AU662911B2 - Use of beta-cell tropin (ACTH 22-39) antagonists in the treatment of hyperinsulinaemia and associated diseases - Google Patents

Description

t OPI DATE.12/01/93 APPLN. ID 20203/92 AOJP DATE 11/03/93 PCT NUMBER PCT/GB92/01079 IIII lllllllll 11111 lll llllll lllllll Ill II IIll AU9220203 Y (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 92/22329 A61K 39/395, 37/02, C07K 7/38 Al (43) International Publication Date: 23 December 1992 (23.12.92) (21) International Application Number: (22) International Filing Date: Priority data: 9113011.2 17 June PCT/GB92/01079 16 June 1992 (16.06.92) 991 (17,06.91) (71) Applicants: POWELL, Judith, Mary (executrix for the deceased inventor) [GB/GB]; c/o Berwin Leighton, Adelaide House, London Bridge, London EC4R 9HA (GB).

CHAIN, Benjamin, Michael (executor for the deceased inventor) [GB/GB]; 829 Finchley Road, London NWII 8AJ (GB).

(71) Applicant (for all designated States except US): SMITH- KLINE BEECHAM PLC [GB/GB]; New Horizons Court, Brentford, Middlesex TW8 9EP (GB).

(72) Inventor; and Inventor/Applicant (for US only) CAWTHORNE, Michael, Anthony [GB/GB]; SmithKline Beecham Pharmaceuticals, Great Burgh, Yew Tree Bottom Road, Epsom, Surrey KT18 5XQ (GB).

(72) Inventor: BELOFF-CHAIN, Anne (deceased).

(74) Agent: RUTTER, Keith; SmithKline Beecham, Corporate Patents, Great Burgh, Yew Tree Bottom Road, Epsom, Surrey KT18 5XQ (GB).

(81) Designated States: AT (European patent), AU, BE (European patent), CA, CH (European patent), DE (European patent), DK (European patent), ES (European patent), FR (European patent), GB (European patent), GR (European patent), IT (European patent), JP, KR, LU (European patent), MC (European patent), NL (European patent), SE (European patent), US.

Published With international search report.

662911 (54)Title: USE OF p-CELL TROPIN (ACTH 22-39) ANTAGONISTS IN THE TREAI AND ASSOCIATED DISEASES 'ENT OF HYPERINSULINAEMIA (57) Abstract Pharmaceuticals, including compounds and compositions and the use of such pharmaceuticals in the treatment of hyperinsulinaemia and conditions associated with hyperinsulinaemia.

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WO 92/22329 PCT/GB92/01079 -1- USE OF a-CELL TROPIN (ACTH 22-39) ANTAGONISTS IN THE TREATMENT OF HYPERINSU- 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 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 inhibit the release of insulin from p-cells within the islets of Langerhan and 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.

P-Cell-tropin (p-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). p-CT has a range of biological activities. It stimulates the ielease of insulin, at substimulatory concentrations of glucose, from the isolated perfused rat pancreas (Dunmore Beloff- PCT/GB92/01079 WO 92/22329 -2- 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. 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). It has an insulin-like action in promoting lipogenesis in adipose tissues (Watkinson Beloff-Chain, Horm. Metab. RRs. (1984) 16, 55-58 (Suppl.)).

P-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 P-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 p-CT modulate the insulin-releasing potency of p-CT Dunmore et al., Biochem. J.

(1987) 244, 797-800).

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

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

P-CT is therefor hyperinsulinaer via increased an resistance and c actions the direi development of e indicated to play a central role in the development of nia by directly increasing insulin release, and indirectly nylin release, causing the development of insulin ompensatory hyperinsulinaemia. In addition to these ct insulin-like action "f P-CT on adipose tissue leads to the obesity.

that the regulation of the formation or release of P-CT or f the biological activity of P-CT provides the potential for It is considered the regulation o WO 92/22329 PCT/GB92/01079 1 -3the treatment of hyperinsulinaemia and conditions associated with hyperinsulinaemia, such as type II diabetes, hyperlipidaemia, hypertension and atherosclerosis, obesity and syndrome X.

j 5 Accordingly, the present invention provides a method for the treatment of ihyperinsulinaemia and conditions associated with hyperinsulinaemia, Isuch as type II diabetes, hyperlipidaemia, hypertension, atherosclerosis, syndrome X and obesity, which method comprises the administration of an effective, non-toxic amount of a compound which regulates the formation, release or biological activity of p-cell tropin (p-CT).

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

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

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

P-CT.

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

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

An example of a competitive p-CT antagonist is a disabled P-CT agonist.

A disabled P-CT agonist is a P-CT agonist which has been structurally modified so that it is still capable of binding to a p-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 P-CT or cross-linked fragments of P-CT.

i- WO 92/22329 PCT/GB92/01079 -4- An example of a disabled agonist is substituted P-CT or a substituted fragment of p-CT. The terms 'substituted P-CT' and 'substituted fragment of P-CT' refer to n-CT or fragments thereof wherein the amino acid sequence has been changed or wherein the amino acids from the normal sequence have been structurally modified.

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

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

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

Disabled p-CT agonists will be prepared by the appropriate conventional procedure: thus for example cross-linked p-CT or cross-linked fragments of P-CT may be prepared by conventional peptide cross-linking techniques such as those described in Biochemistry 12, 1978, 1499, International J.

Peptide Protein Res., 27, 1986, 285-92 and Pierce, 1989 Handbook and General Catalogue (Cross-linking Reagents), p.283-311.

Also, substituted P-CT or substituted fragments of p-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.

WO 92/22329 PCT/GB92/01079 It will be appreciated that a p-CT antagonist will be particularly conveniently prepared when the particular manner by which p-CT interacts with a a-CT receptor has been determined. This first involves the identification of the active site or sites upon p-CT which interact with the p-CT receptor. Once such information has been established, the identity of the relevant amino acid residues of P-CT or the fragments thereof which may be disabled, by substitution or cross-linking, can be Smore easily determined.

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

The structural analysis of the p-CT to p-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 p-CT receptor may be obtained by raising antibodies, such as monoclonal antibodies, in the presence of a source of a P-CT i receptor using appropriate conventional procedures (Proc. Natl. Acad. Sci.

USA (1982), 79, 7312-7316). Thus antibodies to the p-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 P-CT receptor, or with cells with a high concentration of p-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 P-CT receptor. Such activity can be demonstrated by the ability of the antibody to prevent the binding of radiolabelled P-CT 12 5 I-labelled p-CT or 3H--CT) to its receptor.

The monoclonal antibody can then be examined for its antagonistic activity by use of the screening techniques discussed below.

L 1, WO 92/22329 PCT/GB92/01079 -6- A further approach involves the use of anti-idiotype antibodies. Antiidiotype antibodies may be raised against monoclonal antibodies directed against P-CT, such that the anti-idiotype will have complimentary binding affinity for the P-CT receptor site without, of course, the activity promotion associated with p-CT binding. Utilization of anti-idiotype antibodies for blocking viral binding to cells is known Immunol (1983), 131, 2539-2541 or Med. Int. (1987) 317, 219-224.).

Compounds which regulate the biological activity of P-CT may be identified by conventional screening techniques, especially in vitro techniques. Thus, potential -CT antagonists may be identified by determining whether or not they reduce the biological activity of R-CT in the presence of a source of 1-CT receptor. One suitable source of 1-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 R-CT receptor are the perfused pancreas, isolated pancreatic islets, pacreatic 1-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 1-CT antagonist upon the uptake of glucose and/or the synthesis of triacylglycerol by cells in the presence of a source of P-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 1-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 P-CT receptors can be configured in a receptor binding assay whereby the ability of antagonists to prevent the binding of P-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 R-CT it will first be necessary to determine the nature of the metabolic control of the production of p-CT from the pituitary. This will be determined for example, by incubating isolated neurointermediate lobes of the pituitary or whole pituitary and a pituitary cell line with a WO 92/22329 PCT/GB92/01079 -7variety 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 p-CT. The response of the pituitary tissue to the various signals can be determined by the measurement of the formation of p-CT and/or its release into the medium. The ability of molecules to block this mechanism can then be examined using the same techniques.

p-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 p-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 p-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 p-CT.

For example, white or brown adipocytes can be incubated in the presence i of p-CT and labelled glucose with or without the presence of the test i compound. The amount of labelled glucose incorporated as lipid into the I 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 1P-CT it may be prepared by conventional antibody generating methods.

I 30 One particular method involves injecting a test animal as often as necessary with p-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) 4, 125-130) or a pancreatic p-cell line such as HIT or RIN cells with P-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 L M WO 92/22329 PCT/GB92/01079 -8amylin from the islet or cell line can be determined by conventional radioimmunoassay-procedure.

Identification of the -CT receptor site an 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 may be 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 -cell tropin (p-CT) and a pharmaceutically acceptable carrier therefor.

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

Hereinafter a compound which regulates the formation, release or biological function of 1-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 administration, although compositions for administration by other routes, such as by injection and percutaneous absorption are also envisaged.

Particul ,-ly suitable compositions for oral administration are unit dosage

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I

WO 92/22329 PCT/GB92/01079 -9forms 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 ccrtain 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.

i __~anl naPn ui-r,i.

WO 92/22329 PCT/GB92/01079

EXAMPLE

1. DETERMINATI AMYLIN SECRETION ON OF EFFECT OF B-CELL TROPIN UPON METHODS AND MATERIALS:: ANIMALS: Fatty rats (fa/fa) and lean controls (Fa? or Fa/Fa) aged 12 to weeks were used.

PANCREAS PERFUSIONS Pancreata from the male fatty rats and lean littermates described above were isolated and perfused by the method described in Dunmore and Beloff-Chair, J. Endocrinol (1982) 92,15,21. The perfusion buffer was a modified Krebs-Ringer Bicarbonate buffer containing 3% Dextran (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 minute period of "low glucose" perfusion buffer to allow stabilization): 1.

2.

3.

5 minutes of low glucose (5.6 mmol/1) buffer, 5 minutes of buffer as 1. with the addition of 0.5 to 1.0 nmol/1 P-CT 10 minutes of high glucose (16.7 mmol/1) buffer.

i j j i~ Flow-rate was 5.0 ml. min- 1 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 -200 until assayed.

DETERMINATION OF BLOOD GLUCOSE PLASMA INSULIN AND B- 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 KIU/ml), a sample was immediately deproteinized prior to the determmination of glucose concentration, and the remainder centrifuged.

WO 92/22329 PCT/GB92/01079 -11- The supernatant plasma was frozen at -20° until assayed.

J 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 perfusion samples was measured by a conveational radioimmunoassay slightly modified from that described in Dunmore and Beloff-Chain (1982) (loc.

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 1 2 5 iodinated 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 perfusion buffer. Antibody-bound insulin was separated from free insulin by addition of second-antibody (donkey anti-guinea pig Ig) coated cellulose (Sac-Cel, Washington.) and subsequent Scentrifugation. Counts bound were measured in a LKB Rackgamma solid scintillation counter and insulin calculations performed hb: l RIACALC programme (LKB) on a PC-AT computer (Opus PCV) lin:-A. 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 Ig (Sac-Cell, Washington). All standard dilutions (in perfusion buffer aprotinin (400 KIU/ml) and perfusion 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.

L WO 92/22329 PC/GB92/01079 -12-

RESULTS

INSULIN AND AMYLIN SECRETION FROM THE ISOLATED PERFUSED FATTY RAT PANCREAS: EFFECTS OF B-CT AND SUBSEQUENT HIGH GLUCOSE CONCENTRATIONS Perfusion with p-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), loc.cit.). The total insulin secretion in excess of basal release (Ainsulin) was 4.8 fold greater from fa/fa pancreata compared to lean controls Table Amylin secretion (Aamylin) 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 p-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 perfusion with high glucose following a previous p-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 p-CT. Amylin secretion was increased to an even greater extent: fatty secretion was 3.9 fold greater than lean and fold greater than fatty without previous perfusion with p-CT.

2. DEMONSTRATION OF INHIBITION OF O-CELL TROPIN MATERIAL AND METHODS ISOLATION OF BROWN ADIPOCYTES SBrown adipocytes were isolated by the method of Rodbell (1964) with some modifications. Interscapular brown adipose tissue was removed from two male rats weighing ca.150g, whilst under halothane anaesthesia. The tissue was placed in a petri dish containing Krebs-Ringer bicaibonate (KRB, pH 7.4) with 5.6mmole/1 glucose and 4.0% bovine serum albumin. Any adhering white fat was removed and the tissue was finely chopped in KRB (5.6mmole/1 glucose and 4% albumin). The tissue was digested in KRB with 5.6mmnole/l glucose, 4% albumin and WO 92/22329 PCT/GB92/01079 13 collagenase (Serva Chemicals) in a shaking water bath at 37°C for 2 hours. The buffer was gassed (O2/CO 2 95:5) during this period.

This digested tissue was then filtered through a 200pm mesh, resuspended in fresh KRB (5.6mmole/1 glucose and 4% albumin) and cells centrifuged at 800Xg for 1min. 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 (50gl of suspensir. i) were incubated for 1 hour in a shaking water bath at 37 C in KRB (5.6mmole/1 glucose and 4% (w/v) albumin) containing different insulin (14.4nmole/1) or P-cell-tropin (10nmole/1) in the presence of tritiated glucose 3 H] glucose, in the presence or absence of P-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 adir ocytes were gassed continually during this period.

The p-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 5-Cell Tropin Antisera Beta-cell tropin (400nmole) and thyroglobulin (100nmole) were dissolved in lml 0.1M phosphate buffer. Glutaraldehyde (1.5ml of a 0.02M solution) was added dropwise over 15min, 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 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 L WO 92/22329 PCT/GB92/01079 -14tap water and emulsified with 1.5ml Freund's complete adjuvant, and aliquots of 0.lml injected intradermally into multiple sites in the back and flanks of a Dutch halflop rabbit.

For boosting immunisation, the lyophilised conjugate was taken up in 1ml tap water and emulsified with 1.5ml Freund's incomplete adjuvant and aliquots of 0.lml 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 determination 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 radioimmunoassay system.

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

Table 1: Glucose and b-CT induced Insulin and Amylin Secretion0 Group Lean (Fa/R) glucose only f3-CT+ glucose

FATTY

(falfa): glucose only 1-CT+ glucose

NOTES

Insulin Secretion Rate n Basal Glu High Glu [pmol/min] [pmollmin] AInsulin (Total 1-CT induced secn) [pmol] Amylin Secretion Rate n Basal Glu High Glu [pmolmin] [pmol/min] AAmylin (Total 1-CT induced secn) [pmol] 6 0.485 ±0.155 11 0.529 ±0.085 6 4.144 ±0.734 11 5.696 ±1.282 4.105 ±0.340 4.143 ±0.649 6 0.0161 ±0.0044 6 0.0310 ±0.0098 0.0913 ±0.0087 0.0829 ±0.0065 18.614 ±1.830 0.4600 ±0.0570 5.003 ±0.596 8.601 ±1.231 6 0.1337 ±0.0 164 6 0.2002 ±0.0593 0.1628 ±0.0151 0.3245 ±0.0891 89.474 ±13.711 2.0583 ±0.4335 1. Data shown as mean ±SEM.

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

3. Alnsulin and AAmylin are the total 1-CT induced secretion (in excess of basal) observed in the monophasic peak.

Claims (6)

1. A method for the treatment of hyperinsulinaemia and conditions associated with hyperinsulinaemia in mammals, which method comprises the administration of an effective amount of a p-cell-tropin (p-CT) antagonist.

2. A method according to claim 1, wherein the 3-CT antagonist is a non- competitive antagonist.

3. A method according to claim 1 or claim 2, wherein the p-CT antagonist is an antibody specific for P-CT.

4. A method according to claim 1, wherein the p-CT antagonist is a competitive p-CT antagonist.

Lc~ A method according to claim 1 or claim 4, wherein the competitive p-CT antagonist is a cross-linked p-CT or a cross-linked fragment of p-CT.

6. A method according to claim 1, wherein the conditions associated with hyperinsulinaemia are type II diabetes, hyperlipidaemia, hypertension, atherosclerosis, syndrome X and obesity. Dated this 11th day of July, 1995 SmithKline Beecham plc AND Judith Mary POWELL AND Benjamin Michael CHAIN By DAVIES COLLISON CAVE Patent Attorneys for the Applicant(s) 950711,p:\oper\mro,20203-92.clm,16 -i