AU662032B2 - A novel molecule which inhibits neuropeptide tyrosine biological function - Google Patents

Description

21 C1,? OPI DATE 19/07/93 AOJP DATE 16/09/93 APPLN. ID 32506/93 PCT NUMBER PCT/AU92/00673 AU9332506 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 93/12139 C':.7K 7/06, 7/08, 7/10 Al A61K 308,2 (43) International Publication Date: 24 June 1993 (24.06.93) (21) International Application Number: PCT/AU92/00673 (74) Agent: F.B. RICE CO.; 28A Montague Street, Balmain, NSW 2041 (AU).

(22) International Filing Date: 21 December 1992 (21.12.92) (81) Designated States: AT, AU, BB, BG, BR, CA, CH, CS, Priority data: DE, DK, ES, FI, GB, HU, JP, KP, KR, LK, LU, MG, PL0106 19 December 1991 (19.12.91) AU MN, MW, NL, NO, NZ, PL, PT, RO, RU, SD, SE, UA, US, European patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI patent (71) Applicants (for all designated States except US): GARVAN (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, SN, TD, INSTITUTE OF MEDICAL RESEARCH [AU/AU]; St TG).

Vincent's Hospital, 384 Victoria Street, Darlinghurst, NSW 2010 PRINCE OF WALES MEDICAL RE- SEARCH INSTITUTE [AU/AU]; Prince of Wales Hos- Published pital, High Street, Randwick, NSW 2031 With international search report.

(72) Inventors; and Inventors/Applicants (for US only) TSENG, Albert [AU/ AU]; 6 Wyvern Street, Epping, NSW 2121 IN- GLIS, Adam, S. [AU/AU]; 47 Hillsyde Parade, Strath- 6 more, VIC 3041 SELBIE, Lisa [US/AU]; 2/8 Munro Street, McMahons Point, NSW 2060 POT- TER, Erica [AU/AU]; 3 Henry Street, Randwick, NSW 2031 (AU).

(54) Title: A NOVEL MOLECULE WHICH INHIBITS NEUROPEPTIDE TYROSINE BIOLOGICAL FUNCTION (57) 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-YI 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-NH 2 The present invention also relates to compositions including this molecule and to the use of these compositions in treating a range of disease states.

I_

SWO 93/12139 PCT/AU92/00673 A Novel Molecule which Inhibits Nouropeptide Tyrosine Biological Function Field of the Invention The present invention relates to a molecule which 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 .diinistration of this composition.

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 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.

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, 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 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] 1155-1159) suggesting a role for NPY in some forms of At o- i 1 1 WO 93/12139 PCT/AU92/00673 2 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. 260 [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 po entiating the effects of other pressor agents, as well as *resynaptic 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, Y1 and Y2 (Fuhlendorff J et al, Proc. Natl. Acad. Sc. (1990) *.:182-186). The Y2 receptor is suggested to be expressed -ainly 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 Y1 receptors. Both intact N- and C- termini of NPY are required to fully activate the pressor response by this Y1 receptor.

The NPY molecule consists of 36 amino acids and has San 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

!I*

WO93/12139 PCT/AU92/00673 -3amphipathic 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) 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 WO 93/12139 PCT/AU92/00673 4 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 Y1 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

2 (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 i 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-NH 2 was observed. The inhibitory effect of SALRHY-NH 2 is confined to the postsynaptic or Y1 receptor as no significant inhibitory effects are seen on attenuation of cardiac vagal action.

i WO 93/12139 PCT/AU92/00673 5 In vitro experiments demonstrated the hexapeptide does not compete for binding of NPY at the Y1 receptor. In addition, SALRHY-NH 2 inhibits the ability of NPY to induce increases in intracellular calcium in mammalian cells expressing a transfected Y1 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 I neuropeptide Y with an affinity of at least 4 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 1 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: (X -X 2

-X

3

-X

4

-X

5

-X

6

-X

7 -Xg-X 9

-X

10 n in which X1 is null, Cys or R,

X

2 is null, Cys, R 1 or 1 or 2 amino acids,

X

3 is null, Cys, R 1 Ser, Thr, Ala or Gly,

X

4 is Cys, R 1 Ser, Thr, Ala or Gly,

X

5 is Leu, Ile, Val or Norleucine,

X

6 is Arg, Lys or His, X7 is Arg, Lys o0 His, or Val, Leu, Ile, -A or Norleucine,

X

8 is Tyr, Phe, Trp, His, Lys or Prg, Xg is NH 2 ester or 1 or 2 amino acids, X10 is null, Cys or R 2 R1 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 STi

N.

6 (which may be substituted themselves), or aralkyl or aryl optionally substituted as listed for the alkyl and further including alkyl, or R 1 is glycosyl, nucleosyl or lipoyl,

R

2 is -NR 12

R

13 wherein R 12 and R 13 are independently H, straight, branched or cyclic alkyl, aralkyl or aryl optionally substituted as defined for R 1 or N-glycosyl or N-lipoyl or -ORi 4 where R 14 is H, straight, branched or cyclic alkyl, aralkyl or aryl, optionally substituted as defined for R 1 or -O-glycosyl or -0-lipoyl, with the proviso that

X

1 is always and only null when X 2 is null, Cys or

RI,

X

2 is always and only null when X3 is null, Cys or RIr X3 is always and only null when X4 is Cys or R1, is always and only null when Xg is NH 2 or ester.

The amino acids may be D or L isomers, however, generally the peptide will primarily consist of L-amino acids.

In a second aspect, the present invention provides a linear or cyclic peptide molecule of the general formula: (Xi-X 2

-X

3

-X

4

-X

5

-X

6

-X

7

-X

8

-X

9

-X

10 )n in which

X

1 is null, Cys or R 1

X

2 is null, Cys, R 1 or 1 or 2 amino acids,

X

3 is null, Cys, R 1 Ser, Thr, Ala or Gly,

X

4 is Cys, R 1 Ser, Thr, Ala or Gly,

X

5 is Leu, Ile, Val or Norleucine,

X

6 is Arg, Lys or His,

X

7 is Arg, Lys or His, or Val, Leu, Ile, Met or Norleucine,

X

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

X

9 is NH 2 ester or 1 or 2 amino acids,

X

10 is null, Cys or R2, 7

R

1 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, or aralkyl or aryl optionally substituted as listed for the alkyl and further including alkyl, or R 1 is glycosyl, nucleosyl or lipoyl R1 -NR1 2

R

1 3 wherein R 1 2 and R 13 are independently H, straight, branched or cyclic alkyl, aralkyl or aryl optionally substituted as defined for R 1 or N-glycosyl or N-lipoyl or -OR 14 where R 14 is H, straight, branched or cyclic alkyl, aralkyl or aryl, optionally substituted as defined for R 1 or -0-glycosyl or -0-lipoyl, with the proviso that

X

1 is always and only null when X 2 is null, Cys or R1r

X

2 is always and only null when X 3 is null, Cys or

,R

1 20 X 3 is always and only null when X 4 is Cys or R 1 til X 10 is always and only null when X 9 is NH 2 or ester.

Again, 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 according to S. the first or second aspects, may be cyclic, however, it is "t presently preferred that the peptide is linear.

In a referred embodiment of the present invention

X

1 is null, X 2 is hydrogen, X 9 is NH 2 and X 10 is null. In a preferred embodiment of the present invention X 7 is Arg, Lys or His and Xg is Tyr, Phe, Trp or His.

In yet a further preferred embodiment of the present invention X 3 is Ser or Thr, X 4 is Ala or Gly, X 5 is Leu or Ile or Val, X 6 is Arg or lys or His, X 7 is His or Arg or Lys, and Xg is Tyr.

In a further preferred embodiment of the present invention the peptide is of the formula:- Ser-Ala-LeU-Arg-His-Tyr-NH 2 V

I

Aiti ,lb) I mas~~=rp lmsluPi P nnu3 rm3arr~~ 7/1 In a third 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 or second aspect of the present invention and a pharmaceutical carrier.

In a fourth 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 third 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 sulfation, phosphorylation, nitration, halogenation), either conservative or non-conservative n-amino acids, desamino acids) in the peptide Ssequence 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; I F, Y, W, H; and P, Na-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.

WO 93/12139 PCI/AU92/00673 8 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 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-terminus, lead to enhanced stability for proteolytical degradation while not impairing activity. An example is given in the paper "Tritriated D-ala -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- 2 70), 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 ketomethylene, methylsulfide-or retroinverse bonds to WO 93/12139 PCT/AU92/00673 9 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, "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, Johansen, Carlsberg Res. Commun., Volume 44, pp. 37-46 (1979), and Kullmann, "Enzymatic Peptide Synthesis", CRC Press Inc., Boca Raton, Florida 1987 and Widmer, Johansen, J.T. in "Synthetic Peptides in Biology and Medicine:, eds., Alitalo, K., Partanen, Vatieri, 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 WO 93/12139 PCT/AU92/00673 10 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).

i 5 (Synthesis, spectroscopy and computer simulations in peptide research. Proc. llth American Peptide Symposium Spublished in Peptides-Chemistry, Structure and Biolog 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", Escom, Leiden (1991).

Detailed Description of the Present Invention In order that the nature of the present invGntion 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(1-hydroxybenzotriazole) ester activation.

WO 93/12139 PCT/AU92/00673 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 min. in 10ml HF, with 1.3g of phenol as a scavenger.

Peptides were extracted into an aqueous phase 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 Blood Pressure Measurements Adult rats (200-300g) were anaesthetized with sodium pentobarbitone (Nembutal, Abbott; 60mg/kg The trachea was cannulated and the animal artificially ventilated. The femoral vein was cannulated for the presynaptic action of the peptides tested As a WO 93/12139 PCT/AU92/00673 12 measure of postsynaptic action of a peptide we used increase in systolic bloodpressure and duration of the increase in blood pressure following i.v. injection of the peptide.

Figs. la and Ib show the results obtained in the rat of NPY effect in the absence (control or presence of Img of Ser-Ala-Leu-Arg-His-Tyr-NH 2 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 Ic and Id, control YPSKP However, Ser-Ala-Leu-Arg-His-Tyr-NH 2 acetylated at the N-terminus (Ac-SALRHY) was active (Fig. le and If, control 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-Y1 receptor cDNA (Herzog, et al., 1992, Proc.

Natl. Acad. Sci.U.S.A. 89:5794-5798) were suspended in loading media (Modified RPMI, 10mM Hepes, 1% newborn fetal calf serum) and incubated in a spinner flask at 37C for hours at 1x10 cells.ml. Cells were then treated with luM Fura-2-acetoxymethyl ester (Fura-2AM) for minutes at 37C, washed twice with loading media and resuspended at 5x106 cells/ml. Immediately before use in fluoresceice spectroscopy, cells were recovered by centrifugation at 1000 rpm and resuspended at 1x10 6 cells/ml in a modified Krebs Buffer (135 mM NaC1, 4.7 mM KCL, 1.2 mM MgS04, 1.2 mMKH2PO4, 5nM NaHC03, ImM CaC12, 2.8 mM glucose, and 10mM Hepes, Ph 7.4) containing 1mM sulphinpyrazone. Fluorescence recording were made on a I WO 93/12139 PCT/AU92/00673 13 Hitachi fluorescence spectrometer (F4010) at 340nm (excitation) and 505 (emission) over 10 minutes with slit widths of 5nn and a response time of 2 second.

Intracellular calcium levels were quantitated using equation described by Grynkiewicz et al., 1985 Biol.

Chem. 260:3440-3450). Fura-2AM loaded CHO cells expressing the NPY Y1 receptor were stimulated with 5nM or human neuropeptide Y (NPY) (Auspep) after the addition of 40pg/ml SALRHY-NH 2 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 (40pg/ml) does not inhibit the response, shown in Figure 3. Intracellular calcium increases were observed in response to 5nM NPY(N), NPY, and 5nM bombesin from duplicate determinations). 5nM NPY stimulated calcium increases of 43nM and 22nM, 50nM NPY stimulated increases of OnM and and 5nM bombesin stimulated increases of 30nM and 22nM (Figure 3A). Pretreatment with SALRHY-NH2 resulted in increases of calcium in response to 5nM NPY (15nM and 5nM), 50nM NPY (10nM and 9nM), and bombesin (27nM and 19nM) (Figure 3B), while pretreatment with SALRHY-OH resulted in responses to 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 also does not inhibit a subsequent response to NPY, as subsequent responses to 5nM NPY were calcium, to 50nM NPY were OnM calcium, and to bombesin were 29nM calcium (Figure 3D). The inhibitory effect is specific for the NPY response, as bombesin WO 93/12139 PCT/AU92/00673 14 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-NH 2 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 aid not restrictive.

1ULY--I~- Cp

Claims (16)

1. A molecule which binds to one of the helical domains of neuropeptide Y with an affinity of at least 100nM, 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 1 receptor.

2. A molecule as claimed in claim i 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: (X 1 -X 2 -X 3 -X 4 -X -X 6- 7-X g -X in which X 1 is null, Cys or Ri, X 2 is null, Cys, R 1 or 1 or 2 amino acids, X 3 is null, Cys, R 1 Ser, Thr, Ala or Gly, X 4 is Cys, R 1 Ser, Thr, Ala or Gly, is Leu, Ile, Val or Noileucine, X 6 is Arg, Lys or His, X 7 is Arg, Lys or His, or Val, Leu, Ile,A Va- or Norleucine, X 8 is Tyr, Phe, Trp, His, Lys or Arg, X 9 is NH 2 ester or 1 or 2 amino acids, is null, Cys or R2, R 1 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 -(-whi-eh-may- be ubstitutd-t..hems.e.lves) or aralkyl or aryl optionally substituted as listed for the alkyl and further including alkyl, or R is glycosyl, nucleosyl or lipoyl R 2 is -NR 12 R 13 wherein R12 and R 13 are independently H, straight, branched or cyclic alkyl, ST /C B} WO 93/12139 PCT/AU92/00673 16 aralkyl or aryl optionally substituted as defined for R 1 or N-glycosyl or N-lipoyl or -OR 14 where R 14 is H, straight, branched or cyclic alkyl, aralkyl or aryl, optionally substituted as defined for R 1 or -O-glycosyl or -0-lipoyl, with the proviso that X1 is always and only null when X 2 is null, Cys or R,1 X 2 is always and only null when X 3 is null, Cys or R1' X 3 is always and only null when X 4 is Cys or R1, is always and only null when X 9 is NH 2 or ester.

4. A molecule as claimed in claim 3 in which the amino acids are L isomers. 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 1 is null, X 2 is hydrogen, X 9 is NH 2 and X10 is null.

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

8. A molecule as claimed in any one of claims 3 to 7 in which X 3 is Ser or Thr, X 4 is Ala or Gly, X 5 is Leu or Ile or Val, X 6 is Arg or lys or His, X 7 is His or Arg or Lys, and X 8 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-NH 2 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. 4 1* C C I C I 4 CS C 17

11. A method for 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

12. A linear or cyclic peptide molecule of the general formula: (X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X7-X 8 -X 9 -X 10 )n in which X 1 is null, Cys or R 1 .0 X 2 is null, Cys, R 1 or 1 or 2 amino acids, X 3 is null, Cys, R 1 Ser, Thr, Ala or Gly, X 4 is Cys, R 1 Ser, Thr, Ala or Gly, X 5 is Leu, Ile, Val or Norleucine, X 6 is Arg, Lys or His, X 7 is Arg, Lys or His, or Val, Leu, Ile, Met or Norleucine, X 8 is Tyr, Phe, Trp, His, Lys or Arg, Xg is NH 2 ester or 1 or 2 amino acids, X 10 is null, Cys or R 2 !0 R 1 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, or 25 aralkyl or aryl optionally substituted as listed for the alkyl and further including alkyl, or R 1 is glycosyl, nucleosyl or lipoyl R 2 is -NR 1 2 R 1 3 wherein R12 and R 1 3 are independently H, straight, branched or cyclic alkyl, aralkyl or aryl optionally substituted as defined for RI or N-glycosyl or N-lipoyl or -OR 14 where R 14 is H, straight, branched or cyclic alkyl, aralkyl or aryl, optionally substituted as defined for R 1 or -0-glycosyl or -0-lipoyl, with the proviso that t el *i 4 I 4 9T S_ 0 f% N Ct.Zz; 18 X 1 is always and only null when X 2 is null, Cys or R 1 r X 2 is always and only null when X 3 is null, Cys or Rl r i X3 is always and only null when X 4 is Cys or RI, XI0 is always and only null when Xg is NH 2 or ester.

13. A molecule as claimed in claim 12 in which the amino acids are L isomers.

14. A molecule as claimed in claim 12 or claim 13 in which the peptide is linear. A molecule as claimed in any one of claims 12 to 14 in which X 1 is null, X 2 is hydrogen, X 9 is NH 2 and X 10 is null.

16. A molecule as claimed in any one of claims 12 to in which X 7 is Arg, Lys or His and X 8 is Tyr, Phe, Trp or His.

17. A molecule as claimed in any one of claims 12 to 16 in which X 3 is Ser or Thr, X 4 is Ala or Gly, X 5 is Leu or Ile or Val, X 6 is Arg or Lys or His, X 7 is His or Arg or .20 Lys, and X 8 is Tyr.

18. A molecule as claimed in any one of claims 12 to 17 in which the molecule is a peptide of the formula:- Ser-Ala-Leu-Arg-His-Tyr-NH 2

19. 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 12 to 17 and a pharmaceutical carrier. S* 20. A method of treating hypertension, excessive cardiac t t 30 vagal action, obesity, diabetes or Alzheimer-s disease in a subject comprising administering to the subject the S, composition as claimed in claim 19. DATED this 1st day of May 1995 GARVAN INSTITUTE OF MEDICAL RESEARCH and PRINCE OF WALES MEDICAL RESEARCH INSTITUTE Patent Attorneys for the Applicant: F.B. RICE CO. 0iL9