Peptide based PCSK9 vaccine

Abstract

The present invention relates to novel short chain peptides of formula (I) which can be useful as a vaccine when in conjugation with suitable immunogenic carrier and suitable adjuvant. These are useful for the treatment for the PCSK9 mediated diseases.
A-Z.sub.1-Z.sub.2-Z.sub.3-Z.sub.4-Z.sub.5-Z.sub.6-Z.sub.7-Z.sub.8-Z.sub.9-Z.sub.10-Z.sub.11-Z.sub.12-B   Formula (I)

Claims

1. A peptide comprising an amino acid sequence selected from the group consisting of: TABLE-US-00007 [SEQ ID NO: 4] SIPWNLER-Nle-TPC; [SEQ ID NO: 5] SIPWNLER-Aib-TPC; [SEQ ID NO: 6] SIPWNLE-Har-ITPC; [SEQ ID NO: 7] SIPWN-Nle-ERITPC; [SEQ ID NO: 8] SI-Hyp-WNLERITPC; [SEQ ID NO: 10] SIPWN-Aib-ERITPC; [SEQ ID NO: 12] SIPWNLE-Cit-ITPC; [SEQ ID NO: 13] SIPWNLERI-Aib-PC; [SEQ ID NO: 14] Aib-IPWNLERITPC; [SEQ ID NO: 15] S-(N-Me-Ile)-IPWNLERITPC; [SEQ ID NO: 18] SIP-APPA-NLERITPC; and [SEQ ID NO: 19] SIP-Bip(OMe)-NLERITPC.

2. The peptide of claim 1, wherein the amino acid sequence is conjugated with an immunogenic carrier selected from the group consisting of diphtheria toxin (DT), Corynebacterium diphtheriae (CRM) 197, keyhole limpet haemocyanin (KLH), tetanus toxoid (TT) and protein D.

3. The peptide as claimed in claim 1, wherein the amino acid sequence is conjugated with an immunogenic carrier, in combination with one or more pharmaceutically acceptable adjuvants and other pharmaceutical excipients.

4. The peptide as claimed in claim 1, wherein the amino acid sequence is conjugated with an immunogenic carrier and with one or more pharmaceutically acceptable adjuvants, wherein the pharmaceutically acceptable adjuvants are selected from the group of consisting of alum, Toll-like 3 receptors (TLR3) agonist, Poly(I:C), Toll-like 4 receptors (TLR4) agonist, Monophosphoryl Lipid A, Glucopyranosyl Lipid Adjuvant (GLA), Toll-like 5 receptors (TLR5) agonist, Flagellin, Toll-like 7 receptors (TLR7) agonist, Gardiquimod, imiquimod, TLR7/8 agonist, R848, Nucleotide-binding oligomerization domain-containing protein2 (NOD2) agonist, N-glycolyl-muramyl dipeptide (MDP), CpG-containing nucleic acid wherein the cytosine is unmethylated, QS21 (saponin adjuvant), interleukins, and beta-sitosterol.

5. The peptide as claimed in claim 1 suitable for the treatment of health disorders selected from hyperlipidaemia, hypercholesterolemia, atherosclerosis and other cardiovascular diseases.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) In accordance with the present invention, the various novel short-chain peptides having the structural Formula (I), were synthesised and then conjugated with a suitable immunogenic carrier. These vaccines can be used for the treatment of health disorders like hyperlipidemia, hypercholesterolemia, or atherosclerosis.

(2) In one aspect, the present invention thus discloses PCSK9 peptide derivatives having following structure of Formula (I)
A-Z.sub.1-Z.sub.2-Z.sub.3-Z.sub.4-Z.sub.5-Z.sub.6-Z.sub.7-Z.sub.8-Z.sub.9-Z.sub.10-Z.sub.11-Z.sub.12-B   Formula (I)
Wherein,

(3) ‘A’ represents the groups —NH—R.sub.1, R.sub.2—CO—NH—, wherein ‘R.sub.1’ represents hydrogen or optionally substituted linear or branched (C.sub.1-18) alkyl chain; ‘R.sub.2’ is selected from optionally substituted linear or branched (C.sub.1-18) alkyl chain, (C.sub.1-6)alkoxy, (C.sub.3-C.sub.6) cycloalkyl, aryl, heteroaryl or arylalkyl groups;

(4) wherein aryl group is selected from phenyl, naphthyl, indanyl, fluorenyl or biphenyl, groups; the heteroaryl group is selected from pyridyl, thienyl, furyl, imidazolyl, benzofuranyl groups;

(5) ‘B’ represents R.sub.3, —COOR.sub.3, —CONHR.sub.3 or CH.sub.2OR.sub.3, wherein R.sub.3 represents H or suitable amino acids such as serine, cysteine, valine, alpha-methyl-Valine, Lys(Biotin), Lys(alkyl), Lys(acetyl) and the like;

(6) Z.sub.1 is an amino acid residue selected from the group of uncharged amino acid residues, preferably selected from the group of serine, threonine, valine and alanine and their derivatives such as homoserine, O-methyl-threonine, O-methyl-serine, O-methyl-homoserine, etc.

(7) Z.sub.2 is an amino acid residue selected from the group of uncharged amino acid residues, preferably selected from the group of isoleucine, leucine, norleucine, glycine, alanine, Aib and their derivatives such as N-methyl-isoleucine and N-methyl-leucine, etc.

(8) Z.sub.3 is an amino acid residue selected from the group of proline, 1-amino carbocyclic acids and their derivatives such as hydroxyproline, thiaproline, aminoproline, alpha-methyl-proline, 3-fluoroproline, 4-fluoroproline, 1-amino-cyclopropanecarboxylic acid, 1-amino-cyclopentanecarboxylic acid, 1-amino-cyclohexanecarboxylic acid, etc.

(9) Z.sub.4 is an amino acid residue selected from the group preferably of tryptophan, phenylalanine, tyrosine and their derivatives;

(10) Z.sub.5 is an amino acid residue selected from the group of glutamine, histidine, preferably aspargine and their derivatives;

(11) Z.sub.6 is an amino acid residue selected from the group of uncharged amino acid residues, preferably selected from the group of isoleucine, leucine, alanine, threonine, aspartic acid, Aib and their derivatives such as norisoleucine, N-methyl-leucine homoleucine, alpha-methyl-aspartic acid, etc.

(12) Z.sub.7 is an amino acid residue selected from the group of hydrophilic, negatively charged amino acid residue, preferably an amino acid residue selected from the group of glutamic acid, aspartic acid and their derivatives such as alpha-methyl-aspartic acid, alpha-methyl-glutamic acid, homoglutamic acid etc.

(13) Z.sub.8 is an amino acid residue selected from the group of amino acid residues, preferably selected from the group of arginine, homoarginine, citruline, glutamine, aspargine, lysine and their derivatives;

(14) Z.sub.9 is an amino acid residue selected from the group of uncharged amino acid residues, preferably selected from the group of isoleucine, leucine, norleucine, glycine, alanine, Aib and their derivatives such as N-methyl isoleucine, etc.

(15) Z.sub.10 is an amino acid residue selected from the group of polar and uncharged amino acid residues, preferably selected from the group of serine, threonine, valine, alanine and their derivatives;

(16) Z.sub.11 is any amino acid residue, preferably an amino acid residue selected from the group of amino acid residues, preferably selected from the group of proline, and their derivatives such as hydroxyproline, thiaproline, or alpha-methyl-proline, 3-fluoroproline, 4-fluoroproline, 1-amino-cyclopropanecarboxylic acid, 1-amino-cyclopentanecarboxylic acid, 1-amino-cyclohexanecarboxylic acid etc.

(17) Z.sub.12 is any amino acid residue, preferably polar and uncharged amino acid residue selected from the group of cysteine, homocysteine and their derivatives; with the proviso that at least one of Z.sub.1 to Z.sub.12 always represents an unnatural amino acid.

(18) In one of the preferred embodiments, ‘A’ represents the groups —NH—R.sub.1, R.sub.2—CO—NH—, wherein ‘R.sub.1’ is hydrogen or substituted linear or branched (C.sub.1-18) alkyl chain; ‘R.sub.2’ is selected from substituted linear or branched (C.sub.1-18) alkyl chain, (C.sub.1-6) alkoxy, (C.sub.3-C.sub.6) cycloalkyl, aryl, and arylalkyl groups; wherein the aryl group is selected from phenyl, naphthyl, indanyl, fluorenyl or biphenyl, groups; ‘B’ represents R.sub.3, —COOR.sub.3, —CONHR.sub.3 or CH.sub.2OR.sub.3, wherein R.sub.3 is selected from H, serine, cysteine, valine, alpha-methyl-valine, Lys(Biotin), Lys(alkyl), Lys(acetyl), Z.sub.1 is selected from serine, threonine, valine, alanine, Aib, homoserine, O-methyl-threonine, O-methyl-serine or O-methyl-homoserine, Z.sub.2 is selected from isoleucine, leucine, norleucine, glycine, alanine, Aib, N-methyl-isoleucine or N-methyl-leucine, Z.sub.3 is selected from proline, hydroxyproline, thiaproline, aminoproline, 2-thiaproline, 3-hydroxyproline, 3-aminoproline, alpha-methyl-proline, 3-fluoroproline, 4-fluoroproline, 1-amino-cyclopropanecarboxylic acid, 1-amino-cyclopentanecarboxylic acid or 1-amino-cyclohexanecarboxylic acid, Z.sub.4 is selected from tryptophan, phenylalanine, tyrosine, 2-fluorophenylalanine, alpha-methyl phenylalanine, alpha-methyl-2-fluorophenylalanine, alpha-methyl-2,6-difluorophenylalanine, 2-amino-5-phenyl-pentanoic acid, alpha-methyl-2-amino-5-phenyl-pentanoic acid or 2′-ethyl-4′-methoxy-biphenylalanine, Z.sub.5 is selected from glutamine, histidine or aspargine, Z.sub.6 is selected from isoleucine, leucine, alanine, threonine, aspartic acid, Aib, norisoleucine, N-methyl-leucine, homoleucine, beta-alanine or alpha-methyl-aspartic acid, Z.sub.7 is glutamic acid, aspartic acid, alpha-methyl-aspartic acid, alpha-methyl-glutamic acid, 2-amino-4-cyanobutanoic acid or homoglutamic acid, Z.sub.8 is selected from arginine, homoarginine, citruline, glutamine, aspargine or lysine, Z.sub.9 is selected from isoleucine, leucine, norleucine, glycine, alanine, Aib or N-methyl isoleucine, Z.sub.10 is selected from serine, threonine, valine, Aib or alanine, Z.sub.11 is selected from proline, hydroxyproline, thiaproline, aminoproline, alpha-methyl-proline, 3-fluoroproline, 4-fluoroproline, 1-amino-cyclopropanecarboxylic acid, 1-amino-cyclopentanecarboxylic acid or 1-amino-cyclohexanecarboxylic acid, Z.sub.12 is selected from cysteine or Homocysteine.

Definitions

(19) The term ‘natural amino acids’ indicates all those twenty amino acids, which are present in nature. The term ‘unnatural amino acids’ or ‘non-natural amino acids’ preferably represents either replacement of L-amino acids with corresponding D-amino acids such as replacement of L-Ala with D-Ala and the like or suitable modifications of the L or D amino acids, amino alkyl acids, either by α-alkylation such as substitution of Ala with α-methyl Ala (Aib), replacement of Leu with α-methyl Leu; substitution on the side chain of amino acid such as substitution of aromatic amino acid side chain with halogen, (C.sub.1-C.sub.3) alkyl, aryl groups, more specifically the replacement of Phe with halo Phe; β amino acids such as β alanine;

(20) The various groups, radicals and substituents used anywhere in the specification are described in the following paragraphs.

(21) The term “alkyl” used herein, either alone or in combination with other radicals, denotes a linear or branched radical containing one to eighteen carbons, such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, amyl, t-amyl, n-pentyl, n-hexyl, iso-hexyl, heptyl, octyl, decyl, tetradecyl, octadecyl and the like.

(22) The term “cycloalkyl” used herein, either alone or in combination with other radicals, denotes a radical containing three to seven carbons, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like.

(23) Unless otherwise indicated, the term ‘amino acid’ as employed herein alone or as part of another group includes, without limitation, an amino group and a carboxyl group linked to the same carbon, referred to as ‘α’ carbon.

(24) The absolute ‘S’ configuration at the ‘α’ carbon is commonly referred to as the ‘L’ or natural configuration. The ‘R’ configuration at the ‘α’ carbon is commonly referred to as the ‘D’ amino acid. In the case where both the ‘α-substituents’ is equal, such as hydrogen or methyl, the amino acids are Gly or Aib and are not chiral.

(25) The term ‘derivatives’ mentioned anywhere in document indicates any substituted or homologous non-natural amino acid of that particular amino acid.

(26) While the invention has been primarily exemplified in relation to short chain peptides, it will also be understood that the peptide linkage between the residues may be replaced by a non-peptide bond provided that the therapeutic potential is retained. The person skilled in the art will be aware of suitable modifications, such as thioamide bond formation, N-methylation of amide bonds and the like.

(27) Sequences encompassing conservative substitutions of amino acids are also within the scope of the invention, provided that the biological activity is retained.

(28) It is to be clearly understood that the compounds of the invention include peptide amides and non-amides and peptide analogues, including but not limited to the following: a) Compounds in which one or more amino acids is replaced by its corresponding D-amino acid. The skilled person will be aware that retro-inverso amino acid sequences can be synthesized by standard methods; see for example, Chorev M., Acc. Chem. Res., 26, 1993, 266-273; b) Compounds, in which the peptide bond is replaced by a structure more resistant to metabolic degradation. See for example, Olson G. L., et al., J. Med. Chem., 36(21), 1993, 3039-3049 and c) Compounds in which individual amino acids are replaced by analogous structures for example Ala with Aib; Arg with Cit.

(29) Throughout the description the conventional one-letter and three-letter code for natural amino acids are used as well as generally acceptable three-letter codes for other unnatural amino acids such as Hyp (Hydroxyproline), Thz (Thiaproline), Aib (α-amino isobutanoic acid) are used.

(30) Preparation of the Short Chain Peptides:

(31) Several synthetic routes can be employed to prepare the short chain peptides of the present invention well known to one skilled in the art of peptide synthesis. The short chain peptides of formula (I), where all symbols are as defined earlier can be synthesized using the methods described below, together with conventional techniques known to those skilled in the art of peptide synthesis, or variations thereon as appreciated by those skilled in the art. Referred methods include, but not limited to those described below.

(32) The short chain peptides thereof described herein may be produced by chemical synthesis using suitable variations of both the solution-phase (preferably, using Boc-chemistry; M. Bodansky, A. Bodansky, “The practice of peptide synthesis”, Springer-Verlag, Berlim, 1984; E. Gross, J. Meinhofer, “The peptide synthesis, analysis, biology”, Vol. 1, Academic Press, London, 1979) and or solid-phase techniques, such as those described in G. Barany & R. B. Merrifield, “The peptides: Analysis, synthesis, Biology”; Volume 2—“Special methods in peptide synthesis, Part A”, pp. 3-284, E. Gross & J. Meienhofer, Eds., Academic Press, New York, 1980; and in J. M. Stewart and J. D. Young, “Solid-phase peptide synthesis” 2nd Ed., Pierce chemical Co., Rockford, Ill., 1984.

(33) The preferred strategy for preparing the short chain peptides of this invention is based on the use of Fmoc-based SPPS approach, wherein Fmoc (9-fluorenylmethoxycarbonyl) group is used for temporary protection of the α-amino group, in combination with the acid labile protecting groups, such as tert-butoxycarbonyl (Boc), tert-butyl (Bu.sup.t), Trityl (Trt) groups (Figure 1), for temporary protection of the amino acid side chains, if present (see for example E. Atherton & R. C. Sheppard, “The Fluorenylmethoxycarbonyl amino protecting group”, in “The peptides: Analysis, synthesis, Biology”; Volume 9—“Special methods in peptide synthesis, Part C”, pp. 1-38, S. Undenfriend & J. Meienhofer, Eds., Academic Press, San Diego, 1987).

(34) The short chain peptides can be synthesized in a stepwise manner on an insoluble polymer support (resin), starting from the C-terminus of the peptide. In an embodiment, the synthesis is initiated by appending the C-terminal amino acid of the peptide to the resin through formation of an amide, ester or ether linkage. This allows the eventual release of the resulting peptide as a C-terminal amide, carboxylic acid or alcohol, respectively.

(35) In the Fmoc-based SPPS, the C-terminal amino acid and all other amino acids used in the synthesis are required to have their α-amino groups and side chain functionalities (if present) differentially protected (orthogonal protection), such that the α-amino protecting group may be selectively removed during the synthesis, using suitable base such as 20% piperidine solution, without any premature cleavage of peptide from resin or deprotection of side chain protecting groups, usually protected with the acid labile protecting groups.

(36) The coupling of an amino acid is performed by activation of its carboxyl group as an active ester and reaction thereof with unblocked α-amino group of the N-terminal amino acid appended to the resin. After every coupling and deprotection, peptidyl-resin was washed with the excess of solvents, such as DMF, DCM and diethyl ether. The sequence of α-amino group deprotection and coupling is repeated until the desired peptide sequence is assembled (Scheme 1). The peptide is then cleaved from the resin with concomitant deprotection of the side chain functionalities, using an appropriate cleavage mixture, usually in the presence of appropriate scavengers to limit side reactions. The resulting peptide is finally purified by reverse phase HPLC.

(37) The synthesis of the peptidyl-resins required as precursors to the final peptides utilizes commercially available cross-linked polystyrene polymer resins (Novabiochem, San Diego, Calif.). Preferred for use in this invention is Fmoc-PAL-PEG-PS resin, 4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetyl-p-methyl benzhydrylamine resin (Fmoc-Rink amide MBHA resin), 2-chloro-Trityl-chloride resin or p-benzyloxybenzyl alcohol resin (HMP resin) to which the C-terminal amino acid may or may not be already attached. If the C-terminal amino acid is not attached, its attachment may be achieved by HOBt active ester of the Fmoc-protected amino acid formed by its reaction with DIPCDI. In case of 2-Chloro-trityl resin, coupling of first Fmoc-protected amino acid was achieved, using DIPEA. For the assembly of next amino acid, N-terminal protection of peptidyl resin was selectively deprotected using 10-20% piperidine solution. After every coupling and deprotection, excess of amino acids and coupling reagents were removed by washing with DMF, DCM and ether. Coupling of the subsequent amino acids can be accomplished using HOBt or HOAT active esters produced from DIPCDI/HOBt or DIPCDI/HOAT, respectively. In case of some difficult coupling, especially coupling of those amino acids, which are hydrophobic or amino acids with bulky side chain protection; complete coupling can be achieved using a combination of highly efficient coupling agents such as HBTU, PyBOP or TBTU, with additives such as DIPEA.

(38) The synthesis of the short chain peptides described herein can be carried out by using batchwise or continuous flow peptide synthesis apparatus, such as CS-Bio or AAPPTEC peptide synthesizer, utilizing the Fmoc/t-butyl protection strategy. The non-natural non-commercial amino acids present at different position were incorporated into the peptide chain, using one or more methods known in the art. In one approach, Fmoc-protected non-natural amino acid was prepared in solution, using appropriate literature procedures. For example, the Fmoc-protected APPA analogs, described above, were prepared from L-pyroglutamic acid, in good enantiomeric purity, using modified literature procedure (Betsbrugge J. V., et al., Tetrahedron, 54, 1988, 1753-1762).

(39) ##STR00001## ##STR00002## ##STR00003##

(40) The Fmoc-protected α-methylated amino acids were prepared using asymmetric Strecker synthesis (Boesten, W. H. J., et al., Org. Lett., 3(8), 2001, 1121-1124; Cativiela C., Diaz-de-villegas M. D., Tetrahedran Asymmetry, 9, 1988, 3517-3599). The resulting derivative was then used in the step-wise synthesis of the peptide. Alternatively, the required non-natural amino acid was built on the resin directly using synthetic organic chemistry procedures and a linear peptide chain were prepared.

(41) The peptide-resin precursors for their respective short chain peptides may be cleaved and deprotected using suitable variations of any of the standard cleavage procedures described in the literature (King D. S., et al., Int. J. Peptide Protein Res., 1990, 36, 255-266). A preferred method for use in this invention is the use of TFA cleavage mixture, in the presence of water and TIPS as scavengers. Typically, the peptidyl-resin was incubated in TFA/Water/TIPS (95:2.5:2.5) for 1.5-4 hrs at room temperature. The cleaved resin is then filtered off and the TFA solution is concentrated or dried under reduced pressure. The resulting crude peptide is either precipitated or washed with Et.sub.2O or is re-dissolved directly into DMF or 50% aqueous acetic acid for purification by preparative HPLC.

(42) Short chain peptides with the desired purity can be obtained by purification using preparative HPLC. The solution of crude peptide is injected into a semi-Prep column (Luna 10μ; C.sub.18; 100 A°), dimension 250×50 mm and eluted with a linear gradient of ACN in water, both buffered with 0.1% TFA, using a flow rate of 40 ml/min with effluent monitoring by PDA detector at 220 nm. The structures of the purified short chain peptides can be confirmed by Electrospray Mass Spectroscopy (ES-MS) analysis.

(43) All the peptide prepared were isolated as trifluoro-acetate salt, with TFA as a counter ion, after the Prep-HPLC purification. However, some peptides were subjected for desalting, by passing through a suitable ion exchange resin bed, preferably through anion-exchange resin Dowex SBR P(Cl) or an equivalent basic anion-exchange resin. In some cases, TFA counter ions were replaced with acetate ions, by passing through suitable ion-exchange resin, eluted with dilute acetic acid buffer. For the preparation of the hydrochloride salt of peptides, in the last stage of the manufacturing, selected peptides, with the acetate salt was treated with 4 M HCl. The resulting solution was filtered through a membrane filter (0.2 μm) and subsequently lyophilized to yield the white to off-white HCl salt. Following similar techniques and/or such suitable modifications, which are well within the scope of persons skilled in the art, other suitable pharmaceutically acceptable salts of the short chain peptides of the present invention were prepared.

(44) ##STR00004##
General Method of Preparation of Short Chain Peptides, Using SPPS Approach:
Assembly of Short Chain Peptides on Resin:

(45) Sufficient quantity (50-100 mg) of Fmoc-PAL-PEG-PS resin or Fmoc-Rink amide MBHA resin, loading: 0.5-0.6 mmol/g was swelled in DMF (1-10 ml/100 mg of resin) for 2-10 minutes. The Fmoc-group on resin was removed by incubation of resin with 10-30% piperidine in DMF (10-30 ml/100 mg of resin), for 10-30 minutes. Deprotected resin was filtered and washed excess of DMF, DCM and ether (50 ml×4). Washed resin was incubated in freshly distilled DMF (1 ml/100 mg of resin), under nitrogen atmosphere for 5 minutes. A 0.5 M solution of first Fmoc-protected amino acid (1-3 eq.), pre-activated with HOBt (1-3 eq.) and DIPCDI (1-2 eq.) in DMF was added to the resin, and the resin was then shaken for 1-3 hrs, under nitrogen atmosphere. Coupling completion was monitored using a qualitative ninhydrin test. After the coupling of first amino acid, the resin was washed with DMF, DCM and Diethyl ether (50 ml×4). For the coupling of next amino acid, firstly, the Fmoc-protection on first amino acid, coupled with resin was deprotected, using a 10-20% piperidine solution, followed by the coupling the Fmoc-protected second amino acid, using a suitable coupling agents, and as described above. The repeated cycles of deprotection, washing, coupling and washing were performed until the desired peptide chain was assembled on resin, as per general (Scheme 1) above. Finally, the Fmoc-protected peptidyl-resin prepared above was deprotected by 20% piperidine treatment as described above and the peptidyl-resins were washed with DMF, DCM and Diethyl ether. Resin containing desired peptide was dried under nitrogen pressure for 10-15 minutes and subjected for cleavage/deprotection.

(46) Using above protocol and suitable variations thereof which are within the scope of a person skilled in the art, the short chain peptides designed in the present invention were prepared, using Fmoc-SPPS approach. Furthermore, resin bound short chain peptides were cleaved and deprotected, purified and characterized using following protocol.

(47) Cleavage and Deprotection:

(48) The desired short chain peptides were cleaved and deprotected from their respective peptidyl-resins by treatment with TFA cleavage mixture as follows. A solution of TFA/Water/Triisopropylsilane (95:2.5:2.5) (10 ml/100 mg of peptidyl-resin) was added to peptidyl-resins and the mixture was kept at room temperature with occasional starring. The resin was filtered, washed with a cleavage mixture and the combined filtrate was evaporated to dryness. Residue obtained was dissolved in 10 ml of water and the aqueous layer was extracted 3 times with ether and finally the aqueous layer was freeze-dried. Crude peptide obtained after freeze-drying was purified by preparative HPLC as follows:

(49) Preparative HPLC Purification of the Crude Short Chain Peptides:

(50) Preparative HPLC was carried out on a Shimadzu LC-8A liquid chromatography. A solution of crude peptide dissolved in DMF or water was injected into a semi-Prep column (Luna 10μ; C.sub.18; 100 A°), dimension 250×50 mm and eluted with a linear gradient of ACN in water, both buffered with 0.1% TFA, using a flow rate of 15-50 ml/min, with effluent monitoring by PDA detector at 220 nm. A typical gradient of 20% to 70% of water-ACN mixture, buffered with 0.1% TFA was used, over a period of 50 minutes, with 1% gradient change per minute. The desired product eluted were collected in a single 10-20 ml fraction and pure short chain peptides were obtained as amorphous white powders by lyophilisation of respective HPLC fractions.

(51) HPLC Analysis of the Purified Short-Chain Peptides

(52) After purification by preparative HPLC as described above, each peptide was analyzed by analytical RP-HPLC on a Shimadzu LC-10AD analytical HPLC system. For analytical HPLC analysis of short chain peptides, Luna 5μ; C.sub.18; 100 A°, dimension 250×4.6 mm column was used, with a linear gradient of 0.1% TFA and ACN buffer and the acquisition of chromatogram was carried out at 220 nm, using a PDA detector.

(53) Characterization by Mass Spectrometry

(54) Each peptide was characterized by electrospray ionisation mass spectrometry (ESI-MS), either in flow injection or LC/MS mode. Triple quadrupole mass spectrometers (API-3000 (MDS-SCIES, Canada) was used in all analyses in positive and negative ion electrospray mode. Full scan data was acquired over the mass range of quadrupole, operated at unit resolution. In all cases, the experimentally measured molecular weight was within 0.5 Daltons of the calculated monoisotopic molecular weight. Quantification of the mass chromatogram was done using Analyst 1.4.1 software.

(55) Following table 1(i) is the list of short chain peptides synthesized using the SPPS approach as described above. Mentioned Seq. ID. No 1 in the list was taken as a reference from WO 2011027257.

(56) TABLE-US-00001 TABLE 1 (i) List of short chain peptides prepared Seq. ID. No. Sequence of short chain peptides 1 SIPWNLERITPC 2 S-Nle-PWNLERITPC 3 S-Aib-PWNLERITPC 4 SIPWNLER-Nle-TPC 5 SIPWNLER-Aib-TPC 6 SIPWNLE-Har-ITPC 7 SIPWN-Nle-ERITPC 8 SI-Hyp-WNLERITPC 9 SIPWNLERIT-Hyp-C 10 SIPWN-Aib-ERITPC 11 Ac-SIPWNLERITPC 12 SIPWNLE-Cit-ITPC 13 SIPWNLERI-Aib-PC 14 Aib-IPWNLERITPC 15 S-(N-Me-Ile)-IPWNLERITPC 16 SIPWN-(N-Me-Leu)-ERITPC 17 SIPWNLER-(N-Me-Ile)-TPC 18 SIP-APPA-NLERITPC 19 SIP-Bip(OMe)-NLERITPC 20 SI-Hyp-WNLER-Aib-TPC 21 SI-Hyp-WN-Aib-ERITPC 22 SIPWNLE-Cit-Aib-TPC 23 SIPWN-Aib-E-Cit-ITPC 24 SI-Hyp-WNLE-Cit-ITPC 25 SI-Hyp-WN-Aib-E-Cit-ITPC 26 SI-Hyp-WNLE-Cit-Aib-TPC 27 SIPWNLE-Cit-IT-Hyp-C 28 SIPWN-Aib-ERIT-Hyp-C 29 SI-Hyp-WN-Aib-ERIT-Hyp-C 30 SIPWN-Aib-E-Cit-IT-Hyp-C 31 SI-Hyp-WNLE-Cit-IT-Hyp-C 32 SI-(αMe-Pro)-WNLE-Cit-ITPC 33 SI-AC.sub.3C-WNLE-Cit-ITPC 34 SI-AC.sub.5C-WNLE-Cit-ITPC 35 SI-AC.sub.6C-WNLE-Cit-ITPC 36 SI-Hyp-WN-βA1a-E-Cit-ITPC 37 SI-Hyp-WNL-(αMe-Glu)-Cit-ITPC 38 SI-Hyp-WNL-(Homo-Glu)-Cit-ITPC 39 SI-Hyp-WNL-(αMe-Asp)-Cit-ITPC 40 SI-Hyp-WN-HoLeu-E-Cit-ITPC 41 SI-Hyp-WN-(αMe-Asp)-E-Cit-ITPC 42 SI-Thz-WNLE-Cit-ITPC 43 SI-(2-Thz)-WNLE-Cit-ITPC 44 SI-(3-Hyp)-WNLE-Cit-ITPC 45 SI-Amp-WNLE-Cit-ITPC 46 SI-(3-Amp)-WNLE-Cit-ITPC 47 SI-(3-F-Pro)-WNLE-Cit-ITPC 48 SI-(4-F-Pro)-WNLE-Cit-ITPC 49 Ser(OMe)-I-Hyp-WNLE-Cit-ITPC 50 HoSer-I-Hyp-WNLE-Cit-ITPC 51 Thr(OMe)-I-Hyp-WNLE-Cit-ITPC 52 HoSer(OMe)-I-Hyp-WNLE-Cit-ITPC 53 SI-Hyp-(2F-Phe)-NLE-Cit-ITPC 54 SI-Hyp-(αMe-Phe)-NLE-Cit-ITPC 55 SI-Hyp-(αMe-2F-Phe)-NLE-Cit-ITPC 56 SI-Hyp-(αMe-2,6-diF-Phe)-NLE-Cit-ITPC 57 SI-Hyp-(αMe-APPA)-NLE-Cit-ITPC 58 SI-Hyp-WNL-Abu(CN)-Cit-ITPC

(57) In another preferred embodiment the peptides of the present invention can be chemically synthesized by methods which are well known in the art. It is also possible to produce the peptides of the present invention using recombinant methods. The peptides can be produced in microorganisms such as bacteria such as E. coli, B. subtilis, or any other bacterium that is capable of expressing such peptides, yeast such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida, Pichia pastoris or any other yeast capable of expressing peptides or fungi, in eukaryotic cells such as mammalian or insect cells, or in a recombinant virus vector such as adenovirus, poxvirus, herpes virus, Simliki forest virus, baculovirus, bacteriophage, sindbis virus or Sendai virus. Also methods for isolating and purifying recombinantly produced peptides are well known in the art and include e.g. gel filtration, affinity chromatography, ion exchange chromatography etc.

(58) Procedure for DT Conjugation:

(59) Diptheria toxin is a single polypeptide chain consisting of 535 amino acids, containing two subunits linked by disulfide bridges. One of the subunits binds to the cell surface, allowing the more stable subunit to penetrate the host cell. For conjugation, Diptheria toxoid and peptide need to be in equimolar concentration. Concentration of DT and peptide is 2-50 mg/mL. In the first step the Diptheria toxoid is brought into solution by dissolving it in Phosphate buffered saline. Next, EDAC (1-ethyl 3,3 dimethylaminopropyl carbodiimide) was added that provides the first step in cross-linking carboxylic acids. EDAC activates carboxyl groups for direct reaction with primary amines via the amide bond formation, thus primes the Diptheria toxoid for conjugation with peptide. Moreover, EDAC-mediated cross-linking is more effective in acidic pH. Hence, the reaction is allowed to take place by incubating DT with EDAC for one minute in the presence of MES buffer (40-morpholinoethane sulfonic acid) at pH 6.0. Moreover, this EDAC coupling method in the presence of MES improves the efficiency of the conjugation by forming intermediates. Next, ADH (Adipic Acid Dihydrazide) was added, which is a homobifunctional cross-linking reagent that results in relatively stable hydrazone linkages to the DT and peptide. Linking is carried out in a site-specific fashion, by oxidation first and then cross-linking, performed at pH 5.0 (due to the low pKa of the hydrazide). This avoids competition by primary amines. EDAC is again added, and the mixture is incubated for 3 hours at 2-8° C. to allow the conjugation to commence. The above prepared DT conjugated peptide is dialyzed through a 10 kD column and sterile filtered (0.2μ filter) for the removal of impurities, and the pure peptide-DT conjugate is stored at 2-8° C. for at least a week.

(60) In another embodiment, conjugation is done with CRM197 which is genetically detoxified form of diphtheria toxin by following the general procedure given in WO 2011027257 and prior art.

(61) In a further preferred embodiment the vaccine of the present invention may be administered subcutaneously, intramuscularly, intradermally, intravenously (see e.g. “Handbook of Pharmaceutical Manufacturing Formulations”, Sarfaraz Niazi, CRC Press Inc, 2004). The formulation may consist of respective carriers, adjuvants, and/or excipients depending on the route of administration.

(62) Vaccine Formulations:

(63) For the design of more immunopotent and protective vaccines, the final vaccine formulation may contain adjuvants with immunopotentiating properties that can direct the immune responses to humoral or cell-mediated immunity, depending on the type of adjuvant. Traditional adjuvants such as aluminum salts, emulsions, liposomes and virosomes present vaccine antigens to the immune system in an efficient manner and control the release and storage of antigens to increase the specific immune response. The second class of adjuvants is immunostimulants, which affect the immune system and increase the immune responses to antigens. For example, they influence cytokine production through the activation of MHC molecules, co-stimulatory signals, or through related intracellular signaling pathways.

(64) It has been found that many of the currently approved vaccine adjuvants are not always sufficiently potent to induce an efficient protective immune response against different target pathogens, particularly in immunologically hyporesponsive populations such as the elderly and immunocompromised populations, where there is a decreased T-cell dependent antibody response because of compromised T-cell function. The use of adjuvants with immunopotentiating properties yield a more effective response in a large number of vaccines such as hepatitis C virus (HCV), HIV, HBV, HPV, influenza and cancer. A more rational approach to vaccine design therefore is the use of immunostimulant adjuvants that can modulate and enhance cytotoxic T lymphocyte (CTL) responses, and/or affect dendritic cells (DCs) through TLR exploitation. T cells are the most effective arm of the immune response and given the recognized importance of T cells in regulating immune responses to vaccination, use of these novel adjuvants in vaccination strategies seems fitting.

(65) Other Adjuvants that can be Used for the Formulation:

(66) In Order to elicit stronger immune response from the DT-conjugated peptide, additional adjuvants added to the formulation are as below. 1) Alum (aluminum hydroxide gel, 2% wet gel suspension) Alum is an effective adjuvant as the antigen is adjuvanted with insoluble aluminum salts, that remain in the body for a long time. The antigen is slowly released from the insoluble salt particles, allowing prolonged and effective stimulation of the immune system (‘depot effect’) [1]. In addition to or in contrast to the depot effect, insoluble aluminium salts activate innate immune cells in a manner that ultimately results in a T helper 2 (Th2)-type immune response Alum induces a Th2 response by improving antigenic uptake of antigen by antigen-presenting cells (APCs)[2]. 2) Alum with MF-59 AddaVax (also known as MF59) is a squalene-based oil-in-water nano-emulsion. Squalene is an oil more readily metabolized than the paraffin oil used in Freund's adjuvants [3]. Squalene oil-in-water emulsions elicit both cellular (Th1) and humoral (Th2) immune responses [4, 5]. This adjuvant class acts via recruitment and activation of Antigen presenting cells along with stimulation of cytokine and chemokine production by macrophages and granulocytes [3]. 3) Alum with other adjuvants like TLR3 agonist such as Poly(I:C), TLR 4 agonist such as Monophosphoryl Lipid A or GLA-SE, TLR5 agonist such as Flagellin, TLR7 agonist such as Gardiquimod and Imiquimod TLR7/8 agonist such as R848, NOD2 agonist such as N-glycolyl-MDP.CpG-containing nucleic acid (where the cytosine is unmethylated), QS21 (saponin adjuvant), interleukins, beta-sitosterol and the like. An adjuvant used in all the experiments is combination of alum and stable formulation of Monophosphoryl Lipid A.
Affinity Determination of New Peptides:

(67) Affinity parameters of new peptides for recombinant human PCSK9 were analyzed by surface plasmon resonance (SPR), using a Biacore instrument (Biacore T200, GE Healthcare). SPR experiments were performed at 25° C. with a BIACORE T200 apparatus (GE Healthcare, Uppsala, Sweden).

(68) Surface Preparation: (Procedure for Protein Immobilization)

(69) Purified recombinant human PCSK9 (His-tagged, BPS Biosciences) diluted to 100-150 μg/mL in 10 mM acetate buffer, pH 5.0, was immobilized on one of the four flow cells of a Series S Sensor Chip CMS to a level of approximately 6000 to 8000 RU (resonance units) by the use of amine-coupling chemistry. The surface was blocked with 1M ethanolamine, pH 8.5. Whereas one flow cell was immobilized as blank for reference subtraction (no PCSK9). 10 mM Hepes (pH 7.4) with 150 mM NaCl was used as running buffer. The rest two flow cells were saved for future use.

(70) Binding and Kinetics/Affinity Experiments:

(71) 100× Stock solutions of NCE (new peptides) were made in 100% DMSO and were diluted to range of concentrations in 10 mM Hepes, pH7.4+150 mM NaCl so that the final conc of DMSO is kept at 1%. Binding/Kinetics studies were conducted by passing various concentrations of NCEs over blank as well as ligand (protein) immobilized surface. Each cycle consisted of a 60 s or 120 s analyte (diluted small molecule drug) injection at flow rate with between 5 to 30 μL/min (the association phase), followed by a dissociation phase with between 60 to 180 s. If required, regeneration was done using a 30 s (15 μL) injection of 10 mM glycine/HCl (pH 1.5) and 20 s or 30 s (10 or 15 μL) injection of 10 mM NaOH. The data were analyzed using the Biacore T200 Evaluation software. All the curves were reference-subtracted. Baselines were adjusted to zero for all curves and data were presented as binding RU (Average of 5 s window) 5 s before end of sample injection.

(72) For K.sub.D (equilibrium dissociation constant) determinations analyte curves were subtracted from buffer blank and modeled assuming a simple 1:1 interaction to generate the kinetic data.

(73) Immunogenicity Study in BALB/c Mice

(74) Female BALB/c mice of 12-15 weeks of age issued from animal house and kept for 2-3 acclimatization. Mice had access to food and water ad libitum and were kept under a 12 hrs light/dark cycle. On Day-0 (pre-treatment) animals bled and serum was harvested for total cholesterol measurement. Animals were randomized and grouped to various treatments based on their total cholesterol and body weights. On next day of Day-0 (pre-treatment) blood collection animals were immunised with 0.3 ml of vaccine formulations as mentioned Table No. 1 by subcutaneous or intramuscular route. Next booster injection was given on 2 weeks and 4 weeks after first injection and animals were bled for immunogenicity measurement on two weeks thereafter till week 7 and then every four week till 32 week, detailed schedule of blood collection was Day-0 (pre-treatment) from there 5.sup.th, 7.sup.th, 20.sup.th, 24.sup.th, 28.sup.th, 32.sup.nd, 45.sup.th and 57.sup.th week after first vaccine administration. Serum was separated and serum total cholesterol was measured and immunogenicity or antibody confirmation was done using ELISA for PCSK9 antibody titer, binding of serum antibodies with human PCSK9 using Surface plasmon resonance (SPR) assay and LDLR-PCSK9 interaction inhibition assay.

(75) TABLE-US-00002 TABLE NO. 1 Number of Groups Treatment Animals 1 Phosphate Buffer Saline (PBS) 8 2 Placebo (PBS + adjuvant) 8 3 DT conjugated Seq. ID. No. 1 in adjuvant 8 6 DT conjugated Seq. ID. No. 7 in adjuvant 8 7 DT conjugated Seq. ID. No. 8 in adjuvant 8 8 DT conjugated Seq. ID. No. 12 in adjuvant 8
Immunogenicity Study in hApoB100/hCETP Double-Transgenic Mice (dTg)

(76) Male or female hApoB100/hCETP dTg mice of more than 8 weeks of age issued from animal house and kept for 2-3 acclimatization. Mice had access to food and water ad libitum and were kept under a 12 hrs light/dark cycle. On Day-0 (pre-treatment) animals bled and serum was harvested for LDL-cholesterol (LDL-C), total cholesterol, HDL-C and triglycerides measurement. Animals were randomized and grouped to various treatments based on their LDL-C and body weights. On next day of blood collection animals were immunised with 0.3 ml of vaccine formulations by subcutaneous or intramuscular route. Next booster injection was given on 2 weeks and 4 weeks after first injection and animals were bled for immunogenicity measurement on two weeks after third injection. Serum was separated and serum LDL-C, total cholesterol, HDL-C and triglycerides levels were measured and immunogenicity or antibody confirmation was done using ELISA for PCSK9 antibody titer, binding of serum antibodies with human PCSK9 using Surface plasmon resonance (SPR) assay and LDLR-PCSK9 interaction inhibition assay.

(77) Serum LDL-C, HDL-C, total cholesterol and triglycerides levels were determined using commercial kits (Roche Diagnostics, Germany) on a Cobas c311 autoanalyzer (Roche, Germany).

(78) Evaluation of Humoral Response by Anti-PCSK-9 ELISA

(79) At specific intervals of time (15 days post immunization with each booster), serum samples were collected from all groups of mice. 96-well ELISA plates (#442402) were pre-coated with PCSK-9 protein 50 ng/well and plate incubated overnight at 2-8° C. Next day, wells were washed to remove unbound protein and blocked in 2% skimmed milk-PB ST for 2 hours, and washed with PBST. Sera samples were diluted 1:20 with 0.2% skimmed milk made in PBST. Appropriate volume of samples and standards was added to each well, and incubated for 1 hour at 37° C. Anti-mouse IgG secondary HRP-labeled antibody was added to each well and incubated at 37° C. for 1 hour, then washed. OPD-substrate was added to each well and incubated for 20 minutes at room temperature in the dark. Plates were read at 492 nm on a Spectramax microplate reader. Amongst all the DT-conjugated PCSK-9 peptides tested, the one that gave the highest response was treated as the standard, and its placebo was used to calculate the corrected OD. The sera from the mice injected with PBS were used as negative control. By using the negative control value, cut-off value was calculated and the corrected OD's above the cut off value were treated as standards. All test samples gave absolute antibody titres against PCSK-9, extrapolated from the standard curve and values were expressed in log 10.

(80) LDLR-PCSK9 Interaction Inhibition Assay:

(81) Inhibition of LDLR-PCSK9 interaction by antibody (Ab) in serum from vaccine treated animal were assayed using BPS Bioscience in-vitro PCSK9[Biotinylated]-LDLR Binding Assay Kit (BPS Bioscience, San Diego, Calif., USA) with minor modifications. First, LDLR ectodomain is coated on a 96-well plate and plate was incubated overnight. Next, PCSK9-biotin was diluted in 1×PCSK9 assay buffer to obtain 2.5 ng/μl (50 ng/20 μl) concentration and this master mix, test inhibitor (serum samples from animal studies), inhibitor buffer was added to LDL-R coated plate and incubated at room temperature for 2 hours. Finally, the plate is treated with streptavidin-HRP followed by addition of an HRP substrate to produce chemiluminescence, which was then measured using a chemiluminescence reader according to the manufacturer's instructions. Relative inhibition was denoted as the difference in percentage inhibition between the intensity of the PCSK9-LDLR binding in the serum samples from vaccine treated animals and that in placebo treated which was considered as 100%.

(82) Results:

(83) I) Table No. 2 Affinity of new peptides for recombinant human PCSK9 analyzed by surface plasmon resonance (SPR), using a Biacore (Biacore T200, GE Healthcare).

(84) TABLE-US-00003 TABLE NO. 2 Affinity study of new peptides for recombinant human PCSK9. Seq. Binding - Binding- ID. No. Response Unit(RU) Response Unit(RU) 1 7.4 (50 μM) — 4 13.05 (50 μM) 30.00 (100 μM) 5 2.30 (50 μM) 7.10 (100 μM) 6 6.55 (50 μM) 17.50 (100 μM) 7 6.65 (50 μM) 16.00 (100 μM) 8 5.70 (50 μM) 13.25 (100 μM) 10 3.00 (50 μM) 7.50 (100 μM) 12 3.10 (50 μM) 6.70 (100 μM) 13 9.75 (50 μM) 37.35 (100 μM) 14 7.10 (50 μM) 15.00 (100 μM) 15 11.45 (50 μM) 29.85 (100 μM) 18 10.20 (50 μM) 17.85 (100 μM) 19 35.15 (50 μM) 85.90 (100 μM) II) The mean antibody titers against PCSK9 in serum samples of treated animals measured by ELISA assay given in below Table no. 3

(85) TABLE-US-00004 TABLE NO. 3 Mean Antibody titers against PCSK9 in ELISA Assay Antibody Titers against PCSK9 in ELISA Weeks after Vaccine Administration Tests groups WK-5 WK-7 WK-20 WK-24 WK-28 WK-32 WK-45 WK-57 DT conjugation 20664 58298 285 17389 4879 178599 179876 184408 Seq. ID. No. 1 in adjuvant DT conjugation 22773 424261 79448 38510 11553 320988 351529 405753 Seq. ID. No. 7 in adjuvant DT conjugation 39040 199806 100413 80141 14264 297967 356639 780136 Seq. ID. No. 8 in adjuvant DT conjugation 48964 698079 155994 116790 64880 510187 1140238 744605 Seq. ID. No. 12 in adjuvant

(86) All the peptide formulations showed very high levels of antibody titers against human PCSK9 and titers were higher for peptides of Seq. ID. No. 7, 8, 12 as compared to Seq.ID.No.1. Very high antibody response was maintained till 57th week after vaccine administration, demonstrating the possibility of immunogenic response for more than a year for a few of the peptide formulations. III) Inhibition of LDLR-PCSK9 interaction by antibody (Ab) in serum from vaccine treated animal were assayed using in-vitro PCSK9 [Biotinylated]-LDLR Binding Assay Kit as measure of functional assay. The percent inhibition LDLR-PCSK9 interaction by antibody (Ab) was measured once at week-5, given in Table no. 4.

(87) TABLE-US-00005 TABLE NO. 4 LDLR-PCSK9 interaction inhibition by ELISA assay Weeks after Vaccine LDLR- PCSK-9% interaction Inhibition Vs. Placebo Administration Tests groups WK-5 DT conjugation Seq. ID. No. 1 in adjuvant 46 DT conjugation Seq. ID. No. 7 in adjuvant 66 DT conjugation Seq. ID. No. 8 in adjuvant 66 DT conjugation Seq. ID. No. 12 in adjuvant 67

(88) Almost all the peptide formulations showed 40-70% inhibition LDLR-PCSK9 interaction at week 5. The interaction inhibition shown by Seq. ID. No. 7, 8, 12 were significantly higher than that of Seq. ID No.1 IV) The antibody detection in serum samples of treated animals was also confirmed by affinity determination against human PCSK9 using SPR assay given in below Table no. 5

(89) TABLE-US-00006 TABLE NO. 5 Serum Antibody Detection by Affinity Determination against human PCSK9 using SPR assay. Surface plasmon response(SPR) Weeks after Vaccine Administration Response Unit (RU) WK-5 WK-7 WK-20 WK-24 WK-45 WK-57 Tests groups Avg. Avg. Avg. Avg. Avg. Avg. DT conjugation Seq. ID. No. 1 in 19 8 37 35 29 23 adjuvant DT conjugation Seq. ID. No. 7 in 40 29 61 58 41 35 adjuvant DT conjugation Seq. ID. No. 8 in 49 30 59 57 47 42 adjuvant DT conjugation Seq. ID. No. 12 in 48 33 61 61 55 49 adjuvant

(90) The serum of samples from animals treated with peptide formulations showed increase in response unit as compared to placebo. On week 5 and week-57, sequence ID. No. 12 showed significantly higher antibody response and sequence ID. No. 8 showed significantly higher antibody response on week-5 as compared to Seq.ID.No. No. 1. There was very high antibody response maintained till 57.sup.th week after vaccine administration; it clearly demonstrates the possibility of immunogenic response for more than a year for peptide formulations

(91) The peptides of the present invention are administered to a mammal or an individual in an amount of 0.1 ng to 10 mg, preferably of 0.5 to 500 μg, per immunization. In a preferred embodiment these amounts refer to all peptides (if more than one peptide is used in the vaccine) present in the vaccine.

(92) The amount of peptides that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.

(93) The dose of the vaccine may vary according to factors such as the disease state, age, sex and weight of the mammal or individual, and the ability of antibody to elicit a desired response in the individual. Dosage regime may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The dose of the vaccine may also be varied to provide optimum preventative dose response depending upon the circumstances. For instance, the peptides and vaccine of the present invention may be administered to an individual at intervals of several days, one or two weeks or even months or years depending always on the level of antibodies directed to PCSK9.

(94) The peptides of the present invention can act as a vaccine when conjugated with suitable immunogenic carrier. These vaccines are able to form antibodies which bind to PCSK9 upon administration. These vaccines can be suitable for the prevention/treatment of disorders like hyperlipidaemia, hypercholesterolemia or atherosclerosis.

REFERENCES

(95) 1) Glenny A T, Pope C G, Waddington H, Wallace U. Immunological Notes: XVII-XXIV. J Pathol Bacteriol. 1926; 29:31-40. 2) Grun J L, Maurer P H. Different T helper cell subsets elicited in mice utilizing two different adjuvant vehicles: the role of endogenous interleukin 1 in proliferative responses. Cell Immunol. 1989; 121:134-145. 3) Ott G. et al., 2000. The adjuvant MF59: a 10-year perspective. Methods in Molecular Medicine, Vol 42, 211-228. 4) Calabro, S. et al., 2013. The adjuvant effect of MF59 is due to the oil-in-water emulsion formulation, none of the individual components induce a comparable adjuvant effect. Vaccine 31:3363-9. 5) Ott G. et al., 1995. MF59. Design and evaluation of a safe and potent adjuvant for human vaccines. Pharm Biotechnol 6: 277-96.