WO2014075137A1

WO2014075137A1 - Peptides incorporating amino-substituted lactams for treatment of retinopathy

Info

Publication number: WO2014075137A1
Application number: PCT/AU2013/001313
Authority: WO
Inventors: Scott Thomson; Nicholas Ede
Original assignee: Biodiem Limited
Priority date: 2012-11-13
Filing date: 2013-11-13
Publication date: 2014-05-22
Also published as: AU2013204309A1

Abstract

The present invention relates to new therapeutic compounds and the use of these compounds in the treatment of eye diseases involving apoptosis. The compounds of the present invention are useful in the treatment of retinopathy.

Description

PEPTIDES INCORPORATING AMINO-SUBSTITUTED LACTAMS FOR TREATMENT OF RETINOPATHY

Field

5

The present invention relates to new therapeutic compounds and the use of these compounds in the treatment of eye diseases involving apoptosis. The compounds of the present invention are useful in the treatment of retinopathy. 0 Background

Retinopathy generally refers to some form of non-inflammatory damage to the retina of the eyes. The causes of retinopathy are quite varied, including diabetes (i.e., diabetic retinopathy), arterial hypertension (causing hypertensive5 retinopathy), sickle cell disease, direct exposure to sunlight (causing solar retinopathy), phenochromocytoma, cilipathyl and newborn prematurity (causing retinopathy of prematurity, previously known as retrolental fibroplasia).

Many types of retinopathy are progressive, leading to diminished eye sight or0 complete blindness. This is particularly the case if the macular has been affected.

Medications currently available for retinopathy typically depend on the cause and include:

diabetic retinopathy: intravitreal injection of trimacinolone (a corticosteroid); also it5 has been reported that certain Kinase inhibitors and VEGF inhibitors may also be affective;

hypertensive retinopathy: usually treated by lowering the patient's high blood pressure. Accordingly, anti-hypertensives (e.g., and β-blockers, Ca channel blockers, renin-angiotensin acting agents, etc) may be used; and0 retinopathy of prematurity (ROP): intravitreal injection of bevacizumab (Avastin) - but not effective for the zone II disease state when used to treat infants with stage 3+ ROP (see New England Journal of Medicine, 2011 , 364(7): 603-615).

Accordingly, much of the therapeutic advances in this area have been focussed on the underlying cause of the disease state. In addition to these treatments there is a need to provide therapeutics which directly treat retinopathy either by halting the condition or at least minimising its progress. Summary of Invention

The present invention provides compounds represented by formula (I) or salts thereof:

wherein:

A is an amino acid residue selected from the group consisting of Ala, Gly, Val, lie, Leu, Thr, Ser, Asp and Glu;

A₂ is an amino acid residue selected from Glu or Asp;

A3 is absent or is an amino acid residue selected from the group consisting of Ala, Gly, Val, lie, Leu, Thr, Ser, Asp and Glu;

X is a 5-membered heterocycylene moiety selected from:

Y is an amino acid residue selected from the group consisting of Gly, Ser, Val, Ala, Leu, Pro, Asp, Glu, Lys, Arg and His.

In a further aspect the invention provides a method of treating eye disease involving apoptosis comprising the step of administering an effective amount of a compound of formula (I), or a salt thereof. In a further aspect the invention provides a method of treating retinopathy comprising the step of administering an effective amount of a compound of formula (I), or a salt thereof.

In a further aspect the invention provides the use of compound of formula (I), or a salt thereof, in the manufacture of a medicament for treating eye diseases involving apoptosis and/or retinopathy.

In a further aspect the invention provides a compound of formula (I), or a salt thereof, for use in the treatment of eye diseases involving apoptosis and/or retinopathy.

Brief Description of the Figures

Figure 1 Wholemounts of retina from C57BI/6 mice with ROP and and immunolabeled with FITC-conjugated lectin. Scale bar=1mm. A.

Sham control. B. ROP control. C. ROP+BDM-e peptide 1. D. ROP+angiotensin type 1 receptor blocker (candesartan). *, optic disk. Sham controls did not have avascular areas of retina. In ROP mice treated with BDM-E peptide 1 and the angiotensin type 1 receptor blocker, avascular area were both reduced compared to ROP control.

Figure 2 Graph showing mean number (percentage) of avascular area per retina in C57BI/6 mice with ROP. Values are mean+SEM. ^*p<0.05 compared to ROP control. In mice treated (100 μο) with BDM-E peptide 1 (Ala - Glu - Asp - Gly), avascular retina were reduced by 52% respectively compared to ROP control. Detailed Description of the Invention

Without wanting to be bound by any particular theory, this invention is predicated upon the understanding that the 5-membered helerocycylene moiety may play a significant role in increasing the stability of the compounds of the present invention and are therefore beneficial in treating eye diseases involving apoptosis, and in particular retinopathy.

In an embodiment A₂ is Glu. In an embodiment A2 is Asp.

In an embodiment Ai is Ala.

In an embodiment is Gly, Val, lie, Leu, Thr or Ser.

In an embodiment is Asp or Glu. In an embodiment A3, when present, is Ala. In an embodiment A₃, when present, is Gly, Val, lie, Leu, Thr or Ser. ln an embodiment A3, when present, is Asp or Glu. In an embodiment Y is Gly or Ser. In an embodiment Y is Ala, Val, Leu or Pro. In an embodiment Y is Asp, Glu, Lys, Arg or His.

The present invention contemplates all combinations of Ai, A₂, A₃ and Y in the above embodiments.

For instance:

In an embodiment A₂ is Glu and is Ala.

In an embodiment A₂ is Asp and Ai is Ala.

In an embodiment A₂ is Glu and Ai is selected from Gly, Val, lie, Leu, Thr, or Ser. In an embodiment A₂ is Asp and Ai is selected from Gly, Val, He, Leu, Thr or Ser. In an embodiment A₂ is Glu and Ai is selected from Asp or Glu. In an embodiment A₂ is Asp and AT is selected from Asp or Glu.

In an embodiment A₂ is Glu, Ai is Ala, and Y is Gly or Ser.

In an embodiment A₂ is Asp, Ai is Ala, and Y is Gly or Ser. In an embodiment A₂ is Glu, Ai is selected from Gly, Val, He, Leu, Thr, or Ser, and Y is Gly or Ser. In an embodiment A₂ is Asp, is selected from Gly, Val, lie, Leu, Thr, or Ser, and Y is Gly or Ser,

In an embodiment A₂ is Glu, Ai is selected from Asp or Glu, and Y is Gly or Ser,

In an embodiment A₂ is Asp, AT is selected from Asp or Glu, and Y is Gly or Ser.

In one of the above embodiments A₃ may be Ala, or Gly, Val, He, Leu, Thr, or Ser; or Asp or Gin,

In an embodiment the compound is a compound of formula (I) in which A₃ is absent.

In certain preferred embodiments the compounds of formula (I) are represented by formula (la):

Ala - Glu - X - Gly (la) In an embodiment the compounds of formula (I) are represented by formula (lb):

Ala - Glu - X - Ala (lb) In an embodiment the compounds of formula (I) are represented by formula (lc);

Ala - Glu - X - Val (lc) In an embodiment the compounds of formula (I) are represented by formula (Id);

Ala - Glu - X - Leu (Id) ln an embodiment the compounds of formula (I) are represented by formula (le);

Ala - Glu - X - lie (le)

In the above embodiments and also with respect to formula (I) it will be appreciated that the a-amino group of the Y residue forms the nitrogen in the ring of X. In relation to compounds of formulae (I), (la), (lb), (lc) and (le) and each of the embodiments aforementioned described X is preferably selected from:

Accordingly, in another embodiment the compounds of formula (I) may be selected from:

ln an embodiment the N-terminal amino acid is a D-amino acid, preferably D-Ala.

In an embodiment the N-terminal amino acid is a L-amino acid, preferably L-Ala.

·

In one embodiment the N-terminal amino acid residue in the compounds of formulae (I), (la), (lb), (Ic), (Id) and (le) each independently may be derivatised or may be an unsubstituted amine. In one embodiment, the N-terminal amino acid residue may be a -NR"₂, -NC(0)R'^* ₂, -NR"S0₂R", -NR"C(0)NR"₂ or - NR"C(0)OR"; wherein each R" may, independently, represent hydrogen or C C₃ alky I.

In a further embodiment, the N-terminal amino acid residue is independently a primary amine, an acetamide or a pyroglutamide and preferably a primary amine.

In a preferred embodiment the N-terminal amino acid residue is an unsubstituted amino group (i.e., NH₂).

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.

Preferably the carbonyl group is presented in the compounds of the present invention as an unmodified form.

The peptide portion of the compounds of the present invention may be prepared using standard peptide synthetic methods.

For example, the peptide portion may be synthesised by standard solution phase methodology, as described in Hruby, Victor J.; Meyer, Jean-Philippe. Chemical synthesis of peptides. University of Arizona, USA. Editor(s); Hecht, Sidney, M. Bioorganic Chemistry: Peptides and Proteins (1998), pp 27-64, Oxford University Press, New York, N. Y

The linear peptide portion may also be synthesised by solid phase methodology using Boc chemistry, as described by Schnolzer et a/., 1992, Int J Pept Protein Res 40, 180-193. Following deprotection and cleavage from the solid support the reduced peptides are purified using preparative chromatography.

The linear peptide portion may also be synthesised by solid phase methodology using Fmoc chemistry, as described below

1) Peptide is synthesized by Fmoc solid-phase peptide synthesis using an automatic synthesizer.

2) Peptide is synthesized from its C-terminus by stepwise addition of amino acids.

3) the first Fmoc-amino acid is attached to an insoluble support resin via an acid labile linker.

4) After deprotection of Fmoc by treatment with piperidine, the second Fmoc- amino acid is coupled utilizing a pre-activated species or in situ activation.

5) After the desired peptide is synthesized, the resin bound peptide is deprotected and detached from the resin via TFA cleavage.

6) Following deprotection and cleavage from the solid support the reduced peptides are purified using preparative chromatography.

For example, the portion X-Y of the desired product may be prepared first, such that the amino group on X is protected with a Boc group, and the carboxylic acid group on Y is unprotected. X-Y may then be coupled to a resin using an in situ HBTU activation protocol (Schnolzer ef a/., 1992, Int J Pept Protein Res 40, 180- 193). Various resins may be used, depending on the functional group that is desired at the C-terminus of the final product. For example, if the final product has a carboxylic acid group at the C-terminus, then a PAM (4-Hydroxymethyl- phenylacetamidomethyl) resin may be used. Alternatively, if the desired product has an amide group at the C-terminus then a benzhydrylamine resin, for example, may be used. While for these resins the final products may be cleaved from the resin using hydrofluoric acid, in some embodiments resins that are cleavable with trifluoroacetic acid may be used and in these circumstances the amino group on X would be protected with an Fmoc group.

After coupling the portion X-Y to a resin, the next amino acids, e.g., A₂ or A₃, may be added using standard peptide coupling techniques. In one example, Fmoc- protected Ai , A₂ and A₃ may be used in the synthesis. After the on-resin synthesis of the peptide is complete, the resin may be treated with hydrofluoric acid or trifluoroacetic acid, depending on the resin used, to cleave the product. The product may then be purified, for example using reversed-phase HPLC.

It will be appreciated that the compounds of the invention may have at least one asymmetric centre, and therefore are capable of existing in more than one stereoisomeric form. The invention extends to each of these forms individually and to mixtures thereof, including racemates. Where possible the isomers may be separated conventionally by chromatographic methods or using a resolving agent.

Alternatively the individual isomers may be prepared by asymmetric synthesis using chiral intermediates. Where the compound has at least one carbon-carbon double bond, it may occur in Z- and E- forms and all isomeric forms of the compounds being included in the present invention.

The compounds of the present invention include compounds where, for instance, the terminal amino acid residues are in a protected form. Suitable protected forms are well known to those in the art and have been described in many references such a Protecting Groups in Organic Synthesis, Greene T W, Wiley-lnterscience, New York, 1981. Examples of protected forms may include groups which may be added to enhance the solubility or other pharmacological properties of the compounds of the present invention. For instance, a protected group may include a hydrophilic polymer selected from poly(alkylene glycol), poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrolidone), poly(hydroxypropylmethacrylamide), poly(acrylamide), poly(N-isopropylacrylamide), poly(dimethylacrylamide), poly(hydroxyethyl(meth)acrylate), polypeptide molecules, carbohydrates, polynucleic acids, poly(acrylates), poly(poly(alkylene glycol) meth(acrylate)).

In another embodiment the protected form may include a biological recognition motif, including but not limited to, a biotin molecule, a protein or domain or fragment of a protein, an Fc domain of IgG or other antibody, a protein, a molecule (or fragment thereof), a protein G (or fragment thereof), an (oligo or poly) peptide, an (oligo or poly) nucleic acid.

The compounds used or identified according to the present invention may be in the form of a salt or pharmaceutically acceptable derivative thereof. The salts of the compounds of the invention are preferably pharmaceutically acceptable, but it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the present invention, since these are useful as intermediates in the preparation of pharmaceutically acceptable salts or may be useful in some applications, such as probes or assays.

The compounds of formula (I) as represented herein to include where possible "pharmaceutically acceptable derivatives" including pharmaceutically acceptable esters, prodrugs, solvates and hydrates, as well as pharmaceutically acceptable addition salts of such derivatives. Pharmaceutically acceptable derivatives may include any pharmaceutically acceptable hydrate or any other compound or prodrug which, upon administration to a subject, is capable of providing (directly or indirectly) a compounds of the present invention or an active metabolite or residue thereof. "Pharmaceutically acceptable derivatives" also encompasses the protected forms of the peptides as discussed above. The pharmaceutically acceptable salts include acid addition salts, base addition salts, salts of pharmaceutically acceptable esters and the salts of quaternary amines and pyridiniums. The acid addition salts are formed from a compound of the invention and a pharmaceutically acceptable inorganic or organic acid including but not limited to hydrochloric, hydrobromic, sulfuric, phosphoric, methanesulfonic, toluenesulphonic, benzenesulphonic, acetic, propionic, ascorbic, citric, malonic, fumaric, maleic, lactic, salicyclic, sulfamic, or tartartic acids. The counter ion of quarternary amines and pyridiniums include chloride, bromide, iodide, sulfate, phosphate, methansulfonate, citrate, acetate, malonate, fumarate, sulfamate, and tartate. The base addition salts include but are not limited to salts such as sodium, potassium, calcium, lithium, magnesium, ammonium and alkylammonium. Also, basic nitrogen-containing groups may be quaternised with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others. The salts may be made in a known manner, for example by treating the compound with an appropriate acid or base in the presence of a suitable solvent.

The compounds of the invention may be in crystalline form or as solvates (e.g. hydrates) and it is intended that both forms be within the scope of the present invention. The term "solvate" is a complex of variable stoichiometry formed by a solute (in this invention, a compound of the invention) and a solvent. Such solvents should not interfere with the biological activity of the solute. Solvents may be, by way of example, water, ethanol or acetic acid. Methods of solvation are generally known within the art.

The term "pro-drug" is used in its broadest sense and encompasses those derivatives that are converted in vivo to the compounds of the invention. Such derivatives would readily occur to those skilled in the art, and include, for example, compounds where a free amino group is converted into an acylamino, or amino alkylcarbonyl prodrug. Any compound that is a prodrug of a compound of the invention is within the scope and spirit of the invention. Conventional procedures for the preparation of suitable prodrugs according to the invention are described in text books, such as "Design of Prodrugs" Ed. H. Bundgaard, Elsevier, 1985. The term "pharmaceutically acceptable ester" includes biologically acceptable esters of compound of the invention such as sulphonic, phosphonic and carboxylic acid derivatives.

The invention also includes where possible a salt or pharmaceutically acceptable derivative such as a pharmaceutically acceptable salt, ester, solvate and/or prodrug of the above mentioned embodiments.

In a further aspect the invention provides for the use of an effective amount of a compound of Formula (I), (la), (lb), (lc), (Id), or (le) (or mixture thereof) or a pharmaceutically acceptable derivative thereof, and optionally a carrier or diluent in the manufacture of a pharmaceutical composition (medicament) for treating retinopathy.

In another aspect, the present invention provides a pharmaceutical composition for use in treating retinopathy, the composition comprising an effective amount of a compound of Formula (I), (la), (lb), (lc), (Id), or (le) (or a mixture thereof) or a pharmaceutically acceptable derivative thereof, and optionally a carrier or diluent.

As will be readily appreciated by those skilled in the art, the route of administration and the nature of the pharmaceutically acceptable carrier will depend on the nature of the condition and the mammal to be treated. It is believed that the choice of a particular carrier or delivery system, and route of administration could be readily determined by a person skilled in the art. In the preparation of any formulation containing the compound active care should be taken to ensure that the activity is not destroyed in the process and that the compoundis able to reach its site of action without being destroyed. In some circumstances it may be necessary to protect the compound by means known in the art, such as, for example, micro encapsulation. Similarly the route of administration chosen should be such that the compound active reaches its site of action. The pharmaceutical forms suitable for injectable use include sterile injectable solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions. They should be stable under the conditions of manufacture and storage and may be preserved against reduction or oxidation and the contaminating action of microorganisms such as bacteria or fungi.

Those skilled in the art may readily determine appropriate formulations for the compounds of the present invention using conventional approaches. Identification of preferred pH ranges and suitable excipients, for example antioxidants, is routine in the art (see for example Cleland et al, 1993). Buffer systems are routinely used to provide pH values of a desired range and include carboxylic acid buffers for example acetate, citrate, lactate and succinate. A variety of antioxidants are available for such formulations including phenolic compounds such as BHT or vitamin E, reducing agents such as methionine or sulphite, and metal chelators such as EDTA.

The solvent or dispersion medium for the injectable solution or dispersion may contain any of the conventional solvent or carrier systems for peptide actives, and may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about where necessary by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include agents to adjust osmolality, for example, sugars or sodium chloride. Preferably, the formulation for injection will be isotonic with blood. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Pharmaceutical forms suitable for injectable use may be delivered by any appropriate route including intravenous, intramuscular, intracerebral, intrathecal, epidural injection or infusion.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients such as these enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying or freeze-drying of a previously sterile-filtered solution of the active ingredient plus any additional desired ingredients.

Other pharmaceutical forms include oral and enteral formulations of the present invention, in which the active peptide may be formulated with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal or sublingual tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. It will be appreciated that some of these oral formulation types, such as buccal and sublingual tablets, have the potential to avoid liver metabolism. However the compounds of the present invention may also be delivered to the stomach where liver metabolism is likely to be involved. Such compositions and preparations preferably contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations, including those that allow specific delivery of the active peptide to specific regions of the gut.

Liquid formulations may also be administered enterally via a stomach or oesophageal tube. Enteral formulations may be prepared in the form of suppositories by mixing with appropriate bases, such as emulsifying bases or water-soluble bases. It is also possible, but not necessary, for the peptides of the present invention to be administered topically, intranasally, intravaginally, intraocularly and the like. The present invention also extends to any other forms suitable for administration, for example topical application such as creams, lotions and gels, or compositions suitable for inhalation or intranasal delivery, for example solutions, dry powders, suspensions or emulsions. The present invention also extends to parenteral dosage forms, including those suitable for intravenous, intrathecal, and intracerebral or epidural delivery.

The compounds useful according to the present invention may be administered by inhalation in the form of an aerosol spray from a pressurised dispenser or container, which contains a propellant such as carbon dioxide gas, dichlorodifluoromethane, nitrogen, propane or other suitable gas or combination of gases. The compounds may also be administered using a nebuliser.

Pharmaceutically acceptable vehicles and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. It is especially advantageous to formulate the compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable vehicle. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding active materials for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail. As mentioned above the principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable vehicle in dosage unit form. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.25 pg to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.25 pg to about 2000 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients. The conditions to be treated by the methods, uses and compositions of the present invention are generally those characterised by retinopathy. Hence the methods, uses and compositions of the present invention may be generally available for prophylactically or therapeutically treating retinopathy in patients in need thereof.

Thus, the present invention may also provide a method for treating a patient suffering from retinopathy comprising administering to said patient a therapeutically effective dose or amount of a compound of the present invention or a composition comprising a therapeutically effective amount of a compound of the present invention.

Preferably the subject is in need of such treatment, although the compound may be administered in a prophylactic sense. For certain of the abovementioned conditions it is clear that the compounds may be used prophylactically as well as for the alleviation of acute symptoms. Accordingly, references herein to "treatment" or the like are to be understood to include such prophylactic treatment, as well as treatment of acute conditions. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Examples

Synthetic Examples 1. Comparator example - BDME peptide 1 (Ala - Glu-Asp - Gly) (May be prepared according to the methodology in US 6,727,227)

2. Example 1 - Peptide 8

These peptides may be synthesised similarly to the procedure disclosed in Ede ef al. (1994) Int. J. Peptide Protein Res. 44, 568-581.

The dipeptide 9 may be prepared according to the procedures disclosed in Ede et al. (1994) Int. J. Peptide Protein Res. 44, 568-581 and Ede ef al (1991) Peptide Res. 4, 171-176.

9 This dipeptide may be coupled to PAM (4-Hydroxymethyl-pbenylacetamidomethyl) resin using an in situ HBTU activation protocol (Schnolzer et al. , 1992, Int J Pept Protein Res 40, 180-193). After removal of the Boc protecting group with trifluoroacetic acid, Fmoc-Glu(OtBu)-OH and then Fmoc-Ala-OH may be added to the peptide using stepwise synthesis and in situ HBTU activation protocols (Schnolzer et a/., 1992, Int J Pept Protein Res 40, 180-193; Alewood et al., Methods Enzymol. 1997, 289, 14-29), or using BOP, NMM and HOBt in the coupling reactions (as described in Ede et al. (1994) Int, J. Peptide Protein Res. 44, 568-581). The peptides may then be cleaved from the resin with anhydrous HF, and then purified using reversed-phase HPLC to provide^' the desired products.

Biological Data Determination of the reduction of pathological angiogenesis in a mouse model of retinopathy of prematurity (ROP).

Methods Animal groups

• Sham control

• ROP control

• ηθΡ+25Όμ πλον$ /άΒν BDM-E peptide 1 (Ala - Glu - Asp - Gly)

• ROP+250f.ig/mouse/day BDM-E analogue 8

· ROP+ the angiotensin type 1 receptor blocker, candesartan (2.5mg/kg/day).

Represents a positive control.

ROP Model in Mice: All experimental procedures are consistent with the guidelines set by the Australian National Health and Medical Research Council Code of Practice for the Care and Use of Animals for Scientific Purposes. C57BI/6 mice are obtained from the Animal Resource Centre, Perth, Western Australia. Mouse pups and their nursing mothers are housed in a 12-hour light/dark cycle, at room temperature. The mothers are allowed free access to standard rodent chow (GR2 Clark-King and Co., Gladesville, New South Wales, Australia). On postnatal day (P) 7, pups and their mothers are placed in a 75% oxygen environment until P12. The oxygen environment is maintained using a PROOX 110 gas regulator (Reming Bioinstraments Co., Redfield, New York, USA) coupled to a 10m³ medical oxygen cylinder (Linde Gas Pty. Ltd., Yennora, NSW). On P12, pups are housed in room air until P18, ROP pups are treated during the hypoxic room air period, which represents P12 to P18.

Dosing: performed by daily subcutaneous injection.

Pathological Angiogenesis: As described previously (1 ,2,3), on P 8, pups are anaesthetised with intramauscular ketamine (60mg/kg/body weight, Ketamil, 100mg/mL) and xylazine (5mg/kg/body weight). Briefly, eyes were fixed in 4% paraformaldehyde for 30 minutes and retina dissected in 0.1 M phosphate buffered saline, pH 7.4 (PBS) and flatmounted. Retina were stained with FITC-conjugated Griffonia (Bandeiraea) simplicifolia BS-I lectin (1 : 00, Sigma, USA) in 1 % Triton X- 100 (Sigma) in 0.1 M PBS overnight. Retina were washed with PBS for 6x15 minutes and mounted with media (DakoCytomation, Glostrup, Denmark). Images were taken at 100x magnification using AxioCam MRc 6.1.0.0 digital camera attached to a Zeiss Axio X1 microscope (Carl Zeiss, Germany). Retinal montages were constructed using the tiling tool in AxioObserver software (version 5.3, Carl Zeiss). To quantitate avascular retina. BS-I lectin negative areas of retina were traced in the mid-central retina. Results were expressed as the percentage of avascular area/retina (cm²). RESULTS

Peptide 1 reduced neoangiogenesis in C57BI/6 mice with ROP,

Sham control mice had a normal distribution of blood vessels over the entire retina (Figure 1). Mice with ROP exhibited an avascular central retina adjacent to the optic disc (Figures 1 and 2 and Table 1). In mice with ROP, BDM-E peptide reduced neovascular tufts by 72% and avascular retina by 52% (Figures 1 and 2 and Table 1). These findings are comparable to ROP mice treated with the angiotensin type 1 receptor blocker (Table 1),

Table 1 : Percentage of avascular central retina in mice with ROP and treated with compounds of present invention.

Values are mean±SEM *P<0.05 compared to ROP control.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A compound of formula (I) or a salt or a pharmaceutically acceptable derivative thereof:

AT- A2 - A3 - X - Y (I) wherein: i is an amino acid residue selected from the group consisting of Ala, Gly, Val, lie, Leu, Thr, Ser, Asp and Glu;

A₂ is an amino acid residue selected from Glu or Asp; A₃ is absent or is an amino acid residue selected from the group consisting of Ala, Gly, Val, lie, Leu, Thr, Ser, Asp and Glu;

X is a 5-membered heterocycylene moiety selected from:

2. A compound according to claim 1 or a salt or pharmaceutically acceptable derivative thereof, wherein A₂ is Glu.

3. A compound according to claim 1 or a salt or pharmaceutically acceptable derivative thereof, wherein A₂ is Asp. 4. A compound according to claim 1 or 2 or a salt or pharmaceutically acceptable derivative thereof, wherein is Ala.

5. A compound according to claim 1 or 2 or a salt or pharmaceutically acceptable derivative thereof, wherein Ai is Gly, Val, He, Leu, Thr or Ser.

6. A compound according to claim 1 or 2 or a salt or pharmaceutically acceptable derivative thereof, wherein Ai is Asp or Glu.

7. A compound according to any one of claims 1 to 6 or a salt or pharmaceutically acceptable derivative thereof, wherein A₃, when present, is Ala.

8. A compound according to any one of claims 1 to 6 or a salt or pharmaceutically acceptable derivative thereof, wherein A₃, when present, is Gly, Val, lie, Leu, Thr or Ser.

9. A compound according to any one of claims 1 to 6 or a salt or pharmaceutically acceptable derivative thereof, wherein A₃, when present, is Asp or Glu. 10. A compound according to any one of claims 1 to 9 or a salt or pharmaceutically acceptable derivative thereof, wherein Y is Gly or Ser.

11. A compound according to any one of claims 1 to 9 or a salt or pharmaceutically acceptable derivative thereof, wherein Y is Ala, Val, Leu or Pro.

12. A compound according to any one of claims 1 to 9 or a salt or pharmaceutically acceptable derivative thereof, wherein Y is Asp, Glu, Lys, Arg or His. 13. A compound according to any one of claims 1 to 12 or a salt or pharmaceutically acceptable derivative thereof, represented by formula (la):

Ala - Glu - X - Gly (la)

14. A compound according to any one of claims 1 to 13 or a pharmaceutically acceptable derivative thereof, wherein X is selected from:

A compound according to any claim 1 or a salt or pharmaceutically ptable derivative thereof, wherein the compound is selected from:

Ala-GIu- -C „N-Val

-

N-A

16. A compound according to claim 1 , or a salt or pharmaceutically acceptable

derivative thereof, represented by the formula:

17. A method of treating eye disease involving apoptosis comprising the step of administering an effective amount of a compound of formula (I), or a salt or a pharmaceutically acceptable derivative thereof, according to any one of claims 1 to

16.

18. A method of treating retinopathy comprising the step of administering an effective amount of a compound of formula (I), or a or a pharmaceutically acceptable derivative thereof salt thereof, according to any one of claims 1 to 16. 19. The use of compound of formula (I), or a salt or a pharmaceutically acceptable derivative thereof thereof, according to any one of claims 1 to 16 in the manufacture of a medicament for treating eye diseases involving apoptosis and/or retinopathy. 20. A pharmaceutical composition comprising a compound of formula (I), or a salt or a pharmaceutically acceptable derivative thereof thereof, according to any one of claims 1 to 16 and at least one carrier or diluent.