WO1988003536A1

WO1988003536A1 - Pancreastatin

Info

Publication number: WO1988003536A1
Application number: PCT/US1987/002895
Authority: WO
Inventors: Kazuhiko Tatemoto
Original assignee: The Board Of Trustees Of The Leland Stanford Junio
Priority date: 1986-11-06
Filing date: 1987-11-03
Publication date: 1988-05-19

Abstract

A newly discovered pancreatic hormone, called ''pancreastatin'', and biologically active fragments thereof. These polypeptides inhibit glucose-induced secretion of insulin and somatostatin. Polyclonal and monoclonal antibodies to these peptides and pancreastatin antagonist are also described together with diagnostic and therapeutic compositions and methods that employ pancreastatin, its biologically active fragments, or antagonists/antibodies to pancreastatin or such fragements.

Description

PANCREASTATIN

Reference to U.S. Government support The invention was made in the course of research funded in part by the National Institutes of Mental Health and the Office of Naval Research. The U.S. Government accordingly has rights in the invention.

Description

Technical Field

The invention is related to the fields of protein chemistry, endocrinology, immunology and recombinant DNA technology. More particularly, it relates to the purification to homogeneity of a newly discovered polypeptide, called "pancreastatin." that inhibits insulin and soraatostatin secretion, synthetic analogs of pancreastatin that are antagonists of pancreastatin, antibodies to pancreastatin. therapeutic and diagnostic methods employing pancreastatin and/or anti-pancreastatin antibodies, immunoassays for pancreastatin, and recombinant DNA materials and methods for producing pancreastatin.

Background

It is known that in mammalian tissues the C-terminal amide structure occurs only in neuroactive- or hormonally-active polypeptides. About half of the known neuropeptides and hormonal polypeptides have this characteristic terminal structure. Using this characteristic prior workers searched for new neuropeptides and hormone peptides in brain and intestine extracts and isolated neuropeptide Y

(Tatemoto, K., et al. Nature (1982) 296:659-660 and Tatemoto. K., Proc Natl Acad Sci USA (1982) 79:5485-5489), PHI (Tatemoto. K., Proc Natl Acad Sci USA (1981) 78: 6603-6607). peptide YY (Tatemoto. K., Proc Natl Acad Sci USA (1982) 79:2514-2518) and neuropeptide K.

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."

Disclosure of the Invention One aspect of the invention 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.

Synthetic analogs (antagonists) of pancreastatin that lack pancreastatin activity but are able to block pancreastatin activity are another aspect of the invention.

Pharmaceutical compositions that contain pancreastatin, one or more biologically active fragments of pancreastatin, said synthetic analogs, or antibodies thereto are another aspect of the invention.

Still another aspect of the invention is a method of treating an individual for overproduction of pancreastatin comprising administering an amount of anti-pancreastatin antibody to the individual to reduce the level of pancreastatin in the individual to a normal level.

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.

Other aspects of the invention will be apparent from the following disclosure.

Brief Description of the Drawings In the drawings:

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. Modes for Carrying Out the Invention

Definitions

As used herein the term "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.

The term "biologically active fragment of pancreastatin" as used herein 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. The term "pancreastatin antagonist" as used herein 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).

Isolation of Pancreastatin From Pancreas The technique used to isolate pancreastatin from pancreatic tissue was based on the premise that it had a C-terminal amide structure. The extraction protocol was designed to select such polypeptides and is similar to that described in U.S. Pat. No. 3,013,994. 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)

75: 4115-4119. The procedure involved first deactivating 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.

Biologically active fragments of pancreastatin may be obtained by enzymatic digestion of full length pancreastatin. Characterization of Pancreastatin

Terminal analysis of the pure porcine polypeptide indicated that it contains an N-terminal glycine and a C-terminal glycine amide. 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). Stepwise Edman degradation of the fragments of the polypeptide provided partial amino acid sequences from which the complete sequence of the molecule was deduced. The deduced sequence is reported in Figure 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 μg pancreastatin in thioglycerol. mass range 5120-4780 μ, scan rate 200 s/d, 15 scans accumulated).

Applicant believes the primary structure of pancreastatin reported in Figure 1 is different from that of all other known polypeptides. 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-NH₂ structure with vasopressin. It also appears that chromogranin may be a prohormone precursor for pancreastatin (Nature (1987) 325:301).

Bioassays of pancreastatin and its C-terminus fragments show that these molecules suppress both early and late phase insulin release, with the early phase suppression being less pronounced for the fragments. These assays also showed that pancreastatin inhibits glucose-stimulated somatostatin release. These observations indicate that the C-terminal portion of the molecule is important for the insulin secretion inhibiting activity of the peptide and that pancreastatin and/or its C-terminal fragments play an important role in the control of carbohydrate metabolism and hyperglycemia in diabetes.

It is expected that 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 ) .

Antibodies to Pancreastatin

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 10⁶ to 10⁸, preferably about 10⁷ 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.

Surviving hybridomas may be examined for production of antibody to the pancreastatin/fragment by immunoassay using the immunogen as antigen. Positive hybridoma clones may be subcloned by limiting dilution techniques and grown in vitro or in vivo by known techniques. The resulting monoclonal anti-pancreastatin/fragment antibody secreted by the subclones may be separated from the culture medium or body fluid when grown in vivo by known techniques for immunoglobulin separation such as those described above.

Monoclonal antibodies to the pancreastatin/fragment may also be produced by other methods such as with EBV-transformed cell lines.

Pancreastatin Antagonists

As indicated previously the pancreastatin antagonists are pancreastatin analogs that lack the pancreastatic activity (i.e., inhibition of insulin secretion) but are capable of blocking such activity. These analogs may be prepared by derivatizing pancreastatin. by conventional chemical procedures, or biosynthetically in recombinant hosts using genetic engineering techniques (i.e., expression of antagonist genes prepared by oligonucleotide synthesis or site-directed mutagenesis of the pancreastatin gene). In order to deactivate the molecule it is preferable to modify the carboxy terminus (amino acids 33-49) since that region is believed to be critical to activity.

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.

Therapeutic and Diagnostic Methods

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). 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. 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. In 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. In the indirect format, unlabeled antibody to the pancreastatin/fragment (primary antibody) 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. In such use 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.

Synthesis of Pancreastatin

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.

The biosynthesis of pancreastatin/fragment involves obtaining a DNA sequence that encodes the pancreastatin/fragment. Such sequences may be synthesized using codons deduced from the amino acid sequence of pancreastatin, isolated from genomic DNA or prepared from cDNA. When the sequence is synthesized, it is preferred to use 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).

The DNA sequence encoding the pancreastatin/fragment may be cloned and expressed by (1) inserting it into an appropriate expression vector having a replication system, and suitably positioned transcriptional and, as appropriate, translation signals and (2) introducing the resulting expression vector containing the sequence into a compatible host. The sequence may be tailored for insertion by providing it with suitable terminal restriction sites, providing for blunt end ligation, or the like. The expression vector may be a low or high multicopy vector which exists extrachromasomally or integrated into the genome of the host and may provide for secretion or excretion of the pancreastatin/fragment or retention of the pancreastatin/fragment in the cytoplasm or membrane of the host.

A large number of cloning and expression vectors have been described in the published literature and are generally available for use in either 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. Depending upon the host, where secretion is desired, either the native or nonnative secretory leader sequence may be used. 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. If desired, 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.

The invention is further illustrated by the following examples. These examples are not intended to limit the invention in any manner.

Isolation of Native Porcine Pancreastatin

Porcine pancreas (20 kg) was boiled in water for 10 min, frozen, minced and extracted with 0.5 M acetic acid for 24 h. Peptides in the extract were adsorbed onto alginic acid (1 kg wet wt), eluted with 0.2 M HCl and precipitated with NaCl at saturation. The peptide precipitate (18 g wet wt) was redissolved in water (360 ml) and two volumes of ethanol were added to the solution. The pH of the solution was adjusted to 7.2 and the resulting precipitate was removed by filtration. After addition of three volumes of water to the filtrate, peptides in the filtrate were again adsorbed onto alginic acid (250 g wet wt). eluted with 0.2 M HCl and reprecipitated with NaCl at saturation. This second peptide precipitate (4 g wet wt) was applied to a Sephadex G-25 column and eluted with 0.2 M acetic acid. Fractions containing pancreastatin were pooled and lyophilized. The lyophilized material (0.8 g) was further purified by ion-exchange chromatography on CM cellulose with a step-wise elution (0.01-0.2 M) of ammonium bicarbonate. The fraction, eluted by 0.01 M ammonium bicarbonate, was found to contain pancreastatin and this partially-purified preparation (90 mg) was used as the starting material for further purification by RP-HPLC.

The partially-purified 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. In the second RP-HPLC step, the combined 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. Biologically active fragments of pancreastatin were prepared as follows: pancreastatin (1.8 nmoles) was dissolved in 10 μl of 1% ammonium bicarbonate. 2 μl 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.

All fractions were assayed for putative pancreastatin/fragment by the method of Tatemoto and Mutt, supra.

Sequence Analysis of Pancreastatin

Step-wise Edman degradation of the intact molecule (2 nmoles) using a gas-phase sequencer (Applied Biosystems) 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.

Chemical Synthesis of Pancreastatin/Fraqments

Chemical synthesis of pancreastatin and its fragments was carried out using a solid-phase synthesizer. Fully protected pancreastatin (1-49) and the (14-49) and (33-49) 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.

Biological Studies

The inhibiting effect of pancreastatin (1-49), pancreastatin (14-49) and pancreastatin (33-49) on glucose induced insulin and somatostatin production was assessed using a perfused rat pancreas model. 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. In control experiments 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.

The insulin release results are shown in Figure 3 and the somatostatin release results are shown in Figure 4.

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.

It has been suggested that suppression of insulin release upon glucose stimulation, especially in the early phase, is a characteristic feature of manifest type-2 diabetes. Since 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.

The data shown in Figure 4 indicate that pancreastatin and its fragments also inhibited the release of somatostatin upon glucose stimulation from the perfused pancreas.

Modifications of the above-described modes for carrying out the invention that are obvious to those of skill in the fields of protein chemistry, endocrinology, immunology, or recombinant DNA technology are intended to be within the scope of the following claims.

Claims

1. Substantially pure pancreastatin.

2. Pancreastatin substantially free of compounds associated with pancreastatin in the native state.

3. The pancreastatin of claims 1 or 2 having the amino acid sequence shown in Figure 1.

4. The pancreastatin of claims 1 or 2 having an amino acid sequence that is at least 60% homologous to the amino acid sequence shown in Figure 1.

5. A biologically active fragment of pancreastatin.

6. The fragment of claim 5 wherein the fragment includes the C-termmus of pancreastatin.

7. The fragment of claim 5 wherein the fragment consists essentially of amino acid residues 14-49 of Figure 1 or amino acid residues 33-49 of Figure

8. An antibody to pancreastatin or to a biologically active fragment of pancreastatin.

9. The antibody of claim 8 wherein the antibody is a polyclonal antibody or a monoclonal antibody.

10. A composition for treating a condition associated with excessive insulin secretion comprising an insulin secretion-inhibiting amount of pancreastatin or a biologically active fragment of pancreastatin and a pharmaceutically acceptable carrier.

11. A composition for treating a condition associated with excessive pancreastatin comprising an antibody to pancreastatin or a pancreastatin antagonist and a pharmaceutically acceptable carrier.

12. A method of treating a mammalian subject for a condition associated with excessive insulin secretion comprising administering to the patient a insulin secretion-inhibiting amount of pancreastatin or a biologically active fragment of pancreastatin.

13. A method of treating a mammalian subject for a condition associated with excessive pancreastatin or biologically active fragment of pancreastatin comprising administering to the subject an therapeutically effective amount of an antibody to pancreastatin or to said biologically active fragment of pancreastatin or a pancreastatin antagonist.

14. A pancreastatin antagonist.