling means.
  • SEQ ID NO: 1 is an embodiment of a peptide of the present invention which is 9 amino acids in length.
  • SEQ ID NO:l is a partial sequence of human Regenerating islet-derived 1 alpha (Regla).
  • SEQ ID NO: 2 is human Regenerating islet-derived 1 alpha (Regla), also known as human lithostathine-1 -alpha precursor (public accession number NP_002900).
  • SEQ ID NO: 3 is human Regenerating islet-derived lbeta (Reglb), also known as lithostathine-1 -beta precursor (public accession number NP_006498).
  • SEQ ID NO: 4 is an embodiment of a peptide of the present invention which is 8 amino acids in length.
  • SEQ ID NO: 4 is a partial sequence of human Regla, human Reglb, and human Reg3a.
  • SEQ ID NO: 5 is human regenerating islet-derived protein 3-alpha precursor (Reg3a) (public accession number NP_002571).
  • SEQ ID NO: 6 is the human Reg receptor, also known as exostosin-like 3 (public accession number NP_001431).
  • SEQ ID NO: 7 is an embodiment of a peptide of the present invention which is 7 amino acids in length.
  • SEQ ID NO: 7 is a partial sequence of human Regla, human Reglb, human Reg3a, and human Reg4.
  • SEQ ID NO: 8 is an embodiment of a peptide of the present invention which is 8 amino acids in length.
  • SEQ ID NO: 8 is a partial sequence of human Reg3a.
  • SEQ ID NO: 9 is an embodiment of a binding site within the human Reg receptor for peptides of the present invention.
  • SEQ ID NO: 9 is a partial sequence of the human Reg receptor.
  • SEQ ID NO: 10 is human regenerating islet-derived protein 4 isoform 1 precursor (Reg4) (public accession number NP_114433).
  • SEQ ID NO: 11 is a 14-amino Reg 3a peptide sequence (Human Proislet Peptide (HIP)) (SEQ ID NO: 3 of US Patent 7,393,919).
  • SEQ ID NO: 12 is a 15-amino acid peptide within the hamster Reg3gamma peptide sequence (Islet Neogenesis Associated Protein (INGAP)) (amino acid residues 103 to 117 of SEQ ID NO: 2 of US Patent 5,834,590).
  • INGAP Islet Neogenesis Associated Protein
  • SEQ ID NO: 13 is an N-terminal partial sequence of the human Reg receptor.
  • SEQ ID NO: 14 is a 9-amino acid peptide within Human Reg3a.
  • Fig. 1 is a graph depicting the schematic mechanism of action of the human Reg la, Reglb, Reg3a and Reg4 Perle peptides in the generation of new beta cells.
  • Fig. 2 is a photograph of a rodent islet containing beta cells that is budding from a progenitor cell found within the extra-islet exocrine tissue containing ductal, acinar and progenitor cells.
  • Fig. 3 is a graphic depicting the 3-dimensional structures of human Regla,human Reg3a and Hamster Reg 3gamma by Swiss-Prot folding algorithms with the Perle Peptides contained within the Red circled binding arm.
  • Fig. 4 demonstrates to peptide homology between human Reg la, Regla,Reg3a and Reg 4 and the Hamster Reg3gamma. The common sequences are highlighted in red.
  • Fig. 5 demonstrates the 8-amino acid human Reg sequence (SEQ ID: 8) that has 100% homology with Reg sequences found in the chimpanzee, cow, mouse and sheep, which has not been described in the prior art as a beta generation peptide.
  • Fig. 6 is the 919 amino acid sequence of the Reg Receptor with the 20amino acid binding domain (SEQ ID: 9) presented in this invention highlighted in red.
  • Fig. 7 demonstrates the purification and detection of the Reg Receptor for usage in this invention.
  • Fig. 8 demonstrates the labeling of the Reg Receptor on the cell surface (FIG. 8 a) with internalization of the Reg Receptor inside the cell in the presence of Reg (FIG. 8b).
  • Fig. 9 demonstrates the direct binding of the Perle peptides to the Reg Receptor.
  • the 14- amino acid human Reg3a peptide (HIP) demonstrated no direct binding to the Reg Receptor, nor did the scrambled peptide control.
  • the 8 amino-acid Perle peptide demonstrated the greatest binding to the Reg Receptor
  • Fig. 10 is confirmation of nuclear translocation of the Reg Receptor from the cytoplasmic nucleus with the 8-amino acid peptide.
  • Fig. 11 demonstrates the results of studies generating stimulatory antibodies to a 20- amino acid binding site within the Reg Receptor.
  • Fig. 12 provides the protocol for development of stimulatory antibodies to the binding domains within the Reg Receptor.
  • Fig. 13 is the documentation of titer controls and norms for the antibody studies.
  • Fig. 14 is an illustration of the methodology to reverse new onset or existing type 1 diabetes utilizing Perle peptides, formulations, derivatives, and optimized forms including peptidomimetics of the peptides and stimulating antibodies to the Reg receptor.
  • Fig. 15 is an illustration of the methodology to reverse type 2 diabetes utilizing beta agonist therapies including Perle peptides, formulations, and derivatives, optimized forms including peptidomimetics of the peptides and stimulating antibodies to the Reg receptor.
  • Fig. 16 demonstrates the ex-vivo generation of beta cells using Perle peptides, formulations, derivatives, optimized forms including peptidomimetics of the peptides and stimulating antibodies to the Reg receptor to transform human extra-islet tissue including ductal, acinar and progenitor cells, human embryonic stem cells, human adult bone-marrow derived cells, induced pluripotent stem cells, mesenchymal stem cells, umbilical cord stem cells, or other stem cells and may include resident populations of endogenous stem cells that exist within the adult pancreas that are transformed into new beta cells then delivered to patients with diabetes and other conditions of insulin deficiency via the umbilical vein, portal venous systemic, hepatic artery, subcutaneous delivery.
  • Fig. 17 demonstrates the 9-amino acid human Reg sequence (SEQ ID: 1) that has 100% homology with Reg sequences found in other mammals including chimpanzee, rat, golden hamster, white cheeked gibbon, Sumatran orangutan, Lowland gorilla, white-tufted-eared marmoset, which has not been described in the prior art as a beta generation peptide.
  • Fig. 18 demonstrates the 8-amino acid human Reg sequence (SEQ ID: 4) that has 100% homology with Reg sequences found in other mammals including chimpanzee, rat, golden hamster, guinea pig, rabbit, pig, sheep, cow, white-cheeked gibbon, Sumatran orangutan, Lowland gorilla, white-tufted-eared marmoset, which has not been described in the prior art as a beta generation peptide.
  • SEQ ID: 4 8-amino acid human Reg sequence
  • Fig. 19 demonstrates the 8-amino acid human Reg sequences (SEQ ID:8) that has 100% homology with Reg sequences found in other mammals including chimpanzee, rat, mouse, golden hamster, guinea pig, rabbit, pig, sheep, cow, white- cheeked gibbon, Sumatran orangutan, Lowland gorilla, European domestic ferret, which has not been described in the prior art as being beta regeneration peptide.
  • Fig. 20 demonstrates the 7-amino acid human Reg sequences (SEQ ID: 7) that has 100% homology with Reg sequences found in other mammals including chimpanzee, rat, mouse, golden hamster, guinea pig, rabbit, pig, sheep, cow, white- cheeked gibbon, Sumatran orangutan, Lowland gorilla, white-tufted-eared marmoset, European domestic ferret, which has not been described in the prior art as being beta regeneration peptide.
  • SEQ ID: 7 7-amino acid human Reg sequences
  • 21 demonstrates the 9-amino acid human Reg sequences (SEQ ID: 14) that has 100% homology with Reg sequences found in other mammals including chimpanzee, rat, mouse, golden hamster, guinea pig, rabbit, pig, sheep, cow, white- cheeked gibbon, Sumatran orangutan, Lowland gorilla, which has not been described in the prior art as being beta regeneration peptide.
  • Fig. 22 demonstrates the 20-amino acid human sequence that has been identified within the Reg receptor that has 100% homology with Reg receptor sequences found in other mammals including chimp, rat, mouse, guinea pig, rabbit, dog, cow, opossum, galago, gibbon, orangutan, macaque, gorilla, marmoset and horse.
  • beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms of diabetes, diminishment of extent of disease, delay or slowing of disease progression, amelioration, palliation or stabilization of the disease state, and other beneficial results described below.
  • Symptoms of diabetes include low or inadequate levels of insulin or insulin activity, frequent urination, excessive thirst, extreme hunger, unusual weight loss, increased fatigue, irritability, blurry vision, genital itching, odd aches and pains, dry mouth, dry or itchy skin, impotence, vaginal yeast infections, poor healing of cuts and scrapes, excessive or unusual infections, hyperglycemia, loss of glycemic control, fluctuations in postprandial blood glucose, fluctuations in blood glucagon, fluctuations in blood triglycerides. Diabetes may be diagnosed by methods well known to one of ordinary skill in the art. For example, commonly, diabetics have a plasma blood glucose result of greater than 126 mg/dL of glucose.
  • Pre diabetes which may also be treated by the compositions and methods of the invention is commonly diagnosed in patients with a blood glucose result between 100 and 125 mg/dL of glucose.
  • Other symptoms may also be used to diagnose diabetes, related diseases and conditions, and diseases and conditions affected by diminished pancreatic function.
  • "reduction" of a symptom or symptoms means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s).
  • impaired glucose homeostasis is a diminished capacity in a subject for regulating glucose by a system of feedback controls, so as to stabilize health and functioning.
  • Conditions that are associated with or are a risk factor for impaired glucose homeostasis include new onset type 1 and 2 diabetes, previously existing type 1 and 2 diabetes, latent autoimmune diabetes of adulthood (LADA), glutamic acid decarboxylase-65 autoimmunity, prediabetes, metabolic syndrome, hyperglycemia, glucose intolerance, beta cell impairment or deficiency, insulin resistance, obesity, polycystic ovarian syndrome, nonalcoholic steatohepatitis, hyperlipidemia, and hypertriglyceridemia
  • administering or “administration of a drug to a subject (and grammatical equivalents of this phrase) includes both direct administration, including self- administration, and indirect administration, including the act of prescribing a drug.
  • direct administration including self- administration
  • indirect administration including the act of prescribing a drug.
  • a physician who instructs a patient to self-administer a drug and/or provides a patient with a prescription for a drug is administering the drug to the patient.
  • a "subject” or “patient” is a mammal, typically a human, but optionally a mammalian animal of veterinary importance, including but not limited to horses, cattle, sheep, dogs, and cats.
  • a "therapeutically effective amount" of a drug or agent is an amount of a drug or agent that, when administered to a subject with a disease or condition, will have the intended therapeutic effect, e.g., alleviation, amelioration, palliation or elimination of one or more manifestations of the disease or condition in the subject.
  • the full therapeutic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses.
  • a therapeutically effective amount may be administered in one or more administrations.
  • a "therapeutically effective amount" of a drug may also be an amount of a drug that when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of disease or symptoms, or reducing the likelihood of the onset (or reoccurrence) of disease or symptoms.
  • the full prophylactic effect does not necessarily occur by administration of one dose and may occur only after
  • a therapeutically effective amount may be any therapeutically effective amount.
  • Reg is Regenerating islet-derived (as in Regenerating islet-derived protein).
  • Perle Peptides are specific regenerative bioactive peptide sequences contained within Regenerating islet-derived proteins.
  • Type 1 and type 2 diabetes have long been considered diseases resulting from diminished insulin secretion. Research carried out over the past century has more clearly found that generating new beta cells that make insulin, is the key to reversing this disease. At the onset of type 2 diabetes, there is a 50%-80% reduction in beta cell mass compared to the more acute 90% loss found in newly diagnosed type 1 diabetic patients. Although the beta cell mass may expand several fold from birth to adulthood, this is not enough to compensate for the greater loss than generation of new beta cells seen in both type 1 and 2 diabetes.
  • Reg regenerating gene family
  • the Reg genes are associated with islet neogenesis and are upregulated in the pancreas during embryogenesis when the pancreas is being populated with beta cells for the first time.
  • the Reg genes are usually undetectable, but are upregulated in response to acute pancreatic injury including pancreatic stones, pancreatitis, pancreatic duct ligation and wrapping, partial pancreatectomy and pregnancy.
  • the Reg gene proteins are a family of C-type lectin proteins that are expressed almost exclusively by the pancreas.
  • the 15 -amino acid hamster Reg 3 gamma gene protein (INGAP) and the 14-amino acid Reg3a peptide (HIP) have both been shown to stimulate new beta cell formation from extra-islet exocrine progenitors, as well as upregulating growth promoting factors associated with new beta cells including NGN3, PDX-1, NKx6.1, Ins, Sox9 and others.
  • This invention identifies key human Reg peptide sequences that are not contained within either 15-amino acid hamster Reg 3gamma gene protein (INGAP) or the human 14-amino acid Reg3a peptide, Human Pro is let Peptide (HIP), that bind directly to the Reg receptor and stimulate downstream activity resulting in translocation of the Reg Receptor from the cytoplasmic membrane to the nucleus stimulating new beta cell formation. Additionally this invention identifies a specific binding region within the 919-amino acid Reg Receptor from which stimulatory antibodies serving as peptidomimetics have been generated.
  • INGAP 15-amino acid hamster Reg 3gamma gene protein
  • HIP let Peptide
  • FIG 1 is a schematic drawing showing the findings of the present invention
  • FIG. 2 is a photograph of a rodent islet that is formed from the budding from a progenitor cell within surrounding extra-islet ductal tissue containing ducts, acinar and progenitor cells.
  • the first known description of new islet cells arising from pancreatic ductal cells comes from the French histologist, Edouard Laguesse, in 1893.
  • FIG. 3 is an illustration showing the similarities and differences between three dimensional structures of human Regla, Reg3a and the Hamster Reg3 gamma peptides based on their primary amino acid sequences and by Swiss-Prot folding algorithms.
  • FIG 3a shows the three dimensional structure for Human Regla.
  • Fig3b shows the three dimensional structure for Reg3a.
  • FIG 3c shows the three dimensional structure for the hamster Reg3 gamma.
  • the region of the protein that contain the Perle peptides, which bind to Reg Receptor, is circled in Red, The Perle peptides are not contained within the 14-amino acid previously described HIP peptide and the 15-amino acid hamster peptide INGAP.
  • FIG 4 is an alignment of the 166 amino acid sequence for Regla (SEQ ID NO:2), the 166 amino acid sequence for Reglb (SEQ ID NO: 3), the 175-amino acid sequence for Reg3a (SEQ ID NO: 5), the 158 amino acid sequence for Reg4 (SEQ ID NO: 10) and the hamster Reg3gamma protein.
  • a 14-amino Reg 3a peptide sequence (HIP) (SEQ ID NO: 11) and a 15- amino acid peptide within the hamster Reg3gamma peptide sequence INGAP (SEQ ID NO: 12) has been shown in the prior art to interact with the Reg receptor, resulting in downstream generation of new beta cells, but these sequences have not been demonstrated to directly bind to the Reg receptor, which is confirmed in this discovery.
  • the 8 and 7-amino acid sequences that are presented within in this invention bind to the Reg receptor and are not contained within the 14-amino acid Reg3a peptide, HIP, or the hamster Reg3 gamma peptide, INGAP.
  • Figure 5 shows the identification of the 100% homologous sequence (SEQ ID: 8) within the human Reg la, Reglb, Reg3a, Reg4 and the Hamster Reg3gamma peptides that is located within the binding arm of the protein that binds to the Reg Receptor. This exact sequence is also found in the human, chimpanzee, cow, sheep and mouse. This sequence (SEQ ID: 8) and amino-acids within the sequence are not contained within the 14-amino acid human Reg3a HIP peptide or the 15-amino acid hamster Reg3gamma peptide, INGAP.
  • Figure 6 shows the 919-amino acid Reg Receptor (SEQ ID: 6) and the 20- amino acid domain (SEQ ID NO: 9) in red that was identified in this discovery that is a binding domain for the Perle Peptides, and from which stimulatory antibodies to this region of the receptor have been generated.
  • Figure 7 demonstrates the results from studies conducted to demonstrate Reg Receptor expression and purification utilizing 293T cells that were transfected with Reg Receptor expression plasmid DNA. Cells were collected after 72 hours. The Reg Receptor was tagged with FLAG epitope and FLAG resin was utilized to purify out the Reg Receptor. As shown in Fig 7, the Reg Receptor was highly purified. The Reg Receptor was purified by Anti-Flag M2 affinity gel.
  • FIG. 7 demonstrates the use of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) to illustrate the high purity of the Reg Receptor.
  • Figure 7b and 7c are the Western blot results showing that the purified protein is the Reg Receptor using the antibody to the Reg Receptor (CD104). The Reg Receptor is shown In FIG. 7 to be highly purified.
  • the purified Reg receptor was coated onto 96 well plate by using bicarbonate coating buffer, pH 9.6; 4°C overnight at concentration 3ug/ml, lOOul per well. Plates were coated overnight coated plate and washed three times with 0.5X TBST and blocked with 3%BSA and rotated at room temperature for 1 hour. After blocking, plates were washed three times with 0.5X TBST. Peptides were then diluted with TBST buffer and added into wells in duplicate then left to bind at room temperature for 1 hour. After washing three times, lOOng/ml strep-HRP was added into plate at lOOul/well, and rotated at room temperature for one hour.
  • ABST reagents were warmed to room temperature, mixed immediately before using. Then lOOul was added to each well and read after 25 minutes reaction and absorbance was evaluated at 405nm by a Spectramax M5 plate reader. The purified Reg Receptor was coated on plates. Then plates were blocked with BSA solution. Subsequently, the Perle peptides were added into the wells, and HRP- straptavidin and its substrates were added into the wells to reveal the interaction between Receptor and the Perle peptide.
  • the 7-amino acid Perle peptide, the 8-amino acid Perle peptide and the 9-amino acid Perle peptide all bind directly to Reg Receptor.
  • the 8-amino acid peptide appeared to be the strongest in binding to the Reg Receptor.
  • the scrambled control peptide did not bind to Reg Receptor.
  • the 14-amino acid human Reg3a peptide (HIP)
  • the 14-amino acid human Reg3a peptide (HIP) was shown in this study to have the same level of binding as the scrambled peptide (FIG.
  • the 7,8 and 9-amino acid Perle Peptides sequences are not contained within the 14-amino human Reg3a peptide (HIP) sequence or the 15 -amino acid hamster 3 gamma peptide (INGAP).
  • HIP 14-amino human Reg3a peptide
  • INGAP 15 -amino acid hamster 3 gamma peptide
  • FIG. 9 shows immunofluorescent staining of Reg receptor on the cell surface of human pancreatic exocrine ductal cells.
  • Cy3 immunofluorescent staining of Reg receptor in human pancreatic ductal cells in standard medium there is immunofluorescent staining of Reg receptor, which is well-defined at the cell borders indicating surface expression of Reg receptor on the cytoplasmic membrane of cells (FIG 9a)
  • FIG 9b demonstrates the difference in Reg receptor staining when cells are exposed to Reg.
  • Figure 10 demonstrates a confirmation study utilizing Western blot analysis to evaluate the translocation of the Reg Receptor from the cytoplasmic membrane of human extra- islet exocrine tissue inclusive of ductal cells containing progenitor.
  • Western blot analysis identified the presence of the Reg receptor on the cytoplasmic membrane and the movement of the Reg Receptor from the cytoplasmic membrane through the cytoplasm to the nucleus in the presence of the Perle peptides.
  • Cytoplasmic extracts were obtained in lOmM HEPES (pH 8.0), ImM EDTA, 1.5mM MgCl 2 , lOmM KC1, 0.5mM DTT, 200mM sucrose and 0.5% Nonidet P- 40.
  • Nuclear extracts were obtained in 20 mM HEPES (pH 7.9), 0.75mM MgCl 2 , 210mM NaCl, 50mM KC1, ImM EDTA, 10% glycerol, and 0.5mM DTT. Both extraction buffers contained 0.5 mM PMSF, 1 ⁇ g/ml leupeptin, ⁇ g/ml aprotinin, 2.5mM Na 4 P 2 C>7,lmM ⁇ -glycerophosphate, and ImM Na 3 V0 4 . Reg Receptor extracts were size fractionated on SDS-polyacrylamide gels and transferred to nitrocellulose.
  • This invention demonstrates in FIG. 10 that the 8-amino-acid Perle Peptide resulted in the greatest impact on translocation of the Reg Receptor from the cytoplasm to the nucleus both in the shorter and longer exposure times.
  • the impact of the 8-amino acid Perle Peptide was greater than the other peptides tested including the 14-amino acid HIP peptide.
  • Figures 11-13 were from studies undertaken to develop stimulatory antibodies to the Reg Receptor to accelerate the progression of beta cell formation by stimulating potential binding sites on the Reg Receptor.
  • many sequences were evaluated within the N-terminal portion of Reg receptor (amino acids 1-332) (SEQ ID NO:13), which is the amino acid region believed to be the binding domain for the Reg peptides within the Reg receptor that is not contained in the other members of the of Exostoses family, and thus is hypothesized to be the Reg binding domain.
  • the animals were injected with a peptide of SEQ ID NO: 9 and were conjugated to keyhole limpet hemocyanin (KLH).
  • KLH keyhole limpet hemocyanin
  • the screening antigen is not conjugated to KLH so that the response solely to the peptide and not to KLH can be identified.
  • the 50% titer is a dilution value where the signal is half-way between the peak and the baseline, so the higher the dilution value (titer), the greater the response to the antigen.
  • the positive control is an internal control that was generated from ovalbumin antibodies in rabbit. At a dilution of 1 :750,000, the absorbance fell within a range of 0.45 to 0.9.
  • Figure 14 illustrates present invention provides an illustration of the method for treating newly diagnosed or pre-existing type 1 diabetes mellitus in a patient, by a method comprising administering to said patient of an agent that stimulates beta cell regeneration, which includes Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg Receptor Perle peptides, in combination with an immune tolerance agent or combination of immune tolerance agents.
  • an agent that stimulates beta cell regeneration includes, but is not limited to Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg Receptor.
  • Immune tolerance agents may include, but are not limited to, Cyclosporine, the heat shock protein 60, Diapep 277, the Bacillus Calmette-Guerin or Bacille Calmette-Guerin also known as the BCG vaccine and commonly known as the vaccine against tuberculosis, mycophenolate mofetil, daclizumab, rituximab (anti CD20), anti CD3 antibodies including hOKT3 gammal (Ala- Ala), and the monoclonal antibody TRX4 (CbAglyCD3), CTLA4-Ig (abatacept) a selective co-stimulation modulator as it inhibits the co-stimulation of T cells, campath-lH, anti-CD52 antibody, a or humanized monoclonal antibody to T-cells, polyclonal anti-T-lymphocyte globulin (ATG), DiaPep277, GAD antibody vaccine based on the 65 kDa isoform of the recombinant human glutamic
  • This invention provides for specific methodology for administration of Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg Receptor for generation of beta cells in newly diagnosed type 1 patients and in those with existing type 1 diabetes and those with Latent Autoimmune Diabetes of Adulthood.
  • Methods provide for initiation of Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg Receptor on the day of the immune nadir occurring at a different time with each of the above immune agents. Based on this specific timing, which varies with each agent, there is greater ability to protect new beta cells generated by Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg Receptor.
  • Figure 15 illustrates the methodology for reversal of type 2 diabetes and preventing the progression of PreDiabetes to Diabetes utilizing a beta cell agonist such as Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg
  • Perle peptides, formulations, optimized versions, peptidomimetics and stimulating antibodies to a binding region on the Reg receptor identified in this invention would be first- line therapy for patients with type 2 diabetes and PreDiabetes or added to existing diabetes medications regimens including insulin. Based upon glucose levels, other diabetes medications, including insulin could be tapered.
  • Perle peptides, formulations, optimized versions, peptidomimetics and stimulating antibodies to a binding region on the Reg receptor will be used as first- line therapy for new onset and existing type 2 diabetes, as well as PreDiabetes.
  • Perle peptides, formulations, optimized versions, peptidomimetics and stimulating antibodies to a binding region on the Reg receptor may be added to other diabetes agents and including: all types of insulin, Glucagon Like Peptide- 1 (GLP-1) receptor analogs Liraglutide and Exenatide, Dipeptidyl Peptidase-4 Inhibitors, (DPP-4 inhibitors), and including (Sitagliptin, Saxagliptin, Linagliptin), the Amylin, analog, pramlintide, acarbose, orlistat, colesevelam, bromocriptine, oriistat, combination therapies with the biguanide, metformin, and combinations of with thiazolidinediones, sulfonylureas and DPP-4 inhibitors and new agents SGLT2 inhibitors (dapagliflozin and canagliflozin).
  • GLP-1 Glucagon Like Peptide- 1
  • DPP-4 inhibitors Di
  • Figure 16 includes methods for the formation and delivery of new beta cells generated by Perle peptides, derivatives, optimized versions, peptidomimetics and antibodies generated to specific binding regions of the Perle Receptor serving as peptidomimetics when utilized for the ex-vivo generation of new beta cells from progenitor cells, which may include, but are not limited to human extra-islet tissue inclusive of ductal, acinar and progenitor embryonic tissue, human stem cells, human adult bone-marrow derived cells, induced pluripotent stem cells, mesenchymal stem cells, umbilical cord stem cells, or other stem cells and may include resident populations of endogenous stem cells that exist within the adult pancreas that are facilitated to transform into beta cells by the addition of Perle peptides, derivatives, optimized versions, peptidomimetics and antibodies generated to specific binding regions of the Perle Receptor serving
  • progenitor cells which may include, but are not limited to human extra-islet tissue inclusive of ductal, acinar and progen
  • New beta cells are generated ex-vivo transformation using the inventions herein into new beta cells from Perle peptides, derivatives, optimized versions, peptidomimetics and antibodies generated to specific binding regions of the Perle Receptor serving as peptidomimetics.
  • New beta cells are then delivered to patients with PreDiabetes, type 1 and 2 diabetes and other conditions of beta cell deficiency with routes of delivery to include, but are not limited to oral, intravenous, including delivery via the umbilical vein, portal venous systemic, hepatic artic artery, subcutaneous delivery with and without organ specific targeting and may include direct administration to the pancreas or liver (FIG. 16).
  • Perle peptides, derivatives, optimized versions, peptidomimetics and antibodies generated to specific binding regions of the Perle Receptor serving as peptidomimetics are then administered ex-vivo to human extra-islet tissue, inclusive of ductal, acinar and progenitor tissue, embryonic stem cells, human adult bone-marrow derived cells, induced pluripotent stem cells, mesenchymal stem cells, umbilical cord stem cells, or other stem cells and may include resident populations of endogenous stem cells that exist within the adult pancreas to form new beta cells.
  • the new beta cells are generated by the delivery of Perle peptides, derivatives, optimized versions, peptidomimetics and antibodies generated to specific binding regions of the Perle Receptor to ex-vivo cultures of human embryonic stem cells, human adult bone-marrow derived cells, induced pluripotent stem cells, mesenchymal stem cells, umbilical cord stem cells, or other stem cells and may include resident populations of endogenous stem cells that exist within the adult pancreas, accelerate the formation of new beta cells.
  • beta cells are then delivered to patients with PreDiabetes, type 1 and 2 diabetes and other conditions of beta cell deficiency with routes of delivery to include, but are not limited to oral, intravenous, including delivery via the umbilical vein, portal venous systemic, hepatic artic artery, subcutaneous delivery with and without organ specific targeting and may include direct administration to the pancreas or liver.
  • routes of delivery to include, but are not limited to oral, intravenous, including delivery via the umbilical vein, portal venous systemic, hepatic artic artery, subcutaneous delivery with and without organ specific targeting and may include direct administration to the pancreas or liver.
  • This invention includes methods for pancreatic beta cell generation and include both in vivo and ex-vivo beta cell generation and methods for treating new onset and previously existing type 1 and 2 diabetes, Latent Autoimmune Diabetes of Adulthood (LADA), those at risk for type 1 diabetes, including but not limited to those with positive autoimmune antibodies markers including who are Glutamic Acid Decarboxylase-65 antibody, those with PreDiabetes and diseases of hyperglycemia, glucose intolerance and beta cell impairment or deficiency, insulin resistance, associated conditions including, obesity, obesity prior to the development of diabetes, obesity in children leading to PreDiabetes, both type 1 and type 2 diabetes in childhood and adolescence and include, but are not limited to conditions such as polycystic ovarian syndrome, nonalcoholic steatohepatitis, hyperlipidemia and hypertriglyceridemia and other conditions related to the deficiency or lack of effective amounts of insulin.
  • LADA Latent Autoimmune Diabetes of Adulthood
  • Fig. 17 shows the identification of the Perle Peptide 9-amino acid human Reg sequence (SEQ ID: 1) that has 100% homology with sequences found in other mammals including chimpanzee, rat, mouse, golden hamster, guinea pig, rabbit, pig, sheep, cow, white- cheeked gibbon, Sumatran orangutan, Lowland gorilla, white-tufted-eared marmoset, European domestic ferret which has not been described in the prior art as being beta regeneration peptide.
  • SEQ ID: 1 Perle Peptide 9-amino acid human Reg sequence
  • This invention evaluated GenBank, Basic Local Alignment Search Tool (BLAST) algorithm and UniProtKB which produced by the UniProt Consortium which consists of groups from the European Bioinformatics Institute (EBI), the Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR).
  • EBI European Bioinformatics Institute
  • SIB Swiss Institute of Bioinformatics
  • PIR Protein Information Resource
  • Fig. 18 shows the identification of the Perle Peptide 8-amino acid human Reg sequence (SEQ ID: 4) that has 100% homology with sequences found in other mammals including chimpanzee, rat, mouse, golden hamster, guinea pig, rabbit, pig, sheep, cow, white- cheeked gibbon, Sumatran orangutan, Lowland gorilla, and white-tufted-eared marmoset which has not been described in the prior art as being beta regeneration peptide.
  • This invention evaluated GenBank, Basic Local Alignment Search Tool (BLAST) algorithm and UniProtKB which produced by the UniProt Consortium which consists of groups from the European
  • Fig. 19 shows the identification of the Perle Peptide 8-amino acid human Reg sequence (SEQ ID: 8) that has 100% homology with sequences found in other mammals including chimpanzee, rat, mouse, golden hamster, guinea pig, rabbit, pig, sheep, cow, white- cheeked gibbon, Sumatran orangutan, Lowland gorilla, and European domestic ferret which has not been described in the prior art as being beta regeneration peptide.
  • This invention evaluated GenBank, Basic Local Alignment Search Tool (BLAST) algorithm and UniProtKB which produced by the UniProt Consortium which consists of groups from the European Bio informatics Institute (EBI), the Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR).
  • This sequences (SEQ ID: 8) and amino-acids within the sequence are not contained within the 14- amino acid human Reg3a HIP peptide or the 15-amino acid hamster Reg3gamma peptide, ING
  • Fig. 20 shows the identification of the Perle Peptide 7-amino acid human Reg sequence (SEQ ID: 7) that has 100% homology with sequences found in other mammals including chimpanzee, rat, mouse, golden hamster, guinea pig, rabbit, pig, sheep, cow, white- cheeked gibbon, Sumatran orangutan, Lowland gorilla, white-tufted-eared marmoset, European domestic ferret which has not been described in the prior art as being beta regeneration peptide.
  • SEQ ID: 7 Perle Peptide 7-amino acid human Reg sequence
  • This invention evaluated GenBank, Basic Local Alignment Search Tool (BLAST) algorithm and UniProtKB which produced by the UniProt Consortium which consists of groups from the European Bioinformatics Institute (EBI), the Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR).
  • EBI European Bioinformatics Institute
  • SIB Swiss Institute of Bioinformatics
  • PIR Protein Information Resource
  • Fig. 21 shows the identification of the 9-amino acid human Perle Peptide (SEQ ID: 14) that has 100% homology with sequences found in other mammals including chimpanzee, rat, mouse, golden hamster, guinea pig, rabbit, pig, sheep, cow, white-cheeked gibbon, Sumatran orangutan, and Lowland gorilla which has not been described in the prior art as being beta regeneration peptide.
  • This invention evaluated GenBank, Basic Local Alignment Search Tool (BLAST) algorithm and UniProtKB which produced by the UniProt Consortium which consists of groups from the European Bioinformatics Institute (EBI), the Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR).
  • EBI European Bioinformatics Institute
  • SIB Swiss Institute of Bioinformatics
  • PIR Protein Information Resource
  • Fig. 22 demonstrates the 20-amino acid human sequence (SEQ ID NO: 9) that has been identified within the human Reg receptor that has 100% homology with Reg receptor sequences found in chimp, rat, mouse, guinea pig, rabbit, dog, cow, opossum, galago, white-cheeked- gibbon, Sumatran orangutan, macaque, Lowland gorilla, white-tufted-eared marmoset and horse.
  • SEQ ID NO: 9 20-amino acid human sequence
  • Perle Peptide derivatives can be made via altering Perle Peptide sequences by substitutions, insertions, or deletions that provide for functionally equivalent or improved molecules. Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode the same or a substantially similar amino acid sequence as a Perle Peptide or analogs or derivatives thereof may be used in the practice of the present invention. These include, but are not limited to, nucleic acid sequences comprising all or portions of a Perle Peptide that are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.
  • the Perle Peptide derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a Perle Peptide including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity that acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • Perle Peptide derivatives of the invention also include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a Perle Peptide including altered sequences in which amino acid residues are substituted for residues with similar chemical properties. In a specific embodiment, 1, 2, 3, 4, or 5 amino acids are substituted.
  • Perle Peptides include, but are not limited to, those proteins which are substantially homologous to a Perle Peptide or fragments thereof, or whose encoding nucleic acid is capable of hybridizing to the Perle Peptide nucleic acid sequence.
  • chimeric or fusion proteins may be used in the method of the invention.
  • a "chimeric protein” or “fusion protein” comprises Perle a Peptides or an analog or derivative thereof operatively- linked to a non- Perle Peptide or an analog or derivative thereof.
  • the Perle Peptide or analog or derivative thereof can correspond to all or a portion of a Perle Peptide.
  • a Perle Peptide fusion protein comprises at least one biologically-active portion of a Perle Peptide.
  • the Perle Peptide or analog or derivative thereof and the non- Perle Peptide polypeptide are "operatively- linked", that is they are fused in- frame with one another.
  • the non-Perle Peptide polypeptide can be fused to the N-terminus or C-terminus of the Perle Peptide or analog or derivative thereof.
  • the fusion protein may be a Perle Peptide protein containing a heterologous signal sequence at its N-terminus.
  • expression and/or secretion of Perle Peptides or an analog or derivative thereof can be increased through use of a heterologous signal sequence.
  • the fusion protein is a Perle Peptide-immunoglobulin fusion protein in which the Perle Peptide sequences are fused to sequences derived from a member of the immunoglobulin protein family.
  • the Perle Peptide-immunoglobulin fusion proteins can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an immunological response according to the present invention.
  • Perle Peptides, an analog or derivative thereof, or a Perle Peptide-chimeric or fusion protein for use in the methods of the invention may be chemically modified for the purpose of improving bioavailability, and/or increasing efficacy, solubility and stability.
  • the protein may be covalently or non-covalently linked to albumin, transferrin or polyethylene glycol (PEG).
  • Perle Peptides, an or analog or derivative thereof, or a Perle Peptides-chimeric or fusion protein for use in the method of the invention can be produced by standard recombinant DNA techniques in accordance with the teachings of the invention.
  • DNA fragments coding for the different polypeptide sequences may be ligated together in- frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence [see, e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR
  • fusion moiety e.g., a GST polypeptide
  • a Perle Peptide-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in- frame to a Perle Peptide.
  • the fusion protein can be a Perle Peptide protein fused, to a His tag or epitope tag (e.g. V5) to aid in the purification and detection of the recombinant Perle Peptides, or to mask the immune response in a subject.
  • Perle Peptides, an or analog or derivative thereof, or a Perle Peptide-chimeric or fusion protein can be modified so that it has an extended half-life in vivo using any methods known in the art.
  • Fc fragment of human IgG or inert polymer molecules such as high molecular weight polyethyleneglycol (PEG) can be attached to a Perle Peptide or an analog or derivative thereof with or without a multifunctional linker either through site-specific conjugation of the PEG to the N- or C-terminus of the protein or via epsilon-amino groups present on lysine residues. Linear or branched polymer derivatization that results in minimal loss of biological activity will be used.
  • the degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to Perle Peptides or an analog or derivative thereof. Unreacted PEG can be separated from Perle Peptide- PEG conjugates by size-exclusion or by ion-exchange chromatography. PEG-derivatized conjugates can be tested for in vivo efficacy using methods known to those of skill in the art.
  • Derivatives and analogs may be full length or other than full length.
  • Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, or 95% identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993, and below.
  • monoclonal or polyclonal antibodies specific to Perle Peptides or analogs or derivatives thereof can be used in immunoassays to measure the amount of Perle Peptides or analogs or derivatives thereof or used in immunoaffinity purification of a Perle Peptide or analogs or derivatives thereof.
  • a Hopp & Woods hydrophilic analysis (see Hopp & Woods, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828 (1981) can be used to identify hydrophilic regions of a protein, and to identify potential epitopes of a Perle Peptide or analogs or derivatives thereof.
  • the antibodies that immunospecifically bind to a Perle Peptide or analogs or derivatives thereof can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques. (See, e.g., U.S. Publication No. 2005/0084449, which is incorporated herein in its entirety).
  • Polyclonal antibodies immunospecific for Perle Peptides or analogs or derivatives thereof can be produced by various procedures well-known in the art.
  • Perle Peptides or analogs or derivatives thereof can be administered to various host animals, including, but not limited to, rabbits, mice, and rats, to induce the production of sera containing polyclonal antibodies specific for Perle Peptides or analogs or derivatives thereof.
  • adjuvants may be used to increase the immunological response, depending on the host species, including but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • corynebacterium parvum Such adjuvants are also well known in the art.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques, including those known in the art and taught, for example, in Harlow et al.,
  • the term "monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • mice can be immunized with a non-murine antigen, and once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
  • the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with a non-murine antigen with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind to the antigen.
  • Antibody fragments which recognize specific particular epitopes may be generated by any technique known to those of skill in the art.
  • Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • F(ab')2 fragments contain the variable region, the light chain constant region and the CHI domain of the heavy chain.
  • the antibodies of the present invention can also be generated using various phage display methods known in the art.
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of affected tissues).
  • the DNA encoding the VH and VL domains are recombined together with a scFv linker by PCR and cloned into a phagemid vector.
  • the vector is electroporated in E. coli, and the E. coli is infected with helper phage.
  • Phage used in these methods are typically filamentous phage including fd and Ml 3 and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII.
  • Phage expressing an antigen binding domain that binds to a particular antigen can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol.
  • PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences in scFv clones.
  • VH constant region e.g., the human gamma 4 constant region
  • VL constant region e.g., human kappa or lambda constant regions.
  • the vectors for expressing the VH or VL domains comprise an EF- la promoter, a secretion signal, a cloning site for the variable domain, constant domains, and a selection marker such as neomycin.
  • the VH and VL domains may also be cloned into one vector expressing the necessary constant regions.
  • the heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, IgG, using techniques known to those of skill in the art.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also U.S. Pat. Nos. 4,444,887 and 4,716,111; and International publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, W098/16654, WO 96/34096, WO 96/33735, and WO 91/10741.
  • a chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules.
  • Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S. Pat. Nos. 5,807,715; 4,816,567; 4,816,397; and 6,311,415.
  • a humanized antibody is an antibody or its variant or fragment thereof which is capable of binding to a predetermined antigen and which comprises a framework region having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of a non-human immunoglobulin.
  • a humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, Fabc, Fv) in which all or substantially all of the CDR regions correspond to those of a non- human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fe), typically that of a human immunoglobulin.
  • the antibody will contain both the light chain as well as at least the variable domain of a heavy chain.
  • the antibody also may include the CHI, hinge, CH2, CH3, and CH4 regions of the heavy chain.
  • the humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGl, IgG2, IgG3 and IgG4.
  • the constant domain is a complement fixing constant domain where it is desired that the humanized antibody exhibit cytotoxic activity, and the class is typically IgGl.
  • the constant domain may be of the IgG2 class.
  • the humanized antibody may comprise sequences from more than one class or isotope, and selecting particular constant domains to optimize desired effector functions is within the ordinary skill in the art.
  • the framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor CDR or the consensus framework may be mutagenized by substitution, insertion or deletion of at least one residue so that the CDR or framework residue at that site does not correspond to either the consensus or the import antibody. Such mutations, however, will not be extensive.
  • humanized antibody residues will correspond to those of the parental framework region (FR) and CDR sequences, more often 90%, and most preferably greater than 95%.
  • Humanized antibody can be produced using variety of techniques known in the art, including but not limited to, CDR grafting (European Patent No. EP 239,400; International Publication No. WO
  • framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
  • framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature 332:323).
  • any techniques known in the art can be used in purifying Perle Peptides or an analog or derivative thereof, including but not limited to, separation by precipitation, separation by adsorption (e.g., column chromatography, membrane adsorbents, radial flow columns, batch adsorption, high-performance liquid chromatography, ion exchange chromatography, inorganic adsorbents, hydrophobic adsorbents, immobilized metal affinity chromatography, affinity chromatography), or separation in solution (e.g., gel filtration, electrophoresis, liquid phase partitioning, detergent partitioning, organic solvent extraction, and ultrafiltration).
  • separation by precipitation e.g., column chromatography, membrane adsorbents, radial flow columns, batch adsorption, high-performance liquid chromatography, ion exchange chromatography, inorganic adsorbents, hydrophobic adsorbents, immobilized metal affinity chromatography, affinity chromatography
  • separation in solution e.
  • Perle Peptides or an analog or derivative thereof may be monitored by one or more in vitro or in vivo assays.
  • the purity of Perle Peptides or an analog or derivative thereof can be assayed by any methods known in the art, such as but not limited to, gel electrophoresis. See Scopes, supra.
  • Perle Peptides or an analog or derivative thereof employed in a composition of the invention can be in the range of 80 to 100 percent of the total mg protein, or at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% of the total mg protein. In one embodiment, Perle Peptides or an analog or derivative thereof employed in a composition of the invention is at least 99% of the total protein. In another embodiment, Perle Peptides or an analog or derivative thereof is purified to apparent homogeneity, as assayed, e.g., by sodium dodecyl sulfate polyacrylamide gel electrophoresis.
  • a nucleic acid sequence encoding a Perle Peptide or an analog or derivative thereof can be inserted into an expression vector for propagation and expression in host cells.
  • An expression construct refers to a nucleic acid sequence encoding a Perle Peptide or an analog or derivative thereof operably associated with one or more regulatory regions that enable expression of a Perle Peptide or an analog or derivative thereof in an appropriate host cell.
  • "Operably-associated” refers to an association in which the regulatory regions and the Perle Peptide or an analog or derivative thereof to be expressed are joined and positioned in such a way as to permit transcription, and ultimately, translation.
  • the regulatory regions that are necessary for transcription of Perle Peptides or an analog or derivative thereof can be provided by the expression vector.
  • a translation initiation codon may also be provided if a Perle Peptide or an analog or derivative thereof gene sequence lacking its cognate initiation codon is to be expressed.
  • RNA polymerase a promoter which is capable of binding RNA polymerase and promoting the transcription of an operably-associated nucleic acid sequence.
  • Such regulatory regions may include those 5 ' non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • the non-coding region 3 ' to the coding sequence may contain transcriptional termination regulatory sequences, such as terminators and polyadenylation sites.
  • linkers or adapters providing the appropriate compatible restriction sites may be ligated to the ends of the cDNAs by techniques well known in the art (see e.g., Wu et al., 1987, Methods in Enzymol, 152:343- 349). Cleavage with a restriction enzyme can be followed by modification to create blunt ends by digesting back or filling in single-stranded DNA termini before ligation. Alternatively, a desired restriction enzyme site can be introduced into a fragment of DNA by amplification of the DNA using PCR with primers containing the desired restriction enzyme site.
  • An expression construct comprising a Perle Peptide or an analog or derivative thereof sequence operably associated with regulatory regions can be directly introduced into appropriate host cells for expression and production of a Perle Peptide or an analog or derivative thereof without further cloning. See, e.g., U.S. Pat. No. 5,580,859.
  • the expression constructs can also contain DNA sequences that facilitate integration of a Perle Peptide or an analog or derivative thereof sequence into the genome of the host cell, e.g., via homologous recombination. In this instance, it is not necessary to employ an expression vector comprising a replication origin suitable for appropriate host cells to propagate and express Perle Peptides or an analog or derivative thereof in the host cells.
  • a variety of expression vectors may be used, including but are not limited to plasmids, cosmids, phage, phagemids or modified viruses.
  • host-expression systems represent vehicles by which the coding sequences of a Perle Peptide or an analog or derivative thereof gene may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express Perle Peptides or an analog or derivative thereof in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing Perle Peptides or an analog or derivative thereof coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant expression vectors containing Perle Peptides or an analog or derivative thereof coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing Perle Peptides or an analog or derivative thereof coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing Perle Peptides or an analog or derivative thereof coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NSO, and 3T3 cells) harboring
  • bacterial cells such as Escherichia coli and eukaryotic cells are used for the expression of a recombinant Perle Peptide or an analog or derivative thereof.
  • mammalian cells such as Chinese hamster ovary cells (CHO) can be used with a vector bearing promoter element from major intermediate early gene of cytomegalovirus for effective expression of a Perle Peptide or an analog or derivative thereof sequence (Foecking et al., 1986, Gene 45:101 ; and Cockett et al., 1990, Bio/Technology 8:2).
  • a number of expression vectors may be advantageously selected depending upon the use intended for the Perle Peptides or an analog or derivative thereof being expressed. For example, when a large quantity of a Perle Peptide or an analog or derivative thereof is to be produced, for the generation of pharmaceutical compositions of a Perle Peptide or an analog or derivative thereof, vectors that direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • Vectors include, but are not limited to, the E. coli expression vector pCR2.1 TOPO (Invitrogen); pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem.
  • Series of vectors like pFLAG (Sigma), pMAL (NEB), and pET (Novagen) may also be used to express the foreign proteins as fusion proteins with FLAG peptide, malE-, or CBD-protein. These recombinant proteins may be directed into periplasmic space for correct folding and maturation. The fused part can be used for affinity purification of the expressed protein. Presence of cleavage sites for specific proteases like enterokinase allows one to cleave off the Perle Peptide or an analog or derivative thereof.
  • the pGEX vectors may also be used to express foreign proteins as fusion proteins with glutathione 5 -transferase (GST).
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • a Perle Peptide or an analog or derivative thereof coding sequence may be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).
  • a number of viral-based expression systems may be utilized.
  • a Perle Peptide or an analog or derivative thereof coding sequence of interest may be ligated to an adenovirus
  • transcription/translation control complex e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing Perle Peptides or an analog or derivative thereof in infected hosts (see, e.g., Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 8 1:355-359). Specific initiation signals may also be required for efficient translation of inserted Perle Peptides or an analog or derivative thereof coding sequences.
  • These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert.
  • exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, and the like (see, e.g., Bittner et al., 1987, Methods in Enzymol. 153:51-544).
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript and post-translational modification of the gene product, e.g., glycosylation and phosphorylation of the gene product, may be used.
  • Such mammalian host cells include, but are not limited to, PC12, CHO, VERY, BHK, HeLa, COS, MDCK, 293, 313, W138, BT483, Hs578T, HTB2, BT20 and T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030, and HsS78Bst cells.
  • Expression in a bacterial or yeast system can be used if post- translational modifications are found to be non-essential for a desired activity of Perle Peptides or an analog or derivative thereof.
  • Cell lines that stably express Perle Peptides or an analog or derivative thereof may be engineered by using a vector that contains a selectable marker.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the expression construct confers resistance to the selection and may, depending on the vector construct and host cell employed, allow cells to stably integrate the expression construct into their chromosomes and to grow in culture and to be expanded into cell lines. Such cells can be cultured for a long period of time while Perle Peptides or an analog or derivative thereof is expressed continuously.
  • a number of selection systems may be used, including but not limited to, antibiotic resistance (markers like Neo, which confers resistance to geneticine, or G-418 (Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;
  • mutant cell lines including, but not limited to, tk-, hgprt- or aprt-cells, can be used in combination with vectors bearing the corresponding genes for thymidine kinase, hypoxanthine, guanine- or adenine phosphoribosyl- transferase. Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al.
  • the recombinant cells may be cultured under standard conditions of temperature, incubation time, optical density and media composition. However, conditions for growth of recombinant cells may be different from those for expression of Perle Peptides or an analog or derivative thereof. Modified culture conditions and media may also be used to enhance production of Perle Peptides or an analog or derivative thereof. Any techniques known in the art may be applied to establish the optimal conditions for producing Perle Peptides or an analog or derivative thereof.
  • Perle Peptides or a fragment thereof by recombinant techniques or purification from natural sources is peptide synthesis.
  • an entire Perle Peptides or an analog or derivative thereof, or a protein corresponding to a portion of Perle Peptides or an analog or derivative thereof can be synthesized by use of a peptide synthesizer.
  • Conventional peptide synthesis or other synthetic protocols well known in the art may be used.
  • Proteins having the amino acid sequence of Perle Peptide or an analog or derivative thereof or a portion thereof may be synthesized by solid-phase peptide synthesis using procedures similar to those described by Merrifield, 1963, J. Am. Chem. Soc, 85:2149. During synthesis, N-a-protected amino acids having protected side chains are added stepwise to a growing polypeptide chain linked by its C-terminal and to an insoluble polymeric support, i.e., polystyrene beads.
  • the proteins are synthesized by linking an amino group of an N-a- deprotected amino acid to an a-carboxyl group of an N-a-protected amino acid that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide.
  • a reagent such as dicyclohexylcarbodiimide.
  • the attachment of a free amino group to the activated carboxyl leads to peptide bond formation.
  • the most commonly used N-a-protecting groups include Boc, which is acid labile, and Fmoc, which is base labile.
  • Perle Peptides or an analog or derivative thereof is accomplished using conventional procedures, such as preparative HPLC using gel permeation, partition and/or ion exchange chromatography.
  • preparative HPLC using gel permeation, partition and/or ion exchange chromatography.
  • matrices and buffers are well known in the art and so are not described in detail herein.
  • This invention identifies homologous peptide sequences known as "Perle peptides" that are found within the binding arm of the human Regla, Reglb, Reg3a and Reg4 protein and evaluates their role as beta regeneration agents. None of the Perle peptides are contained within the previously described the human 14-amino acid Reg3a HIP peptide or the hamster 15-amino acid Reg3gamma peptide, INGAP. A 20-amino binding region within the 919-amino acid Reg receptor was discovered from which stimulating antibodies were generated to serve as Reg peptidomimetics.
  • peptidomimetics and stimulatory antibodies to the Reg Receptor for the accelerated generation of beta cells, or other beta cells stimulating agents that are specifically initiated on the day of the immune nadir when one or more immunomodulary agents are given to a patient with diabetes.
  • the day of the immune nadir varies by immunomodulatory agent.
  • specific methods are provided for optimizing the glycemic milieu in a patient with type 1 diabetes prior to initiation of Perle peptides, including formulations, derivatives, optimized forms including peptidomimetics and stimulatory antibodies to the Reg
  • Receptor for the accelerated generation of beta cells along with methods tapering insulin as new beta cells are generated.
  • Methods for Reversing new onset and existing type 2 diabetes, PreDiabetes, conditions of insulin resistance, insulin deficiency, beta cell deficiency and/or abnormal glucose metabolism include the use of Perle peptides, including formulations, derivatives, optimized forms including peptidomimetics and stimulatory antibodies to the Reg Receptor for the accelerated generation of beta cells and include specific methods are provided for optimizing the glycemic milieu in a patient with new and existing type 2 diabetes prior to initiation of Perle peptides, including formulations, derivatives, optimized forms including peptidomimetics and stimulatory antibodies to the Reg
  • Receptor for the accelerated generation of beta cells, along with methods tapering oral and subcutaneous diabetes agents including insulin, as new beta cells are generated.
  • Perle peptides including formulations, derivatives, optimized forms including peptidomimetics and stimulatory antibodies to the Reg Receptor for the accelerated generation of beta cells by ex-vivo transformation of human extra-islet tissue including ductal, acinar and progenitor cells, human embryonic stem cells, human adult bone-marrow derived cells, induced pluripotent stem cells, mesenchymal stem cells, umbilical cord stem cells, or other stem cells and may include resident populations of endogenous stem cells that exist within the adult pancreas that are then delivered to patients with diabetes and other conditions of insulin deficiency via the umbilical vein, portal venous systemic, hepatic artery, subcutaneous delivery with and without organ specific targeting and may include direct administration to the pancreas or liver.
  • HIP 14-amino acid human Reg3a peptide
  • INGAP 15-amino acid hamster peptide
  • BrDU labeling considered to be the gold standard in determining whether new beta cells generated are derived from existing beta cells by a process of budding versus the new beta cells being derived from extra-islet exocrine tissue, has demonstrated that not only do both the 14- amino acid human Reg3a peptide (HIP) and the 15-amino acid hamster peptide (INGAP) result in new beta cells, but also the new beta cells are derived from extra-islet exocrine tissue.
  • the mechanism of action of both HIP and INGAP is via their interaction with the Reg Receptor, which is found in human the extra-islet ductal tissue.
  • FIG. 7 demonstrates the results from studies conducted to demonstrate Reg Receptor expression and purification utilizing 293T cells that were transfected with Reg Receptor expression plasmid DNA.
  • Cells were collected after 72 hours.
  • the Reg Receptor was tagged with FLAG epitope and FLAG resin was utilized to purify out the Reg Receptor.
  • the Reg Receptor was highly purified.
  • the Reg Receptor was purified by Anti-Flag M2 affinity gel.
  • Target protein was confirmed by 4-12% SDS-PAGE and Western-blot.
  • Figure 7a demonstrates the use of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) to illustrate the high purity of the Reg Receptor.
  • Figure 7b and 7c are the Western blot results showing that the purified protein is the Reg Receptor using the antibody to the Reg Receptor (CD104).
  • the Reg Receptor is shown In FIG. 7 to be highly purified.
  • the purified Reg receptor was coated onto 96 well plate by using bicarbonate coating buffer, pH 9.6; 4°C overnight at concentration 3ug/ml, lOOul per well. Plates were coated overnight coated plate and washed three times with 0.5X TBST and blocked with 3%BSA and rotated at room temperature for 1 hour. After blocking, plates were washed three times with 0.5X TBST. Peptides were then diluted with TBST buffer and added into wells in duplicate then left to bind at room temperature for 1 hour. After washing three times, lOOng/ml strep-HRP was added into plate at lOOul/well, and rotated at room temperature for one hour.
  • ABST reagents were warmed to room temperature, mixed immediately before using. Then lOOul was added to each well and read after 25 minutes reaction and absorbance was evaluated at 405nm by a Spectramax M5 plate reader. The purified Reg Receptor was coated on plates. Then plates were blocked with BSA solution. Subsequently, the Perle peptides were added into the wells, and HRP- straptavidin and its substrates were added into the wells to reveal the interaction between Receptor and the Perle peptide.
  • the 7-amino acid Perle peptide, the 8-amino acid Perle peptide and the 9-amino acid Perle peptide all bind directly to Reg Receptor.
  • the 8-amino acid peptide appeared to be the strongest in binding to the Reg Receptor.
  • the scrambled control peptide did not bind to Reg Receptor.
  • the 14-amino acid human Reg3a peptide (HIP)
  • the 14-amino acid human Reg3a peptide (HIP) was shown in this study to have the same level of binding as the scrambled peptide (FIG.
  • the 7,8 and 9-amino acid Perle Peptides sequences are not contained within the 14-amino human Reg3a peptide (HIP) sequence or the 15 amino acid hamster 3 gamma peptide (INGAP).
  • Reg proteins and peptides act through a 919-amino acid cell-surface protein. Studies were undertaken to identify the binding site for Perle peptides and develop stimulatory compounds to accelerate the progression of beta cell formation by stimulating the binding sites on the Reg Receptor. For the production of stimulatory antibodies, sequences were evaluated within the N-terminal portion of the putative Reg Receptor (amino acids 1-332) (SEQ ID NO: 13), which is the amino acid region of Reg Receptor that is not contained in the other members of the of Exostoses family, and thus is hypothesized to be the Reg binding domain.
  • the animals were injected with a peptide of SEQ ID NO: 9 and were conjugated to keyhole limpet hemocyanin (KLH).
  • KLH keyhole limpet hemocyanin
  • the screening antigen is not conjugated to KLH so that the response solely to the peptide and not to KLH can be identified.
  • the 50% titer is a dilution value where the signal is half-way between the peak and the baseline, so the higher the dilution value (titer), the greater the response to the antigen.
  • the positive control is an internal control that was generated from ovalbumin antibodies in rabbit. At a dilution of 1 :750,000, the absorbance fell within a range of 0.45 to 0.9.
  • this invention Based on the development of stimulatory antibodies that were generated to a 20-amino acid peptide region within the Reg Receptor (FIG: 11), this invention identifies the potential to utilize stimulatory antibodies for the generation of new beta cells as well as for the development of peptidomimetics utilizing the domain binding site of Perle peptides on the Reg Receptor for the treatment of diabetes and conditions of insulin and/or beta cell insufficiency or loss.
  • Human extra-islet ductal cells were seeded in T75 flasks in Dulbecco's Modified Eagle media containing 10% fetal bovine serum. The cells were incubated at 37 0C, 5% C0 2 for 24 hours and then treated with Perle peptide at the final concentration of 167 nM. This treatment was performed once a day for four days. On the fifth day the cells were broken to obtain the cell lysates. In these cell extracts the total protein levels were determined, and 50 micrograms of total protein were used to perform the western blot analysis. The samples containing 50 micrograms of proteins were diluted in loading buffer and loaded into each well of the gel. For gels run under reducing conditions, the buffer also contained 5 % of the reducing agent beta- mercaptoethanol.
  • Both extraction buffers contained 0.5 mM PMSF, 1 ⁇ g/ml leupeptin, ⁇ g/ml aprotinin, 2.5mM Na 4 P 2 0 7 ,lmM ⁇ -glycerophosphate, and ImM Na 3 V0 4 .
  • Protein extracts were size fractionated on SDS-polyacrylamide gels and transferred to nitrocellulose. After blocking in 3% milk in Tris-buffered saline (pH 7.4), blots were sequentially incubated with rabbit anti-human Reg Receptor antibody overnight at 4°C and appropriate horseradish peroxidase-conjugated secondary antibody. Secondary signals were developed with chemiluminescence substrate and analyzed by autoradiography.
  • Glucose homeostasis requires an adequate number of beta cells, as illustrated by the inability to restore normoglycemia among diabetic patients even when intensive regimens of medications are utilized.
  • the DCCT investigators set, as a major treatment outcome goal, a mean AIC over the trial period of ⁇ 6.05% without an increased risk for hypoglycemia.
  • Sensor- augmented pumps recently were shown to improve AIC levels from 8.3% to 7.5%, over 12 months, with further reductions to 7.4% after an additional 6 months of treatment. These achievements were made without the associated weight gain or hypoglycemia seen in the DCCT.
  • sensor- augmented pump therapy did not improve AIC levels as much as those seen in the DCCT decades ago. Bergenstal RM, N Engl J Med, .
  • diabetes remains the leading cause of new blindness, amputations and kidney failure requiring dialysis or transplantation.
  • Providing and protecting new beta cells from one's own pancreatic progenitor cells provide patients an opportunity to reverse this disease state.
  • FIG. 14 presents a summary of methods to reverse new and existing type 1 diabetes and Latent Autoimmune Diabetes of Adulthood by utilizing beta cell agonists including Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg Receptor.
  • delivery of an immune tolerance agent prior to a beta cell regeneration agent is hypothesized in this invention to improve the ability to preserve new beta formed from Perle peptides.
  • this invention specifically describes the methods and timing for usage of beta cell agonists including Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg Receptor for the treatment of new and existing type 1 diabetes and Latent Autoimmune Diabetes of Adulthood.
  • This invention includes methods that have not previously been described for initiation and usage of beta agonists including Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg Receptor, in type 1 and Latent Autoimmune Diabetes of Adulthood patients at the time of the immune nadir after initiation of an immune modulator, in order to optimally prevent immune attack on the new beta cells generated by beta cell agonists including Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg Receptor.
  • the time of the immune nadir varies with each immunomodulation agent that has been utilized in new onset type 1 diabetes.
  • FIG. 15 illustrates the two-step approach to reverse type 1 diabetes in new onset and existing type 1 diabetes patients and among patients with Latent Autoimmune Diabetes of Adulthood.
  • beta cells As new beta cells form among type 1 patients from the immune agent and Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg Receptor, exogenous insulin must be reduced, because maintaining the glucose milieu within a narrow range is critical to beta cell regeneration. Beta cell formation will not occur in conditions of hypoglycemia. Formation of beta cells will also not optimally occur with extreme
  • the combination therapy of a targeted immune tolerance agent along with an agent that stimulates beta regeneration which is initiated at the immune nadir has the potential to reverse type 1 diabetes in humans.
  • Many of the immune tolerance agents have demonstrated the ability to provide persistent immunity for years following treatment, but despite immunity, which protects remaining beta cells present, the generation of new beta cells is not adequate to render patients insulin- free although some of the immune tolerance agents, including Cyclosporine, anti-CD3 antibodies, GAD vaccine, Diap277 and the BCG vaccine have resulted in improved stimulated c-peptide representing improvement in endogenous insulin. Cyclosporine has rendered 50% of new onset patients with type 1 diabetes, insulin free at 1 year.
  • autoimmunity to newly formed beta cells may have negatively impacted study outcomes in terms of hemoglobin AlC and stimulated C-peptide, a marker of endogenous insulin production.
  • each immune tolerance agent has a unique time in which patients develop an immune nadir. For example, the immune nadir occurs on day 6 following intravenous treatment with cyclosporineanti-CD3 antibody, teplizumab and the immune nadir among those treated with the BCG vaccine occurs week 5, after the second BCG injection.
  • this invention includes the optimal time to initiate usage of a beta cell agonist, including Perle peptides, derivatives, formulations, optimized versions, Perle peptidomimetics and stimulatory antibodies to the Reg receptor, is at the immune nadir for the given immune tolerance agent.
  • This invention includes the initiation of beta agonist including Perle peptides, derivatives, formulations, optimized versions, Perle peptidomimetics and stimulatory antibodies to the Reg receptor specifically during immune nadir with any of the immune tolerance agents which varies between agents utilized.
  • Perle peptides, formulations, derivatives, peptidomimetics, and stimulatory antibodies to the Reg Receptor may be utilized with one or more of many immune modulators to protect new beta cells formed by Perle peptides, formulations, derivatives, peptidomimetics, and stimulatory antibodies to the Reg Receptor.
  • the types of agents include but are not limited to general immunosuppressant agents, which have typically been used in organ transplants, specifically targeted antibodies to those lymphocytes which attack the islets, along with other agents such as Vitamin D, in which a deficiency has been associated with a higher incidence of diabetes.
  • Agents include cyclosporine, mycophenolate mofetil, Rituximab, an anti CD20 agent, which is an FDA approved agent for the treatment of B-lymphocyte lymphoma.
  • Other immune agents include, but are not limited to: cyclosporine, the anti-CD3 antibodies, hOKT3 gammal (Ala- Ala) (teplizumab), and the monoclonal antibody TRX4 (ChAglyCD3).
  • the immune tolerance agent may also include, Polyclonal Anti-T-Lymphocyte Globulin (ATG), CTLA4-Ig
  • Abatacept a selective co-stimulation modulator as it inhibits the co- stimulation of T cells, or Campath-1H, (Anti-CD52 Antibody), a humanized monoclonal antibody to T-cells, or Alpha- 1 antitrypsin (AAT) is a serine proteinase inhibitor.
  • any of the following immune tolerance agents have had limited success, but none have rendered patients insulin- free, and could be used in this method.
  • LAD A Latent Autoimmune Diabetes in Adults
  • Another agent is the GAD antibody vaccine based on the 65 kDa isoform of the recombinant human glutamic acid decarboxylase protein (rhGAD65).
  • CTLA4-Ig (Abatacept) inhibits a crucial stimulatory pathway in the activation of T cells. By this mechanism, the drug is thought to arrest or slow the T cell mediated autoimmune destruction of insulin producing cells and preserves their function.
  • CTLA-4 Ig is being trialed as an intravenous agent begun within three months of diagnosis and then monthly for a total of 25 treatments.
  • CampathHl is another immune tolerance agent being trialed among new onset type 1 diabetes and may be utilized in conjunction with Perle peptides, formulations, optimized versions, peptidomimetics and stimulating antibodies to a binding region on the Reg receptor identified in this invention.
  • Alpha- 1 antitrypsin is a serine proteinase inhibitor is currently in study to preserve beta function among newly diagnosed type 1 patients. Cyclosporine has rendered 50% of new onset patients with type 1 diabetes, insulin free at 1 year by helping retain the 10% or so remaining islets after the initial attack on the beta cells. Bougneres et al., N Engl J Med; 1988 (318)11:663-670. Unfortunately, when these patients were followed out to six years, the beta cell mass declined, but of notable interest, each year that patients remained on cyclosporine, the percentage of antibodies attacking islets was significantly lower each year than patients not treated with cyclosporine. De Fiiippo et al. Diabetes. 1996. 45(l):101-4. The ability to have the Perle Peptides that are enclosed in this invention, given in combination with a beta cell immune protector, may truly reflect a treatment that addresses the underlying pathologies of type 1 diabetes.
  • Optimal glycemic control is critical not only after but also prior to initiating a beta agonist including Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg Receptor.
  • Required is a 4-week period of intensification of glucose control before the initiation of beta agonists including Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg Receptor in which patients should closely monitor both pre and postprandial blood glucose levels.
  • the glucose goal for patients may be between 100 and 200 mg/dL at all times.
  • patients may utilize a medical team with state-of-the-art diabetes tools including subcutaneous continuous monitoring systems. The primary goal will be to ensure that the glucose levels do not fall below 70 mg/dL during the optimization period.
  • Methods for using a Perle Peptide in a newly diagnosed or pre-existing patient with type 1 diabetes would utilize an immune tolerance agent. For example, if the immune tolerance agent given to a patient with new onset type 1 diabetes is selected to be cyclosporine, then patient would receive 7.5 mg/kg/day orally for 7 days before Perle peptides are begun. In order to decrease exposure to high peak concentrations of cyclosporine, half the daily dosage will be given with breakfast and half with dinner. The dosage will be adjusted on days 3,7,15, 30, and 45 of treatment, to maintain trough levels of cyclosporine between 150 and 350 ng per milliliter of as evauated by standard assay.
  • Optmized Perle Peptides will be started on day eight after seven days of treatment cyclosporine and begun at dosages of 1 mg/kg subcutaneously with the two largest meals
  • the immune tolerance agent is teplizumab
  • patients would receive six consecutive days of intravenous teplizumab on a weight based dosage and on day six of treatment, initiation would begin at (1 mg/kg) subcutaneously twice daily of optimized Perle peptide (eg: SEQ ID: 8) modified by blocking (i.e., end-capping of the amino and carboxyl termini of the selected Perle peptide with acetyl and amide groups).
  • SEQ ID: 8 optimized Perle peptide
  • exogenous insulin would be tapered as described below.
  • the immune tolerance agent selected is the heat shock protein 60, DiaPep277
  • initiation would begin of 60 mg of subcutaneous twice daily optimized Perle peptide modified by blocking (i.e., end-capping of the amino and carboxyl termini of both peptides with acetyl and amide groups), of Perle peptide (eg: SEQ ID: 8).
  • Perle peptide eg: SEQ ID: 8
  • the patient would receive another subcutaneous injection of the heat shock protein Diapep277, during which time, based in glucose levels, exogenous insulin would be tapered as described below.
  • exogenous insulin would be tapered as described below.
  • the selected immune tolerance agent selected is the Mycobacterium bovis Bacillus- Calmette-Guerin (BCG) vaccine, also known as the tuberculosis vaccine
  • BCG Mycobacterium bovis Bacillus- Calmette-Guerin
  • BCG Mycobacterium bovis Bacillus- Calmette-Guerin
  • Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg Receptor may be delivered within 15 minutes of two major meals eaten, when at least 30 grams of carbohydrates are consumed. After initiation of the Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg Receptor, based upon the individual's glucose levels, exogenous insulin would be tapered as described below.
  • the patients' insulin dosage may be decreased as required to prevent any episodes of hypoglycemia and to maintain glucose levels in an optimal range for islet neogenesis and may initially be reduced by 1% per day for the first 30 days. This is a total reduction of 1% per day from the preprandial insulin dosages (0.33% per meal reduction of the total premeal insulin dosage from the previous premeal dosage).
  • days 31-60 Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg Receptor there may be a 1% per day reduction in the basal insulin from the previous day.
  • beta agonist therapy including Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg Receptor
  • patients will have daily communication via phone, e-mail or office visits to give feedback on glucose values to the diabetes health care team. Based on the glucose values, more aggressive reduction in basal insulin dosages may occur if premeal glucose levels are less than 100 mg/dL and more aggressive reductions in premeal insulin may occur if 2 hour postprandial levels are less than 140 mg/dL.
  • insulin dosages maybe reduced by 0.5-2.0% per day based upon daily glucose values.
  • Reduction in basal insulin dosages may be required to prevent any episodes of hypoglycemia and to maintain glucose levels in an optimal range for islet neogenesis and may initially be reduced by 0.5% per day if premeal values are 100-125 mg/dL and a 0.6% per day total reduction (0.18% per meal reduction from the previous day) in premeal insulin may occur if 2 hour postprandial levels are 140-160 mg/dl.
  • the dosage of preprandial insulin may be reduced by 2.0% before meals (0.7% per meal in premeal insulin).
  • Beta agonists including Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg Receptor can be discontinued when stimulated C-peptide levels are within the normal range and when optimized glycemic control has been achieved without the usage of other diabetic agents including insulin.
  • Throughout the duration of the treatment patients may have daily communication via phone, e-mail or office visits to give feedback on glucose values to the diabetes health care team. Based on the glucose values, more aggressive reduction in basal and premeal insulin dosages may occur based on premeal and postprandial glucose levels respectively.
  • diabetes remains the leading cause of new blindness, amputations and kidney failure necessitating dialysis.
  • Stimulating new beta cells from one's own pancreatic progenitor cells serves as the best method to reverse type 2 diabetes in adults and children.
  • Perle peptides, formulations, optimized versions, peptidomimetics and stimulating antibodies to a binding region on the Reg receptor will be used as first- line therapy for new onset and existing type 2 diabetes, as well as PreDiabetes.
  • peptidomimetics and stimulating antibodies to a binding region on the Reg receptor may be added to other diabetes agents and including: all types of insulin, Glucagon Like Peptide- 1 (GLP-1) receptor analogs Liraglutide and Exenatide, Dipeptidyl Peptidase-4 Inhibitors, (DPP-4 inhibitors), and including (Sitagliptin, Saxagliptin, Linagliptin), the Amylin, analog, pramlintide, acarbose, orlistat, colesevelam, bromocriptine, orlistat, combination therapies with the biguanide, metformin, and combinations of with thiazolidinediones, sulfonylureas and DPP-4 inhibitors and new agents SGLT2 inhibitors (dapagliflozin and canaglifloziii).
  • GLP-1 Glucagon Like Peptide- 1
  • DPP-4 inhibitors Dipeptidyl Peptidase-4 Inhibi
  • FIG. 15 illustrates a summary of methods to reverse type 2 diabetes utilizing a beta cell agonist including Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg Receptor.
  • Perle peptides, formulations, optimized versions, peptidomimetics and stimulating antibodies to a binding region on the Reg receptor identified in this invention would be first- line therapy for patients with type 2 diabetes and PreDiabetes or added to existing diabetes medications regimens including insulin. Based upon glucose levels, other diabetes medications, including insulin will be tapered as new beta cells are formed as described below.
  • a said patient with PreDiabetes, new onset 2 diabetes or existing type 2 diabetes would undergo a two-week period prior to the initial administration of a beta agonist including Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg Receptor, in which the patients should closely monitor both pre and postprandial blood glucose levels and not have any episodes of symptomatic hypoglycemia or documentation of glucose levels ⁇ 70 mg/dL.
  • diabetic medications should be tapered, and the intensification of glucose levels be reinitiated in order to prevent hypoglycemia, which will inhibit the effects of a beta cell agonist including Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg Receptor.
  • Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg Receptor are added to the patients daily medication regimen in a dosage of 60 mg (1 mg/kg) of subcutaneous twice daily optimized Perle peptide modified by blocking (i.e., end-capping of the amino and carboxyl termini of the selected Perle peptide with acetyl and amide groups), of Perle peptide (SEQ ID: 8).
  • Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg Receptor may be delivered within 15 minutes of two major meals eaten, when at least 30 grams of carbohydrates are consumed. After initiation of the Perle peptide, based on glucose levels, other agents including exogenous insulin may be tapered as described below.
  • the patients' diabetes regimen should be modified when glucose levels fall below 100 mg/dL because hypoglycemia will negate the effects of beta generation due to numerous counter regulatory hormones that prevent new beta cell formation in the presence of hypoglycemia.
  • the patients' diabetes medications may require tapering of agents beginning with insulin should the patient be on insulin.
  • the insulin dosage may be decreased as required to prevent any episodes of hypoglycemia and to maintain glucose levels in an optimal range for beta cell regeneration and if the patient is on insulin therapy, the insulin dosage may initially be reduced by 1% per day for the first 30 days. For patients on insulin, this is a total reduction of 1% per day from the preprandial insulin dosages (0.33% per meal reduction of the total premeal insulin dosage from the previous premeal dosage).
  • days 31-60 Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg Receptor there may be a 1% per day reduction in the basal insulin from the previous day for patient treated with insulin.
  • agents including oral and injectable agents including insulin may be further tapered based on daily glucose values.
  • Reduction in basal insulin dosages may be required to prevent any episodes of hypoglycemia and to maintain glucose levels in an optimal range for islet neogenesis and may initially be reduced by 0.5% per day if premeal values are 100-125 mg/dL and a 0.6% per day total reduction (0.18% per meal reduction from the previous day) in premeal insulin may occur if 2 hour postprandial levels are 140-160 mg/dl.
  • Perle peptides, derivatives, optimized versions, peptidomimetics or stimulatory antibodies to the Reg Receptor can be discontinued when stimulated C-peptide levels are within the normal range and when optimized glycemic control has been achieved without the usage of other diabetic or agents and injectable agents including insulin.
  • Throughout the duration of the treatment patients may have daily communication via phone, e-mail or office visits to give feedback on glucose values to the diabetes health care team. Based on the glucose values, more aggressive reduction in basal and premeal insulin dosages may occur based on premeal and postprandial glucose levels respectively.
  • This invention includes methods for the formation and delivery of new beta cells generated by Perle peptides, derivatives, optimized versions, peptidomimetics and antibodies generated to specific binding regions of the Perle Receptor serving as peptidomimetics when utilized for the ex-vivo generation of new beta cells from progenitor cells, which may include, but are not limited to human extra-islet tissue inclusive of ductal, acinar and progenitor embryonic tissue, human stem cells, human adult bone-marrow derived cells, induced pluripotent stem cells, mesenchymal stem cells, umbilical cord stem cells, or other stem cells and may include resident populations of endogenous stem cells that exist within the adult pancreas that are facilitated to transform into beta cells by the addition of Perle peptides, derivatives, optimized versions, peptidomimetics and antibodies generated to specific binding regions of the Perle Receptor serving as peptidomimetics.
  • progenitor cells may include, but are not limited to human extra-islet tissue inclusive of ductal,
  • New beta cells are generated ex-vivo transformation using the inventions herein into new beta cells from Perle peptides, derivatives, optimized versions, peptidomimetics and antibodies generated to specific binding regions of the Perle Receptor serving as peptidomimetics.
  • New beta cells are then delivered to patients with PreDiabetes, type 1 and 2 diabetes and other conditions of beta cell deficiency with routes of delivery to include, but are not limited to oral, intravenous, including delivery via the umbilical vein, portal venous systemic, hepatic artic artery, subcutaneous delivery with and without organ specific targeting and may include direct administration to the pancreas or liver (FIG. 16).
  • Perle peptides, derivatives, optimized versions, peptidomimetics and antibodies generated to specific binding regions of the Perle Receptor serving as peptidomimetics are then administered ex-vivo to human extra-islet tissue, inclusive of ductal, acinar and progenitor tissue, embryonic stem cells, human adult bone-marrow derived cells, induced pluripotent stem cells, mesenchymal stem cells, umbilical cord stem cells, or other stem cells and may include resident populations of endogenous stem cells that exist within the adult pancreas to form new beta cells.
  • the new beta cells are generated by the delivery of Perle peptides, derivatives, optimized versions, peptidomimetics and antibodies generated to specific binding regions of the Perle Receptor to ex-vivo cultures of human embryonic stem cells, human adult bone-marrow derived cells, induced pluripotent stem cells, mesenchymal stem cells, umbilical cord stem cells, or other stem cells and may include resident populations of endogenous stem cells that exist within the adult pancreas, accelerate the formation of new beta cells.
  • beta cells are then delivered to patients with PreDiabetes, type 1 and 2 diabetes and other conditions of beta cell deficiency with routes of delivery to include, but are not limited to oral, intravenous, including delivery via the umbilical vein, portal venous systemic, hepatic artic artery, subcutaneous delivery with and without organ specific targeting and may include direct administration to the pancreas or liver.
  • routes of delivery to include, but are not limited to oral, intravenous, including delivery via the umbilical vein, portal venous systemic, hepatic artic artery, subcutaneous delivery with and without organ specific targeting and may include direct administration to the pancreas or liver.
  • This invention includes methods for pancreatic beta cell generation and include both in vivo and ex-vivo beta cell generation and methods for treating new onset and previously existing type 1 and 2 diabetes, Latent Autoimmune Diabetes of Adulthood (LADA), those at risk for type 1 diabetes, including but not limited to those with positive autoimmune antibodies markers including who are Glutamic Acid Decarboxylase-65 antibody, those with PreDiabetes and diseases of hyperglycemia, glucose intolerance and beta cell impairment or deficiency, insulin resistance, associated conditions including, obesity, obesity prior to the development of diabetes, obesity in children leading to PreDiabetes, both type 1 and type 2 diabetes in childhood and adolescence and include, but are not limited to conditions such as polycystic ovarian syndrome, nonalcoholic steatohepatitis, hyperlipidemia and hypertriglyceridemia and other conditions related to the deficiency or lack of effective amounts of insulin.
  • LADA Latent Autoimmune Diabetes of Adulthood
  • islet clusters containing beta cells are fixed in formalin and embedded in a paraffin block prior to histological sectioning and immunohistochemical staining for the hormones insulin, glucagon, somatostatin and pancreatic polypeptide, as well as the pancreatic duct marker CK19. Percentages of cells positive for each and CK19 will be counted. A minimum of 6 separate replicates will be determined.
  • a Dose Response Comparison between Placebo and Perle peptides derivatives, formulation, optimized versions and Perle peptide peptidomimetic includes stimulatory antibodies to the Perle Receptor in 2 nd trimester, human fetal pancreatic tissue transplanted into normoglycemic immunodeficient mice to detect and measure any islet neogenesis and beta cells general when Perle peptides derivatives, formulation, optimized versions and Perle peptide peptidomimetic includes stimulatory antibodies to the Perle Receptor are given ex- vivo and compared to placebo.
  • Human fetal pancreatic tissue will be obtained from the therapeutic termination of pregnancies between 13 and 20 weeks gestation. Islet- like cell clusters containing beta cells will be obtained by partial enzymatic digestion of the pancreatic tissue and these will be cultured for 3 days. Next, islet clusters inclusive of beta cells will be transplanted beneath the renal capsule of NOD/SCID mice, and the mice injected daily or a placebo. A minimum of 6 mice will be will be used in each group. Four weeks, after the islet-like cell clusters containing beta cells are transplanted, the mice will be euthanased and grafts removed for analysis. They will be fixed in formalin and embedded in a paraffin block prior to histological sectioning and
  • Percentages of cells positive for each hormone and CK19 will be counted with comparisons between Perle peptide peptidomimetic includes stimulatory antibodies to the Perle Receptor are given ex- vivo and compared to placebo.
  • Perle peptides derivatives, formulation, optimized versions and Perle peptide peptidomimetic includes stimulatory antibodies to the Perle Receptor vs. placebo will be evaluated in 2 nd trimester, human fetal pancreatic tissue transplanted into diabetic immunodeficient mice. The impact of a various dosages of Perle peptide
  • peptidomimetic includes stimulatory antibodies to the Perle Receptor will be given ex-vivo and compared to placebo to detect and measure islet neogenesis and beta cell generation over and above placebo.
  • the disclosed invention would be valuable in the treatment of Diabetes.

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Abstract

La présente invention concerne de nouvelles thérapies pour le traitement du diabète de type 1 et de type 2 nouveau et existant, du prédiabète, du diabète auto-immun latent chez l'adulte, et maladies de l'insulinodéficience, de la déficience en cellules bêta, de l'insulinorésistance et du métabolisme du glucose déficient. En particulier, la présente invention identifie des peptides communs à l'intérieur de Reg1a, Reg1b, Reg3a et Reg4 humains, en tant que peptides signaux pour la génération de cellules bêta agissant par l'intermédiaire du récepteur Reg humain sur la surface du tissu extra-îlot du pancréas humain. Cette invention identifie une région de liaison spécifique du récepteur Reg à partir de laquelle des peptidomimétiques et des anticorps de stimulation ont été développés pour la génération de nouvelles cellules bêta qui peuvent être administrées directement à des patients ayant lesdits états, comprenant le diabète de type 1, le diabète de type 2, le prédiabète et d'autres états de déficience en cellules bêta, et fournit une méthodologie spécifique pour la protection de nouvelles cellules bêta générées pour l'utilisation dans le diabète de type 1 et le diabète auto-immun latent chez l'adulte. Cette invention concerne également la génération ex vivo et l'administration de cellules bêta à l'aide des inventions décrites ici.
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WO2017063657A1 (fr) * 2015-10-14 2017-04-20 Phlogo Aps Antagoniste peptidique dérivé de l'il-1ra
WO2019099368A1 (fr) * 2017-11-16 2019-05-23 Levetan Claresa Compositions et méthodes pour traiter ou prévenir le diabète de type 1 en utilisant un modificateur de la réponse biologique en association avec une ou plusieurs thérapies de type régénération ou remplacement d'îlots de langerhans ou de cellules bêta

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AU2013323462A1 (en) 2015-04-23
JP2015533821A (ja) 2015-11-26

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