US20040266682A1 - Gastrin compositions and formulations, and methods of use and preparation - Google Patents

Gastrin compositions and formulations, and methods of use and preparation Download PDF

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US20040266682A1
US20040266682A1 US10/719,450 US71945003A US2004266682A1 US 20040266682 A1 US20040266682 A1 US 20040266682A1 US 71945003 A US71945003 A US 71945003A US 2004266682 A1 US2004266682 A1 US 2004266682A1
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gastrin
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amino acid
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Antonio Cruz
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Waratah Pharmaceuticals Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/2207—Gastrins; Cholecystokinins [CCK]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1808—Epidermal growth factor [EGF] urogastrone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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

Definitions

  • the invention in various embodiments provides gastrin compositions having longer active function in vivo than gastrin peptides, and methods of making and using the gastrin compositions for treatment of diabetes.
  • Therapeutic agents such as peptides and low molecular weight proteins used in the treatment of diseases suffer from significant limitations. These agents are often eliminated by the kidneys within a short period of time or are destroyed by proteases therefore limiting their bioavailability, resulting in short plasma half-life and lower drug concentrations than are required to be efficacious. A high clearance of a therapeutic agent is not optimal in cases where it is desired to maintain a high serum level over an extended period of time to obtain maximal efficacy. Increased doses or increased frequency of administration often result in a higher therapeutic efficacy but a higher risk of side effects as well, limiting the dose or frequency that can be administered.
  • Gastrin is a peptide hormone that has been shown in combination with other growth factors to be efficacious in the treatment of diabetes.
  • gastrin has a relatively short half life.
  • Gastrin-17 for instance has a circulating half-life of about 5-9 mins while gastrin-34 has a circulating half-life of about 35 mins.
  • compositions containing gastrin compounds that have a protracted or long-acting action.
  • a featured embodiment of the invention provides a pharmaceutical composition comprising a gastrin compound having an extended activity upon administration to a subject in comparison with native gastrin.
  • the gastrin component in this embodiment contains at least amino acids selected from the group of: positions 29-34 of SEQ ID NO:1; positions 29-34 of SEQ ID NO:2; positions 12-17 of SEQ ID NO: 3; and positions 12-17 of SEQ ID NO: 4, and the gastrin is further associated with a protein, a polymer, a lipid or a carbohydrate.
  • An alternative featured embodiment provides a gastrin compound comprising: Z-Y m -X n -AA 1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6 , wherein AA 1 is Tyr or Phe, AA 2 is Gly, Ala, or Ser, AA 3 is Trp, Val, or Ile, AA 4 is Met or Leu, AA 5 is Asp or Glu, and AA 6 is Phe or Tyr the AA 6 being amidated; wherein Z is a polymer which when the polymer is a protein, Z is the amino acid sequence of the protein; Y m is an optional spacer region comprising m amino acid residues of a small neutral amino acid, and X is selected from any consecutive portions of: residues 1-28 of SEQ ID NO: 1, residues 1-28 of SEQ ID NO: 2, residues 1-11 of SEQ ID NO: 3, and residues 1-11 of SEQ ID NO: 4, providing that the gastrin compound binds a gastrin/CCK
  • the AA 1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6 is, for example, Tyr-Gly-Trp-Met-Asp-Phe.
  • the AA 1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6 is Tyr-Gly-Trp-Leu-Asp-Phe.
  • Z can be a protein, for example, Z is human serum albumin.
  • Y m can be an amino acid sequence comprising m residues having glycine alternating with alanine, for example, [Gly-Ala] 5 or having random sequence of glycine and alanine.
  • the gastrin compound further can have a cysteine residue at the amino terminus of Y, when m is greater than 1, or at the amino terminus of X, when m is 0.
  • the gastrin compound can further comprise of a bifunctional crosslinking agent for linkage to Z.
  • m is 0 to about 20 residues. In certain embodiments, if m is 0, wherein X n -AA 1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6 further comprises a bifunctional cross-linking agent for linkage to Z.
  • the X in certain embodiments is selected from the group of sequences: position 1 to position 11 of SEQ ID NO: 3; position 1 to position 11 of SEQ ID NO: 4; position 2 to position 11 of SEQ ID NO: 3; and position 2 to position 11 of SEQ ID NO: 4.
  • the gastrin compound in which Z is a protein can be recombinantly produced.
  • Another embodiment provided herein is a nucleotide sequence encoding the gastrin compound in which the Z is a protein. Further provided is a cell carrying this nucleotide sequence.
  • the cell is a bacterial or a yeast cell.
  • the cell can be, for example, a cell of a species of an Escherichia, a Bacillus, or a Streptomyces.
  • the cell is a yeast cell, it can be a a cell of a species of a Saccharomyces, a Kluyveromyces, a Schizosaccharomyces or a Pichia.
  • the gastrin compound generally contains a minimal gastrin component which is at least amino acids at positions 29-34 of SEQ ID NO:2 or positions 12-17 of SEQ ID NO:4. These are located at the carboxy terminus of gastrins that occur in circulation, and in various embodiments additional amino acids for example from gastrin can be present.
  • the polymer component need not be limited to a protein, but may be a synthetic chemical polymer such as a polyethylene glycol (PEG) or a dextran.
  • PEG polyethylene glycol
  • dextran a polyethylene glycol
  • the polymer is a protein, in various embodiments it can be a serum protein, for example, a serum albumin, for example, human serum albumin.
  • Another embodiment of a gastrin compound provided herein has a structure C-Y m -X, wherein C is Cys or Lys, Y m is an optional spacer region comprising m amino acid residues of a small neutral amino acid, and X is at least six amino acid residues comprising sequences selected from at least positions 12-17 of gastrin-17 (SEQ ID NO: 3 and 4) and at least positions 29-34 of gastrin-34 (SEQ ID NO: 1 and 2).
  • the gastrin compound further is conjugated to a polymer, for example, is conjugated to a polyethylene glycol (PEG) or a dextran.
  • PEG polyethylene glycol
  • the gastrin compound further is alternatively conjugated to a protein.
  • the gastrin compound further includes a bifunctional cross-linking agent wherein a first reactive end of the cross-linking agent is covalently linked to C.
  • a second reactive end of the cross-linking agent is covalently linked to a polymer or protein.
  • the C-Y m -X can be produced recombinantly or it can be synthesized by peptide synthesis.
  • gastrin compounds provided herein are, in certain embodiments, provided in an effective dose.
  • the gastrin compounds provided herein can further include an agent for immune suppression.
  • the gastrin compounds provided herein can further include a hypoglycemic agent.
  • the gastrin compounds provided herein can further include a pharmaceutically acceptable carrier.
  • the gastrin compounds provided herein can further include a growth factor.
  • the growth factor is a glucagon-like peptide 1 receptor ligand.
  • the growth factor is an EGF receptor ligand.
  • inventions which include a method of treating a subject having diabetes, comprising administering a gastrin compound as provided herein.
  • frequency of administering the gastrin compound is less than frequency of administration of a native gastrin.
  • the method may further include measuring a physiological indicator of islet neogenesis, for example, measuring fasting blood glucose (FBG).
  • FBG fasting blood glucose
  • the method may include decreasing insulin dependency.
  • Yet another embodiment of the invention provides a method of making a gastrin compound, the method being associating an amino acid sequence of a gastrin with a carrier composition.
  • the gastrin prior to associating the gastrin with the carrier, the gastrin is modified to comprise a cysteine substitution or an additional cysteine residue.
  • the cysteine substitution is a replacement of pyroglutamate.
  • the gastrin amino acid sequence comprises at least positions selected from the group of: residues 29-34 of amino acid sequence SEQ ID NO: 1; residues 29-34 of amino acid sequence SEQ ID NO: 2; residues 12-17 of amino acid sequence SEQ ID NO: 3; and residues 12-17 of amino acid sequence SEQ ID NO: 4.
  • the cysteine is at the amino terminus of the gastrin.
  • the method includes modifying the gastrin to further include a bifunctional cross-linking agent.
  • Yet another embodiment of the invention provides a method of treating a diabetes patient comprising administering to the patient a modified gastrin capable of covalently reacting with a serum protein.
  • the modified gastrin includes a sequence of a native gastrin capable of binding to the gastrin/CCK receptor and an amino terminal cysteine or lysine. Accordingly, the sequence of the native gastrin is selected from the group of: residues 29-34 of amino acid sequence SEQ ID NO: 1; residues 29-34 of amino acid sequence SEQ ID NO: 2; residues 12-17 of amino acid sequence SEQ ID NO: 3; and residues 12-17 of amino acid sequence SEQ ID NO: 4.
  • Also provided herein is a method for maintaining for an extended period of time an increased gastrin serum level compared with the serum level of a peptide having an amino acid sequence of a gastrin, the method comprising administering a gastrin compound according to any of claim 1, 2 and 23.
  • kits comprising at least one effective dose of a gastrin compound as described above.
  • FIG. 1 shows the effect of unmodified gastrin on fasting blood glucose levels of NOD mice with recent onset diabetes after a 14 day treatment.
  • FIG. 2 shows the effect of unmodified gastrin on pancreatic insulin levels of NOD mice with recent onset diabetes after a 14 day treatment
  • compositions having gastrin-like activity herein referred to as gastrin compounds
  • gastrin compounds means agents that bind to, interacts with or stimulates the gastrin/CCK receptor.
  • Gastrin compounds include gastrin derivatives and conjugates as well as peptide homologs, that are capable of interacting with the gastrin/CCK receptor.
  • derivatives and conjugates as used herein are equivalent, and are used to indicate compositions that are chemically related, and can be prepared by synthetic, biological, recombinant or chemical means.
  • a “modified” gastrin can be prepared and used to treat a patient having a diabetes.
  • diabetes as used herein means any physiologic indication of a shortage of insulin, a production of antibodies against insulin, or an excess of blood sugar or any manifested symptoms of diabetes in any mammal including experimental animal models, and including human forms such as type I and type II diabetes, early stage diabetes, and a pre-diabetic condition characterized by mildly decreased insulin or mildly elevated blood glucose levels.
  • the term “mammal” has the usual meaning of any member of Mammalia, and includes humans.
  • the modified gastrin can be a gastrin derivative or analog comprising a minimal sequence of 6 amino acids (from the C-terminal end), and further having addition of a reactive group such as a cysteine residue capable of undergoing an addition reaction (Refer to SEQ ID 1-4)
  • the gastrin may extend up to 34 amino acids (“Big” Gastrin or Gastrin-34), wherein at least one reactive amino acid such as a cysteine residue or a lysine residue is added or substituted at the N-terminal end.
  • the addition of the reactive amino acid such as a cysteine can be at a terminal region, and in related embodiments, a spacer region can optionally precede the added reactive amino acid.
  • the spacer can be synthesized biologically as part of, or can be chemically attached to the gastrin amino acid sequence, forming a structure which has a gastrin sequence-spacer-cysteine.
  • the spacer region can be a sequence of several amino acids such as alanine or glycine.
  • the sequence of amino acids can be alternating amino acids (e.g. glycine/alanine) or can be non-alternating, i.e., can be a random sequence or a particular sequence.
  • the sequence can consist of at least one amino acid.
  • a bifunctional cross-linking agent which is a reactive component is added to the modified gastrin, particularly to the gastrin having an added reactive group at the amino terminus (e.g., a cysteine), or to a modified gastrin having a spacer, via a homobifunctional or heterobifunctional portion of the crosslinker to generate an a modified gastrin having a reactive group such as a thiol or an amino group at one end.
  • gastrin-spacer-cys-cross-linker-carrier e.g., to form, as listed from the carboxy terminus, a gastrin-spacer-cys-cross-linker-carrier; a gastrin-cys-cross-linker group-carrier; gastrin-spacer-cys/-cross-linker with reactive group exposed, and a gastrin-cys-cross-linker with reactive group exposed.
  • the modified gastrin is then either injected in this state into a patient, or is further conjugated in vitro to one or more plasma components such as whole or fractionated serum obtained from the patient; one ore more purified serum protein(s) such as albumin, transferrin or an immunoglobulin; lipids/lipophilic moieties/hydrophobic moieties; or to polymeric carriers such as dextran or PEG prior to injection.
  • the term polymer as used herein and in the claims includes polymers of amino acids, sugars, nucleosides, synthetic polymers (such as PEG) and mixtures thereof.
  • the polymer can be activated, for instance with a bifunctional crosslinker or via other chemical means prior to conjugation. Administering the activated gastrin compound or the gastrin conjugate results in increased serum half life and/or maintained high plasma concentration of gastrin for an extended period of time, compared with administering native gastrin.
  • the invention in general embodiments provides gastrin compositions which have a moiety that is a gastrin compound and can be associated with a larger molecule such as a polymer, either non-covalently, or as a covalent conjugate, or as a fusion protein to another peptidic compound having an amino acid sequence.
  • the gastrin compounds provided herein have a longer half-life in circulation in a subject animal or patient, and/or maintain higher concentrations in vivo of the gastrin compounds for an extended period of time compared to the native forms of gastrin.
  • the invention in other embodiments provides compositions and methods of making and of using the gastrin compounds, provided to the patient either alone or in combination with at least one growth factor, with a hypoglycemic agent, or with an immunosuppressant, for the treatment of diabetes.
  • growth factors include but are not limited to a EGF receptor ligand such as EGF, a GLP-1 receptor ligand such as GLP-1, prolactin receptor ligand such as prolactin and growth hormone receptor ligand such as growth hormone.
  • immunosuppressants include but are not limited to cyclosporine, FK506, rapamycin, and daclizumab.
  • Hypoglycemic agents include sulfonylureas, meglitinides, biguanides, thiazolidinediones, and alpha-glucosidase inhibitors.
  • a gastrin compound can be bound to a comparatively larger structure or a plurality of structures in the blood and still retain the ability to bind target proteins, i.e., a gastrin/CCK receptor.
  • target proteins i.e., a gastrin/CCK receptor.
  • gastrin which would be otherwise rapidly degraded in the body, is attached to a carrier protein; using this composition, a longer-term of drug efficacy can be achieved.
  • a gastrin compound can be conjugated to a polymeric carrier such as a polyethylene glycol (PEG) or a dextran to achieve similar objectives
  • chemical modification of gastrin is used to provide compounds that react covalently or non-covalently to carrier proteins or polymeric carriers, either in vitro (ex vivo) or in vivo.
  • the non-covalent interaction is electrostatic or hydrophobic.
  • conjugation of modified gastrin to the carrier is carried out prior to injection.
  • the gastrin is modified in such a manner that when injected, will have an enhanced affinity to the carrier in the bloodstream.
  • long acting gastrin compounds are obtained via chemical modification with no requirement for a carrier protein either in vivo or ex vivo.
  • the carrier protein is a plasma protein.
  • the plasma protein is an albumin or an immunoglobulin or components of an immunoglobulin.
  • the immunoglobulin or components of the immunoglobulin can be modified or portions deleted prior to conjugation.
  • the polymeric carrier is polyethylene glycol or dextran.
  • activated PEG can be attached to gastrin compound via an amino group in the gastrin compound (Vernonese, F M. Biomaterials 22(2001)—405-417).
  • the gastrin compound which is a sequence of amino acids is genetically fused with the carrier protein, which is also a sequence of amino acids, prior to injection, using standard recombinant genetic techniques.
  • Gastrin can be fused recombinantly to a carrier protein with or without a linker/spacer, for example, comprising a sequence of small neutral uncharged amino acids.
  • a nucleic acid encoding gastrin can be recombinantly fused or synthesized directly as a fusion to portions or the whole of the carrier protein, and the nucleic acid construct or fusion protein can encode or incorporate a number of additional amino acids to act as a spacer between the two proteins.
  • Recombinant fusion proteins can be expressed in yeast ( Saccharomyces, Pichia ) or in standard bacterial systems, or mammalian or insect cell systems can be used. Following standard procedures for expression and/or purification, the fusion protein can be used therapeutically. Modifications to the sequence of gastrin compound polypeptide can be introduced during construction of the fusion protein if necessary.
  • the gastrin compound is modified to introduce a reactive group such as those present on an amino acid such as a lysine or cysteine so that the reactive group upon further contacting another compound such as a carrier protein or carrier non-proteinaceous polymer, can form covalent interactions with the carrier proteins or polymers.
  • a reactive thiol group can be added to the gastrin molecule through an amino group on lysine, for example, using succinimidyl 3-2-pyridyldithio propionate (SPDP) followed by reduction with DTT to release the active thiol group ((“Protein thiolation and reversible protein-protein conjugation.
  • SPDP succinimidyl 3-2-pyridyldithio propionate
  • N-Succinimidyl 3-(2-pyridyldithio)propionate a new heterobifunctional reagent.” Carlsson J, Drevin H, Axen R. Biochem J 173, 723-737 (1978)).
  • the bifunctional group can also be added after the cysteine or lysine has been added, so that one reactive end of the crosslinking agent will react with cysteine/lysine while the other reactive end at the other end is left exposed or is conjugated to a carrier.
  • Thiols can be also incorporated at carboxylic acid groups by EDAC-mediated reaction with cystamine, followed by reduction of the disulfide with DTT. (“Introduction of sulfhydryl groups into proteins at carboxyl sites.” Lin C M, Mihal K A, Krueger R J. Biochim Biophys Acta 1038, 382-385 (1990).
  • reaction of an amino group on the lysine residue in gastrin with succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate (“Conjugation of glucose oxidase from Aspergillus niger and rabbit antibodies using N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)-maleimide.”
  • Yoshitake S Yamada Y, Ishikawa E, Masseyeff R. Eur J Biochem 101, 395-399 (1979) introduces a thiol reactive group at amino sites of gastrin that can subsequently react with cysteine residues of the carrier protein or free thiol group on the activated polymer.
  • a gastrin compound-carrier complex can include additional modular components including a spacer arm or element or other component that can facilitate preparation or isolation of the gastrin compound-carrier complex or enhance or maintain the functional activity of the gastrin compound.
  • the spacer arm can be one or more amino acids, peptide, a peptidomimetic, or a small organic molecule, and can comprise homobifunctional or heterobifunctional crosslinking agents or chitin oligomers or polyethyelene glycol or related polymers.
  • the carrier and gastrin compound can be covalently crosslinked with or without a spacer arm.
  • non-spacer arms zero-length crosslinkers
  • Homobifunctional crosslinkers that generate a spacer arm can be for instance disuccinimidyl suberate and heterobifunctional crosslinkers that generate a spacer arm can be for instance 2-iminothiolane, succinimidyl 6-[3-(2-pyridyldithio)propionamido] hexanoate (LC-SPDP) and 4-(N-maleimido methyl)cyclohexane-1-carboxylate (SMCC).
  • LC-SPDP succinimidyl 6-[3-(2-pyridyldithio)propionamido] hexanoate
  • SMCC 4-(N-maleimido methyl)cyclohexane-1-carboxylate
  • the gastrin compound is associated with a larger carrier moiety such as a polymer, for example a protein.
  • a carrier moiety such as a polymer, for example a protein.
  • the protein may be considered to be a carrier protein.
  • Classes of carrier proteins can possess the properties of being non-antigenic, i.e., are native human proteins, and are being capable of sustained maintenance in circulation.
  • An ideal carrier protein is one normally found in the human circulatory system.
  • the term “gastrin/CCK receptor ligand” encompasses any compound that binds to, interacts with or stimulates the gastrin/CCK receptor. Examples of such gastrin/CCK receptor ligands are given in U.S. Pat. No. 6,288,301 issued Sep.
  • gastrin such as gastrin 34 (big gastrin), gastrin 17 (little gastrin or small gastrin), gastrin 14, gastrin 13, gastrin-10, and gastrin 8, pentagastrin, tetragastrin; various forms of cholecystokinin such as CCK 58, CCK 33, CCK 22, CCK 12 and CCK 8; and other gastrin/CCK receptor ligands.
  • gastrin/CCK receptor ligands share a carboxy terminal amino acid sequence Trp-Met-Asp-Phe-amide.
  • the aforementioned methionine (Met) can be replaced by a leucine.
  • active analogs, fragments and other modifications of the above including both peptide and non-peptide agonists or partial agonists of the gastrin/CCK receptor such as A71378 (Lin et al., Am. J. Physiol. 258 (4 Pt 1): G648, 1990).
  • gastrin 17 Small forms of gastrin such as gastrin 17 are economically prepared by peptide synthesis, and synthetic peptides are commercially available.
  • Synthetic human gastrin 17 such as human gastrin 17 having methionine or leucine at position 15 are also available from Bachem AG, Bubendorf, Switzerland, and from Researchplus.
  • Gastrin peptides as found in nature are carboxyl-terminally amidated peptides, and amidation of the carboxyl terminus amino acid is within the scope of gastrin compounds herein.
  • Gastrin/CCK receptor ligands include also active analogs, fragments and other modifications of the above ligands, which for example share amino acid sequence with an endogenous mammalian gastrin, for example, share 60% sequence identity, or 70% identity, or 80% identity.
  • Such ligands also include compounds that increase the secretion of endogenous gastrins, cholecystokinins or similarly active peptides from sites of tissue storage. Examples of these are the gastric releasing peptide, omeprazole which inhibits gastric acid secretion, and soya bean trypsin inhibitor which increases CCK stimulation
  • Big gastrin-34 is essentially an extension form of small gastrin-17 having an additional amino acid sequence at the N-terminal end. Big gastrin is cleaved in vivo to release gastrin-17.
  • the symbol “Glp” at the N-terminal end is a pyroglutamate residue, which is a naturally cyclized form of glutamate.
  • gastrins having an N-terminal pyroglutamate residues are modified to contain N-terminal cysteine or lysine residues by either replacing the pyroglutamate with a glutamate or glutamine, or deleting the pyroglutamate.
  • each of a gastrin 34 and gastrin-17 can be used in a modified form that has a methionine or a leucine at position 32 as shown herein in SEQ ID No: 1-2, respectively, or at position 15 as shown in SEQ ID No: 3-4, respectively.
  • N-terminal Glp-Leu-Gly-Pro-Gln-Gly- (SEQ ID NO: 1) Pro-Pro-His-Leu-Val-Ala-Asp-Pro-Ser- Lys-Lys-Gln-Gly-Pro-Trp-Leu-Glu-Glu- Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp- Phe- NH 2 .
  • N-terminal Glp-Leu-Gly-Pro-Gln-Gly- (SEQ ID NO: 2) Pro-Pro-His-Leu-Val-Ala-Asp-Pro-Ser- Lys-Lys-Gln-Gly-Pro-Trp-Leu-Glu-Glu- Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Leu-Asp- Phe- NH 2 .
  • N-terminal Glp-Gly-Pro-Trp-Leu-Glu- (SEQ ID NO: 3) Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met- Asp-Phe- NH 2 .
  • the gastrin compound which is a fusion for example, of a gastrin amino acid sequence with an optional spacer of amino acids at the amino terminus of the gastrin sequence, and having a protein carrier, can be provided transgenically to a subject.
  • a human preprogastrin peptide precursor gene is fused to a gene encoding a carrier protein, with or without a spacer, by techniques similar to those as shown in U.S. Pat. No. 5,885,956.
  • the method for treating diabetes mellitus in an individual in need thereof includes administering to the individual a composition that provides a gastrin compound and a FACGINT such as EGF, GLP-1, prolactin and growth hormone. Included are derivatives, analogs, and conjugates of these FACGINT.
  • a FACGINT means a factor that complements gastrin for islet neogenesis therapy.
  • a FACGINT as used herein can also mean “one or more FACGINTs” or “at least one FACGINT”.
  • FCGINT includes a large variety of growth factors and growth hormones, agents that modify one or more of the factors hormones, and ligands and effectors for one or more receptors involved in binding of these growth hormones and growth factors as these terms are generally understood, exemplified but not limited to: EGF receptor ligand, a PTH-related protein (PTHrP) receptor ligand such as PTHrP (PTHrP; Garcia-Ocana, A. et al., 2001, J. clin. Endocrin. Metab. 86: 984-988); a hepatocyte growth factor (HGF) receptor ligand such as HGF (HGF; Nielsen, J.
  • PTHrP PTH-related protein
  • HGF hepatocyte growth factor
  • FGF fibroblast growth factor
  • KGF keratinocyte growth factor
  • NGF nerve growth factor
  • GIP gastric inhibitory polypeptide
  • TGF ⁇ transforming growth factor beta
  • a laminin receptor ligand such as laminin-1
  • an islet neogenesis associated protein (INGAP) receptor ligand such as INGAP
  • BMP bone morphogenetic factor
  • VIP vasoactive intestinal peptide
  • GLP-1 and exendin-4 glucagon-like peptide 2 (GLP-2) receptor ligand
  • dipeptidyl peptidase IV inhibitors which indirectly affect the levels of GLP-1 (Hughes, T.
  • a REG receptor ligand such as REG protein
  • a Growth hormone (GH) receptor ligand such as GH, a Prolactin (PRL) receptor ligand such as PRL and placental lactogen (PL)
  • GH Growth hormone
  • PRL Prolactin
  • PL placental lactogen
  • IGF1 and IGF-2 an Insulin-like growth factor (Type 1 and 2) receptor ligands such as IGF1 and IGF-2
  • EPO Erythropoietin
  • a betacellulin also considered to be a member of the EGF family
  • an Activin-A receptor ligand such as Activin-A
  • VEGF vascular endothelial growth factor
  • BMP bone morphogenesis factor
  • any of the growth factors, enzymes, enzyme inhibitors, peptide, protein and hormone compounds herein that are indicated to be an exemplary FACGINT all known analogues and derivatives, whether naturally occurring or made by mutagenesis or designed and synthesized shall be considered equivalent to that FACGINT. Also considered among equivalents are conjugates, i.e., compositions derived by addition of one or more of a chemical group, and mixtures thereof. Encoding genes may be altered by, for example, oligonucleotide directed mutagenesis to produce FACGINT analogues thereof, such as the human recombinant analogues. Further, an identity or location of one or more than one amino acid residue may be changed by targeted mutagenesis.
  • the primary amino acid sequence of the protein may be augmented by conjugates, as by glycosylation, acylation, or by addition of any other supplementary molecules, such as one or more of a lipid, a phosphate, a sulphate and/or an acetyl group. Further, individual amino acid residues in the chain may be modified by oxidation, reduction, or other derivatization.
  • the FACGINT may be cleaved to obtain any fragments which retain activity.
  • a prodrug or a metabolite of a FACGINT is equivalent to that FACGINT.
  • the whole polypeptide or protein or any fragment can be fused with any other peptide or protein such as immunoglobulins and other cytokines.
  • Conjugates may include, for example, a composition comprising the FACGINT coupled to a non-naturally occurring polymer comprising a polyethylene glycol moiety.
  • the term also encompasses derivatives obtained by chemically modifying one or more amino acid residues of the parent peptide, for instance by alkylation, acylation, ester formation or amide formation.
  • agents that induce synthesis of the FACGINT or mimic the action of the FACGINT are contemplated as equivalent compounds.
  • the singular form, “FACGINT” may mean any one or more compounds from the exemplary FACGINTs shown herein.
  • derivatives” and “conjugates” as used herein are equivalent, and are used to indicate compositions that are chemically related, and can be prepared by synthetic, biological, or recombinant or chemical means.
  • receptor ligand as used herein in connection with a receptor for a particular ligand shall mean any compositions that binds to, interacts with, or stimulates that receptor.
  • a receptor ligand includes within the scope of the definition a receptor agonist, for the receptor for any particular FACGINT, whether or not the agonist is structurally related to the FACGINT.
  • prolactin as used herein means any polypeptide which shares substantial sequence similarity with an endogenous mammalian prolactin as this term is known in the art of protein factors, for example, human prolactin, and which possesses the activity of a prolactin.
  • Endogenous human prolactin is a 199 amino acid polypeptide produced by the pituitary gland.
  • the term encompasses prolactin analogs which are deletions, insertions, or substitution mutants of endogenous prolactin, and retain the activity, and includes prolactins from other species and naturally occurring variants.
  • the prolactin function includes a composition having agonist activity for the prolactin receptor, as disclosed in U.S. Pat. No.
  • PRL, GH and PL are members of a family of polypeptide hormones that share a structural, immunological and biological functions (reviewed in, “Pancreatic Growth and Regeneration”, Ed. N. Sarvetnick, Ch. 1. Brejie, T. et al., 1997), and therefore referred to herein as the PRL/GH/PL family.
  • PRL and GH are secreted by the anterior pituitary of vertebrate animals.
  • PRL is involved in a broad range of biological functions that include osmoregulation, reproduction, lactation, and immunomodulation.
  • GH is associated with physiological processes related to growth and morphogenesis.
  • the related receptor ligands are referred to as “PRL/GH/PL” receptor ligands.
  • the FACGINTs can be classified into various groups based on structural similarity of the peptides and proteins, functional similarity with respect to complementation of gastrin, functional similarity with respect to binding of one or more receptors, and these groups are each within the scope of various embodiments of the invention.
  • Glucagon-like peptide-1 is synthesized in intestinal endocrine cells in molecular forms GLP-1 (having residues conventionally designated as positions 7-36) which is an amide, and similarly as GLP-1(7-37).
  • GLP-1 having residues conventionally designated as positions 7-36
  • GLP-1(7-37) which is an amide
  • Initial studies of GLP-1 biological activity in utilized the full length N-terminal extended forms of GLP-1 (1-37 and 1-36 which latter is an amide). The larger GLP-1 molecules were generally lacking biological activity. It was later found that removal of the first six amino acids resulted in a shorter version of the GLP-1 molecule having substantially enhanced biological activity.
  • GLP-1(7-36)amide form The majority of circulating biologically active GLP-1 is found in the GLP-1(7-36)amide form, with lesser amounts of the bioactive GLP-1(7-37) form also detectable. See Orskov, C. et al., Diabetes 1994, 43: 335-339. Both peptides show about the same amount of biological function. GLP-1 is secreted from gut endocrine cells in response to nutrient ingestion and plays multiple roles in metabolic homeostasis following nutrient absorption. Regulation of GLP-1 occurs by N-terminal degradation of the peptide by Dipeptidyl Peptidase (DPP-IV)-mediated cleavage at the position 2 alanine residue. For an overview, see DPP-IV.
  • DPP-IV Dipeptidyl Peptidase
  • GLP-1 The biological activities of GLP-1 include stimulation of glucose-dependent insulin secretion and insulin biosynthesis, inhibition of glucagon secretion and gastric emptying, and inhibition of food intake. GLP-1 appears to have a number of additional effects in the GI tract and central nervous system, as reviewed in Drucker, D., Endocrin 142: 521-527, 2001.
  • Exemplary GLP-1 compositions include: BIM 51077 (GLP-1 analog resistant to DPP-IV digestion, available from Beaufour Ipsen); AC2592 (GLP-1, from Amylin, San Diego Calif.); ThGLP-1 (GLP-1, modified amino acids and fatty acid attachment, from Theratechnologies, Saint-Laurent, Quebec, Canada); DAC:GLP-1 (Conjuchem, Montreal, Quebec, Canada); CJC-1131 or DACTM:GLP-1 (GLP-1 analog engineered for covalent coupling to albumin, Conjuchem), LY315902 and sustained release LY315902 (DDP-IV resistant GLP-1 analog from Eli Lilly, Indianapolis, Ind.); low molecular weight GLP-1 mimetic, Albugon (albumin: GLP-1 fusion peptide from Human Genome Sciences, Rockville, Md.); Liraglutide or NN2211 (long acting GLP-1 derivative that is obtained by acylation of the GLP-1 molecule, which upon entering the bloodstream, is extensively bound to
  • Exendin-4 is a novel peptide from Heloderma suspectum (Gila monster) venom, having 53% homology with GLP-1(7-36)amide. It functions as a long-acting potent agonist of the glucagon-like peptide 1 (GLP-1) receptor, as it is resistant to degradation by DDP-IV. Exendin-4 has properties similar to GLP-1, and regulates gastric emptying, insulin secretion, food intake, and slucagon secretion.
  • exendin-4 examples include exenatide (synthetic form also known as AC2993, Amylin); exenatide LAR (long acting form); ZP10 (modified exendin-4 having addition of six lysine residues, Aventis/Zealand Pharma); and AP10 (long acting formulation, Alkermes, Cambridge Mass.).
  • exenatide synthetic form also known as AC2993, Amylin
  • exenatide LAR long acting form
  • ZP10 modified exendin-4 having addition of six lysine residues, Aventis/Zealand Pharma
  • AP10 long acting formulation, Alkermes, Cambridge Mass.
  • Dipeptidyl peptidase IV (DPP-IV) inhibitors refer to compounds that inhibit activity of DPP-IV, a membrane-associated peptidase of 766 amino acids that includes in its substrates GLP-1, GLP-2 and GIP. DPP-IV-mediated inactivation of GLP-1 is a determinant of GLP-1 bioactivity in vivo.
  • DPP-IV inhibitors include isoleucine thiazolidide, valine-purrolidide, NVP-DPP738 (Novartis, Cambridge, Mass.), LAF237 (Novartis), P32/98 (Probiodrug AG, Halle, Germany), and P93/01 (Probiodrug).
  • EGF receptor ligand encompasses compounds that stimulate the EGF receptor such that when gastrin/CCK receptors in the same or adjacent tissue or in the same individual are also stimulated, neogenesis of insulin-producing pancreatic islet cells is induced.
  • EGF receptor ligands include full length EGF, which is EGF1-53, and further include EGF1-48, EGF1-49, EGF1-52, and fragments and active analogs thereof.
  • EGF receptor ligands are TGF ⁇ forms that include 1-48, 1-47, 1-51, and amphiregulin and pox virus growth factor as well as any EGF receptor ligands that demonstrate the same synergistic activity with gastrin/CCK receptor ligands. These include active analogs, fragments and modifications of the above. See also, Carpenter and Wahl, Chapter 4, in Peptide Growth Factors (Eds. Sporn and Roberts), Springer Verlag, 1990.
  • the group of compounds that comprises the EGF receptor ligands further includes “modified EGF”, which includes variants of normal or wild type EGF. Modifications have been shown to affect one or more biological activity such as the rate of clearance of EGF.
  • the term includes peptides having an amino acid sequence substantially similar to that of human EGF, for example, with one or a few amino acid substitutions at various residue positions.
  • EGF forms have been genetically engineered to have alterations in structure and activities, for example, EGF having a methionine at position 21 replaced by a leucine residue has been described (U.S. Pat. No. 4,760,023).
  • Recombinant human EGF (hEGF) having 51 residues, i.e., lacking the two C-terminal residues at positions 52 and 53 of hEGF, and having a neutral amino acid substitution at position 51 retain EGF activity and are more resistant to protease degradation during a microbial production process, and following administration to a subject.
  • a series of nucleic acid molecules have been described that encode a family of proteins that have significant similarity to EGF and TGF ⁇ (WO 00/29438).
  • EGF muteins having histidine at residue 16 replaced with a neutral or acidic amino acid have been described (WO 93/03757), such forms retaining activity at low values of pH.
  • Chemical analogues and fragments of EGF and TGF ⁇ retain ability to bind various members of the EGF receptor family (U.S. Pat. No. 4,686,283).
  • Various modifications of EGF or TGF ⁇ confer advantageous properties affecting one or more of recombinant protein production, in vitro and in vivo stability, and in vivo activity.
  • a exemplary recombinant modified EGF receptor ligand used in the Examples herein is a C-terminus deleted form of human EGF of 51 amino acids in length, having asparagine at position 51 (referred to herein as EGF51 N), which retains substantially full I.N.T.TM activity, and has in vivo and/or in vitro stability that is that is at least about as great or greater than normal or wild type hEGF (S. Magil et al., published May 15, 2003 as PCT/US02/33907, and incorporated by reference herein in its entirety).
  • growth hormone encompasses any polypeptide that shares substantial amino acid sequence identity with an endogenous mammalian growth hormone and possesses a biological activity of a mammalian growth hormone.
  • Human growth hormone is a polypeptide containing 191 amino acids in a single chain, and a molecular weight of about 22 kDal (Goeddel et al., 1979, Nature 281: 544-548; Gray et al., 1985, Gene 39: 247-254).
  • the term encompasses analogs having deletions, insertions or substitutions and growth hormones from other species and naturally occurring variants. See Cunningham et al., 1989, Science 243: 1330-1336, and 1989, Science 244: 1081-1085; and WO 90/05185, and U.S. Pat. No. 5,506,107.
  • diabetes means reducing or eliminating one or more symptoms of diabetes.
  • diabetes means any physiologic indication of a shortage of insulin, a production of antibodies against insulin, or an excess of blood sugar or any manifested symptoms of diabetes in any mammal including experimental animal models, and including human forms such as type I and type II diabetes, early stage diabetes, and a pre-diabetic condition characterized by mildly decreased insulin or mildly elevated blood glucose levels.
  • a “pre-diabetic condition” describes a mammal suspected of having a diabetic or related condition, for example, not formally diagnosed with diabetes, but demonstrating a symptom in terms of insulin or glucose level, and susceptibility to diabetes or a related condition due to family history, genetic predisposition, or obesity in the case of type II diabetes, or has previously had diabetes or a related condition and is subject to risk of recurrence.
  • immunosuppressant or “agent for immune suppression” means any agent that suppresses immune response.
  • exemplary immunosuppressant agents are shown in Table 1, and any derivatives of those agents or functional equivalents are considered appropriate for embodiments of the invention as described herein and in the claims.
  • Immunosuppressive agents in Table 1 or other equivalent agents are administered as supplied by the manufacturers, normalizing to body weight of the subject as is known by one of skill in the pharmacological arts.
  • Tacrolimus is generally administered by injection or orally
  • Sirolimus is generally administered orally
  • TABLE 1 Exemplary agents for immune suppression, and commercial sources Names Company Nature 2-amino-1,3-propanediol Novartis Used for preventing or derivatives treating chronic rejection in a patient receiving an organ or tissue allo-or xeno- transplant 2-amino-2[2-(4- Yoshitomi Immunosuppression, from octylphenyl)ethyl]propane- Pharmaceutical accelerated lymphocyte 1,3-diol hydrochloride Industries, Ltd homing 40-O-(2-hydroxyethyl)- Novartis Sirolimus (rapamycin) rapamycin, SDZ-RAD, Pharmaceuticals derivative, used for acute Everolimus (Certican ®) kidney rejection; reduces rejection and graft vasculopathy following heart transplantation by inhibiting cell proliferation 6-(3-dimethyl- Matsumori Akia Immunosuppressing action aminopropionyl) for
  • Cyclophosphamide (CTX, Generic Immunosuppressant t for Neosar ®, Cytoxan ®, treatment of arthritis and Procytox ®) other auto-immune disorders and cancers Cyclosporine (cyclosporin Novartis 11 amino acid cyclic A, cyclosporin) peptide; blocks helper T- (Sandimmune ®, Neoral ®, cell, immunosuppressant SangCya ®) used in organ transplant therapy and other immune diseases Demethimmunomycin” (L- Merck & Co Treatment of autoimmune 683,742: also described as diseases, infectious diseases 31-desmethoxy-31- and/or prevention of organ hydroxy-L-683,590) transplant rejections Dexamethasone Generic An adrenocorticoid, (Decadron, Dexone, effective Dexasone) immunosuppressant in various disorders Docosahexaenoic acid Not Immunosuppressant by that (
  • IL-2)-dependent T-cell proliferation (kidney transplant) Tacrolimus (Prograf; FK- Fujisawa Interferes with IL-2 TCR 506) communication Antithymocyte SangStat Medical Anti-human thymocyte immunoglobulin Corporation, immunoglobulin; used in (ATGAM, Thymoglobulin ®) Pharmacia and reversal of acute kidney Upjohn transplant rejection and will likely be used off-label for transplant induction therapy efalizumab (Xanelim ®) XOMA T-cell modulator that target T-cells through interactions with adhesion molecules on endothelial cell surface, target migration of T-cells into the skin and target activation of T-cells; Used to treat Psoriasis Daclizumab (Zenapax ®), Protein Design Monoclonal AB inhibits HAT (Humanized Anti- Laboratories/Roche binding of IL-2 to IL-2 Tac), SMART anti-Tac, receptor by binding
  • Hypoglycemic agents or drugs can be insulin response enhancers, and are typically used as for glycemic control in diabetic patients.
  • These agents include but are not limited to sulfonylureas (e.g. acetohexamide, chlorpropamide, tolazamide, tolbutamide, glyburide, glipizide, glimepiride), meglitinides (e.g. repaglinide, nateglinide), biguanides (e.g. metformin), thiazolidinediones (e.g. pioglitazone, rosiglitazone), alpha-glucosidase inhibitors (e.g.
  • Miglitol glucagons antagonists
  • potassium channel openers insulin sensitizers
  • hepatic enzyme inhibitors hepatic enzyme inhibitors
  • glucose uptake modulators compounds modifying lipid metabolism and compounds lowering food intake. Combinations of these agents can be used with the gastrin compounds provided herein.
  • mammal shall include without limitation any members of the Mammalia, such as a human, an ape, a rodent such as a mouse or rat, a dog, a cat, an agriculturally important animal or a protein pig, a goat, a sheep, a horse, or an ape such as a gorilla or a chimpanzee.
  • An individual mammal may be non-diabetic, pre-diabetic, or diabetic, as specified herein.
  • Modes of systemic administration include, but are not limited to, transdermal, intrathecal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, and oral routes.
  • the compounds may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, vaginal, nasal, and intestinal mucosa, etc.), and may be administered together with other biologically active agents.
  • An exemplary route of administration is systemic, for example, by subcutaneous injection.
  • the gastrin compound if administered with a FACGINT or immunosuppressant or a hypoglycemic agent can be administered in a single combined dose, or the components can be administered separately in any order.
  • Gastrin peptides can be produced by any suitable means, such as expression in a recombinant host cell or by chemical synthesis.
  • gastrin peptides for instance can be synthesized by using solid phase Fmoc peptide synthesis on a polydimethylacrylamide gel resin. The polypeptide is then purified by standard methods.
  • Conjugating partners/carriers include plasma components such as serum obtained from the patient, purified serum protein(s) such as albumin, transferrin or an immunoglobulin, red blood cell proteins such as glycophorin A and AE-1, sugar binding proteins such as lectin, inactivated enzymes, phosphate and sulfate binding proteins, cholic acid, lipids binding proteins, lipids/lipophilic moieties/hydrophobic moieties; polymeric carriers such as dextran or polyethylene glycol.
  • purified serum protein(s) such as albumin, transferrin or an immunoglobulin
  • red blood cell proteins such as glycophorin A and AE-1
  • sugar binding proteins such as lectin, inactivated enzymes, phosphate and sulfate binding proteins, cholic acid, lipids binding proteins, lipids/lipophilic moieties/hydrophobic moieties
  • polymeric carriers such as dextran or polyethylene glycol.
  • the carrier may first need to be activated via the introduction of a reactive group.
  • the carrier can already contain at least one reactive group, for instance cysteine 34 on albumin. After activation of the carrier (if necessary), the carrier is conjugated to gastrin compound.
  • carriers can be covalently attached to proteins via reactive groups in the protein chain, such as thiol groups, alpha and epsilon amino groups, carboxyl groups or aromatic rings, all of which may already be present, or can be added by preliminary chemical modification of the protein which can be either incorporated during chemical synthesis or by chemically modifying existing gastrin peptides or by modifying a protein's amino acid sequence, using known molecular biology methods.
  • reactive groups in the protein chain such as thiol groups, alpha and epsilon amino groups, carboxyl groups or aromatic rings, all of which may already be present
  • carrier can be attached to the protein's epsilon amino groups in lysine residues or the thiol groups on cysteine residues located at the N-terminal end.
  • the carrier can be attached to the protein via a heterobifunctional or homobifunctional crosslinking agent.
  • Plasma protein binding can be an effective means of improving the pharmacokinetic properties of otherwise short-lived molecules such as gastrin.
  • One aspect of plasma proteins is a native molecular mass larger than the kidney filtration cutoff ( ⁇ 45kDa), and thus an extended residence time in plasma.
  • Plasma proteins include but are not limited to Albumin, Alpha-1 Acid Glycoprotein, Alpha-1-Antichymotrypsin, Alpha-1-Antitrypsin, Alpha-2-Antiplasmin, Alpha-2-HS-Glycoprotein, Alpha-2-Macroglobulin, Angiotensinogen, Antithrombin III, Apolipoprotein AI (HDL), Apolipoprotein AII (HDL), Apolipoprotein B (LDL), Apolipoprotein CI (VLDL), Apolipoprotein CII (VLDL), Apolipoprotein CIII (VLDL), Apolipoprotein E (VLDL), Apotransferrin,
  • Human serum albumin has a molecular mass of ⁇ 67 kDa, a half-life of 19 days in circulation, and is the most abundant protein in plasma human.
  • Albumin consists of 585 amino acids forming a single polypeptide chain.
  • Albumin interacts with a large number of compounds including physiological ligands such as long chain fatty acids, certain therapeutic drugs such as Warfarin and Valproate and inorganic ligands such as calcium.
  • the antibody/immunoglobulin can include a complete immunoglobulin or fragment thereof, where immunoglobulins include the various classes and isotypes, such as IgA, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereof may include Fab, Fv and F(ab′) 2 , Fab′, and the like. In addition, fragments, aggregates, polymers, or conjugates of immunoglobulins can be used where appropriate.
  • the antibody can be monoclonal or polyclonal and can be prepared by commonly used techniques such as immunization of a host and collection of sera (polyclonal) or by preparing continuous hybrid cell lines and collecting the secreted protein (monoclonal), or by cloning and expressing nucleotide sequences or mutated versions thereof coding at least for the amino acid sequences required for specific binding of natural antibodies.
  • the conjugating partners can also include polymers, which in various embodiments can be naturally occurring or synthetic.
  • polymers include proteins, glycopeptides, polysaccharides such as dextran such as aminodextran or carboxymethyldextran, and bioolymer derivatives of dextran such as dextran sulfate, hydroxyethylstarch, cellulose and cellulose derivatives, including methylcellulose and carboxymethyl cellulose, starch and starch derivatives, inulin, heparin, heparin fragments; synthetic polymers such as polyalkyl glycols (PAG) such as PEG and derivatives thereof, polyoxyethylated polyols (POP) such as polyoxyethylated glycerol (POG), polytrimethylene glycol (PTG), polypropylene glycol (PPG), polyhydroxyethyl methacrylate, polyvinyl alcohol (PVA), polyacrylic acid.
  • PAG polyalkyl glycols
  • POP polyoxyeth
  • polyethyloxazoline polyacrylamide, polyvinylpyrrolidone (PVP), polyamino acids, polyurethane and polyphosphazene, poly(lactic acid-co-ethylene glycol PLA-PEG, poly(D,L-lactic-co-glycolic acid PLGA, poly(orthoester), Poly(lactide-co-glycolide)-block-poly(ethylene glycols, polyoxyethylated polyols, polyvinylpyrrolidone, polyhydroxyethyl methacrylate, polyvinyl alcohols, and polyurethane ol)-block-poly(lactide-co-glycolide) (PLGA-PEG-PLGA), poly(N-isopropylacrylamide, polyoxyethylated polyols, polyhydroxyethyl methacrylate, N-(2-hydroxypropyl)methacrylamide (HPMA), synthetic copolymers of hydrazone, polyvinyl pyrrol
  • Pegylation of peptides and proteins can result in enhanced therapeutic properties with retention of biological function. Conjugation with PEGs (or with other polymers, like dextrans), by increasing molecular size, can reduce the processes of renal filtration, immune response, and degradation by proteolytic enzymes, all of which act to enhance stability in vivo.
  • PEG polyethylene glycol
  • PEG polyethylene glycol
  • mPEG methoxy group
  • PEGs with multiple forks or branches can also be synthesized, allowing for multiple points of attachment for peptides on an individual PEG molecule.
  • Peptides containing a free sulfhydryl group will readily undergo an alkylation reaction with at least one of mPEG modified with maleimide, forming a stable thioester bond.
  • a synthesized gastrin compound with a Cysteine residue introduced in a region of the peptide that is not required for its biological activity can be used.
  • Peptides containing free amines can react with PEGs modified with succinimidyl esters to produce stable amide linkages. Due to the lack of specificity of the chemical reaction, as in the case of gastrin-34, which contains 3 lysine residues, any one, or more, of the lysine residues could react with the modified PEG, resulting in a chemically, and possibly functionally, heterogeneous mixture of conjugated peptides, purification of which may not be trivial.
  • Site-specific PEGylation of a particular lysine residue may be easily attained during peptide synthesis, by introducing a lysine residue in which a PEG is already linked to the ⁇ -amino side chain (as described by Felix (1997)). Felix, A. M. (1997) Site-Specific Poly(ethylene glycol)ylation of Peptides. In “Poly(ethylene glycol). Chemistry and Biological Applications.” Harris, J. M. & Zalipsky S. Eds.
  • the “activated PEG” is any PEG derivative, which can be used as protein modifier, because it contains a functional group capable of reacting with some functional group in the protein/peptide to produce the PEG-protein/peptide conjugates.
  • Activated PEGs can include alkyating PEGs, acylating PEGs and PEG with an amino acid arm.
  • PEG maleimide e.g. mPEG-maleimide-20,000 from Shearwater Corporation, Huntsville, Ala.
  • Amino PEG can be used to pegylate carboxyl groups.
  • Examples of active PEGS include methoxypolyethylene glycol, diaminomethoxy-polyethylene glycol, methoxypolyethylene glycol-p-nitro-phenylcarbonate, methoxypolyethylene glycol succinimidyl succinate, methoxypolyethylene glycol tresylate, methoxy-polyoxyethylene amine (aminoPEG), methoxypolyoxyethylene-carboxylic acid, monomethoxy-PEG p-nitrophenyl carbonate, N-N′-Carbonyldiimidazole-activated PEGand methoxypolyoxyethyleneimidazole-carbonyl.
  • PEGs of different molecular weights are available commercially.
  • the average molecular weight of the reactant PEG can range from between about 5,000 and about 100,000 daltons.
  • the method of attachment is not critical, but preferably does not alter, or only minimally alters, the activity of the biologically active molecule.
  • a preferred method of attachment is via N-terminal linkage to a polypeptide. See Veronese, Biomaterials 22(5)::405 (2001) for a review of the different activation strategies for PEG.
  • PEG can be conjugated to the peptide/protein via glycopegylation by first derivatizing gastrin with sugars molecules prior to conjugation (i.e. glycopegylation approach e.g. using Neose's GlycoPEGylation technology).
  • Dextran is a naturally occurring polymer that consists of mainly a linear polysaccharide of repeating units of D-glucose linked together in glycosidic bonds. Branch points may be present in a dextran polymer and the branch type and degree of branching vary by species. Dextran can be used here to conjugate to gastrin compounds. The average molecular weight of the soluble dextran can range from between about 10,000 and about 500,000 daltons. Dextran polymer contains adjacent hydroxyl groups on each glucose monomer that can be oxidized by sodium periodate to give a functional aldehyde group that can then react with an amino group.
  • Polyaldehyde dextran can conjugate to amine groups by Schiff base formation followed by reductive amination to create stable linkage, for instance to the epsilon amino group on lysine residue.
  • Dextrans can be carboxymethylated by reaction with monochloroacetic acid to give carboxymethyldextran, which can form dextran-hydrazide on condensation with hydrazine, which reacts with the carbonyl group or aldehyde groups.
  • Sulhydryl reactive dextran derivatives can be prepared through the use of heterobifunctional crosslinking agent containing for instance pyridyldisulfide, maleimide or iodoacetyl groups on one end to direct the conjugation reaction to sulhydryl groups.
  • a lipophilic substituent can be attached to a reactive group of an amino acid at the N-terminal end of the gastrin peptide, or optionally to a reactive group generated via a heterobifunctional or homobifunctinal crosslinker group.
  • the carboxyl group of the lipophilic substituent can react with the amino group on lysine.
  • the lipophilic susbtituents are in particular long-chain groups containing e.g. 8-40 carbon atoms.
  • the lipophilic substituents can be a straight chain or branched alkyl group, an acyl group of a straight chain or branched fatty acid, an acyl group of a straight chain or branched alkane ⁇ , ⁇ -dicarboxylic dicarboxylic acid.
  • lipophilic derivatives of gastrin can be synthesized by chemical attachment to an amino group, for instance, at the N-terminal with various fatty acids including lauric acid (n-dodecanoic acid), myristic acid (n-tetradecanoic acid), palmitic acid (n-hexadecanoic acid), palmitoleic acid (n-hexadecenoic acid), stearic acid (n-octadecanoic acid), oleic acid (n-octadencenoic acid), acetic acid, linoleic acid and arachidonic acid.
  • lauric acid n-dodecanoic acid
  • myristic acid n-tetradecanoic acid
  • palmitic acid n-hexadecanoic acid
  • palmitoleic acid n-hexadecenoic acid
  • stearic acid n-octadecanoic acid
  • oleic acid n-o
  • a hydrophobic moiety that is conformationally rigid i.e. presence of a double bond, a triple bond, or a saturated or unsaturated ring
  • a long chain fatty acids comprising of a carboxyl group can be attached to amino groups found for example on lysine residue.
  • crosslinking agents include but are not limited to the following: Amino group directed homobifunctional cross-linking reagents include: Bisimidoesters (Bisimidates), e.g.methyl acetimidate-HCl, dimethyl suberimidate-2HCl; Bis-N-Succinimidyl Derivatives, e.g.bis(sulfosuccinimidyl-suberate (BSSS), succinate bis-(N-hydroxy-succinimide ester); Bifunctional Aryl Halides, e.g.
  • Bifunctional Acylating Agents such as diisocyanates and diisothiocyanates, e.g.1,6,-hexamethylene diisocyanate;bifunctional sulfonyl halides, e.g.phenol-2,4,-disulfonyl-chloride; bis-nitrophenol esters, e.g.bis-(p-nitrophenyl ester) of carboxylic acids; and bifunctional acylazides, e.g.
  • Dialdehydes e.g., glutaraldehyde
  • Diketones e.g.2.5-hexanedione
  • others such as benzoquinone, 2-iminothiolane, erythreitolbiscarbonate, mucobromic acid, mucochloric acid, ethylchloroformate, p-nitrophenylchloroformate, succinimidyl 6-hydrazinonicotinate acetone hydrazone and succinimidyl 4-formylbenzoate (for instance the first modifies an amine with a hydrazine linker and the second modifies an amine to an aldehyde linker, which can then be crosslinked together
  • Another group of bifunctional crosslinking agents are the Sulfhydryl group directed homobifunctional crosslinkers, which include: Mercurial Reagent, e.g. 1,4,-bis(bromomercuri)butane; Disulfide Forming Reagents,
  • Alkylating Agents such as Bio-haloacetyl derivatives, e.g. 1,3,-dibromoacetone; Di-alkyl halides, e.g.
  • di(2-chloroethyl)sulfide di(2-chloroethyl)sulfide
  • S-triazines e.g.2,4,-dichloro-6-methoxy-s-triazine
  • Aziridines e.g.2,3,4-tri(ethyleneimino)-s-triazine
  • Bis-epoxides e.g. 1,2,3,4-diepoxybutane
  • others e.g. divinyl sulfone.
  • cis-dichlorodiaminoplatinum(II) (used to crosslink alpha2-macroglobulin with one or more methionine residues at or near the recognition site, may crosslink complementary strands of DNA adipid acid dihydrazide as well as crosslink glycoproteins, acid phosphatase and invertase), N,N′-bis(b-aminoethyl)-tartramide.
  • Group selective heterobifunctional crosslinkers which include: Amino and sulfhydryl group directed bifunctional reagents, e.g., N-succinimidyl 3-(2-pyridyldithio propionate) (SPDP) and its analogs (LC-SPDP, Sulfo-LC-SPDP), Succinimidyloxycarbonyl- ⁇ -methyl- ⁇ -(2-pyridyldithio)toluene (SMPT) and its analog Sulfosuccinimidyl-6- ⁇ -methyl- ⁇ -(2-pyridyldithio)toluamido]hexanoate (Sulfo-LC-SMPT), Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and its analog, Sulfosuccinidimidyl 4-(N-maleimidomethyl-cyclo
  • SPDP N-succinimi
  • suitable cross-linking agents include: Carboxyl group activating agents, such as carbodiimides e.g., 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) (activates a carboxyl group than can then crosslink with a NH2 group); isoxazloium derivatives; chloroformates; carbonyldiimidazole; and N-carbalkoxydihydroquinolines; Disulfide forming reagent e.g.cupric di(1,10-phenanthroline); Enzymes e.g.transglutaminase, peroxidase (between lysine residues), xanthine oxidase (forms disulfide bonds); and others e.g. pyrroloquinolinequinone (converts lysines to semi-aldeh
  • Carboxyl group activating agents such as carbodiimides e.g.
  • the present invention in various embodiments provides pharmaceutical compositions comprising a therapeutically effective amount of a combination of the gastrin compound alone or the combination of a FACGINT, or an hypoglycemic agent with a gastrin compound.
  • All of the pharmaceutical compositions described herein can be formulated with or without an agent for immune suppression, and with or without components or devices for sustained release, for delivery locally or systemically.
  • a pharmaceutically acceptable carrier or excipient can be added.
  • a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.
  • an “effective amount” as the term is used herein is an amount of a therapeutic agent or combination of agents sufficient to achieve a recognized medical endpoint, in this case, remediation of a symptom of diabetes.
  • the effective amount can be determined empirically by a skilled artisan according to established methods of measurement of relevant parameters, as described herein.
  • compositions herein can further comprise wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • the compositions can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • Various delivery systems are known and can be used to administer a composition of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules and the like.
  • a composition herein is formulated in accordance with routine procedures as a pharmaceutical composition adapted, for example, for subcutaneous administration to human beings.
  • compositions for subcutaneous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic to ameliorate pain at the site of the injection.
  • the ingredients are provided either separately or mixed together in unit dosage form, for example, as a dry, lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette, for example, indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, buffer, or saline.
  • an ampoule of sterile water or saline for injection can be provided so that the ingredients may be mixed prior to administration.
  • the compositions herein can in various components thereof be formulated as suppositories, which contain active ingredient in the range of about 0.5% to about 10% by weight; oral formulations preferably contain about 10% to about 95% active ingredient by weight.
  • a daily dose is administered as a single dose, or is divided into a plurality of smaller fractional doses, to be administered several times during the day.
  • a dosing schedule refers to a protocol for administering any of the compositions comprising for instance a modified gastrin compound as provided herein, or the modified gastrin in combination with a FACGINT or with a hypoglycemic agent and/or with an immunosuppressant, each in an effective dose, administered simultaneously or within a particular interval of each other, for example, within one day of each other, or as a combined preparation, or separately, and includes the amount of the composition delivered per unit time such as per day, and the duration or period of time over which each composition is administered.
  • the invention provides a method for preventing or treating diabetes, the method comprising administering to a mammal in need thereof composition of gastrin compound alone or in combination with a FACGINT a FACGINT or with a hypoglycemic agent and/or with an immunosuppressant, each in an amount sufficient to increase the number of pancreatic insulin secreting ⁇ cells in the mammal; and determining the amount of islet neogenesis, thereby treating or preventing the diabetes. Determining the amount of islet neogenesis is measuring a parameter selected from the group of: blood glucose, serum glucose, blood glycosylated hemoglobin, pancreatic ⁇ cell mass, serum insulin, and pancreatic insulin content.
  • Administering the composition described herein reduces blood glucose compared to blood glucose assayed prior to administering the composition, for example, administering the composition reduces blood glucose by about 50%, or by about 70%, compared to blood glucose assayed prior to administering the composition.
  • Glycosylated hemoglobin concentration is reduced compared to glycosylated hemoglobin concentration in the mammal assayed prior to administering the composition.
  • Serum insulin concentration is increased compared to serum insulin concentration in the mammal assayed prior to administering the composition.
  • Pancreatic insulin concentration is increased compared to pancreatic insulin concentration in the mammal assayed prior to administering the composition.
  • compositions of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • the amount of the therapeutic of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Routine determinations of blood levels of insulin or C peptide, and of fasting levels of glucose or glucose challenges, are determined by one of ordinary skill in the art.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems, by one of ordinary skill in the art of pharmacology.
  • Dosages of the compositions to be administered to a subject are adjusted for known variations from species to species using standard data encompassing criteria for absorption, distribution, half-life kinetics in circulation, metabolism, excretion, and toxicology of the receptor ligands of the embodiments herein.
  • Suitable dosage ranges for administration are generally about 0.01 micrograms to about 10,000 micrograms of each active compound per kilogram body weight per day, for example, about 0.01 micrograms to about 1 microgram/kg, about 0.1 micrograms/kg to about 10 micrograms/kg, about 1 microgram/kg to about 500 micrograms/kg, or about 10 micrograms/kg to about 10 mg/kg of body weight per day.
  • Suitable dosage ranges for administration are thus generally about 0.01 micrograms/kg body weight/day to about 10 mg/kg body weight/day.
  • the invention in other embodiments provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • a container having a unit dosage of the extended use gastrin compound Associated with such container(s) can be various written materials such as instructions for use, or a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • AUC Absolute under the curve, a measure of total exposure to a drug over a period of time
  • Plasmat 1/2 A measure of how long a drug stays in the blood. the time it takes for the plasma concentration to fall 50%
  • mice Male animals were used in the example. Each animal received gastrin by intravenous administration (3 ⁇ g/kg, 10 ⁇ g/kg and 30 ⁇ g/kg). The dosing solutions for intravenous injection were administered as a single bolus dose via the saphenous vein at a dose volume of 1 mL/kg. A 0.2 mL saline flush was administered following dosing in order to ensure administration of the complete dose volume. The actual volume administered to each animal was based on the animal's most recent body weight.
  • Serial blood samples (approximately 0.5 ml per time point) were collected via the brachial or femoral vein at the following time points: 0 (pre-dose), 1, 3, 5, 10, 15, 30 minutes, 1, 2, and 4 hours post dose.
  • Gastrin in plasma samples was measured with a well established competitive radioimmunoassay for the quantitative determination of gastrin (Russell et al., Postgraduate Medical Journal 52:645, 1976).
  • the antibody used in the assay was raised in rabbits against synthetic human gastrin I conjugated with carbodiimide to bovine serum albumin.
  • the antigen was labeled with 125 I and the presence of the antigen:antibody reactions were detected using a gamma counter.
  • FBG fasting blood glucose
  • FIG. 1 shows that in the vehicle-treated control animals, fasting blood glucose levels (FBG) were doubled after 35 days.
  • FBG fasting blood glucose levels
  • treatment with gastrin prevented some of the increase in glucose levels from rising in the diabetic NOD mice, however, the FBG levels remained significantly higher than that observed in normal mice ( ⁇ 3-7 mM).
  • pancreatic insulin levels for vehicle-treated groups decreased at day 35 due to destruction of beta-cells, whereas animals treated with gastrin exhibited significantly elevated levels of pancreatic insulin levels in comparison with pretreatment values. See FIG. 2.
  • the increase of pancreatic insulin levels after treatment of COMPOUND B to about 0.6 ug of insulin/pancreas is still significantly lower than that of normal mice ( ⁇ 12 ug/pancreas).
  • Gastrin peptides may be readily synthesized by anyone with ordinary skills in the art, using standard techniques for solid phase peptide synthesis, for example as described by Steward, J. M. and Young, J. D. (1984) in “Solid Phase Peptide Synthesis”, 2 nd ed., Pierce Chemical Company. Purification of gastrin peptides may be performed using standard techniques, for example using reverse phase HPLC with a volatile binary gradient system consisting of 0.1% TFA in H 2 O and 0.1% TFA in acetonitrile. Monitoring elution by UV absorbance allows for collection of purified peptide, which is then lyophilized to dryness and subsequently dissolved for administration and testing, or for further conjugation reactions where required.
  • Gastrin synthetic peptides may be synthesized containing any consecutive portion of residues 1-28 in addition to residues 29-34 of SEQ ID NO: 1 or 2 or containing any consecutive portion of residues 1-11 in addition to residues 12-17 of SEQ ID NO: 3 or 4. Additionally, gastrin peptides may be synthesized with a spacer region at the N-terminal end comprised of small neutral amino acid residues such as Gly and Ala. Gastrin synthetic peptides, either with or without a spacer region, may also be synthesized with an N-terminal Cys residue.
  • Gastrin peptides modified with Cys at the N-terminal are incubated for about 30 minutes at near neutral conditions (buffered at pH 6.5-7.5) with a molar excess of tris[2-carboxyethyl]phosphine hydrochloride (TCEP; a reducing agent which has no reactivity with maleimide moieties) to ensure that the Cys residue is in the reduced state and available for reaction.
  • TCEP tris[2-carboxyethyl]phosphine hydrochloride
  • a molar excess of mPEG-maleimide-20,000 (average MW 20,000; obtained from Shearwater Corporation, Huntsville Ala., USA) is added gradually with mixing to allow dissolution, and when fully dissolved, the solution is mixed for an additional 1 to 4 hours to allow for completion of the conjugation reaction.
  • Purification of the conjugate may be performed using an anion-exchanger, for example Q-Sepharose at neutral pH, to which both the conjugate and any free unreacted gastrin binds tightly, since the theoretical isoelectric point (pI) for gastrin is 3.4, whereas the neutral unreacted mPEG-maleimide does not bind.
  • the conjugate is readily separated from unreacted gastrin based on the large difference in their molecular weights using size exclusion chromatography, for example using Sephadex G-50, using a buffer suitable for therapeutic use, such as PBS.
  • Serum albumin contains a single accessible, reduced Cysteine residue (Cys-34), which is the most reactive thiol group among human plasma proteins (Pedersen et al (1980) Eur. J. Biochem. 106: 291-295). This property allows specific conjugation of ligands, including peptides, to serum albumin.
  • Human serum albumin is incubated for about 30 minutes at near neutral conditions (buffered at pH 6.5-7.5) with a molar excess of tris[2-carboxyethyl]phosphine hydrochloride (TCEP; a reducing agent which has no reactivity with maleimide moieties) to ensure that Cys-34 residue is in the reduced state and available for reaction.
  • TCEP tris[2-carboxyethyl]phosphine hydrochloride
  • a molar excess of bis-maleimidoethane (a homo-bifunctional cross-linking agent having a short spacer, and reactivity towards sulfhydryl groups) is added to activate Cys-34.
  • gastrin peptides modified with Cys at the N-terminal are added and the resulting solution is mixed for an additional 1 to 4 hours to allow for completion of the conjugation reaction.
  • the conjugate is readily separated from unreacted gastrin (and any gastrin dimers formed) based on the large difference in their molecular weights using size exclusion chromatography, for example using a buffer suitable for therapeutic use, such as PBS. Unreacted HSA was not further separated from HSA conjugated with Gastrin peptides.
  • Rats (groups of three animals) receive the various test compounds as synthesized in Example 3 at the dose level of 10 ⁇ g/kg of gastrin equivalent by iv administration.
  • the following test compounds are used are all of the compounds listed in Table 3 above. Please refer to Table 3 for more details.
  • Serial blood samples (approximately 0.4 ml per time point) were collected at the following time points: 5, 60, 180, 480 minutes after the injection all animals. At each time point, blood was taken from at 3 animals per group.
  • Plasma concentration of gastrin (COMPOUND B) and its different derivatives/conjugates were analyzed using the PK Functions for Microsoft® Excel (Usansky J I, Desai A, Tang-Liu D, Department of Pharmacokinetics and Drug Metabolism, Irvine, Calif.). To assess the PK profiles, various test compounds and the following PK values were calculated: C max , t max , AUC and t 1/2 .
  • gastrins e.g. COMPOUND B
  • baseline values obtained before dosing were subtracted from the plasma levels obtained after COMPOUND B administration. Data are expressed as mean ⁇ SD.
  • modified gastrin compounds with cysteine as functional groups on the N-terminus), or modified gastrin compounds conjugated to PEG or HSA on the N-terminus had significantly longer half lives compared to native gastrin.
  • the modified gastrin compounds/conjugates are present in serum at higher concentrations for longer periods of time compared to the administration of native gastrin 17 or gastrin 34.
  • AUCs also increase with the chemical modification of the gastrin molecules. It is envisioned that the modified gastrin derivatives/conjugates provide the highest concentrations for the longest period of time will have the greatest potential for the efficacy required for islet cell neogenesis and regulation of glucose levels in diabetic animals.
  • Modified gastrin compounds/conjugates used are as follows (Refer to Table 3 for more details): Compound B, which is gastrin prepared as synthetic human gastrin I having 17 amino acid residues with a Leu residue at amino acid position 15, Compound E, Compound G, Compound J, Compound L, Compound O and Compound Q.
  • Non-obese diabetic (NOD) female mice ages 12-14 weeks, are monitored for development of onset of diabetes (fasting blood glucose >8.0 to 15 mmol/l), and within 48 hours after onset of symptoms, the different groups of mice are each treated with 3 or 10 ⁇ g/kg/day of Gastrin equivalent, each treatment administered via the intraperitoneal route daily.
  • NOD non-obese diabetic
  • FBG blood glucose
  • the protocol includes sampling of these mice for data again at 6 weeks, and blood collected for assay of FBG and plasma C-peptide, and the mice are sacrificed for pancreatic insulin determinations and scoring of islet inflammation (insulitis). From the outset of treatment, mice receive neither insulin-replacement treatment nor immunosuppression. The following parameters are assessed: survival rates, pancreatic insulin levels, presence of islet inflammation and fasting blood glucose levels.
  • modified gastrin derivatives/conjugates with a longer half life and AUCs were more effective in preventing hyperglycemia in diabetic NOD mice.
  • the modified gastrin compounds/conjugates completely reverse the glucose levels to normal levels, indicating the stimulation of significant levels of islet neogenesis in this model.
  • Non-obese diabetic (NOD) female mice ages 12-14 weeks, were monitored for development of onset of diabetes (fasting blood glucose >8.0 to 15 mmol/l), and within 48 hours after onset of symptoms, four groups of mice were each treated as follows: one group was treated with vehicle only; and the other group was administered 100 ⁇ g/kg/day of GLP-1, and the remaining groups were treated with combination of GLP-1 (100 ⁇ g/kg/day) and gastrin compound (3 ⁇ g/kg/day gastrin equivalent), each treatment administered via the intraperitoneal route daily.
  • NOD non-obese diabetic
  • FBG blood glucose
  • the protocol includes sampling of these mice for data again at 6 weeks, and blood collected for assay of FBG and plasma C-peptide, and the mice are sacrificed for pancreatic insulin determinations and scoring of islet inflammation (insulitis). From the outset of treatment, mice received neither insulin-replacement treatment nor immunosuppression. The following parameters are assessed: survival rates, pancreatic insulin levels, presence of islet inflammation and fasting blood glucose levels.

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Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020081285A1 (en) * 1992-12-14 2002-06-27 Indu Parikh Treatment for diabetes
US20040229810A1 (en) * 2002-10-22 2004-11-18 Antonio Cruz Gastrin compositions and formulations, and methods of use and preparation
US20060234373A1 (en) * 2002-05-24 2006-10-19 Alex Rabinovitch Treatment for diabetes
US20070066520A1 (en) * 2001-10-23 2007-03-22 Waratah Pharmaceuticals, Inc. Novel epidermal growth factor protein and gene, and methods of use therefor
US20070249005A1 (en) * 2003-03-28 2007-10-25 Stephen Grimes Gastrin hormone immunoassays
US20080039379A1 (en) * 2003-05-27 2008-02-14 Waratah Pharmaceuticals, Inc. Compositions Comprising Gastrin Compounds and Their Use in Diabetes
US20080286301A1 (en) * 1994-05-12 2008-11-20 John Dupre Treatment of diabetes
US7476388B2 (en) 2001-01-12 2009-01-13 Waratah Pharmaceuticals, Inc. Composition comprising a gastrin/CCK receptor ligand and an EGF receptor ligand
US20090054314A1 (en) * 2005-10-07 2009-02-26 Antonio Cruz Combined use of DPP-IV inhibitors and gastrin compounds
US20090117102A1 (en) * 2004-07-01 2009-05-07 Antonio Cruz Methods and compositions using CD3 agonists
US7560425B2 (en) 2002-06-07 2009-07-14 Waratah Pharmaceuticals Inc. Pharmaceutical composition consisting of rapamycine and gastrin 17(LEU15) and a method for treating diabetes
US20090202494A1 (en) * 2004-01-30 2009-08-13 Antonio Cruz Combined use of glp-1 agonists and gastrin for regulating blood glucose levels
WO2009033729A3 (en) * 2007-09-11 2009-09-11 Mondobiotech Laboratories Ag Therapeutic uses of gastrin- 1 and g- pen-grgdspca
US7803766B2 (en) 2002-11-21 2010-09-28 Warath Pharmaceuticals, Inc Gastrin compositions and formulations, and methods of use and preparation
WO2011131646A1 (en) * 2010-04-20 2011-10-27 Novo Nordisk A/S Long-acting gastrin derivatives
CN102260868A (zh) * 2011-07-22 2011-11-30 上海交通大学 高耐蚀性钝化液及其生产工艺
EP2413694A1 (en) * 2009-04-03 2012-02-08 The University of Toledo A peg-albumin composition having at least one protected thiol region as a platform for medications
US8158128B2 (en) 2004-09-22 2012-04-17 Cancer Advances, Inc. Monoclonal antibodies to progastrin
US8343930B2 (en) 2001-05-04 2013-01-01 Cancer Advances, Inc. Combination therapy for the treatment of tumors
US8388966B2 (en) 2001-03-23 2013-03-05 Cancer Advances, Inc. Combination treatment of pancreatic cancer
US11358994B2 (en) * 2017-07-27 2022-06-14 Saint Louis University Fatty acid modified human epidermal growth factor
US11583576B2 (en) 2017-06-15 2023-02-21 Cancer Advances Inc. Compositions and methods for inducing humoral and cellular immunities against tumors and cancer

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100184678A1 (en) * 2007-09-11 2010-07-22 Dorian Bevec Use of a peptide as a therapeutic agent
KR101286721B1 (ko) 2009-06-05 2013-07-16 한국과학기술연구원 폴리-시스테인 펩티드 융합 재조합 알부민 및 이의 제조방법
NZ603169A (en) 2010-04-27 2015-02-27 Zealand Pharma As Peptide conjugates of glp-1 receptor agonists and gastrin and their use
CA2853884A1 (en) * 2011-11-03 2013-05-10 Zealand Pharma A/S Glp-1 receptor agonist peptide gastrin conjugates
WO2013138694A1 (en) * 2012-03-16 2013-09-19 Enzon Pharmaceuticals, Inc. Polymeric conjugates of c-1 inhibitors
AU2014345569B2 (en) 2013-11-06 2020-08-13 Zealand Pharma A/S GIP-GLP-1 dual agonist compounds and methods
BR112016009995B1 (pt) 2013-11-06 2023-04-18 Zealand Pharma A/S Compostos agonistas triplos glucagon-glp-1-gip
CA2965732A1 (en) 2014-10-29 2016-05-06 Zealand Pharma A/S Gip agonist compounds and methods
CN117384274A (zh) 2016-12-09 2024-01-12 西兰制药公司 酰化的glp-1/glp-2双重激动剂
KR102503349B1 (ko) 2019-05-14 2023-02-23 프로벤션 바이오, 인코포레이티드 제1형 당뇨병을 예방하기 위한 방법 및 조성물

Family Cites Families (44)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4464363A (en) * 1979-12-20 1984-08-07 Merck & Co., Inc. Ajuvants for rectal delivery of drug substances
JPS6028994A (ja) * 1983-07-08 1985-02-14 Wakunaga Seiyaku Kk 〔２１―ロイシン〕ヒトウロガストロン
US4686283A (en) * 1985-04-16 1987-08-11 Syntex (U.S.A.) Inc. Analogs of transforming and epidermal growth factor fragments for therapy and diagnosis
EP0284898A3 (de) * 1987-04-02 1990-06-27 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Hinge-Peptid, Verfahren zu seiner Herstellung und seine Verwendung zur Herstellung synthetischer Immunogene
DE69014137T2 (de) * 1989-01-24 1995-06-08 Aphton Corp Immunogenische Zusammensetzungen gegen Gastrin-Peptide.
US5023077A (en) * 1989-01-24 1991-06-11 Aphton Corporation Immunogenic compositions and methods for the treatment and prevention of gastric and duodenal ulcer disease
US4997950A (en) * 1989-04-20 1991-03-05 Richard Finbar Murphy Novel C-terminal gastrin antagonists
US5166322A (en) 1989-04-21 1992-11-24 Genetics Institute Cysteine added variants of interleukin-3 and chemical modifications thereof
SE509359C2 (sv) 1989-08-01 1999-01-18 Cemu Bioteknik Ab Användning av stabiliserade protein- eller peptidkonjugat för framställning av ett läkemedel
US6267964B1 (en) * 1989-08-01 2001-07-31 Affibody Technology Sweden Ab Stabilized protein or peptide conjugates able to bond albumin having extended biological half-lives
IE68593B1 (en) * 1989-12-06 1996-06-26 Sanofi Sa Heterocyclic substituted acylaminothiazoles their preparation and pharmaceutical compositions containing them
DE69231467T2 (de) * 1991-05-10 2001-01-25 Genentech Inc Auswählen von agonisten und antagonisten von liganden
AU2489992A (en) 1991-08-16 1993-03-16 Chiron Corporation Muteins of epidermal growth factor exhibiting enhanced binding at low ph
FR2686899B1 (fr) * 1992-01-31 1995-09-01 Rhone Poulenc Rorer Sa Nouveaux polypeptides biologiquement actifs, leur preparation et compositions pharmaceutiques les contenant.
US5885956A (en) * 1992-12-14 1999-03-23 Research Triangle Pharmaceuticals Treatment for diabetes using a gastrin/CCK receptor ligand and an EGF receptor ligand
US6558952B1 (en) * 1992-12-14 2003-05-06 Waratah Pharmaceuticals, Inc. Treatment for diabetes
DE69435212D1 (de) * 1994-01-24 2009-07-16 Waratah Pharmaceuticals Inc Behandlung von Diabetes
WO1995029690A1 (en) * 1994-04-29 1995-11-09 The Trustees Of The University Of Pennsylvania Biologically active peptides and methods of identifying the same
GB9409496D0 (en) * 1994-05-12 1994-06-29 London Health Ass Method for improving glycaemic control in diabetes
DE19514087A1 (de) * 1995-04-13 1996-10-17 Deutsches Krebsforsch Konjugat aus einem Wirkstoff, einem Polyether und ggfs. einem nicht als körperfremd angesehenen, nativen Protein
WO1999045026A1 (en) * 1998-03-05 1999-09-10 Chiron Corporation Method for increasing the serum half-life of a biologically active molecule
US20040037818A1 (en) 1998-07-30 2004-02-26 Brand Stephen J. Treatment for diabetes
US6420339B1 (en) * 1998-10-14 2002-07-16 Amgen Inc. Site-directed dual pegylation of proteins for improved bioactivity and biocompatibility
AU2153500A (en) 1998-11-19 2000-06-05 Millennium Pharmaceuticals, Inc. Egf-like nucleic acids and polypeptides and uses thereof
PT1180121E (pt) * 1999-05-17 2004-03-31 Conjuchem Inc Peptidos insulinotropicos de longa duracao
US20010046489A1 (en) 1999-12-06 2001-11-29 Habener Joel E. Stem cells of the islets of langerhans and their use in treating diabetes mellitus
EP1265985A4 (en) 2000-02-18 2004-06-23 Inst Medical W & E Hall PANCREAS ISLAND CELL GROWTH FACTORS
EP1305338A2 (en) 2000-08-02 2003-05-02 Theratechnologies Inc. Modified peptides with increased potency
CA2434330A1 (en) * 2001-01-12 2002-07-18 Waratah Pharmaceuticals, Inc. Composition for inducing islet neogenesis, containing gastrin/cck receptor ligands and egf receptor ligands
JP2004526449A (ja) 2001-03-29 2004-09-02 イクシオン・バイオテクノロジー・インコーポレーテッド 非膵性幹細胞の膵分化経路への分化転換法
CN1169827C (zh) 2001-08-07 2004-10-06 沈阳三生制药股份有限公司 一种增强多肽在体内稳定性药物的生产方法及其应用
US7037504B2 (en) * 2001-10-23 2006-05-02 Waratah Pharmaceuticals, Inc. Epidermal growth factor protein and gene, and methods of use therefor
AU2003231864A1 (en) 2002-05-24 2003-12-12 University Of Alberta Treatment for diabetes
WO2004105780A2 (en) 2003-05-27 2004-12-09 Waratah Pharmaceuticals, Inc. Compositions comprising gastrin compounds and their use in diabetes
KR20050048542A (ko) * 2002-06-07 2005-05-24 와라타 파마수티컬즈, 인크. 당뇨병의 치료를 위한 조성물 및 방법
EP1837031B1 (en) 2002-06-07 2009-10-14 Waratah Pharmaceuticals, Inc. Compositions and methods for treating diabetes
US20060189520A1 (en) 2002-10-22 2006-08-24 Brand Stephen J Treatment of diabetes
US20040229810A1 (en) 2002-10-22 2004-11-18 Antonio Cruz Gastrin compositions and formulations, and methods of use and preparation
WO2004096853A1 (en) 2003-04-30 2004-11-11 Waratah Pharmaceuticals, Inc. Combined use of keratinocyte growth factor agonists and gastrin compounds
AU2005207870B2 (en) 2004-01-30 2010-08-19 Waratah Pharmaceuticals, Inc. The combined use of GLP-1 agonists and gastrin for regulating blood glucose levels
CA2571957A1 (en) 2004-07-01 2006-01-12 Waratah Pharmaceuticals, Inc. Methods and compositions using cd3 agonists
US20100144613A1 (en) 2005-10-07 2010-06-10 Antonio Cruz Combined use of DPP-IV Inhibitors and Gastrin Compounds
WO2007062531A1 (en) 2005-12-02 2007-06-07 Waratah Pharmaceuticals, Inc. Combination treatments with gastrin agonists for diabetes and related diseases
WO2007095737A1 (en) 2006-02-21 2007-08-30 Waratah Pharmaceuticals Inc. Combination therapy for the treatment of diabetes comprising an exendin agonist and a gastrin compound

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020081285A1 (en) * 1992-12-14 2002-06-27 Indu Parikh Treatment for diabetes
US20080286301A1 (en) * 1994-05-12 2008-11-20 John Dupre Treatment of diabetes
US20040209816A1 (en) * 1999-01-29 2004-10-21 Indu Parikh Treatment for diabetes
US7476388B2 (en) 2001-01-12 2009-01-13 Waratah Pharmaceuticals, Inc. Composition comprising a gastrin/CCK receptor ligand and an EGF receptor ligand
US8388966B2 (en) 2001-03-23 2013-03-05 Cancer Advances, Inc. Combination treatment of pancreatic cancer
US8343930B2 (en) 2001-05-04 2013-01-01 Cancer Advances, Inc. Combination therapy for the treatment of tumors
US20070066520A1 (en) * 2001-10-23 2007-03-22 Waratah Pharmaceuticals, Inc. Novel epidermal growth factor protein and gene, and methods of use therefor
US20060234373A1 (en) * 2002-05-24 2006-10-19 Alex Rabinovitch Treatment for diabetes
US7560425B2 (en) 2002-06-07 2009-07-14 Waratah Pharmaceuticals Inc. Pharmaceutical composition consisting of rapamycine and gastrin 17(LEU15) and a method for treating diabetes
US20040229810A1 (en) * 2002-10-22 2004-11-18 Antonio Cruz Gastrin compositions and formulations, and methods of use and preparation
US7803766B2 (en) 2002-11-21 2010-09-28 Warath Pharmaceuticals, Inc Gastrin compositions and formulations, and methods of use and preparation
US7964371B2 (en) 2003-03-28 2011-06-21 Cancer Advances, Inc. Gastrin hormone immunoassays
US20070249005A1 (en) * 2003-03-28 2007-10-25 Stephen Grimes Gastrin hormone immunoassays
US20080039379A1 (en) * 2003-05-27 2008-02-14 Waratah Pharmaceuticals, Inc. Compositions Comprising Gastrin Compounds and Their Use in Diabetes
US20090202494A1 (en) * 2004-01-30 2009-08-13 Antonio Cruz Combined use of glp-1 agonists and gastrin for regulating blood glucose levels
US20090117102A1 (en) * 2004-07-01 2009-05-07 Antonio Cruz Methods and compositions using CD3 agonists
US8158128B2 (en) 2004-09-22 2012-04-17 Cancer Advances, Inc. Monoclonal antibodies to progastrin
US8808695B2 (en) 2004-09-22 2014-08-19 Cancer Advances, Inc. Monoclonal antibodies to progastrin
US20090054314A1 (en) * 2005-10-07 2009-02-26 Antonio Cruz Combined use of DPP-IV inhibitors and gastrin compounds
WO2009033729A3 (en) * 2007-09-11 2009-09-11 Mondobiotech Laboratories Ag Therapeutic uses of gastrin- 1 and g- pen-grgdspca
EP2413694A1 (en) * 2009-04-03 2012-02-08 The University of Toledo A peg-albumin composition having at least one protected thiol region as a platform for medications
EP2413694A4 (en) * 2009-04-03 2014-02-26 Univ Toledo PEG-ALBUMIN COMPOSITION HAVING AT LEAST ONE PROTECTED THIOL REGION AS A PLATFORM FOR MEDICAMENTS
WO2011131646A1 (en) * 2010-04-20 2011-10-27 Novo Nordisk A/S Long-acting gastrin derivatives
US20130059781A1 (en) * 2010-04-20 2013-03-07 Novo Nordisk A/S Long-Acting Gastrin Derivatives
US9040660B2 (en) * 2010-04-20 2015-05-26 Novo Nordisk A/S Long-acting gastrin derivatives
CN102260868A (zh) * 2011-07-22 2011-11-30 上海交通大学 高耐蚀性钝化液及其生产工艺
US11583576B2 (en) 2017-06-15 2023-02-21 Cancer Advances Inc. Compositions and methods for inducing humoral and cellular immunities against tumors and cancer
US11358994B2 (en) * 2017-07-27 2022-06-14 Saint Louis University Fatty acid modified human epidermal growth factor

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US7803766B2 (en) 2010-09-28
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EP1837031B1 (en) 2009-10-14
ATE378065T1 (de) 2007-11-15
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