# US 10,117,946 B2 · Self-assembling ultrashort peptides modified with bioactive agents by click chemistry

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- Number: US 10,117,946 B2
- Document ID: US10117946B2
- Status: Granted
- Kind: B2
- Application number: 14/655,223
- Filed: 2013-12-24
- Published: 2015-12-10
- Granted: 2018-11-06
- Earliest claimed: 2012-12-24
- Assignees: Agency for Science, Technology and Research
- Applicants: Agency for Science, Technology and Research
- Inventors: Michael R. Reithofer; Charlotte A. E. Hauser; Kiat Hwa Chan; Archana Mishra
- Web page: https://patentgrep.com/patent/US10117946B2

## CPC

- A61K 47/64: Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent (peptidic linkers A61K47/65)
- A61K 31/282: Platinum compounds
- A61K 31/555: containing heavy metals, e.g. hemin, hematin, melarsoprol
- A61K 38/08: Peptides having 5 to 11 amino acids {(A61K38/043 - A61K38/046 take precedence)}
- A61P 35/00: Antineoplastic agents
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

The present invention relates to hydrogels comprising a first peptide with a covalently linked bioactive agent and optionally a second peptide. The present invention further relates to uses of the hydrogel for delivery of the bioactive agent or as an implant. The present invention further relates to drug delivery devices, implant, pharmaceutical or cosmetic compositions comprising the hydrogel. The present invention further relates to methods of local treatment of diseases and to methods for preparing the first peptide and the hydrogels.

## Claims

### Claim 1

A hydrogel comprising a first peptide having the general formula
B*-Zp-(X)m-(Y)n-Z′q or Zp-(X)m-(Y)n-Z′q-B* or B*-Zp-(X)m-(Y)n-Z′q-B*
wherein
B* is a bioactive agent comprising anti-cancer therapeutic;
X is, at each occurrence, independently selected from an aliphatic amino acid, an aliphatic amino acid derivative and a glycine;
Y is, at each occurrence, independently selected from a polar amino acid and a polar amino acid derivative;
Z is an N-terminal protecting group;
Z′ is a C-terminal protecting group;
m is an integer selected from 2 to 6;
n is selected from 1 or 2; and
p and q are independently selected from 0 or 1,
wherein said hydrogel further comprises a second peptide having the general formula
Z-(X)m-(Y)n-Z′q,
wherein said bioactive agent is covalently linked to the N-terminus and/or the C-terminus of said first peptide by means of a click chemistry reaction and the release of the bioactive agent is triggered via hydrolysis of the hydrogel, and wherein the covalent attachment of the bioactive agent to the first peptide and the release of the bioactive agent via hydrolysis are capable of achieving sustained or controlled release delivery of said bioactive agent wherein said second peptide is used as a matrix, and wherein the hydrogel comprises 10 wt % of the first peptide and 90 wt % of the second peptide.

### Claim 2 (depends on 1)

The hydrogel of claim 1, wherein said first peptide has the general formula
B*-(X)m-(Y)n-Z′q.

### Claim 3 (depends on 1)

The hydrogel of claim 1, wherein said anti-cancer therapeutic is selected from the group consisting of nucleic acids, DNA, RNA, small RNAs, miRNA, mRNA, siRNA, rRNA, snRNA, snoRNA and analogs thereof, (poly)peptides, peptidomimetics, neutral or anionic or cationic polymers, virus particles, (poly)saccharides, oligosaccharides, glycans, vitamins, hormones, steroids, growth factors, sialic acids, antigens, antibiotics, anti-inflammatory molecules, vaccines, drugs, prodrugs, catechols, biotin, lipids and lipid analogs, antibodies, nanoparticles, organometallic compounds and other organic or inorganic compounds, complexes, composites and nanomaterials.

### Claim 4 (depends on 1)

The hydrogel of claim 1, wherein said anti-cancer therapeutic is selected from the group consisting of oxaliplatin, cisplatin, carboplatin, doxorubicin, daunorubicin, clyclophosphamide, 5-fluorouracile, chlorambucil, vincristine, methotrexate, hydroxyurea, bleomecyn, topotecan, irinotecan, dactinomycin, docetaxel, vinblastine, paclitaxel, imatinib, herceptin and other monoclonal or polyclonal antibodies.

### Claim 5 (depends on 1)

The hydrogel of claim 1, wherein said bioactive agent is covalently linked to the N-terminus and/or the C-terminus of said first peptide via a linking group comprising a moiety selected from the group consisting of a triazole group, a cyclohexene group, a thioether group, a succinimide group, an isoxazole group and analogs thereof, a isoxazolidine group and analogs thereof, and a pyrazoline group.

### Claim 6 (depends on 1)

The hydrogel of claim 1, wherein said click chemistry reaction is a [3+2] cycloaddition reaction, a Diels-Alder reaction, a thiol-ene reaction, a thiol-Michael addition reaction, a thiol-vinylsulfone reaction, a Staudinger reaction.

### Claim 7 (depends on 1)

The hydrogel of claim 1, wherein the hydrophobicity decreases from the N-terminus to the C-terminus of said peptide.

### Claim 8 (depends on 1)

The hydrogel of claim 1, wherein said aliphatic amino acid and aliphatic amino acid derivative are selected from the group consisting of alanine (Ala, A), homoallylglycine, homopropargylglycine, isoleucine (Ile, I), norleucine, leucine (Leu, L) and valine (Val, V).

### Claim 9 (depends on 1)

The hydrogel of claim 1, wherein said polar amino acid and polar amino acid derivative are selected from the group consisting of aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), glutamine (Gln, Q), 5-N-ethyl-glutamine (theanine), citrulline, thio-citrulline, cysteine (Cys, C), homocysteine, methionine (Met, M), ethionine, selenomethionine, telluromethionine, serine (Ser, S), homoserine, arginine (Arg, R), homoarginine, threonine (Thr, T), allo-threonine, lysine (Lys, K), hydroxylysine, N(6)-carboxymethyllysine, ornithine (Orn), 2,4-diaminobutyric acid (Dab), 2,4-diaminopropionic acid (Dap), histidine (His, H), azido-alanine, azido-homoalanine, azido-ornithine and azido-lysine.

### Claim 10 (depends on 1)

The hydrogel of claim 1, wherein m is selected from 2 to 5.

### Claim 11 (depends on 1)

The hydrogel of claim 1, wherein m+n is <7 or m+n is <6.

### Claim 12 (depends on 1)

The hydrogel of claim 1, wherein (X)m-(Y)n is selected from the group consisting of
SEQ ID NO: 1
LIVAGDD
SEQ ID NO: 2
LIVAGDE
SEQ ID NO: 3
LIVAGED
SEQ ID NO: 4
LIVAGEE
SEQ ID NO: 5
LIVAGKC
SEQ ID NO: 6
LIVAGSC
SEQ ID NO: 7
AIVAGKC
SEQ ID NO: 8
AIVAGSC
SEQ ID NO: 9
LIVAGC
SEQ ID NO: 10
LIVAGD
SEQ ID NO: 11
ILVAGD
SEQ ID NO: 12
LIVAAD
SEQ ID NO: 13
LAVAGD
SEQ ID NO: 14
AIVAGD
SEQ ID NO: 15
LIVAGE
SEQ ID NO: 16
LIVAGK
SEQ ID NO: 17
LIVAGS
SEQ ID NO: 18
ILVAGS
SEQ ID NO: 19
AIVAGS
SEQ ID NO: 20
LIVAGT
SEQ ID NO: 21
AIVAGT
SEQ ID NO: 22
LIVAD
SEQ ID NO: 23
LIVGD
SEQ ID NO: 24
IVAD
SEQ ID NO: 25
IIID
SEQ ID NO: 26
IIIK
SEQ ID NO: 43
IVD
SEQ ID NO: 44
IID
SEQ ID NO: 45
LVE
SEQ ID NO: 46
IVE
SEQ ID NO: 47
LVD
SEQ ID NO: 48
VIE
SEQ ID NO: 49
VID
SEQ ID NO: 50
VLD
SEQ ID NO: 51
VLE
SEQ ID NO: 52
LLE
SEQ ID NO: 53
LLD
SEQ ID NO: 54
IIE
SEQ ID NO: 55
IVK
SEQ ID NO: 56
IV(Orn)
SEQ ID NO: 57
IV(Dab)
SEQ ID NO: 58
IV(Dap)
SEQ ID NO: 59
IVS
SEQ ID NO: 60
LVS
SEQ ID NO: 61
LVK
SEQ ID NO: 62
LV(Orn)
SEQ ID NO: 63
LV(Dab)
SEQ ID NO: 64
LV(Dap)
SEQ ID NO: 27
ILVAGK
SEQ ID NO: 28
ILVAG(Orn)
SEQ ID NO: 29
ILVAG(Dab)
SEQ ID NO: 30
ILVAG(Dap)
SEQ ID NO: 31
ILVAGS
SEQ ID NO: 32
ILVAGKC
SEQ ID NO: 33
AIVAGK
SEQ ID NO: 34
AIVAG(Orn)
SEQ ID NO: 35
AIVAG(Dab)
SEQ ID NO: 36
AIVAG(Dap)
SEQ ID NO: 37
LIVAG(Orn)
SEQ ID NO: 38
LIVAG(Dab)
SEQ ID NO: 39
LIVAG(Dap)
SEQ ID NO: 40
III(Orn)
SEQ ID NO: 41
III(Dab)
and
SEQ ID NO: 42
III(Dap).

### Claim 13 (depends on 1)

The hydrogel of claim 1, wherein said N-terminal protecting group is an acetyl group.

### Claim 14 (depends on 1)

The hydrogel of claim 1, wherein said N-terminal protecting group is a peptidomimetic molecule, including natural and synthetic amino acid derivatives, wherein the N-terminus of said peptidomimetic molecule may be modified with a functional group selected from the group consisting of carboxylic acid, amide, alcohol, aldehyde, amine, imine, nitrile, an urea analog, thiol, phosphate, carbonate, sulfate, nitrate, maleimide, vinyl sulfone, azide, alkyne, alkene, carbohydrate, imide, peroxide, ester, thioester, aryl, ketone, sulphite, nitrite, phosphonate and silane.

### Claim 15 (depends on 1)

The hydrogel of claim 1, wherein the C-terminal protecting group is an amide group comprising the formula —CONHR or —CONRR′, with R and R′ being selected from the group consisting of H, unsubstituted or substituted alkyls, and unsubstituted or substituted aryls.

### Claim 16 (depends on 1)

The hydrogel of claim 1, wherein the C-terminal protecting group is an ester group comprising the formula —CO2R, with R being selected from the group consisting of H, unsubstituted or substituted alkyls, and unsubstituted or substituted aryls.

### Claim 17 (depends on 1)

The hydrogel of claim 1, wherein said C-terminal protecting group is a peptidomimetic molecule, including natural and synthetic amino acid derivatives, wherein the C-terminus of said peptidomimetic molecule may be modified with a functional group selected from the group consisting of carboxylic acid, amide, alcohol, aldehyde, amine, imine, nitrile, an urea analog, thiol, phosphate, carbonate, sulfate, nitrate, maleimide, vinyl sulfone, azide, alkyne, alkene, carbohydrate, imide, peroxide, ester, thioester, aryl, ketone, sulphite, nitrite, phosphonate and silane.

### Claim 18 (depends on 1)

The hydrogel of claim 1, wherein said second peptide is capable of forming said hydrogel via self-assembly.

### Claim 19 (depends on 1)

The hydrogel of claim 1, wherein said first peptide is capable of forming said hydrogel via self-assembly.

### Claim 20 (depends on 1)

The hydrogel of claim 1, wherein the hydrogel is characterized by a storage modulus G′ to loss modulus G″ ratio that is greater than 2.

### Claim 21 (depends on 1)

The hydrogel of claim 1, wherein the hydrogel is characterized by a storage modulus G′ in the range of from 100 Pa to 100,000 Pa at a frequency in the range of from 0.001 Hz to 16 Hz.

### Claim 22 (depends on 1)

The hydrogel of claim 1, wherein the hydrogel is injectable and gels in situ.

### Claim 23 (depends on 1)

A device for drug delivery comprising a hydrogel of claim 1.

### Claim 24 (depends on 1)

An implant comprising a hydrogel of claim 1.

### Claim 25 (depends on 1)

A pharmaceutical or cosmetic composition comprising a hydrogel of claim 1.

### Claim 26 (depends on 1)

A kit comprising a first container with a first peptide as defined in claim 1 and a second container with an aqueous solution, wherein said first container further contains a second peptide as defined in claim 1, or wherein said kit further comprises a third container with a second peptide as defined in claim 1.

### Claim 27 (depends on 1)

The hydrogel according to claim 1, wherein p is 0.

### Claim 28 (depends on 6)

The hydrogel according to claim 6, wherein said click chemistry reaction further comprises 1,3-dipolar cycloaddition reaction.

### Claim 29 (depends on 28)

The hydrogel according to claim 28, wherein said 1,3-dipolar cycloaddition reaction is selected from the group comprising Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction and strained promoted 1,3-dipolar cycloaddition reaction.

### Claim 30 (depends on 18)

The hydrogel according claim 18, wherein the self-assembly is an antiparallel self-assembly.

### Claim 31 (depends on 19)

The hydrogel according to claim 19, wherein the self-assembly is an antiparallel self-assembly.

### Claim 32 (depends on 21)

The hydrogel according to claim 21, wherein the frequency is in the range of from 0.01 Hz to 0.2 Hz.

### Claim 33 (depends on 23)

The hydrogel according to claim 23, wherein the drug delivery is sustained or controlled release drug delivery.