GB2277519A - Potassium channel modulators - Google Patents

Abstract

This invention concerns peptides (I) having intracellular potassium channel modulating activity comprising the amino acid sequence (N-terminal function)-Met-Ile-Ser-Ser-Val-Cys-Val-Ser-Ser-Tyr- Arg-Gly-Arg-Lys-Ser-Gly-Asn-Lys-Pro-Pro-Ser- Lys-Thr-Cys-Leu-Lys-Glu-Glu-(C-terminal function> in which the cysteines are optionally linked via a disulphide bridge or a variant thereof, with the proviso that excluded is the 13-Lys variant (where 13-Arg is replaced by 13-Lys) in which the cysteines are not linked via a disulphide bridge, which are useful in a test method of screening for compounds having potassium channel modulating activity.

Description

POTASSIUM CHANNEL MODULATORS This invention relates to potassium channel modulators, in particular to linear and cyclic peptides which block potassium channels intracellularly and are useful in screening for potential potassium channel openers having therapeutic utility.

Voltage gated potassium ion (K+) channels which produce outward currents are present in the cell membranes of neurones and serve to repolarise the cell following a depolarisation by opening and allowing potassium ions to flow from the inside of the cell to the outside. They are, therefore, one of the main regulating influences on the nerve cell firing and determine the amount of current reaching the terminal regions of the cells.

This in turn regulates the amount of neurotransrnitter substances released from the nerve terminals. In addition, they help to determine the refractory period of the nerve cell and hence the probability of the cell firing again within a certain time. This governs neuronal excitability and also the tendency of a cell to undergo repetitive firing. An ability to modify the functioning of these channels by chemical means is the aim of current research in the search for therapeutically useful agents.

The present application is particularly concerned with the intracellular block of the K+ channels.

Voltage-dependent potassium (K+) channels open in response to a positive shift in membrane potential. After some variable time these channels close again; certain types of K+ channels close fairly quickly ("inactivate" in several milliseconds) after opening, others remain open for seconds. It was suggested over twenty years ago that the rapid closing mechanism is due to a 'molecular plug' or ball swinging into the open channel thereby blocking the further passage of ions. This simple mechanism has recently been shown to account for the rapid inactivation of several K channels. Initially Aldrich et al., Science 250 568-571 (1990), demonstrated that only 20 amino acids at the N terminal of Shaker B channels acts as the blocking particle.Similarly, Ruppersburg J P et al., Nature 353 657-660 (1991), have recently shown for some mammalian (rat) potassium channels (called Raw3 and RCK4) that the N-tenninal regions act as the natural channel closing particle.

Robertson B., and Owen D., J. Physiol., 459, 92P, 1993 have also shown that noninactivating K channels from a mammalian brain (MK-l) may be blocked by a peptide derived from the N-terminal sequence of the Shaker B channel. This has the overall effect of transforming this previously sustained channel into a rapidly inactivating one.

Rudy, D., et al., J. Neuroscience Res., 29, 401412 (1991), give the amino acid sequence for a human brain inactivating potassium channel (HKShEc), which is extremely homologous to the rat inactivating K channel Raw3.

All of the above known peptide sequences are understood to be 'linear' molecules in so far as there is no intramolecular chemical bonding. However physical intramolecular forces may give the molecule some degree of constraint.

A 28-amino acid peptide derived from the N-terminal sequence of HKShEc was synthesised ('human' 28 mer peptide) and tested in the non-activating MK-l channel to determine if this isolated peptide was capable of blocking the channel. This 'human' 28 mer peptide had the formula: AcetyI-Nle-Ile-Ser-Ser-Val-Cys-Val-Ser-Ser-Tyr- Arg-Gly-Arg-Lys-Ser-Gly-Asn-Lys-Pro-Pro-Ser-Lys Thr-Cys-Leu-Lys-GlU-Glu-NH2 An acetyl group was used to block the terminal NH2- group We have found that the 'human' 28 mer peptide was active in transforming MK-1 into an inactivating channel.

Most surprisingly we have found that the cyclic cysteine-cysteine bridged analogues were also active and apparently more potent than the 'linear' form as K+ channel blockers.

Accordingly this invention provides a peptide (I) having intracellular potassium channel blocking activity comprising the amino acid sequence (terminal function)-Met-Ile-Ser-Ser-Val-Cys-Val-S er- S er-Tyr-Arg-Gly-Arg-Lys-Ser-Gly-Asn-Lys-Pro-Pro-Ser-Lys- Thr-Cys-Leu-Lys-Glu-Glu-(C-terminal function) (I) in which the cysteines are optionally linked via a disulphide bridge and wherein Met represents L-methionine Ile " L-isoleucine Ser " L-serine Val " L-valine Cys " Lcysteine Tyr " L-tyrosine Arg " L-arginine Gly " glycine Lys " L-lysine Asn " L-asparagine Pro " L-proline Thr " L-threonine Leu " L-leucine Glu " L-glutamic acid or a variant of said polypeptide having intracellular potassium channel modulating activity with the proviso that excluded is the 13-Lys variant (where 13-Arg is replaced by 13-Lys) in which the cysteines are not linked via a disulphide bridge.

As an example of a variant mention is made of the l-Nle (norleucine) analogue replacing 1-methionine. Similarly Glu residues may be replaced by aspartic acid (Asp), and Asn residue by Gln (L-glutamine). In addition Arg and Lys residues may be interchanged.

The term variant means any analogue having one (or more) different amino acid residues providing that intracellular potassium channel modulating activity is retained. The term also covers omission or addition of amino acid residues where said intracellular activity is retained.

The a-terminal group may be NH2 (i.e. the N-terminal function is H-) or a substituted amino group, e.g mono- or di- alkyl amino or N-acyl such as N-alkanoyl, e.g N-acetyl.

The C-terminal group may be hydroxy (i.e. the peptide is an acid) or a derivative thereof, e.g an ester function e.g -O-alkyl, or an amide, e.g -NH2, -NHalkyl or -N(alkyl)2.

As used herein 'acyl' refers to carbonyl groups such as alkyl-, aryl- or aralkyl- carbon e.g having 2 to 15 carbon atoms, e.g 2 to 7 for alkyl, 7 to 11 for aryl and 8 to 12 carbon atoms for aralkyl.

Examples of 'alkyl' groups as used herein are straight or branched chain alkyl groups especially those having 1 to 6 carbon atoms e.g methyl, ethyl, propyl, isopropyl and butyl. Examples of 'aryl' are those of 6 to 10 carbon atoms e.g phenyl and naphthyl each optionally substituted. Examples of aralkyl are groups of 7 to 11 carbon atoms e.g benzyl, phenethyl or naphthylmethyl each optionally substituted. The term 'optionally substituted' means optional substitution by groups or radicals commonly used in pharmaceutical chemistry, such as alkyl, alkoxy, hydroxy, halo, nitro, amino, alkylamino, acylamino, carboxy, alkoxycarbonyl, mercapto, haloalkyl and aminocarbonyl.

The term 'aryl' also includes 'heteroaryl', i.e aromatic mono- or bi-cyclic groups having 5 to 10 ring atoms, at least one of which is a heteroatom, e.g oxygen, nitrogen or sulphur.

Examples are furanyl, thienyl, pyrrolyl, pyridyl, quinolyl and isoquinolyl.

The peptides of this invention can be used with a potassium channel, e.g the MK- 1 channel to provide a useful screen for the development of novel drugs designed to interfere with the normal inactivation processes of potassium ion channels. Wholecell recordings of MK-1 channels expressed in cell lines show the modification of MK- 1 current into a rapidly inactivating current as the added peptide dialyses into the cell.

Addition of test compound to the cell can be made from the extracellular or intracellular side to determine whether such molecules hinder the inactivation process, either by binding to the inactivation peptide itself, or its 'receptor' on the ion channel. Compounds which impede the inactivation of potassium ion channels result in more effective braking of cellular activity, and lead to a decrease in both cell excitability and neurotransmitter release, and are potentially useful in preventing epileptiform activity.

Accordingly this invention also provides a test method for screening a test compound for potassium channel modulating activity which comprises administering said test compound to a cell having a potassium channel, said cell containing a peptide of this invention, to determine whether the inactivation process is affected by said test compound.

The cyclic peptides of this invention are prepared by a process comprising oxidising a corresponding linear peptide to form the disulphide bridge.

Accordingly this invention also provides a process for preparing a peptide of formula I as defined above having a disulphide bridge between cysteine residues which comprises oxidising a corresponding linear peptide in which the cysteines each have free SH groups.

Oxidation may be conveniently carried out by air (or gaseous 02) oxidation or by use of potassium ferricyanide.

The corresponding linear peptide precursor can itself be prepared by deprotecting a fully or partially proctected precursor or resin supported precursor as described hereinafter.

For example a fully or partially protected precursor peptide may be represented by the formula (II) x l-Metjie-Ser(R1 )-Ser(R 1 )-Val-Cys(R2)-Val- Ser(R1 )-Ser(R 1 )-Tyr(R3 )-Arg(R4)-Gly-Arg(R4)- Lys(RS)-Ser(Rl)-Gly-Asn-Lys(R5)-Pro-Pro-Ser(R1 )- Lys(R5)-Thr(Rl)-Cys(R2)-Leu-Lys(RS)-Glu(R6)-Glu(R6)-X2 (II) or a variant thereof, where X 1 is hydrogen or an a-amino protecting group, R1 is an hydroxy protecting group for the side chain of Ser or Thr or hydrogen, R2 is a mercapto protecting group or hydrogen, R3 is an hydroxy protecting group for the side chain of Tyr or hydrogen, R4 is an guanyl protecting group for the side chain of Arg or hydrogen, R5 is an amino protecting group for the side chain of Lys or hydrogen, R6 is a carboxy protecting group for the side chain of Glu or hydrogen, X2 is OH, a carboxy protecting group or bond to a solid phase support, e.g X2 = -O-CH2[polystyrene resin support] where the latter group represents one of the many functional groups present in the polystyrene resin; providing at least one protecting group is present when X2 is OH.

When R2 is hydrogen the abovementioned precursor peptide may be cyclised by oxidation (eg. gaseous 02) prior to removal of all protecting groups.

Protecting groups for the a-amino group (X1) are illustrated by (1) acyl type protecting groups such as: formyl, trifluoroacetyl, phthalyl, p-toluenesulfonyl (tosyl), nitrophenylsulfenyl, etc; (2) aromatic urethane type protecting groups such as benzyloxycarbonyl and substituted benzyloxycarbonyl such as e-chlorobenzyloxycarbonyl, n- nitrobenzyloxycarbonyl; (3) aliphatic urethane protecting groups such as tert-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, allyloxycarbonyl.

2,2,2-trichloroethoxycarbonyl, amyloxycarbonyl; (4)cycloalkyl urethane type protecting groups illustrated by cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl; (5) thiourethane type protecting groups such as phenylthiocarbonyl; (6) alkyl type protecting groups such as triphenylmethyl (trityl); (7) trialkylsilane groups such as trimethylsilane.

The side chain nitrogen atoms of arginine, are protected by a group (R4) which may be nitro, tosyl, benzyloxycarbonyl, adamantyloxycarbonyl or tert- butyloxycarbonyl, preferably the tosyl group.

Protection for the side chain amino group of lysine (R5) may be by tosyl, tamyloxycarbonyl, t-butyloxycarbonyl, diisopropyloxycarbonyl, benzyloxycarbonyl, haloberizyloxycarbonyl, nitrobenzyloxycarbonyl, and the like, the 2chlorobenzyloxycarbonyl group being preferred. Protection for the hydroxyl group of tyrosine, threonine and serine (R1, R3) may be with acetyl, benzoyl, tert-butyl, berizyl. The berizyl group is preferred for this purpose.

The protecting group for the sulfydryl group of the cysteinyl amino acid residue (R2) can be a group selected from berizyl; substituted betizyl wherein the substituent is at least one of methyl, methoxy, nitro, or halo (e.g 3,4-dimethylbenzyl, p methoxybenzyl, e-chlorobenzyl, p-nitrobenzyl, etc.); trityl, benzyloxycarbonyl, benzhydryl, p-methoxybenzyloxycarbonyl, benzylthiomethyl, ethylcarbamoyl, thioethyl, tetrahydropyranyl, acetamidomethyl, benzoyl, s-sulfonate salt, etc.; pmethoxybenzyl group being preferred.

The carboxy group of glutamic acid may be protected (R6) by a benzyl or substituted benzyl group.

The corresponding linear protected peptide precursor may be prepared by known methods for building up an amino acid sequence as described in standard textbooks on peptide synthesis. For example solid phase methodology can be used where the peptide is bound to a polystyrene resin support or a benzhydrylamine resin support following techniques generally known in the art for building up amino acid sequences from an initial resin supported amino acid such as illustrated by Merrifield, JACS, 85 2149 (1963).

The resin support employed may be any suitable resin conventially employed in the art for the solid phase preparation of polypeptides, e.g a copolymer of styrene and divinyl benzene in which the degree of crosslinking by the divinyl benzene is from 0.5 to 3%; which resin has been chioromethylated to provide sites for ester formation with the initially introduced protected amino acid. The projected C-terminal (amino protected) amino acid may be coupled to the chloromethylated resin according to the procedure of Gisin, Helv. Chim. Acta., 56 1476(1973). Following the coupling of the first amino protected to the resin support, the amino protecing group may be removed by standard methods employing trifluoroacetic acid in methylene chloride, trifluoroacetic acid alone or HC1 in dioxane.The deprotection may be carried out at a temperature between OOC and room temperature. After removal of the amino protecting group the remaining aamino protected and, if necessary, side chain protected amino acids are coupled, seriatim, in the desired order to obtain the product. Alternatively, multiple amino acid groups may be coupled by the solution method prior to coupling with the resin supported amino acid sequence. The selection of an appropriate coupling reagent is within the skill of the art. Particularly suitable coupling reagents are N,N'-diisopropylcarbodiimide and N,N'-dicyclohexylcarbodiimide.

Each protected amino acid or amino acid sequence is introduced into the solid phase reactor in a two to six fold excess and the coupling is carried out in a medium of dimethylformamide: methylene chloride or in either dimethylformamide or methylene chloride alone. In cases where incomplete coupling occurs the coupling procedure is repeated before removal of the a-amino protecting group, prior to introduction of the next amino acid to the solid phase reactor. The success of the coupling reaction at each stage of the synthesis can be monitored by the ninhydrin reaction as described by E Kaiser et al., Analyt. Biochem., 34, 595 (1970).

The necessary a-amino protecting group employed for each amino acid introduced in the polypeptide is preferably tert-butyloxycarbonyl, although any such protecting group may be employed as long as it is not removed under coupling conditions and is readily removed selectively in relation to the other protecting groups present in the molecule under conditions which otherwise do not affect the formed molecule. Additional examples of such a-amino protecting groups from which selection may be made, after consideration of the rest of the polypeptide molecule, are trityl, phthalyl, tosyl, allyloxycarbonyl, cyclopentyloxycarbonyl, tert-amyloxycarbonyl, benzyloxycarbonyl and o- or p-nitrobenzyloxycarbonyl.

The criteria for selecting protecting groups for R1-R6 are: (a) the protecting group must be stable to the reagent and under the reaction conditions selected for removing the a-amino protecting group at each step of the synthesis, (b) the protecting group must retain its protecting properties (i.e not be split off under coupling conditions), and (c) the protecting group must be readily removable upon conclusion of the polypeptide synthesis, under conditions that do not otherwise affect the polypeptide structure.

Standard methods of removing the protecting groups sequentially or simultaneously may be used.

They may be removed before, after of simultaneously with cleavage from the resin support when present. Preferably cleaving and deprotection are carried out at the same time e.g using hydrogen fluoride and anisole, to obtain the fully deprotected linear peptide. If a protected cyclic precursor peptide is prepared then deprotection gives the final cyclic peptide (I).

Where a variant of the sequence above is desired to be prepared, the variant amino acid(s) protected if required is (are) incorporated at the appropriate stage(s) in the synthesis.

The C-terminal group obtained by cleaving a compound of formula II where X2 is OCH[polystyrene resin support] using HF is the carboxy function, i.e X2 = COOH.

Alternatively the peptide may be removed from the support by ammonolysis with ammonia to give a CONH2 terminal group, or by reaction with an amine such as alkyl NH2 to give a CONH alkyl group. Cleavage by transesterification gives an ester terminal group e.g COOR where R is an organic radical, e.g alkyl or aralkyl as illustrated hereinabove.

The terminal amino acid function if not already modified prior to coupling may be modified after coupling by appropriate reaction of the precursor peptide, e.g acylation using for example an acyl halide.

The wet solution method for preparing the compounds of this invention comprises coupling the requisite amino acids protected, modified and/or activated as necessary in any order of succession to give the desired peptide sequence and thereafter, in any order, removing one or more protecting groups and oxidising if desired to give a disulphide bridge.

The coupling of the amino acids in the above mentioned process may be carried out by the standard methods used in peptide chemistry. Such methods are described in the literature for example in standard textbooks on peptide synthesis - see for example Schroder and Lubke, "The Peptides", Academic Press 1965 and Greenstein and Winitz, "Chemistry of the Amino Acids" Vol. 2, John Wiley and Sons Inc. 1961.

The following Example illustrates the invention: EXAMPLE 1 Acetyl-Nle-Ile-Ser-Ser-Val-Cys-Val-Ser-Ser-Tyr-Arg-Gly-Arg-Lys-Ser-Gly-Asn-Lys- Pro-Pro-Ser-Lys-Thr-Cys-Leu-Lys-Glu-Glu-NH2, 6-Cys-24-Cys disulphide The title compound was prepared by bubbling air through an aqueous solution of the corresponding linear form.

The linear form is prepared according to the processes described above using standard solid phase synthesis.

Analysis of title compound 1H NMR spectrum: (D2O containing TMS as internal reference, 400 MHz) resonances at 0.9-1.1 ppm (27H 9xMe); 1.25 ppm (doublet 1Me); 1.3-2.5 ppm (complex multiplets); 2.9 ppm (multiplet 2H); 3.0-3.1 ppm (multiplet 12H); 3.2-3.3 ppm (multiplet 6H); 3.6-4.1 ppm (multiplet); 4.2-4.6 ppm (multiplet); 6.88 ppm (doublet 2H); 7.2 ppm (doublet 2H).

The terminal methionine residue of the 'human' 28mer peptide was replaced by norleucine in the compound of Example 1 to produce a variant peptide without removing potassium channel activity as shown hereinafter.

The peptide described in Example 1 of this invention was tested for blocking activity on the MK- 1 voltage-activated K+ channel; according to the following standard test procedure: CHO cells stably transfected with cDNA for MK- 1 (Dr B Tempel et. al University of Washington, Nature, 332, 837-839 (1988)) were maintained in tissue culture using standard procedures and media for this cell line. Cells were plated on 35mm plastic dishes and used subsequently for electrophysiology within 3 days.

Currents were recorded using the whole-cell voltage-clamp configuration of the patch clamp technique, using an Axopatch 1C amplifier (Axon Instruments). Patch electrodes were manufactured from aluminosilicate glass tubing and heat polished prior to use. No electrode coating was necessary for whole-cell recording. Signal acquisition and analysis was performed using pClamp software (Axon Instruments). A p/4 subtraction procedure was used to remove leak and capacitive currents on line. A holding potential of -100mV was routinely used.

Two main protocols were used in testing drugs. 1) Current-voltage (I-V) curves were collected, with incrementing steps of either 10 or 20mV. Full I-V curves were obtained both in control and drug solutions. 2);A 'pharmacology' programme, which involved single voltage steps from -lOOmV to +60mV, applied and collected at 20s intervals.

Compounds under investigation were applied via a 'U' tube rapid perfusion system to a small area of the recording chamber. Drug applications were always bracketed by control solutions to ensure reversibility. The recording chamber was continuously perfused at 3ml.min-1. Results are expressed as % of control peak current (at +60mV).

However, where drugs have a time dependent effect on MK- 1, i.e acceleration of decay, results are also expressed as a % of total charge transferred within the duration of the voltage step.

The standard extracellular solution contained (in mM): NaC1 135, KC1 5, MgC12 4, EGTA 1, HEPES 10 and glucose 25, set to pH 7A with NaOH. The intracellular (pipette) solution comprised: K aspartate/K gluconate 120, KC1 20, MgC12, MgATP 2, EGTA 10, HEPES 10, pH at 7.4 with NaOH. This solution was stored in lml aliquots at -4"C, and filtered at 0.2mm. The MK-l current is a slowly rising, very slowly inactivating current, which may reach several nA in amplitude at +60mV.

In the abovementioned test procedure the cyclic peptide of Example 1 was found to have an inactivation time constant of 78 ms + 20 ms at 60mV. This a measure of the blocking potency.

Claims (29)

1. A peptide (I) having intracellular potassium channel modulating activity comprising the amino acid sequence (N-terminal function)-Met-lle-Ser-Ser-Val-Cys-Val-S er-Ser-Tyr- Arg-Gly-Arg-Lys-Ser-Gly-Asn-Lys-Pro-Pro- Lys-Thr-Cys-Leu-Lys-Glu-Glu-(C-terminal function) (I) in which the cysteines are optionally linked via a disulphide bridge and wherein Met represents L-methionine Ile " L-isoleucine Ser " L-serine Val " L-valine Cys " L-cysteine Tyr " L-tyrosine Arg " L-arginine Gly " glycine Lys " L-lysine Asn " L-asparagine Pro " L-proline Thr " L-threonine Leu " L-leucine Glu " L-glutamic acid or a variant thereof, with the proviso that excluded is the 13-Lys variant (where 13-Arg is replaced by 13 Lys) in which the cysteines are not linked via a disulphide bridge.

2. A compound as claimed in Claim 1 in which the N-terminal function is H or acyl.

3. A compound as claimed in Claim 1 or Claim 2 in which the C-terminal group is hydroxy or an ester or amide derivative thereof.

4. A compound as claimed in any one of Claims 1 to 3 in which the N-terminal function is alkylcarbonyl of 2 to 7 carbon atoms, arylcarbonyl of 7 to 11 carbon atoms or aralkylcarbonyl of 8 to 12 carbon atoms.

5. A compound as claimed in any one of Claims 1 to 4 in which the C-terminal function is OH, Oalkyl, NH2 or NHalkyl where alkyl has 1 to 6 carbon atoms.

6. A compound as claimed in Claim 5 in which alkyl is methyl, ethyl, propyl, isopropyl or butyl.

7. A compound as claimed Claim 4 in which aryl is optionally substituted phenyl, naphthyl, furanyl, thienyl, pyrrolyl, pyridyl, quinolyl or isoquinolyl wherein the substituent is selected from one or more of C1-C6 alkyl, C1-C6 alkoxy, hydroxy, halo, nitro, amino, Cl-C6 alkylamino, C2-C7 acylamino, carboxy, C2-C7 alkoxycarbonyl, mercapto, C 1-C6 haloalkyl and aminocarbonyl.

8. A compound as claimed in any one of Claims 1 to 7 having a disulfide bond between the cysteine amino acids.

9. A compound as claimed in Claim 1 which is acetyl-Nle-Ile-Ser-Ser-Val-Cys Val-Ser-Ser-Tyr-Arg-Gly-Arg-Lys-Ser-Gly-Asn-Lys-Pro-Pro-Ser-Lys-Thr-Cys-Leu Lys-Glu-Glu-NH2, 6-Cys-24-Cys disulphide

10. A compound as claimed in Claim 1 which is acetyl-Nle-lle-Ser-Ser-Val-Cys- Val-Ser-Ser-Tyr-Arg-Gly-Arg-Lys-Ser-Gly-Asn-Lys-Pro-Pro-Ser-Lys-Thr-Cys-Leu Lys-Glu-Glu-NH2.

11. A test method for screening a test compound for potassium channel modulating activity which comprises administering said test compound to a cell having a potassium channel, said cell containing a peptide of formula (I): (terminal function)-Met-Ile-Ser-Ser-Val-Cys-Vai -Ser-Ser-Tyr- Arg-Gly-Arg-Lys-Ser-Gly-Asn-Lys-Pro-Pro-Ser Lys-Thr-Cys-Leu-Lys-Glu-Glu-(C-terminal function) (I) in which the cysteines are optionally linked via a disulphide bridge and wherein Met represents L-methionine Ile " L-isoleucine Ser " L-serine Val " L-valine Cys " L-cysteine Tyr " L-tyrosine Arg " L-arginine Gly " glycine Lys " L-lysine Asn " L-asparagine Pro " L-proline Thr " L-threonine Leu " L-leucine Glu " L-glutamic acid or a variant thereof with the proviso that excluded is the 13-Lys variant (where 13-Arg is replaced by 13-Lys) in which the cysteines are not linked via a disulphide bridge, and determining whether the inactivation process is affected by said test compound.

12. A method as claimed in Claim 11 in which the N-terminal function of the peptide of formula I is H or acyl.

13. A method as claimed in Claim 11 or Claim 12 in which the C-terminal function of the peptide of formula I is hydroxy or an ester or amide derivative thereof.

14. A method as claimed in any one of Claims 11 to 13 in which the N-terminal function of the peptide of formula I is mono- or di-alkylcarbonyl of 2 to 7 carbon atoms, arylcarbonyl of 7 to 11 carbon atoms or aralkylcarbonyl of 8 to 12 carbon atoms.

15 A method as claimed in any one of Claims 11 to 14 in which the C-terminal function of the peptide of formula I is OH, Oalkyl, NH2 or NHalkyl where alkyl has 1 to 6 carbon atoms.

16. A method as claimed in Claim 15 in which alkyl is methyl, ethyl, propyl, isopropyl or butyl.

17. A method as claimed Claim 14 in which aryl is optionally substituted phenyl, naphthyl, furanyl, thienyl, pyrrolyl, pyridyl, quinolyl or isoquinolyl in which the substituent is selected from one or more of C1-C6 alkyl, C1-C6 alkoxy, hydroxy, halo, nitro, amino, C1-C6 alkylamino, C2-C7 acylamino, carboxy, C2-C7 alkoxycarbonyl, mercapto, Cl-C6 haloalkyl and aminocarbonyl.

18. A method as claimed in any one of Claims 11 to 17 in which the peptide of formula I has a disulfide bond between the cysteine amino acids.

19. A method as claimed in Claim 11 in which the peptide of formula I is acetyl Nle-Ile-Ser-Ser-Val-Cys-Val-Ser-Ser-Tyr-Arg-Gly-Arg-Lys-Ser-Gly-Asn-Lys-Pro-Pro- Ser-Lys-Thr-Cys-Leu-Lys-Glu-Glu-NH2, 6-Cys-24-Cys disulfide.

20. A method as claimed in Claim 11 in which the peptide of formula I is acetyl Nle-Ile-Ser-Ser-Val-Cys-Val-Ser-Ser-Tyr-Arg-Gly-Arg-Lys-Ser-Gly-Asn-Lys-Pro-Pro- Ser-Lys-Thr-Cys-Leu-Lys-Glu-Glu-NH2.

21. A process for preparing a peptide as claimed in Claim 1 wherein the cysteines are linked via a disulphide bridge, which comprises (a) oxidising a corresponding linear peptide in which the cysteines each have free SH groups or (b) deprotecting a corresponding protected peptide of formula (II) Xl -Met-lle-Ser(R1 )-Ser(R1 )-Val-Cys-Val- Ser(Rl)-Ser(Rl)-Tyr(R3)-Arg(R4)-Gly-Arg(R4)- Lys(R5)-Ser(Rl)-Gly-Asn-Lys(R5)-Pro-Pro-Ser(R 1) Lys(RS)-Thr(Rl)-Cys-Leu-Lys(R5)-Glu(R6)-Glu(R6)-X2 (u) or a variant thereof, where X 1 is hydrogen or an a-amino protecting group, R1 is an hydroxy protecting group for the side chain of Ser or Thr or hydrogen, R3 is an hydroxy protecting group for the side chain of Tyr or hydrogen, R4 is an guanyl protecting group for the side chain of Arg or hydrogen, R5 is an amino protecting group for the side chain of Lys or hydrogen, R6 is a carboxy protecting group for the side chain of Glu or hydrogen, X2 is OH, a carboxy protecting group or bond to a solid phase support, e.g x2 = -O-CH2[polystyrene resin support] where the latter group represents one of the many functional groups present in the polystyrene resin; providing at least one protecting group is present when X2 is OH and the cysteine residues are linked via a disulphide bridge, and cleaving from the polystryene resin support if present.

22. A fully or partially protected peptide represented by the formula (it): X l-Met-lle-Ser(R1 )-S er(R1 )ValCys(R2)-Val Ser(R 1)-Ser(R 1)-Tyr(R3)-Arg(R4)-Gly-Arg(R4)- Lys(R5)-Ser(R1 )-Gly-Asn-Lys(R5)-Pro-ProSer(R1 )- Lys(RS)-Thr(Rl)-Cys(R2)-Leu-Lys(R5)-Glu(R6)-Glu(R6)-X2 (11) or a variant thereof, where X1 is hydrogen or an a-amino protecting group, R1 is an hydroxy protecting group for the side chain of Ser or Thr or hydrogen, R2 is a mercapto protecting group or hydrogen or the R2 groups are joined together to form a disulphide bridge.

R3 is an hydroxy protecting group for the side chain of Tyr or hydrogen, R4 is an guanyl protecting group for the side chain of Arg or hydrogen, R5 is an amino protecting group for the side chain of Lys or hydrogen, R6 is a carboxy protecting group for the side chain of Glu or hydrogen, X2 is OH, a carboxy protecting group or bond to a solid phase support, e.g X2 = -O-CH2[polystyrene resin support] where the latter group represents one of the many functional groups present in the polystyrene resin; providing at least one protecting group is present when X2 is OH.

23. A compound as climed in Claim 22 in which X1 is: formyl, trifluoroacetyl, phthalyl, ttoluene- sulfonyl, nitrophenylsulfenyl, benzyloxycarbonyl p-chlorobenzyloxycarbonyl, tnitrobenzyloxycarbonyl; tert-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, amyloxycarbonyl; cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarb onyl; phenylthiocarbonyl; triphenylmethyl or trimethylsilane.

24. A compound as claimed in Claim 22 or 23 in which the side chain nitrogen atoms of arginine, are protected by a group (R4) selected from nitro, tosyl, benzyloxycarbonyl, adamantyloxycarbonyl and tert- butyloxycarbonyl.

25. A compound as claimed in any one of Claims 22 to 24 in which the protecting group for the side chain amino group of lysine (R5) is selected from tosyl, t-amyloxycarbonyl, t-butyloxycarbonyl, diisopropyloxycarbonyl, benzyloxycarbonyl, halobenzyloxycarbonyl and nitrobenzyloxycarbonyl.

26. A compound as claimed in any one of Claims 22 to 25 in which the hydroxyl protecting groups for tyrosine, threonine and serine (R1, R3) are each selected from acetyl, benzoyl, tert-butyl and benzyl.

27. A compound as claimed in any one of Claims 22 to 26 in which the protecting group for the sulfydryl group of the cysteinyl amino acid residue (R2) is selected from benzyl; substituted berizyl wherein the substituent is at least one of methyl, methoxy, nitro, or halo; trityl, benzyloxycarbonyl, benzhydryl, pmethoxybenzyloxycarbonyl, benzylthiomethyl, ethylcarbamoyl, thioethyl, tetrahydropyranyl, acetamidomethyl, benzoyl, and s-sulfonate salt.

28. A compound as claimed in any one of Claims 22 to 27 in which the carboxy group of glutamic acid is protected (R6) by benzyl.

29. A process for preparing a protected peptide of formula II as shown and defined in Claim 22 which comprises coupling the requisite amino acids or groups of amino acids activated and/or protected if required; and further, if desired, anchored to a solid phase support.