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WO1988003536A1 - Pancreastatine - Google Patents

Pancreastatine Download PDF

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Publication number
WO1988003536A1
WO1988003536A1 PCT/US1987/002895 US8702895W WO8803536A1 WO 1988003536 A1 WO1988003536 A1 WO 1988003536A1 US 8702895 W US8702895 W US 8702895W WO 8803536 A1 WO8803536 A1 WO 8803536A1
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WO
WIPO (PCT)
Prior art keywords
pancreastatin
fragment
antibody
biologically active
amino acid
Prior art date
Application number
PCT/US1987/002895
Other languages
English (en)
Inventor
Kazuhiko Tatemoto
Original Assignee
The Board Of Trustees Of The Leland Stanford Junio
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Board Of Trustees Of The Leland Stanford Junio filed Critical The Board Of Trustees Of The Leland Stanford Junio
Publication of WO1988003536A1 publication Critical patent/WO1988003536A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • pancreastatin Applicants used this characteristic to search porcine pancreas extracts for novel hormonal peptides and located a previously unknown polypeptide which, because of its ability to strongly inhibit glucose-induced insulin release from isolated perfused pancreas, they have named "pancreastatin.”
  • pancreastatin is substantially pure pancreastatin and substantially pure biologically active fragments thereof. Such pancreastatin/fragment is substantially free of compounds associated with pancreastatin in the native state.
  • Antibodies to pancreastatin and its biologically active fragments are another aspect of the invention.
  • the antibodies may be polyclonal or monoclonal.
  • compositions that contain pancreastatin, one or more biologically active fragments of pancreastatin, said synthetic analogs, or antibodies thereto are another aspect of the invention.
  • a method of attenuating pancreastatin activity in an individual comprising administering an effective amount of said synthetic analog to the individual is another aspect of the invention.
  • Figure 1 is the amino acid sequence for porcine pancreastatin.
  • the cleavage sites for the protease Endoproteinase Lys-C are indicated by the arrows;
  • Figures 2a-2d are elution profiles of the reverse phase high performance liquid chromatography (RP-HPLC) purifications of pancreastatin and f ragments thereof described in the examples, infra;
  • Figure 3 is a series of graphs showing the results of the tests described in the examples, infra, on the effect of pancreastatin/pancreastatin fragments on glucose-induced insulin secretion; and
  • Figure 4 is a series of graphs showing the results of the tests described in the examples, infra, on the effect of pancreastatin/pancreastatin fragments on glucose-induced somatostatin secretion.
  • pancreastatin is intended to mean the native 49 residue porcine polypeptide described hereinafter, polypeptides of other mammalian species having in whole or in part such biological activity (i.e., inhibition of insulin secretion), synthetic and recombinant counterparts of such native polypeptides and analogs of such polypeptides that have such activity to any degree provided, however, that such analog does not constitute chromogranin.
  • biologically active fragment of pancreastatin means a portion of any of the above described polypeptides, particularly fragments that include the C-terminus of pancreastatin, which exhibit the ability to inhibit insulin secretion to any degree.
  • pancreastatin antagonist intends a synthetic analog of pancreastatin which by virtue of one or more changes in the chemical composition of pancreastatin such as by derivitization (e.g., alkylation, acylation, or oxidation) of one or more residues, and/or substitution, deletion and/or addition of one or more residues, effectively lacks pancreastatin activity but is able to block pancreastatin activity such as by binding to pancreastatin receptors.
  • the term “substantially” as used herein to denote purity denotes at least about 80% purity and preferably at least 95% purity.
  • the term “substantially free” has corresponding meaning, (i.e., less than about 20% by weight impurity, preferably less than 5% impurity).
  • pancreastatin-containing peptides were identified by the amount of glycine amide released from tryptic digests of the extracts using the chemical method of Tatemoto, K. and Mutt. V., Proc Natl Acad Sci (USA) (1978)
  • pancreatic enzymes by boiling porcine pancreas followed by extracting the peptides using a relatively strong acid.
  • Peptides in the extract were selected by adsorption onto alginic acid.
  • the adsorbed peptides were further fractionated by ethanol precipitation at neutral pH.
  • Peptides were again selected by alginic acid adsorption and then fractionated on a sizing column. Fractions containing the C-terminal amide structure were subjected to cation exchange fractionation.
  • Pancreastatin was purified from the cation exchange eluate by a series of RP-HPLC steps.
  • Pancreastatin from other mammalian species may be isolated in the same manner.
  • pancreastatin Biologically active fragments of pancreastatin may be obtained by enzymatic digestion of full length pancreastatin. Characterization of Pancreastatin
  • Amino acid analysis showed that it consists of 49 amino acid residues: Ala (8), Arg (3), Asp (1), Glu (13), Gly (9), His (1), Leu (1), Lys (2), Met (1), Phe (1), Pro (5), Ser (1), Thr (2) and Trp (1).
  • the theoretical molecular weight of the deduced structure is in agreement with the actual molecular weight of the molecule as measured by FAB mass spectrometry (10 ⁇ g pancreastatin in thioglycerol. mass range 5120-4780 ⁇ , scan rate 200 s/d, 15 scans accumulated).
  • pancreastatin does, however, exhibit primary structure similarities to several known proteins. For instance, it shares the -Glu-Glu-Glu-Glu-Glu-structure (amino acid positions 34-38 in Figure 1) with gastrin and the C-terminal -Arg-Gly-NH 2 structure with vasopressin. It also appears that chromogranin may be a prohormone precursor for pancreastatin (Nature (1987) 325:301).
  • pancreastatin from other mammalian species will have the same or similar primary structure (i.e, be at least about 70% homologous in primary structure as regards the active C-terminus (amino acids 33-49 of Figure 1) and at least about 60% homologous in total) as the porcine molecule and that it may exhibit some interspecies biological activity (i.e., pancreastatin from one species may exhibit activity in another s pecies ) .
  • Polyclonal or monoclonal antibodies to pancreastatin and/or its biologically active fragments may be made by conventional methods.
  • Polyclonal antibodies to the pancreastatin/fragment may be made by immunizing host animals such as a rabbit, guinea pig or mouse, with the pancreastatin/fragment itself, fusion proteins thereof, or conjugates thereof with carrier proteins such as bovine serum albumin or keyhole limpet hemocyanin.
  • Adjuvants may be used with the immunogen.
  • the immunization will typically involve repeated inoculations (normally ip or im administration) with the immunogen. Such inoculation will raise a humoral response to the immunogen resulting in the production of antibodies to the immunogen by the host's immune system. Serum from the immunized host will usually be collected about three to ten days after the final booster.
  • Immunoglobulins may be separated from the serura by conventional methods such as ammonium sulfate precipitation, gel electrophoresis, dialysis, and affinity or other chromatographic techniques.
  • Monoclonal antibodies to the pancreastatin/fragment may be made by the well-known somatic cell hybridization technique using antibody-producing cells such as spleen or lymphoid cells from the immunized host animal as one of the fusion partners.
  • Murine cells are preferred because of the current availability of tumor fusion partners.
  • the antibody-producing cells are hybridized (fused) with an appropriate cancer (myeloma) cell line using a fusogen such as polyethylene glycol of mw 1000-14,000 daltons.
  • a myeloma line that is sensitive to a selective medium such as HAT medium, fuses efficiently, and will support stable high level expression and secretion of antibody by its fusion partner is used. While myelomas from any species may be used, murine lines having such characteristics are currently available and are preferred. Examples of such lines are those derived from the original MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center.
  • a myeloma cell:antibody-producing cell ratio in the range of about 1:10 and about 10:1 will normally be used.
  • the individual cell concentrations will typically be in the range of 10 6 to 10 8 , preferably about 10 7 cells/ml fusion medium.
  • Balanced salt solutions containing 30% to 60% (w/v) fusogen may be used as the fusion medium. After the fusion, the cells are washed with fusogeu-free medium to remove fusogen. They are then seeded and cultivated in the selective medium to eliminate unhybridized parent cells and leave only hybrids that are resistant to the selective medium and possess the immortality of the myeloma parent. The cultivation will normally take about three to five weeks.
  • Monoclonal antibodies to the pancreastatin/fragment may also be produced by other methods such as with EBV-transformed cell lines.
  • Analogs may be screened for activity using the perfused rat pancreas model described hereinafter. An analog's ability to block activity may be assessed by sequential perfusion with the analog and pancreastatin in the model. In this manner, candidate antagonists may be tested and molecules having antagonist activity identified.
  • Isolated pancreastatin or its biologically active fragments may be used for in vitro assays of body fluids for pancreastatin content.
  • Various assay formats may be used.
  • a common format is the competition immunoassay wherein the sample is incubated with labeled pancreastatin/fragment and antibody to pancreastatin. If pancreastatin is present in the sample, it will compete with the labeled pancreastatin for the antibody and can thus be detected.
  • These molecules may also be used in a therapeutic setting to treat conditions associated with excessive insulin production (e.g. hyperglycemia or cancers that cause excessive insulin production) or with excessive somatostatin production (e.g., somatostatin).
  • pancreastatin/fragment When used as therapeutic agents the pancreastatin/fragment will normally be formulated in therapeutically effective amounts as an injectable for parenteral administration (iv, ip, im, ia) with pharmaceutically acceptable injectable vehicles such as water, saline, dextrose solution, and the like.
  • injectable vehicles such as water, saline, dextrose solution, and the like.
  • the dose administered will depend upon the patient and the condition being treated. When used to offset excessive insulin or somatostatin production it will be administered in amounts which will inhibit such production so as to bring the level of insulin/somatostatin in the subject being treated to the desired level (typically levels found in normal subjects who do not suffer from the condition being treated).
  • the dose for an adult human will normally be in the range of 10 to 50 nanomoles of peptide.
  • Antibodies to pancreastatin/fragment may be used in immunoassays of body fluids or tissues for pancreastatin/fragment for the purpose of diagnosing or monitoring conditions associated with abnormal pancreastatin levels.
  • Various assay protocols may be used including direct and indirect formats.
  • direct formats a labeled derivative of the antibody is used to form an immune complex with any pancreastatin/fragment in the test sample and the complex is detected via the label.
  • unlabeled antibody to the pancreastatin/fragment is permitted to form immune complexes with any pancreastatin/fragment in the sample and the resulting complexes are detected via a labeled moiety, such as a labeled second antibody to the primary antibody, that binds to the complex.
  • the antibodies to pancreastatin/fragment and the pancreastatin antagonists may also be used in therapy to block the activity of the pancreastatin/fragment and thus treat conditions that may be associated with excessive pancreastatin, such as certain forms of diabetes.
  • the antibody/ antagonist will normally be administered parenterally as an injectable formulation.
  • the dose of antibody/antagonist required f or therapy will again depend upon the patient and the condition being treated. In general, sufficient antibody/antagonist will be administered to bring the level of pancreastatin/fragment activity in the system to the desired point.
  • Pancreastatin and its biologically active fragments may be isolated from natural sources (pancreas) as described above, synthesized by conventional chemical procedures, or biosynthesized in recombinant hosts using genetic engineering techniques.
  • pancreastatin/fragment involves obtaining a DNA sequence that encodes the pancreastatin/fragment.
  • sequences may be synthesized using codons deduced from the amino acid sequence of pancreastatin, isolated from genomic DNA or prepared from cDNA.
  • codons that are preferred by the host in which the gene is to be expressed (e.g., yeast-preferred codons for yeast hosts, bacteria-preferred codons for bacterial hosts).
  • prokaryotic or eukaryotic hosts including bacteria such as E. coli and B. subtilis. fungi, particularly yeast such as S. cerevisiae. and a wide variety of primary cell lines or immortalized mammalian cells such as 3T3, Vero, Chinese hamster ovary cells.
  • bacteria such as E. coli and B. subtilis.
  • fungi particularly yeast such as S. cerevisiae.
  • primary cell lines such as 3T3, Vero, Chinese hamster ovary cells.
  • the processing signals for cleavage of the secretory leader may be the native signals or the signals associated with the nonnative secretory leader or both.
  • the DNA sequence encoding the pancreastatin/fragment may be introduced in tandem with a gene capable of amplification such as the dihydrofolate reductase (dhfr) gene. Further, the DNA sequence may be linked to other DNA sequences as is known in the art to produce fusion proteins that have multiple functions or activities or include sequences that facilitate the processing or purification of the pancreastatin/fragment.
  • the expression vector may be introduced into the host by conventional techniques depending upon the nature of the host, such as transformation, transfection, and calcium phosphate precipitation. The host may then be cultured in an appropriate medium to produce the pancreastatin/fragment. The product may be isolated from the host or culture medium, as the case may be, by conventional purification procedures.
  • pancreastatin preparation (5 mg) was first fractionated on an Ultrapac TSK ODS 120-T column (4.6 x 250 mm. LKB AB, Sweden) using a gradient of acetonitrile in 0.1% aqueous trifluoroacetic acid at a flow rate of 1.0 ml/min.
  • Figure 2a shows the elution profile of this separation.
  • the pancreastatin-containing fraction (hatched area) was evaporated to dryness using a vacuum centrifuge.
  • a total of 20 mg of the preparation was purified by repeating the RP-HPLC separation and the combined pancreastatin fractions were subjected to a second RP-HPLC purification.
  • pancreastatin fractions were applied to an Ultrapac TSK ODS 120-T column and eluted with a gradient of acetonitrile in 10 mM phosphate buffer pH 6.5 (flow rate: 1.0 ml/min).
  • Figure 2b shows the elution profile of this second RP-HPLC, with the pancreastatin- containing fraction cross-hatched.
  • This fraction was further purified in a third RP-HPLC step on an Ultrasphere ODS column (4.6 x 250 mm, Altex) using the elution conditions of the first RP-HPLC.
  • the elution profile of the third RP-HPLC step is shown in Figure 2c.
  • pancreastatin (1.8 nmoles) was dissolved in 10 ⁇ l of 1% ammonium bicarbonate. 2 ⁇ l of Endoproteinase Lys-C (Boehringer Mannheim) solution (2.mg/ml) was added and the reaction mixture was incubated at room temperature overnight, then at 100°C for 10 min and lyophilized.
  • the enzyme digest was applied to an RP-HPLC column (Ultrapac TSK-ODS 120-T. 4.6 x 250 mm) and eluted at a flow rate of 1 ml/min with the solvent systems used in the first RP-HPLC for full length pancreastatin described above. The elution profile of this fractionation is shown in Figure 2d. The peaks designated I and II represent the fragment-containing fractions.
  • Step-wise Edman degradation of the intact molecule (2 nmoles) using a gas-phase sequencer revealed the identities of the first 25 residues of the N-terminal region. Since amino acid analysis indicated there were only two lysine residues in the molecule, the intact peptide (1.8 nmoles) was treated with a lysine-specific protease (Endoproteinase Lys-C, Boehringer, Mannheim) and the resulting digest was subjected to the RP-HPLC separation as described above (Figure 2d). Analysis of fragment I of Figure 2d) indicated the peptide had an N-terminal threonine and corresponded to the middle fragment (position 14-25) of the intact molecule.
  • the fragment of peak II was found to contain two peptides, one with an N-terminal glycine and the other with an N-terminal glycine and a C-terminal glycine amide, indicating that this peak contained both the N- and C-terminal fragments of the parent molecule. Edman degradation of the peptides in peak II thus yielded two residues for each cycle. Since the structure of the N-terminal fragment (positions 1-13) was already determined, the C-terminal structure at positions 26-49 was deduced from the sequence data by subtraction of the N-terminal sequence. In this way, the complete amino acid sequence ( Figure 1) of porcine pancreastatin was deduced.
  • the calculated mw of the sequence shown in Figure 1 is 5103.46 daltons.
  • the molecular weight of the isolated native molecule determined by FAB mass-spectrometry was 5103.1 daltons.
  • pancreastatin and its fragments were synthesized in a step-wise manner according to Merrifield (J Am Chem Soc (1964) 85: 2149-2154). After complete deprotection and cleavage by hydrofluoric acid, the crude synthetic preparations were purified by semipreparative HPLC. The synthetic pancreastatin (1-49) thus obtained was found to co-elute in RP-HPLC with the native peptide under the conditions shown in Figures 2a and 2c. Furthermore, the results of amino acid sequence analysis and molecular weight (found 5103.4) determination by FAB mass-spectrometry indicated that the synthetic peptide has an identical structure to native pancreastatin.
  • pancreastatin 1-49
  • pancreastatin 14-49
  • pancreastatin 33-49
  • the perfused rat pancreas was prepared as follows:
  • Sprague-Dawley rats weighing 200-250 g, were fed ad libitum. They were anaesthetized by ip injection of 50 mg/kg pentobarbital and the pancreas was isolated.
  • the pancreas was first perfused with Krebs-Ringer buffer containing 20 g/1 of bovine serum albumin and 3.3 mM glucose for 20 min and then perfused for 50 min with the buffer and 16.7 mM glucose.
  • the pancreastatin (10 nM) was added 10 min prior to and during glucose (16.7 mM) administration.
  • the solution was perfused by the use of a perfusion pump through the abdominal aorta. The flow rate of perfusion was 3 ml/min.
  • the perfusate was collected in tubes containing 0. 1 ml of Trasylol. Insulin was measured by radioimmunoassay using a rat insulin standard. The sensitivity of the assay was about 300 ng/1 and coefficient of variation 10%. Somatostatin was measured by radioimmunoassay.
  • pancreastatin As shown in Figure 3, perfusion of glucose (16.7 mM) induced a biphasic insulin release from the. isolated pancreas. Pancreastatin and the two fragments markedly decreased the early phase of insulin release (1-5 rain). The effect on late phase insulin secretion (5-40 min) was less pronounced, but statistically significant for the fragments.
  • pancreastatin strongly suppresses insulin release, particularly in the early phase, abnormalities in the regulation or action of pancreastatin and its receptors may be involved in the pathogenesis of type-2 diabetes.
  • pancreastatin and its fragments also inhibited the release of somatostatin upon glucose stimulation from the perfused pancreas.

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  • Health & Medical Sciences (AREA)
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Abstract

Nouvelle hormone pancréatique, appelée ''pancréastatine'' et ses fragments biologiquement actifs. Ces polypeptides inhibent la sécrétion d'insuline et de somatostatine induite par le glucose. Sont également décrits des anticorps polyclonaux et monoclonaux contre ces peptides et des antagonistes de la pancréastatine, ainsi que des compositions et des procédés diagnostiques et thérapeutiques utilisant la pancréastatine, ses fragments biologiquement actifs, ou les anticorps/antagonistes de la pancréastatine ou de ses fragments.
PCT/US1987/002895 1986-11-06 1987-11-03 Pancreastatine WO1988003536A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US92783686A 1986-11-06 1986-11-06
US927,836 1986-11-06

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WO1988003536A1 true WO1988003536A1 (fr) 1988-05-19

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PCT/US1987/002895 WO1988003536A1 (fr) 1986-11-06 1987-11-03 Pancreastatine

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10157336B4 (de) * 2000-11-24 2007-07-19 Bioserv Analytik Und Medizinprodukte Gmbh Verfahren zur Herstellung eines Testsystems und Diagnoseverfahren zur Erkennung von Pankreasfunktionsstörungen

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3013944A (en) * 1959-12-07 1961-12-19 Jorpes Johan Erik Process for the production of gastrointestinal hormones and hormone concentrate

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3013944A (en) * 1959-12-07 1961-12-19 Jorpes Johan Erik Process for the production of gastrointestinal hormones and hormone concentrate

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
Nature, Volume 285, issued June 1980 (TATEMOTO et al.), "Isolation of Two Novel Candidate Hormones Using a Chemical Method for Finding Naturally Occuring Polypeptides", pages 417-418. *
Nature, Volume 296, issued April 1982 (TATEMOTO et al.), "Neuropeptide Y-A Novel Brain Peptide with Structural Similarities to Peptide YY and Pancreatic Polypeptide", pages 659-660. *
Nature, Volume 324, issued December 1986, (TATEMOTO et al.), "Pancreastatin, a Novel Pancreatic Peptide that Inhibits Insulin Secretion", pages 476-478, see pages 476, 477, 478 in particular. *
Proc. Natl. Acad. Sci. USA, Vol. 75, issued September 1978 (TATEMOTO et al.), "Chemical Determination of Polypeptide Hormones", pgs. 4115-4119. *
Proc. Natl. Acad. Sci. USA, Volume 78, issued November 1981 (TATEMOTO et al.), "Isolation and Characterization of the Intestinal Peptide Porcine PHI (PHI-27), a New Member of the Glucagon - Secretin Family", pages 6603-6607. *
Proc. Natl. Acad. Sci. USA, Volume 79, issued April 1982 (TATEMOTO), "Isolation and Characterization of Peptide YY(PYY), a Candidate Gut Hormone that Inhibits Pancreatic Exocrine Secretion", pages 2514-2518. *
Proc. Natl. Acad. Sci. USA, Volume 79, issued September 1982, (TATEMOTO), "Neuropeptide Y: Complete Amino Acid Sequence of the Brain Peptide", pages 5485-5489. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10157336B4 (de) * 2000-11-24 2007-07-19 Bioserv Analytik Und Medizinprodukte Gmbh Verfahren zur Herstellung eines Testsystems und Diagnoseverfahren zur Erkennung von Pankreasfunktionsstörungen

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