# US 11,305,021 B2 · Membrane-lytic block copolymers

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- Number: US 11,305,021 B2
- Document ID: US11305021B2
- Status: Granted
- Kind: B2
- Application number: 16/323,025
- Filed: 2017-08-04
- Published: 2019-06-13
- Granted: 2022-04-19
- Earliest claimed: 2016-08-04
- Assignees: University of Washington
- Applicants: University of Washington
- Inventors: Yilong Cheng; Suzie H. Pun
- Web page: https://patentgrep.com/patent/US11305021B2

## CPC

- A61K 47/6907: {the form being a microemulsion, nanoemulsion or micelle}
- A61K 47/58: obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
- A61P 35/00: Antineoplastic agents
- A61K 38/10: Peptides having 12 to 20 amino acids {(A61K38/043 - A61K38/046 take precedence)}
- A61K 38/1761: {Apoptosis related proteins, e.g. Apoptotic protease-activating factor-1 (APAF-1), Bax, Bax-inhibitory protein(s)(BI; bax-I), Myeloid cell leukemia associated protein (MCL-1), Inhibitor of apoptosis [IAP] or Bcl-2}
- C08L 33/12: Homopolymers or copolymers of methyl methacrylate
- 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
- C08L: COMPOSITIONS OF MACROMOLECULAR COMPOUNDS (compositions based on polymerisable monomers C08F, C08G; artificial filaments or fibres D01F; textile treating compositions D06)

## Abstract

Membrane-lytic block copolymers, micellar assemblies, pharmaceutical compositions, and related methods are described.

## Claims

### Claim 1

A block copolymer of formula (II):
wherein
each A1 is a hydrophilic monomer-derived unit,
each A2 is a monomer-derived unit that is either independently neutral or charged at pH 10 or less;
each A3 is a monomer-derived unit including a therapeutic agent;
each A4 is a monomer-derived unit with a pKa ranging from about pH 4 to about pH 7;
each A5 is a monomer-derived unit including a therapeutic agent;
A6 is a monomer-derived unit including a membrane-lytic peptide;
T1 is absent or a targeting group;
T2 is absent, a membrane-lytic peptide, or a therapeutic agent;
m is from 1 to about 10,000;
n is from 0 to about 10,000;
o is from 0 to about 10,000;
p is from 1 to about 10,000;
q is from 0 to about 10,000; and
r is from 1 to about 10,000.

### Claim 2 (depends on 1)

The block copolymer of claim 1, wherein A1, A2 or A3 independently comprise repeating units derived from a monomer selected from the group consisting of 2-(dimethylamino)ethyl methacrylate, 2-dimethylaminoethyl acrylate, (3-acrylamidopropyl)trimethylammonium chloride, N-(3-aminopropyl) methacrylamide, N,N-diethylacrylamide, N,N-diethylmethacrylamide, N,N-dimethylacrylamide, N-[3-(dimethylamino)propyl]methacrylamide, 2-aminoethyl methacrylate, 2-(diethylamino)ethyl methacrylate, 2-(dimethylamino)ethylmethacrylate, [2-hydroxy-3-(2-aminoethyl)amino]propyl methacrylate, [3-methacryloylamino)propyl]trimethylammonium chloride, and L-lysine.

### Claim 3 (depends on 1)

The block copolymer of claim 1, wherein A1, A2 or A3 independently comprise repeating units derived from a monomer selected from the group consisting of methacrylic acid, acrylic acid, dimethylmaleic anhydride modified N-(3-aminopropyl) methacrylamide, and 2-aminoethyl methacrylate.

### Claim 4 (depends on 1)

The block copolymer of claim 1, wherein A1, A2 or A3 independently comprise repeating units derived from a monomer selected from the group consisting of oligo(ethylene glycol), hydroxypropylmethacrylamide, 2-hydroxyethyl methacrylate, N-isopropylacrylamide, 3-glucanoamidopropyl methacrylamide, 2-lactobionamidoethyl methacrylamide, betaine, phosphocholine, sulfobetaine and carboxybetaine.

### Claim 5 (depends on 1)

The block copolymer of claim 1, wherein A4, A5 or A6 independently comprise repeating units derived from a monomer selected from the group consisting of 2-diisopropylaminoethyl methacrylate, 2-(pentamethyleneimino)ethyl methacrylate, 2-(hexamethyleneimino)ethyl methacrylate, 2-(dipropylamino) ethyl methacrylate, 2-(dibutylamino) ethyl methacrylate, 2-(dipentylamino) ethyl methacrylate and 2-(ethylpropylamino) ethyl methacrylate.

### Claim 6 (depends on 1)

The block copolymer of claim 1, wherein the membrane-lytic peptide is covalently linked to A6 through a linker.

### Claim 7 (depends on 6)

The block copolymer of claim 6, wherein the linker is selected from a carbon-carbon bond, an oligonucleotide, an ester-containing fragment, an amide-containing fragment or a disulfide-containing fragment.

### Claim 8 (depends on 6)

The block copolymer of claim 6, wherein the linker is a disulfide bridge.

### Claim 9 (depends on 1)

The block copolymer of claim 1, wherein the membrane-lytic peptide is selected from the group consisting of melittin, peptides from adeno virus protein VI, GALA, KALA, EGLA, JTS1, Gramicidin S, HGP peptide (sequence LLGRRGWEVLKYWWNLLQYWSQEL) (SEQ ID NO:1), sHGP peptide (sequence RGWEVLKYWWNLLQY) (SEQ ID NO:2), TAT peptide (sequence GRKKRRQRRRPQ) (SEQ ID NO:3), oligoarginine, CaLL (KWKLFKKIFKRIVQRIKDFLR) (SEQ ID NO:4), hadrurin (GILDTIKSIASKVWNSKTVQDLKRKGINWVANKLGVSPQAA) (SEQ ID NO:5), cupiennin 1a (GFGALFKFLAKKVAKTVAKQAAKQGAKYVVNKQME) (SEQ ID NO:6), crabolin (Fmoc-FLALILRKIVTAL-CONH2) (SEQ ID NO:7), IsCT (ILGKIWEGIKSLF-NH2) (SEQ ID NO:8), HsAP (SGTSEKERESGRLLGVVKRLIVCFRSPFP-NH2) (SEQ ID NO:9), Pandinin2 (FWGALAKGALKLIPSLFSSFSKKD) (SEQ ID NO:10), Ponericin (WLGSALKIGAKLLPSVVGLFKKKKQ) (SEQ ID NO:11), UyCT5 (IWSAIWSGIKGLL-NH2) (SEQ ID NO:12) and hemagglutinin.

### Claim 10 (depends on 1)

The block copolymer of claim 1, having the structure of Formula (IIa):
wherein
each A1 is a hydrophilic monomer-derived unit,
each A2 is a monomer-derived unit that is neutral or charged at pH of 10.0 or less;
each A4 is a monomer-derived unit with a pKa ranging from about pH 4 to about pH 7;
each A3′, A5' and A6′ is independently
wherein
each X independently comprises repeating units comprising an alkylene, arylene, disulfide, alkylene oxide or propane-2,2-diol;
wherein each X is optionally substituted with —CO2R1, L1-D1, L2-D2 or, L3-LP, wherein R1 is a hydrophilic group,
each R is independently hydrogen, —CO2R2, —CN, alkyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, where R2 is a hydrophilic group, L1-D1, L2-D2 or, L3-LP, a is 1- 4, and b is 2- 4;
each of L1, L2 and L3 is independently absent or a linker;
each LP is a membrane-lytic peptide;
each of D1 and D2 is independently a therapeutic agent;
T1 is absent or a targeting group;
T2 is absent, a membrane-lytic peptide, a therapeutic agent;
m is from 1 to about 10,000;
n is from 0 to about 10,000;
o is from 0 to about 10,000;
p is from 1 to about 10,000;
q is from 0 to about 10,000; and
r is from 1 to about 10,000.

### Claim 11 (depends on 10)

The block copolymer of claim 10, having the structure of Formula (IIIb):
wherein m′ is from 1 to about 20.

### Claim 12 (depends on 10)

The block copolymer of claim 10, having the structure of Formula (IVb):
wherein m′ is from about 1 to about 20; and each R′ is independently hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, alkyl-cycloalkyl, alkyl-heterocycloalkyl or alkyl-aryl.

### Claim 13 (depends on 1)

A micellar assembly comprising a plurality of copolymers according to claim 1.

### Claim 14 (depends on 1)

A pharmaceutical composition comprising:
at least one block copolymer according to claim 1; and
a therapeutic agent reversibly associated with at least one of A1, A2 or A3.

### Claim 15 (depends on 14)

A method of intracellularly delivering a therapeutic agent comprising:
administering a pharmaceutical composition according to claim 14 to a subject, wherein the pharmaceutical composition is endocytosed into the endosome and the pharmaceutical composition, thereby, releases the therapeutic agent into the endosome.

### Claim 16

A block copolymer of formula (II):
wherein
each A1 is a hydrophilic monomer-derived unit,
each A2 is a monomer-derived unit that is either independently neutral or charged at pH 10 or less;
each A3 is a monomer-derived unit including a therapeutic agent;
each A4 is a monomer-derived unit with a pKa ranging from about pH 4 to about pH 7;
each A5 is a monomer-derived unit including a therapeutic agent;
A6 is a monomer-derived unit including a membrane-lytic peptide;
T1 is absent or a targeting group;
T2 is a membrane-lytic peptide;
m is from 1 to about 10,000;
n is from 0 to about 10,000;
o is from 0 to about 10,000;
p is from 1 to about 10,000;
q is from 0 to about 10,000; and
r is from 0 to about 10,000.

### Claim 17 (depends on 16)

The block copolymer of claim 16, wherein the membrane-lytic peptide is selected from the group consisting of melittin, peptides from adeno virus protein VI, GALA, KALA, EGLA, JTS1, Gramicidin S, HGP peptide (sequence LLGRRGWEVLKYWWNLLQYWSQEL) (SEQ ID NO:1), sHGP peptide (sequence RGWEVLKYWWNLLQY) (SEQ ID NO:2), TAT peptide (sequence GRKKRRQRRRPQ) (SEQ ID NO:3), oligoarginine, CaLL (KWKLFKKIFKRIVQRIKDFLR) (SEQ ID NO:4), hadrurin (GILDTIKSIASKVWNSKTVQDLKRKGINWVANKLGVSPQAA) (SEQ ID NO:5), cupiennin 1a (GFGALFKFLAKKVAKTVAKQAAKQGAKYVVNKQME) (SEQ ID NO:6), crabolin (Fmoc-FLALILRKIVTAL-CONH2) (SEQ ID NO:7), IsCT (ILGKIWEGIKSLF-NH2) (SEQ ID NO:8), HsAP (SGTSEKERESGRLLGVVKRLIVCFRSPFP-NH2) (SEQ ID NO:9), Pandinin2 (FWGALAKGALKLIPSLFSSFSKKD) (SEQ ID NO:10), Ponericin (WLGSALKIGAKLLPSVVGLFKKKKQ) (SEQ ID NO:11), UyCT5 (IWSAIWSGIKGLL-NH2) (SEQ ID NO:12) and hemagglutinin.

### Claim 18 (depends on 16)

A micellar assembly comprising a plurality of copolymers according to claim 16.

### Claim 19 (depends on 16)

A pharmaceutical composition comprising:
at least one block copolymer according to claim 16; and
a therapeutic agent reversibly associated with at least one of A1, A2 or A3.

### Claim 20 (depends on 19)

A method of intracellularly delivering a therapeutic agent comprising:
administering a pharmaceutical composition according to claim 19 to a subject, wherein the pharmaceutical composition is endocytosed into the endosome and the pharmaceutical composition, thereby, releases the therapeutic agent into the endosome.

### Claim 21

A block copolymer of formula (II):
wherein
each A1 is a hydrophilic monomer-derived unit, each A2 is a monomer-derived unit that is either independently neutral or charged at pH 10 or less;
each A3 is a monomer-derived unit including a therapeutic agent;
each A4 is a monomer-derived unit with a pKa ranging from about pH 4 to about pH 7;
each A5 is a monomer-derived unit including a therapeutic agent;
A6 is a monomer-derived unit including a membrane-lytic entity;
T1 is absent or a targeting group;
T2 is absent, a membrane-lytic entity, or a therapeutic agent;
m is from 1 to about 10,000;
n is from 0 to about 10,000;
o is from 0 to about 10,000;
p is from 1 to about 10,000;
q is from 0 to about 10,000; and
r is from 1 to about 10,000,
wherein the membrane-lytic entity is selected from the group consisting of melittin, peptides from adeno virus protein VI, GALA, KALA, EGLA, JTS1, Gramicidin S, HGP peptide (sequence LLGRRGWEVLKYWWNLLQYWSQEL) (SEQ ID NO:1), sHGP peptide (sequence RGWEVLKYWWNLLQY) (SEQ ID NO:2), TAT peptide (sequence GRKKRRQRRRPQ) (SEQ ID NO:3), oligoarginine, CaLL (KWKLFKKIFKRIVQRIKDFLR) (SEQ ID NO:4), hadrurin (GILDTIKSIASKVWNSKTVQDLKRKGINWVANKLGVSPQAA) (SEQ ID NO:5), cupiennin 1a (GFGALFKFLAKKVAKTVAKQAAKQGAKYVVNKQME) (SEQ ID NO:6), crabolin (Fmoc-FLALILRKIVTAL-CONH2) (SEQ ID NO:7), IsCT (ILGKIWEGIKSLF-NH2) (SEQ ID NO:8), HsAP (SGTSEKERESGRLLGVVKRLIVCFRSPFP-NH2) (SEQ ID NO:9), Pandinin2 (FWGALAKGALKLIPSLFSSFSKKD) (SEQ ID NO:10), Ponericin (WLGSALKIGAKLLPSVVGLFKKKKQ) (SEQ ID NO:11), UyCT5 (IWSAIWSGIKGLL-NH2) (SEQ ID NO:12) and hemagglutinin.