# US 9,750,819 B2 · Lipid nanoparticle compositions and methods of making and methods of using the same

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- Number: US 9,750,819 B2
- Document ID: US9750819B2
- Status: Granted
- Kind: B2
- Application number: 14/403,313
- Filed: 2013-05-23
- Published: 2015-04-30
- Granted: 2017-09-05
- Earliest claimed: 2012-05-23
- Assignees: Ohio State Innovation Foundation
- Applicants: Ohio State Innovation Foundation
- Inventors: Robert J. Lee
- Web page: https://patentgrep.com/patent/US9750819B2

## CPC

- A61K 31/7088: Compounds having three or more nucleosides or nucleotides
- A61K 9/127: Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
- A61K 9/1271: Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers (liposomes as conjugates {A61K47/6911})
- A61K 9/1272: comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers (lipids as modifying agents {A61K47/543})
- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- A61K 38/08: Peptides having 5 to 11 amino acids {(A61K38/043 - A61K38/046 take precedence)}
- A61K 38/10: Peptides having 12 to 20 amino acids {(A61K38/043 - A61K38/046 take precedence)}
- A61K 38/482: {Serine endopeptidases (3.4.21)}
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61K 47/543: {Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine}
- A61K 47/544: {Phospholipids}
- A61K 47/549: {Sugars, nucleosides, nucleotides or nucleic acids}
- A61K 47/59: obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K 47/62: the modifying agent being a protein, peptide or polyamino acid
- A61K 47/643: {Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]}
- A61K 47/6907: {the form being a microemulsion, nanoemulsion or micelle}
- A61K 47/6911: {the form being a liposome}
- A61P 35/00: Antineoplastic agents
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/1135: {against oncogenes or tumor suppressor genes}
- C12N 15/1137: {against enzymes (viral enzymes C12N15/1131; receptors C12N15/1138)}
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- C12N 2310/11: Antisense
- C12N 2310/14: interfering nucleic acids [NA]
- C12Y 304/21064: Peptidase K (3.4.21.64)
- 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
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

Lipid nanoparticle formulations, methods of making, and methods of using same are disclosed.

## Claims

### Claim 1

A nanoparticle comprising:
a combination of tertiary and quaternary amine-based cationic lipids,
wherein said combination consists of the tertiary amine-cationic lipid present at about 40.0 molar percent; wherein the tertiary amine-cationic lipid comprises N-[1-(2, 3-dioleyloyx) propyl]-N—N—N-dimethyl ammonium chloride (DODMA), and the concentration of the quaternary amine cationic lipid present at about 5.0 molar percent;
and at least one single stranded oligonucleotide encapsulated within the lipid combination.

### Claim 2 (depends on 1)

The nanoparticle of claim 1, wherein the tertiary amine-cationic lipid further comprises one or more of: lipids are chosen from 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-CHOL), N,N-dimethylhexadecylamine (DMHDA), and combinations thereof.

### Claim 3 (depends on 1)

The nanoparticle of claim 1, wherein the quaternary amine-cationic lipids are selected from 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2, 3-dioleyloyx) propyl]-N—N—N-trimethyl ammonium chloride (DOTMA), dimethyldioctadecylammonium bromide (DDAB), or combinations thereof.

### Claim 4 (depends on 1)

The nanoparticle of claim 1, wherein the encapsulated single stranded oligonucleotide comprises: antisense oligonucleotides, miRs, anti-miRs, or combinations thereof.

### Claim 5 (depends on 1)

The nanoparticle of claim 1, further comprising a cationic polymer.

### Claim 6 (depends on 5)

The nanoparticle of claim 5, wherein the cationic polymer is selected from the group consisting of: spermine, dispermine, dispermine, tetraspermine, oligospermine, thermine, spermidine, dispermidine, trispermidine, oligospermidine, putrescine, polylysine, polyarginine, a polyethylenimine of branched or linear type, and polyallylamine.

### Claim 7 (depends on 1)

The nanoparticle of claim 1, further comprising a fusogenic peptide covalently bonded to, or conjugated to the lipids.

### Claim 8 (depends on 1)

The nanoparticle of claim 1, wherein the encapsulation rate is 20% or higher.

### Claim 9 (depends on 1)

The nanoparticle of claim 1, wherein the nanoparticle has a diameter under 300 nm.

### Claim 10 (depends on 1)

A nanoparticle having a diameter of less than 300 nm; comprising: the nanoparticle of claim 1; and
at least one peptide mixed with, covalently attached or conjugated to the lipids.

### Claim 11 (depends on 10)

The nanoparticle of claim 10, wherein the peptide is selected from gramicidin A, B, C, D, or S; JTS-1; proteinase K (PrK); trichorovin-Xlla; rabies virus glycoprotein; interleukin-1 β; HIV-Tat; herpes simplex virus VP22 protein; and combinations thereof.

### Claim 12 (depends on 10)

The nanoparticle of claim 10, wherein the peptide comprises an antibiotic.

### Claim 13 (depends on 12)

The nanoparticle of claim 12, wherein the antibiotic is selected from gramicidin A, B, C, D, or S.

### Claim 14 (depends on 10)

The nanoparticle of claim 10, wherein the peptide consists essentially of a lipidated JTS-1 fusogenic peptide.

### Claim 15 (depends on 14)

The nanoparticle of claim 14, wherein the lipidated JTS-1 fusogenic peptide is present at about 0 to about 30 molar percent of the total formulation.

### Claim 16 (depends on 10)

The nanoparticle of claim 10, further comprising proteinase K.

### Claim 17 (depends on 16)

The nanoparticle of claim 16, wherein the proteinase K is present at about 0 to about 30 molar percent of the total formulation.

### Claim 18 (depends on 10)

The nanoparticle of claim 10, wherein the encapsulated single stranded oligonucleotide comprises: antisense oligonucleotides, miRs, anti-miRs, or combinations thereof.

### Claim 19 (depends on 1)

The nanoparticle of claim 1, further comprising a hydrophilic polymer covalently bonded to the lipids, or conjugated to the lipids.

### Claim 20 (depends on 19)

The nanoparticle of claim 19, wherein the hydrophilic polymer is selected from polysorbate 80, D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS), mPEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N (DSPE), methoxypolyethylene glycol (PEG-DMG), dipalmitoylphosphatidlyethanolamine-PEG2000 (DPPE-PEG) or mPEG dimyristoylphosphatidylethanolamine (DMPE).

### Claim 21 (depends on 20)

The nanoparticle of claim 20, wherein the hydrophilic polymer is present at a concentration less than about 10.0 molar percent.

### Claim 22 (depends on 1)

The nanoparticle of claim 1, further comprising a ligand capable of binding to a target cell or a target molecule.

### Claim 23 (depends on 22)

The nanoparticle of claim 22, wherein the ligand is an antibody or an antibody fragment.

### Claim 24 (depends on 22)

The nanoparticle of claim 22, wherein the ligand is selected from cRGD, galatose-containing moieties, transferrin, folate, low density lipoprotein, or epidermal growth factors.

### Claim 25 (depends on 1)

The nanoparticle of claim 1, wherein the nanoparticle further encapsulates an at least one additional therapeutic agent selected from: antineoplastic agents, anti-infective agents, local anesthetics, anti-allergics, antianemics, angiogenesis, inhibitors, beta-adrenergic blockers, calcium channel antagonists, anti-hypertensive agents, anti-depressants, anti-convulsants, anti-bacterial, anti-fungal, anti-viral, anti-rheumatics, anthelminithics, antiparasitic agents, corticosteroids, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, anti-diabetic agents, anti-epileptics, anti-hemmorhagics, anti-hypertonics, antiglaucoma agents, immunomodulatory cytokines, sedatives, chemokines, vitamins, toxins, narcotics, imaging agents, and combinations thereof.

### Claim 26 (depends on 25)

The nanoparticle of claim 25, wherein the encapsulated therapeutic agent comprises a nucleic acid therapeutic agent.

### Claim 27 (depends on 26)

The nanoparticle of claim 26, wherein the nucleic acid therapeutic agent is selected from: pDNA, miRNA, anti-miRNA, antisense oligonucleotide (ASO), and combinations thereof.

### Claim 28 (depends on 26)

The nanoparticle of claim 26, wherein the nucleic acid therapeutic agent is stabilized by modifications to substituent nucleic acid base units and/or by modifying the ribose 2′ position or substituting phosphodiester linkers.

### Claim 29 (depends on 1)

The nanoparticle of claim 1, wherein the lipid nanoparticle has a diameter under about 200 nm.

### Claim 30 (depends on 19)

The nanoparticle of claim 19, wherein the hydrophilic polymer is bound to an external surface of the nanoparticle via direct connection or via a linker.

### Claim 31 (depends on 11)

The nanoparticle of claim 11, wherein the nanoparticle has an encapsulation efficiency of the molecule of at least about 40%.

### Claim 32 (depends on 1)

A pharmaceutical composition comprising the nanoparticle of claim 1, and a pharmaceutically acceptable excipient.

### Claim 33 (depends on 32)

The pharmaceutical composition of claim 32, wherein the pharmaceutical composition is a tablet, an inhalant, or a suppository.

### Claim 34 (depends on 32)

The pharmaceutical composition of claim 32, wherein the pharmaceutical composition is a sterile solution, a sterile suspension, or a lyophilized powder.

### Claim 35 (depends on 1)

A composition comprising anti-miR-221 combined nanoparticle of claim 1.

### Claim 36 (depends on 1)

A composition comprising anti-miR-155 combined with the nanoparticle of claim 1.

### Claim 37 (depends on 36)

The composition of claim 36, wherein the lipid nanoparticle comprises Lac-GLN, wherein Lac-GLN comprises a lipophilic asialoglycoprotein receptor (ASGR) targeting ligand composed of lactobionic acid (LA), bearing a galactose moiety, and linked to a phospholipid.

### Claim 38 (depends on 36)

The composition of claim 36, further including gramicidin A incorporated into the lipid nanoparticle.

### Claim 39 (depends on 36)

The composition of claim 36, wherein the anti-miR-155 has the sequence: 5′-A*C*CCCUAUCACGAUUAGCAUU*A*A-3′, SEQ ID NO. 6.

### Claim 40 (depends on 39)

The composition of claim 39, wherein the sequence contains phosphorothioate linkages (*) and 2′-O-Methyl.