# US 11,992,557 B2 · Collagenase loaded liposomes for enhancing drug delivery > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,992,557 B2 - Document ID: US11992557B2 - Status: Granted - Kind: B2 - Application number: 16/963,648 - Filed: 2019-01-23 - Published: 2021-03-04 - Granted: 2024-05-28 - Earliest claimed: 2018-01-23 - Assignees: Technion Research & Development Foundation Limited - Applicants: Technion Research & Development Foundation Limited - Inventors: Avi Schroeder; Assaf Zinger - Web page: https://patentgrep.com/patent/US11992557B2 ## CPC - A61K 9/127: Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant - A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)} - A61K 9/1271: Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers (liposomes as conjugates {A61K47/6911}) - A61K 9/1277: Preparation processes; Proliposomes - A61K 31/337: having four-membered rings, e.g. taxol - A61K 38/4886: {Metalloendopeptidases (3.4.24), e.g. collagenase} - A61P 35/00: Antineoplastic agents - C12Y 304/24007: Interstitial collagenase (3.4.24.7), i.e. matrix metalloprotease 1 or MMP1 - 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 - C12Y: ENZYMES ## Abstract Pharmaceutical compositions comprising at least one lipid-based particle encapsulating a proteolytic enzyme and a pharmaceutically acceptable carrier, wherein the proteolytic enzyme comprises at least 75% proteolytically active enzyme are provided. Methods of using same and producing same are also provided. ## Claims ### Claim 1 A pharmaceutical composition formulated for administration to a human comprising at least one lipid-based particle encapsulating at least one proteolytic enzyme in solution and a pharmaceutically acceptable carrier, wherein at least 75% of said proteolytic enzyme is proteolytically active and wherein said solution comprises a pH from 8 to 10. ### Claim 2 (depends on 1) The pharmaceutical composition of claim 1, wherein (i) said carrier is substantially devoid of ions that activate said proteolytic enzyme, (ii) said proteolytic enzyme is dissolved in an aqueous solution devoid of ions that activate said proteolytic enzyme or (iii) both (i) and (ii). ### Claim 3 (depends on 1) The pharmaceutical composition of claim 1, wherein said lipid- based particle (i) is a liposome, (ii) comprises a maximum cross-sectional area of less than 70 square microns or (iii) both (i) and (ii). ### Claim 4 (depends on 3) The pharmaceutical composition of claim 3, wherein said liposome comprises between 50 and 60% D1VIPC, 35 and 45% cholesterol and 3 and 7% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-PEG (PEG-D SPE). ### Claim 5 (depends on 1) The pharmaceutical composition of claim 1, wherein said proteolytic enzyme (i) is active against collagen, (ii) is selected from collagenase, papain and bromelain, or (iii) both (i) and (ii). ### Claim 6 (depends on 5) The pharmaceutical composition of claim 5, wherein said proteolytic enzyme is collagenase. ### Claim 7 (depends on 6) The pharmaceutical composition of claim 6, wherein said collagenase is collagenase I, collagenase II or both. ### Claim 8 (depends on 1) A method of decreasing fibrosis in a subject in need thereof, the method comprising administering to said subject the pharmaceutical composition of claim 1, thereby decreasing fibrosis in said subject in need thereof. ### Claim 9 (depends on 1) A method of increasing penetrance of a drug into a tumor in a subject in need thereof, the method comprising: a. administering to the subject a pharmaceutical composition of claim 1; and b. administering said drug to said subject, thereby increasing the penetrance of said drug into said tumor. ### Claim 10 (depends on 9) The method of claim 9, wherein said administering said drug occurs at a time after administering said pharmaceutical composition sufficient for degradation of extra cellular matrix (ECM) around said tumor, wherein said administering said drug occurs at least 24 hours after administering said pharmaceutical composition, wherein administering said pharmaceutical composition comprises administering 2 doses at least 24 hours apart or wherein administering said pharmaceutical composition does not increase the number of circulating tumor cells in said subject. ### Claim 11 (depends on 8) The method of claim 8, wherein said fibrosis is selected from the group consisting of: pancreatic fibrosis, lung fibrosis, liver fibrosis and pterygium. ### Claim 12 A method of producing a pharmaceutical composition comprising a liposome encapsulating at least one proteolytic enzyme, the method comprising: a. providing a thin lipid film; and b. rehydrating said thin lipid film with a solution comprising said proteolytic enzyme, wherein said rehydrating produces liposomes encapsulating said proteolytic enzyme wherein at least 75% of said proteolytic enzyme is proteolytically active and wherein said solution comprises a pH from 8 to 10; thereby producing liposomes encapsulating at least one enzyme. ### Claim 13 (depends on 12) The method of claim 12, wherein at least one of: a. said thin lipid film is produced by evaporating a lipid solution dissolved in an organic solvent and wherein said evaporating is performed at a temperature not higher than 37 degrees Celsius; b. said enzyme is collagenase; c. said solution comprising said enzyme is below the lowest transition temperature of a lipid in thin lipid film, at or below 15 degrees Celsius or both; d. said rehydrating does not comprise vortexing, a temperature above 15 degrees Celsius or both; e. at least 75% of said encapsulated enzyme is enzymatically active; and f. said method is performed at a temperature of not more than 20 degrees Celsius. ### Claim 14 (depends on 12) The method of claim 12, further comprising at least one of: (i) downsizing said liposomes produced in (b) with an extruder, wherein said downsizing is performed at a temperature of not more than 20 degrees Celsius and at a maximal working pressure of not more than 10 bar; and (ii) testing the enzymatic activity of said encapsulated enzyme to ensure that at least 75% of said encapsulated enzyme is enzymatically active. ### Claim 15 (depends on 1) The pharmaceutical composition of claim 1, wherein a proteolytically active enzyme is an enzyme that is capable of cleaving its target. ### Claim 16 (depends on 1) The pharmaceutical composition of claim 1, wherein a proteolytically active enzyme is an enzyme that is active as determined by a collagen cleaving Protease Assay. ### Claim 17 (depends on 1) The pharmaceutical composition of claim 1, formulated for systemic administration. ### Claim 18 (depends on 1) The pharmaceutical composition of claim 1, formulated for intravenous administration. ### Claim 19 (depends on 12) The method of claim 12, wherein said solution: i. is devoid of organic solvents; ii. is devoid of ions that activate said enzyme; iii. comprises a temperature below a transition temperature of a lipid in said thin lipid film; or iv. is a combination thereof. ### Claim 20 A pharmaceutical composition formulated for administration to a human comprising at least one lipid-based particle encapsulating collagenase in solution and a pharmaceutically acceptable carrier, wherein at least 75% of said collagenase is proteolytically active and wherein said solution comprises a pH from 8 to 10.