WO2010038010A1

WO2010038010A1 - Somatostatin analogues

Info

Publication number: WO2010038010A1
Application number: PCT/GB2009/002316
Authority: WO
Inventors: Sunil Shaunak; Ian Teo
Original assignee: Imperial Innovations Limited
Priority date: 2008-10-01
Filing date: 2009-09-30
Publication date: 2010-04-08
Also published as: GB0817978D0; JP2012504409A; BRPI0920777A2; EP2352525A1; CN102264397A

Abstract

The invention relates generally to an isolated peptide consisting of the amino acid sequence X₁CX₂WKX₃CT, wherein X₁ is F or A, X₂ is F or Y and X₃ is T or V in any combination or permutation, and wherein each of the amino acids is in the L- configuration and the peptide contains a disulphide bond between the two cysteine residues. Alternatively, the two sulphur atoms of the two cysteine residues may be linked via three carbon atoms. The invention also relates generally to fusion proteins comprising the peptide, to polynucleotides encoding the peptide or fusion proteins, to methods of producing the peptide or fusion proteins, to pharmaceuticals containing the peptide or fusion proteins, and to uses of the same.

Description

SOMATOSTATIN ANALOGUES

The present invention relates generally to biologically synthesised peptides that do not exist naturally in nature, to polynucleotides encoding the peptides, to methods of producing the peptides, and to uses of the peptides. The peptides of the invention can be used in medicine, for example, in the treatment of hormonal disorders, cancer and bleeding disorders. The present invention also relates generally to a modified version of the peptides having enhanced stability, to methods for manufacturing such modified peptides, and to uses of the modified peptides. In particular, the invention relates to biologically synthesised peptides that can be chemically modified using a polymer such as polyethylene glycol, and to uses of the peptides or modified versions of the peptides in the treatment of acromegaly, tumours including gut hormone secreting tumours and gastro-intestinal bleeding.

Acromegaly is a disabling hormonal disorder characterized by the enlargement of the bones of the head, hands and feet and of soft tissue. It leads to premature death. The incidence of acromegaly is 3 cases per million persons per year. Its prevalence is 60 per million. Since the features of this disease develop insidiously, there is often a delay of 7 to 10 years in making the diagnosis after the onset of clinical symptoms. It is caused by excess secretion of growth hormone (GH) by the pituitary gland, usually due to a pituitary adenoma. The proliferation of pituitary somatotroph cells results in abnormally high levels of growth hormone secretion, which leads to the distinctive clinical features of acromegaly.

Growth hormone is secreted as a 191-amino-acid, 4-helix bundle protein and a less abundant 176-amino-acid form. It enters the circulation in a pulsatile fashion under hypothalamic control via hypothalamic-releasing and hypothalamic-inhibiting hormones that traverse the hypophysial portal circulation. It then acts directly on specific somatotroph cell surface receptors. Growth hormone (i) induces the synthesis of peripheral insulin-like growth factor 1 (IGF-I), (ii) induces circulating (endocrine) and local (autocrine and paracrine) IGF-I induced cell proliferation, and (iii) inhibits apoptosis. The excess morbidity and mortality of acromegaly are the result of the prolonged elevation of GH and IGF-I levels. Meticulous life-long control of the levels of these two hormones improves the patient's well-being and restores a normal life expectancy.

Among the current treatments for acromegaly, surgery is often performed to remove growth hormone secreting micro-adenomas. Radiotherapy is usually reserved for tumours that have recurred or persisted after surgery in patients with resistance to, or intolerance of, medical treatment.

In addition, current treatments include the administration of chemically synthesised analogues of the natural hormone, somatostatin, which suppresses the secretion of growth hormone. Octreotide acetate (commercialized under the name Sandostatin® by Novartis Pharmaceuticals) is one such analogue. It has become a successful and clinically approved treatment for acromegaly over the past two decades. Octreotide acetate (systematic IUPAC name (4RJS, 10S, 13R,16S, 19R)-10-(4-aminobutyl)- 19- [[(2i?)-2-amino-3-phenyl-propanoyl]amino]-16-benzyl-iV-[(2_JR,3i?)-l,3- dihydroxybutan-2-yl]-7-(l-hydroxyethyl)-13-(lH-indol-3-ylmethyl)-6,9,12,15,18- pentaoxo-l,2-dithia-5,8,l l,14,17-pentazacycloicosane-4-carboxamide) is an octapeptide produced by solid-phase chemical synthesis methods (Bauer et al., (1982) Life Sciences, Vol. 31, pp. 1133-1140). The amino acid sequence of chemically synthesised octreotide acetate is:

(D)Phe-c[(L)Cys-(L)Phe-(D)Trp-(L)Lys-(L)Thr-(L)Cys]-(L)Thr(ol)

1 2 3 4 5 6 7 8

Another chemically synthesized somatostatin analogue is lanreotide acetate (Systematic IUPAC name (4,S',75,105',13i?,16S,19₁S}-10-(4-ammobutyl)-19-[[(2i?)-2- amino-3-naphthalen-2-yl-propanoyl]amino]-7v^r-[(ljS',2i?)-l-carbamoyl-2-hydroxy- propyl]-16-[(4-hydroxyphenyl)methyl]-13-(lH-indol-3-ylmethyl)-6,9₃12,15,18- pentaoxo-7-propan-2-yl- 1 ,2-dithia-5,8, 11 , 14, 17-pentazacycloicosane-4-carboxamide). It is commercialized under the name Somatuline LA® by Ipsen Pharmaceuticals. The amino acid sequence of lanreotide acetate is:

Beta-naρthyl-(D)alanine - Cys - Tyr - (D)Trp - Lys - VaI - Cys - Tbr - CONH₂

1 2 3 4 5 6 7 8

Notably, lanreotide acetate contains a (D) alanine in position 1 and a (D) tryptophan in position 4. It also has a disulphide bond across the two cysteine residues. Lanreotide acetate is structurally very closely related to octreotide acetate, exhibits a very similar somatostatin receptor binding profile to octreotide acetate, and has the same medical indications as octreotide acetate (Weckbecker, G. et al., (2003) Nature Reviews, Vol. 2, pp. 999-1017).

It is known that the natural hormone somatostatin has a cyclic part to its conformation because of the presence of an extended antiparallel beta-sheet that is due to the presence of Trp and Lys residues at the corners of the beta-turn. In octreotide acetate and lanreotide acetate, the incorporation of a D-Trp4 acts in the same way to stabilise the pharmacophoric beta-turn.

It is also generally known that cyclization of a peptide by the formation of a disulfide bridge give proteins and peptides (including somatostatin) improved chemical and metabolic stability. For this reason, the bridging unit Cys2-S-S-Cys7 was chemically incorporated into chemically synthesised octreotide acetate and lanreotide acetate. In the case of octreotide acetate, it provides a 3-fold enhancement to the metabolic stability of the peptide (Bauer et al., (1982) Life Sciences, Vol. 31, pp. 1133-1140; Cai et al., (1986) Proc. Natl. Acad. Sci. (USA), Vol. 83, pp. 1896-1900).

Taken together, the insertion of two D-amino acids and the formation of a disulfide bridge increases the plasma half-life of these chemically synthesised 8 amino acid peptides. In the case of octreotide acetate, the increase is from a few minutes to 1.5 hours (Harris AG., (1994) Somatostatin and somatostatin analogues: pharmacokinetics and pharmacodynamic effects. Gut, Vol.35, pp. 1-4). Octreotide acetate and lanreotide acetate bind with high affinity to somatostatin receptor subtype 2 (SST2) and somatostatin receptor subtype 5 (SST5). They bind with moderate affinity to somatostatin receptor subtype 3. They do not bind to somatostatin receptor subtype 1 or somatostatin receptor subtype 4. In addition, octreotide acetate and lanreotide acetate have also been shown to bind to the D2 dopamine receptor.

It is known that only Phe3-Trp4-Lys5-Thr6 in the structure of octreotide acetate are essential for its biological activity. When octreotide acetate binds to somatostatin receptors, it signals the pituitary to suppress the secretion of growth hormone and the proliferation of somatotroph cells, and it acts on the liver to block the synthesis of IGF-I. As a somatostatin receptor ligand for the SST2 and SST5 receptors, it suppresses levels of GH and IGF-I, constrains tumour growth, and inhibits the hepatic mediated binding and action of GH to its receptor. As a GH-receptor antagonist, it prevents GH-receptor signalling, which leads to a reduced level of IGF-I in the peripheral blood.

The suppression of growth hormone secretion in response to octreotide acetate administration in patients with acromegaly is dependent upon somatostatin receptor subtype availability. The direct anti-tumour activities of octreotide acetate and lanreotide acetate have been demonstrated in experimental tumour models to be mediated through the somatostatin receptors expressed in tumour cells. These antiproliferative actions are the result of blocking cell division and the induction of apoptosis. Binding of a chemically synthesised somatostatin analogue to a somatostatin receptor initiates specific signal transduction pathways. In this way, each somatostatin receptor subtype can mediate different biological actions. The receptor subtypes that mediate these mechanisms are SSTl, SST2, SST4 and SST5. Although many human tumours express more than one somatostatin receptor subtype, SST2 is predominant. The somatostatin analogues octreotide acetate and lanreotide acetate have a high affinity for SST2 receptors. Somatostatin and its synthetic analogues also exert a number of indirect anti-tumour actions. These include the inhibition of the release of growth factors and hormones that drive tumour growth. The decrease in tumour growth that results from the indirect effects of these somatostatin analogues include the suppression of synthesis and secretion (and thereby diminution of the actions) of growth factors and hormones that include IGF-I and growth hormone. Somatostatin analogues suppress the growth hormone-IGF-1 axis by both central and peripheral mechanisms. SST2 and SST5 are the primary receptor subtypes mediating the inhibition of pituitary growth hormone release. The somatostatin analogues also inhibit hepatic growth hormone-induced IGF-I production via SST2 mediated activation of a tyrosine phosphatase which leads to dephosphorylation of STAT5b and to a decrease in IGF-I gene transcription (Weckbecker, G. et al., (2003) Nature Reviews, Vol. 2, pp. 999-1017; Susini C & Buscail L, (2006) Ann of Oncology Vol. 17, pp. 1733-1742).

Somatostatin receptor subtype availability, in turn, predicts the long-term effect of octreotide acetate therapy on serum growth hormone and IGF-I concentrations in the patient's blood. Maximal suppression of serum growth hormone and IGF-I is seen with subcutaneous doses of 300 - 600 micrograms/day. Octreotide acetate is initially given to patients as a subcutaneous injection at least three times a day (because its half-life is only 1.5 h) for a period of several months in order to reduce the levels of growth hormone to normal levels. Once the excessive production of growth hormone has been reduced to normal levels, patients are often transferred (over a period of several weeks) to a slow release polymer based complex depot preparation such as Sandostatin® long-acting. It is given by deep intramuscular injection into the gluteal muscle; it cannot be given into the deltoid muscle because of the excessive pain that Sandostatin causes in this muscle as compared to the large gluteal muscle. The slow release of octreotide acetate from this polymer based slow release depot means that it can usually be given (with pain) every two weeks as compared to a weekly basis if it is given by painless subcutaneous injection.

Depot preparations (i.e., long-acting-release octreotide acetate - commercially available as Sandostatin® LAR®) allow for injections to be given every 14 days. These preparations can maintain effective octreotide acetate levels. Reports suggest that 80% of those patients who have been followed up for 9 years whilst on treatment with octreotide acetate have growth hormone levels of less than 2.5 μg/litre and IGF-I levels that are normal. Eugonadism is restored in two thirds of the patients with acromegaly who have hypogonadism (S. Farooqi et al. (1999) Pituitary, Vol. 2, pp. 79-88; A.N. Paisley and PJ. Trainer, (2003) Current opinion in Pharmacology, Vol. 3, pp. 672-677; S. Melmed. (2006) New England Journal of Medicine, Vol. 355, pp. 2558-2573).

Determinants of the efficacy of octreotide acetate include levels of growth hormone before treatment, presence or absence of abundant tumour SST2 and SST5 expression, drug dose, biochemical criteria used to assess status, and adherence to treatment by patients. Although shrinkage of the tumour mass occurs in 50% of patients, it reverses if treatment with octreotide acetate is discontinued.

In addition, surgical debulking of macro-adenomas that are not amenable to total surgical resection enhances the efficacy of subsequent octreotide acetate treatment. More than 80% of patients who receive the drug report an improvement in symptoms, including headache and peripheral soft-tissue swelling. Tumour shrinkage and biochemical control do not necessarily occur in parallel. Octreotide acetate is therefore often administered after surgery that has failed to alter biochemical control of GH and IGF-I levels and after radiation therapy because growth hormone levels remain elevated. Primary medical treatment with octreotide acetate is efficacious and safe. Since equivalent biochemical responses to long-term drug administration can be achieved regardless of whether patients have undergone surgery or irradiation, primary medical treatment can be offered to patients with (i) large extra-sellar tumours who have no evidence of a central compressive effect, (ii) those who are too frail to undergo surgery, and (iii) those who decline surgery.

Octreotide acetate and lanreotide acetate can also be used in the treatment of gut associated carcinoid tumours and of metastatic carcinoid tumours. The overall incidence of gastrointestinal carcinoid has been estimated at 84/million persons per year. Gastrointestinal carcinoid tumours comprise 90% of all carcinoid tumours. Treatment with octreotide acetate improves symptoms and quality of life in 50 - 80% of patients suffering with carcinoid syndrome. In addition, octreotide acetate and lanreotide acetate can halt tumour progression.

Octreotide acetate has also been shown to be therapeutically effective in treating diarrhea in patients with vasoactive intestinal peptide-secreting tumors (VIPomas), and has been used for the treatment of severe, refractory diarrhea from other causes. It is also used in toxicology for the treatment of prolonged recurrent hypoglycemia after sulfonylurea overdose, and has been used in infants with nesidioblastosis to help decrease insulin hypersecretion. In patients with suspected esophageal varices, octreotide acetate can be given to help decrease bleeding. Furthermore, octreotide acetate can be used for treating patients with pain from chronic pancreatitis, in the treatment of thymic neoplasms, and the ocular diseases resulting from diabetes. Lanreotide acetate has the same therapeutic indications as octreotide acetate.

A major practical drawback of all small molecule peptide based medicines that need to be taken regularly for many years, such as octreotide acetate and lanreotide acetate, is the lack of cost-effective, mass production methods for their manufacture as pharmaceutical medicines for global use. At the present time, chemical peptide synthesis is used to manufacture naturally occurring as well as engineered peptides but this approach is very costly. As a result, the clinical use of octreotide acetate in patients is prohibitively expensive. La the case of patients with acromegaly, the cost of treating a single patient in the UK is approximately £16,000/year. When octreotide acetate is used in patients with malignant carcinoid tumours, the cost of treating a single patient in the UK is approximately £32,000/year.

The main obstacle to the industrial preparation of peptides as medicines is economical mass production. In the manufacture of octreotide acetate and lanreotide acetate, a stepwise Fmoc solid-phase chemistry synthesis is required followed by the formation of an intra-molecular disulfide bond. The formation of an intra-molecular disulfide bond to improve the metabolic stability of the peptide by 3 -fold in the presence of a D-Trp can be a major problem. In addition, side-chain protection is also necessary during the chemical synthesis. These technical problems result in a low yield of 14%. Additional pitfalls with the solid-phase synthesis of octreotide acetate include: (i) racemization of the C-terminal Cys residue; (ii) inefficiencies in the assembly of the peptide; (iii) deleterious side reactions; (iv) inefficient disulfide bond formation on- resin; (v) modification of D-Trp during disulfide bond formation; (vi) incomplete and inefficient peptide-resin cleavage by aminolysis, (v) racemization during fragment coupling, and (vi) complicated purification procedures. Furthermore, the cost of chemical peptide based synthesis is considerably increased when (a) an L-Phe has to be replaced by a D-Phe; (b) an L-Trp has to be replaced by a D-Trp; (c) cyclization of the linear peptide is required to create a disulphide bond to increase the stability of the linear peptide. Similar pitfalls exist with the solid-phase synthesis of lanreotide acetate.

Thus, alternative, more economical and more efficient methods of synthesizing a small peptide such as octreotide acetate and lanreotide acetate are needed. In this respect, recombinant DNA methods in bacteria and yeasts are often used. In addition, if glycosylation has been shown to play no role in the biological activity of the protein, there is the considerable cost and manufacturing advantage of carrying out the production of a protein in the commonly used host, E. coli. Many recombinant proteins have been produced in E. coli by this technology.

However, although such systems offer high protein productivity using recombinant DNA technology, the problem for very small naturally occurring peptides is that they have to be produced as part of a larger fusion protein. Thus, the gene for the naturally occurring peptide is joined to that of a larger carrier protein and the fusion protein is then expressed as a single large protein in a host cell such as E. coli. Following synthesis of the protein, the peptide of interest must then be cleaved from the fusion partner. In practice, the major problems with this approach have been that very small peptides are very susceptible to proteolytic degradation (because their very small size prevents them from having a highly ordered tertiary structure) by the enzymes found in the cytoplasm of most microorganisms. Therefore, small peptides are often rapidly degraded within the host cell in which they are produced, and before they can be successfully isolated from other cell cytoplasmic components.

In addition, there is the added problem that many heterologous proteins (i.e., proteins which are not naturally produced in the host cell) have been found to interfere with bacterial/yeast growth due to toxic effects resulting from the production of large amounts of the heterologous protein in the host cell. Furthermore, these foreign peptide products are often unstable in the host cell. This means that a lot of effort is required to stabilize the expression of the peptide when it is expressed as part of a fusion protein in a microorganism. These problems are particularly relevant when the expression of a very short peptide chain is required in a microbial system, even when the peptide sequence of interest is expressed as part of a larger fusion protein.

Even if the production of recombinant peptides using recombinant DNA technology can be achieved on a large scale, and such production can be made economical, the problem of making a cost-effective medicine is compounded by the high costs of isolating and purifying a very small peptide from a bacterial/yeast lysate, and then from the fusion protein. These downstream processing steps for the production of peptides from bacteria or yeast often contribute substantial additional production costs. For example, the initial recovery of the peptide from bacteria or yeast cells can require multiple and distinct processing steps that include cell disruption and lysis, isolation of inclusion bodies (i.e., aggregated and therefore insoluble protein) from the disrupted and lysed cells, dissolving of the isolated inclusion bodies to obtain soluble fusion protein, and fusion protein cleavage followed by separation of the peptide from the carrier protein. It is therefore desirable that all aspects of the production of very small recombinant peptides be improved and optimized in order to make it possible for large scale, cost-effective production to become a reality.

A problem also exists in achieving effective drug delivery for peptide based medicines. One of the critical and inadequately solved issues in the development of controUed-release drug delivery systems for peptides when polymer based matrices are used is the stability of the peptide after it has been incorporated into a biodegradable polymer based matrix. A highly acidic microenvironment (i.e., pH 1.5) is often created inside microspheres containing the peptide by the degradation of the poly(lactic acid) and the poly(lactic-co-glycolic acid) polymers typically used in such microspheres. This is known to be a major source of the instability of the peptides incorporated into these matrices. The peptide's instability is due to the degradation of the poly(lactic acid) and poly(lactic-co-glycolic acid) microspheres which leads to covalent modifications of the incorporated peptides by acylation with the lactic and glycolic acid units produced. Acylation slows the absorption rate of the peptide from its intramuscular depot injection site. Acylation of peptide drugs inside degrading poly(lactic acid) and poly(lactic-co-glycolic acid) microspheres is now regarded by many authorities as the major obstacle that still needs to be overcome for the steady, sustained and successful delivery of bioactive peptide based drugs as new and highly effective pharmaceutical drugs (Werle M. et al., Amino Acids, (2006) Vol. 30; pp. 351-367).

This problem was illustrated in one study by investigating the acylation reaction of octreotide acetate in 3 lactic acid solutions at different concentrations (42.5%, 21.3%, and 8.5%, wt/wt) and pH values (2.25, 1.47, and 1.85, respectively) at 37°C (Na et al., AAPS PharmSciTech., 2003, 4(4): article 72). During these incubations, the acylation products of octreotide acetate were measured in a time-dependent and concentration- dependent manner. In a 42.5% lactic acid solution (pH 2.25), the remaining amount of intact octreotide acetate was only 51% of the starting material after a 30 day incubation. This was the result of increased amounts of biologically inactive acylated octreotide acetate.

Pegylation is an alternative approach to the use of slow-release preparations, which can be used to increase the stability and the half-life of a protein or a peptide. The therapeutically useful effects of pegylating peptides and proteins include better physical and thermal stability, increased circulation half-life, reduced immunogenicity and antigenicity, and decreased toxicity. Pegylated peptides and proteins also show much better stability than native peptides and proteins against exposure to organic solvents. In addition, microspheres of pegylated proteins also exhibit different drug release profiles, with a reduced initial burst of release of the peptide or protein when compared with those of unpegylated peptides and proteins. However, to date, these pegylation based approaches have been limited to protein based drugs with a molecular weight (MWt) of more than 10 kDa. In addition, the focus of stabilization has been primarily to improve the physical stability of the proteins, such as their aggregation and denaturation.

The inventors have now produced polynucleotides encoding novel peptides that do not exist naturally in nature, which can be used to produce the novel peptides. The peptides retain the three dimensional binding site structure of the crucial four amino acids in octreotide acetate. The peptides can be made economically in a host cell, and can be isolated from the host cell in a few cost-effective steps on an industrial scale. The peptides can also undergo a small number of cost-effective additional chemical modifications to substantially enhance their chemical and metabolic stability, thereby substantially increasing their therapeutic efficacy in patients.

In the following description of the invention, reference is made to the accompanying drawings in which:

Figure 1 illustrates the structure of chemically synthesised octreotide acetate. Only the amino acids that are circled (i.e., Phe-(D)Trp-Lys-Thr) bind to the somatostatin cell surface receptor.

Figure 2 shows a molecular model of chemically synthesised octreotide acetate, (molecular weight 1,018 Da) which contains two D-amino acids. The amino acids that are labelled (i.e., Phe-(D)Trp-Lys-Thr) bind to the somatostatin cell surface receptor.

Figure 3 shows a molecular model of biologically synthesised octreotide (containing the L-amino acids Phe-Cys-Phe-Trp-Lys-Thr-Cys-Thr) according to the present invention, superimposed on the molecular model of chemically synthesised octreotide acetate (containing two D-amino acids). The thicker grey line = biologically (all L- amino acids) synthesised octapeptide; the thinner black line = chemically (two D- amino acids) synthesised octreotide acetate.

Figure 4 shows a molecular model of biologically synthesised octreotide according to the present invention, which has then been chemically modified by the insertion of a 3-carbon bridge to which polyethylene glycol is covalently attached.

Figure 5 shows a molecular model of biologically synthesised octreotide according to the present invention, which has then been chemically modified by the insertion of a 3-carbon bridge to which polyethylene glycol is covalently attached. Each chemical component of the pegylated octapeptide is shown: (a) dotted line circle outlines the receptor binding amino acids Phe-Trp-Lys-Thr; (b) dashed line shape outlines the remaining amino acids (i.e., Phe-Cys-disulfide bond-Cys-Thr); (c) continuous line shape outlines polyethylene glycol.

Figure 6 shows a molecular model of biologically synthesised octreotide according to the present invention, which has then been chemically modified by the insertion of a 3-carbon bridge (shown as CCC) across the octapeptide's disulfide bond. Polyethylene glycol can be covalently attached to the 3-carbon bridge.

Figure 7 indicates the flexibility of a biologically synthesised octapeptide of the invention. Using the molecular modelling protocol that has been published (Zloh et al., (2007) Nature Protocols, Vol. 2, pp. 1070-1083), it was shown that the biologically synthesised octapeptide having the amino acid sequence FCFWKTCT exhibits a degree of flexibility that can be monitored by recording the value of the Root Mean Square Deviation (RMSD) of the backbone atoms during a simulation. The range of the RMSD values is between 1 - 4.4 A. This indicates that the octapeptide's flexibility is not obscured by the attachment of polyethylene glycol via a 3-carbon bridge. Therefore, this disulfide site specific pegylated octapeptide will remain sufficiently flexible to be able to interact effectively with its biological target. Figure 8 shows a molecular model of a biologically synthesised octapeptide having the amino acid sequence FCFWKTCT containing L-amino acids according to the present invention, which has then been chemically modified by the insertion of a 3- carbon bridge with polyethylene glycol covalently attached to the 3 -carbon bridge, superimposed on the molecular model of chemically synthesised octreotide acetate containing D-amino acids. The two structures are the lowest energy conformers (i.e., the most stable forms) and they were predicted using the same molecular modelling method as published in Zloh et al., (2007) Nature Protocols, Vol. 2, pp. 1070-1083. The thicker grey line = biologically (all L-amino acids) synthesised octapeptide, the thinner black line = chemically (two D-amino acids) synthesised octreotide acetate.

Figure 9 shows the amino acid sequence and nucleotide sequence used to clone version 1 of the octapeptide plasmid as defined in example 2. The sequences encoding the octapeptide FCFWKTCT are underlined. The sequences encoding the EcoRl and Xhol restriction sites are shown in bold.

Figure 10 illustrates version 1 of a plasmid vector of the invention, and shows the nucleotide sequence alignments of the plasmid octapeptide' s pGex 4.3 clone 5 (containing glutathione transferase linked to a methionine residue followed by the inserted octapeptide encoding sequence), and the pGex 4.3 vector as defined in example 2. Octapeptide* = the octapeptide FCFWKTCT and the cloning sequences. The octapeptide FCFWKTCT coding sequence is underlined. A description of the vector and pGex system is given in the GST-fusion system handbook from GE LifeSciences at http://www4.gelifesciences.com/aptrix/upp00919.nsf/Content/87478CFA7E09E0C7C 1256EB400417E59/$file/l 8115758.pdf .

Figure 11 shows the amino acid sequence and nucleotide sequence used to clone version 2 of the octapeptide plasmid as defined in example 3. This includes a TEV recognition site. X = any amino acid except proline. The octapeptide FCFWKTCT and its coding sequence are underlined. Figure 12 illustrates version 2 of a plasmid vector of the invention. It shows the nucleotide sequence alignments of the plasmid octapeptide's pGex4.3 as defined in example 3 and the octapeptide FCFWKTCT coding sequence with additional TEV cleavage site sequences. The octapeptide FCFWKTCT coding sequence is underlined. TS8v2 contains glutathione transferase linked to a TEV protease recognition sequence followed by the inserted octapeptide encoding sequence.

Figure 13 shows an SDS-PAGE gel of the proteins expressed from the plasmid encoding the SjGST-TM-octapeptide (version 1) as described in Example 2 (Lane 1 : markers; Lane 2 : SjGST-TM-octapeptide as the total bacterial lysate; Lane 3 : SjGST-TM-octapeptide flow through from the glutathione agarose column; Lane 4 : eluted SjGST-TM-octapeptide protein with a MWt of 27 kDa as a single species).

Figure 14 shows an SDS-PAGE gel of the proteins expressed from the plasmid encoding the SjGST-TEV-octapeptide fusion protein (version 2) as described in Example 3. Lane 1: molecular weight marker; Lane 2: total lysate from induced cells; Lane 3: eluted SjGST-TEV-octapeptide fusion protein (MoI Wt = 28.4 kDa)

Figure 15 shows a Matrix Assisted Laser Desorption Ionisation-Time Of Flight Mass Spectroscopy (MALDI-TOF-MS) analysis of chemically synthesized octreotide acetate as defined in example 5. The theoretical mass of chemically synthesised octreotide acetate is 1,018 Da. The experimental mass of chemically synthesised octreotide was found to be 1,019 Da. The theoretical mass of chemically synthesised octreotide Na⁺ is 1,041 Da. The experimental mass of chemically synthesised octreotide Na⁺ was found to be 1,041 Da.

Figure 16 shows a MALDI-TOF-MS analysis of the experimental mass of the SjGST-TM-octapeptide fusion protein version 1 as defined in example 6. The theoretical mass of SjGST is 25,498 Da. The theoretical mass of the octapeptide that is added to the SjGSt is 2,259 Da. This gives a combined theoretical mass of the SjGST-TM-octapeptide version 1 fusion protein of 27,757 Da. The experimental mass of the SjGST-TM-octapeptide was found to be 27,751 Da. The percentage mass error is 0.02%. A percentage mass error of 0.1% between the theoretical mass & experimental mass is acceptable.

Figure 17 shows a MALDI-TOF-MS analysis of the experimental mass of the SjGST protein (version 1) after its cleavage by thrombin as defined in example 7. The purified fusion protein solution was reduced using 100 mM DTT and buffer exchanged by PD-10 gel filtration column to 10 mM sodium phosphate buffer containing 2 mM EDTA, pH 7.8. The solution of the fusion protein was then subjected to thrombin digestion for 24 h at 37 ⁰C in 10 mM sodium phosphate buffer, pH 7.8. To this solution was added 3 mM DTT to prevent the incorrect formation of disulfides. It was then subjected to a MALDI-TOF-MS analysis. The theoretical mass of the SjGST-TM-octapeptide fusion protein version 1 is 27,757 Da. The theoretical mass of the SjGST protein after its cleavage by thrombin is 26,150 Da. The experimental mass of the SjGST protein after its cleavage by thrombin was found to be 26,153 Da. The . percentage mass error is 0.01%. The theoretical mass of the octapeptide after thrombin cleavage is 1,607 Da. The experimental mass of the octapeptide after thrombin cleavage cannot be established from this particular MALDI-TOF spectrum because of its low resolution for small MWt molecules - as shown in Figure 18.

Figure 18 shows a MALDI-TOF-MS analysis of the octapeptide having the sequence FCFWKTCT of the invention after its chemical cleavage by cyanogen bromide at the methionine from the purified SjGST-TM-octapeptide fusion protein version 1 as defined in example 8. The purified protein solution was reduced using 100 mM DTT and buffer exchanged by PD-IO gel filtration column to 10 mM sodium phosphate buffer containing 2 mM EDTA, pH 7.8. The solution was then subjected to cyanogen bromide (i.e., chemical) mediated cleavage of the methionine residue immediately adjacent to the octapeptide for 24 h at 37 ⁰C in 10 mM sodium phosphate buffer, pH 7.8. To the resulting solution was added 3 mM DTT to prevent the incorrect formation of disulfides. It was then subjected to a MALDI-TOF analysis. The theoretical mass of the octapeptide is 1,035 Da. The experimental mass of the biologically synthesised octapeptide after its chemical cleavage by methionine was found to be 1,034 Da. The percentage mass error is 0.09%. The difference in the experimental mass between chemically synthesised octreotide acetate and the biologically synthesised octapeptide FCFWKTCT is 16 Da. This is because octreotide acetate is chemically modified to have a L-threoninol as its C-terminal residue. In the case of the biological octapeptide FCFWKTCT, the naturally occurring amino acid L-threonine is present as the C- terminal residue.

Figure 19 shows the autoinduction of the SjGST-TEV-octapeptide fusion protein (version 2) in E. coli with lactose, and its purification from inclusion bodies as described in example 9. Lane 1: MWt markers; Lane 2: total lysate from the E. coli induced cells; Lane 3: supernatant from the E. coli cell lysate after its sonication; Lane 4: supernatant from the cell pellet after its treatment with 2 M urea and 100 mM Tris pH 12.5; Lane 5: supernatant from the cell pellet after its treatment with 2 M urea and 100 mM Tris pH 12.5 followed by the adjustment of the pH to 8 with HCl; Lane 6: Pellet remaining from the E. coli cells that were lysed in 2 M urea and 100 mM Tris pH 12.5 followed by the adjustment of the pH to 8 with HCl. In each lane, the protein band seen at 17 kDa is the lysozyme that was added to digest the cells.

Figure 20 shows the three carbon bridge pegylation of chemically synthesised octreotide acetate as described in example 10. For comparison, the MALDI-TOF-MS spectrum of octreotide acetate is shown in Figure 15. In Figure 20, the chemically synthesised octreotide acetate is shown covalently linked via a three carbon bridge across the peptide's disulfide bridge to a 5 kDa polyethylene glycol. The theoretical mass of the pegylated octreotide acetate is 6,092 Da (i.e., 5,073 for the polyethylene glycol + 1,019 for the octreotide acetate). The experimental MWt of the polyethylene glycol is 5,073 Da. The experimental MWt of the pegylated octreotide acetate is 6,097

Da. The percentage mass error is 0.08%.

Figure 21 demonstrates the modification of the SjGST-TM-octapeptide fusion protein (version 1) by the chemical insertion of a three carbon bridge to which polyethylene glycol was covalently attached using SDS-PAGE as described in example 11. Lane 1 :

MWt markers; Lane 2: Purified SjGST-TM-octapeptide (version 1) with a theoretical MWt of 27,757 Da; Lane 3: Disulfide site-specific bridging reaction of 5 Da polyethylene glycol with SjGST-TM-OCT for 72 h at 4 ⁰C followed by digestion with thrombin for 24 h at 37 ⁰C. The SjGST-TM-octapeptide protein band at 27.7 kDa has disappeared and a mono-pegylated protein band (i.e., monopegylation of the peptide's disulfide bond), a di-pegylated protein band (i.e., rnono-pegylation of the peptide's disulfide bond and mono-pegylation of a disulfide bond in GST), and a tri-pegylated protein band (i.e., mono-pegylation of the peptide's disulfide bond and di-pegylation of the two disulfide bonds in GST) have appeared; Lane 4: Disulfide site-specific bridging of 5 kDa polyethylene glycol reagent (3 eq. cone.) in the reaction buffer.

Figure 22 demonstrates the modification of the SjGST-TM-octapeptide (version 1) fusion protein by the chemical insertion of a three carbon bridge to which polyethylene glycol has been covalently attached using MALDI-TOF-MS as described in example 12. The figure shows the MALDI-TOF-MS of thrombin digested disulfide site-specific pegylated SjGST-TM-octapeptide (version 1) protein solution. The theoretical mass of the SjGST-TM-octapeptide (version 1) fusion protein is 27,757 Da. The theoretical mass of the large fragment of the thrombin cleaved SjGST is 26,150 Da. The theoretical small cleavage product with thrombin is a fragment of 1,607 Da. This means that the theoretical mass of the polyethylene glycol (5,073 Da) linked to the octapeptide (1,607 Da) = 6,680 Da. The experimental mass of the polyethylene glycol linked to the octapeptide is 6,692 Da. The percentage mass error is 0.1%.

Figure 23 shows the effect of three examples of a peptide of the invention (in comparison with chemically synthesized octreotide acetate) in reducing serum growth hormone (GH) levels in BALB/C mice. Figure 23A shows mouse GH levels (ng/ml). Figure 23B shows the percentage supression of GH compared to chemically synthesized octreotide acetate.

In a first aspect, the invention provides an isolated peptide consisting of the amino acid sequence X₁CX₂WKX₃CT, wherein Xi is F or A, X₂ is F or Y and X₃ is T or V in any combination or permutation, and wherein each of the amino acids is in the L- configuration and the peptide contains a disulphide bond between the two cysteine residues. Thus, the peptide of the invention may have any one of the following amino acid sequences: FCFWKTCT, FCFWKVCT, FCYWKTCT, FCYWKVCT, ACFWKTCT, ACFWKVCT, ACYWKTCT or ACYWKVCT. Preferably, the peptide of the invention consists of the amino acid sequence FCFWKTCT or ACYWKVCT.

Amino acid sequences are defined herein using the standard single letter or three letter amino acid codes, wherein G = Glycine (GIy), P = Proline (Pro), A = Alanine (Ala), V = Valine (VaI), L = Leucine (Leu), I = Isoleucine (He), M = Methionine (Met), C = Cysteine (Cys), F = Phenylalanine (Phe), Y = Tyrosine (Tyr), W = Tryptophan (Trp), H = Histidine (His), K = Lysine (Lys), R = Arginine (Arg), Q = Glutamine (GIn), N = Asparagine (Asn), E = Glutamic Acid (GIu), D = Aspartic Acid (Asp), S = Serine (Ser) and T = Threonine (Thr).

The DNA codons for each amino acid are shown in Table 1 :-

Table 1

The isolated peptides of the invention, wherein each of the amino acids are present in the L-configuration (as opposed to the D-configuration) and which contain a disulphide bond between the two cysteine residues, retain the same three dimensional binding site structure as that of chemically synthesised octreotide acetate and therefore retain the same activity as chemically synthesised octreotide acetate. Whereas octreotide acetate contains D-Phel and D-Trp4 residues and lanreotide acetate contains D-AIaI and D-Trp4 residues, the peptide of the present invention consists entirely of L-enantiomers.

The peptides of the invention may comprise terminal chemical modifications. Such modifications may, for example, increase the stability of the peptides, for example, by reducing their susceptibility to degradation by proteases.

Molecular modelling of the peptide FCFWKTCT of the invention shows that its predicted three-dimensional chemical structure does not significantly differ from that of chemically synthesised octreotide acetate. As shown in Figure 3, the relative positions of the amino acids in the peptide FCFWKTCT are located in a similar position to the amino acids of chemically synthesised octreotide acetate. Most importantly, the Phe3, Trp4, Lys5 and Thr6 are in the same relative positions in the peptide of the invention as they are in octreotide acetate. Although the Iysine5 appears to be in a different orientation, its linear nature means that it is very flexible in solution. This means that the difference between the two positions shown in Figure 3 for Iysine5 is not biologically significant. The amino acids that are labelled in Figure 3 are known to be essential for the biological activity of octreotide; they are responsible for the binding of octreotide acetate to its cell surface receptors. Thus, the peptide FCFWKTCT of the invention has the same three dimensional chemical binding site properties as octreotide acetate and the molecular modelling studies predict that it will act in the same way in biological systems. The three dimensional configuration of each of the peptides of the invention is similarly predicted by molecular modelling studies to retain the same three dimensional chemical binding site properties of octreotide acetate or lanreotide acetate.

The peptide of the invention is preferably made by expression of a polynucleotide encoding the peptide in a biological host, thereby relying on a biological organism's protein expression system to produce the peptide. The peptide is therefore preferably biologically synthesised, in contrast to the chemical synthesis methods used to produce octreotide acetate or lanreotide acetate. Biological synthesis of the peptide of the invention can also result in the spontaneous formation of a disulphide bond between the two cysteine residues. Thus, in another aspect, the invention provides a polynucleotide encoding the peptide of the invention. The polynucleotide preferably encodes the eight amino acids of the octapeptide of the invention in isolation from the remaining amino acids of the natural hormone somatostatin. Thus, natural genomic DNA encoding somatostatin is excluded from the scope of the present invention. Any biological host capable of expressing the peptide can be used. Suitable hosts are known in the art, including bacteria, yeast, insect cells and animal cells.

The polynucleotide encoding the peptide of the invention can be DNA or RNA. Thus, where DNA sequences are described herein, it is to be understood that the equivalent RNA sequences can be used. The polynucleotide encoding the peptide FCFWKTCT of the invention contains the core nucleotide sequence 5'-TTYTGYTTYTGGAARACNTGYACN-S' wherein Y = C or T, wherein R = A or G, and wherein N = A, C, T or G, in any combination or permutation. When the polynucleotide is incorporated into a vector for expression in bacteria, the preferred sequence of the polynucleotide is 5'TTC TGT TTT TGG AAA ACC TGT ACC 3' based upon the most common codon usage in bacteria. Examples of 16 alternative nucleotide sequences encoding the peptide FCFWKTCT are shown in Table 2, in which the underlined sequences correspond to the most commonly used bacterial codons. The polynucleotide can include any one of the sequences shown in Table 2, but is not limited to these sequences. When the polynucleotide encoding the peptide FCFWKTCT is expressed in a yeast host cell, preferred codons can be selected according to the most common codon usage in yeast.

Table 2

The polynucleotides encoding any of the peptides ACYWKVCT, FCFWKVCT, FCYWKTCT, FCYWKVCT, ACFWKTCT, ACFWKVCT or ACYWKTCT contain the core sequences shown in the following Tables 3-9, respectively. In the core sequences shown in Tables 3-9, Y = C or T, R = A or G, and N = A, C, T or G, in any combination or permutation. Examples of 16 alternative nucleotide sequences encoding each peptide are shown in Tables 3-9, in which the underlined sequences correspond to the most commonly used bacterial codons. The polynucleotide of the invention can include any one of the sequences shown in Tables 3-9, but is not limited to these sequences. When the polynucleotide is expressed in a yeast host cell, preferred codons can be selected according to the most common codon usage in yeast.

Table 3 (for the peptide ACYWKVCT)

Table 4 (for the peptide FCFWKVCT)

Table 5 (for the peptide FCYWKTCT)

Table 6 (for the peptide FCYWKVCT)

Table 7 (for the peptide ACFWKTCT)

Table 8 (for the peptide ACFWKVCT)

Table 9 (for the peptide ACYWKTCT)

The polynucleotide encoding the peptide of the invention can be incorporated into a nucleotide vector for transfection into a host cell. The vector may be a DNA or an RNA vector. The vector may be a plasmid vector. Preferably, the vector comprises a promoter sequence operably linked to the polynucleotide encoding the peptide of the invention. Suitable nucleotide vectors include commercially-available plasmid vectors such as pGex 4.3 (GE Healthcare), and other representative examples shown in Table 3. Any of the vectors described in Hosfield T et al., (1998) Biotechniques, Vol. 25, pp. 306-309,di Guan et al., (1988), Gene, 67; 21-30 or Makrides (1996), Microbiol Rev., Vol. 60; 512-538 can also be used.

Thus, the invention provides an expression vector encoding a peptide of the invention. Examples of commercially available expression systems that can be used in the present invention are shown in Table 10, but are not limited to these examples.

Table 10

Preferably, the peptide of the invention is expressed as part of a fusion protein in order to simplify its isolation from a host cell. A fusion protein is a single polypeptide comprising the peptide of the invention and one or more carrier proteins. The carrier protein may be any protein capable of being isolated from an expression system in which the fusion protein is produced.

The fusion protein containing the peptide of the invention can include any carrier protein which can be detected and isolated from a host cell. Preferably, the carrier protein is a heterologous protein (i.e., a protein which is not naturally produced in the host cell). The carrier protein can act as a molecular tag, allowing isolation of the peptide of the invention. For example, the fusion protein can contain any commercially available protein tag (as the carrier protein), such as glutathione-S- transferase (GST) (GE Healthcare), poly-histidine tagged proteins (Clontech), maltose binding protein (New England Biolab), biotinylated fusion protein (Promega), calmodulin binding peptide (Stratagene), beta-lactamase (Gelantis) and others known in the art. In addition, any protein that confers an ionic charge on the fusion protein

(e.g., polyarginine which can be incorporated at the C terminus) to enable the fusion protein's purification by ion exchange chromatography followed by digestion with an appropriate enzyme (e.g., carboxypeptidase B for polyarginine) (Fuchs SM & Raines

RT, (2005) Protein Science Vol. 14; pp 1538-1544). Thus, in another aspect, the invention provides a fusion protein comprising the peptide of the invention and one or more carrier proteins. The invention also provides a polynucleotide encoding a fusion protein comprising the peptide of the invention and a carrier protein. The polynucleotide encoding a fusion protein of the invention can be introduced into any of the vectors described herein. The carrier protein- encoding nucleotide sequence may be located, in frame, immediately upstream of the 5' end of the core nucleotide sequence as described above (or any other sequence encoding a peptide of the invention), or may be located immediately downstream of the core sequence. Alternatively, the nucleotide sequence encoding the fusion protein may also encode a spacer region between the peptide and the carrier protein. This spacer region may provide sufficient space between the peptide and the carrier protein to allow correct folding of the carrier protein.

Furthermore, the spacer region may encode one or more cleavage sites (such as one or more enzymatic or chemical cleavage sites) to enable separation of the peptide from the carrier protein after expression of the fusion protein. Examples of enzymatic cleavage sites which can be incorporated into a fusion protein containing the peptide of the invention include protease recognition sequences such as (a) a TEV protease cleavage site (Glu-Asn-Leu-Tyr-Phe-Gln* X wherein X is any amino acid except Proline, and * represents the site of enzymatic cleavage); (b) an enterokinase cleavage site (Asp-Asp-Asp-Asp-Lys*); (c) a factor Xa protease cleavage site ( Ile-Glu/Asp- Gly-Arg*); (d) an Arg-C proteinase cleavage site (X-X-R*X-X-) (wherein X is any amino acid except Proline and which, when present, upstream of the N-terminus of the peptide will generate a -Phe-Cys-Phe-Trp-Lys-Thr-Cys-Thr sequence without any additional N terminal amino acids when it is cleaved); (e) others known in the art such as the cleavage site for the protease from tobacco vein-mottling virus (TVMV) and intein. Suitable examples of chemical cleavage sites include (a) Met for cleavage by cyanogen bromide; (b) Asp for cleavage by formic acid, and (c) other cleavage sites known in the art.

The DNA codons for the recognition sequences of the cleavage proteases described above are shown in Table 11. Table 11

• *where X is any amino acid.

• (Phe) (Cys) - where Phe or Cys are the preferred amino acids. The nucleotide sequence encoding the cleavage site may be positioned immediately 5' of the core nucleotide sequence encoding a peptide of the invention. Thus, the nucleotide sequence GARAAYTTATAYTTYCAR (encoding a TEV protease cleavage site, wherein Y = C or T and wherein R = A or G, in any combination or permutation) may be positioned immediately 5' of the core nucleotide sequence encoding a peptide of the invention. For example, a polynucleotide of the invention encoding the peptide FCFWKTCT and a TEV protease cleavage site may contain the core nucleotide sequence 5'-GARAAYTTATAYTTYCARTTYTGYTTYTGGAARACNTGYACN-S' wherein Y = C or T, wherein R = A or G, and wherein N = A, C, T or G, in any combination or permutation. When the polynucleotide is incorporated into a vector for expression in bacteria, the preferred sequence is

5' GAA AAC CTG TAT TTT CAG TTC TGT TTT TGG AAA ACC TGT ACC 3' based upon the most common codon usage in bacteria. Alternative sequences comprising preferred yeast codons can be used when the protein is expressed in a yeast host. Examples of alternative nucleotide sequences encoding the peptide portion of the fusion protein are shown in Tables 2-9, in which the underlined sequences correspond to the most commonly used bacterial codons. The polynucleotide encoding a fusion protein of the invention can include any one of the sequences shown in Tables 2-9, but is not limited to these sequences.

Suitable expression and purification systems which can be used to produce the fusion protein of the invention include the commercially available pET system (Novagen), the Ni-NTA purification system (Qiagen), the pMAL protein Fusion and Purification System (New England Biolab), the PinPoint™ Xa Protein Purification System (Promega), the CBP Calmodulin-Binding Peptide Affinity Tag System (Stratagene), the INTEIN™ and INTErN-TWIN System (New England Biolab), the EndoproteinAce system (Gelantis), and others (see, for example, Table 10).

Since the first chemical synthesis of octreotide acetate as a conformationally stabilised analogue of a biologically active fragment of somatostatin in 1982, research into alternative somatostatin analogues has focussed solely on alternative chemically synthesised peptide fragments (Weckbecker, G. et al, (2003) Nature Reviews, Vol. 2, pp. 999-1017). The biological production of the peptide of the present invention provides a cheaper method of manufacturing a peptide that (1) undergoes the spontaneous formation of a disulfide bond in host cells such as E.Coli, (2) retains the three dimensional chemical structure of octreotide acetate, (3) solves the problem of octreotide acetate's cell based toxicity (which is due to the presence of D-amino acids, which impair the growth of organisms such as E. CoIi (Meister, (1965) Biochemistry of the amino acids. New York Academic Press)) and (4) allows the production of the peptide in inclusion bodies that are not toxic to E. coli. Therefore, the present invention enables the high yield and low cost biological production of the peptide.

With specific reference to the toxic effects of D-amino acids in living organisms, it has been shown that several D-amino acids (including D-tryptophan) can be toxic to E.coli (Soutourina et al., (2004) J. Biological Chemistry; VoI 279(41): pp 42560 - 42565). This is because although D-amino acids can be found in the living world, the selectivity of ribosomal protein synthetases ensures that D-amino acids are not incorporated into naturally occurring polypeptides. Aminoacyl-tRNA synthetases are responsible for the first step of exclusion of D-amino acids from naturally occurring polypeptides. This is because D-amino acids lead to cellular toxicity by three different mechanisms. The first mechanism for their cellular toxicity is that their incorporation into polypeptides leads to the formation of non-functional proteins. This is reflected in the stereospecificity of cellular protein translational machinery for L-amino acids. The second mechanism for their cellular toxicity was demonstrated in an experiment in which D-amino acids were incorporated into a 12 amino acid based peptide; it was shown to significantly reduce cellular growth and cellular proliferation as measured by the incorporation of tritrurn-labelled thymidine into living cells (Hayry et al., (1995) The FASEB journal, VoI 9; pp 1336 - 1344). The third mechanism for their cellular toxicity is that peroxisomal D-amino acid oxidase (D-AAO) is crucial for the metabolism and effective elimination of some D-amino acids from living organisms. This is because D-AAO metabolises D-amino acids to their corresponding alpha- ketone acids and NH3. In this process, regeneration of the co-enzyme flavine- adenine-dinucleotide from the reduced to the oxidised state generates cytotoxic hydrogen peroxide, which in turn, produces even more aggressive reactive oxygen species. These free radicals cause severe cellular damage (Krug et al., (2007) Am J. Physiol. Renal Physiol. VoI 293; pp F382 - F390).

In another aspect, the present invention provides a method of producing a peptide of the invention, comprising introducing the polynucleotide or vector of the invention into a host cell capable of expressing the polynucleotide or vector, and isolating the peptide from the host cell. The polynucleotide encoding a peptide of the invention or the polynucleotide encoding a fusion protein of the invention can be used. Production of the peptide of the invention in a host cell ensures that each of the amino acids is provided in the L-configuration. Preferably, the polynucleotide or vector contains a nucleotide sequence encoding a fusion protein comprising the peptide of the invention and a carrier protein, as described above. Thus, the step of purifying the peptide of the invention preferably comprises isolating the fusion protein from the host cell, and isolating the peptide from the fusion protein.

The peptide and fusion protein of the invention can be produced and isolated simply and therefore at low cost. The polynucleotide or vector encoding the peptide or fusion protein of the invention can be introduced into a suitable host cell by any means known to the person skilled in the art. Host cell culture conditions can be optimised to produce the highest yield of peptide or fusion protein. For example, derivatives of the lac promoter (tac, pac, rac) are amongst the strongest bacterial promoters, any of which can be used in the vectors of the present invention. They are frequently used for the induced over-expression of foreign genes in E coli. However, their use in industrial fermentation processes is restricted by the high cost of the inducer iso- propyl-beta-D-thiogalactopyranoside (IPTG). Large scale culture systems suitable for use in the methods of the present invention can use lactose as an inducer because it is capable of inducing vectors of the present invention with the same efficiency as IPTG. hi E. coli, lactose can be utilized as an inducer, and as a carbon/energy source even in the presence of glucose (Neubauer P. et al., (1994) FEMS Microbiol Rev. Vol. 14; pp. 99-102; Vasala A. et al., (2005) J. Biotechnol. Vol. 117; pp. 421-431). This low cost approach can be combined with autoinduction using modified media and modified growth conditions (Studier FW., (2005) Protein Expression and Purification Vol. 41; pp. 207-234). An example of high yield, low cost autoinduction of the SjGST-TEV- octapeptide fusion protein (version 2) using E. coli and lactose, followed by the isolation of the fusion protein from the inclusion bodies in E. coli is given in example 9 and shown in Figure 19.

Chemical modification of the peptide or fusion protein can optionally be performed after expression of the peptide or fusion protein, in order to improve stability of the peptide and also to simplify the method of isolating the peptide.

The peptide can then be isolated from the host cell components by methods known in the art. When the peptide is expressed as part of a fusion protein, the peptide can be isolated from the fusion protein by methods known in the art. Of particular note is the isolation of protein from inclusion bodies because this offers considerable manufacturing and economical process advantages. They are:- (a) expression of a high level of a protein that would be toxic to the host cell when present in large quantities as a soluble protein, but is not toxic when present in inclusion bodies; (b) easy isolation of the inclusion bodies from cells due to differences in their size and density from cellular contaminants; (c) reduced degradation of the expressed protein due to resistance to proteolytic attack by cellular proteases; and (d) homogeneity of the protein of interest in inclusion bodies because of fewer contaminants.

Simply washing the inclusion bodies will remove impurities and improve native protein yield further. These advantages have led to the widespread development of recombinant proteins expressed as inclusion bodies in E. coli for the commercial production of proteins. During the recovery of the bioactive protein from the inclusion bodies, it is necessary to ensure adequate solubilization of the protein aggregates and the subsequent refolding of the solubilised protein into a bioactive form. It has been shown that the protein in the inclusion body exists in an intermediate stage of the folding pathway and that it has a considerable amount of secondary structure. If protein from inclusion bodies can be solubilised without disturbing its existing native- like secondary structure, the extent of protein aggregation during refolding will be low and the recovery of bioactive protein will be high. Mild solubilization of inclusion body aggregates without generating a random coil protein structure is therefore the key to improved recovery of the bioactive protein. One approach involves a pH shock to the protein aggregates distant from the isoelectric point of the protein. This renders the protein soluble in the presence of very low concentrations of denaturants. Once the inclusion body proteins have been solubilised under such mild conditions, the subsequent refolding of the purified protein is easier, and results in a higher recovery of the bioactive protein. In this context, it has been shown that 2 M urea does not unfold a protein's structure and it also preserves the native secondary structure of the protein (Rathore A.S. et al., (2003) Biotechnology Progress Vol. 19; pp. 1541-1546; Lee Y.S. et al., (2003), Biotechnology & Applied Biochemistry Vol. 38; pp. 9-13; DeNardo, SJ. et al., (2003) Clinical Cancer Research Vol. 9; pp. 3854s- 3864s; Pezza J.A. et al., (2004) Chemical Communications pp. 2412-2413; Singh S.R. & Panda AK. (2005) Journal of Bioscience and Bioengineering. Vol. 99; pp. 303-310; Rajan R.S. et al., (2006) Protein Science Vol. 15; pp. 1063-1075; Wang F. et al., (2008) Nuclear Medicine and Biology Vol. 35; pp. 665-671). Thus, the method of producing the peptide of the invention can comprise isolating the peptide of the invention or fusion protein of the invention from inclusion bodies in a host cell.

In addition, the problem of protein aggregation during the traditional solution phase refolding process can be avoided by using a solid phase refolding method that is integrated with expanded bed adsorption chromatography. The result is the correct refolding in high yield of the protein in the inclusion bodies from the cell homogenate (Cho T.H. et al., (2002) Bioseparation Vol. 10, pp. 189-196). This has been demonstrated for human epidermal growth factor produced in E. coli. The expanded bed adsorption chromatography simultaneously captured the protein by cation exchange and it removed the cellular biomass from the diluted culture broth. It could also be carried out at high throughput, resulted in a high yield (>90%), and gave a purification factor of 20-fold to more than 80% purity. This protein purification process was efficient and can be implemented for large-scale and cost-effective manufacturing, for example, of the peptide of the present invention (Lee Y.S. et al., (2003), Biotechnology & Applied Biochemistry Vol. 38; pp. 9-13). After isolation of the fusion protein and/or the chemically modified fusion protein, it can be cleaved via an enzymatic or chemical cleavage site. For example, site-specific cleavage of a fusion protein containing a TEV enzymatic cleavage site can be achieved with a histidine tagged TEV enzyme. The peptide can be purified by removing the histidine tagged TEV protease by passage through a nickel agarose column. Regeneration of the glutathione agarose and the nickel agarose columns enables them to be reused. The full details of the individual steps for each method are well known to the person skilled in the art (Structural Genomics Consortium et al., (2008) Nature Methods, Vol. 5(2), pp. 135-146).

As stated above, the peptide of the invention can be chemically modified to improve its stability, thereby prolonging the peptide's half life and improving its therapeutic efficacy. Such modifications render the peptide particularly suitable as a cost- effective therapeutic product, which can be used as a long-term treatment that is suitable for use over the course of one or more years. The improved efficacy of the modified peptide of the invention renders it particularly suitable for administration to patients over longer time intervals than current dosage regimens for the administration of octreotide acetate or lanreotide acetate allow.

For example, the peptide and/or fusion protein can be pegylated, such as by the method of disulfide site-specific pegylation as described in detail in the following papers: Shaunak et al., (2006) Nature Chemical Biology. Vol. 2, pp. 312-313; Zloh et al., (2006) Physical Chemistry., Vol. F-3-O, pp. 347-349; Brocchini et al., (2006) Nature Protocols, Vol. 1(5), pp. 2241-2252 ; Balan et al., (2007) Bioconjugate Chemistry, Vol. 18, pp. 61-76; Godwin et al., (2007) Theoretical Chemical Accounts, Vol. 117, pp. 259-265; Zloh et al., (2007) Nature Protocols, Vol. 2, pp. 1070-1083 ; Brocchini et al., (2008), Advanced Drug Delivery Reviews, Vol. 60(1), pp. 3-12. The size of the polyethylene glycol can be from 5 IcDa to 40 kDa. The preferred size is 20 IcDa to 30 kDa. Small proteins and peptides tend to have more disulfides than large proteins because the former need to compensate for their relatively low number of hydrophobic interactions. As a solvent accessible disulfide is usually present in most proteins, it is possible to chemically reduce this disulfide to its two free cysteine sulfur atoms, and still maintain the protein's or peptide's tertiary structure. Pegylation can then be accomplished via bis-alkylation to reconnect the two cysteine sulfur atoms via a 3- carbon bridge. The advantage of this approach is that the selective and efficient addition chemistry of thiols can be exploited without the need to recombinantly engineer the protein to introduce a free cysteine (which increases the insolubility of a protein or peptide). More specifically, this methodological approach exploits the chemical reactivity of both of the sulfur atoms in all naturally occurring disulfide bonds.

The preliminary step of inserting a three-carbon bridge between the two sulphur atoms of the two cysteine residues creates a modified peptide which has (a) the particular advantage of enhanced stability; and (b) provides the potential for the further, subsequent modification of the three-carbon bridge.

Thus, the invention also provides an isolated peptide consisting of the amino acid sequence X₁CX₂WKX₃CT, wherein X₁ is F or A, X₂ is F or Y and X₃ is T or V in any combination or permutation, and wherein each of the amino acids is in the L- confϊguration and wherein the two sulphur atoms of the two cysteine residues are linked via three carbon atoms. Thus, the peptide may have any one of the following amino acid sequences: FCFWKTCT, FCFWKVCT, FCYWKTCT, FCYWKVCT, ACFWKTCT, ACFWKVCT, ACYWKTCT or ACYWKVCT. Preferably, the peptide consists of the amino acid sequence FCFWKTCT or ACYWKVCT.

Accordingly, a stabilizing interaction between the two cysteine residues in the peptide of the invention exists. A further stabilizing interaction can be, for example, the modification of a disulphide bond by the insertion of a three-carbon bridge.

Molecular modeling studies carried out by the present inventors show that the peptide FCFWKTCT containing three carbon atoms between the two sulphur atoms of the two cysteine residues retains an extremely similar three dimensional conformation to chemically synthesized octreotide acetate (see Figure 6). Therefore, such a peptide can also be used in the same applications (particularly medical applications) as described herein for the unmodified, biologically synthesized peptide of the invention. The three dimensional configuration of each of the peptides of the invention containing three carbon atoms between the two sulphur atoms of the two cysteine residues is similarly predicted by molecular modeling studies to retain the same three dimensional chemical binding site properties of octreotide acetate or lanreotide acetate. Such peptides therefore retain the same activity as octreotide acetate or lanreotide acetate.

A hydrophilic polymer such as a polyethylene glycol molecule can be covalently attached to the three-carbon bridge. Preferably, the polyethylene glycol molecule has a molecular weight of between 5 kDa and 50 kDa, more preferably has a molecular weight between 10 kDa and 40 kDa and most preferably has a molecular weight between 20 kDa and 30 kDa. Alternatively, hydrophilic polymers other than polyethylene glycol that are known to the person skilled in the art can be covalently attached to the three-carbon bridge.

The pegylation reagents preferably have a substituted propenyl group as the conjugating moiety on the end of the polyethylene glycol reagent. This conjugation moiety may comprise an electron withdrawing group (e.g., carbonyl), an alpha, beta- unsaturated double bond, and an alpha beta sulfonyl group that is prone to elimination as sulfmic acid. The electron-withdrawing group promotes thiol addition and lowers the pKa of the alpha-proton so that the elimination reaction can proceed. This juxtaposition of chemical functionality results in a latently cross-conjugated system. The conjugated double bond in the polyethylene glycol mono-sulfone initiates a sequence of interactive and sequential addition-elimination reactions. The addition of the first thiolate allows the elimination of a sulfmic acid derivative. This generates another conjugated double bond at the alpha, beta'-position for the addition of a second thiolate. If the two thiols are derived from a protein disulfide bond, a 3-carbon bridge is formed between the cysteine sulfur atoms of the original disulfide. This new 3 -carbon disulfide bridge is even more stable to chemical and metabolic degradation, and proteolytic degradation than the original biologically synthesized disulfide bond.

The methods of preparing a pegylated peptide of the invention can be performed in a few steps, at low cost. A fusion protein comprising the peptide of the invention can be produced and subsequently pegylated as described above. The fusion protein can then be purified away from unreacted polyethylene glycol by passing the solution through an ion exchange column or a size exclusion column or an affinity column such as a glutathione agarose affinity purification column for GST-fusion proteins. In the latter case, the GST fusion protein becomes bound to the glutathione agarose column. Endotoxin can then be removed by washing the column (for example, with 50 column volumes of PBS containing 0.1% Triton X-114 followed by 20 column volumes of PBS). Endotoxin elutes in the wash buffer. Preferably all of these procedures are performed at 4⁰C.

Pegylation of the peptide of the invention preferably results in the formation of a mono-pegylated peptide, compared to the mixtures of different pegylated forms of chemically synthesized octreotide acetate that could be synthesized. Pegylated proteins can be separated from the smaller non-pegylated peptide by size exclusion chromatography.

Previously published work has shown that molecular modelling studies can be effectively combined with chemistry based experiments to reliably predict the results of chemical synthesis and the readouts from biological assays; Shaunak et al., (2006) Nature Chemical Biology. Vol. 2, pp. 312-313; Zloh et al., (2006). Physical Chemistry., Vol. F-3-O, pp. 347-349; Brocchini et al., (2006) Nature Protocols, Vol. 1(5), pp. 2241-2252 ; Balan et al., (2007) Bioconjugate Chemistry, Vol. 18, pp. 61-76; Godwin et al., (2007) Theoretical Chemical Accounts, Vol. 117, pp. 259-265; Zloh et al., (2007) Nature Protocols, Vol. 2, pp. 1070-1083 ; Brocchini et al., (2008), Advanced Drug Delivery Reviews, Vol. 60(1), pp. 3-12. Thus, modelling studies can reliably predict the structural effects of inserting a 3-carbon bridge into the accessible disulfide bonds of a protein or peptide. The published protocol has been developed to interrogate protein databases (e.g., Protein Data Bank, PDB - www.pdb.orgj and to find proteins with at least one disulfide bond that is close to the surface of the protein, and which can be chemically modified. Additionally, it can be used to computationally predict whether the insertion of a 3 -carbon bridge will lead to the loss of the protein's tertiary structure. Using this approach, it is possible to accurately determine the changes to the protein's biologically active surface after the insertion of a 3-carbon bridge, and to determine the effect on the protein's biological activity of linking polyethylene glycol to the 3-carbon bridge. The molecular modelling studies described herein were undertaken using the integrated molecular modelling packages Maestro v6.5 and Macromodel v9.1.

It has previously been shown that when polyethylene glycols are in solution, they stabilize the native compact state of human albumin because of their negative preferential interaction with albumin. As the interaction between the polyethylene glycol molecule and albumin is thermodynamically unfavourable (Farruggia et al., (1999), Int. J. Biol. MacromoL, Vol. 26, pp. 23-33), the polyethylene glycol will not obscure the biologically active surface of a protein or a peptide. This has been confirmed computationally; polyethylene glycol folds independently of the peptide of the present invention. The polyethylene glycol molecule does not "wrap" itself around a protein (Zloh et al., (2006) Proceedings of 6^th European Conference on Computational Chemistry, Slovakia). However, due to the significant size difference between the peptide of the present invention and polyethylene glycol, the person skilled in the art would have expected polyethylene glycol to engulf the peptide, thereby impeding the peptide's biological activity. The inventors have surprisingly shown that pegylation of the peptide of the present invention does not impede the peptide's biological activity.

The pegylation process is usually performed with purified proteins. This requires additional multi-step purification processes to harvest the desired pegylated protein. It has been shown that there are benefits for the bioprocessing of a manufactured product if in vitro protein refolding and pegylation can be integrated, especially for inclusion body proteins. Integrating pegylation with a protein renaturation process in order to obtain a bioactive and a pegylated protein directly from an inclusion body significantly improves downstream processing performance. This has been demonstrated for pegylated lipase (Kim M.Y. et al., (2007) Journal of Biotechnology Vol. 131, pp. 177-179; Choi W.C. et al., (2005) Process Biochem. Vol. 40, pp. 1967- 1972). The lipase was denatured with urea and DTT and then modified with polyethylene glycol. The conjugated polyethylene glycol molecules did not hinder the refolding of lipase. It was therefore possible to integrate the pegylation and the protein refolding processes into a single process step. In other words, solubilized protein from inclusion bodies can be pegylated and refolded in a single processing operation. The same methods can be applied in the present invention. Thus, the method of producing a pegylated peptide of the invention can comprise the isolation of a peptide or a fusion protein of the invention from inclusion bodies in a host cell, followed by the combined pegylation and refolding of the pegylated peptide. Covalent attachment of polyethylene glycol to the peptide or a fusion protein of the invention has the additional advantage of increasing the solubility of the peptide or fusion protein considerably and thereby reducing protein aggregate formation (Raj an RS., (2006) Protein Science VoI: 15; pp. 1063-1075). The insolubility of octreotide acetate and lanreotide acetate has been a major product manufacturing problem for industry.

In another aspect, the present invention provides a peptide or a pegylated peptide of the invention, for use in medicine. The peptide or a pegylated peptide of the invention can be used in the treatment of hormonal disorders, and particularly hormonal disorders resulting from an overexpression of growth hormone. Examples of hormonal disorders that can be treated using the peptide or pegylated peptide of the invention include acromegaly and gigantism (S. Farooqi et al., (1999) Pituitary, Vol. 2, pp. 79-88; A.N. Paisley and PJ. Trainer, (2003) Current opinion in Pharmacology, Vol. 3, pp. 672-677; S. Melmed, (2006) New England Journal of Medicine, Vol. 355, pp. 2558-2573).

In addition, the peptide or a pegylated peptide of the invention can be used in the treatment of cancer, and in particular, in the treatment of gut hormone secreting cancers such as the gut associated carcinoid tumours and metastatic carcinoid tumours.

The peptide or a pegylated peptide of the invention can also be used in the treatment of any disease or condition for which octreotide acetate or lanreotide acetate has been shown to be therapeutically effective. For example, the peptide or a pegylated peptide of the invention can be used in the treatment of diarrhea in patients with vasoactive intestinal peptide-secreting tumors (VIPomas) and in the treatment of severe, refractory diarrhea from other causes. The peptide or a pegylated peptide of the invention can also be used in the treatment of prolonged recurrent hypoglycemia after sulfonylurea overdose, to help decrease insulin hypersecretion in infants with nesidioblastosis, and to help decrease bleeding in patients with suspected esophageal varices. The peptide or a pegylated peptide of the invention may also be used for treating patients with pain from chronic pancreatitis, and for treating thymic neoplasms, and the ocular diseases resulting from diabetes (S. Farooqi et al., (1999) Pituitary, Vol. 2, pp. 79-88; A.N. Paisley and PJ. Trainer, (2003) Current opinion in Pharmacology, Vol. 3, pp. 672-677; S. Melmed. (2006) New England Journal of Medicine, Vol. 355, pp. 2558-2573).

Thus, the invention provides a peptide or a pegylated peptide of the invention, for use in the treatment of hormonal disorders (such as acromegaly and gigantism); cancer (such as gut associated carcinoid tumours and of metastatic carcinoid tumours.); diarrhea in patients with vasoactive intestinal peptide-secreting tumors; severe refractory diarrhea; prolonged recurrent hypoglycemia after sulfonylurea overdose; insulin hypersecretion in infants with nesidioblastosis; and bleeding in patients with suspected esophageal varices.

Radiolabeled somatostatin analogues are useful diagnostic tools for somatostatin receptor scintigraphy. They are used to detect and localize small SST-expressing tumours with particular utility in pin pointing primary and metastatic endocrine tumours. The toxicities associated with targeted radiotherapy means that a parallel field of development is now investigating the use of radiolabeled somatostatin analogues for treating those patients who have SST-expressing tumours. In the specific indication of endocrine tumours, new compounds bearing energetic isotopes, such as yttrium 90 (OctreoTher®) or lutetium are expected to provide effective therapy of tumours due to the deep penetration of the radiation. The yttrium-labeled compound DOTATOC has also been found to stabilize disease in 60% of patients, with objective responses in 20% of patients (Weckbecker, G. et al., (2003) Nature Reviews, Vol. 2, pp. 999-1017; Susini C & Buscail L, (2006) Ann of Oncology Vol. 17, pp. 1733-1742; Asnacios A et al. (2008) J. Clin. Oncology Vol. 26, pp. 963-970). The invention also provides a peptide or a pegylated peptide of the invention for use as a tumour visualization agent. For example, a detectable label such as a radiolabel can be attached to a peptide of the invention containing a three-carbon bridge between the two sulphur atoms, thereby allowing detection of the labelled peptide at tumour sites where the peptide binds to its receptor. The label can be attached directly to the three-carbon bridge, or can be attached to a polyethylene glycol molecule attached to the three-carbon bridge. Other detectable labels than radiolabels known in the art can also be used.

Furthermore, the invention provides the use of a peptide or a pegylated peptide of the invention in the manufacture of a medicament for the treatment of hormonal disorders (such as acromegaly and gigantism); cancer (such as gut associated carcinoid tumours and of metastatic carcinoid tumours); diarrhea in patients with vasoactive intestinal peptide-secreting tumors; severe refractory diarrhea; prolonged recurrent hypoglycemia after sulfonylurea overdose; insulin hypersecretion in infants with nesidioblastosis; bleeding in patients with suspected esophageal varices; and the ocular diseases resulting from diabetes.

The invention also provides a method of treating a patient suffering from any of the disorders described above, comprising administering a peptide or a pegylated peptide of the invention to the patient.

The invention also provides a pharmaceutical composition comprising a peptide or a pegylated peptide of the invention and a pharmaceutically acceptable carrier or diluent. The peptide of the invention or pegylated form of the peptide may be provided as a pharmaceutically acceptable salt, such as an acetate. The pharmaceutical composition may be in any suitable form, depending upon the desired method of administering it to a patient. It may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms. The pharmaceutical composition may be formulated for the treatment of any of the disorders mentioned above. The suggested therapeutic dosage can be 300 - 600 μg/day or 20 mg per month with a range from 2 - 200 mg/month. It can be administered daily, three times a week, weekly, every two weeks or monthly.

The pharmaceutical composition may be adapted for administration by any appropriate route, for example by the parenteral (including subcutaneous, intramuscular, intravenous or intradermal), oral (including buccal or sublingual), respiratory, rectal, nasal, or transdermal route. The subcutaneous route of administration is preferred. Such compositions may be prepared by any method known in the art of pharmacy, for example by admixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.

Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solution which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation substantially isotonic with the blood or the tissue of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Excipients which may be used for injectable solutions include water, alcohols, polyols, glycerine and vegetable oils, for example. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets. Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous or non-aqueous liquids; or as edible foams or whips; or as emulsions). Suitable excipients for tablets or hard gelatine capsules include lactose, maize starch or derivatives thereof, stearic acid or salts thereof. Suitable excipients for use with soft gelatine capsules include for example vegetable oils, waxes, fats, semisolid, or liquid polyols etc. For the preparation of solutions and syrups, excipients which may be used include for example water, polyols and sugars. For the preparation of suspensions, oils (e.g., vegetable oils) may be used to provide oil-in-water or water in oil suspensions.

Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3(6):318 (1986).

Another formulation employed in the methods of the present invention uses transdermal delivery devices ("patches"). Such transdermal patches may be used to provide continuous or discontinuous infusion of the peptide or pegylated peptide of the present invention in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.

Another formulation employs the use of biodegradable microspheres that allow controlled, sustained release of the peptides and pegylated peptides of this invention. Such formulations can comprise synthetic polymers or copolymers or hydrogel implants. Such formulations allow for injection, inhalation, nasal or oral administration. The construction and use of biodegradable microspheres for the delivery of pharmaceutical agents is well known in the art (e.g., US Patent No. 6, 706,289).

In addition, incorporation of a disulfide site-specific pegylated octapeptide according to the present invention into biodegradable microspheres would result in a highly effective and efficient controlled-release system with a different release pattern - such as reduced initial burst of the octapeptide followed by prolonged, steady and sustained drug release, as well as stability from inactivation of the octapeptide within the degrading microspheres.

For formulations suitable for subcutaneous, intravenous, intramuscular, and the like, suitable pharmaceutical carriers, and techniques for formulation and administration may be prepared by any of the methods well known in the art (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 20^th edition, 2000).

Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas. Pharmaceutical compositions adapted for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable compositions wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient. Pharmaceutical compositions adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurised aerosols, nebulizers or insufflators.

The pharmaceutical compositions may contain preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colourants, odourants, salts, buffers, coating agents or antioxidants. They may also contain therapeutically active agents in addition to the peptide of the present invention. Dosages of the substances of the present invention can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used. The dosage may be repeated as often as appropriate. If side effects develop the amount and/or frequency of the dosage can be reduced, in accordance with normal clinical practice.

In addition, the invention also provides a method of treating a patient suffering from any of the disorders described above, comprising administering a pharmaceutical composition of the invention to the patient.

Preferred features of each aspect of the invention are as for each of the other aspects mutatis mutandis. The prior art documents mentioned herein are incorporated to the fullest extent permitted by law.

Examples:

The present invention will now be described further with reference to the following non-limiting examples.

Example 1: Molecular modelling of the predicted structure of the peptide of the invention.

The molecular modeling protocol that has been published (Zloh et al., (2007) Nature Protocols, Vol. 2, pp. 1070-1083) was used for these studies. The PDB file was imported into Maestro and the disulfide bond was modified by the insertion of a 3- carbon bridge with an appropriate linker and a 10 kDa polyethylene glycol chain. This model was minimized and subjected to 2 picoseconds of stochastic simulation at 300 K using Macromodel software, OPLS-2005 force field, and GB/SA implicit solvent model. The resulting trajectory of structure snapshots was analysed. It indicated that the biologically synthesized octapeptide did not become wrapped by a 10 kDa PEG (Figures 4 and 5). Importantly, this means that the biologically active surface of the biologically synthesized octapeptide is still available to interact with its biological receptor.

The biologically synthesized octapeptide exhibits a degree of flexibility that can be monitored by recording the value of the Root Mean Square Deviation (RMSD) of the backbone atoms during a simulation (Figure 6). The range of the RMSD values is between 1 - 4.4 A (Figure 7). This indicates that the octapeptide's flexibility is not obscured by the attachment of polyethylene glycol via a 3 -carbon bridge. Therefore, this disulfide site-specific pegylated octapeptide remains sufficiently flexible to be able to interact effectively with its biological receptor binding site.

The effect of pegylation on the conformation of the biologically synthesized octapeptide was evaluated in detail using a conformational search for the biologically synthesized octapeptide and the disulfide site-specific pegylated octapeptide. It was found that the lowest energy (i.e., most stable) conformations of these two peptides are similar with RMSD values of 0.58 A for the backbone atoms of the cyclic part of the octapeptide even though the distance between the two sulphur atoms increases from 2.04 A (biologically synthesized octapeptide) to 3.94 A (disulfide site specific pegylated octapeptide). In addition, the orientation of the amino acids that are involved in the interaction of disulfide site specific pegylated octapeptide with its receptor are extremely similar (Figure 8). This indicates that the biologically relevant surface on the octapeptide will still be available for presentation to the biologically relevant receptor when a large polyethylene glycol molecule is attached.

The results of these modelling studies show that the presence of a polyethylene glycol) molecule on the disulfide bond of the biologically synthesized octapeptide will not prevent the octapeptide's interaction with its receptor binding site.

Example 2: Generation of a pGex4.3 plasmid containing the DNA sequence for the biologically synthesised octapeptide (version 1) The first plasmid that was constructed encoded the following components from its N- terminal to its C-terminal:- Vector with Schisostoma japonicum (Sj) glutathione-S- transferase (GST) + thrombin cleavage site (T) + methionine (M) + octapeptide.

A double stranded 63 bp DNA sequence incorporating nucleotides corresponding to the following 8 amino acids was constructed:

(L)Phe-c[(L)Cys-(L)Phe-(L)Trp-(L)Lys-(L)Thr-(L)Cys]-(L)Thr(COOH)

which also contained EcoRl and Xhol restriction enzyme site recognition sequences. This made it compatible with cloning into a pGex 4.3 fusion protein vector. The plasmid was constructed.

The DNA sequence was based upon the most common bacterial amino acid codon usage. Alternative DNA sequences can be used, for example, as discussed above. In addition, at the 5 'end of the octapeptide DNA sequence, a Met sequence was inserted to facilitate amino acid cleavage by cyanogen bromide. At the 3' end of the DNA sequence, there were 2 tandem stop codons.

The 63 base pair octapeptide DNA sequence shown in Figure 9 was generated by hybridising 2 partially overlapping and complementary oligonucleotides (octapeptide forward: 5' gga tec ccg aat tec atg ttc tgt ttt tgg aaa ace tgt ace 3', and octapeptide reverse: 5'gcg gcc get cga gtc eta tta ggt aca ggt ttt cca aaa aca 3'). Two microlitres of 1 raM aliquots of each oligonucleotide were hybridised in a PCR amplification mix and extended by 5 cycles of PCR amplification in a 20 μL PCR reaction mix (Sigma JumpStart mix).

The double stranded oligonucleotide sequence was then precipitated with 2 volumes of ethanol, resuspended in 20 μL of water and digested with EcoRl and Xhol restriction enzymes for 1 h. The cut sequence was then precipitated with ethanol, resuspended in 20 μL of water, and 2 μL ligated into a pGex 4.3 vector (2 μL) previously cut with EcoRl and Xhol restriction enzymes using a Promega rapid ligation kit. Sequence comparisons of the nucleotide vectors are shown in Figure 10. Following ligation, the construct was transformed into JMl 09 competent cells (Promega).

Transformed colonies were checked by PCR amplification for the presence of the insert using the octapeptide primer 5' gga tec ccg aat tec atg ttc tgt ttt tgg aaa ace tgt ace 3' and the vector pGex 4.3 primer 5 'ccg gga get gca tgt gtc aga gg 3'.

Positive colonies were then grown up, plasmid extracted and the DNA sequences checked by sequencing to verify that the insert was in the correct reading frame. E.Coli BL-21 competent cells (Promega) were then transformed with the pGex-TS8 plasmid.

Example 3: Generation of a pGex4.3 plasmid containing the DNA sequence for octapeptide (version 2):

The second plasmid was constructed and it encoded the following components from its N-terminal to its C-terminal:- Vector with Schisostoma japonicum (Sj) glutathione- S-transferase (GST) + thrombin cleavage site (T) + TEV protease cleavage site + octapeptide.

Version 2 of the plasmid contained a TEV cleavage sequence in front of the TTC TGT TTT TGG AAA ACC TGT ACC sequence. It replaced the Met codon as shown in Figure 11. The 78 bp DNA sequence was generated by hybridising 2 partially overlapping and complementary oligonucleotides (octapeptide TEV forward : 5' GGA TCC CCG AAT TCC GAA AAC CTG TAT TTT CAG TTC TGT TTT TGG 3', and octapeptide TEV reverse : 5'GCG GCC GCT CGA GTC CTA TTA GGT ACA GGT TTT CCA AAA ACA GAA 3'). The oligonucleotide extension, restriction enzyme digestion, ligation and cloning was performed as described for version 1 of the plasmid in Example 1.

Sequence comparisons of the nucleotide vectors are shown in Figure 12. Small scale 50 ml cultures of BL21 E. CoIi cells transfected with plasmid Sj-GST-T- TEV-octapeptide were grown in 2 x TY media. The cultures were induced with IPTG (0.1 mM) for 3 h and harvested. The cells were lysed in a lysis buffer containing 1 mg/ml lysozyme, 50 mM Tris pH 8.5, 10% glycerol, 10 mM DTT, 0.5% sodium deoxycholate and a protease inhibitor cocktail (Sigma). The lysate was sonicated three times for 30 sec with a sonicating probe, centrifuged at 16,00Og for 10 min and the clarified supernatant applied to a GST-agarose column. Following extensive washing with PBS, the fusion protein was eluted with 40 mm glutathione, 50 mM Tris pH 8. The fractions selected were analysed on a 4-20% polyacrylamide gel. Lane 1: molecular weight marker; Lane 2: total lysate from induced cells; Lane 3: eluted fusion protein (MoI Wt = 28.4 kDa)

Example 4:Induction of the octapeptide fusion protein and its purification.

Small scale (20 - 200 ml) and medium scale (1 - 5 L) bacterial cultures transfected with plasmid vectors encoding the octapeptide fusion protein (versions 1 and 2 from Examples 1 and 2) were expressed in E. CoIi BL21. The cultures were grown in 2 x TY media and the protein was induced overnight with 0.1 mM IPTG at room temperature (25⁰C). A total of 46 g of E. coli cells were harvested by centrifugation. They were lysed using a cell disrupter. The lysis buffer contained PBS₃ 10 mM DTT, 10 mM EDTA, 5% glycerol and 1% Triton X-100. The soluble proteins were separated by centrifugation. The insoluble proteins in the inclusion bodies were solubilised by adding 2M urea pH 12.5. When the insoluble proteins had solubilised, they were diluted 1 in 5 with water and the pH of the solution was changed to 7.5 with I N HCl.

The solubilized fusion protein was isolated by passing the supernatant through a glutathione agarose affinity purification column. At this stage, the fusion protein becomes bound to the glutathione agarose column. Endotoxin was then removed by washing the column with 50 column volumes of PBS containing 0.1% Triton X-114 followed by 20 column volumes wash with PBS at 4⁰C. Using this approach, the endotoxin eluted in the wash buffer. The histidine tagged TEV enzyme was then removed by passage through a nickel agarose column. Regeneration of the glutathione agarose and the nickel agarose columns enabled both of these reagents to be reused. Full details of each individual step of the method are known to the person skilled in the art (Structural Genomics Consortium et al, (2008) Nature Methods, Vol. 5(2), pp. 135-146).

A sample of the eluted fractions was resolved on a 5 - 20% pre-cast polyacrylamide gel (Invitrogen) and stained with Coomassie blue protein stain as shown in Figure 13 and Figure 14.

Example 5: Determination of the mass of octreotide acetate using MALDI-TOF-

MS.

This experiment was set up to determine the mass of chemically synthesized octreotide acetate using MALDI-TOF-MS. The theoretical mass of chemically synthesised octreotide acetate is 1,018 Da. As shown in Figure 15, the experimental mass of chemically synthesised octreotide acetate was 1,019 Da.

The theoretical mass of chemically synthesised octreotide Na⁺ is 1,041 Da. As shown in Figure 15, the experimental mass of chemically synthesised octreotide Na⁺ was 1,041 Da.

Example 6: Determination of the mass of isolated SjGST-TM-octapeptide fusion protein (version 1) without any bound glutathione (GSH).

This experiment was set up to determine the mass of purified SjGST-TM-octapeptide fusion protein (version 1) without any bound glutathione (GSH). Dithiothreitol (DTT) (3 mM) was included in the solution of SjGST-TM-octapeptide.

The theoretical mass of the SjGST-TM-octapeptide version 1 fusion protein is 27,757 Da. As shown in Figure 16, the experimental mass of the SjGST-TM-octapeptide version 1 was 27,751 Da. The percentage mass error is 0.02%. A percentage mass error of 0.1% between the theoretical mass & experimental mass is acceptable.

This experiment confirmed that there was no mass increase due to bound glutathione (GSH) of the fusion protein. The theoretical mass of SjGST is 25,498 Da. The theoretical mass increase of 2,259 Da occurs when the additional amino acids and the octapeptide are added to SjGST. The theoretical mass of glutathione, reduced form (GSH), is 307 Da. The theoretical mass of methionine is 131 Da.

Example 7: Identifying the presence of the octapeptide in the purified fusion protein (version 1).

This experiment was set up to determine the presence of the octapeptide in the purified fusion protein (version 1). The purified protein solution was reduced using

100 mM DTT and buffer exchanged by PD-10 gel filtration column to 10 mM sodium phosphate buffer containing 2 mM EDTA, pH 7.8. The solution of the fusion protein was then subjected to thrombin digestion for 24 hours at 37⁰C in 10 mM sodium phosphate buffer, pH 7.8. To this solution was added 3 mM DTT (to prevent the incorrect formation of disulfides) and it was then subjected to a MALDI-TOF-MS analysis.

The theoretical mass of the SjGST-TM-octapeptide version 1 fusion protein is 27,757 Da. The theoretical cleavage product with thrombin is a fragment of 1,607 Da. The theoretical mass of the large fragment of the thrombin cleaved SjGST is 26,150 Da.

As shown in Figure 17, the experimental mass of the large fragment of the thrombin cleaved SjGST was 26,153 Da. The percentage mass error is 0.01%. The theoretical mass of the octapeptide after thrombin cleavage is 1,607 Da. The experimental mass of the octapeptide after thrombin cleavage cannot be established from this particular MALDI-TOF spectrum because of its low resolution for small MWt molecules - as described in Example 8. Example 8: Identifying the presence of the octapeptide in the purified SjGST- TM-octapeptide fusion protein (version 1).

This experiment was set up to determine the presence of the octapeptide in the purified SjGST-TM-octapeptide fusion protein (version 1). The purified protein solution was reduced using 100 mM DTT and buffer exchanged by PD-10 gel filtration column to 10 mM sodium phosphate buffer containing 2 mM EDTA, pH 7.8. The solution was then subjected to cyanogen bromide mediated cleavage of the methionine residue immediately adjacent to the octapeptide for 24 hours at 37 °C in 10 mM sodium phosphate buffer, pH 7.8. To the resulting solution was added 3 mM DTT (to prevent the incorrect formation of disulfides) and it was then subjected to a MALDI-TOF-MS analysis.

The theoretical mass of the octapeptide is 1,035 Da. As shown in Figure 18, the experimental mass of the biologically synthesised octapeptide after its chemical cleavage by methionine was found to be 1,034 Da. The percentage mass error is 0.09%. The difference in the experimental mass between chemically synthesised octreotide acetate and biologically synthesised octapeptide is 16 Da. This is because octreotide acetate is chemically modified to have a L-threoninol as its C-terminal residue. In the case of the biological octapeptide, the naturally occurring amino acid L-threonine is present as the C-terminal residue.

Example 9: Autoinduction of the Si GST-TE V-octapep tide fusion protein (version 2) in E. coli with lactose and its purification from inclusion bodies.

This experiment was set up to demonstrate the autoinduction of the SjGST-TEV- octapeptide fusion protein (version 2) in E. coli with lactose, and its simple purification from inclusion bodies. On day 1, a 5 ml culture of the version" 2 plasmid in BL21 DE3 cells was seeded into LB media and grown at 37⁰C for 8 hours. The culture was then diluted 1/100 into fresh LB media (5 ml) and grown overnight. On day 2, the culture was again diluted 1/100 (5 ml) into LB media and grown at 37⁰C for 8 hours. The culture was then diluted 1/100 into a media containing the following:- 25 niM Na₂HPO₄, 25 mM KH₂PO₄, 50 mM NH₄Cl, 2 mM Na₂SO₄, 2 mM MgSO₄, 0.5% glucose, 0.25% aspartate and it was grown overnight at 37⁰C. On day 3, the culture was seeded into 200 ml of media containing the following:- 1% tryptone, 0.5% yeast extract, 0.05% glucose, 0.2% lactose, 0.5% glycerol, 2 mM MgSO₄, 25 mM Na₂HPO₄, 25 mM KH₂PO₄, 50 mM NH₄Cl, 2 mM Na₂SO₄. The cells were then grown for 26 hours at 28⁰C with shaking and harvested.

Cell pellets from a 50 ml culture were then processed to obtain the inclusion bodies. The pellets were lysed in 5 ml of lysis buffer containing 50 mM Tris pH8.5, 10% glycerol, 10 mM DTT and 0.5% deoxycholate. 500 μl of lysozyme (1 mg/ml) was added and the solution incubated for 10 min at room temperature. The lysate was sonicated 5 times for 30 sec on each occasion and then centrifuged at 16,00Og for 10 min. The supernatant was collected and the pellet resuspended in 5 ml of lysis buffer and sonicated for 30 sec and then centrifuged. This process was repeated three to four times and the supernatants collected after each sonication. The remaining pellet was redissolved in 2 M urea and 100 mM Tris, pH 12.5.

Figure 19 shows the autoinduction of the SjGST-TEV-octapeptide fusion protein (version 2) whose MWt is 28.4 kDa in BL21 DE3 E. coli cells using lactose. Lane 1: MWt markers; Lane 2: total lysate from the E. coli induced cells; Lane 3: supernatant from the E. coli cell lysate after its sonication; Lane 4: supernatant from the cell pellet after its treatment with 2 M urea and 100 mM Tris, pH 12.5; Lane 5: supernatant from the cell pellet after its treatment with 2 M urea and 100 mM Tris, pH 12.5 followed by the adjustment of the pH to 8 with 1 M HCl; Lane 6: Pellet remaining from the E. coli cells that were lysed in 2 M urea and 100 mM Tris, pH 12.5 followed by the adjustment of the pH to 8 with 1 M HCl. In each lane, the protein band seen at 17 kDa is the lysozyme that was added to digest the cells.

Example 10: Three carbon bridge pegylation of chemically synthesised octreotide acetate : To a solution of chemically synthesised octreotide acetate (0.25 mg/niL, 0.25 μmol) that was prepared in 50 mM sodium phosphate buffer containing 10 mM EDTA₅ pH 7.8 was added TCEP HCl (70 μg, 0.25 μmol, 14 μl of a 5 mg/mL solution) for 1 hour at room temperature. Polyethylene glycol δw-sulfone (1.35 mg, 0.25 μmol, 1 equivalent) was then added to the reduced octreotide acetate. The reaction solution was gently swirled to mix and left overnight at 4 °C. The solution was then subjected to desalting against deionised water using a PD-10 desalting column. The conjugated fraction (i.e., the third 1 ml fraction) containing the largest amount of the pegylated octreotide acetate was analysed by MALDI-TOF-MS analysis.

For comparison, the MALDI-TOF-MS spectrum of octreotide acetate is shown in Figure 15. In Figure 20, the chemically synthesised octreotide acetate is shown covalently linked via a three carbon bridge across the peptide's disulfide bridge to a 5 kDa polyethylene glycol.

The theoretical mass of the pegylated octreotide acetate is 6,092 Da (i.e., 5,073 Da for the polyethylene glycol + 1,019 Da for the octreotide acetate). The experimental MWt of the polyethylene glycol is 5,073 Da. The experimental MWt of the pegylated octreotide acetate is 6,097 Da. The percentage mass error is 0.08%.

Example 11: Demonstration by SDS-PAGE of the modification of the SjGST- TM-octapeptide fusion protein (version 1) by the chemical insertion of a three carbon bridge to which polyethylene glycol was covalently attached.

To a purified solution of the SjGST-TM-octapeptide (version 1) (0.5 mg/mL, 1 mL) fusion protein in 50 mM sodium phosphate buffer, pH 7.4 was added 100 mM DTT (15.4 mg). The solution was then incubated for 30 min at room temperature followed by buffer exchange using a Sephadex G-25 PD-10 column to 50 mM sodium phosphate buffer pH 7.8 containing 10 mM EDTA. To this solution was added a disulfide site-specific bridging 5 kDa polyethylene glycol (0.29 mg, 5 μmoles, 3 equivalents). The resulting solution was incubated for 72 hours at 4°C. Bovine thrombin (Sigma-Aldrich, 0.1 equivalent to fusion protein) was then added to the mixture and incubated for 24 hours at 37°C. The resulting solution was then analysed by SDS-PAGE and MALDI-TOF-MS.

In Figure 21, Lane 1: MWt markers; Lane 2: Purified SjGST-TM-octapeptide (version 1) with a theoretical MWt of 27,757 Da; Lane 3: Disulfide site-specific bridging PEG

5 kDa reaction with SjGST-TM-OCT for 72 hours at 4 ⁰C followed by digestion with thrombin for 24 hours at 37⁰C. It shows the disappearance of the purified SjGST-TM- octapeptide (version 1) protein band at 27.7 kDa and the appearance of a mono- pegylated protein band (i.e., monopegylation of the peptide's disulfide bond), a di- pegylated protein band (i.e., mono-pegylation of the peptide's disulfide bond and mono-pegylation of a disulfide bond in GST), and a tri-pegylated protein band (i.e., mono-pegylation of the peptide's disulfide bond and di-pegylation of the two disulfide bonds in GST); Lane 4: Disulfide site-specific bridging 5 kDa polyethylene glycol reagent (3 equivalents concentration) in the reaction buffer.

Example 12: Demonstration by MALDI-TOF-MS of the modification of the SjGST-TM-octapeptide fusion protein (version 1) by the chemical insertion of a three carbon bridge to which polyethylene glycol was covalently attached.

The protein solution described in Example 11 was reduced using 100 mM DTT and buffer exchanged using a PD-IO column to 50 mM sodium phosphate buffer, pH 7.8 containing 10 mM EDTA. The solution of the reduced SjGST-TM-octapeptide (version 1) was then subjected to disulfide site-specific pegylation for 24 hours at 4°C. The resultant solution was subjected to thrombin digestion for 72 hours at 37°C and subjected to MALDI-TOF-MS analysis.

Figure 22 shows the MALDI-TOF-MS of thrombin digested disulfide site-specific pegylated SjGST-TM-octapeptide (version l)_protein solution.

The theoretical mass of the SjGST-TM-octapeptide (version 1) fusion protein is 27,757 Da. The theoretical mass of the large fragment of the thrombin cleaved SjGST is 26,150 Da. The theoretical small cleavage product with thrombin is a fragment of 1,607 Da. This means that the theoretical mass of the polyethylene glycol (5,073 Da) linked to the octapeptide (1,607 Da) = 6,680 Da. The experimental mass of the polyethylene glycol linked to the octapeptide is 6,692 Da. The percentage mass error is 0.1%.

Example 13: Effect of the octapeptides on growth hormone levels in vivo.

Three octapeptides (FCFWKTCT and ACYWKVCT and ACYWKTCT) underwent the chemical insertion of a three carbon bridge to which polyethylene glycol was covalently attached using the chemical processes described in detail in the previous examples. The highest grade chemicals available were used together with ultrapure clinical grade water for the buffer (50 mM sodium phosphate buffer containing 10 mM EDTA, pH 7.8) and for the dialysis. All solutions were purged with argon for 30 min before use. High concentrations of the proteins in the reaction mix were avoided to prevent their aggregation. The proteins were reduced using freshly prepared TCEP HCl (5 mg/mL) for 1 h at room temperature in moisture free conditions. The reduced protein was not exposed to heat or direct sunlight. Vigorous shaking of the reduced protein solution was also avoided. The protein to 30 kDa three carbon bridge polyethylene glycol bis-sulfone ratio was 1:1. The conjugation reaction solution was gently swirled to mix and dissolve the 30 kDa three carbon bridge polyethylene glycol bis-sulfone and the solution was then left in the dark for 12 h at 4°C. The solution was then dialysed using a Pierce dialysis chamber with a 5 - 7 kDa MWt cut-off in order to remove any octapeptide that was not covalently attached to the 30 kDa polyethylene glycol. The dialysis water changes of 1 L each were made after 2 h, 4 h and 6 h. Freeze drying was then used to remove water and other volatiles.

The in vivo animal study used male BALB/C mice weighing approximately 20 grams. Each subcutaneous injection consisted of the pegylated octapeptide dissolved in 250 μL of water for injection. The weight of the octapeptide only that was administered in each injection was 125 μg. Each mouse received a total of four injections at 4 day intervals. The injections were given subcutaneously into the pinched skin at the back of the neck. The animals were killed 30 hours after the last injection. Serum was collected for growth hormone measurements using commercially available enzyme immunoassays. No clinical toxicity was seen.

Figure 23 shows the results (n = 4). All three pegylated octapeptides were more potent than chemically synthesised octreotide in reducing serum growth hormone (GH) levels in this animal study. The pegylated FCFWKTCT octapeptide resulted in a 70% greater reduction in serum growth hormone when compared to chemically synthesised octreotide. The pegylated ACYWKVCT octapeptide resulted in a 78% greater reduction in serum growth hormone when compared to chemically synthesised octreotide. The pegylated ACYWKTCT octapeptide resulted in a 94% greater reduction in serum growth hormone when compared to chemically synthesised octreotide.

Claims

Claims:

1. An isolated peptide consisting of the amino acid sequence X₁CX₂WKlX₃CT, wherein X₁ is F or A, X₂ is F or Y and X₃ is T or V in any combination or permutation, and wherein each of the amino acids is in the L-configuration and the peptide contains a disulphide bond between the two cysteine residues.

2. An isolated peptide consisting of the amino acid sequence X₁CX₂WKX₃CT, wherein X₁ is F or A, X₂ is F or Y and X₃ is T or V in any combination or permutation, wherein each of the amino acids is in the L-configuration and wherein the two sulphur atoms of the two cysteine residues are linked via three carbon atoms.

3. The peptide of claim 2, further comprising a hydrophilic polymer covalently attached to one of the three carbon atoms.

4. The peptide of claim 3, wherein the hydrophilic polymer is polyethylene glycol.

5. A fusion protein comprising the peptide of any one of claims 1-4 and one or more carrier proteins.

6. A polynucleotide comprising a first nucleotide sequence encoding the peptide of claim 1.

7. The polynucleotide of claim 6, wherein the first nucleotide sequence is:

5'-TTYTGYTTYTGGAARACNTGYACN-S ', wherein Y = C or T, wherein R = A or G, and wherein N = A, C, T or G, in any combination or permutation.

8. The polynucleotide of claim 6 or claim 7, further comprising a second nucleotide sequence encoding a carrier protein.

9. A vector comprising the polynucleotide of any one of claims 6-8.

10. The vector of claim 9, further comprising a promoter operably linked to the polynucleotide of any one of claims 6-8.

11. A method of producing the peptide of claim 1, comprising introducing the polynucleotide of any one of claims 6-8 or the vector of claim 9 or claim 10 into a host cell capable of expressing the polynucleotide or vector, and isolating the peptide from the host cell.

12. The method of claim 11, wherein the host cell is a bacterial cell or a yeast cell.

13. A method of producing the peptide of claim 2, comprising:

- introducing the polynucleotide of any one of claims 6-8 or the vector of claim 9 or claim 10 into a host cell capable of expressing the polynucleotide or vector;

- isolating the peptide from the host cell; and

- introducing a three-carbon bridge between the two sulphur atoms of the two cysteine residues.

14. The method of claim 13, further comprising the step of covalently attaching a hydrophilic polymer to one of the three carbon atoms.

15. The method of claim 14, wherein the hydrophilic polymer is polytheylene glycol.

16. The peptide of any one of claims 1 -4 for use in medicine.

17. The peptide of claim 16, for use in the treatment of hormonal disorders, cancer, diarrhea in patients with vasoactive intestinal peptide-secreting tumors, severe refractory diarrhea, prolonged recurrent hypoglycemia, insulin hypersecretion in infants with nesidioblastosis, and bleeding in patients with suspected esophageal varices, and ocular diseases resulting from diabetes.

18. A pharmaceutical composition comprising the peptide of any one of claims 1- and a pharmaceutically acceptable carrier.