GB2436328A

GB2436328A - Peptide derived from colostrinin

Info

Publication number: GB2436328A
Application number: GB0605774A
Authority: GB
Inventors: Antono Polanowski; Tadeusz Wilusz; Jerzy A Georgiades; Marian Kruzel
Original assignee: Regen Therapeutics PLC
Current assignee: Tiziana Life Sciences PLC
Priority date: 2006-03-22
Filing date: 2006-03-22
Publication date: 2007-09-26
Also published as: GB2450660A8; GB0819303D0; GB2450660A; WO2007107759A1; GB0605774D0

Abstract

The amino acid sequence of a new peptide RPKHPIKHQ is disclosed. This peptide is particularly useful in reducing the neurotoxocity, and in the treatment of diseases characterised by neuronal damage.

Description

I

PEPTIDE

The present invention relates to a peptide. The invention also relates to therapeutic uses of the peptide and to antibodies derived therefrom.

Parkinson's Disease (PD) is a neurodegenerative disorder resulting from the progressive loss of dopamine containing neurones in the substantia nigra pars compacta, the part of the brain that controls muscle movement (the basal ganglia and the extrapyramidal area). The cause of the primary PD remains unknown, and the mechanisms involved in cell death remain to be fully elucidated. Factors contributing to this progressive loss of neurones include oxidative stress, complex I inhibition and alterations in protein ubiquitination and protein degradation by the 20S proteasome. Secondaiy PD may be caused by disorders such as a stroke, encephalitis, progressive supranuclear palsy, corticobasal degeneration or multiple systems atrophy, also known to damage the dopamine neurons and produce PD conditions. To date the only treatments are symptomatic, based on dopamine replacement therapy. The current treatments do not prevent the continuing loss of dopamine neurones, and eventually treatment-related side effects result in severe disability. As yet there is no therapy that treats the underlying cause of PD.

The neurotoxin 1 -methy]-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces selective loss of nigral dopaminergic neurones through its metabolite I -rnethyl-4-phenylpyridiniurn (MPP+). MPP+ induces a variety of cytotoxic mechanisms including oxidative stress-induced mitochondrial dysfunction and apoptotic cell death. Insights into the mechanism of cell death induced by MPP+ have contributed to the understanding of the processes involved in cel] death in PD, and thus prpvide a tool with which to search for agents that slow the progression of the disease.

Colostrinin, a complex of proline rich polypeptides derived from colostrurn, has potent antioxidant properties and has been shown to reduce 4-hydroxynoneal-mediated (41-INE) damage in PCI2 cells and also had beneficial effects in Alzheimer's disease (AD). In addition, CLN induces maturation and differentiation of murine thymocytes, promotes proliferation of peripheral blood leukocytes, induces immunomodulator cytokines and ameliorates oxidative stress-mediated activation of c-Jun N1i2-terrninal kinases, and p53 induced by 4-hydroxynonenal. It is, therefore, likely that Colostrinin has neuroprotective effects in other neurodegenerative diseases including PD.

The present invention provides a peptide containing or consisting of one of the amino acid sequences RPKHPIKHQ (i.e. which has been found to be neuroprotective in comparison with colostrinin.

This peptide may be provided in substantially isolated and/or purified form from a natural source. Alternatively, it may be formed by a synthetic process.

The invention further includes any peptide which includes the specified amino acid sequence. The invention further comprises any peptide which includes an amino-terminal amino acid sequence corresponding to the specified sequence. Thus, the invention encompasses any peptide having the N-terminal amino acid sequence RPKI-IPIKBQ. For the avoidance of doubt, it is stated that the amino-terminal end is on the left hand side of the sequence, in accordance with the usual convention. It will be appreciated that the specified amino acid sequence may be provided with an inert amino acid sequence on the amino-terminal and/or the carboxy-terminal end thereof. The invention further includes physiologically acceptable active derivatives of the peptide, including salts thereof The peptide can be obtained by a number of techniques. In one embodiment, it is prepared by a conventional technique for peptide synthesis, such as by solid-phase or liquid-phase peptide synthesis. Alternatively, the gene sequence encoding the peptide can be constructed by known techniques, inserted into expression vectors or plasmids, and transfected into suitable microorganisms that will express the DNA translated sequences as the peptide, whereby the peptide can be later extracted from the medium in which the microorganisms are grown. Thus, the invention also embraces a DNA sequence encoding the peptide described above, and a recombinant vector prepared by inserting said DNA in a vector.

The peptide according to the invention is present as a fragment in the protein alpha S I kappa casein (positions 16 to 26 thereof). We have also found that the peptide can be isolated from the natural product colostrum. It can also be isolated from the proline rich peptide complex known as colostrinin (see W098/1 4473, which also describes the nonapeptide VESYVPLFP).

There are a number of prior patent applications disclosing the peptide constituents of colostrinin, in particular W000/75 173 and W002/4621 1. Despite extensive research over the several years, the peptide according to the invention has not previously been identified. The peptide according to the invention can be isolated using the extraction method described in WO 2004/081038.

The peptide according to the invention has a number of therapeutic uses. In particular, it has been found that the peptide according to the invention inhibits the toxicity of the neurotoxin MPP+. Thus, according to the present invention, there is provided the use of the peptide according to the invention to reduce the toxicity of the neurotoxin MPP+ in a cell.

The ability of the peptide according to the invention to reduce the neurotoxicity of MPP-i-leads to a number of therapeutic applications of the peptide. In particular, the peptide according to the invention is useful in the treatment of diseases characlerised by loss of dopamine neurones.

In one advantageous embodiment, the peptide may be used in the treatment of disorders of the central nervous system, particularly chronic disorders of the central nervous system. The disorders of the central nervous system that may be treated include neurological disorders and mental disorders. Examples of neurological disorders that may, with advantage, be treated include dementia, and also disorders thai cause dementia, such as neurodegenerative disorders.

Neurodegenerative disorders include, for example, senile dementia and motor neurone disease; Parkinson's disease is an example of a motor neurone disease that can be treated. Also, amyotrophic lateral sclerosis (ALS), a progressive disease that attacks motor neurons, can be treated. Multiple sclerosis (MS) is another neurodegenerative disease of the central nervous system identifiably by scattered areas of demyelination affecting the brain, spinal cord and optic 1 5 nerves that can be treated. Alzheimer's disease is another example of a neurodegenerative disease that can be treated. Examples of mental disorders that can be treated by the peptide include psychosis and neurosis. For example, the peptide may be used to treat emotional disturbances, especially the emotional disturbances of psychiatric patients in a state of depression. The peptide may also be used as an auxiliary withdrawal treatment for drug addicts, after a period of detoxification, and in persons dependent on stimulants.

In another advantageous embodiment of the invention, the peptide may be used in the treatment of disorders of the immune system, particularly chronic disorders of the immune system that may occur spontaneously in people of advanced age. The peptide can also be used in the treatment of diseases requiring immuno-modulation. The peptide is useful in the treatment of a variety of diseases with an immunological and infectious basis. For example, it can be used to treat chronic diseases with a bacterial and viral aetiology, and to treat acquired immunological deficiencies that have developed, for example, after chemotherapy or radiotherapy of neoplasms.

The peptide may be used for treating chronic bacterial and viral infections requiring non-specific immunostimulati on and imrnunocorrection.

A chronic disorder is a disorder that has persisted, or is expected to persist, for a long time, i.e., at least 3 months and usually at least 6 months.

The peptide may be used for improving the development of the immune system of a new born child. It is a further feature of the invention to use the peptide to correct immunological deficiencies in a child. These uses of the peptide may be particularly applicable to babies or children who have been deprived of colostrum. This may occur, for example, in babies and children who were not breast fed from birth.

The peptide also has diagnostic and research applications. For example, the synthetic peptide, as well as the corresponding antibodies described below, may he used to recognise pathological processes occurring in a host. These processes may be induced by excessive production or inhibition of the peptide or the antibodies. Once the pathological process associated with a particular level of the peptide or the antibodies is known, measuring the production of the peptide and the antibodies in body fluids may be used to determine pathological processes taking place in the host. This may occur, for example, in lactating mothers during various infections or drug treatments.

According to another aspect of the invention, we provide the use of the peptide as a dietary supplement. This dietary supplement is particularly useful for babies, especially premature babies and babies at term, and for young children to correct deficiencies in the development of their immune system or to maintain the nervous system health. The dietary supplement may also be used as a dietary supplement for adults, including senile persons, who have been subjected to chemotherapy, or have suffered from cahexia, or weight loss due to chronic disease.

In an aspect of the invention, we provide a dietary supplement comprising an orally ingestible blend of the peptide in combination with a physiologically acceptable carrier. The dietary supplement may be provided in liquid or solid form; the dietary supplement may suitably be provided in the form of a tablet. The dietary supplement may be provided in the form of a baby food formula. The dietary supplement may include, as an additive, lactoferrin and/or selenium and/or a group of cytokines containing members of the interferon family.

The peptide may be administered prophylactically in order to help to prevent the development of disorders of the central nervous system and the immune system.

The peptide according to the invention may be used to prevent amyloidosis or to promote the dissolution of bet?-amyloid aggregates (plaques), and, therefore, the peptide may be used prohylactically or in the treatment of any disease which is characterised by the development of bet-aniyloid aggregates.

The peptide according to the invention may be administered in a dosage in the range I ng to 10 mg. A dosage unit of about 3 tg is typical. However, the optimum dosage will, of course, depend upon the condition being treated.

The peptide according to the invention may be formulated for administration in any suitable form. Thus, the invention further provides a composition, especially a pharmaceutical composition, which includes the peptide in combinaton with a physiologically acceptable carrier. The peptide may, for example, be formulated for oral, topical, rectal or parenteral administration. More specifically, the peptide may be formulated for administration by injection, or, preferably, in a form suitable for absorption through the mucosa of the oral/nasopharyngeal cavity, the alimentary canal or any other mucosal surface. The peptide may be formulated for administration intravenously, subcutaneously, or intramuscularly. The oral formulations may be provided in a form for swallowing or, preferably, in a form for dissolving in the saliva, whereby the formulation can be absorbed in the mucous membranes of the oral/nasopharyngeal cavity.

The oral formulations may be in the form of a tablet (i.e. fast dissolving tablets) for oral administration, lozenges (i.e. a sweet-like tablet in a form suitable to be retained in the mouth and sucked), or adhesive gels for rubbing into the gum. The peptide may be formulated as an adhesive plaster or patch, which may be applied to the gums. The peptide may also be formulated for application to mucous-membranes of the genito-urinary organs. The topical formulations may be provided in the form of; for example, a cream or a gel. The peptide may also be formulated as a spray for application to the nasopharyngeal or bronchial mucous surface.

The peptide may be incorporated into products like milk, yogurts, milkshake, ice cream, cheese spread and various beverage products, including sport drinks.

In another aspect, the invention provides an antibody for the peptide, and provides compositions containing said antibodies. In particular the invention provides the antibodies in substantially isolated form. The antibodies can be produced by injecting a suitable subject, such as a rabbit, with the peptide (with a suitable adjuvant), then recovering the antibodies from the subject afier allowing time for them to be produced. This technique is described in detail in Example 3. It is possible to test that the correct antibody has been produced by ELISA (enzyme-linked immunosorbent assay) using the synthetic peptide as antigens. The antibodies can be further tested against the natural peptide in Colostrum or Colostrinin as confirmation that the synthetic peptide does correspond to the natural peptide found in Colostrum or Colostrinin. The antibodies have potential uses in therapy, as a diagnostic tool and as a research tool. The antibodies can be produced in accordance with the methods described in example 3 of The invention also encompasses the selective administration of the peptide, at selected times to a patient, and the selective administration of the antibodies for the peptide in order to switch on or off the activity of the peptide at a selected time.

In some applications it may be desirable to provide a pharmaceutical composition which contains the peptide and the antibodies in combination with a physiologically acceptable carrier.

The invention further embraces the use of one or more of the peptide and/or antibodies in the manufactLlre of a medicament for use in any of the therapeutic applications described above.

Example I

Isolation of RPKHPIIKHQ from mammalian colostrum The method involves (i) the extraction of peptides with 60% methanol, directly from raw Colostrum (MOHS) or after conditioning raw colostrum with 0.125 M EDTA, followed by 0.250 M calcium chloride (CaCI2) (ECa), then (ii) precipitation with 50% ammoniurn sulfate.

Figure 1 show the process, which is further explained in WO 2004/08 1038, the contents of which are incorporated herein by reference. The extract and the pellet resulting from the precipitation (step 2) constitutes full range of peptides known as Colostrinin, including RPKHP]KHQ Alternatively, the peptides extracted from colostrum (step 1) can be further concentrated in the presence of methanol using membrane filtration method (i.e. GE Osmonics 1,000 Daltons cut off) and then diafiltered with water.

Example 2

Purification of RPKI-IPIKHQ from Colostrinin.

Colostrinin obtained according to example I is further dissolved in water, and then subjected to molecular sieving on BlO-GEL P-2 in the presence of EDTA to purify the peptide.

Briefly, 100 mg of Colostrinin (i.e. ECa -colostrum conditioned with EDTA, as described above) is dissolved in 5 ml of 50 mM EDTA and applied onto the Bio-Gel P2 column (2x100 ciii) equilibrated with the same solution. One millilitre (irni) fractions are collected and protein profile is plotted upon protein concentration is measured in each fraction. Figure 2 shows Bio-Gel P-2 protein profile. Both HPLC and SDS PAGE analyses confirmed the presence of the peptide RPKI-IPII(HQ in fractions of Bio-Gel P-2 peak "c". Figures 3 and 4 show HPLC profiles of Bio-Gel P-2 peak "a" and "c" respectively. Figure 5 shows SDS PAGE profile of peptides from peak "a" and "c" further resolved by HPLC.

Example 3

Production of synthetic peptide RPKHPJKI-IQ The peptide is synthesized using automated synthesizer (Advanced ChemTech model ACT 396) and a polystyrene resin (Wang resin) that has the last amino acid attached to it thi-oLigh a linker.

Al] the amino acids are protected at the N-terminus with the FMOC group. The coupling reagents and all amino acids were purchased from NOVABIOCHM/EMD Biosciences, Inc., San Diego, USA. The protocol for the production of the peptide involves the following steps: 1 0 1. The resin is treated with 20% piperidine for I x 5mm and then with I x I 0mm.

2. 4 x I mm wash with dimethylformamide 3. 5x amino acid, 5x coupling reagent (diisopropylcarbodiimide), and 5x Hobt. (Couple for I hour) 4. 4 x 1 mm wash with dimethylformamide 1 5 This cycle is repeated for each amino acid. The last cycle involves three additional steps: 5. 2 x 1 0mm with piperidine to remove the final FMOC 6. 4 x 1 mill wash with Dimethylformamide 7. 4 x 5 mm wash with methanol The peptide is then cleaved form the resin with trifluroacetic acid with the following scavengers: 5% H20, 3% ethanedithiol, 2% thioanisole, and I % triisopropylsilane for 2-3 hours. The peptide is then precipitated in ether and washed 5 more times with ether.

The peptide is analyzed by MALD-TOF mass spectrometry and further purified on a BlO-CAD HPLC from ABI.

Example 4

Production of monospecific antibodies The synthetic peptide from example 3, with the following sequence: Arg-Pro-Lys-His-Pro-Ile-Lys-His-Gin (RPKHPIKJ-iQ), is modified by the attachinent of L-cysteine to the N-terminal end and purified to at least >85% purity. This facilitated peptide conjugation with Keyhole Hemolyrnph (KHL) and subsequently annealing. The antigen (200 micrograms in complete Freund's adjuvant) were given subcutanously and intramuscularly in ten different sites of 5-6 month old rabbits. After day 14, 28 and 42 the rabbits were boosted again with the full dose of antigens. Following 10 weeks of immunization protocol the rabbits were bled and serums were used for the lgG purification, using Protein-A matrix (Sigma, St. Louis, MO, USA). The lgG enriched eluates from the Protein-A column were dialyzed against PBS for 24 hours, then aliquoted and frozen for storage.

Example 5

Effect on TH-and MAP-2 positive cells in rat primary mesencephalic structures Primary neuronal cultures were prepared from rat embrionic mesencephalon (14-1 5 days of gestation) as described in the art -see, for example McNaught K. S. P. and Jenner P. (1999) "Altered glial function causes neuronal death and increases neuronal susceptibility to I -methyl-4-phenylpyridinium-and 6-hydroxydopamine-induced toxicity in astrocytic/ventral mesencephalic co-cultures." Journal of Neurochemistry 73, 2469-2476. Briefly, the embryo- 1 5 derived mesencephalon is triturated using Pasteur pipettes to make a cell suspension. The cells are suspended in DMEM (Fl 2 + G1utaMAX, DMEM (85%) containing 10% foetal calf serum and 1% streptomycin and penicillin, Gibco, UK) and plated on glass coverslips placed in 24 well plates at a density of i05 cells. The cultures are grown in a humidified atmosphere of 5% C02/95% air at 37 C.

Colostrinin, and the peptides RPKHPIKHQ and VESYVPLFP (at concentrations of 0.01, 0.1 and I ug/mi) or vehicle (DMEM) was added to the cultures on DIV 4, 48 hours prior to MPP4 administration (DIV 6). Control experiments (no MPP) were performed using the highest concentration (lug/mI) of Colostrinin RPKHPIKHQ and VESYVPLFP. Cells were then fixed with 4% PFA 24 hours following MPP administration (DIV 7).

Cells were immunostained using the following primary antibodies, anti rat tyrosine hydroxylase (TH) raised in rabbit (Pel Freez, USA, lot numbers 08123, 27525), anti rat MAP2 antibody raised in mouse (Sigma, UK, lot numbers 083K485 1) and the anti rat GAD 65/67 antibody raised in rabbit (Sigma, UK, lot numbers 0721(4845). Briefly, cells were washed once with PBS (0.IM, pH 7.4) and incubated with 70% methanol (VWR International) in distilled water and I % hydrogen peroxide (Sigma, UK) for 30 minutes. Cells were washed with PBS and incubated with 20% normal goat serum in PBS (NGS, Sigma, UK) for 1 hour. Cells were then washed with 1% NGS and 0.05 % Triton-X 1 00 in PBS. They were then incubated with the first primary antibody (either anti rat TH or anti rat GAD, 1:500 dilution) overnight. The cells were then washed twice with I % NGS and then incubated for 1 hour with a biotinylated goal anti rabbit antibody (1:200 dilution, Vector Laboratories, UK). Cells were then washed twice with PBS and then incubated with Vector ABC for 1 hour and then washed twice with PBS and then Tris-HCI. The immunostaining was visualised with a DAB peroxidase kit, (Vector Laboratories, UK) and then washed with Tris. This method was then repeated using the second primary antibody, anti MAP2 but cells were not incubated with 70% methanol and a biotinylated goat anti mouse antibody was used instead (1:500 dilution, Vector Laboratories, UK). Cells positive for MAP2 staining were visualised using a Vector VIP peroxidase kit (Vector Laboratories, UK).

Following immunostaining, the cells were prepared for microscopy by being dehydrated through a series of ascending alcohols, cleared in Histoclear and then mounted onto slides using DPX mounting medium.

For the MPP+ concentration-response curve, TI-I-positive cells were counted in the whole microscopic field using a Zeiss microscope, x20 magnification. Ten random areas per covershp were counted to obtain a mean number of cells.

In the second part of the study, cells positively stained for TH and MAP2 (10 random areas per coverslip) were counted.

MPP+ administration resulted in a concentration-dependent decrease in tyrosine hydroxylase (TH) positive cells (Figure 6). A concentration of 2 tM MPP+ that resulted in approximately 50% decrease in TH-positive neurones was chosen for the further studies.

Administration of MPP+ (2.tM) caused a decrease in TH-and MAP2-positive cells (Figure 7). Pre-treatment with Colostrinin (1, 0.1 and 0.01 p.g/rnl) tended to reduce the loss of TH-positive neurones however, this was not statistically significant (one-way ANOVA). It was noted that in one culture treated with I tg/m1 Colostrinin and MPP+. there was a high degree of clumping which appeared to skew the data. In contrast, RPKHPIKHQ (I tg/ml) pre-treatment protected TH-positive neurones against MPP+ treatment (P < 0.05, bonferroni multiple comparison test). Pre-treatment with VESYVPLFP (1, 0.1 and 0.01 tg/m1) did not prevent the MPP+ induced loss of TH-positive cells (one-way ANOVA).

These results show that the peptide RPKHPIKI-IQ has unexpectedly superior properties than either colostrinin or VESYVPLFP in reducing the loss of TH-posilive neurones. This suggests that the peptide RPKHPJKHQ has improved neuroprotective properties relative to colostrinin or to the peptide VESYVPLFP, which is another peptide that can be isolated from colostrinin.

It will be a.ciated that the invention described h&ein may be modified, within the scope of the claims.

Claims

CLAIMS: I. A peptide which either (i) contains the N-terminal amino

acid sequence RPKHPIKI-JQ; or (ii) substantially consists of the amino-terminal amino acid sequence: RPKI-IPJKHQ, excluding alpha si kappa casein.

2. A peptide which substantially consists of the amino acid sequence X-RPKHPH(HQ-Y, wherein X is an amino acid or peptide, Y is an amino acid or polypeptide, X and Y are the same or different, and wherein: (1) the amino acid of X linked to RPKHPIKHQ is not alanine if the amino acid of Y linked to RPKHPII(HQ is glycine; and (2) the amino acid of Y linked to RPKHPIKI-IQ is not glycine, if the amino acid of X linked to RPKHPII(BQ is alanine.

3. A peptide according to claim 2, wherein X and/or Y is a peptide containing two to ten amino acids.

4. A peptide according to claim 3, wherein X and/or Y is a peptide containing two to five amino acids.

5. A peptide which substantially consists of the amino acid sequence: RPKHPIKHQ.

6. A peptide according to any preceding claim, in substantially isolated form.

7. A peptide according to any preceding claim, in substantially purified form.

8. A peptide according to any preceding claim, when obtained by a synthetic process.

9 A peptide according to any preceding claim, for use as a medicarnent.

10. A peptide according to claim 9, for use in the treatment of chronic disorders of the central nervous system.

11. A peptide according to claim 9, for use in the treatment of neurological disorders and/or mental disorders.

12. A peptide according to claim 9, for use in the treatment of dementia and/or neurodegenerative diseases.

13. A peptide according to claim 9, for use in the treatment of Alzheimer's disease and/or motor neurone disease.

14. A peptide according to claim 9, for use in the treatment of psychosis and/or neurosis.

1 0 15. A peptide according to claim 9, for use in the treatment of chronic disorders of the immune system.

16. A peptide according to claim 9, for use in the treatment of diseases with a bacterial and viral aetiology, and/or for use in the treatment of acquired immunological deficiencies.

17. A peptide according to claim 9, for use in the treatment of chronic bacterial and/or viral infections.

1 8. A peptide according to claim 9, for use in the treatment of diseases characterised by the presence of beta-amyloid plaque.

19. The use of a peptide according to any one of claims 1 to 8, in the manufacture of a medicament for the treatment of chronic disorders of the centra] nervous system.

20. The use of a peptide according to any one of claims 1 to 8 in the manufacture of a medicament for the treatment of chronic disorders of the immune system.

21. The use of a peptide according to any one of claims I to 8 in the manufacture of a medicament for reducing the toxicity of the neurotoxin 1 -methyl-4-phenylpyridiniurn, in a cell.

22. A method of treating disorders of the central nervous system and/or of the immune system, comprising administering a therapeutically effective amount of a peptide according to any one of claims I to 8 to a patient.

23. A pharmaceutical composition comprising a peptide according to any one of claims I to 8, in combination with a physiologically acceptable carrier.

24. A pharmaceutical composition according to claim 23, iii a form suitable for injection.

25. A pharmaceutical composition according to claim 23 or 24, in a form suitable for absorption through the mucosa of the oral/nasopharyngea] cavity and/or in a form suitable for absorption in the alimentary canal.

26. A composition according to claim 23 or 24, in the form of a tablet, lozenge, gel, patch or plaster.

27. A composition according to claim 23 or 24, in a form suitable for topical application.

28. The use of a peptide according to any one of claims 1 to 8, as a dietary supplement.

29. The use of a peptide according to any one of claims 1 to 8, as a dietary supplement for babies, small children, adults who have been subjected to chemotherapy and/or adults who have suffered from cahexia, or weight loss due to chronic disease.

30. A dietary supplement comprising an orally ingestible combination of a peptide according to any one of claims I to 8 in combination with a physiologically acceptable carrier.

31. An antibody which binds to a peptide according to any one of claims I or 8.

32. An antibody obtainable by using a peptide according to any one of claims I to 8 as an antigen.