WO2007025332A1 - A peptide and zinc ion composition for treating diabetes and enhancing immune function - Google Patents

A peptide and zinc ion composition for treating diabetes and enhancing immune function Download PDF

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Publication number
WO2007025332A1
WO2007025332A1 PCT/AU2006/001254 AU2006001254W WO2007025332A1 WO 2007025332 A1 WO2007025332 A1 WO 2007025332A1 AU 2006001254 W AU2006001254 W AU 2006001254W WO 2007025332 A1 WO2007025332 A1 WO 2007025332A1
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Prior art keywords
xaa
zinc
peptide
amino acid
thr
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PCT/AU2006/001254
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French (fr)
Inventor
Mark A Myers
Robyn Gray
Biljana Nikolovski
Paul Zev Zimmet
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Dia-B Tech Limited
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Priority claimed from AU2005904711A external-priority patent/AU2005904711A0/en
Application filed by Dia-B Tech Limited filed Critical Dia-B Tech Limited
Publication of WO2007025332A1 publication Critical patent/WO2007025332A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/30—Zinc; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala

Definitions

  • Zinc deficiency is often found in the elderly, alcoholics, people with anorexia, and individuals on restrictive weight loss diets. Zinc deficiency is also often observed in those people with diseases or disorders that interfere with absorption of nutrients from food, such as irritable bowel syndrome, Coeliac disease and chronic diarrhoea.
  • Zinc also plays an essential role in insulin secretion and action and is also known to have insulin-like activity. Zinc is known to delay insulin release from a site of subcutaneous injection. Insulin forms hexameric complexes with zinc and this is essential for the dense packing of insulin in the secretory granules of beta cells (Blundel et al).
  • Zinc also stimulates lipogenesis and glucose transport in adipocytes (Coulston; May & Contoreggi; Ezaki; Tang & Shay), attenuates hyperglycaemia in db/db (Simon & Taylor) and ob/ob (Chen et al.) mice, activates components of the insulin signalling system including the insulin receptor tyrosine kinase (IRTK) and PD kinase (Tang & Shay; Miranda & Dey; Kim et al), inhibits the phosphatase responsible for inactivating IRTK (Haase and Maret) and increases glycogen synthesis through inhibition of glycogen synthase kinase (Ilouz et al).
  • IRTK insulin receptor tyrosine kinase
  • PD kinase Tang & Shay; Miranda & Dey; Kim et al
  • IRTK insulin receptor tyrosine kinase
  • Xaa is any amino acid
  • Xaai is a hydrophobic amino acid; n ! is 0-10; and n 2 is 0-10; and zinc ions.
  • the peptide is one of the formulae:
  • Xaa, ni and n 2 are as defined above.
  • the composition may further comprise insulin.
  • the present invention provides a complex comprising a peptide of the formula:
  • Xaa is any amino acid; Xaa ! is a hydrophobic amino acid; ni is 0-10; and n 2 is 0-10; and a zinc(II) ion, wherein the ratio of peptide:zinc(II) ion is 1 :1.
  • Xaa, ni and n 2 are as defined above.
  • Especially preferred complexes comprise a tetrapeptide selected from:
  • the peptide:zinc complex is a Val-His-Thr-Asp:Zn 2+ complex having a Circular Dichroism profile with minima at 208 and 222 nm when measured in MiIIiQ water or biological buffers at pH greater than 6 and 20°C.
  • Xaa is any amino acid; Xaai is a hydrophobic amino acid; ni is 0-10; and n 2 is 0-10; and zinc ions.
  • a method of increasing cellular response to zinc ions comprising exposing cells to an effective amount of a combination comprising a peptide of the formula:
  • Xaa is any amino acid; Xaa ! is a hydrophobic amino acid; ni is 0-10; and n 2 is 0-10; and zinc ions.
  • a method of regulating in vivo blood glucose levels in a human or other mammal which comprises administration to said human or other mammal, a combination comprising an effective amount of a peptide of the formula:
  • Xaa is any amino acid
  • Xaaj is a hydrophobic amino acid; ni is 0-10; and n 2 is 0-10; and zinc ions.
  • composition may further comprise insulin, or the combination may be co-administered separately, simultaneously or sequentially with insulin.
  • a method of treating diabetes in a human or other mammal comprising administration to said human or other mammal, a combination comprising an effective amount of a peptide of the formula:
  • Xaa is any amino acid; Xaai is a hydrophobic amino acid; ni is 0-10; and n 2 is 0-10; and zinc ions.
  • the diabetes is Type 1 diabetes. In other embodiments, the diabetes is Type 2 diabetes.
  • the composition may further comprise insulin, or the combination may be co-administered separately, simultaneously or sequentially with insulin.
  • a method of treating or preventing disorders associated with zinc deficiency in a human or other mammal comprising administration to said human or other mammal, a combination comprising an effective amount of a peptide of the formula:
  • Xaa is any amino acid; Xaai is a hydrophobic amino acid; ni is 0-10; and n 2 is 0-10; and zinc ions.
  • a method of enhancing the activity of the immune system of a human or other mammal which comprises administration to said human or other mammal, a combination comprising an effective amount of a peptide of the formula:
  • Xaa is any amino acid
  • Xaai is a hydrophobic amino acid; ni is 0-10; and n 2 is 0-10; and zinc ions.
  • This aspect may be particularly useful in preventing or reducing the severity or duration of infections such as colds and flu.
  • the present invention provides use of a combination comprising a peptide of the formula:
  • Xaa is any amino acid; Xaai is a hydrophobic amino acid;
  • Ri is 0-10; and n 2 is 0-10; and zinc ions, in the manufacture of a medicament for regulating in vivo blood glucose levels in a human or other mammal.
  • the present invention provides use of a combination comprising a peptide of the formula:
  • Xaa is any amino acid; Xaaj is a hydrophobic amino acid; ni is 0-10; and n 2 is 0-10; and zinc ions, in the manufacture of a medicament for treating diabetes in a human or other mammal.
  • the present invention provides use of a combination comprising a peptide of the formula:
  • Xaa is any amino acid
  • Xaai is a hydrophobic amino acid; ni is 0-10; and n 2 is 0-10; and zinc ions, in the manufacture of a medicament for treating or preventing zinc deficiency in a human or other mammal.
  • Xaa is any amino acid
  • Xaaj is a hydrophobic amino acid; m is 0-10; and n 2 is 0-10; and zinc ions, in the manufacture of a medicament for enhancing the activity of the immune system of a human or mammal.
  • the present invention relates to a combination of zinc ions and a class of peptides that may be used to deliver zinc into cells and regulate cellular response to zinc. Delivery of zinc into cells provides a method of treating or preventing zinc deficiency in a subject. Furthermore, such a combination also may enhance the insulin-sensitising and hypoglycaemic effects of the class of peptides. Enhancing the insulin-sensitising and hypoglycaemic effects of the class of peptides provides a method of treating diabetes, particularly in humans.
  • the present invention provides a composition comprising a peptide of the formula:
  • Xaa is any amino acid
  • Xaai is a hydrophobic amino acid; n t is 0-10; and n 2 is 0-10; and zinc ions.
  • the peptide is one of the formulae:
  • the peptide is a tetrapeptide selected from
  • the C-terminus of the peptide and/or the N-terminus of the peptide may be capped with a suitable capping group.
  • the C-terminus of the peptide may be amidated, and/or the N-terminus of the peptide may be acylated, eg. acetylated.
  • the C-terminus of the peptide is amidated.
  • amino acid refers to compounds having an amino group and a carboxylic acid group.
  • An amino acid may be a naturally occurring amino acid or non-naturally occurring amino acid and may be a proteogenic amino acid or a non-proteogenic amino acid.
  • the amino acids incorporated into the amino acid sequences of the present invention may be L-amino acids, D-amino acids, ⁇ -amino acids, ⁇ -amino acids and/or mixtures thereof.
  • Suitable naturally occurring proteogenic amino acids are shown in Table 1 together with their one letter and three letter codes.
  • Suitable non-proteogenic or non-naturally occurring amino acids may be prepared by side chain modification or by total synthesis.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH 4 .
  • the amino group of lysine may also be derivatized by reaction with fatty acids, other amino acids or peptides or labeling groups by known methods of reacting amino groups with carboxylic acid groups.
  • the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.
  • Sulfydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulfides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulfonic acid, phenylmercury chloride, 2-chloromercuri-4- nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carboethoxylation with diethy lpyrocarbonate .
  • Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino-butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
  • Examples of suitable non-proteogenic or non- naturally occurring amino acids contemplated herein is shown in Table 2. TABLE 2
  • Non-conventional Code Non-conventional Code amino acid amino acid
  • one or more of the His, Thr or Asp amino acids in the His-Thr-Asp sequence may be non-naturally occurring His, Thr or Asp.
  • the His, Thr or Asp may be D-amino acids or may be derivatised, for example by N-alkylation such as N-methylation or ⁇ -alkylation such as ⁇ -methylation.
  • Examples of derivatised His, Thr and Asp include, but are not limited to, N-methyl-His, N-methyl-Thr, N-methyl-aspartic acid, ⁇ -methyl-histidine, ⁇ -methyl-threonine or ⁇ -methyl-aspartic acid.
  • the His, Thr and Asp are L-amino acids, and are underivatised.
  • the zinc ions in the composition are preferably zinc (II) ions (Zn 2+ ).
  • the zinc ions may be added to the composition in the form of a salt, in particular, a salt which is pharmaceutically acceptable.
  • suitable zinc salts that provide zinc (II) ions in the composition include, but are not limited to, zinc chloride (ZnCl 2 ), zinc sulfate (ZnSO 4 ), zinc picolinate, zinc citrate, zinc acetate, zinc glycerate and zinc monomethionine, especially zinc chloride.
  • the ratio of zinc ions to peptide in the composition is 1 :1.
  • the complex is formed between the tetrapeptide Val-His-Thr-Asp and Zn ions.
  • the complex has a circular dichroism (CD) profile with minima at 208 and 222 nm when measured in MiIIiQ water or biological buffer at pH greater than 6 and 2O 0 C.
  • the complex has a CD profile as shown in Figure IB.
  • these peptides may be prepared as recombinant peptides using standard recombinant DNA techniques.
  • a recombinant expression vector containing a nucleic acid sequence encoding the peptide and one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed may be introduced into and expressed in a suitable prokaryotic or eukaryotic host cell, as described, for example, in Gene Expression Technology: Methods in Enzymology, 185, Academic Press, San Diego, CA (1990), and Sambrook et al, Molecular Cloning: A Laboratory Manual, 2 nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).
  • the peptide Gly-His-Thr-Asp may also be isolated from human urine by standard protein purification procedures, preferably using reversed-phase high performance liquid chromatography (RP-HPLC). Using these procedures, Gly-His-Thr-Asp is obtained in isolated form.
  • isolated is meant a peptide material that is substantially or essentially freed from components, particularly other proteins and peptides, that normally accompany it in its native state in human urine by at least one purification or other processing step.
  • Such isolated peptide may also be described as substantially pure.
  • substantially pure describes peptide material that has been separated from components that naturally accompany it.
  • peptide material is substantially pure when at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% or even 99% of the total peptide material (by volume, by wet or dry weight, or by mole percent or mole fraction) is the peptide of interest. Purity can be measured by any appropriate method, for example, in the case of peptide material, by chromatography, gel electrophoresis or HPLC analysis.
  • the present invention also extends to methods of regulating in vivo blood glucose levels in a human or other mammal by administering to the human or other mammal, a combination of peptide and zinc ions of the invention as described above.
  • human or other mammal refers to humans and other warm blooded animals that may require regulation of blood glucose.
  • mammals includes domesticated animals such as dogs, cats, horses and the like, livestock animals such as cattle, sheep, pigs and the like, laboratory animals such as mice, rats, rabbits and the like, and captive animals such as those animals held in zoos.
  • the subject is a human.
  • Type 1 diabetes there is a lack of insulin production. This is because the beta cells of the Islets of Langerhans in the pancreas have been destroyed, most often by autoimmune-mediated destruction. Those subjects with Type 1 diabetes require treatment with insulin to replace the insulin that would normally be produced in the pancreas. Since insulin is not produced, a subject with untreated or poorly controlled Type 1 diabetes will have hyperglycaemia.
  • the combination of peptide and zinc ions may also reduce, prevent or slow the progression of complications associated with diabetes.
  • complications include cardiovascular disease and associated complications such as diabetic dyslipidemia; high blood pressure (hypertension); neuropathy and nerve damage; kidney disease; and eye diseases such as glaucoma, cataracts and retinopathy.
  • a variety of administration routes are available. The particular mode selected will depend, of course, upon the particular condition being treated and the dosage required for therapeutic efficacy.
  • the methods of this invention may be practised using any mode of administration that is medically acceptable, meaning any mode that produces therapeutic levels of the active components of the invention without causing clinically unacceptable adverse effects.
  • modes of administration include parenteral (e.g. subcutaneous, intramuscular and intravenous), oral, rectal, topical, nasal and transdermal routes.
  • compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active components which is preferably isotonic with the blood of the recipient.
  • This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in polyethylene glycol and lactic acid.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono-or di-glycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active component, in liposomes or as a suspension in an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, or an emulsion.
  • sustained release delivery systems can include sustained release delivery systems.
  • Preferred sustained release delivery systems are those which can provide for release of the active components of the invention in sustained release pellets or capsules.
  • Many types of sustained release delivery systems are available; these include, but are not limited to: (a) erosional systems in which the active components are contained within a matrix, and (b) diffusional systems in which the active components permeate at a controlled rate through a polymer.
  • increasing cellular response to zinc ions is meant that the function of the cell is improved or upregulated.
  • increasing cellular response includes increasing or upregulating metabolism in a cell. Increased metabolism can be measured, for example, by the acid production of the cells.
  • the cells may be in vivo or in vitro.
  • the cells When the cells are in vitro, the cells may be cells cultured for the purposes of experiment that also requires zinc ions to be present in the cells. Alternatively, the zinc ions may be required in the cells for the purposes of detection in an assay.
  • the method of delivering zinc into cells may be used to treat or prevent zinc deficiency, whether or not the deficiency is associated with diabetes, or alleviate the symptoms of zinc deficiency in humans or other mammals.
  • the symptoms of zinc deficiency include loss of appetite, poor growth, weight loss, impaired taste and smell, poor wound healing, skin abnormalities such as acne, atopic dermatitis and psoriasis, hair loss, lack of menstruation, night blindness, hypogonadism and delayed sexual maturation, white spots on the fingernails and feelings of depression.
  • Zinc deficiency also tends to make a subject more susceptible to a variety of infections.
  • Zinc supplementation can enhance the immune system and protect against a range of infections including colds and upper respiratory infections.
  • a recommended dietary allowance of zinc is between 2-8 mg for children between birth and 13 years of age and between 8-11 mg for people 14 years of age and over. The requirements are on the higher side for children between the ages of 14 to 18 years and also for pregnant women and breastfeeding women.
  • an effective amount of elemental zinc is between 30 and 60 mg per day.
  • a therapeutically effective amount means that amount necessary at least partly to attain the desired effect, or to delay the onset of, inhibit the progression of, or halt altogether, the onset or progression of the particular condition being treated. Such amounts will depend, of course, on the particular condition being treated, the severity of the condition and individual patient parameters including age, physical condition, size, weight and concurrent treatment. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.
  • daily oral doses of active components will be from about 0.01 mg/kg per day to 1000 mg/kg per day. Small doses (0.01-1 mg) may be administered initially, followed by increasing doses up to about 1000 mg/kg per day. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localised delivery route) may be employed to the extent patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.
  • a maximum of 150 mg of elemental zinc is administered to a patient per day, preferably the daily dosage of the combination will result in the administration of less than 100 mg of elemental zinc per patient per day.
  • the amount of elemental zinc administered as part of the combination of the invention is between 20 mg and 70 mg elemental zinc per patient per day, especially 30 mg to 60 mg elemental zinc per patient per day, especially about 50 mg elemental zinc per patient per day.
  • a healthcare provider could readily determine the amount of a composition containing the combination of the invention to administer these amounts of zinc per day or suitable amounts of each component separately to provide a suitable ratio of peptide to zinc, for example a 1 :1 ratio.
  • the amount of combination delivered is determined by the amount of hypoglycaemic effect required. Small doses (0.01-1 mg) may be administered initially and doses may be increased as required. In preferred embodiments, an oral dosage of the combination of the invention will deliver the peptide in an amount of 0.1-100 mg/kg per day. A healthcare provider could readily determine the amount of a composition containing the combination of the invention or separate dosage forms of the peptide and the zinc in the appropriate ratio to provide the required hypoglycaemic effect.
  • the composition may further comprise copper ions.
  • the ratio of zinc ions to copper ions may be in the range of 10:1 to 20:1, especially about 15:1.
  • the source of copper ions may be from any suitable pharmaceutically acceptable salt of copper, for example, copper glycinate.
  • the combination of peptide and zinc ions has a hypoglycaemic effect, but only in those with insulin resistance and/or diabetes. In those with normal insulin sensitivity the peptide exerts a minimal hypoglycaemic effect.
  • the combination of the invention may be used to treat zinc deficiency or enhance immune system activity in patients that are not hyperglycaemic.
  • Figure IA provides a CD spectrum of ISF402 (Val-His-Thr-Asp).
  • Figure IB provides a CD spectrum of ISF402 after addition of ZnCl 2 to 2 mM.
  • Figure 1C provides a CD spectrum of ISF402 and ZnCl 2 to 2 mM after addition of a chelating agent EGTA.
  • Figure 2 graphically represents the titration of ISF402 (320 ⁇ M) with ZnCl 2 (0-680 ⁇ M) as measured by the magnitude of the CD signal at 220 nm.
  • Figure 3 A graphically represents a dose response curve for C2C12 muscle cells exposed to insulin.
  • Cellular response increased with increasing insulin concentrations over the range of 10 "3 to 10OnM. High concentrations, above 10OnM, decreased the cellular response. Results are mean of the percentage maximum stimulation +/- SD.
  • Figure 5B graphically represents the quantitation of cellular fluorescence for cells shown in Figure 5 A equilibrated in the presence of 100 ⁇ M ZnCl 2 and two other cells. Background was measured at the edge of the image and subtracted from the cellular fluorescence and the data expressed as the change in integrated optical density (IOD) ISF402 (closed symbols) and FSI (open symbols) were added between frames 3 and 4. Thirty frames were photographed at 30 second intervals (total time: 15 minutes).
  • IOD integrated optical density
  • Figure 8B graphically represents the titration of GHTD-amide with zinc as measured by the magnitude of the CD signal at 220 nm.
  • the change in CD signal at 220 nm in mdeg was monitored at various zinc ion concentrations and the percent change in signal plotted against zinc concentration.
  • the affinity of interaction was 102 ⁇ 11 ⁇ M and the reaction was saturated at a 1:1 ratio of zinc to peptide.
  • ISF402 was dissolved at a concentration of 1 mg/mL in MiIIiQ water and the pH was raised to 7.4 by the addition of sodium hydroxide (NaOH).
  • Circular dichroism (CD) spectra were measured from 190-250 nm at 20 0 C on a Jasco J-810 spectropolarimeter equipped with a PFD 423/L Peltier type temperature controller. 200 ⁇ L of sample was placed in a quartz cuvette, with a path length of 1 mm, in the spectropolarimeter and the CD spectrum was recorded. Each spectrum represents an average of 3-5 scans performed at 100 nm/min with a band width of 1 nm. The effect of zinc ions was measured by adding an aliquot of stock solution of ZnCl 2 to a solution of ISF402 (to a concentration of 2 mM) and measuring the CD spectrum.
  • C2C12 cells were plated on glass coverslips. After confluency, cells were serum deprived for 24 hours and treated either with ISF402, insulin or a mixture of ISF402 and insulin. Cells were washed with Phosphate buffered saline (PBS) and fixed with 10% formaldehyde for 20 min, washed again and made permeable in 0.1% Triton X-100 for 30 min at room temperature. Cells were blocked with 2% gelatin for 45min at room temperature and incubated for 1 hour with either guinea pig anti-insulin (1:50 Dako, Carpinteria, CA, USA), rabbit anti-ISF402 (1 :500) or both.
  • PBS Phosphate buffered saline
  • Triton X-100 0.1% Triton X-100
  • Coverslips were washed four times in PBS and incubated for 1 hour at room temperature with secondary antibodies FITC anti-guinea pig IgG conjugates (1 :200 Dako, Ca ⁇ interia, CA, USA), and Alexa 568 anti-rabbit IgG conjugates (1 :200 Molecular Probes, Eugene, OR, USA). Coverslips were washed four times with PBS and mounted on glass slides with Dako mounting medium. Confocal images were obtained using a Leica TCSNT7DMRBE confocal microscope (x 63 lense) (Leica Microsystms Pty, Ltd. NSW Australia).
  • C2C12 cells at passage 10 were seeded onto 10cm dishes and differentiated into myotubes for 7 days.
  • Cells were then serum starved overnight in 0.5% FBS DMEM (Gibco BRL) and pre-treated with the PI3-kinase inhibitor Wortmannin (Sigma) at 5OnM for 30 min at 37 0 C.
  • Cells were then treated with Insulin (Sigma) at varying concentrations or ISF402 for 20 minutes at 37 0 C.
  • ISF402 contains a histidine residue.
  • the imidazole side chain of histidine is involved in binding transition metals in many proteins, but only when the imidazole is deprotonated, which generally occurs above pH 6.8.
  • the CD signal at 220 nm was obtained from ISF402 dissolved in MQ water at pH 3 to pH 7 (Table 3).
  • the negative peak at 220 nm does not occur at acidic pH and only appears when the pH reaches 6-7. This is consistent with a requirement for the histidine side chain to be deprotonated for Zn 2+ to induce a change in the structure of ISF402.
  • Table 3 Effect of varying pH on the CD signal at 220 nm from ISF402
  • ISF402 activates the insulin signalling pathway, which is a property shared by zinc.
  • ISF402 stimulates metabolism in muscle cells to produce a change in acid secretion which was detected by the microphysiometer.
  • Addition of zinc (ZnCl 2 ) at 1 ⁇ M also stimulated muscle cells but increasing this to 50 ⁇ M did not significantly increase the cellular response further (Figure 3D) with the response at 1 ⁇ M ZnCl 2 (6.2 ⁇ 2.0 ⁇ Volts/sec) not significantly different to the response at higher ZnCl 2 concentrations (e.g.
  • ISF 402 readily enters C2C12 cells
  • ISF402 may potentiate its biological actions by entering cells.
  • C2C12 cells grown on coverslips, fixed and incubated with the anti-ISF402 and anti-insulin antibody.
  • Second antibodies were anti-rabbit conjugated to Alexa568 and anti-guinea pig conjugated to FITC.
  • Confocal image illustrates a nuclear sybr green stain showing no staining with pre-immune serum, Figure 4A.
  • a second set of images of C2C12 cells were obtained using an Olympus FV500 confocal microscope (xlOO objective).
  • the four panel image illustrates a DAPI nuclear stain (blue) of cells treated with ISF402 (red), Figure 4E, and insulin (green), Figure 4F, demonstrates entry into cells and co-localisation (yellow), Figure 4G.
  • Figure 4H shows a DIC image of the cells. Scale bar is 20 ⁇ m.
  • Confocal images of C2C 12 cells treated with insulin, ISF402 and a mixture of insulin and ISF402 show that insulin (Figure 4A) and ISF402 ( Figure 4B) can enter cells. Treatment of cells with both insulin and ISF402 showed co- localisation of insulin with ISF402 ( Figure 4C) inside the cell.
  • ISF402 increases cellular zinc concentrations
  • ISF402 may facilitate an increasing cellular response to increasing Zn 2+ concentrations by increasing the cellular uptake of Zn 2+ .
  • 10 ⁇ M ISF402 or FSI ISF402 peptide with the sequence of amino acids reversed
  • the cells showed a level of background fluorescence due to the quantities of Zn 2+ that normally reside in cells.
  • FSI no increase in the fluorescent signal was apparent ( Figure 5A, top panel), whereas ISF402 caused a measurable increase in FluoZin fluorescence ( Figure 5A, lower panel).
  • transition metal zinc shows insulin-like effects in many different systems.
  • the binding of zinc ions is also important for the insulin-like and insulin-sensitising effects of ISF402 and other hypoglycaemic peptides of formula (I).
  • Zinc is an important transition metal since it is the second most common transition metal found in the human body (after iron), with 2.3 gm in an average 70 kg person (McCaIl et cd.).
  • ISF402 and other hypoglycaemic peptides of formula (I) bind zinc ions to form metal- peptide complexes. Histidine is involved in the complexation of the hypoglycaemic peptides with zinc as supported by the observation that zinc ions only induced changes in
  • ISF402 secondary structure at pH values above 6 when the imidazole ring of histidine would be uncharged.
  • the complex formed when a peptide solution is fully saturated with zinc ions is one molecule of zinc for every molecule of hypoglycaemic peptide.
  • the interaction between zinc ions and ISF402 occurred at physiological temperature and pH with a dissociation constant of 27 ⁇ M, which is close to the concentration of free zinc in blood (15 ⁇ M). Therefore, a proportion of zinc would be bound to ISF402 in blood.
  • peptides such as ISF402 facilitate an increase in cellular response to zinc at zinc concentrations from 1 to 50 ⁇ M. Furthermore, there is an interplay between zinc ions and the peptides that results in increased transport of zinc into cells in the presence of peptides such as ISF402. Since these cellular effects occurred at concentrations of zinc that are close to physiological concentrations, it is likely that peptides such as ISF402 can facilitate uptake of zinc by cells in vivo. Increasing zinc concentrations within cells will maximise the insulino-mimetic effects of zinc and minimise other effects resulting from zinc deficiency.
  • wortmannin A concentration of wortmannin
  • PB -kinase inhibitor wortmannin is extensively used in molecular signalling studies (Arcaro and Wymann) to block insulin stimulation of signalling intermediates such as Akt (Burgering and Coffer; Kahn).
  • Akt Bodectase Akt
  • wortmannin was used to test whether ISF402 activated the insulin signalling pathway.
  • wortmannin at a sub-maximal concentration, (5OnM) significantly inhibited the cellular response to both insulin and ISF402 in the cytosensor ( Figure 6A and B), suggesting that ISF402 has insulin mimetic activity and can independently activate PI-3 kinase.
  • Glut3 which are also expressed in skeletal muscle cells (Tortorella and Pilch). It is noteworthy that from the microphysiometry and western blotting experiments that ISF402 does not activate PI-3 kinase as efficiently as insulin. ISF402 may only partially stimulate the insulin signalling pathway, perhaps to a level below threshold for triggering many of the downstream cellular events that insulin triggers. In the context of the modest glucose uptake seen in C2C12 muscle cells with insulin, the lesser stimulation of PI-3 kinase by ISF402 may have been insufficient to induce a measurable increase in glucose transport.
  • ISF402 activates PI-3 kinase and increases the response of C2C12 muscle cells to insulin.
  • the glucose lowering activity of ISF402 observed in vivo may be explained by insulin-like activity and insulin sensitisation mediated by PI3-kinase.
  • Zinc activates components of the insulin signalling system including the insulin receptor tyrosine kinase (IRTK) and PI3 kinase (Tang and Shay; Miranda and Dey; Kim et al), and inhibits the phosphatase responsible for inactivating IRTK (Haase and Maret). Therefore, activation of PI3 kinase by ISF402 is likely to be mediated by the ISF402-mediated increase in cellular zinc concentrations.
  • IRTK insulin receptor tyrosine kinase
  • PI3 kinase Tang and Shay; Miranda and Dey; Kim et al
  • GHTD-amide has structural features common to zinc -binding peptides. Histidine is often involved in co-ordinating zinc and less frequently aspartic acid can also contribute (Thickman et al). Generally, tetrapeptides such as GHTD-amide lack secondary structure due to their small size, but interactions with zinc can induce conformational changes (Henin et al). The effect of zinc on the secondary structure of GHTD-amide was examined using far UV circular dichroism (Figure 8). The CD profile of GHTD-amide in the absence of zinc was typical of random coil with a pronounced negative peak at 198 nm ( Figure 8A).
  • Arcaro A. and Wymann M. P. Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses. Biochem. J. 296(Pt2):297-301 (1993).
  • Tortorella L.L. and Pilch P.F. C2C12 myocytes lack an insulin-responsive vesicular compartment despite dexamethasone-induced GLUT4 expression.

Abstract

Compositions comprising a peptide of the formula: (Xaa)n1-Xaa1-His-Thr-Asp-(Xaa)n2 wherein Xaa is any amino acid; Xaa1 is a hydrophobic amino acid; n1 is 0-10; and n2 is 0-10; and zinc ions are described. The invention also relates to methods of delivering zinc ions into cells, methods of regulating in vivo glucose levels in humans and mammals and methods of combating zinc deficiency using the compositions of the invention.

Description

A PEPTIDE AND ZINC ION COMPOSITION FOR TREATING DIABETES AND ENHANCING IMMUNE FUNCTION
Field of the Invention
The present invention relates to compositions comprising a class of peptides and zinc ions. More particularly, the present invention relates to methods of delivering zinc into cells by exposing the cells to compositions comprising a peptide of this class and zinc ions. The combination of peptides and zinc ions is also useful in regulating in vivo glucose levels in humans and other mammals. The combination of peptides and zinc ions therefore has potential for use as anti-diabetic agents and as agents for combating zinc deficiency.
Background of the Invention
Bibliographic details of the publications referred to in this specification are collected at the end of the description.
The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.
Zinc is an essential trace mineral that is obtained from dietary sources. Zinc is stored primarily in muscle cells but is also found in high concentrations in white and red blood cells, the retina, bones, skin, kidneys, liver, pancreas and in men, the prostate gland. Zinc plays a role in the immune system, regulation of appetite, stress level, taste and smell and is essential for normal growth and development and in many aspects of reproduction in both men and women. Zinc also has anti-oxidant effects as it is required for a number of anti-oxidant enzymes.
Low zinc intake or zinc deficiency is often found in the elderly, alcoholics, people with anorexia, and individuals on restrictive weight loss diets. Zinc deficiency is also often observed in those people with diseases or disorders that interfere with absorption of nutrients from food, such as irritable bowel syndrome, Coeliac disease and chronic diarrhoea.
Zinc also plays an essential role in insulin secretion and action and is also known to have insulin-like activity. Zinc is known to delay insulin release from a site of subcutaneous injection. Insulin forms hexameric complexes with zinc and this is essential for the dense packing of insulin in the secretory granules of beta cells (Blundel et al). Zinc also stimulates lipogenesis and glucose transport in adipocytes (Coulston; May & Contoreggi; Ezaki; Tang & Shay), attenuates hyperglycaemia in db/db (Simon & Taylor) and ob/ob (Chen et al.) mice, activates components of the insulin signalling system including the insulin receptor tyrosine kinase (IRTK) and PD kinase (Tang & Shay; Miranda & Dey; Kim et al), inhibits the phosphatase responsible for inactivating IRTK (Haase and Maret) and increases glycogen synthesis through inhibition of glycogen synthase kinase (Ilouz et al).
Increased zinc excretion has been observed in diabetic patients having both Type 1 and Type 2 diabetes, resulting in many diabetic patients having zinc deficiency.
Zinc supplements are often provided in the form of zinc sulfate. However, zinc sulfate is not easily absorbed and may cause stomach upsets. Other forms of zinc, such as zinc picolinate, zinc citrate, zinc acetate, zinc glycerate and zinc monomethione are also available. However, there is a need for zinc supplements that readily transport zinc into cells.
Bioactive peptides have been described in WO 03/002594 as having hypoglycaemic effects. Synthetic analogues of the insulin-sensitising factor (ISF), Gly-His-Thr-Asp-NH2 have been prepared and shown to have insulin-sensitising activity (Zimmet & Ng).
In work leading to the present invention, ISF and its synthetic analogues have been shown to interact with zinc and assist with zinc transport into cells and upregulating cellular response to zinc. Combinations of zinc and ISF and its analogues are therefore useful in combating the symptoms of zinc deficiency.
Furthermore, the interaction between zinc and ISF and its analogues contributes to the insulin enhancing properties of the hypoglycaemic peptides. Combinations of zinc and ISF and its analogues are therefore also useful in the treatment of diabetes.
Summary of the Invention
In one aspect the present invention provides a composition comprising a peptide of the formula:
(Xaa)n i -Xaaj -His-Thr- Asp-(Xaa)n2
wherein
Xaa is any amino acid;
Xaai is a hydrophobic amino acid; n! is 0-10; and n2 is 0-10; and zinc ions.
In preferred embodiments of the invention, the peptide is one of the formulae:
(Xaa)ni-Val-His-Thr-Asp-(Xaa)π2; or (Xaa)ni-Gly-His-Thr-Asp-(Xaa)n2;
wherein Xaa, ni and n2 are as defined above.
Preferably, the peptide is a tetrapeptide selected from
Val-His-Thr-Asp (ISF402); and - A -
Gly-His-Thr-Asp;
especially Val-His-Thr-Asp.
In some embodiments of this aspect of the invention, the composition may further comprise insulin.
In another aspect, the present invention provides a complex comprising a peptide of the formula:
(Xaa)nl-Xaa1-His-Thr-Asp-(Xaa)n2
wherein
Xaa is any amino acid; Xaa! is a hydrophobic amino acid; ni is 0-10; and n2 is 0-10; and a zinc(II) ion, wherein the ratio of peptide:zinc(II) ion is 1 :1.
In preferred embodiments of the invention the peptide in the complex is a peptide of the formula:
(Xaa)nl-Val-His-Thr-Asp-(Xaa)n2; or (Xaa)ni-Gly-His-Thr-Asp-(Xaa)n2;
wherein Xaa, ni and n2 are as defined above.
Especially preferred complexes comprise a tetrapeptide selected from:
Val-His-Thr-Asp; or
Gly-His-Thr-Asp; particularly Val-His-Thr-Asp.
In one embodiment, the peptide:zinc complex is a Val-His-Thr-Asp:Zn2+ complex having a Circular Dichroism profile with minima at 208 and 222 nm when measured in MiIIiQ water or biological buffers at pH greater than 6 and 20°C.
In yet another aspect of the invention, there is provided a method of delivering zinc into cells by exposing the cells to a combination comprising a peptide of the formula:
(Xaa)n i -Xaaj -His-Thr- Asp-(Xaa)n2
wherein
Xaa is any amino acid; Xaai is a hydrophobic amino acid; ni is 0-10; and n2 is 0-10; and zinc ions.
In yet another aspect of the invention, there is provided a method of increasing cellular response to zinc ions comprising exposing cells to an effective amount of a combination comprising a peptide of the formula:
(Xaa)n i -Xaa! -His-Thr- Asp-(Xaa)n2
wherein
Xaa is any amino acid; Xaa! is a hydrophobic amino acid; ni is 0-10; and n2 is 0-10; and zinc ions. In a further aspect of the invention, there is provided a method of regulating in vivo blood glucose levels in a human or other mammal, which comprises administration to said human or other mammal, a combination comprising an effective amount of a peptide of the formula:
(Xaa)n j -Xaa j -His-Thr-Asp-(Xaa)n2
wherein Xaa is any amino acid;
Xaaj is a hydrophobic amino acid; ni is 0-10; and n2 is 0-10; and zinc ions.
In some embodiments of this aspect, the composition may further comprise insulin, or the combination may be co-administered separately, simultaneously or sequentially with insulin.
In yet a further aspect of the invention, there is provided a method of treating diabetes in a human or other mammal comprising administration to said human or other mammal, a combination comprising an effective amount of a peptide of the formula:
(Xaa)n i -Xaaj -His-Thr-Asp-(Xaa)n2
wherein
Xaa is any amino acid; Xaai is a hydrophobic amino acid; ni is 0-10; and n2 is 0-10; and zinc ions. In some embodiments of this aspect of the invention, the diabetes is Type 1 diabetes. In other embodiments, the diabetes is Type 2 diabetes.
In some embodiments where the diabetes is Type 1 diabetes or insulin requiring Type 2 diabetes, the composition may further comprise insulin, or the combination may be co-administered separately, simultaneously or sequentially with insulin.
In another aspect of the invention, there is provided a method of treating or preventing disorders associated with zinc deficiency in a human or other mammal comprising administration to said human or other mammal, a combination comprising an effective amount of a peptide of the formula:
(Xaa)n i -Xaai-His-Thr-Asp-(Xaa)n2
wherein
Xaa is any amino acid; Xaai is a hydrophobic amino acid; ni is 0-10; and n2 is 0-10; and zinc ions.
In yet another aspect, there is provided a method of enhancing the activity of the immune system of a human or other mammal, which comprises administration to said human or other mammal, a combination comprising an effective amount of a peptide of the formula:
(Xaa)n i -Xaai -His-Thr- Asp-(Xaa)n2
wherein Xaa is any amino acid;
Xaai is a hydrophobic amino acid; ni is 0-10; and n2 is 0-10; and zinc ions.
This aspect may be particularly useful in preventing or reducing the severity or duration of infections such as colds and flu.
In yet another aspect, the present invention provides use of a combination comprising a peptide of the formula:
(Xaa)nl-Xaai-His-Thr-Asp-(Xaa)n2
wherein
Xaa is any amino acid; Xaai is a hydrophobic amino acid;
Ri is 0-10; and n2 is 0-10; and zinc ions, in the manufacture of a medicament for regulating in vivo blood glucose levels in a human or other mammal.
In a further aspect, the present invention provides use of a combination comprising a peptide of the formula:
(Xaa)n i -Xaa! -His-Thr- Asp-(Xaa)n2
wherein
Xaa is any amino acid; Xaaj is a hydrophobic amino acid; ni is 0-10; and n2 is 0-10; and zinc ions, in the manufacture of a medicament for treating diabetes in a human or other mammal.
In yet a further aspect, the present invention provides use of a combination comprising a peptide of the formula:
(Xaa)n i -Xaa] -His-Thr- Asp-(Xaa)n2
wherein Xaa is any amino acid;
Xaai is a hydrophobic amino acid; ni is 0-10; and n2 is 0-10; and zinc ions, in the manufacture of a medicament for treating or preventing zinc deficiency in a human or other mammal.
In another aspect, there is provided a use of a combination comprising a peptide of the formula:
(Xaa)nl-Xaa1-His-Thr-Asp-(Xaa)n2
wherein
Xaa is any amino acid;
Xaaj is a hydrophobic amino acid; m is 0-10; and n2 is 0-10; and zinc ions, in the manufacture of a medicament for enhancing the activity of the immune system of a human or mammal.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
Description of the Invention
The present invention relates to a combination of zinc ions and a class of peptides that may be used to deliver zinc into cells and regulate cellular response to zinc. Delivery of zinc into cells provides a method of treating or preventing zinc deficiency in a subject. Furthermore, such a combination also may enhance the insulin-sensitising and hypoglycaemic effects of the class of peptides. Enhancing the insulin-sensitising and hypoglycaemic effects of the class of peptides provides a method of treating diabetes, particularly in humans.
In one aspect the present invention provides a composition comprising a peptide of the formula:
(Xaa)n i -Xaai -His-Thr-Asp-(Xaa)n2
wherein Xaa is any amino acid;
Xaai is a hydrophobic amino acid; nt is 0-10; and n2 is 0-10; and zinc ions.
In a preferred embodiment of this aspect of the invention, the peptide is one of the formulae:
(Xaa)nl-Val-His-Thr-Asp-(Xaa)n2; or (Xaa)ni-Gly-His-Thr-Asp-(Xaa)n2; wherein Xaa, m and n2 are as defined above.
Preferably, the peptide is a tetrapeptide selected from
Val-His-Thr-Asp (ISF402); and
Gly-His-Thr-Asp;
especially Val-His-Thr-Asp.
In some embodiments, the C-terminus of the peptide and/or the N-terminus of the peptide may be capped with a suitable capping group. For example, the C-terminus of the peptide may be amidated, and/or the N-terminus of the peptide may be acylated, eg. acetylated. In preferred embodiments, the C-terminus of the peptide is amidated.
As used herein, the term "amino acid" refers to compounds having an amino group and a carboxylic acid group. An amino acid may be a naturally occurring amino acid or non-naturally occurring amino acid and may be a proteogenic amino acid or a non-proteogenic amino acid. The amino acids incorporated into the amino acid sequences of the present invention may be L-amino acids, D-amino acids, α-amino acids, β-amino acids and/or mixtures thereof.
Suitable naturally occurring proteogenic amino acids are shown in Table 1 together with their one letter and three letter codes.
Table 1
Figure imgf000012_0001
Figure imgf000013_0001
Suitable non-proteogenic or non-naturally occurring amino acids may be prepared by side chain modification or by total synthesis. Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH4. The amino group of lysine may also be derivatized by reaction with fatty acids, other amino acids or peptides or labeling groups by known methods of reacting amino groups with carboxylic acid groups.
The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal. The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.
Sulfydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulfides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulfonic acid, phenylmercury chloride, 2-chloromercuri-4- nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carboethoxylation with diethy lpyrocarbonate .
Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino-butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. Examples of suitable non-proteogenic or non- naturally occurring amino acids contemplated herein is shown in Table 2. TABLE 2
Non-conventional Code Non-conventional Code amino acid amino acid
α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nm gin carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisoleucine Nmile
D-alanine Dal L-N-methylleucine Nmleu
D-arginine Darg L-N-methyllysine Nmlys
D-aspartic acid Dasp L-N-methylmethionine Nmmet
D-cysteine Dcys L-N-methylnorleucine Nmnle
D-glutamine DgIn L-N-methylnorvaline Nmnva
D-glυtamic acid DgIu L-N-methylornithine Nmorn
D-histidine Dhis L-N-methylphenylalanine Nmphe
D-isoleucine DiIe L-N-methylproline Nmpro
D-leucine Dleu L-N-methylserine Nmser
D-lysine Dlys L-N-methylthreonine Nmthr
D-methionine Dmet L-N-methyltryptophan Nmtrp
D-ornithine Dorn L-N-methyltyrosine Nmtyr
D-phenylalanine Dphe L-N-methylvaline Nmval
D-proline Dpro L-N-methylethylglycine Nmetg
D-serine Dser L-N-methyl-t-butylglycine Nmtbug
D-threonine Dthr L-norleucine NIe
D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib
D-valine Dval α-methyl-γ-aminobutyrate Mgabu
D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa
D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen
D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap
D-α-methylaspartate Dmasp α-methylpenicillamine Mpen
D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine NgIu
D-α-methylglutamine Dmgln N-(2-arainoethyl)glycine Naeg
D-α-methylhistidine Dnihis N-(3-aminopropyl)glycine Norn
D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu
D-α-methylleucine Dmleu α-napthylalanine Anap
D-α-methyllysine Dm Iy s N-benzylglycine Nphe
D-α-raethylmethionine Dramet N-(2-carbamylethyl)glycine NgIn
D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn
D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine NgIu
D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp
D-α-methylserine Dmser N-cyclobutylglycine Ncbut
D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep
D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex
D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec
D-α-methylvaline Dmval N-cylcododecylglycine Ncdod
D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct
D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro
D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund
D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm
D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycineNbhe
D-N-methylglutamine Dnmgln N-(3 -guanidinopropyl)glycine Narg
D-N-methylglutamate Dnmglu N-(l-hydroxyethyl)glycine Nthr
D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser
D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis
D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtφ
D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet
D-N-m ethy lornithine Dnmorn N-methylcyclopentylalanine Nmcpen
N-methylglycine NaIa D-N-methylphenylalanine Dnmphe
N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro
N-(I -methylpropyl)glycine Nile D-N-methylserine Dnmser
N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr
D-N-methyltryptophan Dnmtrp N-(l-methylethyl)glycine Nval
D-N-methyltyrosine Dnmtyr N-methyl-napthylalanine Nmanap
D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr
L-/-butylglycine Tbug N-(thiomethyl)glycine Ncys
L-ethylglycine Etg penicillamine Pen
L-homophenylalanine Hphe L-α-methylalanine Mala
L-α-methylarginine Marg L-α-methylasparagine Masn
L-α-methylaspartate Masp L-α-methyl-Z'-butylglycine Mtbug
L-α-methylcysteine Mcys L-methylethylglyciπe Metg
L-α-methylglutamine MgIn ' L-α-methylglutamate MgIu
L-α-methylhistidine Mhis L-α-methylhomophenylalanineMhphe
L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet
L-α-methylleucine Mleu L-α-methyllysine Mlys
L-α-methylmethionine Mmet L-α-methylnorleucine MnIe
L-α-methylnorvaline Mnva L-α-methylornithine Morn
L-α-methylphenylalanine Mphe L-α-methylproline Mpro
L-α-methylserine Mser L-α-methylthreonine Mthr
L-α-methyltryptophan Mtrp L-α-methyltyrosine Mtyr
L-α-methylvaline Mval L-N-methylhomoρhenylalanin Nmhphe
N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine
1 -carboxy- 1 -(2,2-diphenyl Nmbc ethylamino)cyclopropane Suitable β-amino acids include, but are not limited to, L-β-homoalanine, L-β- homoarginine, L-β-homoasparagine, L-β-homoaspartic acid, L-β-homoglutamic acid, L-β- homoglutamine, L-β-homoisoleucine, L-β-homoleucine, L-β-homolysine, L-β- homomethionine, L-β-homophenylalanine, L-β-homoproline, L-β-homoserine, L-β- homothreonine, L-β-homotryptophan, L-β-homotyrosine, L-β-homovaline, 3-amino- phenylpropionic acid, 3-amino-chlorophenylbutyric acid, 3-amino-fluorophenylbutyric acid, 3-amino-bromopheynyl butyric acid, 3-amino-nitrophenylbutyric acid, 3-amino- methylphenylbutyric acid, 3-amino-pentanoic acid, 2-amino-tetrahydroisoquinoline acetic acid, 3-amino-naphthyl-butyric acid, 3 -amino-pentafluorophenyl -butyric acid, 3-amino- benzothienyl-butyric acid, 3-amino-dichlorophenyl-butyric acid, 3-amino-difluorophenyl- butyric acid, 3-amino-iodophenyl-butyric acid, 3-amino-trifluoromethylphenyl-butyric acid, 3-amino-cyanophenyl-butyric acid, 3-amino-thienyl-butyric acid, 3-amino-5- hexanoic acid, 3-amino-furyl-butyric acid, 3-amino-diphenyl-butyric acid, 3-amino-6- phenyl-5-hexanoic acid and 3-amino-hexynoic acid.
As used herein, the term "hydrophobic amino acid" refers to an amino acid with a hydrophobic side chain or no side chain. Suitable hydrophobic amino acids include, but are not limited to, glycine, L-alanine, L-valine, L-phenylalanine, L-isoleucine, L-leucine, L-methionine, L-tyrosine, D-valine, D-phenylalanine, D-isoleucine, D-leucine, D-methionine, D-tyrosine, L-β-homophenylalanine, L-β-homoisoleucine, L-β- homoleucine, L-β-homovaline, L-β-homomethionine, L-β-homotyrosine, cyclohexylalanine, L-norleucine and L-norvaline. Preferred hydrophobic amino acids are glycine, L-valine, L-phenylalanine, L-isoleucine and L-leucine, especially L-valine and glycine.
In some embodiments, one or more of the His, Thr or Asp amino acids in the His-Thr-Asp sequence may be non-naturally occurring His, Thr or Asp. For example, the His, Thr or Asp may be D-amino acids or may be derivatised, for example by N-alkylation such as N-methylation or α-alkylation such as α-methylation. Examples of derivatised His, Thr and Asp include, but are not limited to, N-methyl-His, N-methyl-Thr, N-methyl-aspartic acid, α-methyl-histidine, α-methyl-threonine or α-methyl-aspartic acid. In preferred embodiments, the His, Thr and Asp are L-amino acids, and are underivatised.
The zinc ions in the composition are preferably zinc (II) ions (Zn2+). The zinc ions may be added to the composition in the form of a salt, in particular, a salt which is pharmaceutically acceptable. For example, suitable zinc salts that provide zinc (II) ions in the composition include, but are not limited to, zinc chloride (ZnCl2), zinc sulfate (ZnSO4), zinc picolinate, zinc citrate, zinc acetate, zinc glycerate and zinc monomethionine, especially zinc chloride. In preferred embodiments, the ratio of zinc ions to peptide in the composition is 1 :1.
In embodiments of the invention in which the peptide and the zinc ions are present in a 1 : 1 ratio, the peptide and zinc ions may form a complex in which negative charges on the peptide interact with the positive charge on the zinc ion. In some embodiments, the complex is formed at a pH greater than 6 where the imidazole ring on the histidine in the peptide is charged.
In an especially preferred embodiment, the complex is formed between the tetrapeptide Val-His-Thr-Asp and Zn ions. In this embodiment, the complex has a circular dichroism (CD) profile with minima at 208 and 222 nm when measured in MiIIiQ water or biological buffer at pH greater than 6 and 2O0C. In particular, the complex has a CD profile as shown in Figure IB.
As used herein, the term "biological buffer" refers to any buffer commonly used to mimic biological conditions, such as biological pH. Examples of suitable biological buffers include, but are not limited to, HEPES, MOPS and Tris buffers.
The peptides incorporated in the compositions and complexes of the invention as described above may be synthesised using conventional liquid or solid phase synthesis techniques. For example, reference may be made to solution synthesis or solid phase synthesis as described in Chapter 9, entitled "Peptide Synthesis" by Atherton and Shephard, which is included in the publication entitled "Synthetic Vaccines" edited by Nicholson and published by Blackwell Scientific Publications. Preferably, a solid phase peptide synthesis technique using Fmoc chemistry is used, such as the Merrifield synthesis method (Wellings & Atherton; Merrifield).
Alternatively, these peptides may be prepared as recombinant peptides using standard recombinant DNA techniques. Thus, a recombinant expression vector containing a nucleic acid sequence encoding the peptide and one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed may be introduced into and expressed in a suitable prokaryotic or eukaryotic host cell, as described, for example, in Gene Expression Technology: Methods in Enzymology, 185, Academic Press, San Diego, CA (1990), and Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).
The peptide Gly-His-Thr-Asp may also be isolated from human urine by standard protein purification procedures, preferably using reversed-phase high performance liquid chromatography (RP-HPLC). Using these procedures, Gly-His-Thr-Asp is obtained in isolated form. By "isolated" is meant a peptide material that is substantially or essentially freed from components, particularly other proteins and peptides, that normally accompany it in its native state in human urine by at least one purification or other processing step.
Such isolated peptide may also be described as substantially pure. The term "substantially pure" as used herein describes peptide material that has been separated from components that naturally accompany it. Typically, peptide material is substantially pure when at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% or even 99% of the total peptide material (by volume, by wet or dry weight, or by mole percent or mole fraction) is the peptide of interest. Purity can be measured by any appropriate method, for example, in the case of peptide material, by chromatography, gel electrophoresis or HPLC analysis. While it is possible that, for use in therapy, the combination of peptide and zinc ions may be administered without other additives, it is preferable to present the combination together with one or more pharmaceutically acceptable carriers and/or diluents, and optionally other therapeutic and/or prophylactic agents. The carriers and/or diluents must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient.
As used herein, the term "combination of peptide and zinc ions" may refer to a composition comprising the peptide and zinc ions or a complex comprising the peptide and zinc ions. In relation to methods of administration for therapy, the term ."combination of peptide and zinc ions" includes administration of a composition or complex of the invention and also includes separate administration of a composition containing the peptide and a composition containing zinc, either simultaneously or sequentially, such that the peptide and zinc ions may become associated with each other, for example by formation of a complex, in vivo after administration.
The formulation of such therapeutic compositions is well known to persons skilled in this field. Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art, and it is described, by way of example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as any conventional media or agent is incompatible with the active ingredients, use thereof in the pharmaceutical compositions of the present invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
It is especially advantageous to formulate such compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the human or other mammalian subjects to be treated; each unit contains a predetermined quantity of active ingredients calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and/or diluent. The specifications for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active ingredients and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active ingredient for the particular treatment.
The present invention also extends to methods of regulating in vivo blood glucose levels in a human or other mammal by administering to the human or other mammal, a combination of peptide and zinc ions of the invention as described above.
As used herein, the term "human or other mammal" refers to humans and other warm blooded animals that may require regulation of blood glucose. For example, mammals includes domesticated animals such as dogs, cats, horses and the like, livestock animals such as cattle, sheep, pigs and the like, laboratory animals such as mice, rats, rabbits and the like, and captive animals such as those animals held in zoos. In a preferred embodiment, the subject is a human.
In this aspect of the present invention, without wanting to be bound by theory, the peptide is capable of exerting its therapeutic hypoglycaemic effect and also allows delivery of zinc to cells where the zinc can also contribute to the insulin-enhancing effects of the peptide.
In some aspects of the invention the methods of regulating in vivo blood glucose levels is used in a method of treating diabetes, in a human or other mammal. Both Type 1 diabetes and Type 2 diabetes are associated with reduced plasma zinc concentrations (Chausmer; Ripa; Anderson et al), although it is not clear whether this is a contributing factor to the disease or whether it is a consequence of hyperglycaemia.
In Type 1 diabetes there is a lack of insulin production. This is because the beta cells of the Islets of Langerhans in the pancreas have been destroyed, most often by autoimmune-mediated destruction. Those subjects with Type 1 diabetes require treatment with insulin to replace the insulin that would normally be produced in the pancreas. Since insulin is not produced, a subject with untreated or poorly controlled Type 1 diabetes will have hyperglycaemia.
In Type 2 diabetes, at least at the beginning of the disease, the pancreatic islet cells are capable of making large quantities of insulin. The transport of glucose across a cellular membrane is stimulated by insulin binding to its insulin receptor as part of an insulin signalling pathway. However, in Type 2 diabetes, the insulin signalling pathway malfunctions, a condition called insulin resistance. Although there may be an abundance of insulin in the circulation, there is insufficient transport of glucose into cells and excess glucose production by the liver. This may cause not only hyperglycaemia but also hyperinsulinemia. As the disease progresses, there may be down-regulation of the insulin receptors and possibly even exhaustion of the beta cells. Once the beta cells are exhausted the amount of insulin produced may be too low or may stop and treatment with exogenous insulin may be required temporarily or possibly permanently.
For those forms of diabetes in which insulin is not required, the combination of peptide and zinc ions, may be administered without other therapeutic agents or may be combined with, in a single composition, or administered separately, simultaneously or sequentially with insulin-sensitising agents or compounds that increase insulin secretion from β -cells. Suitable insulin-sensitising agents include, but are not limited to, Metformin (Glucophage™) and thiazolidinediones (also known as glitizones) such as Avandia™ (rosiglitazone) by GlaxoSmithKline and Actos™ (pioglitazone) by Takeda/Eli Lilly. Suitable compounds that increase insulin secretion from β-cells include, but are not limited to, sulfonylureas and meglitinides.
For those forms of diabetes, either Type 1 or Type 2, in which insulin is required, the combination of peptide and zinc ions may be administered in a single composition or administered separately, simultaneously or sequentially with insulin. The insulin may be in fast-acting or slow-acting form. Furthermore, the peptide, zinc ions and insulin combination may be administered in a single composition or administered separately, simultaneously or sequentially with insulin-sensitising agents such as metformin and thiazolidinediones such as rosiglitazone and pioglitazone.
The combination of peptide and zinc ions may also reduce, prevent or slow the progression of complications associated with diabetes. Such complications include cardiovascular disease and associated complications such as diabetic dyslipidemia; high blood pressure (hypertension); neuropathy and nerve damage; kidney disease; and eye diseases such as glaucoma, cataracts and retinopathy.
A variety of administration routes are available. The particular mode selected will depend, of course, upon the particular condition being treated and the dosage required for therapeutic efficacy. The methods of this invention, generally speaking, may be practised using any mode of administration that is medically acceptable, meaning any mode that produces therapeutic levels of the active components of the invention without causing clinically unacceptable adverse effects. Such modes of administration include parenteral (e.g. subcutaneous, intramuscular and intravenous), oral, rectal, topical, nasal and transdermal routes.
The active component may conveniently be presented in unit dosage form and suitable compositions for administration may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing the active components into association with a carrier and/or diluent which may include one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active components into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
Compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active components which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in polyethylene glycol and lactic acid. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active component, in liposomes or as a suspension in an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, or an emulsion.
Other delivery systems can include sustained release delivery systems. Preferred sustained release delivery systems are those which can provide for release of the active components of the invention in sustained release pellets or capsules. Many types of sustained release delivery systems are available; these include, but are not limited to: (a) erosional systems in which the active components are contained within a matrix, and (b) diffusional systems in which the active components permeate at a controlled rate through a polymer.
The present invention extends to methods of delivering zinc to cells and/or increasing cellular response to zinc ions by exposing the cells to the combination of peptide and zinc ions of the invention as described above.
By "delivering zinc into cells" is meant that zinc is transported across the cellular membrane into the cells by zinc transport systems expressed by cells. The delivery or transport of the zinc may be enhanced by the presence of the peptide allowing more zinc to be transported into the cell.
By "increasing cellular response to zinc ions" is meant that the function of the cell is improved or upregulated. For example, increasing cellular response includes increasing or upregulating metabolism in a cell. Increased metabolism can be measured, for example, by the acid production of the cells.
The cells may be in vivo or in vitro. When the cells are in vitro, the cells may be cells cultured for the purposes of experiment that also requires zinc ions to be present in the cells. Alternatively, the zinc ions may be required in the cells for the purposes of detection in an assay.
When the cells are in vivo, the method of delivering zinc into cells may be used to treat or prevent zinc deficiency, whether or not the deficiency is associated with diabetes, or alleviate the symptoms of zinc deficiency in humans or other mammals. The symptoms of zinc deficiency include loss of appetite, poor growth, weight loss, impaired taste and smell, poor wound healing, skin abnormalities such as acne, atopic dermatitis and psoriasis, hair loss, lack of menstruation, night blindness, hypogonadism and delayed sexual maturation, white spots on the fingernails and feelings of depression.
Zinc deficiency also tends to make a subject more susceptible to a variety of infections. Zinc supplementation can enhance the immune system and protect against a range of infections including colds and upper respiratory infections.
A recommended dietary allowance of zinc is between 2-8 mg for children between birth and 13 years of age and between 8-11 mg for people 14 years of age and over. The requirements are on the higher side for children between the ages of 14 to 18 years and also for pregnant women and breastfeeding women. For zinc supplementation and immune system enhancement, an effective amount of elemental zinc is between 30 and 60 mg per day.
The active components are administered in therapeutically effective amounts. A therapeutically effective amount means that amount necessary at least partly to attain the desired effect, or to delay the onset of, inhibit the progression of, or halt altogether, the onset or progression of the particular condition being treated. Such amounts will depend, of course, on the particular condition being treated, the severity of the condition and individual patient parameters including age, physical condition, size, weight and concurrent treatment. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.
Generally, daily oral doses of active components will be from about 0.01 mg/kg per day to 1000 mg/kg per day. Small doses (0.01-1 mg) may be administered initially, followed by increasing doses up to about 1000 mg/kg per day. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localised delivery route) may be employed to the extent patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.
Generally, when orally delivering zinc to cells for treating or preventing zinc deficiency or enhancing the activity of the immune system in a human, a maximum of 150 mg of elemental zinc is administered to a patient per day, preferably the daily dosage of the combination will result in the administration of less than 100 mg of elemental zinc per patient per day. In preferred embodiments for this method, the amount of elemental zinc administered as part of the combination of the invention is between 20 mg and 70 mg elemental zinc per patient per day, especially 30 mg to 60 mg elemental zinc per patient per day, especially about 50 mg elemental zinc per patient per day. A healthcare provider could readily determine the amount of a composition containing the combination of the invention to administer these amounts of zinc per day or suitable amounts of each component separately to provide a suitable ratio of peptide to zinc, for example a 1 :1 ratio.
Generally, when delivering zinc to cells together with the peptide to provide a hypoglycaemic effect, the amount of combination delivered is determined by the amount of hypoglycaemic effect required. Small doses (0.01-1 mg) may be administered initially and doses may be increased as required. In preferred embodiments, an oral dosage of the combination of the invention will deliver the peptide in an amount of 0.1-100 mg/kg per day. A healthcare provider could readily determine the amount of a composition containing the combination of the invention or separate dosage forms of the peptide and the zinc in the appropriate ratio to provide the required hypoglycaemic effect.
In some instances where higher amounts of zinc are being administered, for example, greater than 50 mg of elemental zinc per day, the composition may further comprise copper ions. The ratio of zinc ions to copper ions may be in the range of 10:1 to 20:1, especially about 15:1. The source of copper ions may be from any suitable pharmaceutically acceptable salt of copper, for example, copper glycinate.
At lower doses, the combination of peptide and zinc ions has a hypoglycaemic effect, but only in those with insulin resistance and/or diabetes. In those with normal insulin sensitivity the peptide exerts a minimal hypoglycaemic effect. However, at higher doses, over 100 mg/kg per day, the peptide and zinc combination do not have a hypoglycaemic effect, but the combination retains the ability to transport zinc into cells. Therefore, the combination of the invention may be used to treat zinc deficiency or enhance immune system activity in patients that are not hyperglycaemic.
Further features of the present invention are more fully described in the following Example(s). It is to be understood, however, that this detailed description is included solely for the purposes of exemplifying the present invention, and should not be understood in any way as a restriction on the broad description of the invention as set out above.
Description of the Figures
Figure IA provides a CD spectrum of ISF402 (Val-His-Thr-Asp).
Figure IB provides a CD spectrum of ISF402 after addition of ZnCl2 to 2 mM.
Figure 1C provides a CD spectrum of ISF402 and ZnCl2 to 2 mM after addition of a chelating agent EGTA. Figure 2 graphically represents the titration of ISF402 (320 μM) with ZnCl2 (0-680 μM) as measured by the magnitude of the CD signal at 220 nm.
Figure 3 A graphically represents a dose response curve for C2C12 muscle cells exposed to insulin. Cellular response increased with increasing insulin concentrations over the range of 10"3 to 10OnM. High concentrations, above 10OnM, decreased the cellular response. Results are mean of the percentage maximum stimulation +/- SD.
Figure 3B graphically represents a dose response curve for C2C12 muscle cells exposed to ISF402. Similarly to insulin, cellular response increased with increasing concentrations of ISF402 in the range of 10"3μM to 20μM, and decreased at concentrations above 20μM. The graph shown is representative of 4 independent experiments.
Figure 3C graphically represents the sensitisation of C2C12 muscle cells to insulin by ISF402. The response of C2C12 cells to increasing insulin concentrations with constant ISF402 concentrations in the cytosensor. Exposure of cells to increasing insulin concentrations (closed diamonds) with a constant concentration of ISF402 (0.1 μM) (open squares) enhanced the cellular response particularly at low doses of insulin. Similarly, exposure of cells to increasing ISF402 concentrations with a constant insulin concentration also enhanced the cellular response (data not shown). 0.1 μM ISF402 alone results in an Extracellular Acidification Rate (ECAR) of 5.69% +/- 0.93 SEM. Results are mean ECAR (% of basal) +/- SEM where n=3 for each experiment. The graph shown is representative of 4 independent experiments.
Figure 3D graphically represents a dose response curve for C2C12 cells exposed to increasing concentrations of ZnCl2 with (circles) or without (squares) 10 μM ISF402 as measured by microphysiometry. The data are the mean ± SEM for 3 determinations.
Figure 4 provides a photographic representation of transport of ISF402 into cells. C2C12 cells grown on coverslips, fixed and incubated with the anti-ISF402 and anti-insulin antibody. Second antibodies were anti-rabbit conjugated to Alexa568 and anti-guinea pig conjugated to FITC. Confocal image illustrates a nuclear sybr green stain showing no staining with pre-immune serum A. Cultured muscle cells treated with insulin (green) B and ISF402 (red) C enter the cell and co-localise (yellow) D. Images obtained using Leica TCSNT7DMRBE confocal microscope (*63 objective). A second set of images of C2C12 cells were obtained using an Olympus FV500 confocal microscope (xlOO objective). The four panel image illustrates a DAPI nuclear stain (blue) of cells treated with ISF402 (red) E and insulin (green) F demonstrates entry into cells and co-localisation (yellow) G. H shows a DIC image of the cells. Scale bar is 20μm.
Figure 5A provides photographic representation of transport of zinc into cells by ISF402. Live C2C12 cells loaded with FluoZin-3 and equilibrated with 100 μM ZnCl2 were photographed every 30 seconds. After 1.5 minutes, either FSI (Asp-Thr-His-Val) (negative control, top panel) or ISF402 (bottom panel) was added to the media. Images were artificially coloured according to intensity with the least intense being green (background), intermediate intensity by shades of yellow to red and most intense by black. Addition of FSI had no effect on fluorescence intensity (top.panel) whereas fluorescence in the cell increases after addition of ISF402 (bottom panel). The increasing spread of fluorescence intensity around the cell reflects increasing intensity of fluorescence from the cell refracting through the culture media.
Figure 5B graphically represents the quantitation of cellular fluorescence for cells shown in Figure 5 A equilibrated in the presence of 100 μM ZnCl2 and two other cells. Background was measured at the edge of the image and subtracted from the cellular fluorescence and the data expressed as the change in integrated optical density (IOD) ISF402 (closed symbols) and FSI (open symbols) were added between frames 3 and 4. Thirty frames were photographed at 30 second intervals (total time: 15 minutes).
Figure 5C graphically represents the quantitation of cellular fluorescence for cells equilibrated in the presence of 20 μM ZnCl2 and two other cells. Background was measured at the edge of the image and subtracted from the cellular fluorescence and the data expressed as the change in integrated optical density (IOD) ISF402 (closed symbols) and FSI (open symbols) were added between frames 3 and 4. Thirty frames were photographed at 30 second intervals (total time: 15 minutes).
Figures 6A and B graphically demonstrate that the stimulation of C2C12 cells by insulin or by ISF402 is inhibited by wortmannin. C2C12 cells were pre-treated with 5OnM Wortmannin (PI3-kinase inhibitor) (closed squares) prior to treatment with either insulin or ISF402 and cellular response was then measured in the cytosensor. Wortmannin decreased the cellular response to both insulin (A) and ISF402 (B). Results are expressed as Extracellular Acidification Rate (ECAR) % of basal. Values are mean +/- SEM, where n=4 or 5.
Figure 7 shows that insulin and ISF402 activate PI3 -kinase and increase Akt phosphorylation in C2C12 cells. Western blot analysis of C2C12 lysates treated with insulin and ISF402 in the presence of Wortmannin. (A) Akt phosphorylation decreased in the presence of Wortmannin in insulin treated cells, however the total PI3-kinase levels remained the same across all concentrations (B). (C) Cells treated with ISF402 in the absence of Wortmannin showed an increase in Akt phosphorylation with increasing concentration of ISF402. In the presence of Wortmannin however, the Akt phosphorylation was inhibited. (D) Total PI3-kinase levels remained the same across all concentrations.
Figure 8A provides a CD spectrum of GHTD-amide and the effects of zinc. Zinc induces a conformational change in GHTD-amide. GHTD-amide was dissolved in milliQ water at lmg/ml and pH was raised to 7.4. Far UV CD spectra for GHTD-amide was recorded in the absence (dotted line) and the presence (solid line) of zinc sulphate (2μM). The CD profile shows a reduction around 220 nm, which is consistent with increased alpha helix formation.
Figure 8B graphically represents the titration of GHTD-amide with zinc as measured by the magnitude of the CD signal at 220 nm. The change in CD signal at 220 nm in mdeg was monitored at various zinc ion concentrations and the percent change in signal plotted against zinc concentration. The affinity of interaction was 102 ±11 μM and the reaction was saturated at a 1:1 ratio of zinc to peptide.
EXAMPLES
Peptides
ISF402 (Val-His-Thr-Asp-NH2), the reverse peptide designated FSI (Asp-Thr-His-Val-
NH2) and Gly-His-Thr-Asp-NH2 (GHTD-amide) were synthesised by standard protein synthetic methods. The ISF402 peptide was prepared by solid phase synthesis using F-Moc protection for Asp-NH2 (isoasparagine), Thr, His and VaI. FSI and GHTD-amide were also prepared using solid phase synthesis and standard F-Moc chemistry. Peptides were >95% pure as determined by reverse phase high performance liquid chromatography (RP-HPLC).
Circular dichroism
ISF402 was dissolved at a concentration of 1 mg/mL in MiIIiQ water and the pH was raised to 7.4 by the addition of sodium hydroxide (NaOH). Circular dichroism (CD) spectra were measured from 190-250 nm at 200C on a Jasco J-810 spectropolarimeter equipped with a PFD 423/L Peltier type temperature controller. 200 μL of sample was placed in a quartz cuvette, with a path length of 1 mm, in the spectropolarimeter and the CD spectrum was recorded. Each spectrum represents an average of 3-5 scans performed at 100 nm/min with a band width of 1 nm. The effect of zinc ions was measured by adding an aliquot of stock solution of ZnCl2 to a solution of ISF402 (to a concentration of 2 mM) and measuring the CD spectrum.
Similar experiments were performed with GHTD-amide but the spectra were recorded from 178-240 nm. Microphysiometry
The cytosensor microphysiometer measures the cellular metabolic activity of isolated cells in terms of their rate of production of hydrogen ions. The cytosensor measures the extracellular acidification rate in μvolts s"1 as described previously (Hutchinson et al). Briefly C2C12 cells at passage 10 were seeded onto 12mm transwell inserts (3μm pore size) (Corning Life Sciences, NY, USA) and differentiated into myotubes. 10 days after differentiation the cells were serum deprived in 0.5% fetal bovine serum overnight. The cells were then placed in a Cytosensor (Molecular Devices) in non-buffered pH sensitive RPMI 1640 media. Cells were initially perfused with media for approximately 2 hours to stabilise baseline extracellular acidification rate (EAR) before cumulative concentrations of test sample were added. Flow was stopped for 40 seconds at the end of each 2 minute pump cycle. During this period the rate of acidification (μvolts s'1) was measured for 30 seconds. Once the cells were equilibrated at 370C with RPMI 1640 media (zero control), ISF402, insulin or ZnCl2 at increasing concentrations were added for a duration of 20 minutes, for each treatment. The Cytosensor measured the change in pH. as a response of cells to the treatment. Baseline acidification rates were normalized using cytosoft software (Molecular Devices, Sunnyvale, CA, USA) prior to production of cumulative concentration response curves.
For inhibitor studies, cells were treated with Wortmannin (PI3-kinase specific inhibitor) (Sigma) at 50 nM for 30 minutes prior to treatment with insulin or ISF402.
Confocal Immunofluorescence Microscopy
C2C12 cells were plated on glass coverslips. After confluency, cells were serum deprived for 24 hours and treated either with ISF402, insulin or a mixture of ISF402 and insulin. Cells were washed with Phosphate buffered saline (PBS) and fixed with 10% formaldehyde for 20 min, washed again and made permeable in 0.1% Triton X-100 for 30 min at room temperature. Cells were blocked with 2% gelatin for 45min at room temperature and incubated for 1 hour with either guinea pig anti-insulin (1:50 Dako, Carpinteria, CA, USA), rabbit anti-ISF402 (1 :500) or both. Coverslips were washed four times in PBS and incubated for 1 hour at room temperature with secondary antibodies FITC anti-guinea pig IgG conjugates (1 :200 Dako, Caφinteria, CA, USA), and Alexa 568 anti-rabbit IgG conjugates (1 :200 Molecular Probes, Eugene, OR, USA). Coverslips were washed four times with PBS and mounted on glass slides with Dako mounting medium. Confocal images were obtained using a Leica TCSNT7DMRBE confocal microscope (x 63 lense) (Leica Microsystms Pty, Ltd. NSW Australia).
Detection of Zinc by FluoZin-3 in C2C12 muscle cells
The effect of ISF402 on cellular zinc was analysed in C2C12 cells using FluoZin-3
(Molecular Probes, Eugene, OR, USA). FluoZin-3 is a fluorescent probe with a peak excitation wavelength at 494 nm and a peak emission at 516 nm that increases when bound to zinc. FluoZin-3 (Ka[Zn]=I 5 nM) is useful for measurement of zinc in the 1 to 100 nM range, which encompasses the range of free zinc concentration normally found in cells. Detection of free zinc in C2C12 cells by FluoZin-3 was performed as described previously (Haase and Maret; Qian et ah). Briefly, cells were grown and differentiated into myotubes in 12 well plates (Nalge Nunc International, Rochester, NY, USA). Cells were serum deprived for 6h and loaded with 2μM cell permeant FluoZin-3 AM ester at 280C for 30min in phenol red free DMEM. Cells were washed twice with phenol red free DMEM and incubated for an additional 30min to allow complete de-esterification. Cells were then maintained at 280C on a heated temperature controlled stage on the microscope. Relevant positive and negative controls were initially performed to establish appropriate conditions for FluoZin-3 measurement of zinc in C2C12 cells. As a positive control the ionophore pyrithione (Sigma-Aldrich, St. Louis, CA, USA) at 50μM was added with Zn2+. A high affinity Zn2+ chelator, tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) (Molecular Probes, Eugene, OR, USA) at 50μM was added to FluoZin-3 loaded cells as a negative control. Time-lapse photography of the FluoZin-3 loaded cells was performed using an Olympus 1X81 Inverted Microscope, using a filter for fluoroscein isothiocynate. Images were taken with a Hamamatsu ORCA-ERA camera and integrated through the CellΛR program software. Images were captured every 30sec for 15 min. After the initial 1.5min (frame 3) cells were either treated with ISF402 (lOμM) or a reverse peptide FSI (lOμM) as a negative control, by direct addition to the media. Densitometry of the fluorescence emitted was performed using MCID software (Imaging Research Inc, Ontario, Canada). Cells in the images were outlined and integrated optical density (IOD) across the whole cell measured. Background measurements were performed on a region in the same image that was free of cells and subtracted to give the change in IOD.
Immunoblot Analysis
C2C12 cells at passage 10 were seeded onto 10cm dishes and differentiated into myotubes for 7 days. Cells were then serum starved overnight in 0.5% FBS DMEM (Gibco BRL) and pre-treated with the PI3-kinase inhibitor Wortmannin (Sigma) at 5OnM for 30 min at 370C. Cells were then treated with Insulin (Sigma) at varying concentrations or ISF402 for 20 minutes at 370C. Cells were then washed twice with ice cold PBS and lysed in the presence of Lysis buffer (5OmM Tris/HCl, pH 7.4, 15OmM NaCl, l%(v/v) Triton X-100, ImM EGTA, 2mM EDTA) containing protease inhibitors (ImM AEBSF, ImM Na3VO4, ImM NaF and lug/ml of Leupeptin, Pepstatin A, Aprotinin) (Sigma). Cells were centrifuged at 14K at 40C for 15 minutes. Protein assay was performed on all lysates and normalised across all samples. The cell lysate was then subjected to protein assay and analysed via SDS electrophoresis.
Aliquots of protein were resolved by SDS-PAGE using the Bio-Rad mini Protein II system (10% polyacrylamide gels), transferred to PVDF, blocked with 1% BSA (Sigma) in PBS- Tween(Sigma) and incubated with Anti phospho-Akt (Ser 473) antibody (Cell Signalling) or Anti-PI3 kinase p85 antibody (Cell Signalling). The membranes were then washed and incubated with horseradish peroxidase conjugated anti-rabbit IgG secondary antibody (Chemicon) and proteins were visualized by enhanced chemiluminescent detection reagents (Amersham) on Hypersensitive Film (Amersham).
Example 1
Addition of zinc changes the structure of ISF 402 Circular dichroism spectroscopy is a form of light absorption spectroscopy which measures the difference in the absorbance of right-handed and left-handed polarised light by a substance. The CD spectrum obtained from a native protein and that from a denatured sample of the same protein will appear quite different. Therefore, CD spectroscopy is a useful technique to monitor changes in protein conformation, enabling studies to be made of protein folding and refolding under a variety of conditions. Circular dichroism analysis (190-250 nm) of ISF402 in MQ water, pH 6-7, showed the presence of a more positive region (compared to neighbouring regions) at 216-222 nm (Figure IA). The CD profile of ISF402 shown in Figure IA is typical of a random coil where there is no favoured secondary structure. The value of CD[mdeg] at 220 nm was reduced (from - 1.0 to — 22.0) after the addition of zinc chloride to a concentration of 2 mM (Figure IB), producing a CD profile closely resembling the negative peak characteristic of an alpha helix with minima at 208 nm and 222 nm. The chelator ethyleneglycol-bis(beta-aminoethyl ether)-N,N'- tetraacetic acid (EGTA) was then added and the CD spectrum showed full restoration of the positive region at 216-222 nm (Figure 1C), indicating that ISF402 reverted to a random coil upon removal of Zn2+. These results confirm that the folding of the ISF402 peptide chain is altered by the presence of Zn2+. This alteration in the structural form of ISF402 is due to the presence of Zn2+ since addition of EGTA restores the random coil form of the molecule, as shown by the increase in the value of the CD[mdeg] to +0.9.
Example 2 Deprotonation of Histidine is required for zinc to induce change in ISF402 structure
ISF402 contains a histidine residue. The imidazole side chain of histidine is involved in binding transition metals in many proteins, but only when the imidazole is deprotonated, which generally occurs above pH 6.8. To test the effect of pH on the Zn2+ interaction with ISF402 the CD signal at 220 nm was obtained from ISF402 dissolved in MQ water at pH 3 to pH 7 (Table 3). The negative peak at 220 nm does not occur at acidic pH and only appears when the pH reaches 6-7. This is consistent with a requirement for the histidine side chain to be deprotonated for Zn2+ to induce a change in the structure of ISF402. Table 3: Effect of varying pH on the CD signal at 220 nm from ISF402
Figure imgf000037_0001
Example 3
Each molecule ofISF402 binds to one molecule of zinc The stoichiometry of zinc and ISF402 in the zinc-ISF402 complex was determined by measurement of the CD signal at 220 nm wavelength at increasing zinc concentrations. A 320 μM solution of ISF402 was incubated at 370C with concentrations of ZnCl2 ranging from 0 to 680 μM and the CD signal at a wavelength of 220 nm was determined. The data were fitted using CaLigator software (http://www.bpc.lu.se/staff/personal/ingemar andre.html) (Figure 2). The Kd for zinc binding determined from the fitted curve is 27.0 + 3.5 μM and the stoichiometry of binding is 1 :1 for ISF402:Zn2+.
Example 4 ISF 402 facilitates an increase in cellular response to increasing zinc concentrations
Microphysiometry was used to examine the cellular effects of ISF402 as this technique allows for real-time determination of cellular responses. Insulin and ISF402 both increased extracellular acidification rate (EAR) in C2C12 muscle cells (Figure 3A and B). The cellular response to both agents was "bell shaped". EAR increased with increasing insulin concentrations over the range of 10"3 to 10OnM but decreased at concentrations above
10OnM (Figure 3A). Similarly, EAR increased with increasing concentrations of ISF402 in the range of 10"2μM to lOμM, and decreased at concentrations above 20μM (Figure 3B). To test for insulin sensitisation by ISF402 a concentration of ISF402 (0.1 uM) that caused a sub-maximal stimulation of the cells (12%) was chosen. Exposure of cells to increasing insulin concentrations with a constant 0.1 uM concentration of ISF 402 enhanced the cellular response particularly at low, physiologically relevant insulin concentrations (Figure 3C). Similarly, exposure of cells to increasing ISF 402 concentrations with a constant insulin concentration also enhanced the cellular response with the combined treatment (data not shown).
ISF402 activates the insulin signalling pathway, which is a property shared by zinc. To explore whether zinc binding by ISF402 is associated with the insulin-like activity of ISF402 microphysiometry was used. ISF402 stimulates metabolism in muscle cells to produce a change in acid secretion which was detected by the microphysiometer. Addition of zinc (ZnCl2) at 1 μM also stimulated muscle cells but increasing this to 50 μM did not significantly increase the cellular response further (Figure 3D) with the response at 1 μM ZnCl2 (6.2 ± 2.0 μVolts/sec) not significantly different to the response at higher ZnCl2 concentrations (e.g. 50 μM ZnCl2 9.2 ± 2.7, p = 0.4, Student's t-test). When ISF402 was present, the response significantly increased with increasing ZnCl2 (7.1 ± 1.8 at 1 μM ZnCl2, 12.4 ± 3.6 at 50 μM ZnCl2, p = 0.05, Student's t-test, and Figure 3D). These results are consistent with ISF402 facilitating an increased cellular response to zinc in the C2C12 muscle cells.
Example 5
ISF 402 readily enters C2C12 cells ISF402 may potentiate its biological actions by entering cells. C2C12 cells grown on coverslips, fixed and incubated with the anti-ISF402 and anti-insulin antibody. Second antibodies were anti-rabbit conjugated to Alexa568 and anti-guinea pig conjugated to FITC. Confocal image illustrates a nuclear sybr green stain showing no staining with pre-immune serum, Figure 4A. Cultured muscle cells treated with insulin (green), Figure 4B, and ISF402 (red), Figure 4C, enter the cell and co-localise (yellow), Figure 4D. Images obtained using Leica TCSNT/DMRBE confocal microscope (X63 objective). A second set of images of C2C12 cells were obtained using an Olympus FV500 confocal microscope (xlOO objective). The four panel image illustrates a DAPI nuclear stain (blue) of cells treated with ISF402 (red), Figure 4E, and insulin (green), Figure 4F, demonstrates entry into cells and co-localisation (yellow), Figure 4G. Figure 4H shows a DIC image of the cells. Scale bar is 20μm. Confocal images of C2C 12 cells treated with insulin, ISF402 and a mixture of insulin and ISF402, show that insulin (Figure 4A) and ISF402 (Figure 4B) can enter cells. Treatment of cells with both insulin and ISF402 showed co- localisation of insulin with ISF402 (Figure 4C) inside the cell.
Example 6
ISF402 increases cellular zinc concentrations
ISF402 may facilitate an increasing cellular response to increasing Zn2+ concentrations by increasing the cellular uptake of Zn2+. To test this, either 10 μM ISF402 or FSI (ISF402 peptide with the sequence of amino acids reversed) were added to cells loaded with FluoZin-3 and equilibrated with 100 μM ZnCl2. The cells showed a level of background fluorescence due to the quantities of Zn2+ that normally reside in cells. Upon the addition of FSI no increase in the fluorescent signal was apparent (Figure 5A, top panel), whereas ISF402 caused a measurable increase in FluoZin fluorescence (Figure 5A, lower panel). Quantitation of the fluorescence intensity across the whole cell confirmed that ISF402 increased cellular zinc whereas when FSI was added fluorescence decreased over time, probably due to photo-bleaching of the background fluorescence (Figure 5B). Similar data were obtained for cells equilibrated with 20 μM ZnCl2 prior to addition of peptides (Figure 4C) however the response between cells was more variable. These results show that ISF402 can facilitate transport of zinc from the culture media into the cells.
Discussion for Examples 1 to 6
It has been reported that the transition metal zinc shows insulin-like effects in many different systems. The binding of zinc ions is also important for the insulin-like and insulin-sensitising effects of ISF402 and other hypoglycaemic peptides of formula (I). Zinc is an important transition metal since it is the second most common transition metal found in the human body (after iron), with 2.3 gm in an average 70 kg person (McCaIl et cd.).
ISF402 and other hypoglycaemic peptides of formula (I) bind zinc ions to form metal- peptide complexes. Histidine is involved in the complexation of the hypoglycaemic peptides with zinc as supported by the observation that zinc ions only induced changes in
ISF402 secondary structure at pH values above 6 when the imidazole ring of histidine would be uncharged. The complex formed when a peptide solution is fully saturated with zinc ions is one molecule of zinc for every molecule of hypoglycaemic peptide. The interaction between zinc ions and ISF402 occurred at physiological temperature and pH with a dissociation constant of 27 μM, which is close to the concentration of free zinc in blood (15 μM). Therefore, a proportion of zinc would be bound to ISF402 in blood.
The studies in C2C12 muscle cells shows that peptides such as ISF402 facilitate an increase in cellular response to zinc at zinc concentrations from 1 to 50 μM. Furthermore, there is an interplay between zinc ions and the peptides that results in increased transport of zinc into cells in the presence of peptides such as ISF402. Since these cellular effects occurred at concentrations of zinc that are close to physiological concentrations, it is likely that peptides such as ISF402 can facilitate uptake of zinc by cells in vivo. Increasing zinc concentrations within cells will maximise the insulino-mimetic effects of zinc and minimise other effects resulting from zinc deficiency.
Example 7
To test whether stimulation of C2C12 cells by ISF402 utilised the insulin signalling pathway the PI3-kinase inhibitor wortmannin was used. A concentration of wortmannin
(5OnM) that only partially inhibits PI3 -kinase activity was used to minimise effects on cell viability during the course of the experiment. Wortmannin decreased the cellular response to both insulin and ISF402 by 12% and 8% respectively (Figures 6A and B). Wortmannin
(5OnM) also decreased the cellular response to ISF402. A role for PI3-kinase activation in cellular stimulation by ISF402 was confirmed by western blotting to measure phosphorylation of Akt, a kinase downstream from PI3 -kinase in the insulin signalling pathway. Cells treated with either ISF402 or insulin showed a dose-responsive increase in Akt phosphorylation this was blocked by wortmannin (Figure 7 A and C). The changes in Akt phosphorylation in response to insulin, ISF402 and wortmannin were not due to changes in quantities of PI3-kinase enzyme in the cell lysates (Figure 7B and D) and so reflect the degree of activation or inhibition of PI-3 kinase.
Discussion
The PB -kinase inhibitor wortmannin is extensively used in molecular signalling studies (Arcaro and Wymann) to block insulin stimulation of signalling intermediates such as Akt (Burgering and Coffer; Kahn). Here wortmannin was used to test whether ISF402 activated the insulin signalling pathway. Wortmannin at a sub-maximal concentration, (5OnM), significantly inhibited the cellular response to both insulin and ISF402 in the cytosensor (Figure 6A and B), suggesting that ISF402 has insulin mimetic activity and can independently activate PI-3 kinase. This was confirmed by the observations that phosphorylation of the downstream signalling protein Akt increased in response to increasing cellular stimulation by ISF402, and that this was inhibited by wortmannin (Figure 7C). These results show that ISF402 activates PI3-kinase in C2C12 cells.
The observation in C2C12 cells that ISF402 acts like insulin to activate PI-3 kinase/ Akt and stimulate glycogen synthesis but unlike insulin did not increase glucose uptake suggests that ISF402 is not a true insulin mimetic. Muscle cell lines such as L6, C2C12 and Sol8 express far less Glut4 than muscle tissue and artificially raising Glucose transporter-4 (Glut4) levels by transfection does not increase Glut4 trafficking and glucose uptake (Kotliar and Pilch; Guillet-Deniau et al.\ Wilson et al.) resulting in modest stimulation of glucose uptake by insulin. It has also been noted that even if Glut4 is present at the cell surface, it may not actively transport glucose or its activity could be masked by the actions of Glucose transporter- 1 (Glutl) and/or Glucose transporter-3
(Glut3), which are also expressed in skeletal muscle cells (Tortorella and Pilch). It is noteworthy that from the microphysiometry and western blotting experiments that ISF402 does not activate PI-3 kinase as efficiently as insulin. ISF402 may only partially stimulate the insulin signalling pathway, perhaps to a level below threshold for triggering many of the downstream cellular events that insulin triggers. In the context of the modest glucose uptake seen in C2C12 muscle cells with insulin, the lesser stimulation of PI-3 kinase by ISF402 may have been insufficient to induce a measurable increase in glucose transport.
In conclusion, ISF402 activates PI-3 kinase and increases the response of C2C12 muscle cells to insulin. The glucose lowering activity of ISF402 observed in vivo (Ng et al.) may be explained by insulin-like activity and insulin sensitisation mediated by PI3-kinase. Zinc activates components of the insulin signalling system including the insulin receptor tyrosine kinase (IRTK) and PI3 kinase (Tang and Shay; Miranda and Dey; Kim et al), and inhibits the phosphatase responsible for inactivating IRTK (Haase and Maret). Therefore, activation of PI3 kinase by ISF402 is likely to be mediated by the ISF402-mediated increase in cellular zinc concentrations.
Example 8 Addition of zinc changes the structure of GHTD-amide
GHTD-amide has structural features common to zinc -binding peptides. Histidine is often involved in co-ordinating zinc and less frequently aspartic acid can also contribute (Thickman et al). Generally, tetrapeptides such as GHTD-amide lack secondary structure due to their small size, but interactions with zinc can induce conformational changes (Henin et al). The effect of zinc on the secondary structure of GHTD-amide was examined using far UV circular dichroism (Figure 8). The CD profile of GHTD-amide in the absence of zinc was typical of random coil with a pronounced negative peak at 198 nm (Figure 8A). Upon addition of zinc ions the negative peak displayed a slight "blue" shift to 200 nm and a second negative peak appeared at 220 nm. These changes were reversed by chelation with EDTA (data not shown). The CD profile of GHTD-amide in the presence of zinc was very similar to that previously described for short alpha helices in peptides (Chin et al). Hence, zinc ions promote alpha helical-like conformation in GHTD-amide. The interaction between GHTD-amide and zinc was concentration dependent (Figure 8B), with an apparent Kd of 102 ± 11 uM and saturation at close to equimolar concentrations of zinc and peptide. References
Anderson, R.A. et al. Potential antioxidant effects of zinc and chromium supplementation in people with type 2 diabetes mellitus. Journal of the American College of Nutrition 20, 212-8 (2001).
Arcaro A. and Wymann M. P. Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses. Biochem. J. 296(Pt2):297-301 (1993).
Blundell, T., Dodson, G., Hodghin, G. & Mercola, D. Insulin: the structure in the crystal and its reflection in chemistry and biology. Adv. Protein Chem. 26, 279-402 (1972).
Burgering B.M., Coffer PJ. Protein kinase B (c-Akt) in phosphatidylionositol-3-OH kinase signal transduction. Nature 376:599-602 (1995).
Chausmer, A.B. Zinc, insulin and diabetes. Journal of the American College of Nutrition 17, 109-15 (1998).
Chen, M.D. et al. Effects of zinc supplementation on the plasma glucose level and insulin activity in genetically obese (ob/ob) mice. Biological Trace Element Research 61, 303-11 (1998).
Chin D-H, Woody R. W., Rohl CA. and Baldwin R.L.. Circular dichroism spectra of short, fixed-nucleus alanine helices. Proc. Natl. Acad. Set. USA. 99: 15416-15421 (2002).
Coulston, L.D.P. Insulin-like effect of zinc on adipocytes. Diabetes 29, 665-7 (1980). Ezaki, O. lib group metal ions (Zn2+, Cd2+, Hg2+) stimulate glucose transport activity by post-insulin receptor kinase mechanism in rat adipocytes. Journal of Biological Chemistry 264, 16118 (1989).
Guillet-Deniau I., Leturque A. and Girard J. Expression and cellular localisation of glucose transporters (GLUTl, GLUT3, GLUT4) during differentiation of myogenic cells isolated from rat foetuses. J. Cell Set 107: 487-496 (1994).
Haase, H.M.W. and Maret, W. Intracellular zinc fluctuations modulate protein tyrosine phosphatase activity in insulin/insulin-like growth factor- 1 signaling. Experimental Cell Research 291, 289-98 (2003).
Henin O., Barbier B., Boillot F. and Brack A. Zinc-induced conformational transitions of acidic peptides: characterisation by circular dichroism and electrospray mass spectrometry. Chem. Eur. J. 5:218-226 (1999).
Hutchinson, D. S., Bengtsson, T., Evans, B. A. and Summers, R.J. Mouse beta- 3a and beta 3b-adrenoceptors expressed in Chinese hamster ovary cells display identical pharmacology but utilize distinct signalling pathways, Br J Pharmacol 135, 1903-1914 (2002).
Ilouz, R., Kaidanovich, O., Gurwitz, D. & Eldar-Finkelman, H. Inhibition of glycogen synthase kinase-3beta by bivalent zinc ions: insight into the insulin-mimetic action of zinc. Biochemical & Biophysical Research Communications 295, 102-6 (2002).
Kahn C.R. Diabetes. Causes of insulin resistance. Nature 373:384-385 (1995). Kim, S., Jung, Y., Kim, D., Koh, H. & Chung, J. Extracellular zinc activates p70 S6 kinase through the phosphatidylinositol 3-kinase signaling pathway. Journal of Biological Chemistry 275, 25979-84 (2000).
Kotliar N., Pilch P.F. Expression of the glucose transporter isoform GLUT 4 is insufficient to confer insulin-re gulatable hexose uptake to cultured muscles cells. MoI Endocrinol 6: 337-345 (1992).
May, J.M. & Contoreggi, CS. The mechanism of the insulin-like effects of ionic zinc. Journal of Biological Chemistry 257, 4362-8 (1982).
McCaIl, K.A., Huang, C. & Fierke, CA. Function and mechanism of zinc metalloenzymes. Journal of Nutrition 130, 1437 (2000).
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Qian, W-J., Peters, J.L., Dahlgren, G.M., Gee, K.R. and Kennedy, R.T. Simultaneous monitoring of Zn2+ secretion and intracellular Ca2+ from islet cells by fluorescence microscopy, BioTechniques 37, 922-933. (2004)
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Simon, S.F. & Taylor, CG. Dietary zinc supplementation attenuates hyperglycemia in db/db mice. Experimental Biology & Medicine 226, 43-51 (2001). Tang, X. & Shay, N.F. Zinc has an insulin-like effect on glucose transport mediated by phosphoinositol-3-kinase and Akt in 3T3-L1 fibroblasts and adipocytes. Journal of Nutrition 131, 1414-20 (2001).
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Tortorella L.L. and Pilch P.F. C2C12 myocytes lack an insulin-responsive vesicular compartment despite dexamethasone-induced GLUT4 expression. Am J Physiol Endocrinol Metab 283: E514-E524 (2002).
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Claims

CLAIMS:
1. A composition comprising a peptide of the formula:
(Xaa)nl-Xaa1-His-Thr-Asp-(Xaa)n2
wherein
Xaa is any amino acid;
Xaai is a hydrophobic amino acid; ni is 0-10; and n2 is 0-10; and zinc ions.
2. A composition according to claim 1, wherein the peptide is one of the formulae:
(Xaa)n i - Val-His-Thr- Asp-(Xaa)n2 ; or (Xaa)ni-Gly-His-Thr-Asp-(Xaa)n2;
wherein Xaa, nj and n2 are as defined in claim 1.
3. A composition according to claim 1, wherein the peptide is a tetrapeptide selected from
Val-His-Thr-Asp (ISF402); and Gly-His-Thr-Asp.
4. A composition according to claim 1 further comprising insulin.
5. A composition according to claim 1 , wherein the N-terminus of the peptide is acylated.
6. A composition according to claim 1, wherein the C-terminus of the peptide is amidated.
7. A composition according to claim 1, wherein the zinc ions are derived from a zinc salt selected from zinc chloride (ZnCl2), zinc sulfate (ZnSC^), zinc picolinate, zinc citrate, zinc acetate, zinc glycerate and zinc monomethionine.
8. A composition according to claim 1, wherein the ratio of peptide to zinc ions is 1 : 1.
9. A complex comprising a peptide of the formula:
(Xaa)nl-Xaa1-His-Thr-Asp-(Xaa)n2
wherein
Xaa is any amino acid;
Xaa! is a hydrophobic amino acid; n! is 0-10; and n2 is 0-10; and a zinc(II) ion, wherein the ratio of peptide:zinc(II) ion is 1 : 1.
10. A complex according to Claim 9, wherein the peptide is a peptide of the formula:
(Xaa)ni-Val-His-Thr-Asp-(Xaa)n2; or (Xaa)n i -Gly-His-Thr- Asp-(Xaa)n2 ; wherein Xaa, nj and n2 are as defined in claim 9.
11. A complex according to claim 9, wherein the peptide is a tetrapeptide selected from:
Val-His-Thr-Asp; or Gly-His-Thr-Asp.
12. A method of increasing cellular response to zinc ions comprising exposing cells to an effective amount of a combination comprising a peptide of the formula:
(Xaa)n ] -Xaa1 -His-Thr- Asp-(Xaa)n2
wherein Xaa is any amino acid;
Xaa! is a hydrophobic amino acid; ni is 0-10; and n2 is 0-10; and zinc ions.
13. A method of regulating in vivo blood glucose levels in a human or other mammal, which comprises administration to said human or other mammal, a combination comprising an effective amount of a peptide of the formula:
(Xaa)n i -Xaat -His-Thr- Asp-(Xaa)n2
wherein
Xaa is any amino acid; Xaai is a hydrophobic amino acid; m is 0-10; and n2 is 0-10; and zinc ions.
14. A method according to claim 13, wherein the combination further comprises insulin or is co-administered separately, simultaneously or sequentially with insulin.
15. A method of treating diabetes in a human or other mammal comprising administration to said human or other mammal, a combination comprising an effective amount of a peptide of the formula:
(Xaa)nl-Xaai-His-Thr-Asp-(Xaa)n2
wherein
Xaa is any amino acid; Xaai is a hydrophobic amino acid; ni is 0-10; and n2 is 0-10; and zinc ions.
16. A method according to claim 15, wherein the diabetes is Type 1 diabetes or insulin requiring Type 2 diabetes.
17. A method according to claim 16, wherein the combination further comprises insulin, or is co-administered separately, simultaneously or sequentially with insulin.
18. A method according to claim 17, further comprising co-administration with an insulin sensitising agent.
19. A method according to claim 15, wherein the diabetes is non-insulin requiring Type 2 diabetes.
20. A method according to claim 19, wherein the combination is co-administered in a single composition, or administered separately, simultaneously or sequentially with insulin-sensitising agents or compounds that increase insulin secretion from β-cells.
21. A method of treating or preventing disorders associated with zinc deficiency in a human or other mammal comprising administration to said human or other mammal, a combination comprising an effective amount of a peptide of the formula:
(Xaa)n i -Xaai -His-Thr- Asp-(Xaa)n2
wherein
Xaa is any amino acid;
Xaai is a hydrophobic amino acid;
Figure imgf000051_0001
n2 is 0-10; and zinc ions.
22. A method of enhancing the activity of the immune system of a human or other mammal, which comprises administration to said human or other mammal, a combination comprising an effective amount of a peptide of the formula:
(Xaa)nl-Xaa1-His-Thr-Asp-(Xaa)n2
wherein
Xaa is any amino acid; Xaai is a hydrophobic amino acid; ni is 0-10; and n2 is 0-10; and zinc ions.
23. Use of a combination comprising a peptide of the formula:
(Xaa)n i -Xaaj -His-Thr-Asp-(Xaa)n2
wherein
Xaa is any amino acid; Xaa! is a hydrophobic amino acid;
TLi is 0-10; and n2 is 0-10; and zinc ions, in the manufacture of a medicament for regulating in vivo blood glucose levels in a human or other mammal.
24. Use of a combination comprising a peptide of the formula:
(Xaa)n i -Xaai -His-Thr- Asp-(Xaa)n2
wherein
Xaa is any amino acid;
Xaai is a hydrophobic amino acid;
Figure imgf000052_0001
n2 is 0-10; and zinc ions, in the manufacture of a medicament for treating diabetes in a human or other mammal.
25. Use of a combination comprising a peptide of the formula:
(Xaa)n i -Xaa! -His-Thr- Asp-(Xaa)n2 wherein
Xaa is any amino acid;
Xaai is a hydrophobic amino acid; ni is 0-10; and n2 is 0-10; and zinc ions, in the manufacture of a medicament for treating or preventing zinc deficiency in a human or other mammal.
26. Use of a combination comprising a peptide of the formula:
(Xaa)n i -Xaat -His-Thr- Asp-(Xaa)n2
wherein
Xaa is any amino acid; Xaai is a hydrophobic amino acid; ni is 0-10; and n2 is 0-10; and zinc ions, in the manufacture of a medicament for enhancing the activity of the immune system of a human or mammal.
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WO2018065634A1 (en) * 2016-10-07 2018-04-12 Cyprumed Gmbh Pharmaceutical compositions for the nasal delivery of peptide or protein drugs
US10905744B2 (en) 2015-10-07 2021-02-02 Cyprumed Gmbh Pharmaceutical formulations for the oral delivery of peptide drugs
CN112375729A (en) * 2020-10-09 2021-02-19 北京大学 Method for recovering density of compact core of insulin vesicle

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WO2009026614A1 (en) * 2007-08-24 2009-03-05 Dia-B Tech Limited Hypoglycaemic tripeptide and methods of use thereof
US10905744B2 (en) 2015-10-07 2021-02-02 Cyprumed Gmbh Pharmaceutical formulations for the oral delivery of peptide drugs
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