EP4199906A1 - Systèmes d'administration de lipides permettant l'administration d'acétate palmitate d'oxaliplatine - Google Patents

Systèmes d'administration de lipides permettant l'administration d'acétate palmitate d'oxaliplatine

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Publication number
EP4199906A1
EP4199906A1 EP21762817.1A EP21762817A EP4199906A1 EP 4199906 A1 EP4199906 A1 EP 4199906A1 EP 21762817 A EP21762817 A EP 21762817A EP 4199906 A1 EP4199906 A1 EP 4199906A1
Authority
EP
European Patent Office
Prior art keywords
opa
cancer
delivery system
lipid
liposomes
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP21762817.1A
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German (de)
English (en)
Inventor
Simon Benita
Taher Nassar
Amit BADIHI
Meital NAIM
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bionanosim BNS Ltd
Original Assignee
Bionanosim BNS Ltd
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Filing date
Publication date
Application filed by Bionanosim BNS Ltd filed Critical Bionanosim BNS Ltd
Publication of EP4199906A1 publication Critical patent/EP4199906A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/40—Cyclodextrins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars

Definitions

  • the present disclosure concerns lipid delivery systems for delivery of oxaliplatin palmitate acetate.
  • Pt(II) anticancer drugs are clinically used worldwide in 50- 70% of cancer patients [7-9]; these are cisplatin, carboplatin (approved in 1989), and oxaliplatin (approved in 2002).
  • cisplatin carboplatin
  • carboplatin approved in 1989
  • oxaliplatin approved in 2002.
  • therapeutic outcomes of Pt(II) drugs are seriously affected owing to severe side effects attributed to the reactivity of the Pt(II) compounds with biological nucleophiles prior to reaching the cancerous tissues, as well as inherent or acquired resistance [11].
  • Pt(IV) complexes with two additional axial groups may have advantages over the reactive Pt(II) species.
  • Oxaliplatin a 1,2-diaminocyclohexane (DACH) derivative of cisplatin
  • DACH 1,2-diaminocyclohexane
  • Oxaliplatin is a third-generation Pt(II) drug, active against several lines of colon, ovarian and lung cancer cells.
  • OPA Oxaliplatin palmitate acetate
  • Pt(IV) chemical entity derived from OXA and containing both lipophilic and hydrophilic axial ligands demonstrated at least a 20-time better efficiency in killing cancer cells [17].
  • OPA showed significantly higher tumor growth inhibition compared to OXA in both orthotopic and xenograft mice tumor models. A detailed description of OPA synthesis has been previously reported [22].
  • Oxaliplatin palmitate acetate has demonstrated significantly higher tumor growth inhibition compared to OXA in both orthotopic and xenograft mice tumor models of ovarian, pancreatic, lung and liver. However despite its demonstrated capabilities, OPA was prematurely eliminated before cellular uptake. Even when incorporated in a variety of acceptable nanoparticles, proper retention of OPA in the oil core was not observed. Thus, the inventors of the invention disclosed herein have embarked on the development of a suitable delivery system that would hold or contain OPA over long periods of time and efficiently deliver the drug to a patient. Unlike the nanoparticles proposed in the past, it was surprisingly found that only lipid-based nanocarriers could be loaded with significant amounts of OPA while maintaining their stability over time.
  • OPA Oxaliplatin palmitate acetate
  • Pt(IV) organic complex having the following structural formula:
  • lipids are organic molecules typically comprising a polar “head” and one or more nonpolar “tails”, such that they can be arranged spontaneously into organized structures, typically with the polar heads (that are hydrophilic) oriented toward a water-based medium and their nonpolar tails (that are hydrophobic) shielded from the water.
  • polar heads that are hydrophilic
  • nonpolar tails that are hydrophobic
  • Such structures may be micelles, bilayers or liposomes.
  • the present disclosure provides a lipid-based delivery system comprising OPA and a lipid-based material, wherein the delivery system is in a form of a nanocarrier.
  • a depiction of the delivery system is provided in
  • lipid-based molecular assembly (herein assembly or nanocarrier) intercalating or incorporating a plurality of OPA molecules.
  • the OPA molecules are intercalated between neighboring lipid molecules as depicted in Scheme 1.
  • the lipid-based assembly is a molecular assembly of lipid molecules (at least one lipid) selected from phospholipids, glycerolipids, glycerophospholipids, sphingolipids, and mixtures thereof.
  • the at least one lipid is a phospholipid, which may be fully saturated, unsaturated or partially hydrogenated.
  • the phospholipid may additionally or alternatively be derived from a natural source or may be partially or fully synthetic.
  • Non-limiting examples of phospholipids include phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), as well as lipid derivatives thereof, such as dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), dipalmitoylphosphatidylglycerol (DPPG), and others.
  • PA phosphatidic acid
  • PG phosphatidylglycerol
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • PI phosphatidylinositol
  • PS phosphatidylserine
  • DMPC dim
  • the aliphatic chains can be of various chain lengths, comprising a number of carbon atoms ranging between 12 and 22 carbon atoms, e.g., having a C 12 to C22 aliphatic chain(s).
  • the aliphatic chain has at least 18 carbon atoms.
  • the at least one phospholipid, being fully saturated, unsaturated or partially hydrogenated may be distearoylphosphatidylcholine (DSPC) or hydrogenated soy phosphatidylcholine (HSPC) or mixtures thereof.
  • the at least one phospholipid is not dipalmitoylphosphatidylcholine (DPPC) or dimyristoylphosphatidylcholine (DMPC).
  • DPPC dipalmitoylphosphatidylcholine
  • DMPC dimyristoylphosphatidylcholine
  • Sphingolipids can include lipids having two fatty acid chains, one of which is the hydrocarbon chain of sphingosine. Such also include, for example, glycosphingolipids, which are sphingolipids with one or more sugar residues.
  • Assemblies of the invention are nanocarriers, namely a particulate material that is biocompatible and sufficiently resistant to chemical and/or physical destruction, such that a sufficient amount of the nanocarriers remains substantially intact after administration to a human or an animal and for a time period sufficient to reach the desired target tissue (organ).
  • the nanocarriers are spherical in shape, having an average diameter of up to 500 nm (nanometers). Where the shape of the nanocarrier is not spherical, the diameter refers to the longest dimension of the nanocarrier.
  • the nanocarriers have an average diameter of between about 20 nm and about 500 nm. In some embodiments, the average diameter of the nanocarrier is between about 100 and 200 nm. In other embodiments, the average diameter is between about 200 and 300 nm. In further embodiments, the average diameter is between about 300 and 400 nm, the average diameters between 400 and 500 nm. In other embodiments, the average diameter is between about 50 and 400 nm. In further embodiments, the average diameter is between about 50 and 300 nm. In further embodiments, the average diameter is between about 50 and 200 nm. In further embodiments, the average diameter is between about 50 and 100 nm.
  • the nanocarriers may each be substantially of the same shape and/or size.
  • the nanocarriers have a narrow diameter distribution. In other words, no more than 0.01% to 10% of the particles have a diameter greater than 10% above or below the average diameter noted above, and in some embodiments, such that no more than 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, or 9% of the nanocarriers have a diameter greater than 10% above or below the average diameters noted above.
  • OPA may be intercalated or incorporated in the lipid shell of lipid assembly, as depicted in Scheme 1 and as will be further detailed below.
  • the assembly or nanocarrier may be in a form of a lipid bilayer assembly (e.g. a liposome), a lipid nanocapsule or a lipid nanosphere.
  • the lipids are selected to form a nanocarrier having a lipid bilayer structure.
  • the bilayer structure comprises two layers of lipids, typically arranged such that their hydrophilic heads are appositively directed (directed away from each other) to form external sheets of hydrophilic surfaces, while the hydrophobic tails of the lipids are sandwiched between the two surfaces of the bilayer.
  • the bilayer may be formed or may be provided as a closed spherical bilayer assembly, i.e., as a liposome.
  • the molecular assembly or nanocarrier is in the form of a liposome.
  • the liposome is a closed bilayer structure made of the at least one lipid, and OPA intercalated or incorporated between the lipid molecules in the assembly, as exemplified by the structure of Scheme 1.
  • OPA is lipophilic in nature, and hence may be associated with the lipid bilayer, e.g. incorporated, intercalated or embedded within the lipid bilayer or partially dissolved therein (dispersed at the molecular level and/or partly dispersed as small molecule aggregates within the bilayer).
  • the liposome is a unilamellar liposome, namely structured out of a single lipid bilayer.
  • the bilayer structure (e.g. the liposome) comprises at least one phospholipid.
  • the bilayer structure comprises at least one phospholipid and at least one sterol.
  • Sterols are steroid alcohols and are typically considered a type of lipid. Sterols are derived from steroids, and have a fused rings core structure in which one of the hydrogen atoms is substituted with a hydroxyl group at the 3-position of the A-ring. Sterols are added to the lipids forming the lipid bilayer typically to decrease the bilayer permeability and hence increase its stability.
  • the sterols may be selected from cholesterol, cholesteryl, cholesteryl hemisuccinate, cholesteryl sulfate and other derivatives of cholesterol and combinations thereof.
  • the liposome comprises at least one lipid and at least one sterol, wherein the weight ratio between the lipids and the sterols in a nanocarrier is in the range of between about 1:0.05 and about 1:5.
  • the weight ratio between the lipids and the sterols in nanocarrier may be in the range of between about 1:0.1 and about 1:5, between about 1:0.2 and about 1:5, between about 1:0.3 and about 1:5, between about 1:0.4 and about 1:5, between about 1:0.5 and about 1:5, between about 1:0.6 and about 1:5, between about 1:0.7 and about 1:5, between about 1:0.8 and about 1:5, between about 1:0.9 and about 1:5, or even between about 1:1 and about 1:5.
  • the weight ratio between the lipids and the sterols in nanocarrier may be in the range of between about 1:0.05 and about 1:4.5, between about 1:0.05 and about 1:4, between about 1:0.05 and about 1:3.5, between about 1:0.05 and about 1:3, between about 1:0.05 and about 1:2.5, between about 1:0.05 and about 1:2, between about 1:0.05 and about 1:1.5, or even between about 1:0.05 and about 1:1.
  • the weight ratio between the lipids and the sterols in nanocarrier may be in the range of between about 1:0.1 and about 1:4.5, between about 1:0.3 and about 1:4, between about 1:0.5 and about 1:3, or even between about 1:0.7 and about 1:2.5.
  • the lipid composition of the bilayer may further comprise one or more surfactants.
  • the surfactant(s) can be hydrophilic, hydrophobic, amphiphilic, cationic, anionic, or non-ionic, depending on the lipids used.
  • the lipid composition comprises at least one non-ionic surfactant.
  • the surfactant(s) may be selected from polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monooleate, and polyoxyeyhylene esters of saturated and unsaturated castor oil, ethoxylated monglycerol esters, ethoxylated fatty acids and ethoxylated fatty acids of short and medium and long chain fatty acids and others.
  • the surfactant(s) may be at least one of the polyoxyethylenes, ethoxylated (20EO) sorbitan mono laurate (T20), ethoxylated (20EO) sorbitan monostearate/palmitate (T60), ethoxylated (20EO) sorbitan mono oleate/linoleate (T80), ethoxylated (20EO) sorbitan trioleate (T85), castor oil ethoxylated (20EO to 40EO); hydrogenated castor oil ethoxylated (20 to 40EO), ethoxylated (5-40 EO) monoglyceride stearate/plamitate, polyoxyl 35 and 40 EOs castor oil.
  • ethoxylated (20EO) sorbitan mono laurate T20
  • ethoxylated (20EO) sorbitan monostearate/palmitate T60
  • ethoxylated (20EO) sorbitan mono oleate/linoleate
  • the hydrophilic surfactant may be selected from polyoxyl 35 castor oil, polysorbate 40 (Tween 40), polysorbate 60 (Tween 60), polysorbate 80 (Tween 80), Mirj S40, oleoyl macrogolglycerides, poly glyceryl- 3 dioleate, ethoxylated hydroxyl stearic acid (Solutol HS15), sugar esters such as sucrose monooleate, sucrose monolaurate, sucrose mono stearate, polyglycerol esters such as decaglycerol monooleate or monolaurate, hexaglycerol monolaurate or mono oleate, etc.
  • the surfactant may be at least one of polyethylene glycol 15-hydroxystearate (Solutol HS 15), polysorbate 40 (Tween 40), polysorbate 60 (Tween 60), and polysorbate 80 (Tween 80).
  • the lipid composition may further comprise, by some embodiments, at least one oil at a concentration which does not affect the bilayer structure of the nanocarrier.
  • the at least one oil may be selected from mineral oil, paraffinic oils, vegetable oils, glycerides, fatty acids, esters of fatty acids, liquid hydrocarbons and alcohols thereof, and others.
  • the oil may be selected from medium-chain triglycerides (MCT), long chain triglycerides such as fish oil, safflower oil, soybean oil, cottonseed oil, sesame oil, castor oil, olive oil, and others.
  • MCT medium-chain triglycerides
  • long chain triglycerides such as fish oil, safflower oil, soybean oil, cottonseed oil, sesame oil, castor oil, olive oil, and others.
  • the bilayered nanoparticles may be of a substantially uniform composition not featuring a distinct core/shell structure.
  • These nanocarriers are herein referred to as lipid nanospheres, and comprise a lipid matrix into which OPA is embedded.
  • the lipid matrix of such nanospheres can comprise one or more lipids as disclosed herein.
  • the lipid matrix may also comprise small quantities of injectable oils, e.g. at a quantity between about 0.1 wt% and about 10 wt% of the lipid matrix total weight. Oils which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils.
  • oils include peanut oil, soybean oil, sesame oil, cottonseed oil, com oil, olive oil, fish oil, safflower oil, castor oil.
  • Suitable fatty acids for use in parenteral formulations in small quantities are unsaturated fatty, oleic acid (18: 1), linoleic (18: 2) and linolenic acid (18:3), long- chain omega-3 fatty acids (e.g. docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA)) and others, as well as medium chain fatty acid from C8 to C12, octanoic acid, caprylic acid, etc.
  • DHA docosahexaenoic acid
  • EPA eicosapentaenoic acid
  • the nanocarriers may be surface-associated with at least one non-active agent.
  • surface-associated means a chemical or a physical association of a nanocarrier component(s) to a non-active agent(s) that extends outwards from the surface of the nanocarrier.
  • the term refers to any association between the surface of the nanocarrier and the non-active agent, e.g. ionic bonding, electrostatic bonding, covalent bonding, dipole-dipole interaction, hydrophilic interaction, van der Waal's interaction, hydrogen bonding, physical anchoring, adsorption, or any other suitable attachment mechanism of the non-active agent to the surface of the nanocarrier.
  • the non-active agent may be selected to modulate at least one characteristic of the nanocarrier, such characteristic may for example be one or more of size, polarity, hydrophobicity/hydrophilicity, electrical charge, reactivity, chemical stability, clearance rate, distribution, targeting and others.
  • the non-active agent is a substantially linear carbon chain having at least 5 carbon atoms, and may or may not have one or more heteroatoms in the linear carbon chain.
  • the non-active agent is selected from polyethylene glycols (PEG) of varying chain lengths, fatty acids, amino acids, aliphatic or non-aliphatic molecules, aliphatic thiols, aliphatic amines, and others. The non-active agent may or may not be charged.
  • the non-active agent is polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the PEG may have an average molecular weight in the range of between about 2,000 and 5,000 Da (Daltons).
  • the nanocarrier may be non-PEGylated, i.e. the non- active agent is different from PEG.
  • the nanocarrier is lyophilized.
  • a cryoprotectant may be added to protect and improve the stability of the nanocarriers during the lyophilization process.
  • the cryoprotectant may be selected from lactose, maltose, trehalose, sorbitol, mannitol, sulfobutyl-ether-P-cyclodextrin, polyvinyl alcohols, high molecular weight poloxamers, high molecular weight hyaluronic acid, etc.
  • the assembly constructed of lipid molecules comprises a phospholipid that is distearoylphosphatidylcholine (DSPC) or hydrogenated soy phosphatidylcholine (HSPC) or mixtures thereof.
  • DSPC distearoylphosphatidylcholine
  • HSPC hydrogenated soy phosphatidylcholine
  • a liposome that comprises at least one phospholipid and OPA, wherein said at least one phospholipid is distearoylphosphatidylcholine (DSPC) or hydrogenated soy phosphatidylcholine (HSPC) or mixtures thereof.
  • DSPC distearoylphosphatidylcholine
  • HSPC hydrogenated soy phosphatidylcholine
  • the liposome is surface decorated with a plurality of non-active materials, as defined, e.g., polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the liposome comprises l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), cholesterol, OPA and N-(Carbonyl-methoxypolyethyl- eneglycol-2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-
  • PEG2000 wherein optionally the molar ratio of DSPC:cholesterol:OPA:DSPE- PEG2000 is 5:3:2:0.5, or 5:3:l:0.5, 5:3:0.75:0.5, or 5:3:0.5:0.5 respectively.
  • the liposome comprises hydrogenated soy phosphatidylcholine (HSPC), Cholesterol, OPA and (N-(Carbonyl-methoxypolyethyl- eneglycol-2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine) (DSPE-
  • HSPC:Cholesterol:OPA:DSPE- PEG2000 wherein optionally the molar ratio of HSPC:Cholesterol:OPA:DSPE- PEG2000 is 5:3:2:0.5, or 5:3:l:0.5, 5:3:0.75:0.5, or 5:3:0.5:0.5 respectively.
  • the liposome may comprise hydrogenated soy phosphatidylcholine (HSPC), Cholesterol, OPA and dipalmitoylphosphatidylglycerol sodium salt (DPPG-Na), optionally at a molar ratio of 3 :2: 1 : 1.
  • HSPC hydrogenated soy phosphatidylcholine
  • Cholesterol Cholesterol
  • OPA dipalmitoylphosphatidylglycerol sodium salt
  • DPPG-Na dipalmitoylphosphatidylglycerol sodium salt
  • the liposome may be prepared by thin-film hydration or by ethanol injection, as exemplified herein.
  • this disclosure provides oxaliplatin palmitate acetate (OPA) loaded lipid-based nanocarrier.
  • OPA oxaliplatin palmitate acetate
  • a lipid-based nanocarrier consisting of a lipid material and oxaliplatin palmitate acetate (OPA).
  • OPA oxaliplatin palmitate acetate
  • the nanocarrier is in the form of a lipid bilayer or a liposome. In other embodiments, the nanocarrier is in the form of a uni-lamellar liposome.
  • the lipid is selected from at least one phospholipid, at least one sterol, and combinations thereof.
  • the lipid formulation comprises at least one phospholipid and at least one sterol.
  • the weight ratio between the lipids and the sterol is in the range of between about 1:0.05 and about 1:5.
  • the nanocarrier is surface-associated with at least one non-active agent, e.g. polyethylene glycols (PEG).
  • PEG polyethylene glycols
  • this disclosure provides a composition comprising a lipid-based delivery system or a lipid-based nanocarrier as described herein.
  • the composition is a pharmaceutical composition.
  • composition comprises a therapeutically effective amount of OPA, together with suitable diluents, preservatives, solubilizers, emulsifiers, adjuvant and/or carriers.
  • suitable diluents e.g. tris- HCL, acetate, phosphate
  • pH and ionic strength additives such as albumin or gelatin to prevent absorption to surfaces
  • surfactants e.g. Tween 20, Tween 80, Pluronic F68, bile acid salts
  • solubilizing agents e.g. glycerol, polyethylene glycerol
  • anti-oxidants e.g.
  • compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance.
  • Controlled or sustained release compositions include formulation in lipophilic depots (e.g. fatty acids, waxes, oils).
  • Formulations suitable for parenteral administration include aqueous and nonaqueous formulations, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the compound can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane-4-methanol, ethers, such as poly (ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical
  • Oils which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum oil, and mineral oil. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid.
  • the lipid-based delivery systems of the present disclosure may be made into injectable formulations.
  • the requirements for effective pharmaceutical carriers for injectable compositions are well known to those of ordinary skill in the art. See Pharmaceutics and Pharmacy Practice, J.B. Lippincott Co., Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4 th ed., pages 622-630 (1986).
  • the composition is suitable for administration by injection.
  • the composition is suitable for intravenous administration.
  • the composition is suitable for topical administration, i.e. directly onto at least a portion of a subject's skin (human's or non-human's skin) so as to achieve a desired systemic or local effect.
  • a topical composition comprising the delivery system or nanocarrier of this disclosure may be in any suitable form, e.g. a cream, a lotion, an ointment, an emulsion, a gel, a suspension, a solution, a liquid, an aerosol, a foam, etc.
  • the composition is suitable for ocular administration, e.g. administrated topically to the conjunctiva or the eyelid or administrated parenterally, e.g. intraocular injection to the anterior, posterior and vitreous chambers.
  • the composition may be of any suitable topical delivery form, such as a solution, a suspension, a paste, a cream, a foam, a gel, an ointment, a spray, drops, etc.
  • lipid-based delivery system a nanocarrier or composition as described herein, for use in treating or delaying progression of a proliferative disorder.
  • lipid-based delivery system for the preparation of a medicament for treating or delaying progression of a proliferative disorder.
  • a further aspect of the disclosure provides a method for delivering OPA to a subject in need thereof, the method comprising administering an effective amount of a lipid-based delivery system, a nanocarrier, or a composition as described herein.
  • a further aspect of the disclosure provides a method for treating or delaying or preventing the progression of a proliferative disorder, the method comprising administering an effective amount of a lipid-based delivery system, a nanocarrier, or a composition as described herein.
  • proliferative disorders encompass diseases or disorders that effect a cellular growth, differentiation or proliferation processes.
  • the proliferation disorder is cancer.
  • cancer as used herein encompasses any neoplastic disease which is characterized by abnormal and uncontrolled cell division causing malignant growth or tumor. Cancer may refer to either a solid tumor or tumor metastasis.
  • Non-limiting examples of cancer are ovary cancer, and pancreatic cancer, squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.
  • squamous cell cancer e.g. epithelial squamous cell cancer
  • lung cancer including small-cell lung cancer, non-small cell lung cancer, adeno
  • Solid cancers appear in many forms, for example, breast cancer, prostate cancer, sarcomas, and skin cancer.
  • skin cancer is melanoma.
  • the cancer is selected from lung cancer, colon cancer, pancreatic cancer and ovarian cancer.
  • treatment refers to the administering of a therapeutic amount of the composition of the present disclosure which is effective to ameliorate undesired symptoms associated with a disease, to prevent the manifestation of such symptoms before they occur, to slow down the progression of the disease (also referred to herein as “delaying the progression”), slow down the deterioration of symptoms, to enhance the onset of remission period, slow down the irreversible damage caused in the progressive chronic stage of the disease, to delay the onset of said progressive stage, to lessen the severity or cure the disease, to improve survival rate or more rapid recovery, or to prevent the disease from occurring or a combination of two or more of the above.
  • an effective amount is determined by such considerations as may be known in the art.
  • the amount must be effective to achieve the desired therapeutic effect as described above, depending, inter alia, on the type and severity of the disease to be treated and the treatment regime.
  • the effective amount is typically determined in appropriately designed clinical trials (dose range studies) and the person versed in the art will know how to properly conduct such trials in order to determine the effective amount.
  • an effective amount depends on a variety of factors including the affinity of the ligand to the receptor, its distribution profile within the body, a variety of pharmacological parameters such as half-life in the body, on undesired side effects, if any, on factors such as age and gender, etc.
  • the effective amount of the OPA is provided in the form of a lipid-based delivery system, a nanocarrier, or composition as disclosed herein, and administrated by one or more of the following routes: dermal, ocular, rectal, transmucosal, transnasal, intestinal, parenteral, intramuscular, subcutaneous, intramedullary injections, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • routes dermal, ocular, rectal, transmucosal, transnasal, intestinal, parenteral, intramuscular, subcutaneous, intramedullary injections, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • the term subject refers to a mammal, human or non-human.
  • the term about is meant to encompass deviation of ⁇ 10% from the specifically mentioned value of a parameter, such as temperature, pressure, concentration, etc.
  • Figs. 1A-1G are cryo-TEM images of Blank Lip.l (Figs. 1A-1C) and OPA Lip.l. (Figs. 1D-1G). Lipid composition of DSPC:cholesterol:OPA:DSPE-PEG2000 at ratios 5:3:l:0.5.
  • Figs. 2A-2D are TEM micrographs of uranyl acetate negatively stained non- PEGylated NCs at different areas of the grid: Freshly prepared samples NC7 (Figs. 2A-2B), NC7 nanocapsules (Fig. 2C), NE3 nanoemulsion (Fig. 2D) - after 3 months of lyophilization and reconstitution of the aqueous dispersion.
  • Figs. 3A-3B are cryo-TEM micrographs of non- PEGylated liposomes containing OPA before (Fig. 3A) and after (Fig. 3B) lyophilization. Lipids composition was DSPC:Chol at ratio 2:1.
  • Figs. 4A-4B are cryo-TEM micrographs of PEGylated liposomes containing OPA, before (Fig. 4A) and after (Fig. 4B) lyophilization.
  • Figs. 5A-5B are cryo-TEM micrographs of non-
  • Lipids composition was DSPC:Chol at ratio 2:1.
  • Fig. 12 shows mean body weights of mice in different groups during treatment.
  • Fig. 13 shows mean body weight changes of mice in different groups during treatment.
  • Fig. 14 shows mean body weights of mice in different groups during treatment.
  • Fig. 15 shows mean body weight changes of mice in different groups during treatment.
  • Fig. 16 shows tumor volumes of mice in different groups during treatment of Hep3B model in balb/c nude mice.
  • Fig. 17 shows mean body weights of mice in different groups during treatment of Hep3B model in balb/c nude mice.
  • Fig. 18 shows mean body weight changes of mice in different groups during treatment of Hep3B model in balb/c nude mice.
  • Fig. 19 shows survival curves of mice in different groups during treatment in mouse liver cancer model Hep3B.
  • Fig. 20 shows tumor volumes of mice in different groups during treatment in mouse liver cancer model Hep3B.
  • Fig. 21 shows mean body weights of mice in different groups during treatment in mouse liver cancer model Hep3B.
  • Fig. 22 shows mean body weight changes of mice in different groups during treatment in mouse liver cancer model Hep3B.
  • Fig. 23 shows survival curves of mice in different groups during treatment in mouse liver cancer model Hep3B.
  • Figs. 24A-D show how OPA liposomes and Avastin combination arrest tumor growth and extends survival in ovarian cancer xenograft orthotopic mouse model.
  • luciferase transfected SKOV3-luc cells (2 * 106 cells in 100 pL of PBS) were injected into intraperitoneal cavity of mice. The Tumor growth was measured and quantified by IVIS every week.
  • Fig. 24A Longitudinal detection and quantification of tumor growth. Tumor size is expressed as luminescence intensity of the dorsal images, expressed in radiance units (photons/s/cm2/sr). Results are presented as mean ⁇ S.E.M. (Fig.
  • FIG. 24B Body weight follow-up beginning from tumor inoculation (day 0) through the study period. Changes were recorded as a percentage of the initial body weight observed on the day of tumor cells injection (100% at day 0).
  • FIG. 24C Kaplan-Meier survival curve from tumor cells injection day until death.
  • Fig. 24D Bioluminescent monitoring of orthotopic ovarian SKOV3-luc cancer cells expressing the luciferase gene. Bioluminescent images were acquired 10 min after intraperitoneal injection with luciferin.
  • Fig. 25 shows an illustration of the thin-film hydration method for the preparation of OPA Liposomes.
  • Fig. 26 is an illustration of the ethanol injection method for the preparation of OPA Liposomes.
  • Figs. 27A-B provide Cryo-TEM images of Blank Lip.l. Fig. 27A) image at 1 pm scale and Fig. 27B) image at 100 nm scale.
  • Figs. 28A-B provide Cryo-TEM images of OPA Lip.l. Fig. 28A) image at 1 pm scale and Fig. 28B) image at 100 nm scale.
  • Lipoid PC 14:0/14:0 DMPC
  • Lipoid PC 16:0/16:0 DPPC
  • Lipoid PC 18:0/18:0 DSPC
  • Lipoid PE 18:O/18:O-PEG 2000 DSPE-mPEG2000, sodium salt
  • Lipoid PG 16:0/16:0 DPPG, sodium salt
  • Lipoid S PC-3 HSPC
  • MLV multilamellar vesicles
  • SUV small unilamellar vesicles
  • Tipsonication homogenization Ultrasonic processor, VCX 750, Sonics & Materials, Inc.
  • MLV multilamellar vesicles
  • SUV small unilamellar vesicles
  • tip-sonication homogenization Ultrasonic processor, VCX 750, Sonics & Materials, Inc.
  • PEGylated and Non-PEGylated liposomes were prepared according to the well-established ethanolic injection method (Table 1).
  • the organic phase was 10 ml ethanol and the aqueous phase was 20 ml water.
  • OPA hydrogenated soy phosphatidylcholine
  • MPEG 2000-DSPE N-(carbonyl-methoxypoly ethylene glycol 2000)- 1,2- distearoyl-sn-glycero-3-phosphoethanolamine sodium salt
  • cholesterol cholesterol
  • the aqueous phase and organic phase were heated to 60°C.
  • the organic phase was added dropwise to the aqueous phase while stirring at 800 rpm on a magnetic stirrer. Immediately after addition the mixture was moved to a stirrer at room temperature and continuously stirred at 800 rpm for 30 minutes. Ethanol was evaporated using a rotary evaporator. Liposomes were stored at 4°C.
  • OPA or [Pt(DACH)(OAc)(OPal) (ox)], PLGA/PEG-PLA, Tween 80 and MCT were dissolved in 9 ml acetone.
  • 50 mg/ml Lipoid E80 solution was prepared in methanol. 1 ml of this solution was mixed with 9 ml acetone with the dissolved ingredients and was stirred at 1000 rpm in a magnetic stirrer for 15 min.
  • the organic phase was added dropwise into 20 ml of aqueous phase of Solutol®HS 15 by a needle with an inner diameter of 0.3 mm and was stirred at 1500 rpm for 30 minutes, followed by the evaporation of acetone using a Rota evaporator (Rotavapor, R300, BUCHI, Switzerland).
  • Lyophilization was carried out in Epsilon 2-6d Martin Christ lyophilizer (Gef., Germany) to obtain dry powder.
  • Various sugars were investigated as possible cryoprotectant for the freeze-drying process of OPA NCs and liposomes e.g. Mannitol, sucrose, trehalose, dextrose, lactose, captisol and hydroxypropyl-P-cyclodextrin (HP ⁇ CD) at various concentrations of 2, 3, 4, 5, 6% w/w or at different weight ratios of 1:0.25, 1:0.5, 1:1, 1:2, 1:4 (liposome ingredients: cryoprotectant).
  • the sugars were weighed and directly added to the formulation batches and stirred on magnetic stirrer for 10 min to mix or were added as a solution to the liposome formulation.
  • the vials were rapidly stoppered under vacuum and stored at room temperature.
  • OPA content in NCs and liposomes prepared by the ethanolic injection method was dissolved in ethanol and diluted with acetonitrile, and the OPA concentration was determined using analytical Dionex HPLC consisting of Dionex 3000 Ultimate auto sampler. Separation was performed on a reverse phase Cis column (5pm, 4.6x250 mm) from Agela Technologies, USA. The mobile phase consisted of water: acetonitrile (10:90 v/v), eluted at a flow rate of 1.0 ml/min. The effluent was monitored using a UV detector at 220 nm.
  • liposome sample was diluted x20 with methanol, and the OPA concentration was determined using analytical Thermo HPLC consisting of Thermo Scientific Dionex UltiMate 3000 Autosampler. Separation was performed on a reverse phase C18 column (5pm, 250x4.6mm, Xselect CSH, Waters, USA).
  • the mobile phase consisted of acetonitrile (Eluent A) and water (Eluent B), at a gradient elution (from 60/40 A/B to 80/20 A/B), at a flow rate of ImL/min.
  • the effluent was monitored using a UV detector at 220 nm.
  • Morphological images were recorded on a TEM system (CM12 TEM, Philips) with an acceleration voltage 100 kV after negative staining using 2% uranyl acetate. A diluted suspension of the formulation (1:10) in water was dropped on carbon-coated copper grids (300-mesh), dried and analyzed.
  • Cryo-TEM enables direct imaging of nanostructures in their native, aqueous, environment.
  • the samples were prepared by applying a 3 pL drop onto a glowdischarge TEM grid (300 mesh Cu Lacey substrate, Ted Pella, Ltd.). The excess liquid was blotted, and the specimens were vitrified by a rapid plunging into liquid ethane precooled with liquid nitrogen using Vitrobot Mark IV (FEI).
  • FEI Vitrobot Mark IV
  • the vitrified samples were examined at -177 °C using FEI Tecnai 12 G2 TWIN TEM operated at 120 kV and equipped with a Gatan model 626 cold stage.
  • the images were recorded by a 4K x 4K FEI Eagle CCD camera in low-dose mode (to reduce radiation damage).
  • TIA Tecnai Imaging & Analysis
  • Cytotoxicity was determined in the various cancer cell lines using the colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) assay. Briefly, cells of the different cell lines were seeded in a sterile 96-well plate (3000 cells/well) in the appropriate growth medium and allowed to attach overnight. Then, the cells were treated with the test drugs (OXA, OPA and OPA liposomes) at increasing drug concentrations (0-7 pM for OPA and OPA liposomes, and 0-25 pM for OXA) at 37°C under 5% CO 2 for 72 hr.
  • test drugs OXA, OPA and OPA liposomes
  • Cytotoxicity was determined by the colorimetric MTT (3-(4,5- dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) assay.
  • MTT 3-(4,5- dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide
  • Fresh human blood was collected from at least two donors, and Spiked with test compound at 1 pM in triplicates.
  • the blood-compound mixtures were incubated with gentle shaking for 60 minutes at 37°C, and after incubation, aliquots of blood and plasma were removed for determination of analytes.
  • Relative concentrations of OPA and OXA in samples were assessed based on peak area ratio versus internal standard, and the ratio of compound concentrations in whole blood over plasma (KB/P), the respective drug concentrations in the erythrocytes to plasma (KE/P), and %Recovery in blood were calculated.
  • T60-blood is compound peak area ratios of analyte/intemal standard in whole blood sample at 60 mi
  • T60-plasma is compound peak area ratios of analyte/intemal standard in plasma sample at 60 min
  • TO is compound peak area ratios of analyte/internal standard in whole blood sample at time zero
  • He is the hematocrit of the whole blood used in the determination.
  • OPA intravenous
  • Table 3 An appropriate amount of OPA was accurately weighed and mixed with the appropriate volume of vehicle to get a clear intravenous (IV) solution as detailed in Table 3.
  • the formulations were prepared on the day of dosing, and rats were dosed (via tail vein) up to 4 h after formulations were prepared.
  • IS internal standards
  • Various internal standards were used in the study like 100 ng/ml labetalol & 100 ng/ml dexamethasone & 100 ng/ml tolbutamide & 100 ng/ml verapamil & 100 ng/ml glyburide & 100 ng/ml celecoxib in methanol.
  • An aliquot of 40 pl sample was protein-precipitated with 200 pl IS, the mixture was vortex-mixed well and centrifuged at 12 000 rpm for 15 min, 4°C. 5 pl supernatant was injected for LC- MS/MS analysis.
  • Plasma concentration versus time data was analyzed by non-compartmental approaches using the Phoenix WinNonlin 6.3 software program. Co, Cl, Vdss, Cmax, Tmax, TI/ 2 , AUC(o-t), AUC(o-inf), MRT (o-t), MRT(o-inf) and graphs of plasma concentration versus time profile were reported.
  • OPA-LIP-5 LYO lyophilized liposomes
  • Pharmacokinetic parameters like AUC, Cmax, Tmax, clearance, MRT, ti/2 were calculated using PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel.
  • a ready-to-use OPA-containing liposomes solution was prepared and characterized at BNS, and shipped under controlled conditions to Wuxi facility (shanghai, china), weighted and mixed with the appropriate volume of vehicle to get a clear IV solution as detailed below.
  • the formulation was re-evaluated on the day of dosing, up to 4 hours before rats were dosed (via tail vein). About 200 pL blood per time point was collected from the jugular vein followed by plasma preparation. Dose formulation and sample analysis was performed by LC-MS/MS method. Plasma concentration versus time data was analyzed by non-compartmental approach. Instrument conditions and data analysis were identical to the detailed above.
  • Rats were dosed via jugular vein (5 mg/kg). Approximately 200 pl blood per time point (0.16, 0.5, 1, 2, 4, 6, 10, and 24 h) was collected from the tail vein followed by plasma preparation. Blood samples were processed for plasma by centrifugation at approximately 4°C, 8,000 rpm within half an hour of collection.
  • OPA Liposomes OPA Liposomes (OPA-LIP-5 LYO) double dose, as intravenous injection equivalent to 5 mg/kg of OPA.
  • the second dose was injected after 3 days of the first dose.
  • Rats were dosed via jugular vein (5mg/kg) focula. Approximately 200 pl blood per time point (24, 48, 72, 96, 120, 144, 168 h) were collected from the tail vein followed by plasma preparation. Blood samples were processed for plasma by centrifugation at approximately 4°C, 8,000 rpm within half an hour of collection. Whole blood (100 pl), Plasma and hematocrit each 50 pl samples were stored in glass scintillation vials, and kept at -70 ⁇ 10°C until ICP-MS analysis.
  • a ready-to-use OPA-containing liposomes solutions and OPA-solution were prepared and characterized at BNS, and shipped to Wuxi facility (shanghai, china), weighted and mixed with the appropriate volume of vehicle to get either a milky or clear IV solution as detailed below.
  • the formulations were re-evaluated on the day of dosing, up to 4hr before mice were dosed (via tail vein). Animals were sacrificed per time point (1 and 4 hours) and 100 ⁇ L blood was collected via cardiac puncture followed by plasma preparation. Brain, liver, lung, pancreas and ovaries were harvested and further processed. Dose formulation and sample analysis was performed by LC-MS/MS method. Plasma concentration versus time data was analyzed by non-compartmental approach.
  • the maximum tolerated dose (MTD) of different dosing frequency of test article OPA in non-tumor-bearing female BALB/c mice was evaluated.
  • Table 6.2 Experimental design During routine monitoring, the animals were checked for any effects of treatments on behavior such as mobility, food and water consumption, body weight gain/loss, eye/hair matting and any other abnormalities. Mortality and observed clinical signs were recorded for individual animals in detail. Body weight were measured once a day after randomization. The last measurement of body weights was taken on the day when the study meets the termination criteria or if a mouse is found moribund.
  • the Hep3B tumor cells were maintained in vitro in MEM medium containing O.OlmM NEAA supplemented with 10% heat inactivated fetal bovine serum, at 37°C in an atmosphere of 5% CO 2 in air.
  • the tumor cells were routinely sub-cultured twice weekly.
  • the cells growing in an exponential growth phase were harvested and counted for tumor inoculation.
  • Each mouse was inoculated subcutaneously at the right lower region with Hep3B tumor cells (5xl0 6 ) in 0.1 ml of Matrigel (1:1) for tumor development.
  • mice 50 mice were enrolled in the efficacy study. All animals were randomly allocated to the 5 different study groups. Randomization was performed based on “Matched distribution” randomization method using multi-task method (StudyDirectorTM software, version 3.1.399.19) on Day 1 and treatment was initiated on the same day.
  • the animals were checked daily for morbidity and mortality. At the time of routine monitoring, the animals were checked for any effects of tumor growth and treatments on behavior such as mobility, food and water consumption, body weight gain/loss (body weights will be measured twice per week), and any other abnormalities. Mortality and observed clinical signs were recorded for individual animals.
  • OPA liposomes were prepared using a modified thin film hydration technique and were composed of DSPC (l,2-Distearoyl-sn-glycero-3-phosphocholine), cholesterol, OPA and DSPE-PEG2000 (N-(Carbonyl-methoxypolyethyl-eneglycol- 2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine) at a molar ratio of 5:3:2:0.5, respectively.
  • the lipids and OPA were weighed and transferred to a round -bottom flask.
  • Chloroform was added to the lipid mixture at a total concentration of 12.6 mg/mL lipids and 2.75 mg/mL OPA and heated at 50°C until all the ingredients were completely dissolved. Afterwards, chloroform was evaporated using a rotary evaporator (Rotavapor, R300, BUCHI, Switzerland) at 100 rpm without heating. Consequently, a thin film of lipid cake was obtained and further hydrated, with an appropriate amount of 5% dextrose solution or water pre-heated to 60°C, under rotation for 1 h at 60°C.
  • MLV multilamellar vesicles
  • SUV small unilamellar vesicles
  • Tipsonication homogenization Ultrasonic processor, VCX 750, Sonics & Materials, Inc., USA
  • ovarian adenocarcinoma SKOV-3-luc ovarian adenocarcinoma SKOV-3-luc
  • SKOV-3-luc in McCoy's
  • Cells were kept at 37°C in 5% CO 2 and 95% humidity.
  • Luciferase-transfected cell line SKOV3-luc was purchased from Caliper Life Sciences (Hopkinton, MA, USA). Cytotoxicity
  • SKOV-31uc cells were further treated for 1 h with 0.5 mg/mL MTT in phosphate buffer solution (PBS). The developed dye was dissolved in DMSO and absorbance measured at 570 nm by a microplate reader (Cytation 3, BioTek, USA).
  • mice with severe combined immunodeficiency (SCID)-bg, 8 weeks old (18.5-21.5 gm) were used in accordance with NIH regulations.
  • Luciferase transfected SKOV3-luc cells (2 x 10 6 cells in 100 pL of PBS) were injected directly into intraperitoneal cavity of mice.
  • bioluminescent imaging was performed every 7 days using a CCCD camera (IVIS, Caliper Life Sciences, Xenogen Corporation, USA). 200 pL of 15 mg/mL firefly D- luciferin was injected intraperitoneally and mice were anesthetized by 3% isoflurane. Animals were placed onto black paper in the IVIS imaging box and imaged dorsally.
  • Luminescence was recorded in radiance units (photons/s/cm 2 /sr). The mice were randomly allocated to the 7 study groups according to the tumor size, while verifying that all 7 groups have similar initial Mean Luminescence Intensity (MLI). Randomization was performed 4 days prior to treatment initiation (n 6).
  • the treatment groups were (group 1) vehicle liposomes (dispersed in dextrose); (group 2) free OXA (5mg/kg in dextrose); (group 3) OPA solution (15 mg/kg dissolved in Cremophor EL : ethanol at 1:1 ratio and diluted by 10 in dextrose); (group 4) OPA liposomes (15 mg/kg in dextrose), (group 5) OPA liposomes (30 mg/kg in dextrose), (group 6) OPA liposomes (15 mg/kg in dextrose) and Avastin (10 mg/kg in saline) combination and (group 7) Avastin (10 mg/kg in saline). All the animals were given i.v. bolus doses of the specific formulation to the tail vein, twice a week for four weeks.
  • Data and bars represent the mean ⁇ standard deviation (SD)/standard error mean (S.E.M) of three or more independent experiments. Differences between two groups were analyzed using unpaired Student’s t test. Differences between three or more groups were analyzed using one-way ANOVA. To compare tumor volumes of different groups at a pre-specified day, we first used Bartlett's test to check the assumption of homogeneity of variance across all groups. When the p-value of Bartlett's test was >0.05, we ran one-way ANOVA to test overall equality of means across all groups.
  • Liposomes prepared by the thin-film hydration method Liposomes prepared by the thin-film hydration method
  • Fig. 1 the liposomes exhibit bilayers of phospholipids of small unilamellar vesicles (SUV).
  • Table 10 Characterization of DSPC-lipoid based Blank lip, (without OPA)
  • Table 14 HSPC-lipoid based OPA liposomes prepared for lyophilization optimization; (OPA Lip .8)
  • Table 15 Lyophilization results of OPA Lip.8 According to Table 15, it can be noted that among many cryoprotectants that were used, HP ⁇ CD at a ratio of 1 :2 (added as solution) and 1:4 (added as powder) presented preserved values of mean diameter and PDI before and after lyophilization.
  • Table 19-1 Stability results of DSPC-lipoid based OPA Lip.15-17 as aqueous suspension (aq.) and after lyophilization [T0 Lyo.) _
  • Table 19-1 Stability results of DSPC-lipoid based OPA Lip.15- 17 as aqueous suspension (aq.) and after lyophilization (T0 Lvo.)_
  • Table 19-2 Stability results of HSPC-lipoid based OPA Lip.18-20 as aqueous suspension tag.) and after lyophilization (T0 Lyo.) _
  • Table 19-2 Stability results of HSPC-lipoid based OPA Lip.18-20 as aqueous suspension (aq.) and after lyophilization (T0 Lyo.)
  • Table 19-3 Stability results of DPPC-lipoid based OPA Lip.21-23 as aqueous suspension (aq.) and after lyophilization (T0 Lyo.)
  • Table 19-3 Stability results of DPPC -lipoid based OPA Lip.21-23 as aqueous suspension (aq.) and after lyophilization (T0 Lyo.)
  • Table 19-4 Stability results of DMPC -lipoid based OPA Lip.24-26 as aqueous suspension (aq.) and after lyophilization (T0 Lvo.)
  • Table 20 Summary of stability results of PEGylated OPA liposomes as aqueous suspension
  • OPA Lip.15 In order to increase the shelf-life stability of OPA liposomes, additional formulations were prepared using the same recipe as OPA Lip.15 with two OPA concentrations of 2.5 and 5 mg/ml (OPA Lip.37 and 38, Table 21). For comparison, non-PEGylated liposomes were prepared (OPA Lip. 42a, Table 22). The liposomes were prepared and was then lyophilized. The stability of the liposomes dried powder was evaluated for up to 1 month at 4°C, 25°C and 40°C (accelerated conditions).
  • Table 21 OPA Lip. 37 and OPA Lip.38 formulations
  • Table 22 non-PEGylated OPA Lip. 42a formulation
  • lyophilization of both PEGylated and non- PEGylated OPA liposomes improved their physicochemical stability compared to aqueous suspension. Liposomes samples of all time points and in all temperatures were easily reconstituted obtaining good appearance and size. The liposomes dried powder was found stable after 1 month of storage at 4°C, 25 °C and even at 40°C (accelerated conditions). OPA assay % was above 90% in all lyophilized formulations after 1 month at 40°C (accelerated conditions), indicating the vital effect of lyophilization on enhancing OPA Lip. stability and extending its shelf-life. The various property magnitudes are listed in Table 25. As can be noted in Figs. 2A-5B, the liposomes exhibit bi-layers of phospholipids.
  • Table 23 Stability results of OPA PEGylated Lip.37, 38 as dried powder
  • Table 23 (cont.) Stability results of OPA PEGylated Lip.37, 38 as dried powder
  • Table 25 Properties of OPA liposomes and nanocapsules before and after lyophilization Preliminary stability results of OPA Lip. prepared by the ethanol injection method
  • OPA Lip.EtOH showed promising results regarding appearance, mean diameter and PDI before and after lyophilization and after a storage period of 1 month at room temperature (RT). A decrease in OPA assay % and pH was observed after 1 month of storage at RT (Table 27).
  • Table 26 Description of HSPC-lipoid based OPA liposomes prepared by ethanol injection
  • OXA, OPA and OPA liposomes were tested in human skin squamous cell carcinoma Scl-1, pancreas adenocarcinoma BxPC-3 luc, ovarian cancer cells SKOV-3 luc and rat glioblastoma CNS-1 cell lines by the MTT assay.
  • the 50% growth inhibitory concentration (IC50) values were calculated and summarized in Table 28.
  • OPA showed a unique potency against different cancer cell lines, with higher cytotoxicity than OXA.
  • Table 28 IC 50 ( ⁇ M) values of OPA, OXA and OPA liposomes in various cancer cell lines following 72 h-long treatment
  • IC50 values are drug concentrations required to induce 50% cell death and are the means ⁇ SD of two- four independent experiments with quadruplicates in each.
  • Diclofenac was used as a negative control for low affinity to blood cells in this study.
  • Chloroquine was used as a positive control for high affinity to blood cells for Human.
  • Diclofenac was used as a negative control for low affinity to blood cells in this study.
  • Chloroquine was used as a positive control for high affinity to blood cells for Human.
  • Table 31 Raw data of hematocrit in whole blood
  • PK experiments were performed following administration of OPA at 1.25, 5 and 20 mg/kg in rats.
  • Major PK parameters of intact OPA and oxaliplatin active metabolite originated from OPA biodegradation
  • Table 32 Pharmacokinetic parameters of OPA and oxaliplatin following single dose of increasing doses of OPA to male rats (OXA was formed by the biodegradation of
  • OPA is not a prodrug of oxaliplatin in vivo, irrespective of the injected dose.
  • the formation of OXA from the biodegradation of OPA ranges from 5 to 10% (Table 32), based on the calculated ratio of the respective AUC values regenerated from the pharmacokinetic profiles of both drugs presented in Figs. 6A-6B.
  • the terminal elimination of the intact OPA molecule is rapid, in less than an hour for the ⁇ ti/2 values of OPA and OXA confirming the behavior of most of the platinum drugs and especially oxaliplatin [18].
  • Dose proportionality evaluated from the calculated parameters, was demonstrated between 1.25 and 5 mg/kg but not for the 20 mg/kg.
  • Supra-proportional behavior was recorded between 5 and 20 mg/kg, where, particularly for AUC and ti/2 values of OPA, the data was much more elevated than the proportional values achieved between 1.25 and 5 mg/kg.
  • Oxaliplatin exhibits a complicated PK profile and has several mechanisms of action but cancer cells can develop resistance. OXA exerts its cytotoxic effect mostly through DNA damage. Most papers do not address the PK of OXA per se, but of the Pt content. Shortly after infusion, OXA forms many Pt compounds which bind to blood or cell proteins. Total ultra-filterable plasma Pt is measured by atomic absorption or inductively coupled plasma mass spectrometry (ICP-MS). These techniques result in a codetermination of OXA and other Pt containing complexes due to high propensity of OXA to react with endogenous sulfur-containing compounds. OXA is often administered concomitantly with 5-FU.
  • ICP-MS inductively coupled plasma mass spectrometry
  • Platinum (IV) complexes such as OPA exhibit an advantage over Platinum (Il)-based drugs thanks to their kinetic stability in the body. They remain inert in the blood and only once reaching the tumor sites, they are activated in the cancer cells by a reduction process.
  • the ligand coordination spheres affect the lipophilicity and redox behavior in blood and has a significant impact on their accumulation in red blood cells and their degree of kinetic inertness in blood.
  • the most lipophilic platinum (IV) compounds featuring equatorial Chloro ligands showed a pronounced penetration into blood cells and a rapid reductive biotransformation.
  • Table 33 PK parameters of Pt. following IV administration OPA Solution and lyophilized Liposomes (OPA-LIP-5) at a dose equivalent to 5 mg/kg OPA to rats
  • OPA could not be incorporated within such matrices at significant drug content levels and even low contents of 1 or 2% were incompatible since precipitation and aggregation were observed in the dispersed formulations. Furthermore, it was not possible to prepare nanoemulsions since OPA was not enough soluble within injectable approved oils and precipitated rapidly in the presence of the water continuous phase of the nanoemulsions. Surprisingly, it was possible to prepare appropriate liposomal formulations of OPA at a normal level contents within the bilayers of the phospholipids. Different methods of Liposome manufacturing methods were used, and different formulations were prepared of OPA liposome formulation (PEGylated and non-PEGylated).
  • OPA nanocapsule formulations (PEGylated and non-PEGylated) were prepared followed by lyophilization of all formulations and characterization of the following properties: particle size, PDI and zeta potential analysis, determination of drug content (HPLC), morphology by Cryo TEM and stability at room temperature.
  • PK experiment was performed following administration of OPA-containing liposomes at 7.5 mg/kg in rats.
  • Major PK parameters and another active metabolite, oxaliplatin, are presented in Table 34 and Figs. 8A-8B.
  • AUC and tl/2 levels of OPA were markedly elevated, and clearance value was significantly decreased, compared to IV injection of OPA solution (RND-RPT-007).
  • Table 35 PK parameters of Pt following IV administration OPA Solution and various other formulations at dose equivalent to 5 mg/kg OPA to rats
  • the OPA molecule apparently remains in the liposomes in the plasma over more than 72 hours and even a second injection after 3 days from the first does enhance the levels of Pt the plasma and whole blood but much less in the hematocrit showing minimal release of OPA from the liposomes.
  • Table 36 PK parameters of Pt. following IV administration OPA-LIP-5 (LYQ) as a single dose each equivalent to 5 mg/kg OPA to rats
  • Table 36 shows the various PK parameter values for the first dose only in plasma, whole blood and hematocrit
  • mice PK and organ bio-distribution of OPA and Oxaliplatin after a single intravenous dose of OPA-containing liposomes (60 mg/kg) and OPA solution (15 mg/kg) to female mice is described in Table 37 and Figs. 11A-11D.
  • OPA-containing mPEG-liposomes have the longest circulation residence time, up to 4-fold higher and 4000-fold higher OPA and oxaliplatin normalized concentrations compared to non-PEG liposomes and IV solution, respectively (mPEG-liposomes> Non PEG-liposomes »> OPA solution).
  • organ-to-plasma ratios demonstrate significant differences of tested formulations, presenting dose- normalized organs’ exposures of both compounds. It can be noted that organ-to- plasma ratios of oxaliplatin are much higher than those of OPA for both liposomal preparations. For the IV solution, organ-to-plasma ratios of oxaliplatin are higher than that of OPA in the liver and pancreas, but not in the lungs and ovaries.
  • OPA levels in the liver were relatively similar 1 and 4 hours post administration of PEG-liposomes and non-PEG liposomes.
  • OPA in non-PEGylated liposomes provides highest organ-to-plasma ratios, which may indicate ability of this formulation to provide maximal efficacy of the drug with minimal systemic exposure and, therefore, toxicity, e.g. nephrotoxicity, neurotoxicity, etc.
  • the latter may be predicted as result of lower brain permeability of OPA and OXA following administration of both liposomal formulations, compared to that after OPA solution injection.
  • both liposomal formulations provided high liver exposures.
  • Table 37 Mice PK and organ bio-distribution of OPA and oxaliplatin as metabolite of OPA
  • PK experiment including potential tumor-forming organs bio-distribution, was performed following administration of OPA-containing PEG liposomes, non- PEG liposomes and IV solution in mice.
  • Major PK parameters and another active metabolite, oxaliplatin are reported.
  • concentrations of OPA in organs were in decreasing order: liver>lung>ovary>pancreas, while lung>liver>pancreas>ovary for oxaliplatin.
  • Liposomes-based forms of the OPA DP showed distribution patterns different from those of OPA in IV solution and had impact on overall disposition of the OPA as well as derived OXA molecules.
  • the graphs in Figs.llA-llD demonstrate the advantage of Liposome PEG- formulation in Pt delivery to relevant organs compared to OPA or oxaliplatin solutions.
  • OPA Liposomes can definitely be targeted to treat severe cancer such as ovarian, liver, pancreatic and lung cancers in addition to Glioblastoma multiforme.
  • OPA Liposomes (Lip-42C) administered at 60 mg/kg, i.v., on Day 1, 4, 8 and 11. Body weight loss was observed with -3.69% mean BWL% nadir on Day 2.
  • Tumor volume The tumor growth curves (mean tumor volume over time) of different groups are shown in Fig. 16.
  • the tumor growth inhibition is summarized in Table 38 below.
  • mice Balb/c nude mice
  • moderate to good anti-tumor efficacy was observed in all treatment groups.
  • the most significant anti-tumor efficacy was observed in OPA, 8mg/kg treated group and Cisplatin, 4mg/kg treated group.
  • the tumor growth curves (mean tumor volume over time) of different groups are shown in Fig. 20.
  • the tumor growth inhibition is summarized in Tables 39 and 40 below.
  • Table 39 Antitumor activity of test agents in the treatment of mouse liver cancer model Hep3B on day 18
  • Table 40 Antitumor activity of test agents in the treatment of mouse liver cancer model Hep3B on day 22
  • Table 41 Antitumor activity of various formulations in SKQV3-luc human ovarian cancer orthotopic model in female SCID-bg Mice on Day 43 [Mean ⁇ SEM (mice number)] .
  • Lipids and other ingredients of the lipsomal preparation were weighed and transferred to a round-bottom flask.
  • Tert-butanol was added to the lipids' mixture and heated for few minutes at 50 s C until all ingredients are completely dissolved.
  • the round-bottom flask was frozen under rotation in an ethanol bath for few minutes (Tzabam, medicine school of Hadassah Ein-kerem) followed by an overnight lyophilization (lyophilizer #3, Tzabam, medicine school of Hadassah Ein- kerem). Consequently, a thin-film of lipid cake is obtained and is further hydrated, with an appropriate amount of pre-heated water to 60 s C, under rotation for 1 hour at 60°C.
  • large multilamellar vesicles are obtained and further downsized to small unilamellar vesicles (SUV) using tip-sonication extrusion homogenization method; by inserting a 50 mL tube into an ice bath for 6 min, 40% amplitude.
  • SUV small unilamellar vesicles
  • Lipids of the OPA liposomal preparation were weighed and transferred to a round-bottom flask. Chloroform was added to the lipids' mixture. After a complete dissolution of all lipids chloroform is evaporated using a Rota-evaporator instrument at 100 rpm without heating. Consequently, a thin lipid film is formed on the roundbottom flask and is further hydrated, with an appropriate amount of pre-heated water to 60°C, under rotation for 1 hour at 60°C.
  • the preparation of OPA liposomes using ethanol injection was performed by weighing the ingredients of the liposomal formulation into a 20 mL scintillation vial and dissolving them in 10 mL of ethanol.
  • the aqueous phase was prepared by heating 20 mL of water in a beaker to 60°C. When the temperature of water reaches 55°C; the ethanol solution is heated to 60°C. When the ethanol solution and the aqueous phase reach 60°C, they were removed from heat and mounted on a head-stirrer. The ethanol solution was rapidly injected into the aqueous phase used a needle- syringe 21G and stirred for a 15 min mix at 900 rpm.
  • ethanol was evaporated using a Rota- evaporator instrument and followed by filtering the final preparation using NY 0.1 pm filter, diameter of 30 mm and a glass prefilter.
  • An illustration of the porcess is depicted in Fig. 26.
  • Osmolality (mOsm/Kg) of the prepared liposomes was evaluated using osmometer 3320 purchased from Advanced Instruments, Inc.
  • the sample was prepared by diluting the OPA liposomes sample by 20 (50 pl sample + 950 pl of methanol) containing overall 5% of water. Moreover, working stocks were prepared for OPA quantification at three different concentrations as follows:
  • the Assay percentage is calculated by the following equation:
  • OPA liposomes were lyophilized in order to improve their physicochemical stability over storage period. After lyophilization (Lyo.), a dry powder of OPA liposomes is obtained and is further reconstituted in order to conduct all the required measurements including; mean diameter (nm), PDI, OPA assay%, pH, water content% and osmolality (mOsm/Kg).
  • the lyophilization process was carried out using Christ-lyophilizer, according to the following protocol:
  • the purpose of this stability study was to examine the stability of various OPA liposomal formulations at 4°C over three months in order to identify a stable prototype formulation suitable for efficacy testing and stable over shelf life.
  • OPA liposomes samples were stored in 5 mL clear crimper vials and stored at 4°C.
  • OPA liposome samples were stored in 5 mL clear crimper vials and stored at three different temperatures:
  • DSPC-lipoid based OPA Liposomes preparation In this part, we prepared two Blank Liposomal preparations and three OPA liposomal preparations of the recipe mentioned below (see Table 48), in an effort to learn about OPA Lip. preparation, to determine the appropriate diluent for analytical analysis of OPA and to determine the optimal storage temperature of OPA Liposomes
  • the concentration of OPA in the recipe below is 2.5 mg/mL.
  • Table 51 Preliminary stability test of OPA Lip.1 According to Table 51, it can be noted that a decrease up to 15-20 % in OPA assay % after 1 week of storage was observed at 25 and 40 °C. A decrease up to 50 % in OPA assay % was observed after 2 months of storage at 25 °C. Therefore, 4 °C was selected as the appropriate storage temperature for extending the stability of OPA Liposomes as aqueous suspension.
  • OPA Liposomal preparations Two different OPA Liposomal preparations, with two different concentrations of OPA, were prepared using thin-film hydration method using tert-butanol or chloroform for thin-film preparation in an effort to understand the effect of the solvent on thin-film and on final OPA Liposomal preparation.
  • Table 52 Tert-butanol vs. chloroform in thin-film preparation According to Table 52, it can be noted that no significant effect of tert-butanol or chloroform on OPA lip. preparation was observed.
  • OPA Lip.8 HSPC-lipoid based OPA liposomes (see Table 53), was prepared to optimize the lyophilization protocol of OPA Liposomes after the preparation of OPA Lip.8, different cryoprotectants at different ratios relative to Lipids amounts were used. OPA Lip.8 cakes that were obtained after lyophilization were reconstituted and characterized to determine the appropriate type and ratio of cryoprotectant (see Table 54).
  • Table 56 Description of HSPC-lipoid based OPA liposomes using ethanol injectionrecipe 3
  • OPA Lip In this part of the project, we prepared OPA Lip. using Thin-film Hydration method using different types of Lipoids; including DSPC, HSPC, DPPC and DMPC. Three descending concentrations of OPA were used for each lipoid type. Following preparation, we conducted a preliminary stability study for the aqueous suspensions over storage period of 3 months at 4°C.
  • OPA Lip.15- 17 show physical instability as aqueous suspensions, over time, in accordance to OPA concentration.
  • physico-chemical characterization and OPA assay % the obtained values are promising and apply to specifications of Table 55.
  • high assay % values could be a consequence of vortexing the sample prior to characterization at the different points of storage period.
  • OPA Liposomal preparations were prepared using the same recipe mentioned on Table 58; except for replacing DSPC-lipoid with HSPC-lipoid.
  • OPA Lip.18- 20 show physical instability, after ⁇ 1 month, as aqueous suspensions in accordance to OPA concentration.
  • physico-chemical characterization and OPA assay % the obtained values are promising and apply to specifications on Table 45. However, this could be a consequence of vortexing the sample prior to characterization at the different points of storage period.
  • OPA Lip. preparations were prepared using the same recipe mentioned on Table 58; except for replacing DSPC-lipoid with DPPC-lipoid.
  • OPA Lipsomal preparations were prepared using the same recipe mentioned on Table 58; except for replacing DSPC-lipoid with DMPC-lipoid.
  • OPA Lip. prepared with DMPC present the highest physical instability as aqueous suspensions compared to OPA Lip. prepared with other lipoids.
  • OPA Lip.24 (2.5 mg/mL) was instable after overnight while OPA Lip.25 and 26 (1.87 and 1.25 mg/mL respectively) were instable after 1 month regarding physical appearance and OPA assay %.
  • this formulation was physically instable (precipitated) as an aqueous suspension after an overnight- stand at RT. Therefore, it was crucial to lyophilize it and further evaluate its' stability as a dried-powder.
  • Lyophilization of these preparations was proposed in an effort to extend physico-chemical stability of OPA Lip.
  • Different cryoprotectants were used for optimization of the lyophilization process; HP ⁇ CD at a ratio of 1:2 (Lipoids amounts in OPA Lip. preparation: HP ⁇ CD) was found adequate regarding preservation of physicochemical characteristics and OPA assay % after lyophilization compared to before lyophilization.
  • Lyophilized OPA Lip.EtOH showed promising results regarding physicochemical and OPA assay % stability over a storage period of 1 month at RT.
  • OPA Lip.42a preparation with OPA concentration of 5 mg/mL was found physically instable after an overnight- stand at RT. Lyophilization of OPA Lip.42a showed an enhanced physical stability over a storage period of 1 month at 40°C. Moreover, physico-chemical characterization and OPA assay % were well-preserved and apply to specifications during the stability-test period.

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Abstract

La technologie concerne des systèmes d'administration à base de lipides comprenant de l'OPA et une substance à base de lipides.
EP21762817.1A 2020-08-20 2021-08-18 Systèmes d'administration de lipides permettant l'administration d'acétate palmitate d'oxaliplatine Withdrawn EP4199906A1 (fr)

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