# US 2020/0399311 A1 · Novel peptide based pcsk9 vaccine

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- Number: US 2020/0399311 A1
- Document ID: US20200399311A1
- Status: Published application
- Kind: A1
- Application number: 16/498,084
- Filed: 2018-04-12
- Published: 2020-12-24
- Earliest claimed: 2017-04-12
- Assignees: Cadila Healthcare Limited
- Applicants: Cadila Healthcare Limited
- Inventors: Mukul JAIN; Suresh GIRI; Rajesh BAHEKAR; Cupta GAURAV; Rajendra CHOPADE
- Web page: https://patentgrep.com/patent/US20200399311A1

## CPC

- A61K 47/646: {the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines}
- C07K 7/08: having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68)
- A61K 38/005: {Enzyme inhibitors (protease inhibitors A61K38/55)}
- A61K 38/10: Peptides having 12 to 20 amino acids {(A61K38/043 - A61K38/046 take precedence)}
- A61K 39/0008: {Antigens related to auto-immune diseases; Preparations to induce self-tolerance}
- A61K 39/385: Haptens or antigens, bound to carriers
- A61K 47/6415: {Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins}
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- C12N 9/6454: {Dibasic site splicing serine proteases, e.g. kexin (3.4.21.61); furin (3.4.21.75) and other proprotein convertases}
- A61K 2039/575: {humoral response}
- A61K 2039/6037: {Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]}
- C07K 2319/55: containing a fusion with a toxin, e.g. diphteria toxin
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## 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-Z1—Z2—Z3—Z4—Z5—Z6—Z7—Z8—Z9—Z10—Z11—Z12—B Formula (I)

## Claims

### Claim 1

Peptide comprising amino acids sequence of Formula (I),
A-Z1—Z2—Z3—Z4—Z5—Z6—Z7—Z8—Z9—Z10—Z11—Z12—B Formula (I)
wherein,
‘A’ represents the groups —NH—R1, R2—CO—NH—, wherein ‘R1’ represents hydrogen or substituted linear or branched (C1-18) alkyl chain; ‘R2’ is selected from substituted linear or branched (C1-18) alkyl chain, (C1-6)alkoxy, (C3-C6) cycloalkyl, aryl, heteroaryl or arylalkyl groups;
‘B’ represents R3, —COOR3, —CONHR3 or CH2OR3, wherein R3 represents H or suitable amino acids;
Z1 is an amino acid residue selected from the group of uncharged amino acid residues, selected from the group of serine, threonine, valine, alanine, Aib and their suitable derivatives;
Z2 is an amino acid residue selected from the group of uncharged amino acid residues, selected from isoleucine, leucine, norleucine, glycine, alanine, Aib and their suitable derivatives;
Z3 is an amino acid residue selected from the group of proline, 1-amino carbocyclic acids and their suitable derivatives;
Z4 is an amino acid residue selected from the group of tryptophan, phenylalanine, tyrosine and their suitable derivatives;
Z5 is an amino acid residue selected from the group of glutamine, histidine, aspargine and their suitable derivatives;
Z6 is an amino acid residue selected from the group of uncharged amino acid residues selected from isoleucine, leucine, alanine, threonine, aspartic acid, Aib and their suitable derivatives;
Z7 is an amino acid residue selected from the group of hydrophilic, negatively charged amino acid residues;
Z8 is an amino acid residue selected from the group of arginine, homoarginine, citruline, glutamine, aspargine, lysine and their suitable derivatives;
Z9 is an amino acid residue selected from the group of uncharged amino acid residues, selected from isoleucine, leucine, norleucine, glycine, alanine, Aib and their suitable derivatives;
Z10 is an amino acid residue selected from the group of polar and uncharged amino acid residues selected from serine, threonine, valine, alanine, Aib and their derivatives;
Z11 is an amino acid residue selected from the group of proline, 1-amino carbocyclic acids and their derivatives; and
Z12 is an amino acid residue selected from polar and uncharged amino acid residue, selected from the group of cysteine, homocysteine and their derivatives;
with the proviso that at least one of Z1-Z12 will always represent an unnatural/modified amino acid.

### Claim 2 (depends on 1)

Peptide comprising amino acids sequence of Formula (I), as claimed in claim 1 wherein,
‘A’ represents the groups —NH—R1, R2—CO—NH—,
wherein ‘R1’ is hydrogen or substituted linear or branched (C1-18) alkyl chain; ‘R2’ is selected from substituted linear or branched (C1-18) alkyl chain, (C1-6) alkoxy, (C3-C6) cycloalkyl, aryl, heteroaryl and arylalkyl groups; wherein the aryl group is selected from phenyl, naphthyl, indanyl, fluorenyl or biphenyl, groups; and the heteroaryl group is selected from pyridyl, thienyl, furyl, imidazolyl, benzofuranyl groups; and
‘B’ represents R3, —COOR3, —CONHR3 or CH2OR3, wherein R3 is selected from H and amino acids such as serine, cysteine, valine, alpha-methyl-valine, Lys(Biotin), Lys(alkyl), Lys(acetyl).

### Claim 3 (depends on 1)

The peptide comprising the amino acid sequence of Formula (I), as claimed in claim 1 wherein Z1 is selected from serine, threonine, valine, alanine, Aib, homoserine, O-methyl-threonine, O-methyl-serine or O-methyl-homoserine.

### Claim 4 (depends on 1)

The peptide comprising the amino acid sequence of Formula (I), as claimed in claim 1, wherein Z2 is selected from isoleucine, leucine, norleucine, glycine, alanine or Aib, N-methyl-isoleucine or N-methyl-leucine.

### Claim 5 (depends on 1)

The peptide comprising amino acid sequence of Formula (I), as claimed in claim 1, wherein Z3 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.

### Claim 6 (depends on 1)

The peptide comprising amino acid sequence of Formula (I), as claimed in claim 1, wherein Z4 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.

### Claim 7 (depends on 1)

The peptide comprising amino acids sequence of Formula (I), as claimed in claim 1, wherein Z5 is selected from glutamine, histidine or aspargine.

### Claim 8 (depends on 1)

The peptide comprising amino acids sequence of Formula (I), as claimed in claim 1, wherein Z6 is selected from isoleucine, leucine, alanine, threonine, aspartic acid, Aib, norisoleucine, N-methyl-leucine, homoleucine, beta-alanine or alpha-methyl-aspartic acid.

### Claim 9 (depends on 1)

The peptide comprising amino acids sequence of Formula (I), as claimed in claim 1, wherein Z7 is glutamic acid, aspartic acid, alpha-methyl-aspartic acid, alpha-methyl-glutamic acid, 2-amino-4-cyanobutanoic acid or homoglutamic acid.

### Claim 10 (depends on 1)

The peptide comprising amino acids sequence of Formula (I), as claimed in claim 1, wherein Z8 is selected from arginine, homoarginine, citruline, glutamine, aspargine or lysine.

### Claim 11 (depends on 1)

The peptide comprising amino acids sequence of Formula (I), as claimed in claim 1, wherein Z9 is selected from isoleucine, leucine, norleucine, glycine, alanine, Aib or N-methyl isoleucine.

### Claim 12 (depends on 1)

The peptide comprising amino acids sequence of Formula (I), as claimed in claim 1 wherein Z10 is selected from serine, threonine, valine, Aib or alanine.

### Claim 13 (depends on 1)

The peptide comprising amino acids sequence of Formula (I), as claimed in claim 1, wherein Z11 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.

### Claim 14 (depends on 1)

The peptide comprising amino acids sequence of Formula (I), as claimed in claim 1, wherein Z12 is selected from cysteine or Homocysteine.

### Claim 15 (depends on 1)

The peptide comprising amino acid sequence of Formula (I) as claimed in claim 1, wherein
‘A’ represents the groups —NH—R—R2—CO—NH—, wherein ‘R1’ is hydrogen or substituted linear or branched (C1-18) alkyl chain; ‘R2’ is selected from substituted linear or branched (C1-18) alkyl chain, (C1-6) alkoxy, (C3-C6) cycloalkyl, aryl, and arylalkyl groups; wherein the aryl group is selected from phenyl, naphthyl, indanyl, fluorenyl or biphenyl, groups;
‘B’ represents R3, —COOR3, —CONHR3 or CH2OR3, wherein R3 is selected from H, serine, cysteine, valine, alpha-methyl-valine, Lys (Biotin), Lys (alkyl), Lys (acetyl),
Z1 is selected from serine, threonine, valine, alanine, Aib, homoserine, O-methyl-threonine, O-methyl-serine or O-methyl-homoserine,
Z2 is selected from isoleucine, leucine, norleucine, glycine, alanine, Aib, N-methyl-isoleucine or N-methyl-leucine,
Z3 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,
Z4 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,
Z5 is selected from glutamine, histidine or aspargine,
Z6 is selected from isoleucine, leucine, alanine, threonine, aspartic acid, Aib, norisoleucine, N-methyl-leucine, homoleucine, beta-alanine or alpha-methyl-aspartic acid,
Z7 is glutamic acid, aspartic acid, alpha-methyl-aspartic acid, alpha-methyl-glutamic acid, 2-amino-4-cyanobutanoic acid or homoglutamic acid,
Z8 is selected from arginine, homoarginine, citruline, glutamine, aspargine or lysine,
Z9 is selected from isoleucine, leucine, norleucine, glycine, alanine, Aib or N-methyl isoleucine,
Z10 is selected from serine, threonine, valine, Aib or alanine,
Z11 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, and
Z12 is selected from cysteine or Homocysteine, with the proviso that at least one of Z1-Z12 will always represent an unnatural/modified amino acid.

### Claim 16

The peptide comprising amino acids sequence of formula (I), comprising any of following amino acid sequence:
[SEQ ID NO. 2]
S-Nle-PWNLERITPC;
[SEQ ID NO. 3]
S-Aib-PWNLERITPC;
[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. 9]
SIPWNLERIT-Hyp-C;
[SEQ ID NO. 10]
SIPWN-Aib-ERITPC;
[SEQ ID NO. 11]
Ac-SIPWNLERITPC;
[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-I1e)-IPWNLERITPC;
[SEQ ID NO. 16]
SIPWN-(N-Me-Leu)-ERITPC;
[SEQ ID NO. 17]
SIPWNLER-(N-Me-I1e)-TPC;
[SEQ ID NO. 18]
SIP-APPA-NLERITPC;
[SEQ ID NO. 19]
SIP-Bip(OMe)-NLERITPC;
[SEQ ID NO. 20]
SI-Hyp-WNLER-Aib-TPC;
[SEQ ID NO. 21]
SI-Hyp-WN-Aib-ERITPC;
[SEQ ID NO. 22]
SIPWNLE-Cit-Aib-TPC;
[SEQ ID NO. 23]
SIPWN-Aib-E-Cit-ITPC;
[SEQ ID NO. 24]
SI-Hyp-WNLE-Cit-ITPC;
[SEQ ID NO. 25]
SI-Hyp-WN-Aib-E-Cit-ITPC;
[SEQ ID NO. 26]
SI-Hyp-WNLE-Cit-Aib-TPC;
[SEQ ID NO. 27]
SIPWNLE-Cit-IT-H-C;
[SEQ ID NO. 28]
SIPWN-Aib-ERIT-H-C;
[SEQ ID NO. 29]
SI-Hyp-WN-Aib-ERIT-Hyp-C;
[SEQ ID NO. 30]
SIPWN-Aib-E-Cit-IT-H-C;
[SEQ ID NO. 31]
SI-Hyp-WNLE-Cit-IT-Hyp-C;
[SEQ ID NO. 32]
SI-(αMe-Pro)-WNLE-Cit-ITPC;
[SEQ ID NO. 33]
SI-AC3C-WNLE-Cit-ITPC;
[SEQ ID NO. 34]
SI-AC5C-WNLE-Cit-ITPC;
[SEQ ID NO. 35]
SI-AC6C-WNLE-Cit-ITPC;
[SEQ ID NO. 36]
SI-Hyp-WN-βAla-E-Cit-ITPC;
[SEQ ID NO. 37]
SI-Hyp-WNL-(αMe-Glu)-Cit-ITPC;
[SEQ ID NO. 38]
SI-Hyp-WNL-(Homo-Glu)-Cit-ITPC;
[SEQ ID NO. 39]
SI-Hyp-WNL-(αMe-Asp)-Cit-ITPC;
[SEQ ID NO. 40]
SI-Hyp-WN-HoLeu-E-Cit-ITPC;
[SEQ ID NO. 41]
SI-H-WN-(αMe-Asp)-E-Cit-ITPC;
[SEQ ID NO. 42]
SI-Thz-WNLE-Cit-ITPC;
[SEQ ID NO. 43]
SI-(2-Thz)-WNLE-Cit-ITPC;
[SEQ ID NO. 44]
SI-(3-Hyp)-WNLE-Cit-ITPC;
[SEQ ID NO. 45]
SI-Amp-WNLE-Cit-ITPC;
[SEQ ID NO. 46]
SI-(3-Amp)-WNLE-Cit-ITPC;
[SEQ ID NO. 47]
SI-(3-F-Pro)-WNLE-Cit-ITPC;
[SEQ ID NO. 48]
SI-(4-F-Pro)-WNLE-Cit-ITPC;
[SEQ ID NO. 49]
Ser(OMe)-I-Hyp-WNLE-Cit-ITPC;
[SEQ ID NO. 50]
HoSer-I-Hyp-WNLE-Cit-ITPC;
[SEQ ID NO. 51]
Thr(OMe)-I-Hyp-WNLE-Cit-ITPC;
[SEQ ID NO. 52]
HoSer(OMe)-I-Hyp-WNLE-Cit-ITPC;
[SEQ ID NO. 53]
SI-Hyp-(2F-Phe)-NLE-Cit-ITPC;
[SEQ ID NO. 54]
SI-Hyp-(αMe-Phe)-NLE-Cit-ITPC;
[SEQ ID NO. 55]
SI-Hyp-(αMe-2F-Phe)-NLE-Cit-ITPC;
[SEQ ID NO. 56]
SI-Hyp-(αMe-2,6-diF-Phe)-NLE-Cit-ITPC;
[SEQ ID NO. 57]
SI-Hyp-(αMe-APPA)-NLE-Cit-ITPC;
[SEQ ID NO. 58]
SI-Hyp-WNL-Abu(CN)-Cit-ITPC [[;]].

### Claim 17 (depends on 1)

The peptide comprising amino acids sequence of Formula (I), of claim 1 in conjugation with a suitable immunogenic carrier.

### Claim 18 (depends on 15)

The peptide comprising amino acid sequence of Formula (I) wherein definition of A, B and Z1-Z12 is as defined in claim 15.

### Claim 19 (depends on 17)

The peptide comprising amino acids sequence of Formula (I), in conjugation with the suitable immunogenic carrier as claimed in claim 17 wherein the immunogenic carrier is selected from the group consisting of of diphtheria toxin (DT), modified DT, keyhole limpet haemocyanin (KLH), tetanus toxoid (TT), protein D and any other suitable protein or peptide containing helper T-cell epitopes.

### Claim 20 (depends on 19)

The peptide comprising amino acids sequence of Formula (I), in conjugation with the suitable immunogenic carrier as claimed in claim 19, wherein the immunogenic carrier is diphtheria toxin (DT) and CRM (CRM197).

### Claim 21 (depends on 1)

The peptide comprising amino acids sequence of Formula (I) as claimed in claim 1, conjugated with a suitable immunogenic carrier, in combination with one or more pharmaceutically acceptable adjuvants and other pharmaceutical excipients.

### Claim 22 (depends on 15)

The peptide comprising amino acid sequence of Formula (I) wherein definition of A, B and Z1-Z12 is as defined in claim 15.

### Claim 23 (depends on 21)

The peptide comprising amino acids sequence of Formula (I), conjugated with the suitable immunogenic carrier and with one or more pharmaceutically acceptable adjuvants, as claimed in claim 21, wherein the pharmaceutically acceptable adjuvants are selected from the group of consisting of alum, TLR3 agonist, Poly(I:C), TLR4 agonist, Monophosphoryl Lipid A, GLA, TLR5 agonist, Flagellin, TLR7 agonist, Gardiquimod, Imiquimod, TLR7/8 agonist, R848, NOD2 agonist, N-glycolyl-MDP, CpG-containing nucleic acid (where the cytosine is unmethylated), QS21 (saponin adjuvant), interleukins, and beta-sitosterol.

### Claim 24 (depends on 23)

The peptide comprising amino acids sequence of Formula (I), conjugated with the suitable immunogenic carrier and with one or more pharmaceutically acceptable adjuvants, as claimed in claim 23, wherein one or more pharmaceutically acceptable adjuvants are selected from alum, GLA, Monophosphoryl Lipid A, and combinations thereof.

### Claim 25 (depends on 1)

The peptide comprising amino acids sequence of Formula (I) as claimed in claim 1 suitable for the treatment of health disorders selected from hyperlipidaemia, hypercholesterolemia, or atherosclerosis and other cardiovascular diseases.