# US 10,369,114 B2 · Cholestosome vesicles for incorporation of molecules into chylomicrons

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- Number: US 10,369,114 B2
- Document ID: US10369114B2
- Status: Granted
- Kind: B2
- Application number: 15/603,992
- Filed: 2017-05-24
- Published: 2018-03-01
- Granted: 2019-08-06
- Earliest claimed: 2013-03-14
- Assignees: Therasyn Sensors, Inc.
- Applicants: TheraSyn Sensors, Inc.
- Inventors: Jerome J. Schentag; Mary P. McCourt; Lawrence Mielnicki; Julie Hughes
- Web page: https://patentgrep.com/patent/US10369114B2

## CPC

- A61K 9/51: Nanocapsules; {Nanoparticles; (nanotubes A61K9/0092; polymeric micelles A61K9/1075; polymersomes A61K9/1273; pure drug nanoparticles A61K9/14; drug nanoparticles with adsorbed surface modifiers A61K9/141; conjugates, e.g. between drug and non-active nanoparticles, A61K47/50; preparations for in vivo diagnosis A61K49/00; with radioactive substances A61K51/00)}
- A61K 9/127: Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
- A61K 9/1275: Lipoproteins or protein-free species thereof, e.g. chylomicrons; Artificial high-density lipoproteins [HDL], low-density lipoproteins [LDL] or very-low-density lipoproteins [VLDL]; Precursors thereof
- A61K 31/12: Ketones
- A61K 31/137: Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine {or methadone}
- A61K 31/19: Carboxylic acids, e.g. valproic acid (salicylic acid A61K31/60)
- A61K 31/546: containing further heterocyclic rings, e.g. cephalothin
- A61K 31/7034: attached to a carbocyclic compound, e.g. phloridzin
- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- A61K 38/14: Peptides containing saccharide radicals; Derivatives thereof {, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin}
- A61K 38/28: Insulins
- A61K 39/29: Hepatitis virus
- A61K 39/3955: {against proteinaceous materials, e.g. enzymes, hormones, lymphokines}
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61P 3/06: Antihyperlipidemics
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 5/06: of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
- A61P 5/50: for increasing or potentiating the activity of insulin
- A61P 9/12: Antihypertensives
- A61P 11/06: Antiasthmatics
- A61P 31/04: Antibacterial agents
- A61P 31/12: Antivirals
- A61P 31/14: for RNA viruses
- 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 is directed to a cargo-loaded cholesteryl ester nanoparticle with a hollow compartment (“cholestosome”) consisting essentially of at least one non-ionic cholesteryl ester and one or more encapsulated active molecules which cannot appreciably pass through an enterocyte membrane in the absence of said molecule being loaded into said cholestosome, the cholestosome having a neutral surface and having the ability to pass into enterocytes in the manner of orally absorbed nutrient lipids using cell pathways to reach the golgi apparatus. Pursuant to the present invention, the novel cargo loaded cholestosomes according to the present invention are capable of depositing active molecules within cells of a patient or subject and effecting therapy or diagnosis of the patient or subject.

## Claims

### Claim 1

A pharmaceutical composition comprising a population of cargo-loaded vesicles wherein at least one pharmaceutically active agent is encapsulated in the core of said vesicles by a surface layer, wherein said surface layer comprises at least one cholesteryl ester obtained from cholesterol and a C8-C26 fatty acid and said ester(s) enable intact vesicles to pass through cell membranes by passive diffusion or facilitated diffusion and thereby arrive in an intact form in the cytoplasm of said cells wherein said pharmaceutically active agents are released in said cytoplasm by the intracellular action of cholesteryl ester hydrolase on the surface layer of said vesicles, said cargo-loaded vesicle being capable of delivering said pharmaceutically active agents within body cells of a patient administered said cargo-loaded vesicles to an intracellular concentration level at least 2 times the level obtainable by said pharmaceutically active agent in the absence of said vesicle.

### Claim 2 (depends on 1)

The cargo loaded vesicles of claim 1 adapted for oral administration, wherein said vesicles are stable to stomach acid and are not broken by cholesteryl ester transporters on the surface of duodenal enterocytes during absorption of said cargo-loaded vesicles after oral administration of said vesicles to said patient or subject, whereby said cargo-loaded vesicles enter said duodenal enterocytes intact and said duodenal enterocytes add said intact cargo-loaded vesicles into chylomicrons to produce transformed chylomicrons for transport of said cargo-loaded vesicles to body cells of said patient or subject, and wherein said pharmaceutically active agent(s) in said cargo-loaded vesicle(s) is(are) not detected by said enterocytes during passage of said vesicle through the cell membranes of said enterocytes.

### Claim 3 (depends on 2)

The pharmaceutical composition of claim 2, wherein said composition is contained within a capsule for oral use, and said capsule is optionally enterically coated for release at one or more locations in the human gastrointestinal tract.

### Claim 4 (depends on 2)

The pharmaceutical composition of claim 2, wherein said chylomicrons loaded with said cargo loaded vesicles bind to cell surface APO receptors and following the binding of the chylomicrons to cells, the cell takes intact vesicles into its cytoplasm, whereupon the vesicles release said pharmaceutically active agents in said cell cytoplasm by the action of intracellular cholesteryl ester hydrolases on said surface cholesteryl esters of said vesicles.

### Claim 5 (depends on 1)

The pharmaceutical composition of claim 1 wherein the mass ratio of the pharmaceutically active agent to said one or more cholesteryl esters is between 4:96 to 96:4, said cargo-loaded vesicle being capable of delivering said pharmaceutically active agents within said body cells of said patient to an intracellular concentration level at least 10 times the level obtainable by said pharmaceutically active agent in the absence of said vesicle.

### Claim 6 (depends on 1)

The pharmaceutical composition of claim 1 adapted for intravenous or subcutaneous injection wherein the mass ratio of the pharmaceutically active agent to said one or more cholesteryl esters is between 4:96 to 96:4 wherein said vesicles pass through cell membranes, enter the cytoplasm of the cells and release said one or more pharmaceutically active agents by the of cellular cholesteryl ester hydrolase on the surface layer of said vesicles, said pharmaceutically active agents obtaining an intracellular concentration level at least 10 times the level obtainable in the absence of said vesicle.

### Claim 7 (depends on 6)

The pharmaceutical composition of claim 6 wherein said pharmaceutically active agents arrive at intracellular concentrations between 10 and 100 times the level obtainable in the absence of said vesicle.

### Claim 8 (depends on 1)

The pharmaceutical composition of claim 1 wherein said composition is adapted for topical, intravenous, subcutaneous, oral, inhalation or intravaginal administration.

### Claim 9 (depends on 1)

The pharmaceutical composition according to claim 1, wherein said pharmaceutically active agent is selected from the group consisting of a hydrophilic peptide, a protein, a polypeptide, a polynucleotide and mixtures thereof.

### Claim 10 (depends on 1)

The composition according to any of claims 1 wherein said intact loaded vesicles enter cells in said patient or subject and said cells use cholesteryl ester hydrolases to release said pharmaceutically active agents from said vesicles, and whereby said cells optionally eject a portion of said intact vesicles from said cells into the extracellular fluid surrounding said cells.

### Claim 11

A pharmaceutical composition in oral dosage form of an enterically coated tablet or capsule for release of said composition at one or more intestinal locations to include duodenum, jejunum, ileum and colon comprising a cargo-loaded vesicle having a neutral surface comprising a core containing a pharmaceutically active agent and a surface layer surrounding said core and said active agent, whereby said surface layer consists of one or more non-ionic cholesteryl esters produced from cholesterol and at least one C8-C26 fatty acid, wherein the mass ratio of the pharmaceutically-active agent to said one or more cholesteryl esters in said cargo-loaded vesicle is between about 4:96 to about 96:4, said cargo-loaded vesicle being capable of delivering said active agent within cells of a patient or subject to a concentration level of at least 2 times to at least 1000 times the level obtainable by oral administration of said pharmaceutically active agent that is not contained in said vesicles.

### Claim 12 (depends on 11)

The pharmaceutical composition of claim 11, wherein said pharmaceutical composition comprises unilamellar vesicles in which between 10% to 96% of the vesicles' volume is occupied by one or more of the pharmaceutically-active agents.

### Claim 13 (depends on 1)

The pharmaceutical composition of claim 1, wherein the vesicle has a diameter ranging from 100 nm to 10,000 nm.

### Claim 14 (depends on 1)

A method of delivering one or more pharmaceutically active agents to a target inside a cell of a patient or subject, said method comprising administering to said patient or subject a population of cargo-loaded vesicles according to claim 1, wherein said vesicles are taken up by cells and said pharmaceutically active agents are released intracellularly within the patient or subject, wherein the concentration of said active pharmaceutical agents within the cell being substantially greater than when said agent is delivered to said cells in the absence of said vesicles.

### Claim 15 (depends on 1)

A method of manufacturing a plurality of cholesteryl ester vesicles loaded with pharmaceutically active agent molecules wherein the outer surface coating of said vesicles comprises at least one cholesteryl ester obtained from cholesterol and a C8-C26 fatty acid according to claim 1, whereby said method comprising the steps of:
a. dissolving and mixing said cholesteryl esters in a non-polar solvent;
b. evaporating said non-polar solvent on the inner surface of a round bottom flask;
c. adding an aqueous mixture comprising one or more pharmaceutically active agents to said round bottom flask and continuously sonicating said mixture; and
d. forming a homogenous dispersion of vesicles during sonication of said mixture, wherein said vesicles contain one or more pharmaceutically active agents and said vesicle volume is occupied between about 10% and about 96% of the total vesicle volume by one or more of the pharmaceutically active agent molecules.

### Claim 16 (depends on 15)

The method according to claim 15 wherein said pharmaceutically active agent comprises at least one agent selected from the group consisting of a hydrophilic peptide, human growth hormone, prolactin, oxytocin, calcitonin, bovine growth hormone, porcine growth hormone, Ghrelin, GLP-1, liraglutide, dulaglutide, semaglutide, lixisenatide, albiglutide, or a derivative thereof, PYY36, Oxyntomodulin, an antibiotic, GLP-2, Glucagon, an interferon and an insulin.

### Claim 17 (depends on 16)

The method according to claim 16 wherein said insulin is regular insulin, NPN insulin, Lente insulin, recombinant insulin, insulin glargine, insulin lispro, novolog and insulin degludec.

### Claim 18 (depends on 16)

The method according to claim 16 wherein said pharmaceutically active agent comprises GLP-1 and an insulin and optionally includes an inhibitor of intracellular metabolism of one or both of said GLP-1 and said insulin.

### Claim 19 (depends on 15)

The method according to claim 15 wherein said pharmaceutically active agent comprises one or more monoclonal antibodies selected from the group consisting of Adalimumab; Abciximab; Alemtuzumab; Bevacizumab; Bapineuzumab; Cetuximab; Etaaercept; Elotuzumab; Gemtuzumab; Inotuzumab, mipomersen; MabThera/Rituxan; Natalizumab; Mepolizumab; Necitumumab; Palivizumab; Panitumumab; RN316/Bococizumab; REGN727/Alirocumab; Evolocumab; Solanezumab; Trastuzumab; Tositumomab; T-DM1 linked to trastuzumab); Vemurafenib; Atorolimumab; Belimumab; Brodalumab; Carlumab; Dupilumab; Fresolimumab; Golimumab; ipilimumab, Lerdelimumab; Lirilumab; vrilimumab; Metelimumab; Morolimumab; Namilumab; Oxelumab; Placulumab; Sarilumab; Sifalimumab; Tabalumab; Ipilimumab; Tremelimumab; Nivolumab; Urelumab; Bertilimumab; Zanolimumab; Afelimomab; Elsilimomab; Faralimomab; Gavilimomab; Inolimomab; Maslimomab; Nerelimomab; Odulimomab; Telimomab; Vepalimomab; Zolimomab aritox; Basiliximab; Clenoliximab; Galiximab; Gomiliximab; Infliximab; Keliximab; Lumiliximab; Priliximab; Teneliximab; Vapaliximab; Aselizumab; Apolizumab; Benralizumab; Cedelizumab; Certolizumab pegol; Daclizumab; Eculizumab; Efalizumab; Epratuzumab; Erlizumab; Etrolizumab; Fontolizumab; Itolizumab; Lampalizumab; Ligelizumab; Mepolizumab; Mogamulizumab; Natalizumab; Ocrelizumab; Ofatumumab; Omalizumab; Ozoralizumab; Pascolizumab; Pateclizumab; Pembrolizumab, Pexelizumab; Pidilizumab; Reslizumab; Rontalizumab; Rovelizumab; Ruplizumab; Quilizumab; Samalizumab; Siplizumab; Talizumab; Teplizumab; Tocilizumab; Toralizumab; Tregalizumab; Vatelizumab; Vedolizumab; Visilizumab; Ibalizumab; Otelixizumab; Briakinumab; Canakinumab; Fezakinumab; Secukinumab; Sirukumab; Tralokinumab; Ustekinumab; Anrukinzumab; Clazakizumab; Enokizumab; Gevokizumab; Ixekizumab; Lebrikizumab; Olokizumab; Perakizumab; Tildrakizumab; Besilesomab; Fanolesomab; Lemalesomab; Sulesomab or a mixture thereof.

### Claim 20 (depends on 1)

The pharmaceutical composition of claim 1, wherein said cargo-loaded vesicles are made by a process comprising dissolving one or more of the cholesteryl esters with a nonpolar solvent, removing the non polar solvent under vacuum while introducing an aqueous phase containing said pharmaceutically active agent, and mixing said composition while removing said vesicles wherein between 10% to 96% of the vesicle's volume is occupied by one or more of the pharmaceutically-active agents.

### Claim 21

A pharmaceutical composition in parenteral dosage form comprising a population of cargo-loaded vesicles wherein at least one pharmaceutically active agent is encapsulated in the core of said vesicles by a surface layer, wherein said surface layer comprises at least one cholesteryl ester obtained from cholesterol and a C8-C26 fatty acid and wherein after administration of said vesicles to a patient, said ester(s) enables said vesicles to pass through cell membranes of cells and enter the cytoplasm of said cells wherein said one or more of said pharmaceutically active agents is released into cytoplasm by the intracellular action of cholesteryl ester hydrolase on the surface layer of said vesicles, wherein an intracellular concentration of said active pharmaceutical agents is substantially greater than when said agent is delivered to said cells in the absence of said vesicles.

### Claim 22 (depends on 21)

The pharmaceutical composition according to claim 21, wherein said pharmaceutically active agent is selected from the group consisting of a hydrophilic small molecule, a hydrophilic peptide, a protein, a polypeptide, a nucleotide and mixtures thereof.

### Claim 23 (depends on 11)

The composition according to claim 11 wherein said pharmaceutically active agent comprises at least one agent selected from the group consisting of a hydrophilic peptide, human growth hormone, prolactin, oxytocin, calcitonin, bovine growth hormone, porcine growth hormone, Ghrelin, GLP-1, liraglutide, dulaglutide, semaglutide, lixisenatide, albiglutide, or a derivative thereof, PYY36, Oxyntomodulin, GLP-2, Glucagon, an antibiotic, an interferon and an insulin.

### Claim 24 (depends on 23)

The composition according to claim 23 wherein said antibiotic is a lipopeptide antibiotic.

### Claim 25 (depends on 11)

The composition according to claim 11 wherein said pharmaceutically active agent is WAP-8294A.

### Claim 26 (depends on 21)

The composition according to claim 21 wherein said pharmaceutically active agent is an antibiotic.

### Claim 27 (depends on 26)

The composition according to claim 26 wherein said antibiotic is a lipopeptide antibiotic.

### Claim 28 (depends on 21)

The composition according to claim 21 wherein said pharmaceutically active agent is WAP-8924A.

### Claim 29

A pharmaceutical composition adapted for oral administration to the duodenum of a patient or subject comprising a population of cargo-loaded vesicles wherein at least one pharmaceutically active agent is encapsulated in the core of said vesicles by a surface layer, said surface layer consisting essentially of one or more cholesteryl esters, wherein said vesicles are stable to stomach acid and are not broken by cholesteryl ester transporters on the surface of duodenal enterocytes during absorption of said cargo-loaded vesicles after administration to said patient or subject, wherein said cargo-loaded vesicles enter said duodenal enterocytes intact and said duodenal enterocytes add said intact cargo-loaded vesicles into chylomicrons to produce transformed chylomicrons for transport of said cargo-loaded vesicles to body cells of said patient or subject, wherein said composition is optionally enterically coated, wherein the pharmaceutically active agent is at least one agent selected from the group consisting of a hydrophilic peptide, an insulin composition, human growth hormone, prolactin, oxytocin, calcitonin, GLP-1, PYY36, Oxyntomodulin, GLP-2, Glucagon, interferon, ceftaroline, WAP-8924A, vancomycin, bevacizumab, trastuzumab, adalimumab and an anti-PCSK-9 monoclonal antibody.

### Claim 30 (depends on 29)

The composition according to claim 29 wherein said pharmaceutically active agent is WAP-8924A.