WO2013128391A1 - Synthetic polypeptides and uses thereof - Google Patents

Synthetic polypeptides and uses thereof Download PDF

Info

Publication number
WO2013128391A1
WO2013128391A1 PCT/IB2013/051583 IB2013051583W WO2013128391A1 WO 2013128391 A1 WO2013128391 A1 WO 2013128391A1 IB 2013051583 W IB2013051583 W IB 2013051583W WO 2013128391 A1 WO2013128391 A1 WO 2013128391A1
Authority
WO
WIPO (PCT)
Prior art keywords
cys
seq
synthetic
pro
ala
Prior art date
Application number
PCT/IB2013/051583
Other languages
French (fr)
Inventor
Margaret Anne Brimble
Geoffrey Martyn Williams
Original Assignee
Auckland Uniservices Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Auckland Uniservices Limited filed Critical Auckland Uniservices Limited
Publication of WO2013128391A1 publication Critical patent/WO2013128391A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • 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
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates generally to synthetic peptides and proteins and related compositions and formulations and their preparation and use, and methods for the prevention and treatment of conditions, diseases and disorders that would be improved, eased, or lessened by the administration of a composition of the invention, including but not limited to glucose metabolism diseases and disorders and diseases and disorders and conditions treated or treatable with insulin and other hypoglycemic agents.
  • Diabetes mellitus characterized by hyperglycemia and altered ⁇ -cell function, is a common disorder affecting millions of individuals. According to statistics provided by the American Diabetes Association (ADA), there are 25.8 million people in the United States, or 8.3% of the population, who have diabetes. Direct medical and indirect expenditures attributable to diabetes in 2007 were estimated at $174 billion.
  • ADA American Diabetes Association
  • Type 1 and type 2 diabetes are both diseases of the pancreas characterized by hyperglycemia.
  • pancreatic islet ⁇ -cells which secrete both insulin and amylin, peptide hormones that exert profound effects on glucose metabolism, are destroyed.
  • type 2 diabetes these cells progressively lose function, and often fail in the late stages of the disease. As one would expect given the severity of diabetes, and difficulties associated with it, the islet ⁇ -cells play a major role in physiology.
  • the ADA reports that two out of three people with diabetes die from heart disease and stroke, that diabetes is the leading cause of new cases of blindness in people ages 20-74, that diabetes is the leading cause of end-stage renal disease (kidney failure), accounting for about 44 percent of new cases (with approximately 48,374 people with diabetes initiating treatment for end stage renal disease and 202,290 undergoing dialysis or kidney transplantation in the year 2008), that more than 60 percent of nontraumatic lower-limb amputations in the U.S.
  • Type 1 diabetes is characterized by an early loss of endocrine function in the pancreas due to autoimmune destruction of the pancreatic islet ⁇ -cells, resulting in hypoinsulinemia, hypoamylinemia, and hyperglycemia.
  • Type 2 diabetes is a polygenic and heterogeneous disease resulting from an interaction between genetic factors and environmental influences. See, e.g., Kecha-Kamoun et al., Diabetes Metab Res Rev 17: 146-152 (2001).
  • type 2 diabetes is initially characterized by hyperinsulinemia, peripheral insulin resistance and resulting hyperglycemia characterize type 2 diabetes, ⁇ -cells often compensate for this insulin resistance with both an increase in insulin secretory capacity and ⁇ - cell mass. Levels of insulin eventually decrease as a result of the loss of ⁇ -cell function and eventual ⁇ -cell failure. Thus, there is a progression from normal glucose tolerance, to impaired glucose tolerance, to type 2 diabetes, and to late stage type 2 diabetes, which is associated with altered ⁇ -cell function, ⁇ -cell loss and, eventually, a decline in insulin secretion. See, e.g., Dickson et al., J. Biol. Chem. 276:21110-21120 (2001).
  • ⁇ -cells In other words, hyperglycemia worsens as ⁇ -cells fail to sustain levels of insulin output sufficient to overcome increasing resistance to insulin. Kaytor, et al., J Biol Chem. 16: 16 (2001). Eventual ⁇ -cell failure is primarily a failure in function but later proceeds to ⁇ -cell loss such as that seen in type 1 diabetes.
  • One of the most striking functional ⁇ -cell defects is a loss of acute glucose-induced insulin secretion (GIIS).
  • GIIS acute glucose-induced insulin secretion
  • inventions described and claimed herein relate to the synthesis of polypeptides including polypeptides having insulin agonist activity and their synthetic intermediates.
  • the invention relates to a method of synthesizing a polypeptide comprising a first peptide chain and a second peptide chain, the method comprising providing a first peptide chain and a second peptide chain, forming under conducive conditions one or more interchain disulfide bonds between the first peptide chain and the second peptide chain, and recovering the polypeptide from the reaction medium, wherein the first peptide chain comprises an amino acid sequence corresponding to
  • a chain Gly He Val Glu Glu Cys 3 Cys 4 Phe Arg Ser Cys 5 Asp Leu Ri R 2 Leu Glu R 3
  • the second peptide chain comprises an amino acid sequence corresponding to
  • Ri is Ala, Asn or Leu; R2 is Leu or He; R 3 is Thr or Gin; R 4 is Thr, Ala, Lys, or Val; R 5 is Pro or Ser; Rg is Ala, Val, or Pro; R 7 is Lys or Glu, R 8 is Ser or Ala, R 9 is Glu or Ala, R 10 is absent or is Ala, Glu, or Asp, Rn is Tyr, Ala, or Val, R 12 is Arg, Gly, or Ala, Ri 3 is Pro, Thr, Ser, or Leu, Ri 4 is Ser, Gly, Ala, or Glu, R15 is Thr or Ala, Ri 6 is Val or He, R 17 is Gly, Ser, Glu, or Ala, Ri 8 is Asp or Glu, R19 is Ser or Val, R 20 is Arg, Leu, or Ser, R 2 i is Pro or Lys, R22 is Ala, Ser, Gly, Val, or Thr, R 23 is Ser, Gly, or Val, R 19
  • Rj - R2 include conservative amino acid variants for the amino acids listed above.
  • Ri is Ala, Asn, Leu, or a conservative variant of either
  • R 2 is Leu, He, or a conservative variant of either
  • R 3 is Thr, Gin, or a conservative variant of either
  • R 4 is Thr, Ala, Lys, Val, or a conservative variant of either
  • R5 is Pro, Ser, or a conservative variant of either
  • Rg is Ala, Val, Pro, or a conservative variant thereof
  • R 7 is Lys, Glu, or a conservative variant of either
  • R 8 is Ser, Ala, or a conservative variant of either
  • R is Glu, Ala, or a conservative variant of either
  • Rio is absent or is Ala, Glu, Asp, or a conservative variant thereof
  • Rn is Tyr, Ala, Val, or a conservative variant of either
  • R 12 is Arg, Gly, Ala, or a conservative variant thereof
  • Ri 3 is Pro, Thr, Ser
  • first peptide chain and the second peptide chain are each provided separately.
  • first peptide chain and the second peptide chain are provided together in the form of a polypeptide comprising the first peptide chain and the second peptide chain, wherein the first peptide chain is bound to the second peptide chain by one interchain disulfide bond.
  • the thiol group of each cysteine residue present in the first and second chains may be protected with a suitable protecting group, provided that at least one cysteine residue in the first peptide chain and at least one cysteine residue in the second peptide chain are available to form the one or more interchain disulfide bonds.
  • one or more of the cysteine residues present in either the first peptide chain, the second peptide chain, or in both the first peptide chain and the second peptide chain are protected with one or more suitable protecting groups. In one embodiment, one or more of the cysteine residues present in both the first peptide chain and the second peptide chain are protected with one or more suitable protecting groups.
  • Cys 4 and Cysi are each independently protected with a suitable protecting group or Cys 2 and Cys 6 are each independently protected with a suitable protecting group. In one exemplary embodiment, Cys 4 and Cysi are each independently protected with a suitable protecting group. In another embodiment, Cys 2 and Cys 6 are each independently protected with a suitable protecting group.
  • Cys 3 or Cys 5 are each independently protected with a suitable protecting group or bound together in an intrachain disulfide bond.
  • Cys 3 or Cys 5 are each independently protected with a suitable protecting group or bound together in an intrachain disulfide bond; and Cys 4 and Cysi are each independently protected with a suitable protecting group or bound together in an interchain disulfide bond; or Cys 2 and Cys 6 are each independently protected with a suitable protecting group or bound together in an interchain disulfide bond.
  • Cys 3 or Cys 5 are each bound together in an intrachain disulfide bond; and Cys 4 and Cysi are each independently protected with a suitable protecting group or Cys 2 and Cys 6 are each independently protected with a suitable protecting group.
  • Cys 3 or Cyss are each bound together in an intrachain disulfide bond and Cys 4 and Cysi are each independently protected with a suitable protecting group.
  • Cys 3 or Cys 5 are each bound together in an intrachain disulfide bond and Cys 2 and Cys 6 are bound together in an interchain disulfide bond.
  • one or more of the cysteine residues in the first peptide chain, the second peptide chain, or in both the first peptide chain and the second peptide chain are protected with one or more suitable protecting groups removable under the same conditions.
  • the suitable protecting groups removable under the same reaction conditions are identical.
  • two or more of the cysteine residues present in either the first peptide chain, the second peptide chain, or in both the first peptide chain and the second peptide chain are with a suitable protecting group.
  • the two or more cysteine residues are differentially protected with two or more suitable protecting groups such that one or more protecting groups may be selectively removed in the presence of the other protecting groups (i.e. without removing the other protecting groups) by the judicious choice of reaction conditions.
  • Cys 4 is differentially protected with respect to Cys 3 and Cys 5 .
  • Cys 6 is differentially protected with respect to Cys 3 and Cys 5 .
  • Cysi and Cys 4 are differentially protected with respect to Cys 3 and Cys 5 .
  • Cys 2 and Cys 6 are differentially protected with respect to Cys 3 and Cys 5 .
  • Cysi is differentially protected with respect to Cys 4 or Cys 2 is differentially protected with respect to Cys 6 .
  • the protecting groups for the cysteine residues are selected from the group consisting of trityl, acetamidomethyl, tert-butyl, tert-butylthio, xanthyl, picolyl, and 4- methoxytrityW-methylbenzyl.
  • the protecting groups are selected from the group consisting of trityl, acetamidomethyl, tert-butyl, tert-butylthio, and 4-methoxytrityl4- methylbenzyl.
  • the protecting groups are selected from the group consisting of trityl, acetamidomethyl, and tert-butyl.
  • Cysi and Cys 4 are each protected by an acetamidomethyl group.
  • Cys 6 is protected with a tert-butyl group.
  • Cys 3 and Cyss are each protected by a trityl.
  • one or more of the amino acids other than cysteine in the first peptide chain, the second peptide chain, or in both the first peptide chain and the second peptide chain are protected with one or more suitable protecting groups.
  • the one or more suitable protecting groups can be selectively removed without removing one or more cysteine protecting groups.
  • At least one cysteine residue in the first peptide chain and at least one cysteine residue in the second peptide chain must be available to form an interchain disulfide bond.
  • one cysteine residue in the first peptide chain and one cysteine residue in the second peptide chain are available to form an interchain disulfide bond.
  • the other cysteine residues present in the chains are protected with one or more suitable protecting groups or bound in one or more intrachain or interchain disulfide bonds.
  • the other cysteine residues present in the chains protected with one or more protecting groups are differentially protected with two or more protecting groups.
  • Cys 4 and Cysi are each independently protected with a suitable protecting group or are bound together in an interchain disulfide bond and Cys 2 and Cys 6 are available to form an interchain disulfide bond; or Cys 2 and Cys 6 are each independently protected with a suitable protecting group or bound in an interchain disulfide bond and Cys 4 and Cysi are available to form an interchain disulfide bond. In one embodiment, Cys 4 and Cysi are each independently protected with a suitable protecting group and Cys 2 and Cys 6 are available to form an interchain disulfide bond; or Cys 2 and Cys 6 are each independently protected with a suitable protecting group and Cys 4 and Cysi are available to form an interchain disulfide bond.
  • Cys 4 and Cysi are each independently protected with a suitable protecting group and Cys 2 and Cys 6 are available to form an interchain disulfide bond. In another exemplary embodiment, Cys 4 and Cysi are available to form an interchain disulfide bond and Cys 2 and Cys 6 are bound together in an interchain disulfide bond.
  • Cys 3 and Cyss are each independently protected with one or more suitable protecting groups or are bound together in an intrachain disulfide bond.
  • Cys 3 and Cys 5 are bound together in an intrachain disulfide bond; Cys 4 and Cysi are each independently protected with a suitable protecting group; and Cys 2 and Cys 6 are available to form an interchain disulfide bond.
  • Cys 3 and Cys 5 are bound together in an intrachain disulfide bond; Cys 4 and Cysi are available to form an interchain disulfide bond; and Cys 2 and Cys 6 are bound together in an interchain disulfide bond.
  • At least one of the cysteine residues in the first and second peptide chains available to form a disulfide bond are in the form of a thiol. In one exemplary embodiment, both of the cysteine residues in the first and second peptide chains available to form a disulfide bond are in the form of a thiol. In another exemplary embodiment, one of the cysteine residues in the first and second peptide chains available to form a disulfide bond is in the form of a thiol and the other is in a form activated towards formation of a disulfide bond. In one specifically contemplated embodiment, the activated form is a pyridyldisulfide derivative of the thiol group.
  • the first chain comprises an intrachain disulfide bond. In one specifically contemplated embodiment, the first chain comprises an intrachain disulfide bond between Cys 3 and Cys 5 .
  • the first peptide chain, the second peptide chain or both the first peptide chain and the second peptide chain additionally comprises one or more solubilising groups. In one exemplary embodiment, the first peptide chain comprises one or more solubilising groups.
  • the solubilising group enhances the solubility of the peptide chain in the reaction medium. In another embodiment, the solubilising group prevents or inhibits intrachain association.
  • the one or more solubilising groups are linked to the C-termini of the peptide chain, optionally via a suitable linker.
  • linkers examples include aryl linkers, for example 4-hydroxymethyl benzoic acid or 4-hydrazinobenzoic acid. Other suitable linkers will be apparent to those skilled in the art.
  • the linker is bound to the C-termini of the peptide via a ester, thioester, or amide bond. The nature of the bond will depend on the linker used.
  • the solubilising group is a polycationic amino acid sequence.
  • the cationic amino acids are arginine or lysine residues.
  • the sequence comprises from 2 to 20, 2 to 15, 2 to 10, 3 to 7, or 3 to 5 amino acids.
  • the solubilising group is a poly-lysine or poly-arginine tag.
  • the solubilising group is a tri-, terra-, penta-, hexa-, or hepta- lysine or arginine tag.
  • the solubilising group is a pentalysine tag.
  • the a pentalysine tag is linked to the C-termini of the peptide chain via a 4- hydroxymethyl benzoic acid (HMBA) linker.
  • HMBA 4- hydroxymethyl benzoic acid
  • first peptide chain, the second peptide chain, or both the first peptide chain and the second peptide chain are bound to a solid phase support, optionally via a suitable linker.
  • linkers examples include the Rink amide linker, phenylacetamido (PAM) linker, Sheppard's linker, and Wang ester linker. Other suitable linkers will be apparent to those skilled in the art.
  • the peptide chain is bound to the solid phase support via a solubilising group linked to the C-termini of the peptide chain.
  • the solubilising group is bound to the C-termini of the peptide chain via a suitable linker.
  • the solubilising group is bound to the solid phase support via a suitable linker.
  • one of the one or more interchain bonds is formed between Cysi and Cys 4 , or is formed between Cys 2 and Cys 6 .
  • the method comprises forming an interchain disulfide bond between Cysi and Cys 4 and between Cys 2 and Cys 6 .
  • the method comprises first forming an interchain disulfide bond between Cysi and Cys 4 , optionally followed by an interchain disulfide bond between Cys 2 and Cys 6 .
  • the method comprises first forming an interchain disulfide bond between Cys 2 and Cys 6 , optionally followed by forming an interchain disulfide bond between Cysi and Cys 4 .
  • the method comprises first forming an interchain disulfide bond between Cys 2 and Cys 6 , then forming an interchain disulfide bond between Cysi and Cys 4 .
  • the method comprises first forming an intrachain disulfide bond between Cys 3 and Cyss, then forming an interchain disulfide bond between Cys 2 and Cys 6 or forming an interchain disulfide bond between Cysi and Cys 4 , and then forming an interchain disulfide bond between the other of Cysi and Cys 4 or Cys 2 and Cys 6 .
  • the method comprises first forming an interchain disulfide bond between Cys 2 and Cys 6 or forming an interchain disulfide bond between Cysi and Cys 4 , and then forming an interchain disulfide bond between the other of Cysi and Cys 4 or Cys 2 and Cys 6 , and then forming an intrachain disulfide bond between Cys 3 and Cys 5 .
  • the method comprises first forming an interchain disulfide bond between Cys 2 and Cys 6 or forming an interchain disulfide bond between Cysi and Cys 4 , then forming an intrachain disulfide bond between Cys 3 and Cys 5 , and then forming an interchain disulfide bond between the other of Cysi and Cys 4 or Cys 2 and Cys 6 ,
  • the method comprises first forming an intrachain disulfide bond between Cys 3 and Cyss, then forming an interchain disulfide bond between Cys 2 and Cys 6 , and then forming an interchain disulfide bond between Cysi and Cys 4 .
  • the one or more interchain disulfide bonds are formed under oxidative conditions.
  • Any suitable oxidant or combination of oxidants may be used to provide the oxidative conditions.
  • suitable oxidants include dipyridyldisulfide, iodine, thallium(III) trifluoroacetate, molecular oxygen, dimethylsulfoxide, and the like.
  • the oxidant is dipyridyldisulfide or iodine.
  • the oxidant is dipyridyldisulfide.
  • the oxidant is iodine.
  • the reaction medium is a liquid reaction medium.
  • the liquid reaction medium comprises one or more suitable solvents.
  • suitable solvents include dimethylformamide, dichloromethane, chloroform, carbon tetrachloride, water, methanol, ethanol, dimethylsulfoxide, trifluoroacetic acid, acetic acid, acetonitrile, and mixtures thereof.
  • the liquid reaction medium comprises one or more buffers, for example a phosphate, citrate, guanidine, 2-amino-2-hydroxymethyl-propane-l,3-diol (Tris) buffer, carbonate, or 4-(2-hydroxyethyl)-l-piperzineethanesulfonic acid (HEPES).
  • buffers for example a phosphate, citrate, guanidine, 2-amino-2-hydroxymethyl-propane-l,3-diol (Tris) buffer, carbonate, or 4-(2-hydroxyethyl)-l-piperzineethanesulfonic acid (HEPES).
  • the reaction medium is at a temperature below ambient temperature. In one embodiment, the reaction medium is at a temperature from -75 to 15 °C, from -50 to 10 °C, or from -20 to 5 °C. In one embodiment, the reaction medium is at a temperature less than 15 °C, 10 °C, 5 °C, less than 0 °C, less than -10 °C, or less than -20 °C. In one embodiment, the reaction medium is at a temperature from -10 to 5 °C, for example 0 °C.
  • the reaction medium may be cooled by any suitable method known in the art, for example, immersing a vessel containing the reaction medium in an ice bath.
  • the reaction medium is at a temperature above ambient temperature. In one embodiment, the reaction medium is at a temperature from 40 to 200 °C, from 50 to 150 °C, from 60 to 100 °C, from 65 to 90 °C, or from 70 to 80 °C. In one embodiment, the reaction medium is at a temperature greater than 40 °C, greater than 50 °C, greater than 75 °C, greater than 100 °C, or greater than 150 °C.
  • the reaction medium may be heated using any suitable method known in the art, for example, immersing a vessel containing the reaction medium in a heated oil bath. The temperature used may depend on, for example, the boiling points and degradation of solvents present in the reaction medium.
  • the reaction medium is irradiated with microwave irradiation. In another embodiment, the reaction medium is irradiated with ultraviolet light.
  • the disulfide bonds are formed under an atmosphere of ambient gas.
  • the ambient gas is selected from the group consisting of nitrogen and argon.
  • the disulfide bonds are formed under an atmosphere of oxygen gas.
  • the reaction medium is mixed.
  • the reaction medium may be mixed by any suitable method known in the art, for example, using a magnetic stirrer in the reaction medium or agitating a vessel containing the reaction medium, for example using a vortex mixer.
  • the progress of the disulfide bond forming reactions may be monitored by any suitable means, for example HPLC.
  • the reaction is allowed to proceed to completion, as monitored by the consumption of at least one of the starting materials by HPLC. In one embodiment, the reaction is allowed to proceed for a period of time from 1 minute to 7 days, 5 minutes to 72 hours, 10 minutes to 48 hours, 15 minutes to 24 hours. In another embodiment, the reaction is allowed to proceed for a period of time less than 72 h, less than 48 h, less than 24 h, less than 12 h, less than 6 h, less than 4 h, less than 2 h, or less than 1 h.
  • the conducive conditions for forming each interchain disulfide bond are different. In some embodiments where the method comprises forming more than one interchain disulfide bond, the conducive conditions for forming each interchain disulfide bond are the same. In some embodiments where the method comprises forming more than one interchain disulfide bond, more than one interchain disulfide bonds are formed in the same reaction.
  • the method comprises forming under conducive conditions an intrachain disulfide bond in the first peptide chain.
  • the intrachain disulfide may be formed under any of the conditions conducive to formation of the one or more interchain disulfide bonds described herein.
  • the method comprises removing one or more cysteine protecting groups in the in the first peptide chain, second peptide chain, or both the first peptide chain and the second peptide chain to provide one or more thiol groups.
  • the method comprises converting the thiol group of one or more cysteine residues in the first peptide chain, second peptide chain, or both the first peptide chain and the second peptide chain, into a form activated towards formation of a disulfide bond.
  • the method comprises recovering and optionally purifying the activated form.
  • the method comprises cleaving the solubilising group and optional linker.
  • the method comprises cleaving the solid phase support and optional linker.
  • the polypeptide may be recovered from the reaction medium by any suitable method known in the art.
  • the polypeptide is recovered after forming one interchain disulfide bond and, optionally, purified. In one embodiment, the polypeptide is recovered after forming two interchain disulfide bonds and, optionally purified.
  • the polypeptide is recovered and, optionally, purified, after forming each interchain disulfide bond. In another embodiment, the polypeptide is recovered after forming an intrachain disulfide bond in the first peptide chain and two interchain disulfide bonds between the first peptide chain and the second peptide chain, and optionally purified. In one embodiment, the polypeptide is recovered and optionally purified after forming an intrachain disulfide bond in the first peptide chain and after forming each interchain disulfide bond between the first peptide chain and the second peptide chain.
  • the method comprises recovering and optionally purifying the first peptide chain after forming the intrachain disulfide bond.
  • recovering the polypeptide from the reaction medium comprises precipitating the polypeptide and optionally separating the polypeptide from the reaction medium.
  • the precipitated polypeptide may be separated from the reaction medium by for example, centrifuging and decanting or filtering the reaction medium.
  • recovering the polypeptide comprises separating the solid phase support from the reaction medium, for example by decanting or filtering the reaction medium. In one embodiment, recovering the polypeptide comprises cleaving the polypeptide from the solid phase support.
  • recovering the polypeptide comprises cleaving the solubilising group from the polypeptide.
  • recovering the polypeptide comprises cleaving the peptide chain from the linker.
  • the method comprises purifying the polypeptide after recovering the polypeptide from the reaction medium.
  • the polypeptide is purified by HPLC using one or more suitable solvents.
  • the first peptide chain, the second peptide chain, or both the first peptide chain and the second peptide chain are synthesized using solid phase peptide synthesis.
  • the peptide chains are synthesized by stepwise solid phase peptide synthesis or sequential solid phase fragment condensation. In exemplary embodiments, the peptide chains are synthesized by stepwise solid phase peptide synthesis. In one embodiment, the peptide chains are synthesized by Fmoc or Boc solid phase peptide synthesis.
  • the synthesis comprises assembling the amino acid sequences of the peptide chains on a suitable solid phase support.
  • the solid phase support is a polyethylene glycol resin or a polystyrene resin.
  • the amino acid sequence of the peptide chain is assembled on the solid phase support via a suitable linker.
  • the synthesis comprises binding the linker to the solid phase support.
  • the synthesis comprises assembling the amino acid sequence of the peptide chain and incorporating one or more solubilising groups. In one embodiment, the synthesis comprises binding the solubilising group, optionally via a suitable linker, to the solid phase support and assembling the amino acid sequence of the peptide chain on the solubilising group. In one embodiment, the synthesis comprises assembling the amino acid sequence of the peptide chain on the solubilising group via a suitable linker.
  • the side chains of the amino acids incorporated into the peptide chain may be protected by one or more suitable protecting groups.
  • the protecting groups are selected having regard to the overall strategy for synthesizing the polypeptide, for example, for example the conditions used for synthesising the peptide chain, cleaving the peptide from the solid phase support, and forming the one or more interchain disulfide bonds.
  • one or more of the cysteine residues of the peptide chain are differentially protected with one or more suitable protecting groups.
  • the synthesis comprises cleaving the peptide chain from the solid phase support.
  • the peptide chain is bound to the solid phase support via a suitable linker, the peptide chain is cleaved from the solid phase support by cleaving the peptide chain from the linker.
  • the synthesis comprises cleaving the solubilising group from the solid phase support.
  • the solubilising group is bound to the solid phase support via a suitable linker, the solubilising group is cleaved from the linker.
  • the synthesis comprises cleaving the one or more solubilising groups.
  • the solubilising group is linked to the peptide chain via a suitable linker, the peptide chain is cleaved from the linker.
  • the synthesis comprises removing the Na-amino protecting group of the N-terminal amino acid of the peptide chain. In one embodiment, the Na-amino protecting group of the N-terminal amino acid of the peptide chain is removed on cleaving the peptide from the linker bound to the solid phase support.
  • the synthesis comprises removing one or more amino acid side chain protecting groups.
  • the one or more protecting groups are removed while the peptide chain is bound to the solid phase support.
  • the one or more protecting groups are removed on cleaving the peptide chain from the solid phase support.
  • the one or more protecting groups are removed after cleaving the peptide chain from the solid phase support.
  • the synthesis comprises one or more purification steps.
  • the peptide chain is purified after it has been cleaved from the solid phase support.
  • the peptide chain is purified by HPLC using one or more suitable solvents.
  • synthesis of the first peptide chain or the second peptide chain comprises converting the thiol group of one or more cysteine residues into a form activated towards formation of an interchain disulfide bond.
  • the first peptide chain is synthesized using solid phase peptide synthesis.
  • the first peptide is synthesized using Fmoc solid phase peptide synthesis.
  • the synthesis comprises assembling the amino acid sequence of the first peptide on the solid phase support, optionally via a suitable linker.
  • the synthesis comprises assembling the amino acid sequence of the first peptide and incorporating one or more solubilising groups. In one embodiment, the synthesis comprises binding the solubilising group to the solid phase support, optionally via a suitable linker, and assembling the amino acid sequence of the first peptide chain on the solubilising group. In one embodiment, synthesis comprises assembling the amino acid sequence of the peptide chain on the solubilising group via a suitable linker.
  • the amino acid sequence of the first peptide chain is assembled using amino acids optionally protected with one or more suitable protecting groups.
  • each cysteine in the amino acid sequence is optionally protected with one or more suitable protecting groups.
  • at least Cys 4 and Cys 6 are differentially protected with respect to Cys 3 and Cys 5 .
  • Cys 4 and Cys 6 are differentially protected.
  • the synthesis comprises cleaving the first peptide chain from the solid phase support. In some embodiments where the first peptide chain is bound to the solid phase support via a suitable linker, the first peptide chain is cleaved from the linker. In some embodiments where the first peptide chain comprises a solubilising group bound to the solid phase support via a suitable linker, the synthesis comprises cleaving the solubilising group from the solid phase support to provide a first peptide chain comprising the solubilising group.
  • the synthesis comprises cleaving the solubilising group from the first peptide chain.
  • the amino acid sequence of the first peptide chain is bound to the solubilising group via a suitable linker, the first peptide chain is cleaved from the linker.
  • the synthesis comprises removing one or more amino acid side chain protecting groups, including one or more cysteine protecting groups prior to, during, or after cleavage of the first peptide from the solid phase support. In one embodiment, the synthesis comprises selectively removing one or more amino acid side chain protecting groups without removing one or more cysteine protecting groups.
  • the one or more amino acid side chain protecting groups are selected from the group consisting of acetamidomethyl, 2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), tert-butyl, trityl, and tert-butyloxycarbonyl.
  • the side chains of one or more arginine residues are protected with one or more Pbf groups; the side chains of one or more aspartic acid, glutamic acid, serine, threonine, or tyrosine residues are protected with one or more tert-butyl groups; the side chains of one or more asparagine, glutamine, or histadine residues are protected with one or more trityl groups; the side chains of one or more cysteine residues are protected with one or more trityl, acetamidomethyl, or tert-butyl groups; and the side chains of one or more tryptophan or lysine residues are protected with one or more tert-butylcarbonyloxy groups.
  • the one or more cysteine protecting groups are selected from the group consisting of tert-butyl, acetamidomethyl, and trityl.
  • the synthesis comprises removing one or more cysteine protecting groups.
  • the synthesis comprises converting the thiol group of one or more cysteine residues in the first peptide chain into a form activated towards formation of a disulfide bond.
  • the method comprises recovering and optionally purifying the activated form.
  • the synthesis comprises purifying the first peptide after cleaving the peptide from the solid phase support. In one embodiment, purification is carried out by HPLC using a suitable solvent system.
  • the synthesis of the first peptide chain comprises forming an intrachain disulfide bond.
  • the intrachain disulfide bond is between Cys 3 and Cys 5 .
  • the first peptide chain is synthesized using solid phase peptide synthesis as described herein in the examples.
  • the second peptide chain is synthesized using solid phase peptide synthesis. In one embodiment, the second peptide is synthesized using Boc solid phase peptide synthesis. In one embodiment, the synthesis comprises assembling the amino acid sequence of the second peptide chain on the solid phase support, optionally via a suitable linker.
  • the amino acid sequence of the second peptide chain is assembled using amino acids optionally protected with one or more suitable protecting groups.
  • each cysteine in the amino acid sequence is optionally protected with one or more suitable protecting groups.
  • at least one of Cysi and Cys 2 is protected with one or more suitable protecting groups.
  • Cysi and Cys 2 are differentially protected with one or more suitable protecting groups.
  • Cysi is protected with a suitable protecting group.
  • the synthesis comprises cleaving the second peptide chain from the solid phase support.
  • the synthesis comprises removing one or more amino acid side chain protecting groups, including one or more cysteine protecting groups prior to, during, or after cleavage of the second peptide chain from the solid phase support.
  • the synthesis comprises removing one or more cysteine protecting groups.
  • the one or more amino acid side chain protecting groups are selected from the group consisting of xanthyl, tosyl, benzyl, 2-bromobenzyl, cyclohexyl, 4- methylbenzyl, acetamidomethyl.
  • the side chains of one or more asparagine residues is protected with one or more xanthyl groups; the side chains of one or more arginine residues is protected with one or more tosyl groups; the side chains of one or more serine or threonine residues is protected with one or more benzyl groups; the side chains of one or more tyrosine residues is protected with one or more 2-bromobenzyl groups; the side chains of one or more aspartic acid or glutamic acid residues is protected with one or more cyclohexyl groups; the side chains of one or more cysteine groups is protected with one or more 4-methylbenzyl or acetamidomethyl groups.
  • the one or more cysteine protecting groups are selected from the group consisting of tert-butyl, acetamidomethyl, 4-methylbenzyl, and trityl. In one embodiment, the one or more cysteine protecting groups are selected from the group consisting of tert-butyl, acetamidomethyl, and trityl. In one embodiment, the one or more cysteine protecting groups are selected from the group consisting of 4-methylbenzyl and acetamidomethyl. In one embodiment, the one or more cysteine protecting groups are acetamidomethyl groups.
  • the synthesis comprises purifying the cleaved second peptide chain.
  • purification is carried out by HPLC using a suitable solvent system.
  • the solvent system comprises formic acid.
  • the second peptide chain is synthesized using solid phase peptide synthesis as described herein in the examples.
  • the invention relates to a method of synthesizing a polypeptide comprising an amino acid sequence corresponding to A chain : Gly He Val Glu Glu Cys 3 Cys 4 Phe Arg Ser Cys 5 Asp Leu Ri R 2 Leu Glu R 3 Tyr Cys 6 Ala R4 R 5 Rg R 7 Rg R 9 (SEQ ID NO: 1)
  • Ri is Ala, Asn or Leu; R 2 is Leu or He; R 3 is Thr or Gin; R 4 is Thr, Ala, Lys, or Val; R 5 is Pro or Ser; R 6 is Ala, Val, or Pro; R 7 is Lys or Glu, R 8 is Ser or Ala, and R9 is Glu or Ala, the method essentially as described herein in the examples.
  • the invention relates to a method of synthesizing a polypeptide comprising an amino acid sequence corresponding to
  • R 10 is absent or is Ala, Glu, or Asp, Rn is Tyr, Ala, or Val, Ri 2 is Arg, Gly, or Ala, R13 is Pro, Thr, Ser, or Leu, Ri 4 is Ser, Gly, Ala, or Glu, R15 is Thr or Ala, Ri 6 is Val or He, Rn is Gly, Ser, Glu, or Ala, Ri 8 is Asp or Glu, R1 is Ser or Val, R 2 o is Arg, Leu, or Ser, R 21 is Pro or Lys, R 22 is Ala, Ser, Gly, Val, or Thr, R 23 is Ser, Gly, or Val, R 24 is Arg, Pro or Gly, R 25 is Ala, Arg, Val, He, Leu, Asn, Ser, or Gly, R 26 is Ser, Asn, or Arg, R 27 is absent or is Arg, Ser, Asn, R 28 is absent or is Val, R 2 is absent or is Ser, the method essentially as described herein in
  • a synthetic polypeptide comprising an amino acid sequence corresponding to
  • a chain Gly He Val Glu Glu Cys 3 Cys 4 Phe Arg Ser Cys 5 Asp Leu Ri R 2 Leu Glu R 3
  • Ri is Ala, Asn or Leu; R 2 is Leu or He; R 3 is Thr or Gin; R 4 is Thr, Ala, Lys, or Val; R 5 is Pro or Ser; R 6 is Ala, Val, or Pro; R 7 is Lys or Glu, Rg is Ser or Ala, and R9 is Glu or Ala; or an amino acid sequence corresponding to
  • R 10 is absent or is Ala, Glu, or Asp, Rn is Tyr, Ala, or Val, Ri 2 is Arg, Gly, or Ala, R13 is Pro, Thr, Ser, or Leu, Ri 4 is Ser, Gly, Ala, or Glu, R15 is Thr or Ala, Ri 6 is Val or He, Rn is Gly, Ser, Glu, or Ala, Rig is Asp or Glu, R19 is Ser or Val, R 2 o is Arg, Leu, or Ser, R 2 i is Pro or Lys, R 22 is Ala, Ser, Gly, Val, or Thr, R 23 is Ser, Gly, or Val, R 24 is Arg, Pro or Gly, R 25 is Ala, Arg, Val, He, Leu, Asn, Ser, or Gly, R 26 is Ser, Asn, or Arg, R 27 is absent or is Arg, Ser, Asn, R 2 g is absent or is Val, R 2 9 is absent or is Ser.
  • Ri 10 is
  • the synthetic polypeptide is bound to a solid phase support, optionally via a suitable linker.
  • the invention provides a synthetic polypeptide wherein the synthetic polypeptide comprises an A chain as described herein bound to a B chain as described herein via at least one interchain disulfide bond, wherein either the A chain or the B chain, or both the A chain and the B chain is bound to a solid phase support, optionally via a suitable linker.
  • one or more amino acids of the polypeptide are protected by one or more suitable protecting groups.
  • Ri- R 2 include conservative amino acid variants for the amino acids listed above.
  • Ri is Ala, Asn, Leu, or a conservative variant of either
  • R 2 is Leu, He, or a conservative variant of either
  • R 3 is Thr, Gin, or a conservative variant of either
  • R 4 is Thr, Ala, Lys, Val, or a conservative variant of either
  • R 5 is Pro, Ser, or a conservative variant of either
  • R ⁇ is Ala, Val, Pro, or a conservative variant thereof
  • R 7 is Lys, Glu, or a conservative variant of either
  • R 8 is Ser, Ala, or a conservative variant of either
  • R is Glu, Ala, or a conservative variant of either
  • Rio is absent or is Ala, Glu, Asp, or a conservative variant thereof
  • R11 is Tyr, Ala, Val, or a conservative variant of either
  • Ri 2 is Arg, Gly, Ala, or a conservative variant thereof
  • Ri 3 is Pro, Thr, Ser, Le
  • the synthetic polypeptides of the invention may be pure or purified, or substantially pure.
  • the synthetic polypeptide has insulin agonist activity.
  • insulin agonist activity is a capability of binding to a receptor to which insulin binds, or eliciting a response mediated by a receptor to which insulin binds.
  • the synthetic polypeptide binds a receptor to which insulin binds with at least about 10%, at least about 15%, at least about 20%, or at least about 25% the affinity as does insulin.
  • the receptor to which insulin binds is the insulin receptor.
  • the synthetic polypeptide binds a receptor to which insulin binds with a binding affinity of at least 10 7 , 10 8 , 10 9 , or 10 10 M "1 .
  • the synthetic polypeptide has an EC 5 o for effecting a response mediated by the insulin receptor (such as, for example, an effect on carbohydrate metabolism or an effect on cell growth/proliferation and cytoprotection) less than about two hundred-fold that of insulin.
  • synthetic vesiculin A chain polypeptides are provided, for example, human synthetic vesiculin A chain polypeptides. Also provided are synthetic vesiculin A chain polypeptide intermediates.
  • synthetic vesiculin B chain polypeptides are provided, for example, human synthetic vesiculin B chain polypeptides. Also provided are synthetic vesiculin B chain polypeptide intermediates.
  • Synthetic vesiculin A and B chain polypeptides and intermediates may be pure or purified, or substantially pure.
  • synthetic vesiculin and “synthetic vesiculin polypeptide(s)” are used interchangeably herein and refer to a synthetic polypeptide comprising a two chain peptide having insulin agonist activity, comprising a synthetic vesiculin A chain polypeptide and a synthetic vesiculin B chain polypeptide.
  • the naturally-occuring human vesiculin sequence corresponding to that of one embodiment of a synthetic vesiculin as contemplated herein, may be represented as follows:
  • a chain Gly He Val Glu Glu Cys 3 Cys 4 Phe Arg Ser Cys 5 Asp Leu Ala Leu Leu Glu
  • Alai is either present or absent.
  • Variants of synthetic vesiculin include, for example: A chain : Gly He Val Glu Glu Cys 3 Cys 4 Phe Arg Ser Cys 5 Asp Leu Ala Leu Leu Glu
  • Alai is either present or absent; Ri is Gly or Arg; R 2 is Gly or Ser; R 3 is Gly or Ser; R 4 is Ser or Ala; R 5 is He or Val or Ala either; R 6 is Asn or Ser; and R 7 is Thr or Ala.
  • Alai is either present or absent; Ri is Gly or Arg, or a conservative variant of either; R 2 is Gly or Ser, or a conservative variant of either; R 3 is Gly or Ser, or a conservative variant of either; R 4 is Ser or Ala, or a conservative variant of either; R5 is He or Val or Ala, or a conservative variant of either; R 6 is Asn or Ser, or a conservative variant of either; and R 7 is Thr or Ala, or a conservative variant of either.
  • Additional vesiculin variants include, for example:
  • a chain Gly He Val Glu Glu Cys 3 Cys 4 Phe Arg Ser Cys 5 Asp Leu Ri R 2 Leu Glu R 3
  • Ri is Ala, Asn or Leu; R 2 is Leu or He; R 3 is Thr or Gin; R4 is Thr, Ala, Lys, or Val; R 5 is Pro or Ser; R5 is Ala, Val, or Pro; R 7 is Lys or Glu, R 8 is Ser or Ala, R 9 is Glu or Ala, R 10 is absent or is Ala, Glu, or Asp, Rn is Tyr, Ala, or Val, R12 is Arg, Gly, or Ala, R13 is Pro, Thr, Ser, or Leu, Ri 4 is Ser, Gly, Ala, or Glu, R15 is Thr or Ala, Ri 6 is Val or He, Rn is Gly, Ser, Glu, or Ala, Ri 8 is Asp or Glu, R19 is Ser or Val, R 20 is Arg, Leu, or Ser, R 2 i is Pro or Lys, R 22 is Ala, Ser, Gly, Val, or Thr, R 23 is Ser, Gly, or Val, R 19
  • Ri- R29 include conservative amino acid variants for the amino acids listed above.
  • Ri is Ala, Asn, Leu, or a conservative variant of either
  • R 2 is Leu, He, or a conservative variant of either
  • R 3 is Thr, Gin, or a conservative variant of either
  • R 4 is Thr, Ala, Lys, Val, or a conservative variant of either
  • R 5 is Pro, Ser, or a conservative variant of either
  • R ⁇ is Ala, Val, Pro, or a conservative variant thereof
  • R 7 is Lys, Glu, or a conservative variant of either
  • R 8 is Ser, Ala, or a conservative variant of either
  • R9 is Glu, Ala, or a conservative variant of either
  • R 10 is absent or is Ala, Glu, Asp, or a conservative variant thereof
  • Rn is Tyr, Ala, Val, or a conservative variant of either
  • Ri 2 is Arg, Gly, Ala, or a conservative variant thereof
  • R13 is Pro, Thr, Ser
  • the synthetic vesiculin comprises A and B chains joined by at least one inter-chain disulfide bond.
  • the synthetic vesiculin may include disulfide bonds formed between any one of Cysi, Cys 2 , Cys 3 , Cys 4 , Cys 5 and Cys 6 residues.
  • the synthetic vesiculin comprises A and B chains joined by two inter-chain disulfide bonds.
  • the synthetic vesiculin includes disulfide bonds formed between residues Cysi and Cys 4 , and Cys 2 and Cys 6 .
  • the synthetic vesiculin comprises an intra-chain disulfide bond in chain A between residues Cys 3 and Cys 5 .
  • the synthetic vesiculin comprises A and B chains joined by one or more inter-chain disulfide bonds and an A-chain intra-chain disulfide bond.
  • the synthetic vesiculin includes disulfide bonds formed between residues Cysi and Cys 4 , and Cys 2 and Cys 6 , and Cys 3 and Cys 5 .
  • the vesiculin variant comprises one or more solubilising groups bound to the amino acid sequence of the A chain, the B chain, or both the A chain and the B chain, optionally via a suitable linker.
  • the vesiculin variant is bound to a solid phase support, optionally via a suitable linker.
  • one or more amino acids of the A chain, the B chain, or both the A chain and the B chain are protected by one or more suitable protecting groups.
  • the invention relates to one or more synthetic vesiculin polypeptide intermediates, wherein the one or more synthetic vesiculin polypeptide intermediates comprises a resin-bound polypeptide comprising amino acid sequence corresponding to
  • a chain Gly He Val Glu Glu Cys 3 Cys 4 Phe Arg Ser Cys 5 Asp Leu Ri R 2 Leu Glu R 3
  • the synthetic vesiculin polypeptide intermediate includes a disulfide bond formed between residues Cys 3 and Cys 5 .
  • the invention relates to one or more synthetic vesiculin polypeptide intermediates, wherein the one or more synthetic vesiculin polypeptide intermediates comprises a resin-bound polypeptide comprising or consisting of amino acid sequence corresponding to
  • R 10 is absent or is Ala, Glu, or Asp, Rn is Tyr, Ala, or Val, Ri 2 is Arg, Gly, or Ala, Ri 3 is Pro, Thr, Ser, or Leu, Ri 4 is Ser, Gly, Ala, or Glu, R15 is Thr or Ala, Ri 6 is Val or He, R 17 is Gly, Ser, Glu, or Ala, Ri 8 is Asp or Glu, R19 is Ser or Val, R20 is Arg, Leu, or Ser, R 21 is Pro or Lys, R22 is Ala, Ser, Gly, Val, or Thr, R 23 is Ser, Gly, or Val, R 24 is Arg, Pro or Gly, R25 is Ala, Arg, Val, He, Leu, Asn, Ser, or Gly, R26 is Ser, Asn, or Arg, R 27 is absent or is Arg, Ser, Asn, R2 8 is absent or is Val, R2 is absent or is Ser.
  • synthetic vesiculin intermediates include those comprising or consisting of at least 12 amino acids having an amino acid sequence corresponding to the 12 C- terminal amino acids of a vesiculin polypeptide A chain.
  • exemplary synthetic vesiculin human A chain intermediates include, for example, the following polypeptides:
  • ALLETYCATPAKSE (SEQ ID NO:9);
  • CDLALLETYCATPAKSE (SEQ ID NO: 12);
  • RSCDLALLETYCATPAKSE SEQ ID NO: 14
  • FRSCDLALLETYCATPAKSE SEQ ID NO: 15
  • VEECCFRSCDLALLETYC ATP AKSE (SEQ ID NO:20);
  • Additional specifically contemplated synthetic vesiculin intermediates include those comprising or consisting of at least 12 amino acids having an amino acid sequence corresponding to the 12 C-terminal amino acids of a vesiculin polypeptide A chain, wherein the N-terminal amino acid is Na-protected by a protecting group.
  • the N-terminal amino acid is Na-protected by a protecting group.
  • the N- terminal amino acid is ⁇ -protected with Fmoc.
  • the functional groups in the side chains of the amino acids may also be protected with one or more protecting groups.
  • further exemplary synthetic vesiculin human A chain intermediates include, for example, the following polypeptides:
  • (pro)- is one or more protecting group, including a protecting group selected from the following: acetyl (Ac), amide, a 3 to 20 carbon alkyl group, Fmoc, 9-fluoreneacetyl group, l-fiuorenecarboxylic group, 9-fluorenecarboxylic group, 9-fluorenone-l-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4- dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4- methylbenzyl (Me
  • the Na-position of the N-terminal amino acid is protected with a (pro)- group.
  • the (pro)- is Fmoc.
  • the synthetic vesiculin human A chain intermediates comprise one or more solubilising groups bound to the amino acid sequence of the polypeptide chain, optionally via a suitable linker.
  • the synthetic vesiculin human A chain intermediates are bound to a solid phase support, optionally via a suitable linker.
  • one or more amino acids of the synthetic vesiculin human A chain intermediates are protected by one or more suitable protecting groups.
  • a chain intermediates from other species, and synthetic vesiculins comprising amino acid sequences corresponding to the vesiculin of other species incorporating those intermediates, are also provided by the invention.
  • synthetic vesiculin intermediates include those comprising or consisting of at least 12 amino acids having an amino acid sequence corresponding to the 12 C-terminal amino acids of a vesiculin polypeptide B chain.
  • exemplary synthetic vesiculin human B chain intermediates include, for example, the following polypeptides:
  • RGFYFSRPASRVS (SEQ ID NO:24);
  • GDRGFYFSRPASRVS (SEQ ID NO:26);
  • CGDRGFYFSRPASRVS SEQ ID NO:27
  • VCGDRGFYFSRPASRVS SEQ ID NO:28
  • FVCGDRGFYFSRPASRVS (SEQ ID NO:29);
  • DTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:33);
  • VDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:34);
  • ELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:36);
  • GGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:38);
  • Additional specifically contemplated synthetic vesiculin intermediates include those comprising or consisting of at least 12 amino acids having an amino acid sequence corresponding to the 12 C-terminal amino acids of a vesiculin polypeptide B chain, wherein the N-terminal amino acid is ⁇ -protected by a protecting group.
  • the N-terminal amino acid is ⁇ -protected with Boc.
  • the functional groups in the side chains of the amino acids may also be protected with one or more protecting groups.
  • further exemplary synthetic vesiculin human B chain intermediates include, for example, the following polypeptides:
  • (pro)- is one or more protecting group, including a protecting group selected from the following: acetyl (Ac), amide, a 3 to 20 carbon alkyl group, Fmoc, 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-fluorenecarboxylic group, 9-fluorenone-l-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4- dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4- methylbenzyl (MeBzl),
  • the synthetic vesiculin human B chain intermediates comprise one or more solubilising groups bound to the amino acid sequence of the polypeptide chain, optionally via a suitable linker.
  • the synthetic vesiculin human B chain intermediates are bound to a solid phase support, optionally via a suitable linker.
  • one or more amino acids of the synthetic vesiculin human B chain intermediates are protected by one or more suitable protecting groups.
  • Resin-bound intermediates such as the human synthetic vesiculin intermediates specifically disclosed herein, are also specifically contemplated.
  • vesiculin variants having an amino acid sequence that is at least about 60% identical to a vesiculin, for example, a human vesiculin.
  • a synthetic vesiculin variant may contain an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, or at least about 99% identical to a vesiculin, for example, a human vesiculin.
  • the synthetic vesiculin polypeptide or variant or derivative or synthetic intermediate thereof has a serine at position 33 of the B chain. In other embodiments, the synthetic vesiculin polypeptide or variant or derivative or synthetic intermediate thereof has an amino acid other than arginine at position 33 of the B chain. In another embodiment, the synthetic vesiculin polypeptide or variant or derivative or synthetic intermediate thereof has a serine at position 36 of the B chain. In other embodiments, the synthetic vesiculin polypeptide or variant or derivative or synthetic intermediate thereof has an amino acid other than lysine at position 36 of the B chain. In other embodiments, the synthetic vesiculin polypeptide or variant or derivative or synthetic intermediate thereof has serine residues at positions 33 and 36 of the B chain.
  • the synthetic vesiculin polypeptide or variant or derivative or synthetic intermediate thereof has an amino acid other than arginine at position 33 of the B chain, and additionally has an amino acid other than lysine at position 36 of the B chain. (The position numbering corresponds to that of human vesiculin.)
  • the invention also provides a method of determining whether a synthetic polypeptide, variant, or intermediate of the invention is useful as a therapeutic agent by determining whether the synthetic polypeptide has insulin agonist activity.
  • the method comprises contacting the synthetic polypeptide, variant, or intermediate and a receptor to which insulin binds, and determining a capability of binding to the receptor.
  • the method comprises contacting the synthetic polypeptide, variant, or intermediate and a receptor to which insulin binds, and determining a capability of eliciting a response mediated by the receptor.
  • a capability of eliciting a response mediated by the receptor is determined by a determination of the EC50 for effecting a response mediated by the insulin receptor.
  • the response is an effect on glucose incorporation into glycogen.
  • insulin agonist activity is identified by assay systems, including the soleus muscle assay, which measures the effect an agent, such as a synthetic vesiculin polypeptide, variant, or intermediate has on glucose incorporation into glycogen. Exemplary methods are described herein in the examples.
  • the invention in another aspect, relates to a method of modulating blood glucose levels in a subject.
  • the method comprises administering an effective amount of one or more of a synthetic vesiculin, or a synthetic vesiculin variant, derivative, or synthetic intermediate, or a salt of any of them, to a subject in need thereof.
  • the invention includes methods for treating and/or preventing, in whole or in part, various diseases, disorders, and conditions, including for example, impaired glucose tolerance; impaired fasting glucose; prediabetes; diabetes and/or its complications, including type 1 and type 2 diabetes and their complications; insulin resistance; Syndrome X; obesity and other weight related disorders; fatty liver disease, including non alcoholic and/or alcoholic fatty liver disease; glucose metabolism diseases and disorders; diseases, disorders or conditions that are treated or treatable with insulin; diseases, disorders or conditions that are treated or treatable with a hypoglycemic agent; diseases, disorders, and conditions characterized at least in part by hyperglycemia; diseases, disorders, and conditions characterized at least in part by hypoinsulinemia and/or diseases, disorders, and conditions characterized at least in part by hyperinsulinemia.
  • the invention includes methods for treating a subject having or suspected of having or predisposed to, or at risk for, for example, any diseases, disorders and/or conditions characterized in whole or in part by hyperglycemia, insulin resistance, impaired glucose tolerance, and/or impaired fasting glucose, comprising administering a composition comprising a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, a synthetic vesiculin derivative and/or a synthetic vesiculin intermediate or a salt thereof.
  • diseases, disorders and/or conditions include but are not limited to those described or referenced herein.
  • Such compounds may be administered in amounts, for example, that are effective to (1) lower serum glucose, (2) lower blood glucose, (3) lower urine glucose, (4) lower fructosamine, (5) lower glycosylated hemoglobin (HbAi c ) levels, (6) lower postprandial glycemia, (7) ameliorate impaired glucose tolerance, (8) ameliorate impaired fasting glucose, and/or (9) lower the rate and/or severity of hypoglycemic events, including severe hypoglycemic events.
  • Such compositions include, for example, formulations for delivery by injection, transdermal patch, inhalation, and other non-oral delivery methods.
  • the invention includes methods for regulating glycemia in a subject having or suspected of having or predisposed to diseases, disorders and/or conditions characterized in whole or in part, for example, by hyperglycemia, insulin resistance, impaired glucose tolerance, and/or impaired fasting glucose, comprising administering a composition comprising a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, a vesiculin derivative and/or a synthetic vesiculin synthetic or a salt thereof.
  • diseases, disorders and/or conditions include but are not limited to those described or referenced herein.
  • Such compounds may be administered in amounts, for example, that are effective to (1) lower serum glucose, (2) lower blood glucose, (3) lower urine glucose, (4) lower fructosamine, (5) lower glycosylated hemoglobin (HbAi c ) levels, (6) lower postprandial glycemia, (7) ameliorate impaired glucose tolerance, (8) ameliorate impaired fasting glucose, and/or (9) lower the rate and/or severity of hypoglycemic events, including severe hypoglycemic events.
  • Such compositions include, for example, formulations for delivery by injection, transdermal patch, inhalation, and other non-oral delivery methods.
  • the invention relates to a use of one or more of a synthetic vesiculin, or a synthetic vesiculin variant, derivative, or synthetic intermediate, or a salt of any of them, in the preparation of a medicament, including medicaments for modulating blood glucose levels in a subject.
  • the invention relates to a method of modulating glucose incorporation into glycogen in a subject. The method comprises administering an effective amount of one or more of a synthetic vesiculin, or a synthetic vesiculin variant, derivative, or synthetic intermediate thereof, or a salt of any of them, to a subject in need thereof.
  • the invention is directed to the use of an effective amount of one or more of a synthetic vesiculin, or a synthetic vesiculin variant, derivative, or synthetic intermediate, or a salt of any of them, in the manufacture, with or without other pharmaceutically acceptable materials (such as, for example, excipients, diluents or the like, and/or within a dosage unit defining vessel), of a dosage unit effective for use in a method of the invention or for any of the purposes herein described or provided.
  • pharmaceutically acceptable materials such as, for example, excipients, diluents or the like, and/or within a dosage unit defining vessel
  • the invention also includes synthetic vesiculin, synthetic vesiculin variants, derivatives or synthetic intermediates produced by protein synthesis techniques, followed by isolation and/or purification, as disclosed herein.
  • the invention further includes a pharmaceutical composition which comprises a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, or a synthetic vesiculin, synthetic vesiculin A chain, or synthetic vesiculin B chain variant or derivative, or an synthetic intermediate thereof, or salts or derivatives of the above.
  • the synthetic vesiculins, synthetic vesiculin A and B chains, and variants and derivatives and intermediates thereof for use in the methods of the invention may be formulated in a manner suitable for administration to a subject, for example, a human. Administration is preferably, for example, parenteral via routes such as subcutaneous (s.c), intradermal (i.d.), intravenous (i.v.), intraperitoneal (i.p.) or transdermal, although other delivery form are envisioned, including oral, nasal, and pulmonary, for example.
  • routes such as subcutaneous (s.c), intradermal (i.d.), intravenous (i.v.), intraperitoneal (i.p.) or transdermal, although other delivery form are envisioned, including oral, nasal, and pulmonary, for example.
  • FIGURE 1 is a schematic overview of the synthesis of vesiculin showing the stepwise installation of firstly the intrachain disulfide bond of the A-chain followed by the interchain disulfides that cross-link the A and B chains, as described herein in Example 1.
  • LI is the Rink linker and L2 is the 4-hydroxymethyl benzoic acid (HMBA) linker, through which the solubilising pentalysine tag is attached and which is hydrolysed at the final step.
  • Figure 2 shows HPLC analysis of crude A-chain. Column: Phenomenex Gemini CI 8, 5 ⁇ , 11 OA, 4.6 x 150mm; Eluent A: water/0.1% TFA, B: MeCN/0.1% TFA; Gradient: 1-51%B over 25 min.
  • Figure 3 shows HPLC profiles for formation of the Cys6-Cysl 1 disulfide bond of the A-chain (* dithiol 3; ** disulfide 4) and the low-resolution mass spectrum for the disulfide, which indicates the M+3H + (1284.7) and higher ionisation states.
  • Figure 4 shows the HPLC profile of crude murine B-chain synthesised using Boc SPPS and low-resolution mass spectrum of the main peak, showing the M+3H + (1324.7) and higher ionisation states.
  • Figure 5 shows HPLC profiles for deprotection of Cys(tBu) of the A-chain 4 (*) and in- situ conversion to the activated SSPyr disulfide 6 (**), together with low-resolution mass spectrum of the product showing the M+3H + (1302.3) and higher ionisation states.
  • the strong peak at 17 min is excess dipyridyldisulfide.
  • Figure 6 shows HPLC profiles for crosslinking the A- and murine B-chains via an interchain disulfide bond, and low-resolution mass spectrum of the product 7 showing the required M+5H + (1553.9) and higher ionisation states.
  • Figure 7 shows HPLC profiles for removal of the Acm groups from murine 7 (*) and concomitant formation of the second interchain disulfide bond to give 8 (**), confirmed by low- resolution mass spectrum of the product showing the required M+5H + (1525.2) and higher ionisation states.
  • Figure 8 shows HPLC profiles for hydrolytic removal of the pentalysine tag of 8 (*) to afford murine Vesiculin 9 (**), with the low-resolution mass spectrum of the product showing the required M+4H + (1712.4) and higher ionisation states.
  • Figure 9 shows the HPLC profile of human Vesiculin and low-resolution mass spectrum of the product showing the required M+4H + (1733.6) and higher ionisation states.
  • Figure 10 shows the effect of synthetic murine vesiculin and pharmacological additive on blood glucose 60 min after administration, relative to fasting, as described in Example 3 herein.
  • FIGURE 11 shows an assay of the hypoglycaemic potential of synthetic murine vesiculin, as described in Example 3 herein.
  • This invention relates in one aspect to one or more of a synthetic polypeptide having insulin agonist activity, such as a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, synthetic vesiculin A chain and synthetic vesiculin B chain variants, and synthetic vesiculin, synthetic vesiculin A chain and synthetic vesiculin B chain derivatives, and salts thereof.
  • a synthetic polypeptide having insulin agonist activity such as a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, synthetic vesiculin A chain and synthetic vesiculin B chain variants, and synthetic vesiculin, synthetic vesiculin A chain and synthetic vesiculin B chain derivatives, and salts thereof.
  • Amino acid sequences for mouse, human and rat vesiculin include the following:
  • Amino acid sequences for vesiculins of other species include:
  • GIVEECCFRSCDLNLLEQYCAKPAKSE (SEQ ID NO. 66)
  • GIVEECCFRSCDLNLLEQYCAKPAKSE (SEQ ID NO. 68)
  • the synthetic vesiculin has been synthesized in accordance with the following description including the synthetic scheme outlined herein.
  • Synthetic vesiculin intermediates also include those having an A chains with from one to five N-terminal amino acid residue deletions and a B chain with from one to eight N-terminal amino acid residue deletions. These include synthetic human vesiculin intermediates including an A chain intermediate and a B chain intermediate, as well as synthetic intermediates of other vesiculin species including an A chain intermediate and a B chain intermediate. They also include vesiculin intermediates having an A chain intermediate from one species combined with a B chain intermediate from another species.
  • Synthetic vesiculin intermediates also include those having an A chain with from one to five N-terminal amino acid residue deletions and a full length B chain. These include synthetic human vesiculin intermediates with an A chain intermediate and a full length B chain, as well as similar molecules from other vesiculin species. They also include vesiculin intermediates having an A chain intermediate from one species combined with a full length B intermediate from another species.
  • Synthetic vesiculin intermediates also include those having a full length A chain and a B chain with from one to eight N-terminal amino acid residue deletions. These include synthetic human vesiculin intermediates with a full length A chain and a B chain intermediate, as well similar molecules from other vesiculin species. They also include vesiculin intermediates having a full length A chain from one species combined with a B chain intermediate from another species.
  • synthetic vesiculin variants, derivatives and synthetic intermediates including, for example, synthetic vesiculin variants, derivatives and synthetic intermediates having a synthetic vesiculin B chain from any species (for example, human, rat, mouse, etc.) and a synthetic vesiculin A chain having the sequence GIVEECCFRSCDLALLETYCATPAKSE (SEQ ID NO. 4).
  • vesiculin intermediates with a full length A chain and a B chain intermediate, as well similar molecules from other vesiculin species. They also include vesiculin intermediates having a full length A chain from one species combined with a B chain intermediate from another species.
  • a “conservative amino acid substitution” is one in which an amino acid residue is replaced with another residue having a chemically similar or derivitized side chain.
  • Families of amino acid residues having similar side chains, for example, have been defined in the art. These families include, for example, amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenyla
  • Amino acid analogs e.g., phosphorylated amino acids
  • peptides substituted with non-naturally occurring amino acids including but not limited to D-amino acids, ⁇ amino acids, and ⁇ amino acids.
  • purified does not require absolute purity; rather, it is intended as a relative term where the subject protein or other substance is more pure than in its natural environment within a cell or other environment, such as a manufacturing environment. In practice the material has typically, for example, been subjected to fractionation to remove various other components, and the resultant material has substantially retained its desired biological activity or activities.
  • substantially purified refers to peptides that are at least about 60% free, preferably at least about 75% free, and most preferably at least about 90%> free, at least about 95% free, at least about 98% free, or more, from other components with which they may be associated naturally or during manufacture.
  • a pharmaceutical composition that contains an effective amount of one or more of a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, synthetic vesiculin A chain and synthetic vesiculin B chain variants, and synthetic vesiculin, synthetic vesiculin A chain and synthetic vesiculin B chain derivatives, and synthetic intermediates thereof, and salts of any of them, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier (adjuvant or vehicle) that may be administered to a subject together with one or more of a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, synthetic vesiculin A chain and synthetic vesiculin B chain variants, and synthetic vesiculin, synthetic vesiculin A chain and synthetic vesiculin B chain derivatives, and salts of any of them.
  • Pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions described above include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-a-tocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block
  • Cyclodextrins such as ⁇ -, ⁇ -, and ⁇ -cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl-P-cyclodextrins, or other solubilized derivatives may also be advantageously used to enhance delivery of compounds of the formulae described herein.
  • Oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents, which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions.
  • an “effective amount” is an amount sufficient to effect beneficial or desired results including clinical results.
  • An effective amount can be administered in one or more administrations by various routes of administration.
  • a “biological sample” encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay.
  • the definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom, and the progeny thereof.
  • the definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as proteins or polynucleotides.
  • biological sample encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples.
  • treatment is an approach for obtaining beneficial or desired results including clinical results although the term also encompasses prophylactic and/or therapeutic treatments
  • polypeptide and “peptide” and the like are used interchangeably herein to refer to any polymer of amino acid residues of any length.
  • the polymer can be linear or nonlinear (e.g., branched), it can comprise modified amino acids or amino acid analogs, and it can be interrupted by chemical moieties other than amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.
  • an "active fragment" of a polypeptide is a subsequence of the polypeptide that performs a function that is required for the biological activity and/or provides a three dimensional structure of the polypeptide.
  • the term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof capable of performing any one or more of the methods herein described, particularly with reference to modulating glucose.
  • a "fragment" of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides that is capable of specific hybridization to a target of interest, for example, a sequence that is at least about 15 nucleotides in length.
  • the polynucleotide fragment of the invention comprise about 15 nucleotides, preferably at least about 20 nucleotides, more preferably at least about 30 nucleotides, more preferably at least about 40 nucleotides, more preferably at least about 50 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, at least about 80 nucleotides, at least about 90 nucleotides, at least about 100 nucleotides, at least about 1 10 nucleotides, at least about 120 nucleotides, and most preferably at least about 130 nucleotides of contiguous nucleotides of a polynucleotide of the invention.
  • a fragment of a polynucleotide sequence can be used in antisense, gene silencing, triple helix or ribozyme technology, or as a primer, a probe, included in a microarray, or used in polynucleotide -based selection methods of the invention.
  • a "fragment" of a polypeptide is a subsequence of the polypeptide, typically one that performs a function that is required for the activity of the polypeptide, such as the enzymatic or binding activity, and/or provides part of three dimensional structure of the polypeptide.
  • variants refers to polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the inventive polypeptides and polynucleotides possess biological activities that are the same or similar to those of the inventive polypeptides or polynucleotides.
  • vesiculin variant(s) include, for example, vesiculins having amino acid deletions or substitutions, including conservative amino acid substitutions, wherein one or more biological activities are retained, in whole or in part.
  • Variant polynucleotide sequences preferably exhibit at least about 50%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, more preferably at least about 95%, more preferably at least about 98%, and most preferably at least about 99% identity to a specified polynucleotide. Identity is found over a comparison window of at least about 20 nucleotide positions, preferably at least about 50 nucleotide positions, more preferably at least about 100 nucleotide positions or more of the entire length of a polynucleotide of the invention.
  • An exemplary family of programs useful for identifying variants in sequence databases is the BLAST suite of programs (version 2.2.5 [Nov 2002]) including BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX, which are publicly available from (ftp://ftp.ncbi.nih.gov/blast/) or from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894 USA.
  • NCBI National Center for Biotechnology Information
  • the NCBI server also provides the facility to use the programs to screen a number of publicly available sequence databases.
  • BLASTN compares a nucleotide query sequence against a nucleotide sequence database.
  • BLASTP compares an amino acid query sequence against a protein sequence database.
  • BLASTX compares a nucleotide query sequence translated in all reading frames against a protein sequence database.
  • tBLASTN compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames.
  • tBLASTX compares the six-frame translations of a nucleotide query sequence against the six- frame translations of a nucleotide sequence database.
  • the BLAST programs may be used with default parameters or the parameters may be altered as required to refine the screen.
  • the use of the BLAST family of algorithms, including BLASTN, BLASTP, and BLASTX is described in the publication of Altschul et al, Nucleic Acids Res. 25:3389-3402, (1997).
  • BLASTN, BLASTP, BLASTX, tBLASTN, tBLASTX, or a similar algorithm align and identify similar portions of sequences.
  • the hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.
  • Polynucleotide sequence identity can be determined in the following manner.
  • the subject polynucleotide sequence is compared to a candidate polynucleotide sequence using BLASTN (from the BLAST suite of programs, version 2.2.5 [Nov 2002]) in bl2seq (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250), which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/).
  • the default parameters of bl2seq are utilized except that filtering of low complexity parts should be turned off.
  • polynucleotide sequences may be examined using the following UNIX command line parameters: bl2seq -i nucleotideseql -j nucleotideseq2 -F F -p blastn
  • the parameter -F F turns off filtering of low complexity sections.
  • the parameter -p selects the appropriate algorithm for the pair of sequences.
  • Polynucleotide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs (for example Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453).
  • Needleman- Wunsch global alignment algorithm is found in the needle program in the EMBOSS package (Rice, P. Longden, I. and Bleasby, A. EMBOSS: The European Molecular Biology Open Software Suite, Trends in Genetics June 2000, vol 16, No 6. pp.276-277) which can be obtained from http://www.hgmp.mrc.ac.uk/Software/EMBOSS/.
  • the European Bioinformatics Institute server also provides the facility to perform EMBOSS-needle global alignments between two sequences on line at http:/www.ebi. ac.uk/emboss/align/.
  • GAP Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.
  • BLASTN as described above is preferred for use in the determination of sequence identity for polynucleotide variants according to the present invention.
  • Polynucleotide variants also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance.
  • sequence similarity with respect to polynucleotides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/).
  • the parameter -F F turns off filtering of low complexity sections.
  • the parameter -p selects the appropriate algorithm for the pair of sequences.
  • the BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX algorithms also produce "Expect" values for alignments. These programs find regions of similarity between the sequences and for each such region reports an Expect value (E value) which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences.
  • the E value is used as a significance threshold for determining whether the hit to a database indicates true similarity.
  • the size of this database is set by default in the bl2seq program.
  • E value is approximately the probability of such a random match.
  • an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size of the database screened, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance.
  • sequences having an E value of 0.01 or less over aligned and matched portions the probability of finding a match by chance in that database is 1% or less using the BLASTN, BLASTP, BLASTX, tBLASTN or tBLASTX algorithm.
  • Variant polynucleotide sequences preferably exhibit an E value of less than about 1 x 10 "5 , more preferably less than about 1 x 10 "6 , more preferably less than about 1 x 10 "9 , more
  • variant polynucleotides hybridize to the specified polynucleotide sequence, or a complement thereof under stringent conditions.
  • hybridize under stringent conditions refers to the ability of a polynucleotide molecule to hybridize to a target polynucleotide molecule (such as a target polynucleotide molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot) under defined conditions of temperature and salt concentration.
  • the ability to hybridize under stringent hybridization conditions can be determined by initially hybridizing under less stringent conditions then increasing the stringency to the desired stringency.
  • Tm melting temperature
  • Typical stringent conditions for polynucleotide of greater than 100 bases in length would be hybridization conditions such as pre -washing in a solution of 6X SSC, 0.2% SDS; hybridizing at 65°C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in IX SSC, 0.1% SDS at 65° C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65°C.
  • exemplary stringent hybridization conditions are 5 to 10° C below Tm.
  • Tm of a polynucleotide molecule of length less than 100 bp is reduced by approximately (500/oligonucleotide length) 0 C.
  • Tm values are higher than those for DNA-DNA or DNA- RNA hybrids, and can be calculated using the formula described in Giesen et al., Nucleic Acids Res. 26(21):5004-6 (1998).
  • Exemplary stringent hybridization conditions for a DNA-PNA hybrid having a length less than 100 bases are 5 to 10° C below the Tm.
  • Variant polynucleotides also encompasses polynucleotides that differ from the sequences of the invention but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide having similar activity to a polypeptide encoded by a polynucleotide of the present invention.
  • a sequence alteration that does not change the amino acid sequence of the polypeptide is a "silent variation.” Except for ATG (methionine) and TGG (tryptophan), other codons for the same amino acid may be changed by art recognized techniques, for example, to optimize codon expression in a particular host organism.
  • Variant polynucleotides due to silent variations and conservative substitutions in the encoded polypeptide sequence may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/) via the tblastx algorithm as previously described.
  • Polypeptide sequence identity can also be determined in the following manner.
  • the subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, version 2.2.5 [Nov 2002]) in bl2seq, which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/).
  • BLASTP from the BLAST suite of programs, version 2.2.5 [Nov 2002]
  • bl2seq which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/).
  • the default parameters of bl2seq are utilized except that filtering of low complexity regions should be turned off.
  • Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs. EMBOSS-needle (available at http:/www.ebi.
  • BLASTP as described above is preferred for use in the determination of polypeptide variants according to the present invention.
  • Polypeptide variants also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance.
  • sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/).
  • the similarity of polypeptide sequences may be examined using the following UNIX command line parameters:
  • Variant polypeptide sequences preferably exhibit an E value of less than 1 x 10 "5 , more preferably less than 1 x 10 "6 , more preferably less than 1 x 10 "9 , more preferably less than 1 x 10
  • the parameter -F F turns off filtering of low complexity sections.
  • the parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. For small E values, much less than one, this is approximately the probability of such a random match.
  • a "subject” refers to a vertebrate that is a mammal, for example, a human. Mammals include, but are not limited to, humans, farm animals, sport animals, pets, primates, mice and rats. Peptides of the invention, including, for example, synthetic vesiculins and variants thereof may be generated by synthetic methods (including, for example, single or fusion polypeptides), such as by solid phase peptide synthesis.
  • SPPS solid phase peptide synthesis
  • the amino acid to be coupled to the resin is protected at its Na-terminus by a chemical protecting group.
  • the amino acid may also have a chemical side-chain protecting group.
  • Such protecting groups prevent undesired or deleterious reactions from taking place during the process of forming the new peptide bond between the carboxyl group of the amino acid to be coupled and the unprotected Na-amino group of the peptide chain attached to the resin.
  • the amino acid to be coupled is reacted with the unprotected Na-amino group of the N-terminal amino acid of the peptide chain, increasing the chain length of the peptide chain by one amino acid.
  • the carboxyl group of the amino acid to be coupled may be activated with a suitable chemical activating agent to promote reaction with the ⁇ -amino group of the peptide chain.
  • the Na- protecting group of N-terminal amino acid of the peptide chain is then removed in preparation for coupling with the next amino acid residue. This technique consists of many repetitive steps making automation attractive whenever possible.
  • the peptide is cleaved from the solid phase support at the linker molecule.
  • SPPS may be carried out using a continuous flow method or a batch flow method.
  • Continuous flow is useful because it permits real-time monitoring of reaction progress via a spectrophotometer.
  • continuous flow has two distinct disadvantages in that the reagents in contact with the peptide on the resin are diluted, and scale is more limited due to physical size constraints of the solid phase resin.
  • Batch flow occurs in a filter reaction vessel and is useful because reactants are accessible and can be added manually or automatically.
  • Further options involve the identity of the protecting group used for protecting the N- alpha-amino terminus.
  • One protecting group is known as "Boc" (tert-butyloxycarbonyl).
  • Reagents for the Boc method are relatively inexpensive, but they are highly corrosive and require expensive equipment and more rigorous precautions to be taken.
  • the typically preferred alternative is the "Fmoc” (9-fluorenylmethyloxycarbonyl) protection scheme, which uses less corrosive, although more expensive, reagents.
  • the solid phase support used for synthesis can be a synthetic resin, a synthetic polymer film or a silicon or silicate surface, e.g. controlled pore glass (CPG), suitable for synthesis purposes.
  • CPG controlled pore glass
  • a resin is used, and commonly polystyrene suspensions, or polystyrene-polyethyleneglycol, or polymer supports for example polyamide are used.
  • 2-chlortrityl resin an acid labile resin, is commonly used to cleave a product from the resin without cleaving the protective groups.
  • Photolable resins are useful because cleavage is carried out without using acidic or basic conditions and therefore basic- and acid-lable side chain protective groups remain stable. Brominated Wang resin, ANP resin and Fmoc-photolable resin are examples of this class.
  • resins functionalized with linkers suitable for Boc-chemistry include PAM resin, oxime resin SS, phenol resin, brominated Wang resin and brominated PPOA resin.
  • resins suitable for Fmoc chemistry include AMPB-BHA resin, Sieber amide resin, Rink acid resin, Tentagel S AC resin, 2-chlorotrityl chloride resin, 2-chlorotrityl alcohol resin, TentaGel S Trt-OH resin, Knorr-2-chlorotrityl resin, hydrazine-2-chlorotrityl resin, ANP resin, Fmoc photolable resin, HMBA-MBHA resin, TentaGel S HMB resin, Aromatic Safety Catch resinBAl resin and Fmoc-hydroxylamine 2 chlorotrityl resin.
  • Other suitable resins include PL Cl- Trt resin, PL-Oxime resin and PL-HMBA Resin.
  • the solid phase is a Rink acid resin or a HMTP
  • Preparation of the solid phase support includes solvating the support in an appropriate solvent (dimethyl formamide (DMF), for example).
  • DMF dimethyl formamide
  • the solid phase tyically increases in volume during solvation, which in turn increases the surface area available to carry out peptide synthesis.
  • Linker molecules are then attached to the support for connecting the peptide chain to the solid phase support.
  • Linker molecules are generally designed such that eventual cleavage provides either a free acid or amide at the C-terminus.
  • Linkers are generally not resin-specific. Examples of linkers include peptide acids for example 4-hydroxymethylphenoxyacetyl-4'- methylbenzyhydrylamine (HMP), or peptide amides for example benzhydrylamine derivatives.
  • HMP 4-hydroxymethylphenoxyacetyl-4'- methylbenzyhydrylamine
  • peptide amides for example benzhydrylamine derivatives.
  • the first amino acid of the peptide sequence may be attached to the linker after the linker is attached to the solid phase support or attached to the solid phase support using a linker that includes the first amino acid of the peptide sequence.
  • Linkers that include amino acids are commercially available.
  • the next step is to deprotect the Na-amino group of the first amino acid.
  • deprotection of the Na-amino group may be carried out with a mild base treatment (piperazine or piperidine, for example). Side-chain protecting groups may be removed by moderate acidolysis (trifluoroacetic acid (TFA), for example).
  • TFA trifluoroacetic acid
  • deprotection of the ⁇ -amino group may be carried out using for example TFA.
  • the amino acid chain extension, or coupling proceeds by the formation of peptide bonds.
  • This process requires activation of the C-alpha-carboxyl group of the amino acid to be coupled. This is typically accomplished using, for example, in situ reagents, preformed symmetrical anhydrides, active esters, acid halides, or urethane-protected N- carboxyanhydrides.
  • in situ reagents preformed symmetrical anhydrides, active esters, acid halides, or urethane-protected N- carboxyanhydrides.
  • the in situ method allows concurrent activation and coupling; the most popular type of coupling reagents are carbodiimide derivatives, for example ⁇ , ⁇ '- dicyclohexylcarbodiimide or N,N-diisopropylcarbodiimide.
  • the peptide is cleaved from the resin.
  • the conditions used in this process depend on the sensitivity of the amino acid composition of the peptide and the side-chain protecting groups. Generally, however, cleavage is carried out in an environment containing a plurality of scavenging agents to quench the reactive carbonium ions that originate from the protective groups and linkers. Common cleaving agents include for example TFA and hydrogen fluoride (HF).
  • SPPS the principle of SPPS is the iterative steps of deprotecting, activating, and coupling each amino acid, followed by the final step of cleavage to separate the completed peptide from the solid support.
  • protective groups in SPPS is well established in the art. Examples of common protective groups are listed together with their abbreviations below: acetamidomethyl (Acm), acetyl (Ac), adamantyloxy (AdaO), benzoyl (Bz), benzyl (Bzl), 2-bromobenzyl, benzyloxy (BzlO), benzyloxycarbonyl (Z), benzyloxymethyl (Bom), 2-bromobenzyloxycarbonyl (2-Br-Z), tert-butoxy (tBuO), tert-butoxycarbonyl (Boc), tert-butoxymethyl (Bum), tert-butyl (tBu), tert-buthylthio (tButhio), 2-chlorobenzyloxycarbonyl (2-Cl-Z), cyclohexyloxy (cHxO), 2,6-dichlorobenzyl (2,6-DiCl-
  • one or more of the side chains of the amino acids of the peptide contains additional functional groups, such as for example additional carboxylic, amino, hydroxy or thiol groups
  • additional functional groups such as for example additional carboxylic, amino, hydroxy or thiol groups
  • further protective groups may be necessary.
  • Mtr, Pmc, Pbf may be used for the protection of Arg
  • Trt, Tmob may be used for the protection of Asn and Gin
  • Boc may be used for the protection of Trp and Lys
  • tBu may be used for the protection of Asp, Glu, Ser, Thr and Tyr
  • Acm, tBu, tButhio, Trt and Mmt may be used for the protection of Cys.
  • the Fmoc group is used as a first Na-protective group during the synthesis of synthetic vesiculin A chain polypeptides, and can be cleaved using piperidine.
  • the side chains of one or more amino acids in the synthesis of synthetic vesiculin A chain polypeptides are protected with one or more suitable protectin groups.
  • the one or more protecting groups are removeable using trifluoroacetic acid.
  • one or more of the cysteine residues are protected with one or more suitable protecting groups.
  • the one or more suitable protecting groups are not removable using trifluoroacetic acid.
  • the Boc group is used as a first Na-protective group during the synthesis of synthetic vesiculin B chain polypeptides, and can be cleaved using trifluoroacetic acid.
  • Confirmation of the identity of the newly synthesized vesiculin polypeptides and vesiculin variants is conveniently achieved by amino acid analysis, mass spectroscopy, Edman degradation, or for functional variants or intermediates by assessing biological function (i.e., stimulating glucose incorporation into glycogen, or ⁇ -cell mitogenesis).
  • Synthetic variants of vesiculin may also be made by substituting amino acids which do not substantially alter the bioactivity of the synthetic vesiculin variant relative to the parent vesiculin ⁇ e.g., conservative substitutions). Selection of amino acids for substitution can depend on the size, structure, charge, and can be either an amino acid found in nature or synthetic amino acid. Generally, amino acids which have a similar charge ⁇ e.g., hydrophobic for hydrophobic) or similar size ⁇ e.g., isoleucine for leucine) can be selected for substitution. One or more substitutions can be made in a stepwise fashion or concurrently. Variations in the residues included in the peptide are also both possible and contemplated. For example, it is possible to substitute amino acids in a sequence with equivalent amino acids using conventional techniques. Groups of amino acids known normally to be equivalent are:
  • synthetic vesiculin variants can be achieved by substituting one or more amino acids.
  • the synthetic vesiculin variants can be tested for biological function, such as for example, to stimulate glucose incorporation into glycogen, whether in vivo or in vitro.
  • the biological activity of a synthetic vesiculin variant is generally at least about 25% of a vesiculin, preferably at least about 35%, preferably at least about 50%, preferably at least about 60%, preferably at least about 75%, preferably at least about 85%, and more preferably at least about 95%.
  • the invention also encompasses synthetic intermediates with vesiculin bioactive functionality.
  • Such synthetic intermediates may be obtained during synthesis of vesiculin, by for example, the methods of the present invention. Synthetic intermediates may be ascertained by stepwise isolation and assay. If an amino acid is omitted and the bioactivity of vesiculin is not substantially reduced, then the amino acid need not comprise a portion of the synthetic intermediate.
  • polypeptides comprising a synthetic intermediate of vesiculin or vesiculin variant(s) are also encompassed in the invention.
  • synthetic intermediates of vesiculin may comprise about 10 contiguous amino acids of the amino acids of the amino acid sequence of either or both the A-chain and/or B-chain of vesiculin, more preferably about 15 contiguous amino acids, more preferably about 20 contiguous amino acids, more preferably about 25 contiguous amino acids, more preferably about 30 contiguous amino acids, about 40 contiguous amino acids, about 50 contiguous amino acids, about 60 contiguous amino acids, or more preferably 61 contiguous amino acids.
  • Additions and/or deletions of amino acids may also be made as long as the resulting synthetic polypeptide is immunologically cross-reactive with and/or has substantially the same function or functions as a vesiculin.
  • a fusion protein may also be constructed that facilitates purification or identification.
  • components for these fusion proteins include, but are not limited to myc, HA, FLAG, or His-6.
  • Longer fusion partners typically used in recombinant methods of production, such as glutathione S-transferase, maltose binding protein or the Fc portion of immunoglobulin, are generally considered too large for sensible use in synthetic methods, except where such fusion partners can be coupled to the synthetic vesiculin polypeptide, variant or intermediate without a need for synthesis of the partner in situ.
  • polypeptides are at least substantially purified or isolated from other cellular constituents.
  • the polypeptides are preferably at least about 80% pure, and free of pyrogens and other contaminants. Methods of protein purification are known in the art and are not described in detail herein.
  • Proteins can be classified according to their sequence relatedness to other proteins in the same genome (paralogies) or a different genome (orthologues).
  • Orthologous genes are genes that evolved by speciation from a common ancestral gene and normally retain the same function as they evolve.
  • Paralogous genes are genes that are duplicated within a genome and genes may acquire new specificities or modified functions which may be related to the original one. Phylogenetic analysis methods are reviewed in Tatusov, et al., 1997, Science 278, 631-637,).
  • polypeptide variants may be identified by physical methods, for example by screening expression libraries using antibodies raised against polypeptides of the invention (Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987) or by identifying polypeptides from natural sources with the aid of such antibodies.
  • Variants according to the invention also include the homologues of vesiculin from species other than human, rat or mouse.
  • homologues can be readily identified using, for example, nucleic acid probes based upon the conserved regions of the polynucleotides which encode human, rat and mouse vesiculin.
  • the invention includes methods for treating and/or preventing, in whole or in part, various diseases, disorders, and conditions, including for example, impaired glucose tolerance; impaired fasting glucose; prediabetes; diabetes and/or its complications, including type 1 and type 2 diabetes and their complications; insulin resistance; Syndrome X; obesity and other weight related disorders; fatty liver disease, including nonalcoholic alcoholic fatty liver disease; glucose metabolism diseases and disorders; diseases, disorders or conditions that are treated or treatable with insulin; diseases, disorders or conditions that are treated or treatable with a hypoglycemic agent; diseases, disorders, and conditions characterized at least in part by hyperglycemia; diseases, disorders, and conditions characterized at least in part by hypoinsulinemia and/or diseases, disorders, and conditions characterized at least in part by hyperinsulinemia.
  • the invention includes methods for treating a subject having or suspected of having or predisposed to, or at risk for, for example, any diseases, disorders and/or conditions characterized in whole or in part by hyperglycemia, insulin resistance, impaired glucose tolerance, and/or impaired fasting glucose, comprising administering a composition comprising a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, a synthetic vesiculin derivative and/or a synthetic vesiculin intermediate or a salt thereof.
  • diseases, disorders and/or conditions include but are not limited to those described or referenced herein.
  • Such compounds may be administered in amounts, for example, that are effective to (1) lower serum glucose, (2) lower blood glucose, (3) lower urine glucose, (4) lower fructosamine, (5) lower glycosylated hemoglobin (HbAi c ) levels, (6) lower postprandial glycemia, (7) ameliorate impaired glucose tolerance, (8) ameliorate impaired fasting glucose, (9) lower the rate and/or severity of hypoglycemic events, including severe hypoglycemic events, and/or (10) and/or stimulate glucose disposal.
  • Such compositions include, for example, formulations for delivery by injection, transdermal patch, inhalation, and other non- oral delivery methods.
  • the invention includes methods for regulating glycemia in a subject having or suspected of having or predisposed to diseases, disorders and/or conditions characterized in whole or in part, for example, by hyperglycemia, insulin resistance, impaired glucose tolerance, and/or impaired fasting glucose, comprising administering a composition comprising a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, a synthetic vesiculin derivative and/or a synthetic vesiculin intermediate or a salt thereof.
  • diseases, disorders and/or conditions include but are not limited to those described or referenced herein.
  • Such compounds may be administered in amounts, for example, that are effective to (1) lower serum glucose, (2) lower blood glucose, (3) lower urine glucose, (4) lower fructosamine, (5) lower glycosylated hemoglobin (HbAi c ) levels, (6) lower postprandial glycemia, (7) ameliorate impaired glucose tolerance, (8) ameliorate impaired fasting glucose, (9) lower the rate and/or severity of hypoglycemic events, including severe hypoglycemic events.
  • Such compositions include, for example, formulations for delivery by injection, transdermal patch, inhalation, and other non-oral delivery methods, and/or (10) stimulate glucose disposal.
  • a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, a synthetic vesiculin derivative, and/or a synthetic vesiculin intermediate, or salts thereof may be used in pure or substantially pure form.
  • Synthetic vesiculins, synthetic vesiculin A chains, synthetic vesiculin B chains, synthetic vesiculin variants, synthetic vesiculin derivatives, and/or synthetic vesiculin active fragments, or salts thereof may be presented as a pharmaceutical composition.
  • compositions may comprise one or more of a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, a synthetic vesiculin derivative, and/or a synthetic vesiculin intermediate, or salts thereof, for example, together with one or more pharmaceutically acceptable carriers and optionally other ingredients where desirable.
  • a synthetic vesiculin a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, a synthetic vesiculin derivative, and/or a synthetic vesiculin intermediate, or salts thereof, for example, together with one or more pharmaceutically acceptable carriers and optionally other ingredients where desirable.
  • Formulations for parenteral and non parenteral drug delivery are known in the art and are set forth, for example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing (1990).
  • the carrier is acceptable in the sense of being compatible with the peptide being administered and not over
  • the composition should not include substances with which peptides are known to be incompatible.
  • conventional non-toxic carriers include, for example mannitol, lactose, starch, magnesium stearate, magnesium carbonate, sodium saccharin, talcum, cellulose, glucose, sucrose, pectin, dextrin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, a low- melting wax, cocoa butter, and the like may be used.
  • the active compound as defined above may be formulated as suppositories using, for example, polyalkylene glycols, for example, propylene glycol as a carrier.
  • a solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, or tablet disintegrating agents; it can also be an encapsulating material.
  • cachets or transdermal systems are included.
  • the carrier is a finely divided solid which is in a mixture with the finely divided active component.
  • the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
  • Liquid form preparations include solutions, suspensions, or emulsions suitable, for example, for parenteral administration.
  • Aqueous solutions for parenteral administration can be prepared by dissolving the subject peptide in water and adding other suitable agents, stabilizers, buffers, etc., as desired.
  • Actual methods of preparing such dosage forms are known, or will be apparent to those skilled in this art; for example, see Remington's Pharmaceutical Sciences.
  • composition or formulation to be administered will preferably contain a quantity of the active compound in an amount effective to (1) lower serum glucose, (2) lower blood glucose, (3) lower urine glucose, (4) lower fructosamine, (5) lower glycosylated hemoglobin (HbAi c ) levels, (6) lower postprandial glycemia, (7) ameliorate impaired glucose tolerance, (8) ameliorate impaired fasting glucose, (9) lower the rate and/or severity of hypoglycemic events, including severe hypoglycemic events, and/or (10) stimulate glucose disposal.
  • HbAi c glycosylated hemoglobin
  • An effective amount of a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, a synthetic vesiculin derivative, and/or a synthetic vesiculin intermediate, or a salt thereof may include, for example, from about 0.01 nmol/kg/day to about 100 nmol/kg/day, from about 0.02 nmol/kg/day to about 75 nmol/kg/day, from about 0.025 nmol/kg/day to about 50 nmol/kg/day, from about 0.03 nmol/kg/day to about 40 nmol/kg/day, from about 0.04 nmol/kg/day to about 30 nmol/kg/day, from about 0.05 nmol/kg/day to about 25 nmol/kg/day, from about 0.07 nmol/kg/day to about 20 nmol/kg/day, from about 0.08 nmol/kg/day to about 15 nmol/kg/
  • An effective amount of a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, a synthetic vesiculin derivative, and/or a synthetic vesiculin intermediate, or a salt thereof may also include, for example, from about 120 ng/kg/day to about 1.2 mg/kg/day, from about 240 ng/kg/day to about 900 ⁇ g/kg/day, from about 300 ng/kg/day to about 600 ⁇ g/kg/day, from about 360 ng/kg/day to about 480 ⁇ g/kg/day, from about 480 ng/kg/day to about 400 ⁇ g/kg/day, from about 600 ng/kg/day to about 300 ⁇ g/kg/day, from about 840 ng/kg/day to about 240 ⁇ g/kg/day, from about 960 ng/kg/day to about 180 ⁇ g/kg/day, from about 1.2 ⁇ g/kg
  • the dosage administered may vary from individual to individual. It is also understood that the dosage may be administered in a single dose or optionally multiple doses (e.g., two, three, or four doses per day).
  • a clinician or physician will determine the dosage needed for individuals. A clinician or physician may monitor factors including but not limited to glucose level, vesiculin level (either circulating or resident in tissues), insulin levels (either circulating or resident in tissues), level of depletion of pancreatic ⁇ -cells, presence or absence of polydipsia, presence or absence of polyphagia, presence or absence of polyuria, levels of glycated hemoglobin, levels of glycated albumin, and levels of fructosamine.
  • compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredients into association with a carrier which constitutes one or more accessory ingredients.
  • composition may be injected parenterally, for example, intravenously into the blood stream of the patient being treated.
  • the route can vary, and can be intravenous, subcutaneous, transcutaneous, intramuscular, intradermal, intraarticular, intrathecal, intraperitoneal, enterally, transdermally, transmucously, sustained release polymer compositions (for example a lactide polymer or copolymer microparticle or implant), perfusion, pulmonary (for example, inhalation), nasal, oral, etc.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspension, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients include, for example, water, saline, aqueous dextrose, glycerol, ethanol or the like.
  • compositions suitable for parenteral and in particular subcutaneous administration are preferred.
  • Other suitable administration routes are intravenous administration and intramuscular administration.
  • Such compositions conveniently comprise sterile aqueous solutions of a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, a synthetic vesiculin derivative, and/or a synthetic vesiculin intermediate, or a salt thereof.
  • compositions can be isotonic with the blood of the patient to be treated.
  • Such compositions may be conveniently prepared by dissolving a vesiculin, a vesiculin A chain, a vesiculin B chain, a vesiculin variant, a vesiculin derivative, and/or a vesiculin active fragment, or a salt thereof in water to produce an aqueous solution and rendering this solution sterile.
  • the composition may then be presented in unit or multi-dose containers, for example sealed ampoules or vials.
  • One particularly preferred composition is a vesiculin, for example, a human vesiculin, in a physiological buffered solution suitable for injection.
  • compositions suitable for sustained release parenteral administrations are also well known in the art. See, for example, US Patent Nos. 3,773,919 and 4,767,628 and PCT Publication No. WO 94/15587.
  • oral delivery forms are equally acceptable, one example of oral delivery forms of tablet, capsule, lozenge, or the like form, or any liquid form such as syrups, aqueous solutions, emulsion and the like, capable of protecting the therapeutic protein from degradation prior to eliciting an effect, e.g., in the alimentary canal if an oral dosage form.
  • oral delivery forms for transdermal delivery include transdermal patches, transdermal bandages, and the like.
  • topical dosage forms any lotion, stick, spray, ointment, paste, cream, gel, etc., whether applied directly to the skin or via an intermediary such as a pad, patch or the like.
  • Examples of dosage forms for suppository delivery include any solid or other dosage form to be inserted into a bodily orifice (particularly those inserted rectally, vaginally and urethrally).
  • Examples of dosage units for transmucosal delivery include depositories, solutions for enemas, pessaries, tampons, creams, gels, pastes, foams, nebulised solutions, powders and similar formulations containing in addition to the active ingredients such carriers as are known in the art to be appropriate.
  • dosage units for depot administration include pellets or small cylinders of active agent or solid forms wherein the active agent is entrapped in a matrix of biodegradable polymers, microemulsions, liposomes or is microencapsulated.
  • implantable infusion devices include any solid form in which the active agent is encapsulated within or dispersed throughout a biodegradable polymer or synthetic, polymer such as silicone, silicone rubber, silastic or similar polymer.
  • dosage forms for infusion devices may employ liposome delivery systems.
  • Examples of dosage units for delivery via bolus include single or multiple administrations by intravenous injection, subcutaneous, subdermal, and intramuscular administration or oral administration.
  • dosage units for inhalation or insufflation include compositions comprising solutions and/or suspensions in pharmaceutically acceptable, aqueous, or organic solvents, or mixtures thereof and/or powders.
  • vesiculin it is also convenient for synthetic vesiculin to be converted to be in the form of a salt.
  • a salt will generally be physiologically acceptable, and can be formed using any method well known in the art.
  • Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable non-toxic inorganic and organic acids and bases.
  • Suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, benzoic, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, gluconic, glutamic, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, isethionic, lactate, maleate, malonate, malic, mandelic, methanesulfonate, mucic, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, pantothenic, pectinate
  • Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl) 4 + salts.
  • alkali metal e.g., sodium
  • alkaline earth metal e.g., magnesium
  • ammonium e.g., ammonium
  • N-(alkyl) 4 + salts e.g., ammonium
  • This invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products may be obtained by such quaternization. Hydrochloride and acetate salts are preferred.
  • Synthetic vesiculin salts formed by combination of synthetic vesiculin with anions of organic acids are particularly preferred.
  • Such salts include, but are not limited to, malate, acetate, propionate, butyrate, oxaloacetate, citrate, isocitrate, a-ketoglutarate, succinate, fumarate and trifluoroacetate salts.
  • the salts thus formed can also be formulated into pharmaceutical compositions for therapeutic administration where desired.
  • the present invention provides a synthetic vesiculin and synthetic vesiculin A and B chains (including but not limited to their human, rat and mouse forms), a synthetic vesiculin variant, or a synthetic vesiculin derivative, or synthetic variant thereof.
  • Synthetic vesiculins are shown herein to play a role, for example, in the stimulation of glucose incorporation into glycogen.
  • the invention therefore also provides methods by which glucose incorporation into glycogen can be modulated.
  • modulation will usually involve administration of a synthetic vesiculin or synthetic vesiculin-related polypeptide as described herein.
  • a vesiculin agonist is a compound which can, for example, promote or potentiate the effect of vesiculin on glucose incorporation into glycogen.
  • a vesiculin antagonist is a compound which competes with vesiculin or otherwise interacts with vesiculin to block or reduce the effect of vesiculin, for example, on glucose incorporation into glycogen.
  • Vesiculin agonists and antagonists can be identified by assay systems, including the soleus muscle assay, which measure the effect synthetic vesiculin has on glucose incorporation into glycogen in the presence and absence of a test compound.
  • a vesiculin agonist or vesiculin antagonist be employed in modulating, for example but not limited to, glucose incorporation into glycogen
  • the agonist/antagonist can be administered as a substantially pure compound or formulated as a pharmaceutical composition as described above for vesiculin.
  • Synthetic vesiculin polypeptides include use in vaccines and for generation of antibodies, including monoclonal antibodies.
  • Synthetic vesiculin polypeptides are used as immunogens to immunize mice.
  • Splenocytes including lymphocytes
  • Hybridomas are prepared from the lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C, Nature 256:495-497 (1975).
  • Other modified methods for example by Buck, D. W., et al., In Vitro 18:377-381 (1982) may also be used.
  • myeloma lines include but are not limited to X63-Ag8.653 and those from the Salk Institute, Cell Distribution Center, San Diego, Calif, USA, may be used in the hybridization.
  • the technique involves fusing the myeloma cells and lymphoid cells using a fusogen such as polyethylene glycol, or by electrical means well known to those skilled in the art. After the fusion, the cells are separated from the fusion medium and grown in a selective growth medium, such as HAT medium, to eliminate unhybridized parent cells. Any of the media described herein, supplemented with or without serum, can be used for culturing hybridomas that secrete monoclonal antibodies.
  • EBV immortalized B cells are used to produce the monoclonal antibodies of the subject invention.
  • the hybridomas are expanded and subcloned, if desired, and supernatants are assayed for anti-immunogen activity by conventional immunoassay procedures ⁇ e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).
  • Hybridomas that produce such antibodies may be grown in vitro or in vivo using known procedures.
  • the monoclonal antibodies may be isolated from the culture media or body fluids, by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration, if desired.
  • Undesired activity if present, can be removed, for example, by running the preparation over adsorbants made of the immunogen attached to a solid phase and eluting or releasing the desired antibodies off the immunogen.
  • Synthetic vesiculin and synthetic vesiculin-related polypeptides may also be used as immunogens to immunize other animals ⁇ e.g., rats and rabbits) to generate polyclonal antibodies.
  • Methods of producing polyclonal antibodies and the subsequent isolation and purification thereof are well known in the art. See, for example, Harlow et al, supra.
  • Other suitable techniques for preparing antibodies involve in vitro exposure of lymphocytes to the antigen or alternatively to selection of libraries of antibodies in phage or similar vectors.
  • recombinant antibodies may be produced using procedures known in the art. See, for example, US Patent 4,816,567.
  • the antibodies may be used with or without modification. Frequently, antibodies will be labeled by joining, either covalently or non-covalently a substance which provides a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in the literature. Antibodies can be used to monitor the presence of vesiculin in a patient or in vesiculin quantification assays. Further, anti-vesiculin antibodies, for example, can be used to measure levels of vesiculin in an individual, either at one fixed time point or over a period of time to monitor fluctations in circulating vesiculin levels. Anti-vesiculin antibodies can also be used to measure levels of vesiculin in an individual to whom drugs have been administered. In such assays, any convenient immunological format can be employed. Such formats include immunohistochemical assays, RIA, IRMA and ELISA assays.
  • the assays can be conducted in relation to any biological fluid which does, or should, contain vesiculin.
  • biological fluids include blood, serum, plasma, urine and cerebrospinal fluid.
  • Antibodies, monoclonal or polyclonal, against synthetic vesiculin may be used for diagnosis or for therapeutic purposes. Antibodies may be used by themselves or attached to a solid substrate, such as a column or a plate. Antibodies which are attached to a solid substrate may be used for assays, for example ELISA, or as a standard in other assays. Antibodies against synthetic vesiculin are also useful for vesiculin isolation, vesiculin purification, and vesiculin quantitation.
  • kits can contain, in addition, a number of optional but conventional components, the selection of which will be routine to the art skilled worker.
  • additional components will however generally include a vesiculin reference standard, which may be vesiculin itself or a variant (such as an intermediate).
  • antibodies such as described above can be used as vesiculin antagonists by binding to vesiculin and partly or completely interfering with vesiculin activity.
  • compositions useful in the invention are prepared by mixing the ingredients following generally accepted procedures.
  • the selected components may be mixed in a blender or other standard device to produce a concentrated mixture which may then be adjusted to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to control tonicity.
  • a blender or other standard device to produce a concentrated mixture which may then be adjusted to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to control tonicity.
  • This example describes a SPPS method for the production of synthetic murine vesiculin polypeptides.
  • Figure 1 An outline of the synthetic scheme is presented in Figure 1 which shows how the requisite disulfide bridges can be constructed in a stepwise and unambiguous manner using an orthoganol Cys- protecting group strategy.
  • the A-chain was synthesised using Fmoc SPPS on a Liberty microwave-enhanced peptide synthesiser (CEM corp) using a PEG-based ChemMatrixTM resin .
  • CEM corp Liberty microwave-enhanced peptide synthesiser
  • the Rink linker was used as the point of attachment (LI, scheme 1) of the peptide to the resin. Once the linker was installed, a sequence of five lysine residues was then added followed by the
  • HMBA linker L2, figure 1
  • Fmoc-Glu residue The HMBA-pentalysine motif is retained after acidic cleavage of the completed peptide from resin at LI .
  • the tag enhances the solubility of the attached peptide and facilitates purification over subsequent steps. Following completion of the synthesis the tag is removed hydrolytically. The synthesis was continued, incorporating the following protecting groups on the cysteine residues: Trityl on Cys 3 and Cys 5 , Acm on Cys 4 and tert- utyl on Cys 6 to afford 2.
  • the peptide was cleaved from the resin using a trifluoroacetic acid-based cocktail and precipitated in ether/hexane. HPLC analysis showed one main peak (Figure 2) with a mass corresponding to that required for the A-chain sequence.
  • the B-chain was synthesised using Boc-based, manual SPPS on a polystyrene resin derivatised with the PAM linker.
  • the peptide was cleaved from resin using neat hydrofluoric acid and HPLC analysis of the crude material showed one main peak ( Figure 4) with a mass corresponding to that required for the B-chain sequence 5. Isolation of 5 was achieved by dissolving the crude product in water/MeCN containing 0.1% formic acid and purifiying aliquots of the solution by HPLC using a Gemini semi-preparative (10 x 250 mm) column.
  • Residual DPDS was separated from the product material by HPLC using a Phenomenex Jupiter C4 semi-prep column.
  • the final step entailed hydrolysis of the HMBA linker from 8 under basic conditions to remove the pentalysine tail. Hydrolysis was achieved with sodium bicarbonate buffers at pH 9.5 after 70 hours. Hydrolysis was also achieved with 0.1 M aqueous NaOH at 0°C in 5 minutes, affording murine vesiculin 9 in 80% yield after purification (**, Figure 8), with the identity confirmed by MS.
  • HBTU hexafluorophosphate 3 -oxide
  • ChemMatrix resin was purchased from PCAS Biomatrix (Canada). Methanol, diethyl ether, and dichloromethane
  • DIPEA 2,2'-dipyridyl disulfide
  • TFMSA trifiuoromethanesulfonic acid
  • the side chain protecting groups of trifunctional amino acids (Fmoc-Arg: Pbf (2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl); Asp, Glu, Ser, Thr, and Tyr: tert- vXy ⁇ ; Asn, Gin, and His: trityl; Cys: trityl, acetamidomethyl, and tert-bvXy ⁇ ; Trp and Lys: Boc (tert- butoxycarbonyl)) were TFA-labile except for acetamidomethyl (Acm)-protected and tert-butyl (tBu)-protected cysteines in the indicated positions.
  • the peptides were synthesized on a 0.1 mmol scale using instrument default protocols with either a 4- or 5 -fold molar
  • S-(Acm)-protected cysteine was introduced into the Vesiculin A-chain at position Cys4 and S- (tBu)-protected cysteine at position Cys 6 .
  • the peptide was cleaved by treatment of the resin with 10 mL of a mixture comprised of TIPS (1%), DODT (2.5%), water (2.5%) and TFA (94%) for 2.5 h.
  • the resin was filtered away and the filtrate diluted with 8 volumes of ice-cooled 1 : 1 ether/hexane to precipitate the crude peptide. After centrifugation at 4000 rpm for 3 min the supernatant was discarded and the peptide pellet resuspended in cold ether, centrifuged and the ether again discarded.
  • the peptide was allowed to air-dry before being dissolved in 70% aqueous MeCN containing 0.1% tfa and lyophilized.
  • A-chain 4 Intrachain CyS3-Cyss disulfide bond formation
  • A-chain 6 Activation of the A-chain
  • the B-chain was synthesised using Boc-SPPS conditions, using a PAM linker.
  • the side chains of the trifunctional amino acid residues used were protected as follows: Boc-Asn: xanthyl; Arg: tosyl; Ser and Thr: benzyl (ether); Tyr: 2-bromobenzyl (ether); Asp and Glu: cyclohexyl (ester); Cys: 4-methylbenzyl (thioether) and acetamidomethyl (Acm).
  • Aminomethyl polystyrene resin of loading 1 mmol/g of (200 mg, 0.2 mmol) was swollen in DMF and drained.
  • A-chain(-SS-, SAcm) - B-chain(-SAcm) 7 formation of the first inter-chain disulfide
  • a sample of activated A-chain 6 (45 mg, 11.5 //mol) was dissolved in 6M guanidine.HCl (9 mL), Tris.HCl buffer (2.0 mL of a 1 M aqueous solution, pH 8.1) added and the stirred solution cooled in an ice-water bath. A solution of the B-chain 5 (50 mg, 12.5 mol) in 6 M
  • guanidine.HCl (8 mL) was also cooled in ice-water and then added in a dropwise manner to the solution of A-chain over a period of 15 min. Stirring was then continued for a further 30 min after which time a sample was analysed by HPLC. This showed the reaction was complete, as evidenced by disappearance of the A-chain component.
  • the solution was acidified with neat trifluoroacetic acid (200 //L) and 2 mL aliquots of the resulting solution (containing about 10 mg peptide) were purified using a Phenomenex Gemini C18 column (5//, 110 A, 10x250mm) eluting with a suitable gradient (A: water/0.1% tfa; B: MeCN/0.1% tfa). The pooled product-containing fractions were then lyophilised to give 64 mg product (71%).
  • A-chain(-SS-) - B-chain 8 formation of the second inter-chain disulfide
  • Vesiculin 9 hydrolysis of the pentalysine tag
  • a sample of A-B(Lys 5 ) 8 (10 mg) was dissolved in water (1.6 mL) and cooled in an ice-water bath. An ice-cold solution of sodium hydroxide (1.6 mL of 0.2M in water) was added and the resulting solution agitated at 0°C for 5 minutes. Neat trifluoroacetic acid (25 L) was added (ensuring pH ⁇ 2 for the solution) and the resulting crude Vesiculin 9 purified using a
  • This example describes the synthesis of synthetic human vesiculin.
  • Example 1 The process outlined above in Example 1 was repeated for human vesiculin in an identical manner, with requisite alterations in the addition of amino acids in accordance with the differing B chain sequence.
  • the low-resolution mass spectrum of the desired product, synthetic human vesiculin, is shown in Figure 9.
  • This Example relates to the characterization of blood glucose lowering effects of synthetic murine vesiculin in mice.
  • FVBN mice Proc Natl Acad Sci U S A. 1991 March 15; 88(6): 2065-2069 were obtained from the Animal Resource Centre (Canning Vale, WA, Australia). Synthetic vesiculin was prepared as described above in Example 1. Actrapid Insulin was obtained from Novo Nordisk Limited.
  • any of the terms “comprising”, “consisting essentially of, and “consisting of may be replaced with either of the other two terms in the specification.
  • the terms “comprising”, “including”, containing”, etc. are to be read expansively and without limitation.
  • the methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and that they are not necessarily restricted to the orders of steps indicated herein or in the claims. It is also that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.
  • a reference to "a host cell” includes a plurality (for example, a culture or population) of such host cells, and so forth.
  • the patent be interpreted to be limited to the specific examples or embodiments or methods specifically or otherwise expressly disclosed herein.
  • the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is expressly and specifically, without qualification or reservation, adopted in a responsive writing by Applicants.
  • the terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Diabetes (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Endocrinology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Emergency Medicine (AREA)
  • Hematology (AREA)
  • Obesity (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The invention relates generally to methods of synthesizing polypeptides and intermediates thereof, and fragments, variants and derivatives thereof, having insulin agonist activity, related compositions and formulations and their preparation and use, and methods for the prevention and treatment of conditions, diseases and disorders that would be improved, eased, or lessened by the administration of a composition of the invention, including but not limited to glucose metabolism diseases.

Description

SYNTHETIC POLYPEPTIDES AND USES THEREOF
FIELD OF THE INVENTION
The invention relates generally to synthetic peptides and proteins and related compositions and formulations and their preparation and use, and methods for the prevention and treatment of conditions, diseases and disorders that would be improved, eased, or lessened by the administration of a composition of the invention, including but not limited to glucose metabolism diseases and disorders and diseases and disorders and conditions treated or treatable with insulin and other hypoglycemic agents.
BACKGROUND OF THE INVENTION
The following includes information that may be useful in understanding the present inventions. It is not an admission that any of the information provided herein is prior art, or relevant, to the presently described or claimed inventions, or that any publication or document that is specifically or implicitly referenced is prior art.
Diabetes mellitus, characterized by hyperglycemia and altered β-cell function, is a common disorder affecting millions of individuals. According to statistics provided by the American Diabetes Association (ADA), there are 25.8 million people in the United States, or 8.3% of the population, who have diabetes. Direct medical and indirect expenditures attributable to diabetes in 2007 were estimated at $174 billion.
Type 1 and type 2 diabetes are both diseases of the pancreas characterized by hyperglycemia. In type 1 diabetes the pancreatic islet β-cells, which secrete both insulin and amylin, peptide hormones that exert profound effects on glucose metabolism, are destroyed. In type 2 diabetes these cells progressively lose function, and often fail in the late stages of the disease. As one would expect given the severity of diabetes, and difficulties associated with it, the islet β-cells play a major role in physiology.
While therapeutic regimens exist to replace insulin and amylin function, diabetic individuals remain prone to complications which are a major threat to both the quality and the quantity of life. Many patients with diabetes die early, often as a result of cardiovascular or renal complications, preceded by many years of crippling and debilitating disease beforehand. It is estimated that diabetic individuals have a 25 -fold increase in the risk of blindness, a 20-fold increase in the risk of renal failure, a 15- to 40-fold increase in the risk of amputation as a result of gangrene, and a 2- to 6-fold increased risk of coronary heart disease and ischemic brain damage. See, Klein R., et al., Diabetes Care 8:311-5 (1985). The ADA reports that two out of three people with diabetes die from heart disease and stroke, that diabetes is the leading cause of new cases of blindness in people ages 20-74, that diabetes is the leading cause of end-stage renal disease (kidney failure), accounting for about 44 percent of new cases (with approximately 48,374 people with diabetes initiating treatment for end stage renal disease and 202,290 undergoing dialysis or kidney transplantation in the year 2008), that more than 60 percent of nontraumatic lower-limb amputations in the U.S. occur among people with diabetes (with more than 65,700 amputations performed among people with diabetes in 2006), that people with diabetes are two to four times more likely to suffer strokes (and once having had a stroke, are two to four times as likely to have a recurrence), that deaths from heart disease in women with diabetes have increased 23 percent over the past 30 years (compared to a 27 percent decrease in women without diabetes), and that deaths from heart disease in men with diabetes have decreased by only 13 percent (compared to a 36 percent decrease in men without diabetes).
Type 1 diabetes is characterized by an early loss of endocrine function in the pancreas due to autoimmune destruction of the pancreatic islet β-cells, resulting in hypoinsulinemia, hypoamylinemia, and hyperglycemia. Type 2 diabetes is a polygenic and heterogeneous disease resulting from an interaction between genetic factors and environmental influences. See, e.g., Kecha-Kamoun et al., Diabetes Metab Res Rev 17: 146-152 (2001).
Although type 2 diabetes is initially characterized by hyperinsulinemia, peripheral insulin resistance and resulting hyperglycemia characterize type 2 diabetes, β -cells often compensate for this insulin resistance with both an increase in insulin secretory capacity and β- cell mass. Levels of insulin eventually decrease as a result of the loss of β-cell function and eventual β-cell failure. Thus, there is a progression from normal glucose tolerance, to impaired glucose tolerance, to type 2 diabetes, and to late stage type 2 diabetes, which is associated with altered β-cell function, β-cell loss and, eventually, a decline in insulin secretion. See, e.g., Dickson et al., J. Biol. Chem. 276:21110-21120 (2001). In other words, hyperglycemia worsens as β-cells fail to sustain levels of insulin output sufficient to overcome increasing resistance to insulin. Kaytor, et al., J Biol Chem. 16: 16 (2001). Eventual β-cell failure is primarily a failure in function but later proceeds to β-cell loss such as that seen in type 1 diabetes. One of the most striking functional β-cell defects is a loss of acute glucose-induced insulin secretion (GIIS). β- cells initially adapt to increased demand for insulin but then decompensate as type 2 diabetes worsens. One hypothesis is that β-cells can become de-differentiated, leading to a loss of specialized functions, such as GIIS. Weir et al., Diabetes, 50 Supplement 1, S154-S159 (2001). It is understood that integrated networks of signaling events act in concert to control β- cell mass adaptation to insulin demand, and there is some evidence to suggest that increased β- cell growth might in some part be due to a circulating growth factor. See, e.g., Flier et al. (2001) Proc. Nat. Acad. Sci. USA, 98:7475-7480, which reported that transplantation of normal islets into the pancreas or kidney capsule of insulin resistant mice led to a marked increase in β-cell mass.
Despite decades of research into diabetes and its causes, despite enormous research on β-cells themselves and the proteins they produce, and despite the existence and use of therapies for the treatment of people with type 1 and type 2 diabetes, serious problems remain.
The identification of other treatment options for diabetes would be of great benefit in the continuing effort to further improve the lives of people with diabetes including, for example, treatments based on the proteins that increase β-cell mass and address issues relating to hyperglycemia and the long-term complications of diabetes and the loss of circulating proteins as a result of β-cell destruction.
It is an object of the present invention to provide methods for synthesizing polypeptides amenable to use in the treatment of diseases, such as diabetes, and to provide the synthetic polypeptides themselves, or to at least provide the public with useful choice.
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date.
BRIEF DESCRIPTION OF THE INVENTION
The inventions described and claimed herein have many attributes, aspects, and embodiments including, but not limited to, those set forth or described or referenced in this Brief Summary. It is not intended to be all-inclusive and the inventions described and claimed herein are not limited to or by the features or embodiments identified in this Brief Summary, which is included for purposes of illustration only and not restriction.
The inventions described and claimed herein relate to the synthesis of polypeptides including polypeptides having insulin agonist activity and their synthetic intermediates.
In a first aspect, the invention relates to a method of synthesizing a polypeptide comprising a first peptide chain and a second peptide chain, the method comprising providing a first peptide chain and a second peptide chain, forming under conducive conditions one or more interchain disulfide bonds between the first peptide chain and the second peptide chain, and recovering the polypeptide from the reaction medium, wherein the first peptide chain comprises an amino acid sequence corresponding to
A chain : Gly He Val Glu Glu Cys3 Cys4 Phe Arg Ser Cys5 Asp Leu Ri R2 Leu Glu R3
Tyr Cys6 Ala R4 R5 Rg R7 R8 R9 (SEQ ID NO : 1 )
and wherein the second peptide chain comprises an amino acid sequence corresponding to
B chain : R10 Rn Ri2 Ri3 Ri4 Glu Thr Leu Cysi Gly Gly Glu Leu Val Asp Ri5 Leu Gin
Phe Ri6 Cys2 Rn Rig Arg Gly Phe Tyr Phe Rl9 R20 R21 R22 R23 R24 R25 26 R27 R28 R29 (SEQ ID NO :2)
wherein Ri is Ala, Asn or Leu; R2 is Leu or He; R3 is Thr or Gin; R4 is Thr, Ala, Lys, or Val; R5 is Pro or Ser; Rg is Ala, Val, or Pro; R7 is Lys or Glu, R8 is Ser or Ala, R9 is Glu or Ala, R10 is absent or is Ala, Glu, or Asp, Rn is Tyr, Ala, or Val, R12 is Arg, Gly, or Ala, Ri3 is Pro, Thr, Ser, or Leu, Ri4 is Ser, Gly, Ala, or Glu, R15 is Thr or Ala, Ri6 is Val or He, R17 is Gly, Ser, Glu, or Ala, Ri8 is Asp or Glu, R19 is Ser or Val, R20 is Arg, Leu, or Ser, R2i is Pro or Lys, R22 is Ala, Ser, Gly, Val, or Thr, R23 is Ser, Gly, or Val, R24 is Arg, Pro or Gly, R25 is Ala, Arg, Val, He, Leu, Asn, Ser, or Gly, R26 is Ser, Asn, or Arg, R2-7 is absent or is Arg, Ser, Asn, R28 is absent or is Val, R2 is absent or is Ser.
In certain embodiments, Rj - R2 include conservative amino acid variants for the amino acids listed above. Thus, Ri is Ala, Asn, Leu, or a conservative variant of either, R2 is Leu, He, or a conservative variant of either, R3 is Thr, Gin, or a conservative variant of either, R4 is Thr, Ala, Lys, Val, or a conservative variant of either, R5 is Pro, Ser, or a conservative variant of either, Rg is Ala, Val, Pro, or a conservative variant thereof, R7 is Lys, Glu, or a conservative variant of either, R8 is Ser, Ala, or a conservative variant of either, R is Glu, Ala, or a conservative variant of either, Rio is absent or is Ala, Glu, Asp, or a conservative variant thereof, Rn is Tyr, Ala, Val, or a conservative variant of either, R12 is Arg, Gly, Ala, or a conservative variant thereof, Ri3 is Pro, Thr, Ser, Leu, or a conservative variant thereof, Ri4 is Ser, Gly, Ala, Glu, or a conservative variant thereof, R15 is Thr, Tyr, Ala, or a conservative variant thereof, Ri6 is Val, He, or a conservative variant thereof, R17 is Gly, Ser, Glu, Ala, or a conservative variant thereof, Ri8 is Asp, Glu, or a conservative variant of either, R1 is Ser, Val, or a conservative variant of either, R20 is Arg, Leu, Ser, or a conservative variant thereof, R21 is Pro, Lys, or a conservative variant of either, R22 is Ala, Ser, Gly, Val, Thr, or a conservative variant thereof, R23 is Ser, Gly, Val, or a conservative variant thereof, R24 is Arg, Pro, Gly, or a conservative variant thereof, R25 is Ala, Arg, Val, He, Leu, Asn, Ser, Gly, or a conservative variant thereof, R26 is Ser, Asn, Arg, or a conservative variant thereof, R27 is absent or is Arg, Ser, Asn, or a conservative variant thereof, R28 is absent or is Val, or a conservative variant thereof, R2 is absent or is Ser, or a conservative variant thereof.
In one embodiment, the first peptide chain and the second peptide chain are each provided separately. In another embodiment, the first peptide chain and the second peptide chain are provided together in the form of a polypeptide comprising the first peptide chain and the second peptide chain, wherein the first peptide chain is bound to the second peptide chain by one interchain disulfide bond.
The thiol group of each cysteine residue present in the first and second chains may be protected with a suitable protecting group, provided that at least one cysteine residue in the first peptide chain and at least one cysteine residue in the second peptide chain are available to form the one or more interchain disulfide bonds.
In one embodiment, one or more of the cysteine residues present in either the first peptide chain, the second peptide chain, or in both the first peptide chain and the second peptide chain are protected with one or more suitable protecting groups. In one embodiment, one or more of the cysteine residues present in both the first peptide chain and the second peptide chain are protected with one or more suitable protecting groups.
In one embodiment, Cys4 and Cysi are each independently protected with a suitable protecting group or Cys2 and Cys6 are each independently protected with a suitable protecting group. In one exemplary embodiment, Cys4 and Cysi are each independently protected with a suitable protecting group. In another embodiment, Cys2 and Cys6 are each independently protected with a suitable protecting group.
In one embodiment, Cys3 or Cys5 are each independently protected with a suitable protecting group or bound together in an intrachain disulfide bond.
In one embodiment, Cys3 or Cys5 are each independently protected with a suitable protecting group or bound together in an intrachain disulfide bond; and Cys4 and Cysi are each independently protected with a suitable protecting group or bound together in an interchain disulfide bond; or Cys2 and Cys6 are each independently protected with a suitable protecting group or bound together in an interchain disulfide bond. In one embodiment, Cys3 or Cys5 are each independently protected with a suitable protecting group or bound together in an intrachain disulfide bond; and Cys4 and Cysi are each independently protected with a suitable protecting group or Cys2 and Cys6 are each independently protected with a suitable protecting group. In one exemplary embodiment, Cys3 or Cys5 are each bound together in an intrachain disulfide bond; and Cys4 and Cysi are each independently protected with a suitable protecting group or Cys2 and Cys6 are each independently protected with a suitable protecting group. In one exemplary embodiment, Cys3 or Cyss are each bound together in an intrachain disulfide bond and Cys4 and Cysi are each independently protected with a suitable protecting group. In another embodiment, Cys3 or Cys5 are each bound together in an intrachain disulfide bond and Cys2 and Cys6 are bound together in an interchain disulfide bond.
In one embodiment, one or more of the cysteine residues in the first peptide chain, the second peptide chain, or in both the first peptide chain and the second peptide chain are protected with one or more suitable protecting groups removable under the same conditions. In one embodiment, the suitable protecting groups removable under the same reaction conditions are identical.
In one embodiment, two or more of the cysteine residues present in either the first peptide chain, the second peptide chain, or in both the first peptide chain and the second peptide chain are with a suitable protecting group. In one embodiment, the two or more cysteine residues are differentially protected with two or more suitable protecting groups such that one or more protecting groups may be selectively removed in the presence of the other protecting groups (i.e. without removing the other protecting groups) by the judicious choice of reaction conditions.
In one embodiment, Cys4 is differentially protected with respect to Cys3 and Cys5. In another embodiment, Cys6 is differentially protected with respect to Cys3 and Cys5. In another embodiment, Cysi and Cys4 are differentially protected with respect to Cys3 and Cys5. In another embodiment, Cys2 and Cys6 are differentially protected with respect to Cys3 and Cys5.
In one embodiment, Cysi is differentially protected with respect to Cys4 or Cys2 is differentially protected with respect to Cys6.
In one embodiment, the protecting groups for the cysteine residues are selected from the group consisting of trityl, acetamidomethyl, tert-butyl, tert-butylthio, xanthyl, picolyl, and 4- methoxytrityW-methylbenzyl. In another embodiment, the protecting groups are selected from the group consisting of trityl, acetamidomethyl, tert-butyl, tert-butylthio, and 4-methoxytrityl4- methylbenzyl. In another embodiment, the protecting groups are selected from the group consisting of trityl, acetamidomethyl, and tert-butyl. In one exemplary embodiment, Cysi and Cys4 are each protected by an acetamidomethyl group. In another exemplary embodiment, Cys6 is protected with a tert-butyl group. In another exemplary embodiment, Cys3 and Cyss are each protected by a trityl.
In one embodiment, one or more of the amino acids other than cysteine in the first peptide chain, the second peptide chain, or in both the first peptide chain and the second peptide chain are protected with one or more suitable protecting groups. In one embodiment, the one or more suitable protecting groups can be selectively removed without removing one or more cysteine protecting groups.
At least one cysteine residue in the first peptide chain and at least one cysteine residue in the second peptide chain must be available to form an interchain disulfide bond.
In one embodiment, one cysteine residue in the first peptide chain and one cysteine residue in the second peptide chain are available to form an interchain disulfide bond. In one embodiment, the other cysteine residues present in the chains are protected with one or more suitable protecting groups or bound in one or more intrachain or interchain disulfide bonds. In one embodiment, the other cysteine residues present in the chains protected with one or more protecting groups are differentially protected with two or more protecting groups.
In one embodiment, Cys4 and Cysi are each independently protected with a suitable protecting group or are bound together in an interchain disulfide bond and Cys2 and Cys6 are available to form an interchain disulfide bond; or Cys2 and Cys6 are each independently protected with a suitable protecting group or bound in an interchain disulfide bond and Cys4 and Cysi are available to form an interchain disulfide bond. In one embodiment, Cys4 and Cysi are each independently protected with a suitable protecting group and Cys2 and Cys6 are available to form an interchain disulfide bond; or Cys2 and Cys6 are each independently protected with a suitable protecting group and Cys4 and Cysi are available to form an interchain disulfide bond. In one exemplary embodiment, Cys4 and Cysi are each independently protected with a suitable protecting group and Cys2 and Cys6 are available to form an interchain disulfide bond. In another exemplary embodiment, Cys4 and Cysi are available to form an interchain disulfide bond and Cys2 and Cys6 are bound together in an interchain disulfide bond.
In one embodiment, Cys3 and Cyss are each independently protected with one or more suitable protecting groups or are bound together in an intrachain disulfide bond. In one exemplary embodiment, Cys3 and Cys5 are bound together in an intrachain disulfide bond; Cys4 and Cysi are each independently protected with a suitable protecting group; and Cys2 and Cys6 are available to form an interchain disulfide bond. In another exemplary embodiment, Cys3 and Cys5 are bound together in an intrachain disulfide bond; Cys4 and Cysi are available to form an interchain disulfide bond; and Cys2 and Cys6 are bound together in an interchain disulfide bond.
In one embodiment, at least one of the cysteine residues in the first and second peptide chains available to form a disulfide bond are in the form of a thiol. In one exemplary embodiment, both of the cysteine residues in the first and second peptide chains available to form a disulfide bond are in the form of a thiol. In another exemplary embodiment, one of the cysteine residues in the first and second peptide chains available to form a disulfide bond is in the form of a thiol and the other is in a form activated towards formation of a disulfide bond. In one specifically contemplated embodiment, the activated form is a pyridyldisulfide derivative of the thiol group.
In one embodiment, the first chain comprises an intrachain disulfide bond. In one specifically contemplated embodiment, the first chain comprises an intrachain disulfide bond between Cys3 and Cys5.
In one embodiment, the first peptide chain, the second peptide chain or both the first peptide chain and the second peptide chain additionally comprises one or more solubilising groups. In one exemplary embodiment, the first peptide chain comprises one or more solubilising groups.
In one embodiment, the solubilising group enhances the solubility of the peptide chain in the reaction medium. In another embodiment, the solubilising group prevents or inhibits intrachain association.
In one embodiment, the one or more solubilising groups are linked to the C-termini of the peptide chain, optionally via a suitable linker.
Examples of suitable linkers include aryl linkers, for example 4-hydroxymethyl benzoic acid or 4-hydrazinobenzoic acid. Other suitable linkers will be apparent to those skilled in the art. In one embodiment, the linker is bound to the C-termini of the peptide via a ester, thioester, or amide bond. The nature of the bond will depend on the linker used.
In one embodiment, the solubilising group is a polycationic amino acid sequence. In one embodiment, the cationic amino acids are arginine or lysine residues. In one embodiment, the sequence comprises from 2 to 20, 2 to 15, 2 to 10, 3 to 7, or 3 to 5 amino acids. In one embodiment, the solubilising group is a poly-lysine or poly-arginine tag. In one embodiment, the solubilising group is a tri-, terra-, penta-, hexa-, or hepta- lysine or arginine tag. In one specifically contemplated example, the solubilising group is a pentalysine tag. In one exemplary embodiment, the a pentalysine tag is linked to the C-termini of the peptide chain via a 4- hydroxymethyl benzoic acid (HMBA) linker.
In one embodiment, the first peptide chain, the second peptide chain, or both the first peptide chain and the second peptide chain are bound to a solid phase support, optionally via a suitable linker.
Examples of suitable linkers include the Rink amide linker, phenylacetamido (PAM) linker, Sheppard's linker, and Wang ester linker. Other suitable linkers will be apparent to those skilled in the art.
In one embodiment, the peptide chain is bound to the solid phase support via a solubilising group linked to the C-termini of the peptide chain. In one embodiment, the solubilising group is bound to the C-termini of the peptide chain via a suitable linker. In another embodiment, the solubilising group is bound to the solid phase support via a suitable linker.
In one embodiment, one of the one or more interchain bonds is formed between Cysi and Cys4, or is formed between Cys2 and Cys6. In one specifically contemplated embodiment, the method comprises forming an interchain disulfide bond between Cysi and Cys4 and between Cys2 and Cys6. In one embodiment, the method comprises first forming an interchain disulfide bond between Cysi and Cys4, optionally followed by an interchain disulfide bond between Cys2 and Cys6. In a further specifically contemplated embodiment, the method comprises first forming an interchain disulfide bond between Cys2 and Cys6, optionally followed by forming an interchain disulfide bond between Cysi and Cys4. In a further specifically contemplated embodiment, the method comprises first forming an interchain disulfide bond between Cys2 and Cys6, then forming an interchain disulfide bond between Cysi and Cys4.
In one specifically exemplified embodiment, the method comprises first forming an intrachain disulfide bond between Cys3 and Cyss, then forming an interchain disulfide bond between Cys2 and Cys6 or forming an interchain disulfide bond between Cysi and Cys4, and then forming an interchain disulfide bond between the other of Cysi and Cys4 or Cys2 and Cys6. In another embodiment, the method comprises first forming an interchain disulfide bond between Cys2 and Cys6 or forming an interchain disulfide bond between Cysi and Cys4, and then forming an interchain disulfide bond between the other of Cysi and Cys4 or Cys2 and Cys6, and then forming an intrachain disulfide bond between Cys3 and Cys5. In another embodiment, the method comprises first forming an interchain disulfide bond between Cys2 and Cys6 or forming an interchain disulfide bond between Cysi and Cys4, then forming an intrachain disulfide bond between Cys3 and Cys5, and then forming an interchain disulfide bond between the other of Cysi and Cys4 or Cys2 and Cys6,
In one specifically contemplated example, the method comprises first forming an intrachain disulfide bond between Cys3 and Cyss, then forming an interchain disulfide bond between Cys2 and Cys6, and then forming an interchain disulfide bond between Cysi and Cys4.
In one embodiment, the one or more interchain disulfide bonds are formed under oxidative conditions. Any suitable oxidant or combination of oxidants may be used to provide the oxidative conditions. Examples of suitable oxidants include dipyridyldisulfide, iodine, thallium(III) trifluoroacetate, molecular oxygen, dimethylsulfoxide, and the like. In one specifically contemplated embodiment, the oxidant is dipyridyldisulfide or iodine. In one exemplary embodiment, the oxidant is dipyridyldisulfide. In another exemplary embodiment, the oxidant is iodine.
In one embodiment, the reaction medium is a liquid reaction medium. In one embodiment, the liquid reaction medium comprises one or more suitable solvents. Examples of suitable solvents include dimethylformamide, dichloromethane, chloroform, carbon tetrachloride, water, methanol, ethanol, dimethylsulfoxide, trifluoroacetic acid, acetic acid, acetonitrile, and mixtures thereof.
In one embodiment, the liquid reaction medium comprises one or more buffers, for example a phosphate, citrate, guanidine, 2-amino-2-hydroxymethyl-propane-l,3-diol (Tris) buffer, carbonate, or 4-(2-hydroxyethyl)-l-piperzineethanesulfonic acid (HEPES).
In one embodiment, the reaction medium is at a temperature below ambient temperature. In one embodiment, the reaction medium is at a temperature from -75 to 15 °C, from -50 to 10 °C, or from -20 to 5 °C. In one embodiment, the reaction medium is at a temperature less than 15 °C, 10 °C, 5 °C, less than 0 °C, less than -10 °C, or less than -20 °C. In one embodiment, the reaction medium is at a temperature from -10 to 5 °C, for example 0 °C. The reaction medium may be cooled by any suitable method known in the art, for example, immersing a vessel containing the reaction medium in an ice bath.
In one embodiment, the reaction medium is at a temperature above ambient temperature. In one embodiment, the reaction medium is at a temperature from 40 to 200 °C, from 50 to 150 °C, from 60 to 100 °C, from 65 to 90 °C, or from 70 to 80 °C. In one embodiment, the reaction medium is at a temperature greater than 40 °C, greater than 50 °C, greater than 75 °C, greater than 100 °C, or greater than 150 °C. The reaction medium may be heated using any suitable method known in the art, for example, immersing a vessel containing the reaction medium in a heated oil bath. The temperature used may depend on, for example, the boiling points and degradation of solvents present in the reaction medium.
In one embodiment, the reaction medium is irradiated with microwave irradiation. In another embodiment, the reaction medium is irradiated with ultraviolet light.
In one embodiment, the disulfide bonds are formed under an atmosphere of ambient gas. In one embodiment, the ambient gas is selected from the group consisting of nitrogen and argon.
In another embodiment, the disulfide bonds are formed under an atmosphere of oxygen gas.
In one embodiment, the reaction medium is mixed. The reaction medium may be mixed by any suitable method known in the art, for example, using a magnetic stirrer in the reaction medium or agitating a vessel containing the reaction medium, for example using a vortex mixer.
The progress of the disulfide bond forming reactions may be monitored by any suitable means, for example HPLC.
In one embodiment, the reaction is allowed to proceed to completion, as monitored by the consumption of at least one of the starting materials by HPLC. In one embodiment, the reaction is allowed to proceed for a period of time from 1 minute to 7 days, 5 minutes to 72 hours, 10 minutes to 48 hours, 15 minutes to 24 hours. In another embodiment, the reaction is allowed to proceed for a period of time less than 72 h, less than 48 h, less than 24 h, less than 12 h, less than 6 h, less than 4 h, less than 2 h, or less than 1 h.
In some embodiments where the method comprises forming more than one interchain disulfide bond, the conducive conditions for forming each interchain disulfide bond are different. In some embodiments where the method comprises forming more than one interchain disulfide bond, the conducive conditions for forming each interchain disulfide bond are the same. In some embodiments where the method comprises forming more than one interchain disulfide bond, more than one interchain disulfide bonds are formed in the same reaction.
In one embodiment, the method comprises forming under conducive conditions an intrachain disulfide bond in the first peptide chain. The intrachain disulfide may be formed under any of the conditions conducive to formation of the one or more interchain disulfide bonds described herein.
In one embodiment, the method comprises removing one or more cysteine protecting groups in the in the first peptide chain, second peptide chain, or both the first peptide chain and the second peptide chain to provide one or more thiol groups. In one embodiment, the method comprises converting the thiol group of one or more cysteine residues in the first peptide chain, second peptide chain, or both the first peptide chain and the second peptide chain, into a form activated towards formation of a disulfide bond. In one embodiment, the method comprises recovering and optionally purifying the activated form.
In some embodiments where the first peptide chain, second peptide chain, or both the first peptide chain and the second peptide chain, comprise one or more solubilising groups, optionally bound via a suitable linker, the method comprises cleaving the solubilising group and optional linker.
In some embodiments where a solid phase support is bound to the first peptide chain, second peptide chain, or both the first peptide chain and the second peptide chain, optionally via a suitable linker, the method comprises cleaving the solid phase support and optional linker.
The polypeptide may be recovered from the reaction medium by any suitable method known in the art.
In one embodiment, the polypeptide is recovered after forming one interchain disulfide bond and, optionally, purified. In one embodiment, the polypeptide is recovered after forming two interchain disulfide bonds and, optionally purified.
In one embodiment, the polypeptide is recovered and, optionally, purified, after forming each interchain disulfide bond. In another embodiment, the polypeptide is recovered after forming an intrachain disulfide bond in the first peptide chain and two interchain disulfide bonds between the first peptide chain and the second peptide chain, and optionally purified. In one embodiment, the polypeptide is recovered and optionally purified after forming an intrachain disulfide bond in the first peptide chain and after forming each interchain disulfide bond between the first peptide chain and the second peptide chain.
In some embodiments where an intrachain disulfide bond in the first peptide chain is formed before the one or more interchain disulfide bonds between the first peptide chain and the second peptide chain, the method comprises recovering and optionally purifying the first peptide chain after forming the intrachain disulfide bond.
In one embodiment, recovering the polypeptide from the reaction medium comprises precipitating the polypeptide and optionally separating the polypeptide from the reaction medium. The precipitated polypeptide may be separated from the reaction medium by for example, centrifuging and decanting or filtering the reaction medium.
In some embodiments where the first peptide chain, the second peptide chain, or both the first and the second peptide chain are bound to a solid phase support, recovering the polypeptide comprises separating the solid phase support from the reaction medium, for example by decanting or filtering the reaction medium. In one embodiment, recovering the polypeptide comprises cleaving the polypeptide from the solid phase support.
In some embodiments where the first peptide chain, the second peptide chain, or both the first and the second peptide chain comprise one or more solubilising groups, recovering the polypeptide comprises cleaving the solubilising group from the polypeptide. In some embodiments where the solubilising group is bound to the peptide chain via a suitable linker, recovering the polypeptide comprises cleaving the peptide chain from the linker.
In one embodiment, the method comprises purifying the polypeptide after recovering the polypeptide from the reaction medium. In specifically contemplated embodiments, the polypeptide is purified by HPLC using one or more suitable solvents.
In one embodiment, the first peptide chain, the second peptide chain, or both the first peptide chain and the second peptide chain are synthesized using solid phase peptide synthesis.
In one embodiment, the peptide chains are synthesized by stepwise solid phase peptide synthesis or sequential solid phase fragment condensation. In exemplary embodiments, the peptide chains are synthesized by stepwise solid phase peptide synthesis. In one embodiment, the peptide chains are synthesized by Fmoc or Boc solid phase peptide synthesis.
The synthesis comprises assembling the amino acid sequences of the peptide chains on a suitable solid phase support. In one embodiment, the solid phase support is a polyethylene glycol resin or a polystyrene resin.
In one embodiment, the amino acid sequence of the peptide chain is assembled on the solid phase support via a suitable linker. In one embodiment, the synthesis comprises binding the linker to the solid phase support.
In some embodiments, the synthesis comprises assembling the amino acid sequence of the peptide chain and incorporating one or more solubilising groups. In one embodiment, the synthesis comprises binding the solubilising group, optionally via a suitable linker, to the solid phase support and assembling the amino acid sequence of the peptide chain on the solubilising group. In one embodiment, the synthesis comprises assembling the amino acid sequence of the peptide chain on the solubilising group via a suitable linker.
The side chains of the amino acids incorporated into the peptide chain may be protected by one or more suitable protecting groups. The protecting groups are selected having regard to the overall strategy for synthesizing the polypeptide, for example, for example the conditions used for synthesising the peptide chain, cleaving the peptide from the solid phase support, and forming the one or more interchain disulfide bonds. In one embodiment, one or more of the cysteine residues of the peptide chain are differentially protected with one or more suitable protecting groups.
In one embodiment, the synthesis comprises cleaving the peptide chain from the solid phase support. In some embodiments where the peptide chain is bound to the solid phase support via a suitable linker, the peptide chain is cleaved from the solid phase support by cleaving the peptide chain from the linker.
In some embodiments where the peptide chain is bound to the solid phase support via a solubilising group, the synthesis comprises cleaving the solubilising group from the solid phase support. In some embodiments where the solubilising group is bound to the solid phase support via a suitable linker, the solubilising group is cleaved from the linker.
In some embodiments where the peptide chain comprises one or more solubilising groups, the synthesis comprises cleaving the one or more solubilising groups. In some embodiments where the solubilising group is linked to the peptide chain via a suitable linker, the peptide chain is cleaved from the linker.
In one embodiment, the synthesis comprises removing the Na-amino protecting group of the N-terminal amino acid of the peptide chain. In one embodiment, the Na-amino protecting group of the N-terminal amino acid of the peptide chain is removed on cleaving the peptide from the linker bound to the solid phase support.
In one embodiment, the synthesis comprises removing one or more amino acid side chain protecting groups. In one embodiment the one or more protecting groups are removed while the peptide chain is bound to the solid phase support. In another embodiment, the one or more protecting groups are removed on cleaving the peptide chain from the solid phase support. In another embodiment, the one or more protecting groups are removed after cleaving the peptide chain from the solid phase support.
In one embodiment, the synthesis comprises one or more purification steps. In specifically contemplated embodiments, the peptide chain is purified after it has been cleaved from the solid phase support. In one embodiment, the peptide chain is purified by HPLC using one or more suitable solvents.
In one embodiment, synthesis of the first peptide chain or the second peptide chain comprises converting the thiol group of one or more cysteine residues into a form activated towards formation of an interchain disulfide bond. In one embodiment, the first peptide chain is synthesized using solid phase peptide synthesis. In one embodiment, the first peptide is synthesized using Fmoc solid phase peptide synthesis. In one embodiment, the synthesis comprises assembling the amino acid sequence of the first peptide on the solid phase support, optionally via a suitable linker.
In one embodiment, the synthesis comprises assembling the amino acid sequence of the first peptide and incorporating one or more solubilising groups. In one embodiment, the synthesis comprises binding the solubilising group to the solid phase support, optionally via a suitable linker, and assembling the amino acid sequence of the first peptide chain on the solubilising group. In one embodiment, synthesis comprises assembling the amino acid sequence of the peptide chain on the solubilising group via a suitable linker.
In one embodiment, the amino acid sequence of the first peptide chain is assembled using amino acids optionally protected with one or more suitable protecting groups. In one embodiment, each cysteine in the amino acid sequence is optionally protected with one or more suitable protecting groups. In one embodiment, at least Cys4 and Cys6 are differentially protected with respect to Cys3 and Cys5. In one embodiment, Cys4 and Cys6 are differentially protected.
In one embodiment, the synthesis comprises cleaving the first peptide chain from the solid phase support. In some embodiments where the first peptide chain is bound to the solid phase support via a suitable linker, the first peptide chain is cleaved from the linker. In some embodiments where the first peptide chain comprises a solubilising group bound to the solid phase support via a suitable linker, the synthesis comprises cleaving the solubilising group from the solid phase support to provide a first peptide chain comprising the solubilising group.
In one embodiment, the synthesis comprises cleaving the solubilising group from the first peptide chain. In some embodiments where the amino acid sequence of the first peptide chain is bound to the solubilising group via a suitable linker, the first peptide chain is cleaved from the linker.
In one embodiment, the synthesis comprises removing one or more amino acid side chain protecting groups, including one or more cysteine protecting groups prior to, during, or after cleavage of the first peptide from the solid phase support. In one embodiment, the synthesis comprises selectively removing one or more amino acid side chain protecting groups without removing one or more cysteine protecting groups.
In one embodiment, the one or more amino acid side chain protecting groups are selected from the group consisting of acetamidomethyl, 2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), tert-butyl, trityl, and tert-butyloxycarbonyl. In one embodiment, the side chains of one or more arginine residues are protected with one or more Pbf groups; the side chains of one or more aspartic acid, glutamic acid, serine, threonine, or tyrosine residues are protected with one or more tert-butyl groups; the side chains of one or more asparagine, glutamine, or histadine residues are protected with one or more trityl groups; the side chains of one or more cysteine residues are protected with one or more trityl, acetamidomethyl, or tert-butyl groups; and the side chains of one or more tryptophan or lysine residues are protected with one or more tert-butylcarbonyloxy groups.
In one embodiment, the one or more cysteine protecting groups are selected from the group consisting of tert-butyl, acetamidomethyl, and trityl.
In one embodiment, the synthesis comprises removing one or more cysteine protecting groups.
In one embodiment, the synthesis comprises converting the thiol group of one or more cysteine residues in the first peptide chain into a form activated towards formation of a disulfide bond. In one embodiment, the method comprises recovering and optionally purifying the activated form.
In one embodiment, the synthesis comprises purifying the first peptide after cleaving the peptide from the solid phase support. In one embodiment, purification is carried out by HPLC using a suitable solvent system.
In one embodiment, the synthesis of the first peptide chain comprises forming an intrachain disulfide bond. In one embodiment, the intrachain disulfide bond is between Cys3 and Cys5.
In one exemplary embodiment, the first peptide chain is synthesized using solid phase peptide synthesis as described herein in the examples.
In one embodiment, the second peptide chain is synthesized using solid phase peptide synthesis. In one embodiment, the second peptide is synthesized using Boc solid phase peptide synthesis. In one embodiment, the synthesis comprises assembling the amino acid sequence of the second peptide chain on the solid phase support, optionally via a suitable linker.
In one embodiment, the amino acid sequence of the second peptide chain is assembled using amino acids optionally protected with one or more suitable protecting groups. In one embodiment, each cysteine in the amino acid sequence is optionally protected with one or more suitable protecting groups. In one embodiment, at least one of Cysi and Cys2 is protected with one or more suitable protecting groups. In one embodiment, Cysi and Cys2 are differentially protected with one or more suitable protecting groups. In one specifically contemplated embodiment, Cysi is protected with a suitable protecting group.
In one embodiment, the synthesis comprises cleaving the second peptide chain from the solid phase support.
In one embodiment, the synthesis comprises removing one or more amino acid side chain protecting groups, including one or more cysteine protecting groups prior to, during, or after cleavage of the second peptide chain from the solid phase support.
In one embodiment, the synthesis comprises removing one or more cysteine protecting groups.
In one embodiment, the one or more amino acid side chain protecting groups are selected from the group consisting of xanthyl, tosyl, benzyl, 2-bromobenzyl, cyclohexyl, 4- methylbenzyl, acetamidomethyl. In one embodiment, the side chains of one or more asparagine residues is protected with one or more xanthyl groups; the side chains of one or more arginine residues is protected with one or more tosyl groups; the side chains of one or more serine or threonine residues is protected with one or more benzyl groups; the side chains of one or more tyrosine residues is protected with one or more 2-bromobenzyl groups; the side chains of one or more aspartic acid or glutamic acid residues is protected with one or more cyclohexyl groups; the side chains of one or more cysteine groups is protected with one or more 4-methylbenzyl or acetamidomethyl groups.
In one embodiment, the one or more cysteine protecting groups are selected from the group consisting of tert-butyl, acetamidomethyl, 4-methylbenzyl, and trityl. In one embodiment, the one or more cysteine protecting groups are selected from the group consisting of tert-butyl, acetamidomethyl, and trityl. In one embodiment, the one or more cysteine protecting groups are selected from the group consisting of 4-methylbenzyl and acetamidomethyl. In one embodiment, the one or more cysteine protecting groups are acetamidomethyl groups.
In one embodiment, the synthesis comprises purifying the cleaved second peptide chain. In one embodiment, purification is carried out by HPLC using a suitable solvent system. In one embodiment, the solvent system comprises formic acid.
In one exemplary embodiment, the second peptide chain is synthesized using solid phase peptide synthesis as described herein in the examples.
In another aspect, the invention relates to a method of synthesizing a polypeptide comprising an amino acid sequence corresponding to A chain : Gly He Val Glu Glu Cys3 Cys4 Phe Arg Ser Cys5 Asp Leu Ri R2 Leu Glu R3 Tyr Cys6 Ala R4 R5 Rg R7 Rg R9 (SEQ ID NO: 1)
wherein Ri is Ala, Asn or Leu; R2 is Leu or He; R3 is Thr or Gin; R4 is Thr, Ala, Lys, or Val; R5 is Pro or Ser; R6 is Ala, Val, or Pro; R7 is Lys or Glu, R8 is Ser or Ala, and R9 is Glu or Ala, the method essentially as described herein in the examples.
In another aspect, the invention relates to a method of synthesizing a polypeptide comprising an amino acid sequence corresponding to
B chain : R10 Rn Ri2 R13 Ri4 Glu Thr Leu Cysi Gly Gly Glu Leu Val Asp Ri5 Leu Gin
Phe Ri6 Cys2 Rn Rig Arg Gly Phe Tyr Phe Rl9 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 (SEQ ID NO :2)
wherein R10 is absent or is Ala, Glu, or Asp, Rn is Tyr, Ala, or Val, Ri2 is Arg, Gly, or Ala, R13 is Pro, Thr, Ser, or Leu, Ri4 is Ser, Gly, Ala, or Glu, R15 is Thr or Ala, Ri6 is Val or He, Rn is Gly, Ser, Glu, or Ala, Ri8 is Asp or Glu, R1 is Ser or Val, R2o is Arg, Leu, or Ser, R21 is Pro or Lys, R22 is Ala, Ser, Gly, Val, or Thr, R23 is Ser, Gly, or Val, R24 is Arg, Pro or Gly, R25 is Ala, Arg, Val, He, Leu, Asn, Ser, or Gly, R26 is Ser, Asn, or Arg, R27 is absent or is Arg, Ser, Asn, R28 is absent or is Val, R2 is absent or is Ser, the method essentially as described herein in the examples.
In another aspect a synthetic polypeptide is provided, wherein the synthetic polypeptide comprises an amino acid sequence corresponding to
A chain : Gly He Val Glu Glu Cys3 Cys4 Phe Arg Ser Cys5 Asp Leu Ri R2 Leu Glu R3
Tyr Cys6 Ala R4 R5 R, R7 R8 R9 (SEQ ID NO: 1);
wherein Ri is Ala, Asn or Leu; R2 is Leu or He; R3 is Thr or Gin; R4 is Thr, Ala, Lys, or Val; R5 is Pro or Ser; R6 is Ala, Val, or Pro; R7 is Lys or Glu, Rg is Ser or Ala, and R9 is Glu or Ala; or an amino acid sequence corresponding to
B chain : Rio Rn R12 R13 Ri4 Glu Thr Leu Cysi Gly Gly Glu Leu Val Asp R15 Leu Gin
Phe Ri6 Cys2 Rn Rig Arg Gly Phe Tyr Phe Rl9 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 (SEQ ID NO:2);
wherein R10 is absent or is Ala, Glu, or Asp, Rn is Tyr, Ala, or Val, Ri2 is Arg, Gly, or Ala, R13 is Pro, Thr, Ser, or Leu, Ri4 is Ser, Gly, Ala, or Glu, R15 is Thr or Ala, Ri6 is Val or He, Rn is Gly, Ser, Glu, or Ala, Rig is Asp or Glu, R19 is Ser or Val, R2o is Arg, Leu, or Ser, R2i is Pro or Lys, R22 is Ala, Ser, Gly, Val, or Thr, R23 is Ser, Gly, or Val, R24 is Arg, Pro or Gly, R25 is Ala, Arg, Val, He, Leu, Asn, Ser, or Gly, R26 is Ser, Asn, or Arg, R27 is absent or is Arg, Ser, Asn, R2g is absent or is Val, R29 is absent or is Ser. In one embodiment, the synthetic polypeptide comprises one or more solubilising groups bound to the amino acid sequence of the polypeptide, optionally via a suitable linker.
In another embodiment, the synthetic polypeptide is bound to a solid phase support, optionally via a suitable linker.
Thus in one specifically contemplated embodiment, the invention provides a synthetic polypeptide wherein the synthetic polypeptide comprises an A chain as described herein bound to a B chain as described herein via at least one interchain disulfide bond, wherein either the A chain or the B chain, or both the A chain and the B chain is bound to a solid phase support, optionally via a suitable linker.
In one embodiment, one or more amino acids of the polypeptide are protected by one or more suitable protecting groups.
In certain embodiments, Ri- R2 include conservative amino acid variants for the amino acids listed above. Thus, Ri is Ala, Asn, Leu, or a conservative variant of either, R2 is Leu, He, or a conservative variant of either, R3 is Thr, Gin, or a conservative variant of either, R4 is Thr, Ala, Lys, Val, or a conservative variant of either, R5 is Pro, Ser, or a conservative variant of either, R^ is Ala, Val, Pro, or a conservative variant thereof, R7 is Lys, Glu, or a conservative variant of either, R8 is Ser, Ala, or a conservative variant of either, R is Glu, Ala, or a conservative variant of either, Rio is absent or is Ala, Glu, Asp, or a conservative variant thereof, R11 is Tyr, Ala, Val, or a conservative variant of either, Ri2 is Arg, Gly, Ala, or a conservative variant thereof, Ri3 is Pro, Thr, Ser, Leu, or a conservative variant thereof, Ri4 is Ser, Gly, Ala, Glu, or a conservative variant thereof, R15 is Thr, Tyr, Ala, or a conservative variant thereof, Ri6 is Val, He, or a conservative variant thereof, Ri7 is Gly, Ser, Glu, Ala, or a conservative variant thereof, Ri8 is Asp, Glu, or a conservative variant of either, R19 is Ser, Val, or a conservative variant of either, R20 is Arg, Leu, Ser, or a conservative variant thereof, R2i is Pro, Lys, or a conservative variant of either, R22 is Ala, Ser, Gly, Val, Thr, or a conservative variant thereof, R23 is Ser, Gly, Val, or a conservative variant thereof, R24 is Arg, Pro, Gly, or a conservative variant thereof, R25 is Ala, Arg, Val, He, Leu, Asn, Ser, Gly, or a conservative variant thereof, R26 is Ser, Asn, Arg, or a conservative variant thereof, R27 is absent or is Arg, Ser, Asn, or a conservative variant thereof, R28 is absent or is Val, or a conservative variant thereof, R¾ is absent or is Ser, or a conservative variant thereof.
The synthetic polypeptides of the invention may be pure or purified, or substantially pure.
In one embodiment, the synthetic polypeptide has insulin agonist activity. In one embodiment, insulin agonist activity is a capability of binding to a receptor to which insulin binds, or eliciting a response mediated by a receptor to which insulin binds.
In one embodiment, the synthetic polypeptide binds a receptor to which insulin binds with at least about 10%, at least about 15%, at least about 20%, or at least about 25% the affinity as does insulin. In one embodiment the receptor to which insulin binds is the insulin receptor.
In one embodiment, the synthetic polypeptide binds a receptor to which insulin binds with a binding affinity of at least 107, 108, 109, or 1010 M"1.
In one embodiment, the synthetic polypeptide has an EC5o for effecting a response mediated by the insulin receptor (such as, for example, an effect on carbohydrate metabolism or an effect on cell growth/proliferation and cytoprotection) less than about two hundred-fold that of insulin.
In one embodiment synthetic vesiculin A chain polypeptides are provided, for example, human synthetic vesiculin A chain polypeptides. Also provided are synthetic vesiculin A chain polypeptide intermediates.
In yet another embodiment synthetic vesiculin B chain polypeptides are provided, for example, human synthetic vesiculin B chain polypeptides. Also provided are synthetic vesiculin B chain polypeptide intermediates.
Synthetic vesiculin A and B chain polypeptides and intermediates may be pure or purified, or substantially pure.
As used herein, the terms "synthetic vesiculin" and "synthetic vesiculin polypeptide(s)" are used interchangeably herein and refer to a synthetic polypeptide comprising a two chain peptide having insulin agonist activity, comprising a synthetic vesiculin A chain polypeptide and a synthetic vesiculin B chain polypeptide. The naturally-occuring human vesiculin sequence, corresponding to that of one embodiment of a synthetic vesiculin as contemplated herein, may be represented as follows:
A chain : Gly He Val Glu Glu Cys3 Cys4 Phe Arg Ser Cys5 Asp Leu Ala Leu Leu Glu
Thr Tyr Cys6 Ala Thr Pro Ala Lys Ser Glu (SEQ ID NO:3)
B chain : Alai Tyr Arg Pro Ser Glu Thr Leu Cysi Gly Gly Glu Leu Val Asp Thr Leu
Gin Phe Val Cys2 Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala Ser Arg Val Ser (SEQ ID NO:4)
wherein Alai is either present or absent.
Variants of synthetic vesiculin are also provided and include, for example: A chain : Gly He Val Glu Glu Cys3 Cys4 Phe Arg Ser Cys5 Asp Leu Ala Leu Leu Glu
Thr Tyr Cys6 Ala R7 Pro Ala Lys Ser Glu (SEQ ID NO:5)
B chain : Alai Tyr Ri Pro R2 Glu Thr Leu Cysi Gly Gly Glu Leu Val Asp Thr Leu Gin
Phe Val Cys2 R3 Asp Arg Gly Phe Tyr Phe Ser Arg Pro R4 Ser Arg R5 R, (SEQ ID NO:6)
wherein Alai is either present or absent; Ri is Gly or Arg; R2 is Gly or Ser; R3 is Gly or Ser; R4 is Ser or Ala; R5 is He or Val or Ala either; R6 is Asn or Ser; and R7 is Thr or Ala. In yet another embodiment, Alai is either present or absent; Ri is Gly or Arg, or a conservative variant of either; R2 is Gly or Ser, or a conservative variant of either; R3 is Gly or Ser, or a conservative variant of either; R4 is Ser or Ala, or a conservative variant of either; R5 is He or Val or Ala, or a conservative variant of either; R6 is Asn or Ser, or a conservative variant of either; and R7 is Thr or Ala, or a conservative variant of either.
Additional vesiculin variants include, for example:
A chain : Gly He Val Glu Glu Cys3 Cys4 Phe Arg Ser Cys5 Asp Leu Ri R2 Leu Glu R3
Tyr Cys6 Ala R4 R5 R, R7 R8 R9 (SEQ ID NO: 1)
B chain : R10 Rn Ri2 R13 Ri4 Glu Thr Leu Cysi Gly Gly Glu Leu Val Asp Ri5 Leu Gin
Phe Ri6 Cys2 Rn Rig Arg Gly Phe Tyr Phe Rl9 R20 R21 R22 R23 R24 R25 R26 27 28 29 (SEQ ID NO:2)
wherein Ri is Ala, Asn or Leu; R2 is Leu or He; R3 is Thr or Gin; R4 is Thr, Ala, Lys, or Val; R5 is Pro or Ser; R5 is Ala, Val, or Pro; R7 is Lys or Glu, R8 is Ser or Ala, R9 is Glu or Ala, R10 is absent or is Ala, Glu, or Asp, Rn is Tyr, Ala, or Val, R12 is Arg, Gly, or Ala, R13 is Pro, Thr, Ser, or Leu, Ri4 is Ser, Gly, Ala, or Glu, R15 is Thr or Ala, Ri6 is Val or He, Rn is Gly, Ser, Glu, or Ala, Ri8 is Asp or Glu, R19 is Ser or Val, R20 is Arg, Leu, or Ser, R2i is Pro or Lys, R22 is Ala, Ser, Gly, Val, or Thr, R23 is Ser, Gly, or Val, R24 is Arg, Pro or Gly, R25 is Ala, Arg, Val, He, Leu, Asn, Ser, or Gly, R26 is Ser, Asn, or Arg, R27 is absent or is Arg, Ser, Asn, R28 is absent or is Val, R29 is absent or is Ser. In certain embodiments, Ri- R29 include conservative amino acid variants for the amino acids listed above. Thus, Ri is Ala, Asn, Leu, or a conservative variant of either, R2 is Leu, He, or a conservative variant of either, R3 is Thr, Gin, or a conservative variant of either, R4 is Thr, Ala, Lys, Val, or a conservative variant of either, R5 is Pro, Ser, or a conservative variant of either, R^ is Ala, Val, Pro, or a conservative variant thereof, R7 is Lys, Glu, or a conservative variant of either, R8 is Ser, Ala, or a conservative variant of either, R9 is Glu, Ala, or a conservative variant of either, R10 is absent or is Ala, Glu, Asp, or a conservative variant thereof, Rn is Tyr, Ala, Val, or a conservative variant of either, Ri2 is Arg, Gly, Ala, or a conservative variant thereof, R13 is Pro, Thr, Ser, Leu, or a conservative variant thereof, R14 is Ser, Gly, Ala, Glu, or a conservative variant thereof, R15 is Thr, Tyr, Ala, or a conservative variant thereof, Ri6 is Val, He, or a conservative variant thereof, Ri7 is Gly, Ser, Glu, Ala, or a conservative variant thereof, Rig is Asp, Glu, or a conservative variant of either, R19 is Ser, Val, or a conservative variant of either, R2o is Arg, Leu, Ser, or a conservative variant thereof, R2i is Pro, Lys, or a conservative variant of either, R22 is Ala, Ser, Gly, Val, Thr, or a conservative variant thereof, R23 is Ser, Gly, Val, or a conservative variant thereof, R24 is Arg, Pro, Gly, or a conservative variant thereof, R25 is Ala, Arg, Val, He, Leu, Asn, Ser, Gly, or a conservative variant thereof, R26 is Ser, Asn, Arg, or a conservative variant thereof, R27 is absent or is Arg, Ser, Asn, or a conservative variant thereof, R28 is absent or is Val, or a conservative variant thereof, R¾ is absent or is Ser, or a conservative variant thereof.
In one embodiment the synthetic vesiculin comprises A and B chains joined by at least one inter-chain disulfide bond. For example, the synthetic vesiculin may include disulfide bonds formed between any one of Cysi, Cys2, Cys3, Cys4, Cys5 and Cys6 residues.
In another embodiment the synthetic vesiculin comprises A and B chains joined by two inter-chain disulfide bonds. For example, in one embodiment the synthetic vesiculin includes disulfide bonds formed between residues Cysi and Cys4, and Cys2 and Cys6.
In another embodiment the synthetic vesiculin comprises an intra-chain disulfide bond in chain A between residues Cys3 and Cys5.
In another embodiment the synthetic vesiculin comprises A and B chains joined by one or more inter-chain disulfide bonds and an A-chain intra-chain disulfide bond. For example, in one embodiment the synthetic vesiculin includes disulfide bonds formed between residues Cysi and Cys4, and Cys2 and Cys6, and Cys3 and Cys5.
Also provided are synthetic vesiculin variants, derivatives or intermediates having a vesiculin B chain from any species (for example, human, rat, mouse, etc.) and a vesiculin A chain having the sequence GIVEECCFRSCDLALLETYCATPAKSE. (SEQ ID NO:3).
In one embodiment, the vesiculin variant comprises one or more solubilising groups bound to the amino acid sequence of the A chain, the B chain, or both the A chain and the B chain, optionally via a suitable linker.
In another embodiment, the vesiculin variant is bound to a solid phase support, optionally via a suitable linker.
In one embodiment, one or more amino acids of the A chain, the B chain, or both the A chain and the B chain are protected by one or more suitable protecting groups. In one embodiment, the invention relates to one or more synthetic vesiculin polypeptide intermediates, wherein the one or more synthetic vesiculin polypeptide intermediates comprises a resin-bound polypeptide comprising amino acid sequence corresponding to
A chain : Gly He Val Glu Glu Cys3 Cys4 Phe Arg Ser Cys5 Asp Leu Ri R2 Leu Glu R3
Tyr Cys6 Ala R4 R5 Rg R7 R8 R9 (SEQ ID NO: 1)
wherein Ri is Ala, Asn or Leu; R2 is Leu or He; R3 is Thr or Gin; R4 is Thr, Ala, Lys, or Val; R5 is Pro or Ser; R6 is Ala, Val, or Pro; R7 is Lys or Glu, R8 is Ser or Ala, R9 is Glu or Ala. In one embodiment, the synthetic vesiculin polypeptide intermediate includes a disulfide bond formed between residues Cys3 and Cys5.
In one embodiment, the invention relates to one or more synthetic vesiculin polypeptide intermediates, wherein the one or more synthetic vesiculin polypeptide intermediates comprises a resin-bound polypeptide comprising or consisting of amino acid sequence corresponding to
B chain : Rio Rn Ri2 Ri3 Ri4 Glu Thr Leu Cysi Gly Gly Glu Leu Val Asp Ri5 Leu Gin
Phe Ri6 Cys2 Rn Rig Arg Gly Phe Tyr Phe Rl9 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 (SEQ ID NO:2)
wherein R10 is absent or is Ala, Glu, or Asp, Rn is Tyr, Ala, or Val, Ri2 is Arg, Gly, or Ala, Ri3 is Pro, Thr, Ser, or Leu, Ri4 is Ser, Gly, Ala, or Glu, R15 is Thr or Ala, Ri6 is Val or He, R17 is Gly, Ser, Glu, or Ala, Ri8 is Asp or Glu, R19 is Ser or Val, R20 is Arg, Leu, or Ser, R21 is Pro or Lys, R22 is Ala, Ser, Gly, Val, or Thr, R23 is Ser, Gly, or Val, R24 is Arg, Pro or Gly, R25 is Ala, Arg, Val, He, Leu, Asn, Ser, or Gly, R26 is Ser, Asn, or Arg, R27 is absent or is Arg, Ser, Asn, R28 is absent or is Val, R2 is absent or is Ser.
Specifically contemplated synthetic vesiculin intermediates include those comprising or consisting of at least 12 amino acids having an amino acid sequence corresponding to the 12 C- terminal amino acids of a vesiculin polypeptide A chain. Exemplary synthetic vesiculin human A chain intermediates include, for example, the following polypeptides:
LETYCATPAKSE (SEQ ID NO:7);
LLETYCATPAKSE (SEQ ID NO: 8);
ALLETYCATPAKSE (SEQ ID NO:9);
LALLETYCATPAKSE (SEQ ID NO: 10);
DLALLETYCATPAKSE (SEQ ID NO: 11);
CDLALLETYCATPAKSE (SEQ ID NO: 12);
SCDLALLETYCATPAKSE (SEQ ID NO: 13);
RSCDLALLETYCATPAKSE (SEQ ID NO: 14); FRSCDLALLETYCATPAKSE (SEQ ID NO: 15);
CFRSCDLALLETYCATPAKSE (SEQ ID NO: 16);
CCFRSCDLALLETYCATPAKSE (SEQ ID NO: 17);
EC CFRS CDL ALLET YC ATP AKSE (SEQ ID NO: 18);
EECCFRSCDLALLETYCATPAKSE (SEQ ID NO: 19);
VEECCFRSCDLALLETYC ATP AKSE (SEQ ID NO:20);
IVEECCFRSCDLALLETYCATPAKSE (SEQ ID NO:21).
Additional specifically contemplated synthetic vesiculin intermediates include those comprising or consisting of at least 12 amino acids having an amino acid sequence corresponding to the 12 C-terminal amino acids of a vesiculin polypeptide A chain, wherein the N-terminal amino acid is Na-protected by a protecting group. In one embodiment, the N- terminal amino acid is Να-protected with Fmoc. The functional groups in the side chains of the amino acids may also be protected with one or more protecting groups. Accordingly, further exemplary synthetic vesiculin human A chain intermediates include, for example, the following polypeptides:
(pro)-LETYCATPAKSE (SEQ ID NO:7);
(pro)-LLETYC ATP AKSE (SEQ ID NO: 8);
(pro)-ALLETYCATPAKSE (SEQ ID NO:9);
(pro)-LALLETYCATPAKSE (SEQ ID NO: 10);
(pro)-DLALLETYC ATP AKSE (SEQ ID NO: 11);
(pro)-CDLALLETYCATPAKSE (SEQ ID NO: 12);
(pro)-SCDLALLETYCATPAKSE (SEQ ID NO: 13);
(pro)-RSCDLALLETYCATPAKSE (SEQ ID NO: 14);
(pro)-FRSCDLALLETYCATPAKSE (SEQ ID NO: 15);
(pro)-CFRSCDLALLETYC ATP AKSE (SEQ ID NO: 16);
(pro)-CCFRSCDLALLETYCATPAKSE (SEQ ID NO: 17);
(pro)-ECCFRSCDLALLETYCATPAKSE (SEQ ID NO: 18);
(pro)-EECCFRSCDLALLETYCATPAKSE (SEQ ID NO: 19);
(pro)-VEECCFRSCDLALLETYC ATP AKSE (SEQ ID NO:20);
(pro)-IVEECCFRSCDLALLETYCATPAKSE (SEQ ID NO:21);
(pro)-GIVEECCFRSCDLALLETYCATPAKSE (SEQ ID NO:22);
wherein (pro)- is one or more protecting group, including a protecting group selected from the following: acetyl (Ac), amide, a 3 to 20 carbon alkyl group, Fmoc, 9-fluoreneacetyl group, l-fiuorenecarboxylic group, 9-fluorenecarboxylic group, 9-fluorenone-l-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4- dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4- methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys), l-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-C1--Z), 2- bromobenzyloxycarbonyl (2-Br~Z), Benzyloxymethyl (Bom), tert-butyloxycarbonyl (Boc), cyclohexyloxy (cHxO),t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), and Trifiuoroacetyl (TFA).
In some embodiments of the A chain intermediates, the Na-position of the N-terminal amino acid is protected with a (pro)- group. In particularly contemplated embodiments of A chain intermediates, the (pro)- is Fmoc.
In one embodiment, the synthetic vesiculin human A chain intermediates comprise one or more solubilising groups bound to the amino acid sequence of the polypeptide chain, optionally via a suitable linker.
In another embodiment, the synthetic vesiculin human A chain intermediates are bound to a solid phase support, optionally via a suitable linker.
In one embodiment, one or more amino acids of the synthetic vesiculin human A chain intermediates are protected by one or more suitable protecting groups.
Corresponding A chain intermediates from other species, and synthetic vesiculins comprising amino acid sequences corresponding to the vesiculin of other species incorporating those intermediates, are also provided by the invention.
Further specifically contemplated synthetic vesiculin intermediates include those comprising or consisting of at least 12 amino acids having an amino acid sequence corresponding to the 12 C-terminal amino acids of a vesiculin polypeptide B chain. Exemplary synthetic vesiculin human B chain intermediates include, for example, the following polypeptides:
GFYFSRPASRVS (SEQ ID NO:23);
RGFYFSRPASRVS (SEQ ID NO:24);
DRGFYFSRPASRVS (SEQ ID NO:25);
GDRGFYFSRPASRVS (SEQ ID NO:26);
CGDRGFYFSRPASRVS (SEQ ID NO:27); VCGDRGFYFSRPASRVS (SEQ ID NO:28);
FVCGDRGFYFSRPASRVS (SEQ ID NO:29);
QFVCGDRGFYFSRPASRVS (SEQ ID NO:30);
LQFVCGDRGFYFSRPASRVS (SEQ ID N0:31);
TLQFVCGDRGFYFSRPASRVS (SEQ ID NO:32);
DTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:33);
VDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:34);
LVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:35);
ELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:36);
GELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:37);
GGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:38);
CGGEL VDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:39);
LCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:40);
TLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID N0:41);
ETLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:42);
SETLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:43);
PSETLCGGEL VDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:44);
RPSETLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:45);
YRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:46).
Additional specifically contemplated synthetic vesiculin intermediates include those comprising or consisting of at least 12 amino acids having an amino acid sequence corresponding to the 12 C-terminal amino acids of a vesiculin polypeptide B chain, wherein the N-terminal amino acid is Να-protected by a protecting group. In one embodiment, the N- terminal amino acid is Να-protected with Boc. The functional groups in the side chains of the amino acids may also be protected with one or more protecting groups. Accordingly, further exemplary synthetic vesiculin human B chain intermediates include, for example, the following polypeptides:
(pro)-GFYFSRPASRVS (SEQ ID NO:23);
(pro)-RGFYFSRPASRVS (SEQ ID NO:24);
(pro)-DRGFYFSRPASRVS (SEQ ID NO:25);
(pro)-GDRGFYFSRPASRVS (SEQ ID NO:26);
(pro)-CGDRGFYFSRPASRVS (SEQ ID NO:27);
(pro)-VCGDRGFYFSRPASRVS (SEQ ID NO:28); (pro)-FVCGDRGFYFSRPASRVS (SEQ ID NO:29);
(pro)-QFVCGDRGFYFSRPASRVS (SEQ ID NO:30);
(pro)-LQFVCGDRGFYFSRPASRVS (SEQ ID NO:31);
(pro)-TLQFVCGDRGFYFSRPASRVS (SEQ ID NO:32);
(pro)-DTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:33);
(pro)-VDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:34);
(pro)-LVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:35);
(pro)-ELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:36);
(pro)-GELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:37);
(pro)-GGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:38);
(pro)-CGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:39);
(pro)-LCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:40);
(pro)-TLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:41);
(pro)-ETLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:42);
(pro)-SETLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:43);
(pro)-PSETLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:44);
(pro)-RPSETLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:45);
(pro)-YRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:46);
(pro)-AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:47);
wherein (pro)- is one or more protecting group, including a protecting group selected from the following: acetyl (Ac), amide, a 3 to 20 carbon alkyl group, Fmoc, 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-fluorenecarboxylic group, 9-fluorenone-l-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4- dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4- methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys), l-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-C1--Z), 2- bromobenzyloxycarbonyl (2-Br~Z), Benzyloxymethyl (Bom), tert-butyloxycarbonyl (Boc), cyclohexyloxy (cHxO),t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), and Trifluoroacetyl (TFA). In some embodiments of the B chain intermediates, the Na-position of the N-terminal amino acid is protected with a (pro)- group. In particularly contemplated embodiments of B chain intermediates, the (pro)- is Boc.
In one embodiment, the synthetic vesiculin human B chain intermediates comprise one or more solubilising groups bound to the amino acid sequence of the polypeptide chain, optionally via a suitable linker.
In another embodiment, the synthetic vesiculin human B chain intermediates are bound to a solid phase support, optionally via a suitable linker.
In one embodiment, one or more amino acids of the synthetic vesiculin human B chain intermediates are protected by one or more suitable protecting groups.
Corresponding B chain intermediates from other species, and vesiculins comprising amino acid sequences corresponding to the vesiculin of other species incorporating those fragments, are also provided by the invention.
Resin-bound intermediates, such as the human synthetic vesiculin intermediates specifically disclosed herein, are also specifically contemplated.
Other embodiments include synthetic vesiculin variants having an amino acid sequence that is at least about 60% identical to a vesiculin, for example, a human vesiculin. In other embodiments, a synthetic vesiculin variant may contain an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, or at least about 99% identical to a vesiculin, for example, a human vesiculin.
In one embodiment, the synthetic vesiculin polypeptide or variant or derivative or synthetic intermediate thereof has a serine at position 33 of the B chain. In other embodiments, the synthetic vesiculin polypeptide or variant or derivative or synthetic intermediate thereof has an amino acid other than arginine at position 33 of the B chain. In another embodiment, the synthetic vesiculin polypeptide or variant or derivative or synthetic intermediate thereof has a serine at position 36 of the B chain. In other embodiments, the synthetic vesiculin polypeptide or variant or derivative or synthetic intermediate thereof has an amino acid other than lysine at position 36 of the B chain. In other embodiments, the synthetic vesiculin polypeptide or variant or derivative or synthetic intermediate thereof has serine residues at positions 33 and 36 of the B chain. In other embodiments, the synthetic vesiculin polypeptide or variant or derivative or synthetic intermediate thereof has an amino acid other than arginine at position 33 of the B chain, and additionally has an amino acid other than lysine at position 36 of the B chain. (The position numbering corresponds to that of human vesiculin.)
The invention also provides a method of determining whether a synthetic polypeptide, variant, or intermediate of the invention is useful as a therapeutic agent by determining whether the synthetic polypeptide has insulin agonist activity. In one embodiment, the method comprises contacting the synthetic polypeptide, variant, or intermediate and a receptor to which insulin binds, and determining a capability of binding to the receptor. In another embodiment, the method comprises contacting the synthetic polypeptide, variant, or intermediate and a receptor to which insulin binds, and determining a capability of eliciting a response mediated by the receptor.
In one embodiment, a capability of eliciting a response mediated by the receptor is determined by a determination of the EC50 for effecting a response mediated by the insulin receptor.
In one embodiment, the response is an effect on glucose incorporation into glycogen. In certain embodiments, insulin agonist activity is identified by assay systems, including the soleus muscle assay, which measures the effect an agent, such as a synthetic vesiculin polypeptide, variant, or intermediate has on glucose incorporation into glycogen. Exemplary methods are described herein in the examples.
In another aspect, the invention relates to a method of modulating blood glucose levels in a subject. The method comprises administering an effective amount of one or more of a synthetic vesiculin, or a synthetic vesiculin variant, derivative, or synthetic intermediate, or a salt of any of them, to a subject in need thereof.
The invention includes methods for treating and/or preventing, in whole or in part, various diseases, disorders, and conditions, including for example, impaired glucose tolerance; impaired fasting glucose; prediabetes; diabetes and/or its complications, including type 1 and type 2 diabetes and their complications; insulin resistance; Syndrome X; obesity and other weight related disorders; fatty liver disease, including non alcoholic and/or alcoholic fatty liver disease; glucose metabolism diseases and disorders; diseases, disorders or conditions that are treated or treatable with insulin; diseases, disorders or conditions that are treated or treatable with a hypoglycemic agent; diseases, disorders, and conditions characterized at least in part by hyperglycemia; diseases, disorders, and conditions characterized at least in part by hypoinsulinemia and/or diseases, disorders, and conditions characterized at least in part by hyperinsulinemia. The invention includes methods for treating a subject having or suspected of having or predisposed to, or at risk for, for example, any diseases, disorders and/or conditions characterized in whole or in part by hyperglycemia, insulin resistance, impaired glucose tolerance, and/or impaired fasting glucose, comprising administering a composition comprising a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, a synthetic vesiculin derivative and/or a synthetic vesiculin intermediate or a salt thereof. Such diseases, disorders and/or conditions include but are not limited to those described or referenced herein. Such compounds may be administered in amounts, for example, that are effective to (1) lower serum glucose, (2) lower blood glucose, (3) lower urine glucose, (4) lower fructosamine, (5) lower glycosylated hemoglobin (HbAic) levels, (6) lower postprandial glycemia, (7) ameliorate impaired glucose tolerance, (8) ameliorate impaired fasting glucose, and/or (9) lower the rate and/or severity of hypoglycemic events, including severe hypoglycemic events. Such compositions include, for example, formulations for delivery by injection, transdermal patch, inhalation, and other non-oral delivery methods.
The invention includes methods for regulating glycemia in a subject having or suspected of having or predisposed to diseases, disorders and/or conditions characterized in whole or in part, for example, by hyperglycemia, insulin resistance, impaired glucose tolerance, and/or impaired fasting glucose, comprising administering a composition comprising a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, a vesiculin derivative and/or a synthetic vesiculin synthetic or a salt thereof. Such diseases, disorders and/or conditions include but are not limited to those described or referenced herein. Such compounds may be administered in amounts, for example, that are effective to (1) lower serum glucose, (2) lower blood glucose, (3) lower urine glucose, (4) lower fructosamine, (5) lower glycosylated hemoglobin (HbAic) levels, (6) lower postprandial glycemia, (7) ameliorate impaired glucose tolerance, (8) ameliorate impaired fasting glucose, and/or (9) lower the rate and/or severity of hypoglycemic events, including severe hypoglycemic events. Such compositions include, for example, formulations for delivery by injection, transdermal patch, inhalation, and other non-oral delivery methods.
In another aspect, the invention relates to a use of one or more of a synthetic vesiculin, or a synthetic vesiculin variant, derivative, or synthetic intermediate, or a salt of any of them, in the preparation of a medicament, including medicaments for modulating blood glucose levels in a subject. In another aspect, the invention relates to a method of modulating glucose incorporation into glycogen in a subject. The method comprises administering an effective amount of one or more of a synthetic vesiculin, or a synthetic vesiculin variant, derivative, or synthetic intermediate thereof, or a salt of any of them, to a subject in need thereof.
In another aspect the invention is directed to the use of an effective amount of one or more of a synthetic vesiculin, or a synthetic vesiculin variant, derivative, or synthetic intermediate, or a salt of any of them, in the manufacture, with or without other pharmaceutically acceptable materials (such as, for example, excipients, diluents or the like, and/or within a dosage unit defining vessel), of a dosage unit effective for use in a method of the invention or for any of the purposes herein described or provided.
The invention also includes synthetic vesiculin, synthetic vesiculin variants, derivatives or synthetic intermediates produced by protein synthesis techniques, followed by isolation and/or purification, as disclosed herein.
The invention further includes a pharmaceutical composition which comprises a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, or a synthetic vesiculin, synthetic vesiculin A chain, or synthetic vesiculin B chain variant or derivative, or an synthetic intermediate thereof, or salts or derivatives of the above.
The synthetic vesiculins, synthetic vesiculin A and B chains, and variants and derivatives and intermediates thereof for use in the methods of the invention may be formulated in a manner suitable for administration to a subject, for example, a human. Administration is preferably, for example, parenteral via routes such as subcutaneous (s.c), intradermal (i.d.), intravenous (i.v.), intraperitoneal (i.p.) or transdermal, although other delivery form are envisioned, including oral, nasal, and pulmonary, for example.
These and other features, objects, and advantages of the inventions, which are not limited to or by the information in this Brief Summary, will be apparent from the further description provided herein, including the drawings and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 is a schematic overview of the synthesis of vesiculin showing the stepwise installation of firstly the intrachain disulfide bond of the A-chain followed by the interchain disulfides that cross-link the A and B chains, as described herein in Example 1. LI is the Rink linker and L2 is the 4-hydroxymethyl benzoic acid (HMBA) linker, through which the solubilising pentalysine tag is attached and which is hydrolysed at the final step. Figure 2 shows HPLC analysis of crude A-chain. Column: Phenomenex Gemini CI 8, 5μ, 11 OA, 4.6 x 150mm; Eluent A: water/0.1% TFA, B: MeCN/0.1% TFA; Gradient: 1-51%B over 25 min.
Figure 3 shows HPLC profiles for formation of the Cys6-Cysl 1 disulfide bond of the A-chain (* dithiol 3; ** disulfide 4) and the low-resolution mass spectrum for the disulfide, which indicates the M+3H+ (1284.7) and higher ionisation states.
Figure 4 shows the HPLC profile of crude murine B-chain synthesised using Boc SPPS and low-resolution mass spectrum of the main peak, showing the M+3H+ (1324.7) and higher ionisation states. Column: Phenomenex Gemini CI 8, 5μ, 1 ΙθΑ, 4.6 x 150mm; Eluent A:
water/0.1% TFA, B: MeCN/0.1% TFA; Gradient: 1-61%B over 25 min.
Figure 5 shows HPLC profiles for deprotection of Cys(tBu) of the A-chain 4 (*) and in- situ conversion to the activated SSPyr disulfide 6 (**), together with low-resolution mass spectrum of the product showing the M+3H+ (1302.3) and higher ionisation states. The strong peak at 17 min is excess dipyridyldisulfide. Column: Phenomenex Gemini CI 8, 5μ, I IOA, 4.6 x 150mm; Eluent A: water/0.1% TFA, B: MeCN/0.1% TFA; Gradient: 1-51%B over 25 min.
Figure 6 shows HPLC profiles for crosslinking the A- and murine B-chains via an interchain disulfide bond, and low-resolution mass spectrum of the product 7 showing the required M+5H+ (1553.9) and higher ionisation states. Column: Phenomenex Gemini CI 8, 5μ, 11 OA, 4.6 x 150mm; Eluent A: water/0.1% TFA, B: MeCN/0.1% TFA; Gradient: 1-51%B over 25 min.
Figure 7 shows HPLC profiles for removal of the Acm groups from murine 7 (*) and concomitant formation of the second interchain disulfide bond to give 8 (**), confirmed by low- resolution mass spectrum of the product showing the required M+5H+ (1525.2) and higher ionisation states. Column: Phenomenex Gemini CI 8, 5μ, 1 ΙθΑ, 4.6 x 150mm; Eluent A:
water/0.1% TFA, B: MeCN/0.1% TFA; Gradient: 1-61%B over 30 min.
Figure 8 shows HPLC profiles for hydrolytic removal of the pentalysine tag of 8 (*) to afford murine Vesiculin 9 (**), with the low-resolution mass spectrum of the product showing the required M+4H+ (1712.4) and higher ionisation states. Column: Phenomenex Gemini CI 8, 5μ, 11 OA, 4.6 x 150mm; Eluent A: water/0.1% TFA, B: MeCN/0.1% TFA; Gradient: 1-61%B over 30 min.
Figure 9 shows the HPLC profile of human Vesiculin and low-resolution mass spectrum of the product showing the required M+4H+ (1733.6) and higher ionisation states. Column: Phenomenex Gemini C18, 5μ, 1 ΙθΑ, 4.6 x 150mm; Eluent A: water/0.1% TFA, B: MeCN/0.1% TFA; Gradient: 1-61%B over 30 min.
Figure 10 shows the effect of synthetic murine vesiculin and pharmacological additive on blood glucose 60 min after administration, relative to fasting, as described in Example 3 herein.
FIGURE 11 shows an assay of the hypoglycaemic potential of synthetic murine vesiculin, as described in Example 3 herein.
DETAILED DESCRIPTION OF THE INVENTION
This invention relates in one aspect to one or more of a synthetic polypeptide having insulin agonist activity, such as a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, synthetic vesiculin A chain and synthetic vesiculin B chain variants, and synthetic vesiculin, synthetic vesiculin A chain and synthetic vesiculin B chain derivatives, and salts thereof.
Amino acid sequences for mouse, human and rat vesiculin include the following:
AYGPGETLCGGELVDTLQFVCSDRGFYFSRPSSRAN (SEQ ID NO: 48)
GIVEECCFRSCDLALLETYCATPAKSE (SEQ ID NO: 49)
AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO: 50) GIVEECCFRSCDLALLETYCATPAKSE (SEQ ID NO: 3)
I I
AYRPSETLCGGELVDTLQFVCSDRGFYFSRPSSRAN (SEQ ID NO: 51)
GIVEECCFRSCDLALLETYCATPAKSE (SEQ ID NO: 52)
I I Amino acid sequences for vesiculins of other species include:
Cow/Deer
AYRPSETLCGGELVDTLQFVCGDRGFYFSRPSSRIN (SEQ ID NO. 55) GIVEECCFRSCDLALLETYCATPAKSE (SEQ ID NO. 54)
Sheep
AYRPSETLCGGELVDTLQFVCGDRGFYFSRPSSRIN (SEQ ID NO. 55) GIVEECCFRSCDLALLETYCAAPAKSE (SEQ ID NO. 56)
Kangaroo
AYRPSETLCGGELVDTLQFVCGDRGFYFSLPGRPLSRVS (SEQ ID NO. 57) GIVEECCFRSCDLALLETYCATPAKSE (SEQ ID NO. 58)
I I
Horse
AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO. 59) GIVEECCFRSCDLALLETYCATPAKSE (SEQ ID NO. 60) Chicken
AYGTAETLCGGELVDTLQFVCGDRGFYFSRPVGRNN (SEQ ID NO. 25) GIVEECCFRSCDLALLETYCAKSVKSE (SEQ ID NO. 66)
Salmon/Trout
EVASAETLCGGELVDALQFVCEDRGFYFSRPTSRSNS (SEQ ID NO. 61) GIVEECCFRSCDLNLLEQYCAKPAKSE (SEQ ID NO. 64) Eel
DAGSGETLCGGELVDALQFVCEDRGFYFSRPTSRANS (SEQ ID NO. 65)
GIVEECCFRSCDLNLLEQYCAKPAKSE (SEQ ID NO. 66)
Gilthead Bream
EVASAETLCGGELVDALQFVCEDRGFYFSRPTSRGNN (SEQ ID NO. 67)
GIVEECCFRSCDLNLLEQYCAKPAKSE (SEQ ID NO. 68)
I I
Shark
EARLEETLCGSELVDTLQFICAERGFYFVSKVVGRRS (SEQ ID NO. 69)
GI VEEC CFRS CDLLILET YC AVPPE AA (SEQ ID NO. 70)
As broadly defined above, the synthetic vesiculin has been synthesized in accordance with the following description including the synthetic scheme outlined herein.
Synthetic vesiculin intermediates also include those having an A chains with from one to five N-terminal amino acid residue deletions and a B chain with from one to eight N-terminal amino acid residue deletions. These include synthetic human vesiculin intermediates including an A chain intermediate and a B chain intermediate, as well as synthetic intermediates of other vesiculin species including an A chain intermediate and a B chain intermediate. They also include vesiculin intermediates having an A chain intermediate from one species combined with a B chain intermediate from another species.
Synthetic vesiculin intermediates also include those having an A chain with from one to five N-terminal amino acid residue deletions and a full length B chain. These include synthetic human vesiculin intermediates with an A chain intermediate and a full length B chain, as well as similar molecules from other vesiculin species. They also include vesiculin intermediates having an A chain intermediate from one species combined with a full length B intermediate from another species.
Synthetic vesiculin intermediates also include those having a full length A chain and a B chain with from one to eight N-terminal amino acid residue deletions. These include synthetic human vesiculin intermediates with a full length A chain and a B chain intermediate, as well similar molecules from other vesiculin species. They also include vesiculin intermediates having a full length A chain from one species combined with a B chain intermediate from another species.
Also provided are synthetic vesiculin variants, derivatives and synthetic intermediates, including, for example, synthetic vesiculin variants, derivatives and synthetic intermediates having a synthetic vesiculin B chain from any species (for example, human, rat, mouse, etc.) and a synthetic vesiculin A chain having the sequence GIVEECCFRSCDLALLETYCATPAKSE (SEQ ID NO. 4).
These include synthetic human vesiculin intermediates with a full length A chain and a B chain intermediate, as well similar molecules from other vesiculin species. They also include vesiculin intermediates having a full length A chain from one species combined with a B chain intermediate from another species.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Animal Cell Culture (R.I. Freshney, ed., 1987); Handbook of Experimental Immunology (D.M. Weir & C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller & M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); The Immunoassay Handbook (David Wild, ed., Stockton Press NY, 1994); Antibodies: A Laboratory Manual (Harlow et al., eds., 1987); and Methods of Immunological Analysis (R. Masseyeff, W.H. Albert, and N.A. Staines, eds., Weinheim: VCH Verlags gesellschaft mbH, 1993).
A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another residue having a chemically similar or derivitized side chain. Families of amino acid residues having similar side chains, for example, have been defined in the art. These families include, for example, amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Amino acid analogs (e.g., phosphorylated amino acids) are also contemplated in the present invention, as are peptides substituted with non-naturally occurring amino acids, including but not limited to D-amino acids, β amino acids, and γ amino acids.
As used herein "purified" does not require absolute purity; rather, it is intended as a relative term where the subject protein or other substance is more pure than in its natural environment within a cell or other environment, such as a manufacturing environment. In practice the material has typically, for example, been subjected to fractionation to remove various other components, and the resultant material has substantially retained its desired biological activity or activities. The term "substantially purified" refers to peptides that are at least about 60% free, preferably at least about 75% free, and most preferably at least about 90%> free, at least about 95% free, at least about 98% free, or more, from other components with which they may be associated naturally or during manufacture.
Also within the scope of this invention is a pharmaceutical composition that contains an effective amount of one or more of a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, synthetic vesiculin A chain and synthetic vesiculin B chain variants, and synthetic vesiculin, synthetic vesiculin A chain and synthetic vesiculin B chain derivatives, and synthetic intermediates thereof, and salts of any of them, and a pharmaceutically acceptable carrier.
The term "pharmaceutically acceptable carrier" refers to a carrier (adjuvant or vehicle) that may be administered to a subject together with one or more of a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, synthetic vesiculin A chain and synthetic vesiculin B chain variants, and synthetic vesiculin, synthetic vesiculin A chain and synthetic vesiculin B chain derivatives, and salts of any of them.
Pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions described above include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-a-tocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Cyclodextrins such as α-, β-, and γ-cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl-P-cyclodextrins, or other solubilized derivatives may also be advantageously used to enhance delivery of compounds of the formulae described herein. Oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents, which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions.
An "effective amount" is an amount sufficient to effect beneficial or desired results including clinical results. An effective amount can be administered in one or more administrations by various routes of administration.
A "biological sample" encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay. The definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom, and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as proteins or polynucleotides. The term "biological sample" encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples.
As used herein, "treatment" is an approach for obtaining beneficial or desired results including clinical results although the term also encompasses prophylactic and/or therapeutic treatments
The terms "polypeptide" and "peptide" and the like are used interchangeably herein to refer to any polymer of amino acid residues of any length. The polymer can be linear or nonlinear (e.g., branched), it can comprise modified amino acids or amino acid analogs, and it can be interrupted by chemical moieties other than amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.
An "active fragment" of a polypeptide is a subsequence of the polypeptide that performs a function that is required for the biological activity and/or provides a three dimensional structure of the polypeptide. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof capable of performing any one or more of the methods herein described, particularly with reference to modulating glucose.
A "fragment" of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides that is capable of specific hybridization to a target of interest, for example, a sequence that is at least about 15 nucleotides in length. The polynucleotide fragment of the invention comprise about 15 nucleotides, preferably at least about 20 nucleotides, more preferably at least about 30 nucleotides, more preferably at least about 40 nucleotides, more preferably at least about 50 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, at least about 80 nucleotides, at least about 90 nucleotides, at least about 100 nucleotides, at least about 1 10 nucleotides, at least about 120 nucleotides, and most preferably at least about 130 nucleotides of contiguous nucleotides of a polynucleotide of the invention. A fragment of a polynucleotide sequence can be used in antisense, gene silencing, triple helix or ribozyme technology, or as a primer, a probe, included in a microarray, or used in polynucleotide -based selection methods of the invention.
A "fragment" of a polypeptide is a subsequence of the polypeptide, typically one that performs a function that is required for the activity of the polypeptide, such as the enzymatic or binding activity, and/or provides part of three dimensional structure of the polypeptide.
As used herein, the term "variant" refers to polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the inventive polypeptides and polynucleotides possess biological activities that are the same or similar to those of the inventive polypeptides or polynucleotides. The term "variant" with reference to polynucleotides and polypeptides encompasses all forms of polynucleotides and polypeptides as defined herein. Thus, "vesiculin variant(s)" include, for example, vesiculins having amino acid deletions or substitutions, including conservative amino acid substitutions, wherein one or more biological activities are retained, in whole or in part.
Variant polynucleotide sequences preferably exhibit at least about 50%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, more preferably at least about 95%, more preferably at least about 98%, and most preferably at least about 99% identity to a specified polynucleotide. Identity is found over a comparison window of at least about 20 nucleotide positions, preferably at least about 50 nucleotide positions, more preferably at least about 100 nucleotide positions or more of the entire length of a polynucleotide of the invention.
An exemplary family of programs useful for identifying variants in sequence databases is the BLAST suite of programs (version 2.2.5 [Nov 2002]) including BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX, which are publicly available from (ftp://ftp.ncbi.nih.gov/blast/) or from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894 USA. The NCBI server also provides the facility to use the programs to screen a number of publicly available sequence databases. BLASTN compares a nucleotide query sequence against a nucleotide sequence database. BLASTP compares an amino acid query sequence against a protein sequence database. BLASTX compares a nucleotide query sequence translated in all reading frames against a protein sequence database. tBLASTN compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames. tBLASTX compares the six-frame translations of a nucleotide query sequence against the six- frame translations of a nucleotide sequence database. The BLAST programs may be used with default parameters or the parameters may be altered as required to refine the screen. The use of the BLAST family of algorithms, including BLASTN, BLASTP, and BLASTX, is described in the publication of Altschul et al, Nucleic Acids Res. 25:3389-3402, (1997).
The "hits" to one or more database sequences by a queried sequence produced by
BLASTN, BLASTP, BLASTX, tBLASTN, tBLASTX, or a similar algorithm, align and identify similar portions of sequences. The hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.
Polynucleotide sequence identity can be determined in the following manner. The subject polynucleotide sequence is compared to a candidate polynucleotide sequence using BLASTN (from the BLAST suite of programs, version 2.2.5 [Nov 2002]) in bl2seq (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250), which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The default parameters of bl2seq are utilized except that filtering of low complexity parts should be turned off.
The identity of polynucleotide sequences may be examined using the following UNIX command line parameters: bl2seq -i nucleotideseql -j nucleotideseq2 -F F -p blastn
The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. The bl2seq program reports sequence identity as both the number and percentage of identical nucleotides in a line "Identities = ".
Polynucleotide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs (for example Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). A full implementation of the Needleman- Wunsch global alignment algorithm is found in the needle program in the EMBOSS package (Rice, P. Longden, I. and Bleasby, A. EMBOSS: The European Molecular Biology Open Software Suite, Trends in Genetics June 2000, vol 16, No 6. pp.276-277) which can be obtained from http://www.hgmp.mrc.ac.uk/Software/EMBOSS/. The European Bioinformatics Institute server also provides the facility to perform EMBOSS-needle global alignments between two sequences on line at http:/www.ebi. ac.uk/emboss/align/.
Alternatively the GAP program may be used which computes an optimal global alignment of two sequences without penalizing terminal gaps. GAP is described in the following paper: Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.
Use of BLASTN as described above is preferred for use in the determination of sequence identity for polynucleotide variants according to the present invention.
Polynucleotide variants also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polynucleotides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/).
The similarity of polynucleotide sequences may be examined using the following UNIX command line parameters:
bl2seq -i nucleotideseql -j nucleotideseq2 -F F -p tblastx
The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. The BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX algorithms also produce "Expect" values for alignments. These programs find regions of similarity between the sequences and for each such region reports an Expect value (E value) which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. The E value is used as a significance threshold for determining whether the hit to a database indicates true similarity. The size of this database is set by default in the bl2seq program. For small E values, much less than one, the E value is approximately the probability of such a random match. For example, an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size of the database screened, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance. For sequences having an E value of 0.01 or less over aligned and matched portions, the probability of finding a match by chance in that database is 1% or less using the BLASTN, BLASTP, BLASTX, tBLASTN or tBLASTX algorithm.
Variant polynucleotide sequences preferably exhibit an E value of less than about 1 x 10 "5, more preferably less than about 1 x 10 "6, more preferably less than about 1 x 10 "9, more
-12 -15
preferably less than about 1 x 10 " , more preferably less than about 1 x 10 " , more preferably
-18 -21
less than about 1 x 10 " and most preferably less than about 1 x 10 " when compared with any one of the specifically identified sequences.
Alternatively, variant polynucleotides hybridize to the specified polynucleotide sequence, or a complement thereof under stringent conditions. The term "hybridize under stringent conditions", and grammatical equivalents thereof, refers to the ability of a polynucleotide molecule to hybridize to a target polynucleotide molecule (such as a target polynucleotide molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot) under defined conditions of temperature and salt concentration. The ability to hybridize under stringent hybridization conditions can be determined by initially hybridizing under less stringent conditions then increasing the stringency to the desired stringency.
With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization conditions are no more than 25 to 30° C (for example, 10° C) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publishing,). Tm for polynucleotide molecules greater than about 100 bases can be calculated by the formula Tm = 81. 5 + 0. 41% (G + C-log (Na+). (Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Bolton and McCarthy, 1962, PNAS 84: 1390). Typical stringent conditions for polynucleotide of greater than 100 bases in length would be hybridization conditions such as pre -washing in a solution of 6X SSC, 0.2% SDS; hybridizing at 65°C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in IX SSC, 0.1% SDS at 65° C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65°C.
With respect to polynucleotide molecules having a length less than 100 bases, exemplary stringent hybridization conditions are 5 to 10° C below Tm. On average, the Tm of a polynucleotide molecule of length less than 100 bp is reduced by approximately (500/oligonucleotide length)0 C.
With respect to the DNA mimics known as peptide nucleic acids (PNAs) (Nielsen et al., Science 254(5037): 1497-500 (1991)) Tm values are higher than those for DNA-DNA or DNA- RNA hybrids, and can be calculated using the formula described in Giesen et al., Nucleic Acids Res. 26(21):5004-6 (1998). Exemplary stringent hybridization conditions for a DNA-PNA hybrid having a length less than 100 bases are 5 to 10° C below the Tm.
Variant polynucleotides also encompasses polynucleotides that differ from the sequences of the invention but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide having similar activity to a polypeptide encoded by a polynucleotide of the present invention. A sequence alteration that does not change the amino acid sequence of the polypeptide is a "silent variation." Except for ATG (methionine) and TGG (tryptophan), other codons for the same amino acid may be changed by art recognized techniques, for example, to optimize codon expression in a particular host organism.
Polynucleotide sequence alterations resulting in conservative substitutions of one or several amino acids in the encoded polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, for example, Bowie et al, Science 247: 1306 (1990)).
Variant polynucleotides due to silent variations and conservative substitutions in the encoded polypeptide sequence may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/) via the tblastx algorithm as previously described.
Polypeptide sequence identity can also be determined in the following manner. The subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, version 2.2.5 [Nov 2002]) in bl2seq, which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The default parameters of bl2seq are utilized except that filtering of low complexity regions should be turned off. Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs. EMBOSS-needle (available at http:/www.ebi. ac.uk/emboss/align/) and GAP (Huang, X., "On Global Sequence Alignment," Computer Applications in the Biosciences 10:227-235 (1994)) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity.
Use of BLASTP as described above is preferred for use in the determination of polypeptide variants according to the present invention.
Polypeptide variants also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The similarity of polypeptide sequences may be examined using the following UNIX command line parameters:
bl2seq -i peptideseql -j peptideseq2 -F F -p blastp
Variant polypeptide sequences preferably exhibit an E value of less than 1 x 10 "5, more preferably less than 1 x 10 "6, more preferably less than 1 x 10 "9, more preferably less than 1 x 10
-12 -15 -18
, more preferably less than 1 x 10 " , more preferably less than 1 x 10 " and most preferably
-21
less than 1 x 10 " when compared with any one of the specifically identified sequences.
The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. For small E values, much less than one, this is approximately the probability of such a random match.
Conservative substitutions of one or several amino acids of a described polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, for example, Bowie et ah, 1990, Science 247, 1306).
A "subject" refers to a vertebrate that is a mammal, for example, a human. Mammals include, but are not limited to, humans, farm animals, sport animals, pets, primates, mice and rats. Peptides of the invention, including, for example, synthetic vesiculins and variants thereof may be generated by synthetic methods (including, for example, single or fusion polypeptides), such as by solid phase peptide synthesis.
Solid phase peptide synthesis
The basic principle for solid phase peptide synthesis (SPPS) is a stepwise addition of amino acids to a growing polypeptide chain anchored via a linker molecule to a solid phase support, typically a resin particle, which allows for cleavage and purification once the polypeptide chain is complete. Briefly, a solid phase resin support and a starting amino acid are attached to one another via a linker molecule. Such resin-linker-acid matrices are commercially available (e.g., Calbiochem, a brand of EMD Biosciences, an affiliate of Merck KGaA of Darmstadt, Germany; or ORPEGEN Pharma of Heidelberg, Germany, for example).
The amino acid to be coupled to the resin is protected at its Na-terminus by a chemical protecting group. The amino acid may also have a chemical side-chain protecting group. Such protecting groups prevent undesired or deleterious reactions from taking place during the process of forming the new peptide bond between the carboxyl group of the amino acid to be coupled and the unprotected Na-amino group of the peptide chain attached to the resin. The amino acid to be coupled is reacted with the unprotected Na-amino group of the N-terminal amino acid of the peptide chain, increasing the chain length of the peptide chain by one amino acid. The carboxyl group of the amino acid to be coupled may be activated with a suitable chemical activating agent to promote reaction with the Να-amino group of the peptide chain. The Na- protecting group of N-terminal amino acid of the peptide chain is then removed in preparation for coupling with the next amino acid residue. This technique consists of many repetitive steps making automation attractive whenever possible.
When the desired sequence of amino acids is achieved, the peptide is cleaved from the solid phase support at the linker molecule.
A number of options exist for the various steps of SPPS. SPPS may be carried out using a continuous flow method or a batch flow method. Continuous flow is useful because it permits real-time monitoring of reaction progress via a spectrophotometer. However, continuous flow has two distinct disadvantages in that the reagents in contact with the peptide on the resin are diluted, and scale is more limited due to physical size constraints of the solid phase resin. Batch flow occurs in a filter reaction vessel and is useful because reactants are accessible and can be added manually or automatically. Further options involve the identity of the protecting group used for protecting the N- alpha-amino terminus. One protecting group is known as "Boc" (tert-butyloxycarbonyl). Reagents for the Boc method are relatively inexpensive, but they are highly corrosive and require expensive equipment and more rigorous precautions to be taken. The typically preferred alternative is the "Fmoc" (9-fluorenylmethyloxycarbonyl) protection scheme, which uses less corrosive, although more expensive, reagents.
For SPPS, a wide variety of solid support phases are available. The solid phase support used for synthesis can be a synthetic resin, a synthetic polymer film or a silicon or silicate surface, e.g. controlled pore glass (CPG), suitable for synthesis purposes. Generally, a resin is used, and commonly polystyrene suspensions, or polystyrene-polyethyleneglycol, or polymer supports for example polyamide are used. For example, 2-chlortrityl resin, an acid labile resin, is commonly used to cleave a product from the resin without cleaving the protective groups. Photolable resins are useful because cleavage is carried out without using acidic or basic conditions and therefore basic- and acid-lable side chain protective groups remain stable. Brominated Wang resin, ANP resin and Fmoc-photolable resin are examples of this class.
Examples of resins functionalized with linkers suitable for Boc-chemistry include PAM resin, oxime resin SS, phenol resin, brominated Wang resin and brominated PPOA resin. Examples of resins suitable for Fmoc chemistry include AMPB-BHA resin, Sieber amide resin, Rink acid resin, Tentagel S AC resin, 2-chlorotrityl chloride resin, 2-chlorotrityl alcohol resin, TentaGel S Trt-OH resin, Knorr-2-chlorotrityl resin, hydrazine-2-chlorotrityl resin, ANP resin, Fmoc photolable resin, HMBA-MBHA resin, TentaGel S HMB resin, Aromatic Safety Catch resinBAl resin and Fmoc-hydroxylamine 2 chlorotrityl resin. Other suitable resins include PL Cl- Trt resin, PL-Oxime resin and PL-HMBA Resin. In one embodiment, the solid phase is a Rink acid resin or a HMBA-MBHA resin.
For each resin appropriate coupling conditions are known in the literature for the attachment of the starting monomer or sub-unit.
Preparation of the solid phase support includes solvating the support in an appropriate solvent (dimethyl formamide (DMF), for example). The solid phase tyically increases in volume during solvation, which in turn increases the surface area available to carry out peptide synthesis.
A linker molecule is then attached to the support for connecting the peptide chain to the solid phase support. Linker molecules are generally designed such that eventual cleavage provides either a free acid or amide at the C-terminus. Linkers are generally not resin-specific. Examples of linkers include peptide acids for example 4-hydroxymethylphenoxyacetyl-4'- methylbenzyhydrylamine (HMP), or peptide amides for example benzhydrylamine derivatives.
The first amino acid of the peptide sequence may be attached to the linker after the linker is attached to the solid phase support or attached to the solid phase support using a linker that includes the first amino acid of the peptide sequence. Linkers that include amino acids are commercially available.
The next step is to deprotect the Na-amino group of the first amino acid. For Fmoc SPPS, deprotection of the Na-amino group may be carried out with a mild base treatment (piperazine or piperidine, for example). Side-chain protecting groups may be removed by moderate acidolysis (trifluoroacetic acid (TFA), for example). For Boc SPPS, deprotection of the Να-amino group may be carried out using for example TFA.
Following deprotection, the amino acid chain extension, or coupling, proceeds by the formation of peptide bonds. This process requires activation of the C-alpha-carboxyl group of the amino acid to be coupled. This is typically accomplished using, for example, in situ reagents, preformed symmetrical anhydrides, active esters, acid halides, or urethane-protected N- carboxyanhydrides. The in situ method allows concurrent activation and coupling; the most popular type of coupling reagents are carbodiimide derivatives, for example Ν,Ν'- dicyclohexylcarbodiimide or N,N-diisopropylcarbodiimide.
After the desired amino acid sequence has been synthesized, the peptide is cleaved from the resin. The conditions used in this process depend on the sensitivity of the amino acid composition of the peptide and the side-chain protecting groups. Generally, however, cleavage is carried out in an environment containing a plurality of scavenging agents to quench the reactive carbonium ions that originate from the protective groups and linkers. Common cleaving agents include for example TFA and hydrogen fluoride (HF).
Thus, the principle of SPPS is the iterative steps of deprotecting, activating, and coupling each amino acid, followed by the final step of cleavage to separate the completed peptide from the solid support.
The use of protective groups in SPPS is well established in the art. Examples of common protective groups are listed together with their abbreviations below: acetamidomethyl (Acm), acetyl (Ac), adamantyloxy (AdaO), benzoyl (Bz), benzyl (Bzl), 2-bromobenzyl, benzyloxy (BzlO), benzyloxycarbonyl (Z), benzyloxymethyl (Bom), 2-bromobenzyloxycarbonyl (2-Br-Z), tert-butoxy (tBuO), tert-butoxycarbonyl (Boc), tert-butoxymethyl (Bum), tert-butyl (tBu), tert-buthylthio (tButhio), 2-chlorobenzyloxycarbonyl (2-Cl-Z), cyclohexyloxy (cHxO), 2,6-dichlorobenzyl (2,6-DiCl-Bzl), 4,4'-dimethoxybenzhydryl (Mbh), l-(4,4-dimethyl-2,6-dioxo- cyclohexylidene)3 -methyl-butyl (ivDde), 4- {N-[ 1 -(4,4-dimethyl-2,6-dioxo-cyclohexylidene)3- methylbutyl] -amino) benzyloxy (ODmab), 2,4-dinitrophenyl (Dnp), fluorenylmethoxycarbonyl (Fmoc), formyl (For), mesitylene-2-sulfonyl (Mts), 4-methoxybenzyl (MeOBzl), 4-methoxy- 2,3,6-trimethyl-benzenesulfonyl (Mtr), 4-methoxytrityl (Mmt), 4-methylbenzyl (MeBzl), 4- methyltrityl (Mtt), 3-nitro-2-pyridinesulfenyl (Npys), 2,2,4,6, 7-pentamethyldihydrobenzofurane- 5-sulfonyl (Pbf), 2,2,5,7,8-pentamethyl-chromane-6-sulfonyl (Pmc), tosyl (Tos), trifluoro acetyl (Tfa), trimethylacetamidomethyl (Tacm), trityl (Trt) and xanthyl (Xan).
In the event one or more of the side chains of the amino acids of the peptide contains additional functional groups, such as for example additional carboxylic, amino, hydroxy or thiol groups, further protective groups may be necessary. For example, if the Fmoc strategy is used, Mtr, Pmc, Pbf may be used for the protection of Arg; Trt, Tmob may be used for the protection of Asn and Gin; Boc may be used for the protection of Trp and Lys; tBu may be used for the protection of Asp, Glu, Ser, Thr and Tyr; and Acm, tBu, tButhio, Trt and Mmt may be used for the protection of Cys. A person skilled in the art will appreciate that there are numerous other suitable combinations.
In a specifically contemplated embodiment of the present invention the Fmoc group is used as a first Na-protective group during the synthesis of synthetic vesiculin A chain polypeptides, and can be cleaved using piperidine.
In a specifically contemplated embodiment, the side chains of one or more amino acids in the synthesis of synthetic vesiculin A chain polypeptides are protected with one or more suitable protectin groups. In one embodiment, the one or more protecting groups are removeable using trifluoroacetic acid. In a specifically contemplated embodiment, one or more of the cysteine residues are protected with one or more suitable protecting groups. In one embodiment, the one or more suitable protecting groups are not removable using trifluoroacetic acid.
In a specifically contemplated embodiment of the present invention the Boc group is used as a first Na-protective group during the synthesis of synthetic vesiculin B chain polypeptides, and can be cleaved using trifluoroacetic acid.
The methods for SPPS outlined above are well known in the art. See, for example, Atherton and Sheppard, "Solid Phase Peptide Synthesis: A Practical Approach," New York: IRL Press, 1989; Stewart and Young: "Solid-Phase Peptide Synthesis 2nd Ed.," Rockford, Illinois: Pierce Chemical Co., 1984; Jones, "The Chemical Synthesis of Peptides," Oxford: Clarendon Press, 1994; Merrifield, J. Am. Soc. 85:2146-2149 (1963); Marglin, A. and Merrifield, R.B. Annu. Rev. Biochem. 39:841-66 (1970); and Merrifield R.B. JAMA. 210(7): 1247-54 (1969); and "Solid Phase Peptide Synthesis - A Practical Approach" (W.C. Chan and P.D. White, eds. Oxford University Press, 2000). Equipment for automated synthesis of peptides or polypeptides is readily commercially available from suppliers such as Perkin Elmer/ Applied Biosystems (Foster City, CA) and may be operated according to the manufacturers instructions.
Confirmation of the identity of the newly synthesized vesiculin polypeptides and vesiculin variants is conveniently achieved by amino acid analysis, mass spectroscopy, Edman degradation, or for functional variants or intermediates by assessing biological function (i.e., stimulating glucose incorporation into glycogen, or β-cell mitogenesis).
Synthetic variants of vesiculin may also be made by substituting amino acids which do not substantially alter the bioactivity of the synthetic vesiculin variant relative to the parent vesiculin {e.g., conservative substitutions). Selection of amino acids for substitution can depend on the size, structure, charge, and can be either an amino acid found in nature or synthetic amino acid. Generally, amino acids which have a similar charge {e.g., hydrophobic for hydrophobic) or similar size {e.g., isoleucine for leucine) can be selected for substitution. One or more substitutions can be made in a stepwise fashion or concurrently. Variations in the residues included in the peptide are also both possible and contemplated. For example, it is possible to substitute amino acids in a sequence with equivalent amino acids using conventional techniques. Groups of amino acids known normally to be equivalent are:
(a) Ala, Ser, Thr, Pro, and Gly;
(b) Asn, Asp, Glu, and Gin;
(c) His, Arg, and Lys;
(d) Met, Glu, He, and Val; and
(e) Phe, Tyr, and Trp.
The substitution of one amino acid for another in the same group as shown above is an example of a conservative amino acid substitution as used herein. Synthetic vesiculin variants produced by one or more conservative amino acid substitutions are herein termed "conservative synthetic variants."
It is understood that many synthetic vesiculin variants can be achieved by substituting one or more amino acids. The synthetic vesiculin variants can be tested for biological function, such as for example, to stimulate glucose incorporation into glycogen, whether in vivo or in vitro. The biological activity of a synthetic vesiculin variant is generally at least about 25% of a vesiculin, preferably at least about 35%, preferably at least about 50%, preferably at least about 60%, preferably at least about 75%, preferably at least about 85%, and more preferably at least about 95%.
The invention also encompasses synthetic intermediates with vesiculin bioactive functionality. Such synthetic intermediates may be obtained during synthesis of vesiculin, by for example, the methods of the present invention. Synthetic intermediates may be ascertained by stepwise isolation and assay. If an amino acid is omitted and the bioactivity of vesiculin is not substantially reduced, then the amino acid need not comprise a portion of the synthetic intermediate. Further, polypeptides comprising a synthetic intermediate of vesiculin or vesiculin variant(s) are also encompassed in the invention. For example, synthetic intermediates of vesiculin may comprise about 10 contiguous amino acids of the amino acids of the amino acid sequence of either or both the A-chain and/or B-chain of vesiculin, more preferably about 15 contiguous amino acids, more preferably about 20 contiguous amino acids, more preferably about 25 contiguous amino acids, more preferably about 30 contiguous amino acids, about 40 contiguous amino acids, about 50 contiguous amino acids, about 60 contiguous amino acids, or more preferably 61 contiguous amino acids.
Additions and/or deletions of amino acids may also be made as long as the resulting synthetic polypeptide is immunologically cross-reactive with and/or has substantially the same function or functions as a vesiculin.
Whilst not typically necessary for synthetic methods, a fusion protein may also be constructed that facilitates purification or identification. Examples of components for these fusion proteins include, but are not limited to myc, HA, FLAG, or His-6. Longer fusion partners typically used in recombinant methods of production, such as glutathione S-transferase, maltose binding protein or the Fc portion of immunoglobulin, are generally considered too large for sensible use in synthetic methods, except where such fusion partners can be coupled to the synthetic vesiculin polypeptide, variant or intermediate without a need for synthesis of the partner in situ.
If used for diagnostic purposes, the polypeptides are at least substantially purified or isolated from other cellular constituents. The polypeptides are preferably at least about 80% pure, and free of pyrogens and other contaminants. Methods of protein purification are known in the art and are not described in detail herein.
Proteins can be classified according to their sequence relatedness to other proteins in the same genome (paralogies) or a different genome (orthologues). Orthologous genes are genes that evolved by speciation from a common ancestral gene and normally retain the same function as they evolve. Paralogous genes are genes that are duplicated within a genome and genes may acquire new specificities or modified functions which may be related to the original one. Phylogenetic analysis methods are reviewed in Tatusov, et al., 1997, Science 278, 631-637,).
In addition to the computer/database methods described above, polypeptide variants may be identified by physical methods, for example by screening expression libraries using antibodies raised against polypeptides of the invention (Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987) or by identifying polypeptides from natural sources with the aid of such antibodies.
Variants according to the invention also include the homologues of vesiculin from species other than human, rat or mouse. Such homologues can be readily identified using, for example, nucleic acid probes based upon the conserved regions of the polynucleotides which encode human, rat and mouse vesiculin.
Administration of vesiculin
The invention includes methods for treating and/or preventing, in whole or in part, various diseases, disorders, and conditions, including for example, impaired glucose tolerance; impaired fasting glucose; prediabetes; diabetes and/or its complications, including type 1 and type 2 diabetes and their complications; insulin resistance; Syndrome X; obesity and other weight related disorders; fatty liver disease, including nonalcoholic alcoholic fatty liver disease; glucose metabolism diseases and disorders; diseases, disorders or conditions that are treated or treatable with insulin; diseases, disorders or conditions that are treated or treatable with a hypoglycemic agent; diseases, disorders, and conditions characterized at least in part by hyperglycemia; diseases, disorders, and conditions characterized at least in part by hypoinsulinemia and/or diseases, disorders, and conditions characterized at least in part by hyperinsulinemia.
The invention includes methods for treating a subject having or suspected of having or predisposed to, or at risk for, for example, any diseases, disorders and/or conditions characterized in whole or in part by hyperglycemia, insulin resistance, impaired glucose tolerance, and/or impaired fasting glucose, comprising administering a composition comprising a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, a synthetic vesiculin derivative and/or a synthetic vesiculin intermediate or a salt thereof. Such diseases, disorders and/or conditions include but are not limited to those described or referenced herein. Such compounds may be administered in amounts, for example, that are effective to (1) lower serum glucose, (2) lower blood glucose, (3) lower urine glucose, (4) lower fructosamine, (5) lower glycosylated hemoglobin (HbAic) levels, (6) lower postprandial glycemia, (7) ameliorate impaired glucose tolerance, (8) ameliorate impaired fasting glucose, (9) lower the rate and/or severity of hypoglycemic events, including severe hypoglycemic events, and/or (10) and/or stimulate glucose disposal. Such compositions include, for example, formulations for delivery by injection, transdermal patch, inhalation, and other non- oral delivery methods.
The invention includes methods for regulating glycemia in a subject having or suspected of having or predisposed to diseases, disorders and/or conditions characterized in whole or in part, for example, by hyperglycemia, insulin resistance, impaired glucose tolerance, and/or impaired fasting glucose, comprising administering a composition comprising a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, a synthetic vesiculin derivative and/or a synthetic vesiculin intermediate or a salt thereof. Such diseases, disorders and/or conditions include but are not limited to those described or referenced herein. Such compounds may be administered in amounts, for example, that are effective to (1) lower serum glucose, (2) lower blood glucose, (3) lower urine glucose, (4) lower fructosamine, (5) lower glycosylated hemoglobin (HbAic) levels, (6) lower postprandial glycemia, (7) ameliorate impaired glucose tolerance, (8) ameliorate impaired fasting glucose, (9) lower the rate and/or severity of hypoglycemic events, including severe hypoglycemic events. Such compositions include, for example, formulations for delivery by injection, transdermal patch, inhalation, and other non-oral delivery methods, and/or (10) stimulate glucose disposal.
For administration to a patient, a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, a synthetic vesiculin derivative, and/or a synthetic vesiculin intermediate, or salts thereof may be used in pure or substantially pure form. Synthetic vesiculins, synthetic vesiculin A chains, synthetic vesiculin B chains, synthetic vesiculin variants, synthetic vesiculin derivatives, and/or synthetic vesiculin active fragments, or salts thereof, may be presented as a pharmaceutical composition. Such compositions may comprise one or more of a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, a synthetic vesiculin derivative, and/or a synthetic vesiculin intermediate, or salts thereof, for example, together with one or more pharmaceutically acceptable carriers and optionally other ingredients where desirable. Formulations for parenteral and non parenteral drug delivery are known in the art and are set forth, for example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing (1990). The carrier is acceptable in the sense of being compatible with the peptide being administered and not overtly harmful to the subject to be treated. Desirably, the composition should not include substances with which peptides are known to be incompatible. For solid compositions, conventional non-toxic carriers include, for example mannitol, lactose, starch, magnesium stearate, magnesium carbonate, sodium saccharin, talcum, cellulose, glucose, sucrose, pectin, dextrin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, a low- melting wax, cocoa butter, and the like may be used. The active compound as defined above may be formulated as suppositories using, for example, polyalkylene glycols, for example, propylene glycol as a carrier.
A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, or tablet disintegrating agents; it can also be an encapsulating material. In a similar manner, cachets or transdermal systems are included. In powders, the carrier is a finely divided solid which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
Liquid form preparations include solutions, suspensions, or emulsions suitable, for example, for parenteral administration. Aqueous solutions for parenteral administration can be prepared by dissolving the subject peptide in water and adding other suitable agents, stabilizers, buffers, etc., as desired. Actual methods of preparing such dosage forms are known, or will be apparent to those skilled in this art; for example, see Remington's Pharmaceutical Sciences.
The composition or formulation to be administered will preferably contain a quantity of the active compound in an amount effective to (1) lower serum glucose, (2) lower blood glucose, (3) lower urine glucose, (4) lower fructosamine, (5) lower glycosylated hemoglobin (HbAic) levels, (6) lower postprandial glycemia, (7) ameliorate impaired glucose tolerance, (8) ameliorate impaired fasting glucose, (9) lower the rate and/or severity of hypoglycemic events, including severe hypoglycemic events, and/or (10) stimulate glucose disposal. An effective amount of a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, a synthetic vesiculin derivative, and/or a synthetic vesiculin intermediate, or a salt thereof may include, for example, from about 0.01 nmol/kg/day to about 100 nmol/kg/day, from about 0.02 nmol/kg/day to about 75 nmol/kg/day, from about 0.025 nmol/kg/day to about 50 nmol/kg/day, from about 0.03 nmol/kg/day to about 40 nmol/kg/day, from about 0.04 nmol/kg/day to about 30 nmol/kg/day, from about 0.05 nmol/kg/day to about 25 nmol/kg/day, from about 0.07 nmol/kg/day to about 20 nmol/kg/day, from about 0.08 nmol/kg/day to about 15 nmol/kg/day, from about 0.1 nmol/kg/day to about 10 nmol/kg/day, from about 0.2 nmol/kg/day to about 5 nmol/kg/day.
An effective amount of a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, a synthetic vesiculin derivative, and/or a synthetic vesiculin intermediate, or a salt thereof may also include, for example, from about 120 ng/kg/day to about 1.2 mg/kg/day, from about 240 ng/kg/day to about 900 μg/kg/day, from about 300 ng/kg/day to about 600μg/kg/day, from about 360 ng/kg/day to about 480 μg/kg/day, from about 480 ng/kg/day to about 400 μg/kg/day, from about 600 ng/kg/day to about 300 μg/kg/day, from about 840 ng/kg/day to about 240 μg/kg/day, from about 960 ng/kg/day to about 180 μg/kg/day, from about 1.2 μg/kg/day to about 120 μg/kg/day, and from about 2.4 μg/kg/day to about 60 μg/kg/day.
It is understood that the dosage administered may vary from individual to individual. It is also understood that the dosage may be administered in a single dose or optionally multiple doses (e.g., two, three, or four doses per day). A clinician or physician will determine the dosage needed for individuals. A clinician or physician may monitor factors including but not limited to glucose level, vesiculin level (either circulating or resident in tissues), insulin levels (either circulating or resident in tissues), level of depletion of pancreatic β-cells, presence or absence of polydipsia, presence or absence of polyphagia, presence or absence of polyuria, levels of glycated hemoglobin, levels of glycated albumin, and levels of fructosamine.
The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredients into association with a carrier which constitutes one or more accessory ingredients.
The precise form the composition will take will largely be dependent upon the administration route chosen. For example, a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, a synthetic vesiculin derivative, and/or a synthetic vesiculin intermediate, may be injected parenterally, for example, intravenously into the blood stream of the patient being treated. However, it will be readily appreciated by those skilled in the art that the route can vary, and can be intravenous, subcutaneous, transcutaneous, intramuscular, intradermal, intraarticular, intrathecal, intraperitoneal, enterally, transdermally, transmucously, sustained release polymer compositions (for example a lactide polymer or copolymer microparticle or implant), perfusion, pulmonary (for example, inhalation), nasal, oral, etc. Injectables can be prepared in conventional forms, either as liquid solutions or suspension, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients include, for example, water, saline, aqueous dextrose, glycerol, ethanol or the like. In addition, if desired, the pharmaceutical compositions may also contain minor amounts of non-toxic substances such as wetting or emulsifying agents, auxiliary pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, etc. Compositions suitable for parenteral and in particular subcutaneous administration are preferred. Other suitable administration routes are intravenous administration and intramuscular administration. Such compositions conveniently comprise sterile aqueous solutions of a synthetic vesiculin, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, a synthetic vesiculin derivative, and/or a synthetic vesiculin intermediate, or a salt thereof. The solutions can be isotonic with the blood of the patient to be treated. Such compositions may be conveniently prepared by dissolving a vesiculin, a vesiculin A chain, a vesiculin B chain, a vesiculin variant, a vesiculin derivative, and/or a vesiculin active fragment, or a salt thereof in water to produce an aqueous solution and rendering this solution sterile. The composition may then be presented in unit or multi-dose containers, for example sealed ampoules or vials. One particularly preferred composition is a vesiculin, for example, a human vesiculin, in a physiological buffered solution suitable for injection.
Compositions suitable for sustained release parenteral administrations {e.g. biodegradable polymer formulations) are also well known in the art. See, for example, US Patent Nos. 3,773,919 and 4,767,628 and PCT Publication No. WO 94/15587.
It is envisaged oral delivery forms are equally acceptable, one example of oral delivery forms of tablet, capsule, lozenge, or the like form, or any liquid form such as syrups, aqueous solutions, emulsion and the like, capable of protecting the therapeutic protein from degradation prior to eliciting an effect, e.g., in the alimentary canal if an oral dosage form. Examples of dosage forms for transdermal delivery include transdermal patches, transdermal bandages, and the like.
Included within the topical dosage forms are any lotion, stick, spray, ointment, paste, cream, gel, etc., whether applied directly to the skin or via an intermediary such as a pad, patch or the like.
Examples of dosage forms for suppository delivery include any solid or other dosage form to be inserted into a bodily orifice (particularly those inserted rectally, vaginally and urethrally). Examples of dosage units for transmucosal delivery include depositories, solutions for enemas, pessaries, tampons, creams, gels, pastes, foams, nebulised solutions, powders and similar formulations containing in addition to the active ingredients such carriers as are known in the art to be appropriate.
Examples of dosage units for depot administration include pellets or small cylinders of active agent or solid forms wherein the active agent is entrapped in a matrix of biodegradable polymers, microemulsions, liposomes or is microencapsulated.
Examples of implantable infusion devices include any solid form in which the active agent is encapsulated within or dispersed throughout a biodegradable polymer or synthetic, polymer such as silicone, silicone rubber, silastic or similar polymer. Alternatively dosage forms for infusion devices may employ liposome delivery systems.
Examples of dosage units for delivery via bolus include single or multiple administrations by intravenous injection, subcutaneous, subdermal, and intramuscular administration or oral administration.
Examples of dosage units for inhalation or insufflation include compositions comprising solutions and/or suspensions in pharmaceutically acceptable, aqueous, or organic solvents, or mixtures thereof and/or powders.
It is also convenient for synthetic vesiculin to be converted to be in the form of a salt. Such a salt will generally be physiologically acceptable, and can be formed using any method well known in the art. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable non-toxic inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, benzoic, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, gluconic, glutamic, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, isethionic, lactate, maleate, malonate, malic, mandelic, methanesulfonate, mucic, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, pantothenic, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate, undecanoate and the like. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts. Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)4 + salts. This invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products may be obtained by such quaternization. Hydrochloride and acetate salts are preferred.
Synthetic vesiculin salts formed by combination of synthetic vesiculin with anions of organic acids are particularly preferred. Such salts include, but are not limited to, malate, acetate, propionate, butyrate, oxaloacetate, citrate, isocitrate, a-ketoglutarate, succinate, fumarate and trifluoroacetate salts. The salts thus formed can also be formulated into pharmaceutical compositions for therapeutic administration where desired.
Methods for using vesiculin polypeptides
As described above, the present invention provides a synthetic vesiculin and synthetic vesiculin A and B chains (including but not limited to their human, rat and mouse forms), a synthetic vesiculin variant, or a synthetic vesiculin derivative, or synthetic variant thereof. Synthetic vesiculins are shown herein to play a role, for example, in the stimulation of glucose incorporation into glycogen.
The invention therefore also provides methods by which glucose incorporation into glycogen can be modulated. Such modulation will usually involve administration of a synthetic vesiculin or synthetic vesiculin-related polypeptide as described herein.
A vesiculin agonist is a compound which can, for example, promote or potentiate the effect of vesiculin on glucose incorporation into glycogen. In contrast, a vesiculin antagonist is a compound which competes with vesiculin or otherwise interacts with vesiculin to block or reduce the effect of vesiculin, for example, on glucose incorporation into glycogen. Vesiculin agonists and antagonists can be identified by assay systems, including the soleus muscle assay, which measure the effect synthetic vesiculin has on glucose incorporation into glycogen in the presence and absence of a test compound.
Where it is desired that a vesiculin agonist or vesiculin antagonist be employed in modulating, for example but not limited to, glucose incorporation into glycogen, the agonist/antagonist can be administered as a substantially pure compound or formulated as a pharmaceutical composition as described above for vesiculin.
Other uses for synthetic vesiculin polypeptides include use in vaccines and for generation of antibodies, including monoclonal antibodies. Synthetic vesiculin polypeptides are used as immunogens to immunize mice. Splenocytes (including lymphocytes) are obtained from the immunized mice. Hybridomas are prepared from the lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C, Nature 256:495-497 (1975). Other modified methods, for example by Buck, D. W., et al., In Vitro 18:377-381 (1982) may also be used. Available myeloma lines, include but are not limited to X63-Ag8.653 and those from the Salk Institute, Cell Distribution Center, San Diego, Calif, USA, may be used in the hybridization. The technique involves fusing the myeloma cells and lymphoid cells using a fusogen such as polyethylene glycol, or by electrical means well known to those skilled in the art. After the fusion, the cells are separated from the fusion medium and grown in a selective growth medium, such as HAT medium, to eliminate unhybridized parent cells. Any of the media described herein, supplemented with or without serum, can be used for culturing hybridomas that secrete monoclonal antibodies. As another alternative to the cell fusion technique, EBV immortalized B cells are used to produce the monoclonal antibodies of the subject invention. The hybridomas are expanded and subcloned, if desired, and supernatants are assayed for anti-immunogen activity by conventional immunoassay procedures {e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).
Hybridomas that produce such antibodies may be grown in vitro or in vivo using known procedures. The monoclonal antibodies may be isolated from the culture media or body fluids, by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration, if desired. Undesired activity if present, can be removed, for example, by running the preparation over adsorbants made of the immunogen attached to a solid phase and eluting or releasing the desired antibodies off the immunogen.
Synthetic vesiculin and synthetic vesiculin-related polypeptides, including synthetic vesiculin intermediates, may also be used as immunogens to immunize other animals {e.g., rats and rabbits) to generate polyclonal antibodies. Methods of producing polyclonal antibodies and the subsequent isolation and purification thereof are well known in the art. See, for example, Harlow et al, supra. Other suitable techniques for preparing antibodies involve in vitro exposure of lymphocytes to the antigen or alternatively to selection of libraries of antibodies in phage or similar vectors. Also, recombinant antibodies may be produced using procedures known in the art. See, for example, US Patent 4,816,567.
The antibodies may be used with or without modification. Frequently, antibodies will be labeled by joining, either covalently or non-covalently a substance which provides a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in the literature. Antibodies can be used to monitor the presence of vesiculin in a patient or in vesiculin quantification assays. Further, anti-vesiculin antibodies, for example, can be used to measure levels of vesiculin in an individual, either at one fixed time point or over a period of time to monitor fluctations in circulating vesiculin levels. Anti-vesiculin antibodies can also be used to measure levels of vesiculin in an individual to whom drugs have been administered. In such assays, any convenient immunological format can be employed. Such formats include immunohistochemical assays, RIA, IRMA and ELISA assays.
The assays can be conducted in relation to any biological fluid which does, or should, contain vesiculin. Such fluids include blood, serum, plasma, urine and cerebrospinal fluid.
Antibodies, monoclonal or polyclonal, against synthetic vesiculin may be used for diagnosis or for therapeutic purposes. Antibodies may be used by themselves or attached to a solid substrate, such as a column or a plate. Antibodies which are attached to a solid substrate may be used for assays, for example ELISA, or as a standard in other assays. Antibodies against synthetic vesiculin are also useful for vesiculin isolation, vesiculin purification, and vesiculin quantitation.
The antibodies can also be included in assay kits. Such kits can contain, in addition, a number of optional but conventional components, the selection of which will be routine to the art skilled worker. Such additional components will however generally include a vesiculin reference standard, which may be vesiculin itself or a variant (such as an intermediate).
It will also be appreciated that antibodies such as described above can be used as vesiculin antagonists by binding to vesiculin and partly or completely interfering with vesiculin activity.
Compositions useful in the invention are prepared by mixing the ingredients following generally accepted procedures. For example, the selected components may be mixed in a blender or other standard device to produce a concentrated mixture which may then be adjusted to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to control tonicity. Other applications relating to the discovery of vesiculin will be apparent to those persons skilled in the art, who will appreciate that the above description is provided by way of example only and that the invention is not limited thereto.
A better understanding of the invention will be gained by reference to the following non-limiting experimental section which is illustrative and are not intended to limit the invention or the claims in any way. EXAMPLE 1
This example describes a SPPS method for the production of synthetic murine vesiculin polypeptides.
An outline of the synthetic scheme is presented in Figure 1 which shows how the requisite disulfide bridges can be constructed in a stepwise and unambiguous manner using an orthoganol Cys- protecting group strategy.
Synthetic overview
A-chain
The A-chain was synthesised using Fmoc SPPS on a Liberty microwave-enhanced peptide synthesiser (CEM corp) using a PEG-based ChemMatrix™ resin . The Rink linker was used as the point of attachment (LI, scheme 1) of the peptide to the resin. Once the linker was installed, a sequence of five lysine residues was then added followed by the
hydroxymethylbenzoic acid (HMBA) linker (L2, figure 1) and the terminal Fmoc-Glu residue to give 1. The HMBA-pentalysine motif is retained after acidic cleavage of the completed peptide from resin at LI . The tag enhances the solubility of the attached peptide and facilitates purification over subsequent steps. Following completion of the synthesis the tag is removed hydrolytically. The synthesis was continued, incorporating the following protecting groups on the cysteine residues: Trityl on Cys3 and Cys5, Acm on Cys4 and tert- utyl on Cys6 to afford 2.
The peptide was cleaved from the resin using a trifluoroacetic acid-based cocktail and precipitated in ether/hexane. HPLC analysis showed one main peak (Figure 2) with a mass corresponding to that required for the A-chain sequence.
The intrachain disulfide bond of the crude A-chain was formed prior to purification. Adding a methanolic solution of dipyridyldisulfide to an aqueous solution of the A-chain 3 caused rapid formation of the disulfide bond between Cys3 and Cys5 to give 4 (Figure 3), as evidenced by the formation of an earlier-eluting peak under the given conditions with the desired mass.
The crude material was firstly concentrated and freeze-dried, then purified by HPLC using the over-loading method in which approximately half of the isolated crude peptide (400mg on this case) was dissolved in water/MeCN and loaded on to a Gemini semi-preparative (10 x 250 mm) column which was eluted with a very shallow gradient, with numerous small fractions being collected and analysed. Those fractions containing the required material were then pooled and lyophilised. Performing a single run in this manner afforded a reasonable quantity of 4 with a purity of >90%. B-Chain
The B-chain was synthesised using Boc-based, manual SPPS on a polystyrene resin derivatised with the PAM linker. The peptide was cleaved from resin using neat hydrofluoric acid and HPLC analysis of the crude material showed one main peak (Figure 4) with a mass corresponding to that required for the B-chain sequence 5. Isolation of 5 was achieved by dissolving the crude product in water/MeCN containing 0.1% formic acid and purifiying aliquots of the solution by HPLC using a Gemini semi-preparative (10 x 250 mm) column.
Synthetic murine vesiculin
The protected A- and B-chains were combined in the manner outlined in Scheme 1.
Firstly, Cys20 of the A-chain 4 was activated by treating a solution of
dipyridyldisulfide and the peptide in trifluoroacetic acid with a mixture of tfa and triflic acid for a few minutes. This treatment caused removal of the tert-butyl group and concomitant formation of the pyridyl disulfide 6 (Figure 5). The reaction was then halted by adding ether to precipitate the peptide.
Residual DPDS was separated from the product material by HPLC using a Phenomenex Jupiter C4 semi-prep column.
Generation of the inter-chain disulfide bond between Csy2 and Cys6 was achieved by dissolving 6 in Tris-buffered Guanidine.HCl, pH 8.1 and adding a solution of the 5, also in Guanidine.HCl. Reaction was evidenced by the rapid disappearance of 5 and 6 (Figure 6).
Formation of the required product 7 was confirmed by mass spectrometry.
Generation of the interchain disulfide bond between Cysi and Cys4 required the removal of the Acm protecting groups. Incubating a solution of the substrate peptide in 80%> acetic acid at 0°C with lOmM iodine for 50 minutes removed the Acm groups and
simultaneously generated the required disulfide bond (Figure 7), giving 8 in 75% yield after purification. Heating the substrate in trifluoroacetic acid at approximately 60°C was also found to remove the Acm groups.
The final step entailed hydrolysis of the HMBA linker from 8 under basic conditions to remove the pentalysine tail. Hydrolysis was achieved with sodium bicarbonate buffers at pH 9.5 after 70 hours. Hydrolysis was also achieved with 0.1 M aqueous NaOH at 0°C in 5 minutes, affording murine vesiculin 9 in 80% yield after purification (**, Figure 8), with the identity confirmed by MS.
Experimental
Materials. 9-Fluorenylmethoxycarbonyl (Fmoc) protected L-R-amino acids and l-[bis(dimethylamino)methylene]-lHbenzotriazolium
hexafluorophosphate 3 -oxide (HBTU) were
purchased from GL Biochem (Shanghai, China). Dimethylformamide (DMF, Scharlau), was purchased from Global Scientific. Piperidine and diisopropylamine were purchased from Sigma- Aldrich.
ChemMatrix resin was purchased from PCAS Biomatrix (Canada). Methanol, diethyl ether, and dichloromethane
were from Merck (New Zealand), 3,6-Dioxa-l,8-octanedithiol
(DODT), triisopropylsilane (TIPS), diisopropylethylamine
(DIPEA), 2,2'-dipyridyl disulfide (DPDS), and trifiuoromethanesulfonic acid (TFMSA) were purchased from Sigma-Aldrich.
Acetonitrile was obtained Unichrom.
All other reagents were from Sigma-Aldrich. Methods. Fmoc SPPS Peptide Synthesis.
Peptides C-terminally linked to a solubilizing tag were assembled ChemMatrix PEG-based resin via a RINK linker, so that the polycationic tag itself possessed a C-terminal amide. SPPS employed Fmoc chemistry on the following instrument:
CEM Liberty microwave peptide synthesiser
The side chain protecting groups of trifunctional amino acids (Fmoc-Arg: Pbf (2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl); Asp, Glu, Ser, Thr, and Tyr: tert- vXy\; Asn, Gin, and His: trityl; Cys: trityl, acetamidomethyl, and tert-bvXy\; Trp and Lys: Boc (tert- butoxycarbonyl)) were TFA-labile except for acetamidomethyl (Acm)-protected and tert-butyl (tBu)-protected cysteines in the indicated positions. The peptides were synthesized on a 0.1 mmol scale using instrument default protocols with either a 4- or 5 -fold molar
excess of Fmoc-protected amino acids (0.4 or 0.5 mmol) that were activated by using 4- or 5- fold excess of HBTU or HCTU in the presence of DIPEA (0.8 or 1.0 mmol). NR-Fmoc protecting groups were removed by treating the resin-attached peptide with piperazine (5%w/v) in DMF. Using the microwave synthesizer, the coupling and deprotection were carried out at 75°C using 25 W microwave power for 5 min and 60 W microwave power for 3 min, respectively.
Fmoc-Glu- [HMB A] -KKKKK- [RINK] - [ChemMatrix] 1 (i) A sample of amino-methyl ChemMatrix resin (0.4 mmol) was was swollen in 1 : 1 v/v
DMF/DCM and aggregates gently broken up. The resin was drained and washed with DMF. Fmoc-Rink linker (432 mg, 0.8 mmol) and HCTU (331 mg, 0.8 mmol) were combined, dissolved in DMF (3 mL) and the solution transferred to the resin, which was then agitated for 1 hour. After draining and washing, the procedure was repeated and the resin then washed with DMF.
(ii) The resin was treated with 20% v/v piperidine in DMF (15 mL) for 12 min, drained and washed well with DMF. Solid Fmoc-Lys(Boc)-OH (646 mg, 1.38 mmol) and HCTU (537 mg, 1.3 mmol) were combined and dissolved in DMF (3 mL). Neat N-methylmorpholine (220 //L, 2 mmol) was added and the resulting solution transferred to the resin, which was then agitated for 1 hour, drained and washed. This deprotection-coupling cycle was repeated four times to create the pentalysine tag.
(iii) The resin was treated with 20% v/v piperidine in DMF (15 mL) for 12 min, drained and washed well with DMF. Solid 4-Hydroxymethylbenzoic acid (HMBA, 146 mg, 0.92 mmol) and HOBt (141 mg, 0.92 mmol) were combined and dissolved in DMF (4 mL). The solution was transferred to the resin neat DIC (142 /L, 0.92 mmol) added and the mixture agitated for 1 hour. The resin was drained and washed and the coupling procedure repeated until a negative Kaiser assay indicated complete loading of the HMBA linker.
(iv) Solid Fmoc-Glu(tBu)-OH (1700 mg, 4 mmol and 4-DMAP (48 mg, 0.4 mmol) were added to the drained resin and minimum volume of DMF (approx. 4 mL) added to dissolve the solids and give a free-flowing suspension of resin. Neat DIC (620 //L, 4 mmol) was added and the mixture agitated for 2 hours. The resin was then drained and washed.
GIVEECC(Acm)FRSCDLALLETYC(tBu)ATPAKSE-[HMBA]-KKKKK-NH2 3
The synthesis was performed on the Liberty MW peptide synthesiser on a 0.1 mmol scale using recommended instrument conditions. Coupling entailed use of a 5 -fold excess of HBTU in the presence of DIPEA (1.0 mmol) for 15 min; 25 W microwave power, 75°C max. Mx-Fmoc protecting groups were removed by treating the resin with piperazine (5%w/v) in DMF for 3 min; 60 W microwave power, 75°C max.
S-(Acm)-protected cysteine was introduced into the Vesiculin A-chain at position Cys4 and S- (tBu)-protected cysteine at position Cys6.
On completion of the solid-phase synthesis, the peptide was cleaved by treatment of the resin with 10 mL of a mixture comprised of TIPS (1%), DODT (2.5%), water (2.5%) and TFA (94%) for 2.5 h. The resin was filtered away and the filtrate diluted with 8 volumes of ice-cooled 1 : 1 ether/hexane to precipitate the crude peptide. After centrifugation at 4000 rpm for 3 min the supernatant was discarded and the peptide pellet resuspended in cold ether, centrifuged and the ether again discarded. The peptide was allowed to air-dry before being dissolved in 70% aqueous MeCN containing 0.1% tfa and lyophilized.
A-chain 4: Intrachain CyS3-Cyss disulfide bond formation
The crude A-chain[Cys4(Acm), Cys6(tBu)] with pentalysine tag (900 mg, 233 //mol) was dissolved in deionised water (900 mL) and dipyridyldisulfide (DPDS, 62 mg, 280 /mol) dissolved in methanol (60 mL) added in one portion. The solution was agitated gently for 1.5 hours at room temperature, after which time HPLC analysis showed the reaction was complete. After acidification with TFA (ImL), the resulting solution was divided into four equal volumes of ca. 250 mL and each of these aliquots (containing approximately 225 mg of crude peptide) was concentrated by passage through a Phenomenex Gemini CI 8 (10 x 250mm) column equilibrated in 1% aqueous MeCN (O. P/oTFA) using the C-line of a semiprep HPLC, then switching to 55% aqueous MeCN (0.1%TFA) to strip the bound peptide from the column in a relatively small volume. This process was repeated for the remaining aliquots and the concentrates then pooled and lyophilised to give 800 mg of crude material. This was purified as follows:
A solution of 400 mg of this crude material in aqueous 0.1% formic acid (9 mL) was injected on to a Phenomenex Gemini C18, 1 ΙθΑ, 5μ (10 x 250mm) column equilibrated in 1 % aqueous MeCN containing 0.1 %TF A.
- Eluents for chromatography: A, water/0.1 %TFA; eluent B, MeCN/0.1 %TFA.
Gradient: 1%B to 15%B over 15 minutes then 15%B to 31%B over 150 minutes at 5mL/min.
After 27 minutes, 2.5 mL fractions were collected and analysed, with the desired product eluting in fractions 33-65.
Fractions of suitable purity were pooled and lyophilised.
The process was repeated with the remaining 400 mg the crude peptide to give lOOmg of lyophilised material of >92% purity (Figure 6).
A-chain 6: Activation of the A-chain
Solid A-chain (Cys4(Acm), Cys6(tBu)) (99.5 mg, 25.8 /mol) and dipyridinedisulfide (45.5 mg, 207 //mol) were combined and anisole (380 //L) added. The mixture was then dissolved in trifluoroacetic acid (3.4 mL) and the resulting clear solution cooled with vigorous stirring in an ice-water bath. A freshly prepared solution of 4: 1 v/v trifluoroacetic acid/trif ic acid (1.9 mL) was added quickly in one portion and the stirring continued for 3 minutes exactly, during which time a faint pink colour developed. Chilled hexane/ether (45mL, 1 : 1 v/v) was added and, after mixing and centrifugation, the precipitated pellet washed with hexane/ether. The white solid was allowed to air-dry then dissolved in 1 : 1 MeCN/water (0.1% tfa) and lyophilised. The material was dissolved in water (0.1% tfa) (10 mL) and a series of 1500 /L aliquots purified using a Phenomenex Jupiter C4 column (10//, 30θΑ, 10x250mm) eluting with a suitable gradient* (A: water/0.1% tfa; B: MeCN/0.1% tfa). The fractions containing the desired material were pooled and lyophilised, affording 47 mg (46%>) desired peptide.
*0-l min 5%B - 5%B (5 mL/min); 1-6 min 5%B - 15%B; 6-21 min 15%B - 40%B; 21-21.1 min 40%B - 75%B, 21.1 -22 min 75%B - 75%B; 22-24 min 75%B - 5%B; 24-29 min 5%B - 5%B.
B-chain 5 (SAcm, SH)
The B-chain was synthesised using Boc-SPPS conditions, using a PAM linker. The side chains of the trifunctional amino acid residues used were protected as follows: Boc-Asn: xanthyl; Arg: tosyl; Ser and Thr: benzyl (ether); Tyr: 2-bromobenzyl (ether); Asp and Glu: cyclohexyl (ester); Cys: 4-methylbenzyl (thioether) and acetamidomethyl (Acm). (i) Aminomethyl polystyrene resin of loading 1 mmol/g of (200 mg, 0.2 mmol) was swollen in DMF and drained. Solid Boc- Asn-PAM-OH (Polypeptide Group; 114 mg, 0.3 mmol) and HCTU (122 mg, 0.295 mmol) were combined and dissolved in DMF (1.5 mL). DIPEA (105 //L, 0.6 mmol) was then added and the resulting solution transferred to the moist (DMF) resin, which was agitated for 80 min then drained and washed (DMF). A negative Kaiser test indicated complete coupling.
(ii) The resin was washed with DCM (10 mL) then treated with neat trifluoroacetic acid (10-12 mL) for 2 minutes. The resin was drained and washed sequentially with DCM (10 mL) and DMF (3x10 mL). In a separate vessel the Boc-amino acid (1.0 mmol) was dissolved in a solution of HCTU (2.4 mL of a 0.406 M solution in DMF, 0.97 mmol) and DIPEA (418 L, 2.4 mmol) then added and the solution transferred to the resin, which was agitated for 20 min, drained and washed with DMF (10 mL). The process was repeated with the requisite amino acids to build up the peptide chain. On completion of the synthesis, the final Boc group was removed and the peptide cleaved from half the resin (0.1 mmol) in 10 mL HF at 0°C (0.5 mL /?-cresol as scavenger) for 1 hour then precipited using chilled ether. The crude peptide was dissolved in 1 : 1 v/v MeCN/water (0.1% formic acid) and lyophilised.
(iii) Crude B-chain (300 mg) was dissolved in 25 mL of 10% MeCN in water (0.1% formic acid). A series of 1300 /L aliquots were purified using a Phenomenex Gemini C18 column (5μ, 1 ΙθΑ, 10x250mm) eluting with a suitable gradient* (A: water/0.1% tfa; B: MeCN/0.1% tfa). The fractions containing the desired material were pooled and lyophilised.
*0-l min 5%B - 5%B (5 mL/min); 1-6 min 5%B - 25%B; 6-21 min 25%B - 40%B; 21-21.1 min 40%B - 75%B, 21.1-22 min 75%B - 75%B; 22-24 min 75%B - 5%B; 24-29 min 5%B - 5%B.
A-chain(-SS-, SAcm) - B-chain(-SAcm) 7: formation of the first inter-chain disulfide
A sample of activated A-chain 6 (45 mg, 11.5 //mol) was dissolved in 6M guanidine.HCl (9 mL), Tris.HCl buffer (2.0 mL of a 1 M aqueous solution, pH 8.1) added and the stirred solution cooled in an ice-water bath. A solution of the B-chain 5 (50 mg, 12.5 mol) in 6 M
guanidine.HCl (8 mL) was also cooled in ice-water and then added in a dropwise manner to the solution of A-chain over a period of 15 min. Stirring was then continued for a further 30 min after which time a sample was analysed by HPLC. This showed the reaction was complete, as evidenced by disappearance of the A-chain component. The solution was acidified with neat trifluoroacetic acid (200 //L) and 2 mL aliquots of the resulting solution (containing about 10 mg peptide) were purified using a Phenomenex Gemini C18 column (5//, 110 A, 10x250mm) eluting with a suitable gradient (A: water/0.1% tfa; B: MeCN/0.1% tfa). The pooled product-containing fractions were then lyophilised to give 64 mg product (71%).
*0-l min 5%B - 5%B (5 mL/min); 1-6 min 5%B - 25%B; 6-21 min 25%B - 40%B; 21-21.1 min 40%B - 75%B, 21.1-22 min 75%B - 75%B; 22-24 min 75%B - 5%B; 24-29 min 5%B - 5%B.
A-chain(-SS-) - B-chain 8: formation of the second inter-chain disulfide
A sample of A(SS, SAcm) - B(SAcm) 7 ( 5.1 mg, 0.66 /mol) was dissolved in chilled
(refrigerated ) 80% aqueous acetic acid (5 mL), cooled in an ice-water bath and iodine (100 //L of 0.5M in MeOH) added in one portion. After mixing, the homogeneous, brown solution was incubated on ice for 50 min with gentle agitation every 5 minutes or so. Aqueous 0.5M ascorbic acid (100 //mol) was then added, causing the colour to fade to pale-brown, and the solution diluted with water (20 mL), giving a completely colourless mixture. Neat trifluoroacetic acid (250 //L) was added and, following centrifugation, the solution was passed through a
Phenomenex Gemini C18 (5//, 1 ΙθΑ, 10x250mm) column using the C-line of a preparative RP- HPLC instrument. The peptide was then eluted using a suitable gradient to afford the desired material (3.75 mg, 75%), which was then lyophilised. *0-l min 5%B - 5%B (5 mL/min); 1-6 min 5%B - 25%B; 6-21 min 25%B - 40%B; 21-21.1 min 40%B - 75%B, 21.1-22 min 75%B - 75%B; 22-24 min 75%B - 5%B; 24-29 min 5%B - 5%B.
Vesiculin 9: hydrolysis of the pentalysine tag
A sample of A-B(Lys5) 8 (10 mg) was dissolved in water (1.6 mL) and cooled in an ice-water bath. An ice-cold solution of sodium hydroxide (1.6 mL of 0.2M in water) was added and the resulting solution agitated at 0°C for 5 minutes. Neat trifluoroacetic acid (25 L) was added (ensuring pH<2 for the solution) and the resulting crude Vesiculin 9 purified using a
Phenomenex Gemini C18 column (5μ, 1 ΙθΑ, 10x250mm) eluting with a suitable gradient* (A: water/0.1% tfa; B: MeCN/0.1% tfa) and lyophilised to give 8 mg product (80%).
*0-l min 5%B - 5%B (5 mL/min); 1-6 min 5%B - 25%B; 6-21 min 25%B - 40%B; 21-21.1 min 40%B - 75%B, 21.1-22 min 75%B - 75%B; 22-24 min 75%B - 5%B; 24-29 min 5%B - 5%B. EXAMPLE 2 - SYNTHESIS OF SYNTHETIC HUMAN VESICULIN
This example describes the synthesis of synthetic human vesiculin.
The process outlined above in Example 1 was repeated for human vesiculin in an identical manner, with requisite alterations in the addition of amino acids in accordance with the differing B chain sequence. The low-resolution mass spectrum of the desired product, synthetic human vesiculin, is shown in Figure 9.
Conclusion
This experiment shows that the preparative method described herein for murine vesiculin is amenable to the production of vesiculins of other species, in this case human vesiculin.
EXAMPLE 3 - CHARACTERISATION OF SYNTHETIC VESICULIN
This Example relates to the characterization of blood glucose lowering effects of synthetic murine vesiculin in mice.
Materials and Methods
In vivo studies:
FVBN mice (Proc Natl Acad Sci U S A. 1991 March 15; 88(6): 2065-2069) were obtained from the Animal Resource Centre (Canning Vale, WA, Australia). Synthetic vesiculin was prepared as described above in Example 1. Actrapid Insulin was obtained from Novo Nordisk Limited.
Doses of synthetic vesiculin (50, 62, 75, 100, 150 and 200pmol/g in 0.1% n-Dodecyl β- D-Maltopyranoside DDM) or insulin (0.37, 0.55, 0.74, 1.47, 1.84, 2.21pmol/g (0.1, 0.15, 0.2, 0.4, 0.5, 0.6mU) in saline) were given LP. to FVBN male mice approx. 85 days old, caged separately and fasted for 6 hours prior to treatment. Dose range was based on pilot data as was the concentration of DDM to use as an excipient (pharmaceutical additive) for the synthetic peptide, as seen in Figure 10. Blood glucose measurements were taken at 5, 15, 30, 60, 90, and 120 minutes post treatment. Baselines (fasting blood glucose) were taken 5 minutes prior to injection. Maximal blood glucose lowering was achieved at 60 minutes post LP. drug delivery. Results
Administration of synthetic vesiculin results in significant blood glucose lowering, as shown in Figures 10 and 11. Figure 10 shows that there is no statistically significant difference in the lowering of blood glucose using synthetic vesiculin compared to insulin. As can be seen in Figure 11, synthetic vesiculin of the invention lowers blood glucose in a similar manner to insulin, with approximately 100 times higher dose for the same blood glucose response.
Conclusion
This example clearly demonstrates the biological efficacy of synthetic vesiculin polypeptides of the invention, and the capacity of the synthetic methods described herein to produce safe synthetic polypeptides having full insulin agonist activity.
From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for the purpose of illustration, various modifications may be made without deviating from the spirit and scope of the invention. The foregoing description is intended to illustrate and not limit the scope of the invention. Accordingly, the present invention is not limited except as by the appended claims.
All patents, patent applications, publications, scientific articles, web sites, and other documents and materials referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the inventions pertain, and each such referenced document and material is hereby incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. The written description portion of this patent includes all claims. Furthermore, all claims, including all original claims as well as all claims from any and all priority documents, are hereby incorporated by reference in their entirety into the written description portion of the specification, and Applicants reserve the right to physically incorporate into the written description or any other portion of the application, any and all such claims. Thus, for example, under no circumstances may the patent be interpreted as allegedly not providing a written description for a claim on the assertion that the precise wording of the claim is not set forth in haec verba in written description portion of the patent. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such patents, applications, publications, scientific articles, web sites, electronically available information, and other referenced materials or documents.
The specific methods and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the inventions. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the inventions as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the inventions disclosed herein without departing from the scope and spirit of the inventions. The inventions illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. Thus, for example, in each instance herein, in embodiments or examples of the present inventions, any of the terms "comprising", "consisting essentially of, and "consisting of may be replaced with either of the other two terms in the specification. Also, the terms "comprising", "including", containing", etc. are to be read expansively and without limitation. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and that they are not necessarily restricted to the orders of steps indicated herein or in the claims. It is also that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host cell" includes a plurality (for example, a culture or population) of such host cells, and so forth. Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically or otherwise expressly disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is expressly and specifically, without qualification or reservation, adopted in a responsive writing by Applicants. The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. Thus each is to be read as including the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group, which also form a part of the written description. It is also to be understood that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise, the term "X and/or Y" means "X" or "Y" or both "X" and "Y", and the letter "s" following a noun designates both the plural and singular forms of that noun.
The claims will be interpreted according to law. However, and notwithstanding the alleged or perceived ease or difficulty of interpreting any claim or portion thereof, under no circumstances may any adjustment or amendment of a claim or any portion thereof during prosecution of the application or applications leading to this patent be interpreted as having forfeited any right to any and all equivalents thereof that do not form a part of the prior art.
All of the features disclosed in this specification may be combined in any combination. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Thus, from the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for the purpose of illustration, various modifications may be made without deviating from the spirit and scope of the invention.

Claims

A method of synthesizing a polypeptide comprising a first peptide chain and a second peptide chain, the method comprising providing a first peptide chain and a second peptide chain, forming under conducive conditions one or more interchain disulfide bonds between the first peptide chain and the second peptide chain, and recovering the polypeptide from the reaction medium, wherein the first peptide chain comprises an amino acid sequence corresponding to
A chain : Gly He Val Glu Glu Cys3 Cys4 Phe Arg Ser Cys5 Asp Leu Ri R2 Leu Glu R3
Tyr Cys6 Ala R4 R5 Rg R7 R8 R9 (SEQ ID NO: 1)
and wherein the second peptide chain comprises an amino acid sequence corresponding to
B chain : Rio Rn Ri2 Ri Ri4 Glu Thr Leu Cysi Gly Gly Glu Leu Val Asp Ri5 Leu Gin
Phe Ri6 Cys2 Rn Rig Arg Gly Phe Tyr Phe Rl9 R20 R21 R22 R23 R24 R25 R26 27 28 29 (SEQ ID NO:2)
wherein Ri is Ala, Asn or Leu; R2 is Leu or He; R3 is Thr or Gin; R4 is Thr, Ala, Lys, or Val; R5 is Pro or Ser; R6 is Ala, Val, or Pro; R7 is Lys or Glu, R8 is Ser or Ala, R is Glu or Ala, Rio is absent or is Ala, Glu, or Asp, Rn is Tyr, Ala, or Val, R12 is Arg, Gly, or Ala, Ri3 is Pro, Thr, Ser, or Leu, Ri4 is Ser, Gly, Ala, or Glu, R15 is Thr or Ala, Ri6 is Val or He, Rn is Gly, Ser, Glu, or Ala, Ri8 is Asp or Glu, R1 is Ser or Val, R20 is Arg, Leu, or Ser, R21 is Pro or Lys, R22 is Ala, Ser, Gly, Val, or Thr, R23 is Ser, Gly, or Val, R24 is Arg, Pro or Gly, R25 is Ala, Arg, Val, He, Leu, Asn, Ser, or Gly, R26 is Ser, Asn, or Arg, R2-7 is absent or is Arg, Ser, Asn, R28 is absent or is Val, R2 is absent or is Ser, wherein Ri- R2 each individually includes a conservative amino acid variant for the recited amino acids. The method according to claim 1 wherein the first peptide chain and the second peptide chain are each provided separately.
The method according to claim 1 wherein the first peptide chain and the second peptide chain are provided together in the form of a polypeptide comprising the first peptide chain and the second peptide chain, wherein the first peptide chain is bound to the second peptide chain by one interchain disulfide bond.
The method according to claim 1 wherein one or more of the cysteine residues present in either the first peptide chain, the second peptide chain, or in both the first peptide chain and the second peptide chain are protected with one or more suitable protecting groups.
5. The method according to claim 1 wherein one or more of the cysteine residues present in both the first peptide chain and the second peptide chain are protected with one or more suitable protecting groups.
6. The method according to claim 1 wherein Cys4 and Cysi are each independently protected with a suitable protecting group or Cys2 and Cys6 are each independently protected with a suitable protecting group.
7. The method according to claim 1 wherein Cys4 and Cysi are each independently protected with a suitable protecting group.
8. The method according to claim 1 wherein Cys2 and Cys6 are each independently protected with a suitable protecting group.
9. The method according to claim 1 wherein Cys3 or Cyss are each independently protected with a suitable protecting group or bound together in an intrachain disulfide bond.
10. The method according to claim 1 wherein Cys3 or Cys5 are each independently protected with a suitable protecting group or bound together in an intrachain disulfide bond; and Cys4 and Cysi are each independently protected with a suitable protecting group or bound together in an interchain disulfide bond; or Cys2 and Cys6 are each independently protected with a suitable protecting group or bound together in an interchain disulfide bond.
11. The method according to claim 1 wherein Cys3 or Cys5 are each independently protected with a suitable protecting group or bound together in an intrachain disulfide bond; and Cys4 and Cysi are each independently protected with a suitable protecting group or Cys2 and Cys6 are each independently protected with a suitable protecting group.
12. The method according to claim 1 wherein Cys3 or Cys5 are each bound together in an intrachain disulfide bond; and Cys4 and Cysi are each independently protected with a suitable protecting group or Cys2 and Cys6 are each independently protected with a suitable protecting group.
13. The method according to claim 1 wherein Cys3 or Cys5 are each bound together in an intrachain disulfide bond and Cys4 and Cysi are each independently protected with a suitable protecting group.
14. The method according to claim 1 wherein Cys3 or Cys5 are each bound together in an intrachain disulfide bond and Cys2 and Cys6 are bound together in an interchain disulfide bond.
15. The method according to claim 1 wherein one or more of the cysteine residues in the first peptide chain, the second peptide chain, or in both the first peptide chain and the second peptide chain are protected with one or more suitable protecting groups removable under the same conditions.
16. The method according to claim 1 wherein the suitable protecting groups removable under the same reaction conditions are identical.
17. The method according to claim 1 wherein two or more of the cysteine residues present in either the first peptide chain, the second peptide chain, or in both the first peptide chain and the second peptide chain are with a suitable protecting group.
18. The method according to claim 1 wherein the two or more cysteine residues are differentially protected with two or more suitable protecting groups such that one or more protecting groups may be selectively removed in the presence of the other protecting groups (i.e. without removing the other protecting groups) by the judicious choice of reaction conditions.
19. The method according to claim 4 wherein Cys4 is differentially protected with respect to Cys3 and Cys5.
20. The method according to claim 4 wherein Cys6 is differentially protected with respect to Cys3 and Cys5.
21. The method according to claim 4 wherein Cysi and Cys4 are differentially protected with respect to Cys3 and Cys5.
22. The method according to claim 4 wherein Cys2 and Cys6 are differentially protected with respect to Cys3 and Cys5.
23. The method according to claim 4 wherein Cysi is differentially protected with respect to Cys4 or Cys2 is differentially protected with respect to Cys6.
24. The method according to claim 4 wherein the protecting groups for the cysteine residues are selected from the group consisting of trityl, acetamidomethyl, tert-butyl, tert-butylthio, xanthyl, picolyl, and 4-methoxytrityl4-methylbenzyl.
25. The method according to claim 4 wherein the protecting groups are selected from the group consisting of trityl, acetamidomethyl, tert-butyl, tert-butylthio, and 4-methoxytrityl4- methylbenzyl.
26. The method according to claim 4 wherein the protecting groups are selected from the group consisting of trityl, acetamidomethyl, and tert-butyl.
27. The method according to claim 4 wherein Cysi and Cys4 are each protected by an acetamidomethyl group.
28. The method according to claim 4 wherein Cys6 is protected with a tert-butyl group.
29. The method according to claim 4 wherein Cys3 and Cys5 are each protected by a trityl.
30. The method according to claim 1 wherein one or more of the amino acids other than cysteine in the first peptide chain, the second peptide chain, or in both the first peptide chain and the second peptide chain are protected with one or more suitable protecting groups.
31. The method according to claim 30 wherein the one or more suitable protecting groups can be selectively removed without removing one or more cysteine protecting groups.
32. The method according to claim 30 or 31 wherein at least one cysteine residue in the first peptide chain and at least one cysteine residue in the second peptide chain must be available to form an interchain disulfide bond.
33. The method according to claim 30 wherein one cysteine residue in the first peptide chain and one cysteine residue in the second peptide chain are available to form an interchain disulfide bond.
34. The method according to claim 33 wherein the other cysteine residues present in the chains are protected with one or more suitable protecting groups or bound in one or more intrachain or interchain disulfide bonds.
35. The method according to claim 33 or 34 wherein the other cysteine residues present in the chains protected with one or more protecting groups are differentially protected with two or more protecting groups.
36. The method according to claim 1 wherein Cys4 and Cysi are each independently protected with a suitable protecting group or are bound together in an interchain disulfide bond and Cys2 and Cys6 are available to form an interchain disulfide bond; or Cys2 and Cys6 are each independently protected with a suitable protecting group or bound in an interchain disulfide bond and Cys4 and Cysi are available to form an interchain disulfide bond.
37. The method according to claim 1 wherein Cys4 and Cysi are each independently protected with a suitable protecting group and Cys2 and Cys6 are available to form an interchain disulfide bond; or Cys2 and Cys6 are each independently protected with a suitable protecting group and Cys4 and Cysi are available to form an interchain disulfide bond.
38. The method according to claim 1 wherein the first peptide chain, the second peptide chain or both the first peptide chain and the second peptide chain additionally comprises one or more solubilising groups.
39. The method according to claim 38 wherein the first peptide chain comprises one or more solubilising groups.
40. The method according to claim 38 wherein the solubilising group enhances the solubility of the peptide chain in the reaction medium.
41. The method according to claim 38 the solubilising group prevents or inhibits intrachain association.
42. The method according to claim 38 wherein, the one or more solubilising groups are linked to the C-termini of the peptide chain, optionally via a suitable linker.
43. The method according to claim 38 wherein, the solubilising group is a polycationic amino acid sequence.
44. The method according to claim 38 wherein, the cationic amino acids are arginine or lysine residues.
45. The method according to claim 38 wherein, the sequence comprises from 2 to 20, 2 to 15, 2 to 10, 3 to 7, or 3 to 5 amino acids.
46. The method according to claim 38 wherein, the solubilising group is a tri-, terra-, or penta- lysine or arginine tag.
47. The method according to claim 38 wherein the solubilising group is a pentalysine tag.
48. The method according to claim 38 wherein the pentalysine tag is linked to the C-termini of the peptide chain via a 4-hydroxymethyl benzoic acid (HMBA) linker.
49. The method according to claim 1 wherein, the first peptide chain, the second peptide chain, or both the first peptide chain and the second peptide chain are bound to a solid phase support, optionally via a suitable linker.
50. The method according to claim 49 wherein the linker is selected from the group comprising the Rink amide linker, phenylacetamido (PAM) linker, Sheppard's linker, and
Wang ester linker.
51. The method according to claim 49 wherein the peptide chain is bound to the solid phase support via a solubilising group linked to the C-termini of the peptide chain.
52. The method according to claim 49 wherein the solubilising group is bound to the C-termini of the peptide chain via a suitable linker.
53. The method according to claim 49 wherein the solubilising group is bound to the solid phase support via a suitable linker.
54. The method according to claim 1 wherein the method comprises first forming an intrachain disulfide bond bytween Cys3 and Cys5, then forming an interchain disulfide bond between Cys2 and Cys6, and then forming an interchain disulfide bond between Cysi and Cys4.
55. The method according to claim 1 wherein the one or more interchain disulfide bonds are formed under oxidative conditions.
56. The method according to claim 55 wherein the oxidant is dipyridyldisulfide or iodine.
57. The method according to claim 55 wherein the oxidant is dipyridyldisulfide.
58. The method according to claim 55 wherein the oxidant is iodine.
59. The method according to claim 55 wherein, the reaction medium is a liquid reaction medium.
60. The method according to claim 1 wherein the first peptide chain is synthesized using solid phase peptide synthesis as described herein in the examples.
61. The method according to claim 1 wherein the second peptide chain is synthesized using solid phase peptide synthesis.
62. The method according to claim 1 wherein the second peptide is synthesized using Boc solid phase peptide synthesis.
63. The method according to claim 1 wherein the second peptide chain is synthesized using solid phase peptide synthesis as described herein in the examples.
64. A method of synthesizing a polypeptide comprising an amino acid sequence corresponding to
A chain : Gly He Val Glu Glu Cys3 Cys4 Phe Arg Ser Cys5 Asp Leu Ri R2 Leu Glu R3
Tyr Cys6 Ala R4 R5 Rg R7 R8 R9 (SEQ ID NO: 1)
wherein Ri is Ala, Asn or Leu; R2 is Leu or He; R3 is Thr or Gin; R4 is Thr, Ala, Lys, or Val; R5 is Pro or Ser; R6 is Ala, Val, or Pro; R7 is Lys or Glu, R8 is Ser or Ala, and R9 is Glu or Ala, the method essentially as described herein in the examples.
65. A method of synthesizing a polypeptide comprising an amino acid sequence corresponding to
B chain : Rio Rn Ri2 Ri3 Ri4 Glu Thr Leu Cysi Gly Gly Glu Leu Val Asp R15 Leu Gin
Phe Ri6 Cys2 Rn Ris Arg Gly Phe Tyr Phe Ri9 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 (SEQ ID NO:2)
wherein Ri0 is absent or is Ala, Glu, or Asp, Rn is Tyr, Ala, or Val, Ri2 is Arg, Gly, or Ala, Ri3 is Pro, Thr, Ser, or Leu, Ri4 is Ser, Gly, Ala, or Glu, R15 is Thr or Ala, Ri6 is Val or He, Rn is Gly, Ser, Glu, or Ala, Ri8 is Asp or Glu, R19 is Ser or Val, R20 is Arg, Leu, or Ser, R2i is Pro or Lys, R22 is Ala, Ser, Gly, Val, or Thr, R23 is Ser, Gly, or Val, R24 is Arg, Pro or Gly, R25 is Ala, Arg, Val, He, Leu, Asn, Ser, or Gly, R26 is Ser, Asn, or Arg, R27 is absent or is Arg, Ser, Asn, R28 is absent or is Val, R2g is absent or is Ser, the method essentially as described herein in the examples.
66. A synthetic polypeptide wherein the synthetic polypeptide comprises an amino acid sequence corresponding to
A chain : Gly He Val Glu Glu Cys3 Cys4 Phe Arg Ser Cys5 Asp Leu Ri R2 Leu Glu R3
Tyr Cys6 Ala R4 R5 Rg R7 R8 R9 (SEQ ID NO: 1); wherein Ri is Ala, Asn or Leu; R2 is Leu or He; R3 is Thr or Gin; R4 is Thr, Ala, Lys, or Val; R5 is Pro or Ser; R6 is Ala, Val, or Pro; R7 is Lys or Glu, R8 is Ser or Ala, and R9 is Glu or Ala;
or an amino acid sequence corresponding to
B chain : Rio Rn Ri2 R13 Ri4 Glu Thr Leu Cysi Gly Gly Glu Leu Val Asp R15 Leu Gin
Phe Ri6 Cys2 Rn Rig Arg Gly Phe Tyr Phe Rl9 R20 R21 R22 R23 R24 R25 R26 27 28 29 (SEQ ID NO:2);
wherein R10 is absent or is Ala, Glu, or Asp, Rn is Tyr, Ala, or Val, Ri2 is Arg, Gly, or Ala, Rn is Pro, Thr, Ser, or Leu, Ri4 is Ser, Gly, Ala, or Glu, R15 is Thr or Ala, Ri6 is Val or He, Rn is Gly, Ser, Glu, or Ala, Ri8 is Asp or Glu, R1 is Ser or Val, R20 is Arg, Leu, or Ser, R2i is Pro or Lys, R22 is Ala, Ser, Gly, Val, or Thr, R23 is Ser, Gly, or Val, R24 is Arg, Pro or Gly, R25 is Ala, Arg, Val, He, Leu, Asn, Ser, or Gly, R26 is Ser, Asn, or Arg, R27 is absent or is Arg, Ser, Asn, R28 is absent or is Val, R2 is absent or is Ser, and wherein Ri- R29 each individually includes a conservative amino acid variant for the recited amino acids.
67. The synthetic polypeptide of claim 66 wherein the polypeptide is pure or purified, or substantially pure.
68. The polypeptide according to claim 66 wherein the synthetic polypeptide has insulin agonist activity.
69. The polypeptide according to claim 66 wherein the insulin agonist activity is a capability of binding to a receptor to which insulin binds, or eliciting a response mediated by a receptor to which insulin binds.
70. The polypeptide according to claim 66 wherein the synthetic polypeptide binds a receptor to which insulin binds with at least about 10%, at least about 15%, at least about 20%, or at least about 25% the affinity as does insulin.
71. The polypeptide according to claim 66 wherein the receptor to which insulin binds is the insulin receptor.
72. The polypeptide according to claim 66 wherein the synthetic polypeptide binds a receptor to which insulin binds with a binding affinity of at least 10 7, 108, 109, or 1010 M -"1.
73. The polypeptide according to claim 66 wherein the synthetic polypeptide has an EC50 for effecting a response mediated by the insulin receptor less than about two hundred-fold that of insulin.
74. One or more synthetic vesiculin polypeptide intermediates, wherein the one or more synthetic vesiculin polypeptide intermediates comprises a resin-bound polypeptide comprising amino acid sequence corresponding to
A chain : Gly He Val Glu Glu Cys3 Cys4 Phe Arg Ser Cys5 Asp Leu Ri R2 Leu Glu R3
Tyr Cys6 Ala R4 R5 Rg R7 R8 R9 (SEQ ID NO: 1)
wherein Ri is Ala, Asn or Leu; R2 is Leu or He; R3 is Thr or Gin; R4 is Thr, Ala, Lys, or Val; R5 is Pro or Ser; R6 is Ala, Val, or Pro; R7 is Lys or Glu, R8 is Ser or Ala, R9 is Glu or Ala, and wherein R1-R9 include conservative amino acid variants of the recited amino acids.
75. The synthetic vesiculin polypeptide according to claim 74 comprising or consisting of one of the following sequences:
LETYCATPAKSE (SEQ ID NO:7);
LLETYCATPAKSE (SEQ ID NO: 8);
ALLETYCATPAKSE (SEQ ID NO:9);
LALLETYCATPAKSE (SEQ ID NO: 10);
DLALLETYCATPAKSE (SEQ ID NO: 11);
CDLALLETYCATPAKSE (SEQ ID NO: 12);
SCDLALLETYCATPAKSE (SEQ ID NO: 13);
RSCDLALLETYCATPAKSE (SEQ ID NO: 14);
FRSCDLALLETYCATPAKSE (SEQ ID NO: 15);
CFRSCDLALLETYCATPAKSE (SEQ ID NO: 16);
CCFRSCDLALLETYCATPAKSE (SEQ ID NO: 17);
EC CFRS CDLALLETYCATPAKSE (SEQ ID NO: 18);
EECCFRSCDLALLETYCATPAKSE (SEQ ID NO: 19);
VEECCFRSCDLALLETYCATPAKSE (SEQ ID NO:20);
rVEECCFRSCDLALLETYCATPAKSE (SEQ ID NO:21).
76. The synthetic vesiculin intermediates of claim 74 or 75 wherein the N-terminal amino acid is Na-protected by a protecting group.
77. The intermediate according to claim 76 wherein the N-terminal amino acid is Na-protected with Fmoc.
78. The intermediate according to claim 74 wherein comprising or consisting of one of the following sequences:
(pro)-LETYCATPAKSE (SEQ ID NO:7);
(pro)-LLETYCATPAKSE (SEQ ID NO: 8); (pro)-ALLETYCATPAKSE (SEQ ID NO:9);
(pro)-LALLETYCATPAKSE (SEQ ID NO: 10);
(pro)-DLALLETYCATPAKSE (SEQ ID NO: l 1);
(pro)-CDLALLETYCATPAKSE (SEQ ID NO: 12);
(pro)-SCDLALLETYCATPAKSE (SEQ ID NO: 13);
(pro)-RSCDLALLETYCATPAKSE (SEQ ID NO: 14);
(pro)-FRSCDLALLETYCATPAKSE (SEQ ID NO: 15);
(pro)-CFRSCDLALLETYCATPAKSE (SEQ ID NO: 16);
(pro)-CCFRSCDLALLETYCATPAKSE (SEQ ID NO: 17);
(pro)-ECCFRSCDLALLETYCATPAKSE (SEQ ID NO: 18);
(pro)-EECCFRSCDLALLETYCATPAKSE (SEQ ID NO: 19);
(pro)-VEECCFRSCDLALLETYCATPAKSE (SEQ ID NO:20);
(pro)-IVEECCFRSCDLALLETYCATPAKSE (SEQ ID NO:21);
(pro)-GIVEECCFRSCDLALLETYCATPAKSE (SEQ ID NO:22);
wherein (pro)- is one or more protecting group, including a protecting group selected from the following: acetyl (Ac), amide, a 3 to 20 carbon alkyl group, Fmoc, 9- fluoreneacetyl group, 1-fluorenecarboxylic group, 9-fluorenecarboxylic group, 9- fluorenone-l-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4- methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4- methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2- pyridinesulphenyl (Npys), l-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), 2,6- dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-C1--Z), 2- bromobenzyloxycarbonyl (2-Br~Z), Benzyloxymethyl (Bom), tert-butyloxycarbonyl (Boc), cyclohexyloxy (cHxO),t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), and Trifluoroacetyl (TFA).
79. The intermediate of claim 78 wherein the (pro)- is Fmoc.
80. The intermediate of claim 74 wherein the intermediate comprises one or more solubilising groups bound to the amino acid sequence of the polypeptide chain, optionally via a suitable linker.
81. One or more synthetic vesiculin polypeptide intermediates, wherein the one or more synthetic vesiculin polypeptide intermediates comprises a resin-bound polypeptide comprising or consisting of amino acid sequence corresponding to B chain : Rio Rn R12 R13 Ri4 Glu Thr Leu Cysi Gly Gly Glu Leu Val Asp R15 Leu Gin Phe Ri6 Cys2 Rn Ris Arg Gly Phe Tyr Phe Ri9 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 (SEQ ID NO:2)
wherein Ri0 is absent or is Ala, Glu, or Asp, Rn is Tyr, Ala, or Val, Ri2 is Arg, Gly, or Ala, Rn is Pro, Thr, Ser, or Leu, R14 is Ser, Gly, Ala, or Glu, R15 is Thr or Ala, Ri6 is Val or He, Rn is Gly, Ser, Glu, or Ala, Rig is Asp or Glu, R19 is Ser or Val, R20 is Arg, Leu, or Ser, R2i is Pro or Lys, R22 is Ala, Ser, Gly, Val, or Thr, R23 is Ser, Gly, or Val, R24 is Arg, Pro or Gly, R25 is Ala, Arg, Val, He, Leu, Asn, Ser, or Gly, R26 is Ser, Asn, or Arg, R27 is absent or is Arg, Ser, Asn, R28 is absent or is Val, R2 is absent or is Ser.
The intermediate according to claim 81 comprising or consisting of one of the following sequences:
GFYFSRPASRVS (SEQ ID NO:23);
RGFYFSRPASRVS (SEQ ID NO:24);
DRGFYFSRPASRVS (SEQ ID NO:25);
GDRGFYFSRPASRVS (SEQ ID NO:26);
CGDRGFYFSRPASRVS (SEQ ID NO:27);
VCGDRGFYFSRPASRVS (SEQ ID NO:28);
FVCGDRGFYFSRPASRVS (SEQ ID NO:29);
QFVCGDRGFYFSRPASRVS (SEQ ID NO:30);
LQFVCGDRGFYFSRPASRVS (SEQ ID NO:31);
TLQFVCGDRGFYFSRPASRVS (SEQ ID NO:32);
DTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:33);
VDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:34);
LVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:35);
ELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:36);
GELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:37);
GGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:38);
CGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:39);
LCGGEL VDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:40);
TLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:41);
ETLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:42);
SETLCGGEL VDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:43);
PSETLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:44);
RPSETLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:45); YRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:46).
83. The synthetic vesiculin intermediates of claim 82 wherein the N-terminal amino acid is Na-protected by a protecting group.
84. The intermediate according to claim 83 wherein the N-terminal amino acid is Na-protected with Boc.
85. The intermediates of claim 82 comprising or consisting of one of the following sequences (pro)-GFYFSRPASRVS (SEQ ID NO:23);
(pro)-RGFYFSRPASRVS (SEQ ID NO:24);
(pro)-DRGFYFSRPASRVS (SEQ ID NO:25);
(pro)-GDRGFYFSRPASRVS (SEQ ID NO:26);
(pro)-CGDRGFYFSRPASRVS (SEQ ID NO:27);
(pro)-VCGDRGFYFSRPASRVS (SEQ ID NO:28);
(pro)-FVCGDRGFYFSRPASRVS (SEQ ID NO:29);
(pro)-QFVCGDRGFYFSRPASRVS (SEQ ID NO:30);
(pro)-LQFVCGDRGFYFSRPASRVS (SEQ ID NO:31);
(pro)-TLQFVCGDRGFYFSRPASRVS (SEQ ID NO:32);
(pro)-DTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:33);
(pro)-VDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:34);
(pro)-LVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:35);
(pro)-ELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:36);
(pro)-GELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:37);
(pro)-GGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:38);
(pro)-CGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:39);
(pro)-LCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:40);
(pro)-TLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:41);
(pro)-ETLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:42);
(pro)-SETLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:43);
(pro)-PSETLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:44);
(pro)-RPSETLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:45);
(pro)-YRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:46);
(pro)-AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVS (SEQ ID NO:47);
wherein (pro)- is one or more protecting group, including a protecting group selected from the following: acetyl (Ac), amide, a 3 to 20 carbon alkyl group, Fmoc, 9- fluoreneacetyl group, 1-fluorenecarboxylic group, 9-fluorenecarboxylic group, 9- fluorenone-l-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4- methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4- methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2- pyridinesulphenyl (Npys), l-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), 2,6- dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-C1--Z), 2- bromobenzyloxycarbonyl (2-Br— Z), Benzyloxymethyl (Bom), tert-butyloxycarbonyl (Boc), cyclohexyloxy (cHxO),t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), and Trifluoroacetyl (TFA).
86. The intermediate according to claim 85 wherein of the B chain intermediates, the Na- position of the N-terminal amino acid is protected with a (pro)- group.
87. The intermediate of claim 85 wherein the (pro)- is Boc.
88. A method of determining whether a synthetic polypeptide, variant, or intermediate of claim 1 is useful as a therapeutic agent by determining whether the synthetic polypeptide has insulin agonist activity.
89. The method according to claim 88 wherein the method comprises contacting the synthetic polypeptide, variant, or intermediate and a receptor to which insulin binds, and determining a capability of binding to the receptor.
90. The method according to claim 88 wherein the method comprises contacting the synthetic polypeptide, variant, or intermediate and a receptor to which insulin binds, and determining a capability of eliciting a response mediated by the receptor.
91. The method according to claim 88 wherein a capability of eliciting a response mediated by the receptor is determined by a determination of the EC50 for effecting a response mediated by the insulin receptor.
92. The method according to claim 88 wherein the response is an effect on glucose incorporation into glycogen.
93. A method of modulating blood glucose levels in a subject, the method comprising administering an effective amount of one or more of a synthetic vesiculin according to claim 66, or a synthetic vesiculin variant, derivative, or synthetic intermediate, or a salt of any of them, to a subject in need thereof.
94. A method for treating a subject having or suspected of having or predisposed to, or at risk for any diseases, disorders and/or conditions characterized in whole or in part by hyperglycemia, insulin resistance, impaired glucose tolerance, and/or impaired fasting glucose, comprising administering a composition comprising a synthetic vesiculin according to claim 66, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, a synthetic vesiculin derivative and/or a synthetic vesiculin intermediate or a salt thereof.
95. A method for regulating glycemia in a subject having or suspected of having or predisposed to diseases, disorders and/or conditions characterized in whole or in part by hyperglycemia, insulin resistance, impaired glucose tolerance, and/or impaired fasting glucose, comprising administering a composition comprising a synthetic vesiculin according to claim 66, a synthetic vesiculin A chain, a synthetic vesiculin B chain, a synthetic vesiculin variant, a vesiculin derivative and/or a synthetic vesiculin synthetic or a salt thereof.
96. The use of one or more of a synthetic vesiculin according to claim 66, or a synthetic vesiculin variant, derivative, or synthetic intermediate, or a salt of any of them, in the preparation of a medicament for modulating blood glucose levels in a subject.
97. A method of modulating glucose incorporation into glycogen in a subject, comprising administering an effective amount of one or more of a synthetic vesiculin according to claim 66, or a synthetic vesiculin variant, derivative, or synthetic intermediate thereof, or a salt of any of them, to a subject in need thereof.
98. The use of an effective amount of one or more of a synthetic vesiculin according to claim 66, or a synthetic vesiculin variant, derivative, or synthetic intermediate, or a salt of any of them, in the manufacture, with or without other pharmaceutically acceptable materials of a dosage unit effective for use in a method of any one of claims 88-95 and 97.
99. A pharmaceutical composition which comprises a synthetic vesiculin according to claim 66, a synthetic vesiculin A chain, a synthetic vesiculin B chain, or a synthetic vesiculin, synthetic vesiculin A chain, or synthetic vesiculin B chain variant or derivative, or an synthetic intermediate thereof, or salts or derivatives of the above.
PCT/IB2013/051583 2012-02-28 2013-02-28 Synthetic polypeptides and uses thereof WO2013128391A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201261604352P 2012-02-28 2012-02-28
US61/604,352 2012-02-28

Publications (1)

Publication Number Publication Date
WO2013128391A1 true WO2013128391A1 (en) 2013-09-06

Family

ID=49081720

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2013/051583 WO2013128391A1 (en) 2012-02-28 2013-02-28 Synthetic polypeptides and uses thereof

Country Status (1)

Country Link
WO (1) WO2013128391A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106432472A (en) * 2016-10-24 2017-02-22 合肥国肽生物科技有限公司 Solid-phase synthesis method for insulin

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006112737A1 (en) * 2005-04-20 2006-10-26 Protemix Discovery Limited Vesiculins

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006112737A1 (en) * 2005-04-20 2006-10-26 Protemix Discovery Limited Vesiculins

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
AKAJI, K. ET AL.: "Total synthesis of human insulin by regioselective disulfide formation using the silyl chloride-sulfoxide method", J. AM. CHEM. SOC., vol. 115, no. 24, 1993, pages 11384 - 11392 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106432472A (en) * 2016-10-24 2017-02-22 合肥国肽生物科技有限公司 Solid-phase synthesis method for insulin
CN106432472B (en) * 2016-10-24 2020-01-03 合肥国肽生物科技有限公司 Solid-phase synthesis method of insulin

Similar Documents

Publication Publication Date Title
JP4519324B2 (en) Covalently cross-linked insulin dimer
US20060252916A1 (en) Modified glucagon-like peptide-1 analogs
CN102171244A (en) Analogues of glucose-dependent insulinotropic polypeptide
JP7513707B2 (en) GLP-1 agonist polypeptide compounds, their salts, methods of synthesis, and uses
CN104031140A (en) Truncated analogues of glucose-dependent insulinotropic polypeptide
Ji et al. Covalent structures of BmK AS and BmK AS-1, two novel bioactive polypeptides purified from Chinese scorpion Buthus martensi Karsch
HRP970416A2 (en) Method for the synthesis of analogs of parathyroid hormone and parathyroid hormone related peptide
TW201940505A (en) Modified lipidated Relaxin B chain peptides and their therapeutic use
US20140088024A1 (en) Vesiculins
KR101417872B1 (en) Analogues of insulin-like growth factor-1 (igf-1) having amino acid substitution at position 59
CZ158895A3 (en) Insulin analog, process of its preparation and pharmaceutical composition containing thereof
WO1996003437A1 (en) PTH OR PTHrP ANTAGONISTS
Vita et al. Synthesis of charybdotoxin and of two N‐terminal truncated analogues: Structural and functional characterisation
WO2013128391A1 (en) Synthetic polypeptides and uses thereof
EP3888667B1 (en) Glucagon analog and methods of use thereof
EP0785947B1 (en) Peptides endowed with antiinflammatory activity
JP2001186887A (en) Antimicrobial peptide originating from pandinus imperator
Zaykov et al. Synthesis and Characterization of the R27S Genetic Variant of Insulin‐like Peptide 5
AU668170B2 (en) Calcium channel blocking polypeptides from filistata hibernalis
WO2016090628A1 (en) Oxyntomodulin (oxm) analogs, synthesis and use thereof
TWI796339B (en) Peptide compound, application thereof and composition containing the same
US9073975B2 (en) Cyclic peptides, their preparation and their use as inhibitors of the platelet adhesion
AU736207B2 (en) Peptides which promote activation of latent TGF-beta and method of screening TGF-beta activity-regulating compounds
Velentza et al. Synthesis and biological activity of adipokinetic hormone analogues with modifications in the 4–8 region
JPH03255095A (en) Casein peptide

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13755494

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13755494

Country of ref document: EP

Kind code of ref document: A1