FIELD OF INVENTION

The invention relates to a ophthalmic compositions and methods of inhibiting degeneration of cells of the retina. In particular, the invention relates to a method and

composition for use in the treatment of diabetics and retina transplantation.

BACKGROUND ART

Neurodegeneration of the retina is a complication of a number of diseases, including diabetes. In diabetic retinopathy the gradual loss of neurons suggests that progress of the disease is ultimately irreversible, since these cells cannot usually be replaced (Barber (2003)).

The vision of people with diabetes will only be protected when a means to prevent the gradual, but constant loss of neurons within the inner retina has been developed.

Neurodegeneration is one of the many factors limiting the transplantation of retinas from donors to recipients.

Ischemic induced oxidative stress is known to trigger glial activation and apoptotic cell death in in vitro models of neurodegeneration .

A need exists for treatments that inhibit neurodegeneration as a complication of diseases such as diabetes and in the transplantation of retinas. It is an object of this invention to provide a method and composition for use in such treatment or at least to provide a useful choice. STATEMENT OF INVENTION

In a first aspect the invention provides a composition comprising one or more ophthalmologically acceptable

excipients and an effective amount of a peptide comprising the sequence:

GlyArgArgAlaAlaProGlyArgAibGlyGly (SEQ ID NO:l)

In one embodiment the peptide consists of the 11 amino acid residue sequence:

GlyArgArgAlaAlaProGlyArgAibGlyGly and ophthalmologically acceptable derivatives thereof. One such ophthalmically acceptable derivative includes the sequence wherein the C-terminus of the peptide is amidated to give: GlyArgArgAlaAlaProGlyArgAibGlyGly-NH ₂ (SEQ ID NO:2) .

In a second aspect the invention provides a method of inhibiting degeneration of cells of the retina of a subject comprising the step of:

• Administering to the subject an effective amount of the peptide SEQ ID N0:1 and/or SEQ ID NO: 2. In one embodiment the administration step to the subject is by way of an intraocular injection.

In one embodiment the subject has been diagnosed as having the metabolic disorder diabetes mellitus.

In one embodiment the subject is selected from the group consisting of: humans and companion animals.

In another embodiment of the second aspect the invention provides a method of inhibiting degeneration of cells of the retina of a diabetic subject by administering to the subject by way of an intraocular injection a composition comprising ophthalmologically acceptable excipients and an effective amount of at least one of the peptides selected from SEQ ID NO:l and SEQ ID NO: 2. In a third aspect the invention provides a method of

transplanting a retina from a donor subject to a recipient subject comprising the step of intravitreal administration of an effective amount of at least one of the peptides selected from SEQ ID NO:l and SEQ ID NO: 2 to the eye of the donor prior to transplantation of the retina.

In one embodiment the subject is selected from the group consisting of: humans and companion animals.

It will be understood from the following description that the effective amount is indicated to be in the range of pmol amounts. Because the administration is to the internal volume of the eye the effective amount will be dependent, at least in part, on the internal volume of the eye of the subj ect .

In the description and claims of this specification the following acronyms, terms and phrases have the meaning provided:

"Effective amount" means an amount effective to inhibit degeneration of cells of the retina.

"Functionally similar amino acid" means an amino acid with similar properties according to the following groupings:

Neutral-weakly hydrophobic (Ala, Gly, Pro, Ser, Thr)

Hydrophilic-Acid Amine (Asn, Asp, Gin, Glu)

Hydrophilic-Basic (Arg, His, Lys)

Hydrophobic (lie, Met, Leu, Val) Hydrophobic-Aromatic (Phe, Trp, Tyr)

Cross-linking (Cys)

"Intraocular injection" means injection into the eyeball and specifically includes injection into the vitreous humour (intravitreal administration) .

"Ophthalmologically acceptable derivatives" means derivatives of the peptide defined in SEQ ID NO:l obtained by amidation, acylation, alkylation, carboxylation, glycosylation,

phosphorylation, prenylation, salification, sulfation, or a combination thereof, that are suitable for inclusion in a composition for administration to the eye.

"Ophthalmologically acceptable excipients" means excipients selected from stabilizing agents, surfactants, buffering agents, chelating agents, viscosity agents, tonicity agents and preservative agents that are suitable for inclusion in a composition for administration to the eye.

In the description and claims of this specification the nucleotides and amino acids of biosequences (nucleic acids and peptides) are identified in accordance with Tables 1 to 4 of Annex C, Appendix 2 of the PCT Administrative Instructions (as in force from January 1, 2010) .

The terms "first", "second", "third", etc. used with

reference to elements, features or integers of the subject matter defined in the Statement of Invention and Claims, or when used with reference to alternative embodiments of the invention are not intended to imply an order of preference.

The invention will now be described with reference to

embodiments or examples and the figures of the accompanying drawings pages. BRIEF DESCRIPTION OF DRAWINGS

Figure 1. Comparison of ex vivo ischemic induced glial activation in retinal tissue following: no treatment (A) and three daily intravitreal administrations of 2 pmol SEQ ID NO: 2 (B) (ONL - Outer Nuclear Layer; INL - Inner Nuclear Layer; IPL - Inner Plexiform Layer; GCL - Ganglionic Cell Layer; NFL - Nerve Fibre Layer) .

Figure 2. Comparison of ex vivo ischemic induced apoptotic cell death in retinal tissue following: no treatment (A) and three daily intravitreal administrations of 2 pmol SEQ ID NO: 2 (B) . Figure 3. Composite of Figure 1 and Figure 2 comparing ex vivo ischemic induced glial activation and apoptotic cell death in retinal tissue following: no treatment (A) and three daily intravitreal administrations of 2 pmol SEQ ID NO: 2 (B) .

DETAILED DESCRIPTION Whole vascular permeability is a target that is often

considered for new treatments of diabetic retinopathy.

Barber (2003) has suggested that apoptosis of neurons is also an essential target for pharmacological studies.

Harris et al (2009) have described a number of synthetic peptides that are effective to promote neural regeneration, migration, proliferation, differentiation and/or axonal outgrowth .

It has now been demonstrated that the synthetic peptide designated here as SEQ ID NO: 2 can be administered

prophylactically to inhibit neurodegeneration in the retina.

A composition comprising 667n SEQ ID NO: 2 in 0.9% NaCl phosphate buffer was prepared as a stock solution. An amount of 2 pmol of SEQ ID NO: 2 was injected into the left eye of a Wistar rat daily for three days by administering 3μ1 of the stock solution (treatment). A volume of 3μ1 of 0.9% NaCl phosphate buffer was administered to the right eye (control) . Twenty four hours after the third injection the rats are sacrificed by injection of ketamine and the retinal tissue extracted and placed into HEPES-buffered extraction medium under a nitrogen atmosphere (oxygen and glucose-free

containing 5% C0 ₂ and 95% N ₂ ) containing all essential amino acids and lipids for a period of 40 min (ischemia) .

Subsequently, the retinal tissue was fixed, cut into slices by the use of a microtome and incubated with anti-glial fibrillary acid protein (anti-GFAP) antibodies and analysed with the Terminal deoxynucleotidyl transferase dUTP Nick End Labelling (TUNEL) kit.

The distribution of the Mueller glial retinal marker GFAP in the control and treatment samples is presented in the

fluorescent photomicrographs of Figure 3. Far more

pronounced glial inflammatory reaction was observed in the control, i.e. untreated, sample.

Analysis with the TUNEL kit is presented in the fluorescent photomicrographs of Figure 4. A far higher number of apoptotic neurons is observed in the inner retinal layer of the control sample.

The treatment sample shows a significantly reduced glial cell-inflammatory response and no apoptotic cell death. It appears that daily administration of 2 pmol SEQID NO: 1 was effective to inhibit degeneration of cells of the retina under ischemic stress.

The ability to induce prolonged survival of different retinal neuronal cell types (receptor cells (cones and rods) , ganglionic cells, amacrine and horizontal cells) will be advantageous in mitigating degeneration of the retina, such as that induced by disease or trauma, including retinal transplantation . In retinal transplantation the time factor between the potential choice of the right donor tissue and the

preparedness of the recipient in a clinical setting is a considerable factor for a successful transplantation

strategy. It is anticipated that administration of SEQ IDs NO: 1 and 2 will improve the survival of donor retinal tissue .

Although the invention has been described with reference to an embodiment or example it should be appreciated that variations and modifications may be made to this embodiment or example without departing from the scope of the invention.

Where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to in this specification. In particular, it is anticipated that functionally similar peptide sequences may be obtained by substitution of one or more amino acids of the biosequence with a functionally similar amino acid. It is suggested that the functionality of similar peptide sequences may be confirmed without undue additional experimentation by use of the method disclosed in this specification.

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