WO2000078805A1 - Peptide ayant une fonctionnalite preptine - Google Patents

Peptide ayant une fonctionnalite preptine Download PDF

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Publication number
WO2000078805A1
WO2000078805A1 PCT/NZ2000/000102 NZ0000102W WO0078805A1 WO 2000078805 A1 WO2000078805 A1 WO 2000078805A1 NZ 0000102 W NZ0000102 W NZ 0000102W WO 0078805 A1 WO0078805 A1 WO 0078805A1
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Prior art keywords
pro
preptin
asp
analog
thr
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PCT/NZ2000/000102
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English (en)
Inventor
Christina Maree Buchanan
Garth James Smith Cooper
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Protemix Corporation Limited
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Application filed by Protemix Corporation Limited filed Critical Protemix Corporation Limited
Priority to AU57178/00A priority Critical patent/AU759203B2/en
Priority to JP2001505563A priority patent/JP2003503019A/ja
Priority to EP00942575A priority patent/EP1185558A4/fr
Priority to CA002375207A priority patent/CA2375207A1/fr
Priority to US09/745,078 priority patent/US20030050434A1/en
Publication of WO2000078805A1 publication Critical patent/WO2000078805A1/fr
Priority to HK02103568.4A priority patent/HK1042307A1/zh
Priority to US10/374,624 priority patent/US20030166561A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to a bioactive peptide.
  • it relates to a peptide secreted by the pancreatic islet ⁇ -cell that stimulates insulin secretion.
  • Pancreatic islet ⁇ -cells play a major regulatory role in physiology, mainly through their secretion of insulin, a peptide hormone which exerts profound effects on intermediary metabolism (Draznm et al (1994))
  • a second ⁇ -cell hormone, amylin may also cont ⁇ bute to ⁇ -cell regulatory function through its actions on insulin secretion and tissue insulin sensitivity (Cooper, G ( 1994); Hettiarachchi et al (1997)).
  • hormones are packaged in secretory granules, which undergo regulated release in response to signals such as fuels (eg. glucose, ammo acids) or neurohormonal stimuli.
  • fuels eg. glucose, ammo acids
  • neurohormonal stimuli e.g. glucose, ammo acids
  • These granules contain dense cores ⁇ ch in insulin and Zn, while smaller amounts of insulin C-peptide, amylin, promsuhn, chromogramn- de ⁇ ved peptides, proteases and other proteins are found in the granule matrix (Hutton, J ( 1989)).
  • pancreatic islet ⁇ -cells secrete yet a further regulatory peptide.
  • this peptide enhances glucose-mediated insulin secretion.
  • the present invention provides the peptide preptin or an analog thereof.
  • preptin a peptide of 34 ammo acids, the sequence of which is as follows.
  • R 2 is Gin or Pro; Rs is Ala or Thr; t is Asp or Asn;
  • Rs is Gin or Lys
  • Re is Tyr or Phe
  • R7 is Arg or Lys; Rs is Ala or Thr; and or an analog thereof.
  • the invention provides human preptin having the amino acid sequence:
  • the invention provides rat preptin having the amino acid sequence:
  • the invention provides mouse preptin having the amino acid sequence:
  • amino acid sequence corresponds to Asp69-Leu ⁇ o 2 of the proIGF-II E-peptide in each mammal.
  • the present invention provides a polynucleotide which encodes preptin or an analog thereof.
  • the invention provides a vector or cell-line which includes a polynucleotide which encodes preptin or an analog thereof and which is capable of expressing preptin or said analog.
  • Preptin salts which are preferably physiologically acceptable, are also provided.
  • the invention further provides a pharmaceutical composition which comprises preptin or an analog thereof, or preptin salts.
  • the invention provides a method of stimulating insulin secretion for a therapeutic or prophylactic purpose which comprises the step of administering to a patient in need of such therapy or prophylaxis an effective amount of preptin or an analog thereof.
  • the invention provides the use of preptin or an analog thereof or a salt thereof in the preparation of a medicament, particularly for stimulating insulin secretion.
  • the invention provides a method of modulating glucose mediated insulin secretion which comprises the step of administering to a patient an effective amount of preptin, a preptin analog, a preptin agonist or a preptin antagonist.
  • the invention provides antibodies which bind preptin or its analogs, assays which employ such antibodies and assay kits which contain such antibodies.
  • Figure 1 shows purification and characterisation of preptin.
  • Figure 2 shows cellular preptin secretion, a) Preptin R1A standard curve, b) RIA charactensation of preptm-hke immunoreactive matenal (PLIM) in RP-HPLC fractions of 24-h ⁇ TC6-F7 conditioned medium and mtra-granular fractions from Figure lb. c) MALDI-TOF MS of the major PLIM containing fraction secreted from ⁇ TC6-F7 cells. Peak corresponds to munne preptin (M + H + ) with 0.07% error.
  • PLIM preptm-hke immunoreactive matenal
  • FIG 4 shows the immunohistochemistry of munne pancreas.
  • Pancreas harvested from adult FVB/n mice was sectioned and stained with haematoxylin and polyclonal rabbit antisera using lmmunoperoxidase-conjugated goat-anti-rabbit second antibody.
  • Figure 5 shows the RIA characterisation of preptin-like immunoreactive material (PLIM) in RP-HPLC fractions from rat islets or ⁇ TC6-F7 granule fractions (standard; Fig. lb).
  • PLIM prept
  • Figure 6 shows preptin and insulin co-secretion from ⁇ TC6-F7 cells and isolated rat islets.
  • a,b Glucose-mediated co-secretion of preptin with insulin from a, ⁇ TC6-F7 cells and b, isolated rat islets.
  • Figure 7 shows the effects of preptin on insulin secretion, a, b, Purity and mass of purified a, rabbit anti-rat preptin ⁇ -globulin and b, non-immune rabbit ⁇ -globulin.
  • 1 light chain IgG, M + H + ; 2: whole IgG, M + 4H + ; 3: heavy chain IgG, M + H + ; 4: whole IgG, M + 2H + ; whole IgG, M + H + .
  • the present invention is directed to a novel peptide which has been found in pancreatic islet ⁇ -cell granules.
  • This peptide, preptin has been determined to stimulate glucose-evoked insulin secretion.
  • preptin was identified using a single-step density- gradient centrifugal method to purify secretory granules from cultured munne ⁇ TC6-F7 cells with purity being confirmed by marker- protein analysis (Figure la). Insulin was used to track purification of granule-cores, whereas amylin, which is present in the granule- matrix (Johnson, K (1988)), was measured to verify granule-membrane integrity ( Figure la). Soluble granule components were then separated using reversed-phase HPLC (A 2 ⁇ ; Figure lb). Peptide-identity was determined by mass spectrometry and NH 2 - terminal amino-acid sequencing.
  • Preptin is flanked NH 2 - terminally by a recognised Arg cleavage-site, and COOH- terminally by a putative dibasic (Arg-Arg) cleavage motif (Bell et at, (1985)) (Fig. le). These residues are highly conserved between species, and are likely to serve as post- translational processing signals.
  • amino acid sequence of mouse preptin is as follows:
  • Preptin is encoded by polynucleotides having the following nucleotide sequences:
  • Preptin may be generated by synthetic or recombinant means.
  • preptin may be synthesised using any of the commercially available solid phase techniques such as the Merryfield solid phase synthesis method, where ammo acids are sequentially added to a growing amino acid chain (see Merryfield, J. Am. Soc. 85:2146-2149 ( 1963)).
  • Equipment for automative synthesis of peptides is also commercially available from suppliers such as Perkm Elmer/Applied Biosystems, Inc and may be operated according to the manufacturers instructions.
  • Preptin may also be produced recombinantly by inserting a polynucleotide (usually DNA) sequence that encodes the protein into an expression vector and expressing the peptide in an appropnate host.
  • a polynucleotide usually DNA
  • Any of a vanety of expression vectors known to those of ordinary skill m the art may be employed.
  • Expression may be achieved in any appropnate host cell that has been transformed or transfected with an expression vector containing a DNA molecule which encodes the recombinant peptides. Suitable host cells include prokaryotes, yeasts and higher eukaryotic cells.
  • Vectors and/or cells lines which express preptin have utility in their own nght and also form part of the invention.
  • Analogs of preptin and of its encoding polynucleotides are also within the scope of the present invention. Such analogs include functional equivalents of preptm and of the polynucleotides descnbed above. In terms of preptin itself, functional equivalents include all proteins which are immunologically cross-reactive with and have substantially the same function as preptin. That equivalent may, for example, be a fragment of preptin containing from 6 to 33 amino acids (usually representing a C-terminal truncation) and including a preptin active site or sites, a substitution, addition or deletion mutant of preptin, or a fusion of preptin or a fragment or a mutant with other amino acids.
  • the six amino acids forming the smallest fragment can be from any part of the sequence, provided they are consecutive in that sequence and fulfil the functional requirement. It is of course also possible (and expressly contemplated) that the bioactive peptide include any one of those hexapeptides, or indeed be or include any heptapeptide, octapeptide, nonapeptide, or decapeptide from the sequence.
  • Peptides which are, or include a hexapeptide, heptapeptide, octapeptide, nonapeptide or decapeptide from human preptin are particularly preferred.
  • Additions and/or deletions of amino acids may also be made as long as the resulting peptide is immunologically cross-reactive with and has substantially the same function as preptin.
  • Equivalent polynucleotides include nucleic acid sequences that encode proteins equivalent to preptin as defined above. Equivalent polynucleotides also include nucleic acid sequences that, due to the degeneracy of the nucleic acid code, differ from native polynucleotides in ways that do not effect the corresponding amino acid sequences. A prediction of whether a particular polynucleotide or polypeptide is equivalent to those given above can be based upon homology. Polynucleotide or polypeptide sequences may be aligned, and percentage of identical nucleotides in a specified region may be determined against another sequence, using computer algorithms that are publicly available.
  • Two exemplary algorithms for aligning and identifying the similarity of polynucleotide sequences are the BLASTN and FASTA algorithms.
  • the similarity of polypeptide sequences may be examined using the BLASTP algorithm.
  • Both the BLASTN and BLASTP software are available on the NCBI anonymous FTP server (ftp://ncbi.nlm.nih.gov) under /blast/execu tables/.
  • the BLASTN algorithm version 2.0.4 [Feb-24- 1998] set to the default parameters described in the documentation and distributed with the algorithm, is preferred for use in the determination of variants according to the present invention.
  • BLAST family of algorithms, including BLASTN and BLASTP, is described at NCBI's website at URL http://www.ncbi.nlm.nih.gov/BLAST/newblast.html and in the publication of Altschul, Stephen F, et al (1997). "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402.
  • the computer algorithm FASTA is available on the Internet at the ftp site ftp://ftp.virginia.edu.pub/fasta/.
  • Analogs according to the invention also include the homologues of preptin from species other than human, rat or mouse. Such homologues can be readily identified using, for example, nucleic acid probes based upon the conserved regions of the polynucleotides which encode human, rat and mouse preptin.
  • Preptin or its analogs can also be present in various degrees of purity.
  • the preptin/analog component makes up at least 50% by weight of the preparation, more preferably at least 80% by weight, still more preferably at least 90% by weight, still more preferably at least 95% by weight and yet more preferably at least 99% by weight.
  • the preptin or analog be present in a pure or substantially pure form.
  • preptin or preptin analogs may also be presented as a pharmaceutical composition.
  • Such compositions may comprise preptin or preptin analogs together with one or more pharmaceutically acceptable carriers therefor and optionally other therapeutic ingredients where desirable.
  • the carrier must be acceptable in the sense of being compatible with the preptin or preptin analog and not deleterious to the patient to be treated. Desirably, the composition should not include substances with which peptides are known to be incompatible.
  • compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredients into association with a carrier which constitutes one or more accessory ingredients.
  • preptin or preptin analogs may be injected parenterally, eg. intravenously into the blood stream of the patient being treated.
  • the route can vary, and can be intravenous, subcutaneous, intramuscular, intraperitoneal, enterally, transdermally, transmucously, sustained release polymer compositions (eg. a lactide polymer or co-polymer microparticle or implant), perfusion, pulmonary (eg. inhalation), nasal, oral, etc.
  • compositions suitable for parenteral and in particular intravenous administration are presently preferred.
  • Such compositions conveniently comprise sterile aqueous solutions of preptin or the preptin analog.
  • the solutions are isotonic with the blood of the patient to be treated.
  • Such compositions may be conveniently prepared by dissolving the preptin or analog in water to produce an aqueous solution and rendering this solution sterile.
  • the composition may then be presented in unit or multi-dose containers, for example sealed ampoules or vials.
  • compositions suitable for sustained release parenteral administrations are also well known in the art. See, for example, US Patent ⁇ os. 3,773,919 and 4,767,628 and PCT Publication No. WO 94/ 15587.
  • preptin it is also convenient for preptin to be converted to be in the form of a salt.
  • a salt will generally be physiologically acceptable, and can be formed using any convenient art standard approach.
  • Preptin salts formed by combination of preptin with anions of organic acids are particularly preferred.
  • Such salts include, but are not limited to, malate, acetate, propionate, butyrate, oxaloacetate, citrate, isocitrate, ⁇ -ketoglutarate, succinate, fumarate and trifluoroacetate salts.
  • the salts this formed can also be formulated into pharmaceutical compositions for therapeutic administration where this is desired.
  • Granule purification ⁇ -cells at passages 55-60 from 8- 12 triple flasks were harvested by trypsinization, yielding on average 2.5-4.0 ml of pure cells (1.6 - 2.4 x 10 9 ), which then were concentrated (1700 x g, 5 min), washed twice with PBS, and once with Homogenisation Medium (0.3 M sucrose/ 10 mM MES K (Sigma) /l mM K 2 EGTA/ 1 mM Mg2S0 /pH 6.5), then homogenised on ice in the same medium at 1 :5 (v/v).
  • Homogenisation Medium 0.3 M sucrose/ 10 mM MES K (Sigma) /l mM K 2 EGTA/ 1 mM Mg2S0 /pH 6.5
  • Solutions (v/v) of 13% and 31% OptiPrepTM (Nycomed) were prepared by dilution with Homogenisation Medium, and 6 x 10 ml continous gradients (31%- 13% OptiPrep) poured (Auto Densi-Flow II, Haakebuchler) into Ultra- Clear tubes (Beckman).
  • Integrity of granule preparations was monitored using radioimmunoassays for insulin (crystalline granule core), amylin (granule matrix); purity by functional assays for aryl sulphatase (lysosomes) and citrate synthase (mitochondria) ; and total protein content using Bicinchoninic acid (Pierce) .
  • Granule proteins were purified in two sequential RP-HPLC runs (A: 0.08% TFA v/v; B: 80% acetonitrile with 20% A; Applied Biosystems 140B/785A/ 1 12A system;
  • Secretory granule material was initially centrifuged ( 16,000 x g, 20 min) before loading. An initial 15 min isocratic step was employed, and sequential 30s fractions collected from 19 min post-injection. Slightly different gradients were used sequentually to purify proteins; the first semi-purified granule proteins, whereas the second was slightly flatter, to increase resolution and purity.
  • Purified peptides were identified by N-terminal sequence determination (automated Edman method; ABI ProciseTM) combined with accurate mass determination by MALDI-TOF MS. For complete sequence verification, purified mouse preptin isolates were cleaved using Lys-C (Boehringer Mannheim), and the resulting peptide fragments repurified by RP-HPLC.
  • Peptide molecule weights were determined by MALDI-TOF MS (Hewlett-Packard G2025A; 337 nm-emission nitrogen laser/ 150 ⁇ j maximum output/3 ns pulsewidth/30 kV ion acceleration potential) fitted with a 500 MHz digital oscilloscope (G2030AA, LeCroy) using an -CHC matrix with recombinant human insulin ( ⁇ ovo ⁇ ordisk; M + H + , 5808.66 Da; M + 2H + , 2904.83) and somatostatin (Bachem; M + H + , 1638.91 , M + ⁇ a + , 1660.90) mass standards.
  • MALDI-TOF MS Hewlett-Packard G2025A; 337 nm-emission nitrogen laser/ 150 ⁇ j maximum output/3 ns pulsewidth/30 kV ion acceleration potential
  • MS was performed under high vacuum ( ⁇ 1.0 ⁇ Torr) and data acquired (ChemStation; 0-20PS method positive polarity in the 0-20 kDa range) with external mass calibration in "single shots" mode. Accurate molecular weights of purified peptides were confirmed by interpolation with external mass standardisation.
  • Rat preptin was chemically synthesised (Auspep Pty, Australia), according to the predicted sequence, using Fmoc chemistry on an Advanced Chem Tech 396 Robotics Peptide Synthesiser starting with FmocLeu- Wang resin.
  • the peptide was deprotected and cleaved from the resin with a solution of 92.5% TFA: 2.5% water:2.5% triisopropylsilane: 2.5% dithiothreitol for 3 h.
  • the peptide was precipitated from the TFA solution by addition of diisopropyl ether and the precipitate dissolved in 30% acetonitrile: water, lyophilised, and purified by RP- HPLC. Purity was confirmed as >99% by analytical RP-HPLC (rat preptin eluted at 47% B), while MALDI-TOF MS validated the mass as 3932.4 Da + 0.026%.
  • Synthetic rat preptin was conjugated to the carrier, ovalbumin, using the single step glutaraldehyde method at pH 7.0, then used to raise polyclonal antisera in NZW rabbits.
  • Preptin was 125 I-radiolabelled using the chloramine-T method, and [ 12 I)preptin (362 ⁇ Ci/ ⁇ g) purified by Sephadex G- 10 chromatography (50 mM phosphate buffer, pH 7.5). An optimised RIA for preptin was then developed, with B/F separation by the PEG-assisted second antibody (goat- anti- rabbit method).
  • Preptin secretion was studied in ⁇ TC6-F7 cells (passage #52), cultured otherwise as above in 24-well plates at 4 x 10 5 cells per well. Preptin stimulation was performed after 3 d growth, at 80% confluence. Cells were washed twice in HEPES-buffered KRB before commencement of secretion studies, then preincubated for 1 h in 1 ml/well incubation buffer (0 mM glucose; 0. 1% w/w Fraction V BSA (Sigma) dissolved in HEPES-KRB) 500 ⁇ l/well was then removed, and replaced with an equivalent volume of fresh incubation buffer containing various concentrations of glucose.
  • preptin-like immunoreacti ⁇ e material Since preptin is a cleavage product of the E-peptide of IGF-II, and other cleavage products from a similar region have been isolated from serum in the past (Hylka (1985); Daughaday ( 1992); Liu ( 1993)), quantitation by preptin RIA was insufficient to characterise the nature of the secreted and circulating peptide.
  • a combined RP- HPLC/preptin RIA method was therefore developed to further characterise PLIM.2 ml aliquots of separated plasma from a human donor, and ⁇ TC6-F7 conditioned medium, were acidified with 0.1 ml of 4M acetic acid and applied to a C- 18 Sep Pak (Waters, 1 ml volume) which had been pre-equilibrated with 10 ml of 100% methanol and 20 ml of 4% (v/v) acetic acid.
  • peripheral tissues including skeletal muscle.
  • preptin to alter glucose uptake and incorporation into muscle glycogen was investigated using isolated incubated stripped soleus muscle as a model tissue. All animal methods were carried out with appropriate permission from the Institutional Animal Ethics Committee.
  • 18-h fasted rats were anaesthetised (45 mg/kg Pentobarbitone sodium) then sacrificed by cervical dislocation, and soleus mucles dissected under carboxygenated-KHB (O2:C0295: 5 v/v), then incubated in nDMEM supplemented with various concentrations of insulin and preptin. Muscles were teased longitudinally into 3 equal strips with a final radius of approximately 1.5 mm [(U) 14 C] D(+)-glucose ( 1 mCi/ml, Amersham) was diluted 1 : 20 (v/v) in 70% ethanol to yield a final concentration of 0.5 ⁇ Ci/ 10 ⁇ l.
  • Actrapid® Recombinant Human Insulin 100 U/ml, ⁇ ovo ⁇ ordisk was diluted 1/ 1000 in 10 ml nDMEM.
  • 60 ⁇ g rat preptin was dissolved in 1526 ⁇ l of nDMEM to a concentration of 10 ⁇ M, then further diluted in nDMEM to give stock solutions of 1 ⁇ M, 100 nM, 10 nM, 100 pM and 1 pM.
  • Two different experimental paradigms were employed to determine whether preptin (i) stimulated the rate of glucose incorporation into glycogen, or (ii) acted as an antagonist of insulin-evoked glucose incorporation into glycogen.
  • Preptin agonist protocol All methods were as described above, except that strips were incubated in the absence of insulin (except for the positive control, at 23.7 nM) and final preptin concentrations of 0, 0.1, 1 , 10 and 100 nM. Effect of preptin on insulin secretion
  • Insulin and amylin are known to modulate ⁇ -cell insulin secretion via presumed autocnne mechanisms.
  • the effect of preptin on insulin secretion was therefore tested using a ⁇ -cell secretagogue protocol ⁇ TC6-F7 cells were subcultured at passage #52 into 24- well plates at 4 x 10 5 cells/well.
  • Mouse preptin is a 34 ammo acid peptide which corresponds to Asp69-Leu ⁇ o2 of munne proIGF-II E- peptide
  • Preptin was present in granules at 1 :8 the content of insulin, but 2: 1 that of amylin (mol/mol), as determined by integration of RP-HPLC peak- areas.
  • Preptin is flanked NH 2 -term ⁇ nally by a recognised Arg cleavage site, and COOH-terminally by a putative dibasic (Arg-Arg) cleavage motif (Bell ( 1984)) ( Figure le). These residues likely serve as post-translational processmg signals, and are highly conserved between species Many prohormone precursors incorporate more than one hormone with differential proteolytic processing often being tissue specific (Martinez ( 1989)). The above results indicated that proIGF-II is a prohormone with more than one peptide- hormone product.
  • IGF-II is a member of the insulin family that regulates cell growth, differentiation and metabolism (De Chiara et al (1990). It is a single polypeptide chain denved from the BCA and D domains of proIGF-II (see Figure le) and is widely synthesised in fetal and adult tissues. Insulin expression, on the other hand, is almost completely confined to ⁇ -cells. In mammalian genomes, the IGF-II gene is contiguous with those of insulin (Bell ( 1985)) and recent studies in humans have identified a V ⁇ TR polymorphism upstream of the INS and IGF-II genes, which may contribute to differential regulation of both genes (Ong ( 1999)).
  • preptin is synthesised in islet ⁇ -cells and packaged in secretory granules. Further, it is co-secreted with insulin in a glucose-dependent manner.
  • insulin secretion may be modulated by islet ⁇ -cell hormones, including insulin (Kulkami ( 1999); Elahi ( 1982); Argoud ( 1987)), amylin (Waggoner et al (1993); Silvestre ( 1996); Degano et al ( 1993)), and pancreastatin (Tatemoto (1986)).
  • insulin Kulkami ( 1999); Elahi ( 1982); Argoud ( 1987)
  • amylin Waggoner et al (1993); Silvestre ( 1996); Degano et al ( 1993)
  • pancreastatin Tatemoto (1986)
  • preptin is a physiological regulator of insulin secretion, which acts in a newly recognised feed-forward autocnne loop to enhance glucose-stimulated insulin secretion, and may function to counterbalance the inhibitory effects of other ⁇ -cell hormones on insulin secretion.
  • preptin acts to recruit, pnme and co-ordinate the glucose-responsive activity of ⁇ -cells m a local manner, amplifying the glucose- evoked signal to the ⁇ -cell organ. This action would be similar to the feed-forward mechanism effected in platelets by the thrombm- elicited release of thromboxane A2 (Barntt ( 1992)).
  • preptin biology will be important in type 2 diabetes melhtus, which is charactensed by a complex impairment of msulm secretion (De Fronzo et al ( 1992)).
  • a defect in preptin synthesis, secretion, or action could contnbute to the defective glucose-mediated msulm secretion in this condition and preptin administration may be advantageous for the treatment of type 2 diabetes melhtus or other disorders associated with diminished ⁇ -cell insulin secretion.
  • V ⁇ TR vanable number of tandem repeat polymorphism upstream of the adjacent insulin (INS) and IGF-II genes regulates expression of both genes, and is associated with an increased tendency to both type 2 diabetes melhtus and polycystic ovary syndrome.
  • Section B Preptin is co-packaged with insulin in islet tissue
  • PLIM Preptin-like immunoreactive material
  • insulin-like immunoreactive material were co-localised in islet ⁇ -cells (Figs. 4a,b).
  • Competition studies showed that PLIM-staining was suppressed by pre-incubating preptin antiserum with synthetic preptin in a concentration dependent manner (Figs. 4b-d).
  • Pancreatic islets were isolated from normal adult male Wistar rats, and the contents extracted with acid ethanol according to a modification of published methods (Wollheim and Sharp ( 1981), Romanus ( 1988)).
  • preptin levels were much lower than in ⁇ TC6-F7 cells, the major peak of PLIM co-eluted with intra-granular preptin, indicating that preptin is the dominant physiological component of PLIM in normal islets (Fig. 5).
  • Pancreases were perfused with KHB supplemented with 4% dextran, 0.5% BSA, 3 mM-arginine and 5.5 mM glucose (final concentrations). Perfusate was gassed with a mixture of 95% U2/5% CO2 and infused by peristaltic pump at 2.7 ml.min 1 without re-circulation. Pancreases were perfused and equilibrated for 20-min prior to each 70-min perfusion.
  • Isolated perfused pancreases were infused with anti-preptin or control ⁇ -globulin and subjected to square-wave stimulation by 20 mM glucose (Fig. 7d).
  • preptin is a previously unknown, pancreatic islet ⁇ -cell hormone. It is produced from the E-peptide of pro-IGF-II, is present in islet ⁇ -cell granules in significant amounts, is co-secreted with insulin in a regulated manner, enhances glucose-stimulated insulin secretion, and may act in a feed-forward autocrine loop, probably via binding to a ⁇ -cell surface receptor.
  • the present invention provides preptin (including in its human, rat and mouse forms) and analogs of preptin.
  • Preptin and its analogs play a physiological role in the stimulation of glucose evoked insulin secretion.
  • the invention therefore also provides methods by which glucose-evoked insulin secretion can be modulated.
  • modulation will usually involve administration of preptin and its analogs as described above.
  • modulation can also be achieved by use of preptin agonists and antagonists.
  • a preptin agonist is a compound which promotes or potentiates the effect of preptin on insulin secretion.
  • a preptin antagonist is a compound which competes with preptin or otherwise interacts with preptin to block or reduce the effect of preptin on insulin secretion.
  • Preptin agonists and preptin antagonists can be identified by assay systems which measure the effect preptin has on insulin secretion in the presence and absence of a test compound.
  • assay systems which measure the effect preptin has on insulin secretion in the presence and absence of a test compound.
  • the assay systems described in the experimental section herein can be used.
  • the agonist/ antagonist can be administered as a pure compound or formulated as a pharmaceutical composition as described above for preptin.
  • immunological reagents which bind preptin.
  • Such reagents (which can be polyclonal antibodies) can be generated using art standard techniques, including those described in the experimental section.
  • Monoclonal antibodies can also be provided. Such antibodies will typically be made by standard procedures as described, eg. in Harlow and Lane 1988. Briefly, appropriate animals are selected and the desired immunisation protocol followed. After the appropriate period of time, the spleens of such animals are excised and individual spleen cells fused, typically, to immortalised myeloma cells under appropriate selection conditions. Thereafter, the cells are clonally separated and the supernatants of each clone tested for the production of an appropriate antibody specific for the desired region of the immunising antigen. Other suitable techniques for preparing antibodies involve in vitro exposure of lymphocytes to the antigen or alternatively, to selection of libraries of antibodies in phage or similar vectors. See, for example Huse et al 1989.
  • recombinant antibodies may be produced using procedures known in the art. See, for example, US Patent 4,816,567.
  • the antibodies may be used with or without modification. Frequently, antibodies will be labelled by joining, either covalently or non-covalently a substance which provides a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in the literature.
  • Antibodies as above to preptin can therefore be used to monitor the presence of preptin in a patient or in preptin quantification assays.
  • any convenient immunological format can be employed. Such formats include immunohistochemical assays, RIA, IRMA and ELISA assays.
  • the assays can be conducted in relation to any biological fluid which does, or should, contain preptin.
  • Such fluids include blood, serum, plasma, urine and cerebrospinal fluid.
  • kits can contain, in addition, a number of optional but conventional components, the selection of which will be routine to the art skilled worker.
  • additional components will however generally include a preptin reference standard, which may be preptin itself or an analog (such as a fragment).
  • antibodies such as described above can, if some circumstances, also function as preptin antagonists by binding to preptin and partly or completely interfering with preptin activity.
  • a diagnostic or prognostic method will involve detection of mutations in the gene coding for preptin and/or the preptin secretory mechanism.
  • Detection can occur using any one of a number of art standard techniques including Single Stranded Confirmation Analysis (Orita et al ( 1989)) or the Amplification Refractory Mutation System (ARMS) as disclosed in European Patent Application Publication No 0 332 435.
  • Draznin, B. and LeRoith, D. Molecular biology of diabetes II. Insulin action, effects on gene expression and regulation, and glucose transport (Humana Press Inc., New Jersey ( 1994)).

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  • Health & Medical Sciences (AREA)
  • Diabetes (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
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  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
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Abstract

L'invention porte sur un peptide mammalien bioactif, et notamment sur un peptide sécrété par la cellule β des îlots pancréatiques qui stimule la sécrétion de l'insuline, appelée preptine. L'invention porte entre autre sur des analogues de la preptine, sur des compositions pharmaceutiques contenant la preptine ou ses analogues et sur leur utilisation comme médicaments.
PCT/NZ2000/000102 1999-06-18 2000-06-19 Peptide ayant une fonctionnalite preptine WO2000078805A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
AU57178/00A AU759203B2 (en) 1999-06-18 2000-06-19 Peptide having preptin functionality
JP2001505563A JP2003503019A (ja) 1999-06-18 2000-06-19 プレプチン機能を有するペプチド
EP00942575A EP1185558A4 (fr) 1999-06-18 2000-06-19 Peptide ayant une fonctionnalite preptine
CA002375207A CA2375207A1 (fr) 1999-06-18 2000-06-19 Peptide ayant une fonctionnalite preptine
US09/745,078 US20030050434A1 (en) 1999-06-18 2000-12-20 Peptide
HK02103568.4A HK1042307A1 (zh) 1999-06-18 2002-05-11 具有刺激胰島素分泌功能的肽
US10/374,624 US20030166561A1 (en) 1999-06-18 2003-02-24 Peptide

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NZ336359 1999-06-18
NZ33635999 1999-06-18

Publications (1)

Publication Number Publication Date
WO2000078805A1 true WO2000078805A1 (fr) 2000-12-28

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Country Status (7)

Country Link
EP (1) EP1185558A4 (fr)
JP (1) JP2003503019A (fr)
CN (1) CN1296384C (fr)
AU (1) AU759203B2 (fr)
CA (1) CA2375207A1 (fr)
HK (1) HK1042307A1 (fr)
WO (1) WO2000078805A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004012760A1 (fr) * 2002-08-01 2004-02-12 Auckland Uniservices Limited Procedes d'utilisation de la preptine
WO2004012761A1 (fr) * 2002-08-01 2004-02-12 Protemix Corporation Limited Methodes d'utilisation de composes avec une fonction preptine
WO2006112737A1 (fr) * 2005-04-20 2006-10-26 Protemix Discovery Limited Vesiculines
US7459446B2 (en) 1998-09-25 2008-12-02 John Richard Baker Treatment of diabetes with copper binding compounds
US7582796B2 (en) 2004-07-19 2009-09-01 Protemix Corporation Limited Synthesis of triethylenetetramines
US8034799B2 (en) 2002-03-08 2011-10-11 Philera New Zealand Limited Preventing and/or treating cardiovascular disease and/or associated heart failure
US8691760B2 (en) 2009-02-02 2014-04-08 Ramot At Tel-Aviv University Peptides, pharmaceutical compositions comprising same and uses thereof
US8901074B2 (en) 2010-08-04 2014-12-02 Ramot At Tel-Aviv University Methods of treating autoimmune diseases of the central nervous system (CNS) and neurodegenerative diseases
US9339479B2 (en) 2002-08-20 2016-05-17 Philera New Zealand Limited Dosage forms and related therapies

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2157588A (en) * 1987-08-26 1989-07-20 Amylin Corporation Treatment of diabetes mellitus
AU2949489A (en) * 1988-01-11 1989-08-01 Amylin Corporation Treatment of type 2 diabetes mellitus
AU5953790A (en) * 1989-07-10 1991-02-06 Amylin Pharmaceuticals, Inc. Treatment of obesity and essential hypertension and related disorders
AU1328692A (en) * 1991-01-10 1992-08-17 Amylin Pharmaceuticals, Inc. Hyperglycemic compositions

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61502657A (ja) * 1984-07-13 1986-11-20 チロン　コ−ポレイシヨン プレプロインシュリン様成長因子１及び２

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2157588A (en) * 1987-08-26 1989-07-20 Amylin Corporation Treatment of diabetes mellitus
AU2949489A (en) * 1988-01-11 1989-08-01 Amylin Corporation Treatment of type 2 diabetes mellitus
AU5953790A (en) * 1989-07-10 1991-02-06 Amylin Pharmaceuticals, Inc. Treatment of obesity and essential hypertension and related disorders
AU1328692A (en) * 1991-01-10 1992-08-17 Amylin Pharmaceuticals, Inc. Hyperglycemic compositions

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE CHEMCATS 10 June 1999 (1999-06-10), "Advanced ChemTech Product Catalog", Database accession no. 1998:22309 *
DATABASE REGISTRY [online] XP002954928, accession no. STN *
See also references of EP1185558A4 *

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7459446B2 (en) 1998-09-25 2008-12-02 John Richard Baker Treatment of diabetes with copper binding compounds
US7928094B2 (en) 1998-09-25 2011-04-19 Philera New Zealand Limited Treatment of diabetes with copper binding compounds
US8987244B2 (en) 2002-03-08 2015-03-24 Philera New Zealand Limited Preventing and/or treating cardiovascular disease and/or associated heart failure
US8034799B2 (en) 2002-03-08 2011-10-11 Philera New Zealand Limited Preventing and/or treating cardiovascular disease and/or associated heart failure
WO2004012761A1 (fr) * 2002-08-01 2004-02-12 Protemix Corporation Limited Methodes d'utilisation de composes avec une fonction preptine
WO2004012760A1 (fr) * 2002-08-01 2004-02-12 Auckland Uniservices Limited Procedes d'utilisation de la preptine
US11419831B2 (en) 2002-08-20 2022-08-23 Philera New Zealand Limited Dosage forms and related therapies
US10543178B2 (en) 2002-08-20 2020-01-28 Philera New Zealand Limited Dosage forms and related therapies
US9993443B2 (en) 2002-08-20 2018-06-12 Philera New Zealand Limited Dosage forms and related therapies
US9339479B2 (en) 2002-08-20 2016-05-17 Philera New Zealand Limited Dosage forms and related therapies
US7582796B2 (en) 2004-07-19 2009-09-01 Protemix Corporation Limited Synthesis of triethylenetetramines
US8912362B2 (en) 2004-07-19 2014-12-16 Philera New Zealand Limited Synthesis of triethylenetetramines
US9556123B2 (en) 2004-07-19 2017-01-31 Philera New Zealand Limited Synthesis of triethylenetetramines
US8394992B2 (en) 2004-07-19 2013-03-12 Philera New Zealand Limited Synthesis of triethylenetetramines
US11795150B2 (en) 2004-07-19 2023-10-24 Philera New Zealand Limited Synthesis of triethylenetetramines
US8618051B2 (en) 2005-04-20 2013-12-31 Auckland Uniservices Limited Vesiculins
WO2006112737A1 (fr) * 2005-04-20 2006-10-26 Protemix Discovery Limited Vesiculines
US8691760B2 (en) 2009-02-02 2014-04-08 Ramot At Tel-Aviv University Peptides, pharmaceutical compositions comprising same and uses thereof
US8901074B2 (en) 2010-08-04 2014-12-02 Ramot At Tel-Aviv University Methods of treating autoimmune diseases of the central nervous system (CNS) and neurodegenerative diseases

Also Published As

Publication number Publication date
HK1042307A1 (zh) 2002-08-09
AU5717800A (en) 2001-01-09
EP1185558A4 (fr) 2006-02-15
EP1185558A1 (fr) 2002-03-13
AU759203B2 (en) 2003-04-10
CN1296384C (zh) 2007-01-24
JP2003503019A (ja) 2003-01-28
CA2375207A1 (fr) 2000-12-28
CN1355813A (zh) 2002-06-26

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