WO1992022329A1

WO1992022329A1 - USE OF β-CELL TROPIN (ACTH 22-39) ANTAGONISTS IN THE TREATMENT OF HYPERINSULINAEMIA AND ASSOCIATED DISEASES

Info

Publication number: WO1992022329A1
Application number: PCT/GB1992/001079
Authority: WO
Inventors: Michael Anthony Cawthorne; Anne Beloff-Chain
Original assignee: Smithkline Beecham Plc; POWELL, Judith, Mary; Chain, Benjamin, Michael
Priority date: 1991-06-17
Filing date: 1992-06-16
Publication date: 1992-12-23
Also published as: AU662911B2; EP0589989A1; MX9202932A; IE921965A1; GB9113011D0; ZA924428B; PT100595A; NZ243176A; JPH06508615A; AU2020392A; CA2111584A1

Abstract

Pharmaceuticals, including compounds and compositions and the use of such pharmaceuticals in the treatment of hyperinsulinaemia and conditions associated with hyperinsulinaemia.

Description

USE OF (S-CELL TROPIN (ACTH 22-39) ANTAGONISTS IN THE TREATMENT OF HYPERINS LINEMIA AND ASSOCIATED DISEASES .

The invention relates to pharmaceuticals, including compounds and compositions, and the use of such pharmaceuticals in a treatment of hyperinsulinaemia and conditions associated with hyperinsulinaemia.

Hyperinsulinaemia is a component of a number of diseases particularly Type II diabetes (or non-insulin-dependent diabetes) which is a serious and common metabolic disease state of humans and other mammals such as dogs and cats. At present there is no marketed medicament which provides effective treatment for reducing hyperinsulinaemia. Hyperinsulinaemia is also associated with obesity and obesity prone hypertension and atherosclerosis and hyperlipidaemia. Recently, hyperinsulinaemia has been described as a component of Syndrome X (G.M. Reaven, Diabetes, Vol. 37, 1988, 1595-1607). In each of these disorders it is believed that hyperinsulinaemia is a component in the progression of the disease and normalisation of hyperinsulinaemia will provide an effective treatment.

Amylin is a polypeptide recently isolated from amyloid masses extracted from Type II diabetic pancreases, (Proc. Natl. Acad. Sci. USA. Vol. 84, p. 8628-8632, Dec. 1987). Amylin has been stated to play an active part in an endocrine homeostatic mechanism in the body for controlling the distribution of carbohydrate energy (as glucose). In particular Amylin is stated to (1) inhibit the release of insulin from β-cells within the islets of Langerhan and (2) inhibit the basal and insulin stimulated glycogen synthesis in skeletal muscle by causing muscle cells to ignore the insulin signal (WO 89/06135). Type II diabetes and in particular insulin resistance is stated to be an effect of the over-secretion of amylin. A consequence of the insulin resistance is often a compensatory hyperinsulinaemia .

β-Cell-tropin (β-CT) is a peptide which was originally isolated from perifusates (superfusates) of the isolated pituitary neurointermediate lobe of the genetically obese (ob/ob) mouse (Beloff-Chain et al., FEBS Lett., (1980) 117, 303-307). β-CT has a range of biological activities. (1) It stimulates the release of insulin, at substimulatory concentrations of glucose, from the isolated perfused rat pancreas (Dunmore & Beloff- Chain, J. Endocrinol. (1982) 92, 15-21) and from perifused islets of Langerhans (Billingham et al., J. Endocrinol. (1982) 94, 125-130) eliciting a monophasic response. (2) It potentiates the biphasic release of insulin induced by high concentrations of glucose from the perfused pancreas (Beloff-Chain et al., Biochem. Soc. Trans. (1981) 9, 522-524). (3) It has an insulin-like action in promoting lipogenesis in adipose tissues (Watkinson & Beloff-Chain, Horm. Metab. Res. (1984) 16, 55-58 (Suppl.)).

β-CT has been characterized as adrenocorticotropin (ACTH)-(22-39) (Beloff-Chain et al., Nature (London), 301, (1983) 255-258) and evidence of its hormonal nature is provided by its presence in the plasma of the obese mouse (Billingham et al., 1982) and human plasma (Salvatoni et al., J. Endocrinol. (1986) 110, 303-307). Synthetic β-CT has also been prepared using mild solid phase procedures (Biochem. and Biophys. Res. Comm. (1983), vol. 114, No. 2, 763-766).

It is known that certain modifications to the structure of β-CT modulate the insulin-releasing potency of β-CT (S. Dunmore et al., Biochem. J. (1987) 244, 797-800).

It has now been discovered that the insulin-like lipogenic activity of β-CT can be regulated.

It has also been discovered that β-CT may play an important role in the regulation of the release of amylin. In particular it is indicated that β-CT can be causitive in increasing amylin release thereby contributing to the development of insulin resistance and the consequential hyperinsulinaemia.

β-CT is therefore indicated to play a central role in the development of hyperinsulinaemia by directly increasing insulin release, and indirectly via increased amylin release, causing the development of insulin resistance and compensatory hyperinsulinaemia. In addition to these actions tiie direct insulin-like action of β-CT on adipose tissue leads to the development of obesity.

It is considered that the regulation of the formation or release of β-CT or the regulation of the biological activity of β-CT provides the potential for the treatment of hyperinsulinaemia and conditions associated with hyperinsulinaemia, such as type II diabetes, hyperlipidaemia, hypertension and atherosclerosis, obesity and syndrome X.

Accordingly, the present invention provides a method for the treatment of hyperinsulinaemia and conditions associated with hyperinsulinaemia, such as type II diabetes, hyperlipidaemia, hypertension, atherosclerosis, syndrome X and obesity, which method comprises the aclministration of an effective, non-toxic amount of a compound which regulates the formation, release or biological activity of β-cell tropin (β-CT).

One compound is that which regulates the formation of β-cell tropin. One compound is one which regulates the release of β-cell tropin. One compound is that which regulates the biological activity of β-cell tropin.

A particular compound which regulates the biological activity of β-CT is a compound which inhibits the biological activity of β-CT, such as a β-CT antagonist.

One example of a β-CT antagonist is a non-competitive antagonist such as an antibody specific for β-CT, suitably a monoclonal antibody specific for β-CT.

A further example of a β-CT antagonist is a competitive β-CT antagonist.

A competitive β-CT antagonst is an antagonist which competes with β-CT or a β-CT agonist for a β-CT receptor.

An example of a competitive β-CT antagonist is a disabled β-CT agonist. A disabled β-CT agonist is a β-CT agonist which has been structurally modified so that it is still capable of binding to a β-CT receptor but which ellicits a reduced biological response, suitably it ellicits no pharmacologically relevant biological response.

An example of a disabled agonist is a cross-linked agonist.

Suitable cross-linked agonists include cross-linked β-CT or cross-linked fragments of β-CT. An example of a disabled agonist is substituted β-CT or a substituted fragment of β-CT. The terms Substituted β-CT^* and Substituted fragment of β-CT' refer to β-CT or fragments thereof wherein the a ino acid sequence has been changed or wherein the amino acids from the normal sequence have been structurally modified.

A further example of a competitive β-CT antagonist is an antibody, suitably monoclonal, directed to a β-CT receptor.

In a further aspect, the present invention provides a compound which regulates the formation, release or biological activity of β-cell tropin (β-CT), for use as an active therapeutic substance.

In particular, the present invention provides a compound which regulates formation, release or the biological activity of β-cell tropin (β-CT), for use in the treatment of hyperinsulinaemia and conditions associated with hyperinsulinaemia, such as type II diabetes, hyperlipidaemia, hypertension, atherosclerosis, syndrome X and obesity.

In yet a further aspect, the present invention provides the use of a compound which regulates the formation, release or biological activity of β-cell tropin (β-CT) for the manufacture of a medicament for the treatment of hyperinsulinaemia and conditions associated with hyperinsulinaemia, such as type IE diabetes, hyperhpidaemia, hypertension, atherosclerosis, syndrome X and obesity.

Disabled β-CT agonists will be prepared by the appropriate conventional procedure: thus for example cross-linked β-CT or cross-linked fragments of β-CT may be prepared by conventional peptide cross-linking techniques such as those described in Biochemistry 17_, 1978, 1499, International J. Peptide & Protein Res., 2__, 1986, 285-92 and Pierce, 1989 Handbook and General Catalogue (Cross-linking Reagents), p.283-311.

Also, substituted β-CT or substituted fragments of β-CT may be prepared by conventional peptide chemical techniques such as those described in Solid Phrase Peptide Synthesis - a Practical Approach, E. Atherton, R.C. Sheppard Pub. IRL Press. It will be appreciated that a β-CT antagonist will be particularly conveniently prepared when the particular manner by which β-CT interacts with a β-CT receptor has been determined. This first involves the identification of the active site or sites upon β-CT which interact with the β-CT receptor. Once such information has been established, the identity of the relevant amino acid residues of β-CT or the fragments thereof which may be disabled, by substitution or cross-linking, can be more easily determined.

Crystalographic analysis of the structure of β-CT co-crystalized with its receptor, or part of its receptor, will allow analysis of the interaction between β-CT and its receptor. Such analysis will allow the determination of those amino acid residues that are of primary importance in the interaction between β-CT and its receptor and in turn will indicate which residues may, for example, be substituted or cross-linked in order to produce an antagonist.

The structural analysis of the β-CT to β-CT receptor interaction will also allow the determination of the likely molecular shape of and other structural features necessary for a peptide or non-peptide organic inhibitor.

An antibody to a β-CT receptor may be obtained by raising antibodies, such as monoclonal antibodies, in the presence of a source of a β-CT receptor using appropriate conventional procedures (Proc. Natl. Acad. Sci. USA (1982), 79, 7312-7316). Thus antibodies to the β-CT receptor can be raised in an appropriate test animal, such as BALB/c or other similar strains of mice, by immunization with purified or partially purified preparations of the β-CT receptor, or with cells with a high concentration of β-CT receptors. The spleens of the animals can be removed, and their lymphocytes fused to a mouse myeloma cell line. After screening of hybrids by known techniques, a stable hybrid will be isolated that produces antibodies against the β-CT receptor. Such activity can be demonstrated by the ability of the antibody to prevent the binding of radiolabelled β-CT (e.g. l-^I-labelled β-CT or 3H-β-CT) to its receptor. The monoclonal antibody can then be examined for its antagonistic activity by use of the screening techniques discussed below. A further approach involves the use of anti-idiotype antibodies. Anti- idiotype antibodies may be raised against monoclonal antibodies directed against β-CT, such that the anti-idiotype will have complimentary binding affinity for the β-CT receptor site without, of course, the activity promotion associated with β-CT binding. Utilization of anti-idiotype antibodies for blocking viral binding to cells is known (J. Immunol (1983), 131, 2539-2541 or Med. Int. (1987) 317, 219-224.).

Compounds which regulate the biological activity of β-CT may be identified by conventional screening techniques, especially in vitro techniques. Thus, potential β-CT antagonists may be identified by determining whether or not they reduce the biological activity of β-CT in the presence of a source of β-CT receptor. One suitable source of β-CT receptor is considered to be adipose tissue or adipocytes or appropriate cell lines such as 3T3-L1 or 3T3-F442 cells. Other suitable sources of β-CT receptor are the perfused pancreas, isolated pancreatic islets, pacreatic β-cells or an appropriate cell line such as HIT or RIN cells.

An example of an in vitro screen involves determining the effect of a potential β-CT antagonist upon the uptake of glucose and/or the synthesis of triacylglycerol by cells in the presence of a source of β-CT and in the presence or absence of insulin: the decrease in uptake relative to a control provides an indication of antagonistic activity. A further example of an in vitro screen is the inhibition of β-cell tropin induced insulin and/or amylin release, by potential antagonists.

Alternatively, plasma membranes from adipose tissue(s) or pancreatic islet cells or a suitable cell line containing β-CT receptors can be configured in a receptor binding assay whereby the ability of antagonists to prevent the binding of β-CT to its receptor can be monitored by conventional radio-immunoassay, Elisa or time-resolved fluorescent technology.

To determine the nature of compounds which regulate the formation or release of β-CT it will first be necessary to determine the nature of the metabolic control of the production of β-CT from the pituitary. This will be determined for example, by incubating isolated neurointeπnediate lobes of the pituitary or whole pituitary and a pituitary cell line with a variety of concentrations of candidate molecules both intermediary metabolites and signal molecules such as biologically active peptides and non-peptide hormones to determine which molecules exert a positive effect and which a negative effect on the formation and/or release of β-CT. The response of the pituitary tissue to the various signals can be determined by the measurement of the formation of β-CT and/or its release into the medium. The ability of molecules to block this mechanism can then be examined using the same techniques.

β-CT may be produced by methods indicated above, such as by the specific cleavage of precursor molecules such as ACTH and corticotrophin-like intermediary peptide (CLIP; ACTH 18-39). One method of controlling the production of β-CT involves inhibiting the action of the enzyme(s) involved and/or by supressing the synthesis of the enzyme(s).

Test methods for determining that a particular compound regulates the the biological activity of β-CT are analogous to conventional test methods: for example the procedures used may be analogous to conventional procedures used to determine the lipogenic activity of β-CT.

For example, white or brown adipocytes can be incubated in the presence of β-CT and labelled glucose with or without the presence of the test compound. The amount of labelled glucose incorporated as lipid into the adipocytes with or without the presence of the test compound may then be determined by conventional scintillation counting methods.

The particular compound used may be prepared by any appropriate method. For example when the compound is an antibody raised against β-CT it may be prepared by conventional antibody generating methods. One particular method involves injecting a test animal as often as necessary with β-CT and then after an appropriate time, for example 18 weeks, harvesting the antibodies produced in the test animal.

Another example of a test procedure involves the incubation of isolated pancreatic islets (Billingham et. al or J. Endocrinol (1982) f_4, 125-130) or a pancreatic β-cell line such as HIT or RIN cells with β-CT with or without the presence of test substance and determining the effect of the test substance on insulin and/or amylin release. The release of insulin and amylin from the islet or cell fine can be determined by conventional radio- immunoassay-procedure.

Identification of the β-CT receptor site on pancreas islet and on adipose tissue will make it possible to provide for direct antagonism of its activity.

A compound which regulates the formation, release or biological activity of the invention maybe administered per se or, preferably, as a pharmaceutical composition also comprising a pharmaceutically acceptable carrier.

Accordingly, the present invention also provides a pharmaceutical composition comprising a compound which regulates the formation, release or biological activity of β-cell tropin (β-CT) and a pharmaceutically acceptable carrier therefor.

A compound which regulates the formation, release or biological activity of β-cell-tropin is also considered to be part of the present invention.

Hereinafter a compound which regulates the formation, release or biological function of β-cell tropin will be referred to as a compound of the invention'.

As used herein the term 'pharmaceutically acceptable' embraces compounds, compositions and ingredients for both human and veterinary use.

Suitable non-human mammals include dogs and cats.

The composition may, if desired, be in the form of a pack accompanied by written or printed instructions for use.

Usually the pharmaceutical compositions of the present invention will be adapted for oral aclministration, although compositions for administration by other routes, such as by injection and percutaneous absorption are also envisaged.

Particularly suitable compositions for oral administration are unit dosage forms such as tablets and capsules. Other fixed unit dosage forms, such as powders presented in sachets, may also be used.

In accordance with conventional pharmaceutical practice the carrier may comprise a diluent, filler, disintegrant, wetting agent, lubricant, colourant, flavourant or other conventional adjuvant.

Typical carriers include, for example, microcrystalline cellulose, starch, sodium starch glycollate, polyvinylpyrrolidone, polyvinylpolypyrrolidone, magnesium stearate, sodium lauryl sulphate or sucrose.

Most suitably the composition will be formulated in unit dose form. Such unit dose will normally contain an amount of the active ingredient in the range of from 0.1 to 1000 mg, more usually 0.1 to 500 mg, and more especially 0.1 to 250 mg.

Conveniently, the compound of the invention may be administered as a pharmaceutical composition hereinbefore defined, and this forms a particular aspect of the present invention.

In the treatment of hyperinsulinaemia or conditions associated with hyperinsulinaemia, the compound of the invention may be taken in doses, such as those described above, one to six times a day in a manner such that the total daily dose for a 70 kg adult will generally be in the range of from 0.1 to 6000 mg, and more usually about 1 to 1500 mg.

The following Example illustrates the invention but does not limit it in any way.

EXAMPLE

1. DETERMINATION OF EFFECT OF β-CELL TR PTN UPON AMYT.IN SECRETT N

METHODS AND MATERIALS::

ANIMALS: Fatty rats (fa/fa) and lean controls (Fa/? or Fa/Fa) aged 12 to 20 weeks were used.

PANCREAS PERFUSIONS

Pancreata from the male fatty rats and lean littermates described above were isolated and perfused by tiie method described in Dunmore and Beloff-Chain, J. Endocrinol (1982) 22,15,21. The perfiision buffer was a modified Rrebs-Ringer Bicarbonate buffer containing 3% Dextran T40 (Pharmacia) and 1% high purity BSA as described in Dunmore and Beloff- Chain (1982) (loc. lit.).

The isolated preparation was then perfused with the following (after a 15 minute period of 'low glucose" perfiision buffer to allow stabilization):

1. 5 minutes of low glucose (5.6 mmol/1) buffer,

2. 5 minutes of buffer as 1. with the addition of 0.5 to 1.0 nmol/1 β-CT 3. 10 minutes of high glucose (16.7 mmol/1) buffer.

Flow-rate was 5.0 ml. min"****, with collection of 1 minute fractions. Fractions were collected on ice in tubes to which 1800 KIU aprotinin (Traysylol, Bayer) had been added. Fractions were stored at -20° until assayed.

DETERMINATION OF BLOOD GLUCOSE. PLASMA INSULIN AND β- CELL TROPIN

Blood was taken from the tail vein (approx. 5 ml) into tubes containing heparin (Pularin, approx. 200 units/ml) and aprotin (Trasylol, 400 iπU/ml), a sample was immediately deproteinized prior to the determmination of glucose concentration, and the remainder centrifuged. The supernatant plasma was frozen at -20° until assayed.

ASSAYS;

GLUCOSE ASSAY: Glucose was determined spectrophotometricallly using the glucose oxidase/peroxidase method on blood deproteinized with uranyl acetate (URAC)(Boehringer.).

INSULIN ASSAY: The insulin present in plasma and perfiision samples was measured by a conventional radioimmunoassay slightly modified from that described in Dunmore and Beloff-Chain (1982) Goc. cit.). In brief, samples were assayed in triplicate with a suitable dilution of a high titre (1:30000 giving 50% binding) guinea-pig insulin antiserum and l²5_odinated bovine insulin (iodinated by the Chloramine T method). Rat insulin (Novo.) was used as a standard (diluted in either plasma assay buffer (Sodium Phosphate.) or perfiision buffer. Antibody-bound insulin was separated from free insulin by addition of second-antibody (donkey anti-guinea pig lg) coated cellulose (Sac-Cel, Washington.) and subsequent centrifugation. Counts bound were measured in a LKB Rackgamma solid scintillation counter and insulin calculations performed by a RIACALC programme (LKB) on a PC-AT computer (Opus PCV) linked to the counter.

AMYLIN ASSAY: Amylin was radioimmunoassayed using lyophilized reagents supplied by Peninsula Laboratories Ltd. All reagents were reconstituted fresh for each assay. The supplier's method of assay was followed with the exception of separation of bound and free amylin which was accomplished using donkey anti-rabbit lg (Sac-Cell, Washington). All standard dilutions (in perfiision buffer + aprotinin (400 KlU/ml) and perfiision samples were assayed in triplicate. Counting and calculation was performed as for the insulin RIA.

STATISTICAL ANALYSES: Student's t-test for unpaired observations or for paired observations, were used as appropriate. RESULTS

INSULIN AND AMYLIN SECRETION FROM THE ISOLATED PERFUSED FATTY RAT PANCREAS: EFFECTS OF β-CT AND SUBSEQUENT HIGH GLUCOSE CONCENTRATIONS

Perfiision with β-CT (0.5-1.0 nmol/1) for 5 minutes preceding switch over to "high" glucose, resulted in a monophasic release of insulin as has previously been reported (Dunmore and Beloff-Chain (1982), loccit.). The total insulin secretion in excess of basal release (Δinsulin) was 4.8 fold greater from fa/fa pancreata compared to lean controls Table 1). Amylin secretion (Δamylin) was very similar to that of insulin, with a similar difference (4.5 fold) between the two groups. The insulin:amylin molar ratio increased slightly during β-CT stimulation of isolated pancreata in leans from 42:1 to 48:1 and in fa/fa from 31:1 to 49:1.

The effects of perfiision with high glucose following a previous β-CT stimulation differed from the glucose-only experiments discussed above. High glucose induced a 2.1 fold greater release of insulin from fatty pancreas than from lean and 1.7 fold greater than from fatty pancreas not previously stimulated with β-CT. Amylin secretion was increased to an even greater extent: fatty secretion was 3.9 fold greater than lean and 2.0 fold greater than fatty without previous perfiision with β-CT.

2. DEMONSTRATION OF INHIBITION OF β-CELL TROPIN

MATERIAL AND METHODS

ISOLATION OF BROWN ADIPOCYTES

Brown adipocytes were isolated by the method of Rodbell (1964) with some modifications. Interscapular brown adipose tissue was removed from two male rats weighing ca.l50g, whilst under halothane anaesthesia. The tissue was placed in a petri dish containing Krebs-Ringer bicarbonate (KRB, pH 7.4) with 5.6mmole/l glucose and 4.0% (w/v) bovine serum albumin. Any adhering white fat was removed and the tissue was finely chopped in KRB (5.6mmole l glucose and 4% albumin). The tissue was digested in KRB with 5.6mmnole/l glucose, 4% albumin and 1.0mg/ml collagenase (Serva Chemicals) in a shaking water bath at 37°C for 2 hours. The buffer was gassed (O2/CO2 95:5) during this period.

This digested tissue was then filtered through a 200μm mesh, resuspended in fresh KRB (5.6mmole/l glucose and 4% albumin) and cells centrifuged at 800Xg for lmin. The buffer wash was removed and the cell resuspended in 5ml of fresh buffer. A sample of this initial cell suspension was used for determination of cell number using a haemocytometer.

INCUBATION

The brown adipocytes (50μl of suspension) were incubated for 1 hour in a shaking water bath at 37°C in KRB (5.6mmole/l glucose and 4% (w/v) albumin) containing different insulin (14.4nmole/l) or β-cell- tropin (lOnmole/1) in the presence of tritiated glucose (D-[3-^H] glucose, 0.5μCi/vial) in the presence or absence of β-cell-tropin antiserum (dilution 1 in 80). Glucose incorporation into lipid was determined by overnight extraction into non-aqueous scintillation fluid (Betafluor, National Diagnostics) and measurement of beta emission on a scintillation counter. The adipocytes were gassed continually during this period.

The β-cell-tropin used in this study was prepared from the incubation of mouse neurointermediate lobes by the method described by Beloff-Chain et al (1980).

Raising of β-Cell Tronin Antisera

Beta-cell tropin (400nmole) and thyroglobulin (lOOnmole) were dissolved in 1ml 0.1M phosphate buffer. Glutaraldehyde (1.5ml of a 0.02M solution) was added dropwise over lδmin, and the mixture incubated overnight at 4°C. The conjugate was then dialysed overnight against distilled water to remove glutaraldehyde, and the conjugate was lyophilised in aliquots of 50%, 25% and 25% of the total, being enough to provide a primary immunisation and two booster injections respectively. This quantity of conjugate was sufficient to immunise a single rabbit.

For primary immunisation the lyophilised conjugate was taken up in 1ml tap water and emulsified with 1.5ml Freund's complete adjuvant, and aliquots of 0.1ml injected intradermally into multiple sites in the back and flanks of a Dutch halflop rabbit.

For boosting immunisation, the lyophihsed conjugate was taken up in 1ml tap water and emulsified with 1.5ml Freund's incomplete adjuvant and aliquots of 0.1ml injected intradermally into multiple sites at 12 weeks and 18 weeks following the primary innoculation. Starting 3 days after the second booster immunisation, blood samples were obtained daily for deteπ ination of antibody titre.

Blood was allowed to clot for 4hours at room temperature and then overnight at 4°C. The antibody titre was determined in a conventional radioi mmun oassay system.

Results

β-cell tropin produced a similar stimulation to insulin of glucose incorporation into lipids i.e. Iipogensis. The differences between insulin treated cells and controls and β-CT treated cells and controls were both significant statistically (P<0.05). There was no significant difference between the effect of insulin and β-cell tropin. The β-CT antibody completely blocked the β-CT response and significantly reduced the basal rate of lipogenesis.

Table 1: Glucose and b-CT induced Insulin and Amylin Secretion

Δinsulin Insulin Secretion Rate (Total β-CT

Group n Basal Glu High Glu induced sec¹¹) n

[pmol/min] [pmol/min] [pmol]

Lean (Fa/?) glucose 6 only

β-CT+ 11 glucose

FATTY (fa/fa): glucose only

β-CT+ 11 glucose

NOTES

1. Data shown as mean ±SEM.

2. InsuUn and amylin secretion rates are average secretion rates over period of perfusion with basal (5.6mmol/l) or high (16.7mmol l) glucose.

3. Δinsulin and ΔAmylin are the total β-CT induced secretion (in excess of basal) observed in the monophasic peak.

Claims

1. A compound which regulates the formation, release or biological activity of β-cell-tropin.

2. A pharmaceutical composition comprising a compound which regulates the formation, release or biological activity of β-cell tropin (β-CT) and a pharmaceutically acceptable carrier therefor.

3. A method for the treatment of hyper sulinaemia and conditions associated with h_/perinsulinaemia in mammals, which method comprises the administration of an effective, non- toxic amount of a compound which regulates the formation, release or biological activity of β-cell tropin (β-CT).

4. A method according to claim 3, wherein the conditions associated with h3 erinsulinaemia are type II diabetes, hyperlipidaemia, hypertension, atherosclerosis, syndrome X and obesity.

5. A method according to claim 3 or claim 4, wherein the compound which regulates the biological activity of β-CT is a β-CT antagonist.

6. A method according to claim 5, wherein the β-CT antagonist is a non-competitive antagonist.

7. A method according to claim 5 or claim 6, wherein the β-CT antagonist is an antibody specific for β-CT.

8. A method according to claim 5, wherein the β-CT antagonist is a competitive β-CT antagonist.

9. A method according to claim 8, wherein the competitive β-CT antagonist is cross-linked β-CT or a cross-linked fragment of β-CT.

10. A compound which regulates the formation, release or biological activity of β-cell tropin (β-CT), for use as an active therapeutic substance.

11. A compound according to claim 12, for use in the treatment of hyperinsulinaemia and conditions associated with hyperinsulinaemia.

12. A compound according to claim 11, wherein the conditions associated with hyperinsulinaemia are type II diabetes, hyperlipidaemia, hypertension and atherosclerosis, syndrome X, and obesity.

13. A compound according to any one of claims 10 to 12, wherein the compound which regulates the biological activity of β-CT is a β-CT antagonist.

14. A compound according to claim 13, wherein the β-CT antagonist is a non-competitive antagonist.

15. A compound according to claim 13 or claim 14, wherein the β-CT antagonist is an antibody specific for β-CT.

16. A compound according to claim 13,wherein the β-CT antagonist is a competitive β-CT antagonist.

17. A compound according to claim 16, wherein the competitive β-CT antagonist is a cross-linked β-CT or a cross-linked fragment of β-CT.

18. The use of a compound which regulates the formation, release or biological activity of β-cell tropin (β-CT) for the manufacture of a medicament for the treatment of hyperinsulinaemia and conditions associated with hyperinsulinaemia.