WO1993012139A1

WO1993012139A1 - A novel molecule which inhibits neuropeptide tyrosine biological function

Info

Publication number: WO1993012139A1
Application number: PCT/AU1992/000673
Authority: WO
Inventors: Albert Tseng; Adam S. Inglis; Lisa Selbie; Erica Potter
Original assignee: Garvan Institute Of Medical Research; Prince Of Wales Medical Research Institute
Priority date: 1991-12-19
Filing date: 1992-12-21
Publication date: 1993-06-24
Also published as: CA2126212A1; JPH08501055A; EP0672054A4; EP0672054A1

Abstract

The present invention provides a novel molecule which inhibits the biological activity of neuropeptide tyrosine (NPY). The molecule of the present invention binds with an affinity of at least 100 nM to one of the helical domains of NPY in a manner such that NPY with the molecule bound thereto cannot bind to the NPY-Y1 receptor. It is preferred that the molecule of the present invention is a peptide and preferably a peptide of the sequence Ser-Ala-Leu-Arg-His-Tyr-NH2. The present invention also relates to compositions including this molecule and to the use of these compositions in treating a range of disease states.

Description

A Novel Molecule which Inhibits Neuropeptide

Tyrosine Biological Function Field of the Invention

The present invention relates to a molecule which

<- 5 inhibits the biological activity of Neuropeptide Tyrosine (NPY) . In addition, the present invention relates to pharmaceutical compositions including, as the active ingredient, this molecule and to methods of treatment involving the administration of this composition.

10 Background of the Invention

Neuropeptide tyrosine (NPY) was first isolated and sequenced from porcine brain in 1982 by Tatemoto and colleagues (Tatemoto et al, Proc. Natl. Acad. Sci. 79. 5485-5489). Subsequently it has been isolated from

15 peripheral nevrves and is found in most sympathetic postganglionic neurons co-localised with noradrenaline. It is found in high concentrations in human plasma when activity in the sympathetic nervous system is increased either physiologically for example during exercise (e.g.

20 Morris et al, Clin. Exp. Pharm. Physiol 13 [1986] 437-440 or pathologically following myocardial infarction (Hulting et al, Cardiovas. Res. 24 [1990] 102-108), in mycocardial ischaemia or angina (Ullman et al, J. Int. Med. 228 [1990] 583-589), in some forms of hypertension (Edvinsson et al,

25 Regul Pep. 32 [1991] 279-287) as well as in tumors of the sympathetic nervous system e.g. phaeochromocytoma (Adrian et al. Lancet ii [1983] 540-542). Because of its widespread presence in sympathetic nerves and its release following sympathetic nervous system activation, NPY is

30 implicated as an important sympathetic neurotransmitter in the cardiovascular system NPY is also found in sympathetic nerve fibres in pancreatic islets (Ekblad et al. Front hormone Res. 12[1984]85-90) and has been shown to inhibit insulin release (Moltz & McDonald, Peptides 6 [1985]

35 1155-1159) suggesting a role for NPY in some forms of diabetes. NPY is also found in many areas of the central nervous system and increases in the level of NPY in some central neurons are found in Alzheimer's disease (Allen et al, J. Neurol. Sci £4 [1984] 325-331, Chan-Palay, J. Comp. Neurol. 160. [1987], 201-223). Injection of NPY into discrete regions of the central nervous system can evoke powerful behavioural and hormonal changes. One effect studied in some detail is the stimulation of feeding by injection of NPY into the paraventricular nucleus of the hypothalamus. Such injections can lead to a six-fold increase in rate of weight gain and a three-fold increase in body fat (Stanley et al, Peptides, 7[1986] 1189-1192) and from this a role has been suggested for neuropeptide Y in hyperphagia and obesity. In the cardiovascular system neuropeptide Y has been shown to have a potent vasoconstrictor activity both directly and by potentiating the effects of other pressor agents, as well as presynaptic inhibition of noradrenaline and acetycholine release in anaesthetized rats (Potter E et al. Regulatory Peptides (1989) 25, 167-177).

NPY binds specifically to at least two receptors, Yl and Y2 (Fuhlendorff J et al, Proc. Natl. Acad. Sc. (1990) 87:182-186). The Y2 receptor is suggested to be expressed mainly prejunctionally and requires the C-terminal fragment of NPY (2-36 to 22-36) to mediate the presynaptic effect of attenuated cardiac vagal action. The NPY-evoked pressor response is mainly mediated by postsynaptic or Yl receptors. Both intact N- and C- termini of NPY are required to fully activate the pressor response by this Yl receptor.

The NPY molecule consists of 36 amino acids and has an amidated C-terminal tyrosine residue. Its proposed structure, based on the crystal structure of the related pancreatic polypeptide, comprises a poly-proline helical structure (residues 2-8) as part of one side and an amphipathic alpha-helix (residues 13-32) as the other. Residues 33-36 most likely are not involved in the helical structure. Hydrophobic interactions between the two helices appear to provide the main source of stability to the active three-dimensional structure of the molecule overall (Potter E et al. Regulatory Peptides (1989) 25, 167-177) .

Boublik et al. in US patent US5026685 described a blood pressure lowering or hypotensive action of the fragment NPY18-36 in high dose. However, this action has since been shown to be an indirect effect of NPY18-36 mediated by stimulation of afferent vagal fibres (probably by NPY18-36 -evoked histamine release) to cause a reflex hypotension. In in vitro experiments NPY18-36 has been shown to have no direct effect on arterioles (Potter et al (1991) J Auto. Nerv. Syst. in press, Michel et al (1990) Am. J. Physiol. 290 E131-139).

The functional importance of NPY structure has been supported by a recent report of Schwartz et al, "Central & Peripheral Significance of NPY and its Related Peptides" Annal N.Y. Acad. Sc Vol 611 (1990) 35-37. They demonstrated that the NPY molecule retained full binding affinity to mouse brain membranes, as well as its ability to suppress the stimulated formation of cAMP, when the loop of the hairpin (residues 8-17) was replaced with

8-amino-octanoic acid and the structure was stabilized by introduction of a disulphide bridge between the two sides. This experiment reinforces the importance of the association of the two helical limbs to the native NPY molecule for correct presentation at the receptor surface, and hence, biological activity.

However, in the native molecule there is no disulphide bond to restrict the movement of the two helical limbs which are held together by the hydrophobic and hydrogen bonds. These can be dissociated completely by the addition of an excess of a denaturing molecule such as urea.

The present inventors hypothesised that the amino acids in one helix will also interact with the same segment of chain in the second helix if they are present in the solution as a separate peptide. Since both ends of NPY are required for pressor response, and a precise tertiary structural definition is presumably important for docking with the receptor, the addition of a high concentration of a synthetic molecule with a high affinity towards one of the helical domains of the native molecule would be expected to interact sufficiently to alter the orientation of the binding amino acids of the Yl receptor; and so decrease the pressor response. Since proteins in general depend both on the amino acid sequence and correct folding of the chain for their biological activities, the present inventors propose, as a principle, that small segments of the structure or analogues of it that are vital for providing the correct functional confirmation also have the potential, because of their specific affinity for the active site(s), to distort this conformation and inhibit activity when they are present in excess in a solution of the protein.

As an example, using synthetic peptide chemistry, the present inventors prepared a hexapeptide amide SALRHY-NH, (Ser-Ala-Leu-Arg-His-Tyr amide) which corresponds to residues 22-27 of the NPY molecule, portion of the amphipathic helix in NPY. In a series of experiments in anaesthetized rats significant inhibition of the NPY-evoked pressor response and a decrease in resting blood pressure levels 60 minutes after administration of SALRHY-NH₂ was observed. The inhibitory effect of SALRHY-NH- is confined to the postsynaptic or Yl receptor as no significant inhibitory effects are seen"on attenuation of cardiac vagal action. In vitro experiments demonstrated the hexapeptide does not compete for binding of NPY at the Yl receptor. In addition, SALRHY-NH- inhibits the ability of NPY to induce increases in intracellular calcium in mammalian cells expressing a transfected Yl receptor. Summary of the Invention

In a first aspect the present invention consists in a molecule which binds to one of the helical chains of neuropeptide Y with an affinity of at least 400nM, the binding of the molecule to neuropeptide Y being such that the neuropeptide Y with the molecule bound thereto will not bind to the neuropeptide Y - Y₁ receptor.

In a preferred embodiment of the present invention the molecule is a linear or cyclic peptide. In yet a further preferred embodiment of the present invention the linear or cyclic peptide is of the general formula:

(x₁-x--x₃-x₄-x₅-x--x₇-x₈-x--x₁₀)_n in which

X. is null, Cys or R-,

X- is null, Cys, R.. , or 1 or 2 amino acids,

X- is null, Cys, R-, Ser, Thr, Ala or Gly,

X. is Cys, R., Ser, Thr, Ala or Gly,

X_ is Leu, lie, Val or Norleucine, X, is Arg, Lys or His,

X- is Arg, Lys or His, or Val, Leu, lie, Val or

Norleucine,

X- is Tyr, Phe, Trp, His, Lys or Arg,

X_Q is NH-, ester or 1 or 2 amino acids.

10 is null, Cys or R-,

R., is H or R-CO, where R is H, straight, branched or cyclic alkyl up to C20, optionally containing double bonds and/or substituted with halogen, nitro, amino, hydroxy, sulfo, phospho or carboxyl groups (which may be substituted themselves), or aralkyl or aryl optionally substituted as listed for the alkyl and further including alkyl, or R.. is glycosyl, nucleosyl or lipoyl, R- is -NR₁₂R₁₃, wherein R..- and R_l3 are independently H, straight, branched or cyclic alkyl, aralkyl or aryl optionally substituted as defined for R.. or N-glycosyl or N-lipoyl or -OR,^, where R-. is H, straight, branched or cyclic alkyl, aralkyl or aryl, optionally substituted as defined for R., or -O-glycosyl or -O-lipoyl, with the proviso that

X, is always and only null when X- is null, Cys or R_χ, - is always and only null when X- is null, Cys or R_χf

X- is always and only null when X. is Cys or R-, X.- is always and only null when X_g is NH- or ester. The amino acids may be D or L isomers, however, generally the peptide will primarily consist of L-amino acids.

The peptide of the present invention may be cyclic, however, it is presently preferred that the peptide is linear.

In a preferred embodiment of the present invention X₁ is null, X- is hydrogen, X_g is NH₂ and X-_Q is null. In a preferred embodiment of the present invention X- is Arg, Lys or His and X- is Tyr, Phe, Trp or His. In yet a further preferred embodiment of the present invention X- is Ser or Thr, X. is Ala or Gly, X- is Leu or lie or Val, X- is Arg or lys or His, X_ is His or Arg or Lys, and X- is Tyr. In a further preferred embodiment of the present invention the peptide is of the formula:- Ser-Ala-Leu-Arg-His-Tyr-NH-.

In a second aspect the present invention consists in a composition for use in anti-hypertensive therapy, cardiovascular therapy, anti-obesity and anti-diabetic therapy or as an anti-psychotic, the composition comprising the molecule of the first aspect of the present invention and a pharmaceutical carrier. In a third aspect the present invention consists in a method of treating hypertension, excessive cardiac vagal activity, e.g. syncope, obesity, diabetes or Alzheimer's disease in a subject comprising administering to the subject the composition of the second aspect of the present invention.

It will be appreciated by those skilled in the art that when the molecule of the present invention is a peptide that a number of modifications may be made to the peptide without deleteriously effecting the biological activity of the peptide. This may be achieved by various changes, such as insertions, deletions and substitutions (e.g., sulfation, phosphorylation, nitration, halogenation) , either conservative or non-conservative (e.g., ω-amino acids, desamino acids) in the peptide sequence where such changes do not substantially altering the overall biological activity of the peptide. By conservative substitutions the intended combinations are:-

G, A; V, I, L, M; D, E; N, Q; S, T; K, R, H; F, Y, W, H; and P, Nα-alkylamino acids. It may also be possible to add various groups to the peptide to confer advantages such as increased potency or extended half-life in vivo, without substantially altering the overall biological activity of the peptide.

The term peptide is to be understood to embrace peptide bond replacements and/or peptide mimetics, i.e. pseudopeptides, as recognised in the art (see for example: Proceedings of the 20th European Peptide Symposium, edt. G. Jung. E. Bayer, pp. 289-336, and references therein), as well as salts and pharmaceutical preparations and/or formulations which render the bioactive peptide(s) particularly suitable for delivery. Such salts, formulations, amino acid replacements and pseudopeptide structures may be necessary and desirable to enhance the stability, formulation, deliverability (e.g., slow release, prodrugs), or to improve the economy of production, and they are acceptable, provided they do not negatively affect the required biological activity of the peptide.

Apart from substitutions, three particular forms of peptide mimetic and/or analogue structures of particular relevance when designating bioactive peptides, which have to bind to a receptor while risking the degradation by proteinases and peptidases in the blood, tissues and elsewhere, may be mentioned specifically, illustrated by the following examples: Firstly, the inversion of backbone chiral centres leading to D-amino acid residue structures may, particularly at the N- erminus, lead to enhanced stability for proteolytical degradation while not impairing activity. An example is given in the paper "Tritriated D-ala¹-Peptide T Binding", Smith, C.S. et al. Drug Development Res. 15, pp. 371-379 (1988). Secondly, cyclic structure for stability, such as N to C interchain imides and lactames (Ede et al in Smith and Rivier (Eds) "Peptides: Chemistry and Biology", Escom, Leiden (1991), p268-270), and sometimes also receptor binding may be enhanced by forming cyclic analogues. An example of this is given in "Confirmationally restricted thymopentin-like compounds", U.S. pat. 4,457,489 (1985), Goldstein, G. et al. Finally, the introduction of keto ethylene, methylsulfide or retroinverse bonds to replace peptide bonds, i.e. the interchange of the CO and NH moieties may both greatly enhance stability and potency. An example of the latter type is given in the paper "Biologically active retroinverso analogues of thymopentin", Sisto A. et al in Rivier, J.E. and Marshall, G.R. (eds.) "Peptides, Chemistry, Structure and Biology", Escom, Leiden (1990), p.722-773.

Where the molecule of the present invention is a peptide the peptide can be synthesized by various methods which are known in principle, namely by chemical coupling methods (cf. Wunsch, E.: "Methoden der organischen Chemie", Volume 15, Band 1 + 2, Synthese von Peptiden, Thieme Verlag, Stuttgart (1974), and Barrany, G.; Merrifield, R.B: "The Peptides", eds. E. Gross, J. Meienhofer., Volume 2, Chapter 1, pp. 1-284, Academic Press (1980)), or by enzymatic coupling methods (cf. Widmer, F., Johansen, J.T., Carlsberg Res. Commun., Volume 44, pp. 37-46 (1979), and Kullmann, W. : "Enzymatic Peptide Synthesis", CRC Press Inc., Boca Raton, Florida (1987), and Widmer, F., Johansen, J.T. in "Synthetic Peptides in Biology and Medicine:, eds., Alitalo, K. , Partanen, P., Vatieri, A., pp. 79-86, Elsevier, Amsterdam (1985)), or by a combination of chemical and enzymatic methods if this is advantageous for the process design and economy.

It will be seen that one of the alternatives embraced in the general formula set out above is for a cysteine residue to be positioned at both the amino and carboxy terminals of the peptide. This will enable the cylisation of the peptide by the formation of di-sulphide bond.

It is intended that such modifications to the peptide of the present invention which do not result in a decrease in biological activity are within the scope of the present invention. The three-dimensional structure and function of the biologically active peptides can be simulated by other compounds, some not even peptidic in nature, but which mimic the activity of such peptides. This field of science is summarised in a review by Goodman, M. (1990). (Synthesis, spectroscopy and computer simulations in peptide research. Proc. 11th American Peptide Symposium published in Peptides-Chemistry, Structure and Biology pp 3-29. Ed Rivier, J.E. and Marshall, G.R. Publisher ESCOM.) As will be recognized by those skilled in the art, armed with the disclosure of this application, it will be possible to produce peptide and non-peptide compounds having the same three-dimensional structure as the peptide of the present invention. These "functionally equivalent structures" or "peptide mimics" will react with antibodies raised against the peptide of the present invention. It is intended that such "peptide mimics" are included within the scope of the present invention.

More detail regarding pharmacophores can be found in Bolin et al. p 150, Polinsky et al. p 287, and Smith et al. p 485 in Smith and Rivier (Eds) "Peptides: Chemistry and Biology", Esco , Leiden (1991).

Detailed Description of the Present Invention In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following examples. Peptide Synthesis:

Peptides were synthetisized on the Applied Biosytem (ABI) Peptide Synthesizer Model 430A. T-boc(t-butyloxycarbonyl) chemistry was used. Peptide synthesis was carried out in solid phase on PAm-resin (PAM-phenylacetamidomethyl) or MBHA-resin (MBHA=pp-methylbenzhydrylamino) supplied by ABI. Peptide bonds were formed either viasymmetric anhydride coupling or via HOB5(l-hydroxybenzotriazole) ester activation. *.^* ~

- 11 -

Cleavage : hydrogen fluoride (HF) cleavage of the fully protected peptide from the solid support was performed by Auspep Pty Ltd, Melbourne. The resin was treated for 60 min. in 10ml HF, with 1.3g of phenol as a scavenger. Peptides were extracted into an aqueous phase (30% acetonitrile/water, v/v) and scavengers were washed out with ether. The aqueous extracts were then lyophilized to yield the crude product. Side chain protection groups chosed for each amino acid was removed during the cleavage process.

Purification:

Crude synthetic products were subjected to HPLC (both ion exchange and reverse phase chromatography) , and a major peak representing the desired peptide was observed.

Purity Determination:

To confirm peptide purity and identity, purified peptides were then subjected to both sequence and amino acid analysis. Amino acid analysis was performed routinely on a Millipore Waters Picotag system using HC1 vapour phase hydrolysis and PITC as the derivatising agent. The peptide-resin, crude peptide, as well as the final product were checked. The procedure often give low figures for cysteine and occasionally give flow figures for Tyrosine due to partial oxidation. For all other amino acids, a good analysis give figures within 10% of the theoretical and where the noise level is high, results can deviate from the theoretical of up to 15%. Blood Pressure Measurements

Adult rats (200-300g) were anaesthetized with sodium pentobarbitone (Nembutal, Abbott; 60mg/kg i.p.). The trachea was cannulated and the animal artificially ventilated. The femoral vein was cannulated for the presynaptic action of the peptides tested [9]. As a measure of postsynaptic action of a peptide we used increase in systolic blood pressure and duration of the increase in blood pressure following i.v. injection of the peptide. Figs, la and lb show the results obtained in the rat of NPY effect in the absence (control • ■■ •) or presence o o of lmg of Ser-Ala-Leu-Arg-His-Tyr-NH₂. Equivalent doses of a peptide with sequence derived from a different region of the NPY molecule (residues 1-5) with the amino acid sequence Tyr-Pro-Ser-Lys-Pro-OH had no effect (Figure lc and Id, control o -o; YPSKP • ■ ■•) . However,

Ser-Ala-Leu-Arg-His-Tyr-NH₂ acetylated at the N-terminus (Ac-SALRHY) was active (Fig. le and If, control o——o;

AC-SALRHY • •) . In addition to the marked effect shown in Figs, la and lb a decrease in the resting blood pressure of the animals of 14 ± 5 mm Hg (mean + SEM for 5 rats; range 5-30 mm Hg) was observed 1 hour after administration of the peptide. In vitro Calcium releasing Experiment:

Chinese hamster ovary cells (CHO) expressing the human NPY-Yl receptor cDNA (Herzog, et al., 1992, Proc. Natl. Acad. Sci.U.S.A. 89:5794-5798) were suspended in loading media (Modified RPMI, lOmM Hepes, 1% newborn fetal calf serum) and incubated in a spinner flask at 37C for 2.5 hours at 1x10 cells.ml. Cells were then treated with luM Fura-2-acetoxymethyl ester (Fura-2AM) for 30 minutes at 37C, washed twice with loading media and resuspended at 5x10 cells/ml. Immediately before use in fluorescence spectroscopy, cells were recovered by centrifugation at 1000 rpm and resuspended at 1x10 cells/ml in a modified rebs Buffer (135 mM NaCl, 4.7 mM KCL, 1.2 mM MgS04, 1.2 mMKH2P04, 5nM NaHC03, ImM CaC12, 2.8 mM glucose, and lOmM Hepes, Ph 7.4) containing ImM sulphinpyrazone. Fluorescence recording were made on a ... _*

- 13 -

Hitachi fluorescence spectrometer (F4010) at 340nm (excitation) and 505 (emission) over 10 minutes with slit widths of 5nm and a response time of 2 second. Intracellular calcium levels were quantitated using equation described by Grynkiewicz et al., 1985 (J. Biol. Che . 16^:3440-3450). Fura-2AM loaded CHO cells expressing the NPY Yl receptor were stimulated with 5nM or 50nM human neuropeptide Y (NPY) (Auspep) after the addition of 40]_g/ml SALRHY-NH₂ hexapeptide. Increases in calcium induced by the endogenous bombesin receptor were measured by the addition of 5nM bombesin. The results are shown in Fig. 2 and indicate that pretreatment with the SALRHY Peptide can inhibit the subsequent response to NPY. The action of the SALRHY Peptide is dependent on the presence of an amide modification at the C-terminus, as the peptide SALRHY-OH (40μg/ml) does not inhibit the response, shown in Figure 3. Intracellular calcium increases were observed in response to 5nM NPY(N) , 50nM NPY, and 5nM bombesin (B)( from duplicate determinations). 5nM NPY stimulated calcium increases of 43nM and 22nM, 50nM NPY stimulated increases of OnM and 5nM, and 5nM bombesin stimulated increases of 3OnM and 22nM (Figure 3A) . Pretreatment with SALRHY-NH2 (40μg/ml) resulted in increases of calcium in response to 5nM NPY (15nM and 5nM) , 50nM NPY (lOnM and 9nM) , and 5nM bombesin (27nM and 19nM) (Figure 3B) , while pretreatment with SALRHY-OH resulted in responses to 5nM NPY(27nM and 25nM, 50nM NPY(OnM and 29nM) and bombesin (22nM) (Figure 3C) . In addition, pretreatment with a pentapeptide derived from NPY sequence residues 1-5 YPSKP-OH(40μg/ml) also does not inhibit a subsequent response to NPY, as subsequent responses to 5nM NPY were 25nM calcium, to 5OnM NPY were OnM calcium, and to 5nM bombesin were 29nM calcium (Figure 3D) . The inhibitory effect is specific for the NPY response, as bombesin responses were relatively unaffected.

As is clear from the above examples, the present inventors have synthesized a novel peptide which inhibits NPY pressor function. The hexapeptide amide, SALRHY-NH₂ is part of the native NPY molecule. The peptide of interest is a small molecule readily synthesized, purified and characterised. Like monoclonal antibodies, they can interact with specific regions for protein/peptide molecule to affect its activity, but are much easier to prepare purified. The interaction of the peptide could be due to various known non-covalent forces and other similar peptides many also have a similar effect. It would also be expected that the peptide of the present invention would not be immunogenic due to its small size. The native peptide inactivator theory developed by the present inventors could be applicable to biologically active proteins or polypeptides in general. It is envisaged that peptide segments which play a positive role in interacting with, and stabilising intramolecular structures that are important for biological or enzymic function will also be active in a negative way and cause distortion of the structure if they are able to compete for it in the form of free peptides in solution. It is also envisaged that this principle can be applied to receptors in general: segments of chains that play a structural role in maintaining the confirmation of the receptor docking sites for reaction with the biologically active molecule would be expected to play a negative role if in solution as free peptide chains. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

CLAIMS : -

1. A molecule which binds to one of the helical domains of neuropeptide Y with an affinity of at least lOOnM, the binding of the molecule to neuropeptide Y being such that the neuropeptide Y with the molecule bound thereto will not bind to the neuropeptide Y - Y. receptor.

2. A molecule as claimed in claim 1 in which the molecule is a linear or cyclic peptide.

3. A molecule as claimed in claim 2 in which the linear or cyclic peptide is of the general formula:

(x₁-x₂-x₃-x₄-x₅-x₆-x₇-x--x--x₁₀)_n in which

X.. is null, Cys or R-,

X₂ is null, Cys, R-, or 1 or 2 amino acids, X- is null, Cys, R., Ser, Thr, Ala or Gly,

X. is Cys, R-, Ser, Thr, Ala or Gly,

X_ is Leu, lie, Val or Norleucine,

X- is Arg, Lys or His,

X- is Arg, Lys or His, or Val, Leu, lie, Val or Norleucine,

X_p is Tyr, Phe, Trp, His, Lys or Arg,

X_q is NH-, ester or 1 or 2 amino acids.

X _1Q is null, Cys or R-,

R. is H or R-CO, where R is H, straight, branched or cyclic alkyl up to C20, optionally containing double bonds and/or substituted with halogen, nitro, amino, hydroxy, sulfo, phospho or carboxyl groups (which may be substituted themselves), or aralkyl or aryl optionally substituted as listed for the alkyl and further including alkyl, or R.. is glycosyl, nucleosyl or lipoyl

R₂ is -NR₁₂R₁₃, wherein R₁₂ and R₁₃ are independently H, straight, branched or cyclic alkyl. aralkyl or aryl optionally substituted as defined for R» or N-glycosyl or N-lipoyl or -OR-., where R.. is H, straight, branched or cyclic alkyl, aralkyl or aryl, optionally substituted as defined for R. or -O-glycosyl or -O-lipoyl, with the proviso that

X.. is always and only null when X₂ is null, Cys or R_λ, ₂ is always and only null when X₃ is null, Cys or R_χ,

X_ is always and only null when X. is Cys or R., , _{1 n} is always and only null when X_g is NH- or ester.

4. A molecule as claimed in claim 3 in which the amino acids are L isomers.

5. A molecule as claimed in claim 3 or claim 4 in which the peptide is linear.

6. A molecule as claimed in any one of claims 3 to 5 in which X-. is null, X- is hydrogen, X_g is NH₂ and X_1Q is null.

7. A molecule as claimed in any one of claims 3 to 6 in which X- is Arg, Lys or His and X„ is Tyr, Phe, Trp or His.

8. A molecule as claimed in any one of claims 3 to 7 in which X- is Ser or Thr, X. is Ala or Gly, X- is Leu or lie or Val, X- is Arg or lys or His, X_ is His or Arg or Lys, and X- is Tyr.

9. A molecule as claimed in any one of claims 1 to 8 in which the molecule is a peptide is of the formula:- Ser-Ala-Leu-Arg-His-Tyr-rø-.

10. A composition for use in anti-hypertensive therapy, cardiovascular therapy, anti-obesity and anti-diabetic therapy or as an anti-psychotic, the composition comprising a molecule as claimed in any one of claims 1 to 8 and a pharmaceutical carrier.

11. A method of treating hypertension, excessive cardiac vagal action, obesity, diabetes or Alzheimer's disease in a subject comprising administering to the subject the composition as claimed in claim 9.