WO2020058892A1 - Deformable liposomes containing micelles - Google Patents

Deformable liposomes containing micelles Download PDF

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WO2020058892A1
WO2020058892A1 PCT/IB2019/057899 IB2019057899W WO2020058892A1 WO 2020058892 A1 WO2020058892 A1 WO 2020058892A1 IB 2019057899 W IB2019057899 W IB 2019057899W WO 2020058892 A1 WO2020058892 A1 WO 2020058892A1
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drug
micelles
deformable
deformable liposome
bilayer
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PCT/IB2019/057899
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French (fr)
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Francesco Cilurzo
Paola Minghetti
Silvia FRANZE'
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Pharmafilm Srl
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    • 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
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • 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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • 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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/07—Retinol compounds, e.g. vitamin A
    • 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
    • A61K31/12—Ketones
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/196—Carboxylic acids, e.g. valproic acid having an amino group the amino group being directly attached to a ring, e.g. anthranilic acid, mefenamic acid, diclofenac, chlorambucil
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
    • 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
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • 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—Liposomes

Definitions

  • DL Deformable liposomes
  • nanotechnology in transdermal drug delivery field is growing in interest and several nanocarries (e.i. polymeric and metallic nanoparticles, liposomes, dendrimers, nanogels etc) have been studied for breaching the skin barrier (Roberts, 2017 reference 6).
  • This interest born with the hope to develop a shell able to maintain the drug payload whilst squeezing through the pores re sulting from the imperfect overlapping of the cell membranes of corneocytes.
  • each nanosystem has an its own structure and a diameter of 100-200 nm and that the pores of stratum corneum have a maximum opening of 36 nm in full hydration conditions, as a matter of fact almost all nanocarries used in drug delivery suffer of limited skin penetration.
  • the only nanocarriers that seem to be able to cross the stratum corneum are the deformable vesicles.
  • lipo somes formed by the combination of phospholipids and single chain surfactants, that act as desta bilizing agents of the lipid bilayer appear of particular interest.
  • the surfactants may relocate in the areas of maximum curvature when the vesicles undergo an anisotropic stress. This behavior should allow the vesicle to reversibly modify its morphology elongating in the narrow pores of the stratum corneum.
  • deformable vesicle One of the major drawbacks of deformable vesicle is the difficile balance between the flexibility, re quired for skin penetration, and the physico-chemical stability.
  • the fluidity of the lipid bilayer of the deformable vesicle favors the leakage of the drug out of the bilayer. This decreases the shelf life of the product and therefore limits the pos sibility to reach the market.
  • the for mer consists in the development of particular active loading processes that, using peculiar charac teristics of a specific drug (such as acid/base equilibrium), create a gradient that determines the crystallization of the drug in the core of the liposome.
  • the entrapment of doxorubi cin can be enhanced by using an ammonium solphate gradient.
  • An alternative method which in theory is not significantly influenced by the naive characteristics of the drug, may consist in the decoration of the surface of the deformable liposome with hydrophilic moieties. Nevertheless, this approach determines an increase of the stiffness of the lipid bilayer which slows down the permea tion of the vesicle through the stratum corneum.
  • the present invention deals with new deformable liposome compositions such as: micelles in de formable liposome (MiL) and drug in micelles in deformable liposome (DiMiL), in particular:
  • - micelles selected from the group comprising: amphiphilic monomers or amphiphilic polymers
  • a deformable liposomal vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deformable liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably characterized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm,
  • (DiMiL) a deformable liposome composition
  • a drug such as a poorly permeable drug or a water-insoluble drug, incorporated in micelles:
  • said micelles selected from the group comprising: amphiphilic monomers or amphiphilic polymers,
  • a deformable liposomal vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deformable liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably characterized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm,
  • deformable liposome compositions MiL and DiMiL being useful (trans)dermal delivery systems for a hydrophobic compound or for a poorly permeable drug or a water-insoluble drug.
  • Figure 1 shows graphical representation of: deformable liposome composition as micelles in de formable liposome (MiL) wherein deformable liposome vesicle, made of bilayer comprising phos pholipid or lipid forming bilayer and single chain surfactant as an example of fluidizing agent, en- capsules micelles according to the present invention;
  • Figure 2 shows graphical representation of deformable liposome composition as drug in micelles in deformable liposome (DiMiL) wherein deformable liposome vesicle, made of bilayer comprising phospholipid or lipid forming bilayer and single chain surfactant as an example of fluidizing agent, en-capsules micelles incorporating hydrophobic drug according to the present invention;
  • deformable liposome vesicle made of bilayer comprising phospholipid or lipid forming bilayer and single chain surfactant as an example of fluidizing agent, en-capsules micelles incorporating hydrophobic drug according to the present invention
  • Figure 3 shows the graphics dealing with the in vitro drug release profile, as the percentage release vs time, of nifedipine (NIF) (A) and piroxicam (PRX) (B) loaded in the lipid bilayer of the deforma ble liposome composition (DL) system wherein the lipid vesicle is composed of phospholipid egg- phosphatidylcholine and fluidizing agent polisorbate 80 or Tween® 80 (T80) at 95:5 or 85:15 w/w ratios, and nifedipine (NIF) (A) and piroxicam (PRX) (B) encapsulated as drug in micelles in de formable liposome composition (DiMiL) system wherein the lipid vesicle is composed of phospho lipid egg-phosphatidylcholine and fluidizing agent polisorbate 80 or Tween® 80 (T80) at 95:5 or 85:15 w/w ratios, and the micelles component is PEG-hydroxyste
  • Figure 4 shows the graphics dealing with the in vitro permeation profile of A) nifedipine and B) piroxicam through human epidermis after application thereto of: deformable liposome composition (DL) loading in the lipid bilayer of the vesicle thereof nifedipine and piroxicam, respectively, de formable liposome composition as drug in micelles in deformable liposome (DiMiLs) wherein the micelles component PEG-hydroxystearate (Kolliphor® HS 15) incorporates nifedipine (NIF) and piroxicam (PRX), respectively; and PEG-hydroxystearate (Kolliphor® HS 15) micelles incorporating nifedipine (NIF) and piroxicam (PRX), respectively.
  • DL deformable liposome composition
  • DIMiLs deformable liposome
  • the micelles component PEG-hydroxystearate (Kolliphor® HS 15) incorporates nifedipine (NIF)
  • micelles selected from the group comprising amphiphilic monomers or amphiphilic polymers are used to solubilize hydrophobic compounds in the aqueous core of the deformable liposomes. This allows to accommodate hydrophobic drugs in the aqueous core of de formable liposomes through micelles entrapment, in order to avoid drug leakage.
  • deformable liposome composition as drug in micelles in deformable lipo some (DiMiLs) revealed to have superior properties of enhancing drug permeation with respect to both conventional deformable liposomes and surfactant based micelles alone.
  • US 6 143 321 A discloses liposomes containing micelles wherein it is mandatory the use of stabilizing agents in the bilayer or on the bilayer of the liposome vescicle.
  • stabilizing agents are cholesterol and grafted PEG, respectively.
  • US 2012/231069 Al discloses the feasibility to allow the gastro-intestinal absorption of nanoparticu late drug delivery systems by using the active transport of vitamin B12 or a derivative thereof and, using the same targeting moiety or other molecules, to deliver the cargo of the carrier to diseased cells in the body [007]
  • the vitamin B12 or a derivative thereof (hereinafter targeting moiety) is at tached to the surface of the nanoparticle [008] or to micelle [009] loaded in a nanoparticle.
  • the targeting moiety can be attached to the surface of liposomes.
  • the stability of liposome is due to the fact that the vitamin B12 or a derivative thereof must be grafted on the lip osome surface.
  • GILLET ET AL "Liposomes and parameters affecting their skin penetration behaviour", JOUR NAL OF DRUG DELIVERY SCIENCE AND TECHNOLOGY, vol. 21 , no. 1 , 1 January 201 1 , pages 35-42 does not disclose not even suggests the possibility to produce a deformable liposome con taining micelles in its aqueous core.
  • Liposomes are lipid vesicles composed of one or more lipid bilayers enclosing aqueous compart ments.
  • the main components of liposomes are phospholipids or other lipids able to form bilayers, and cholesterol. The last is required to reduce bilayer fluidity and to allow the control of the drug re lease and the liposome stability.
  • the surface of lipo somes can bear hydrophilic chains, generally, but not exclusively, of polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • deformable liposomes, according to the present invention are free of cholesterol or other stabilizing materials in/on the bilayer since they have to be“soft” structures to pass through the tight skin barrier.
  • deformable liposomes contain at least one fluidizing agent (generally in a concentration of 5-20% w/w with respect to the main bilayer forming lipid).
  • the fluid izing agent is a substance able to confer flexibility to the lipid vesicles.
  • a non-ionic sin gle chain surfactant, a skin penetration enhancer, ethanol or a combination thereof can be used.
  • the deformable liposome according to the present invention such as: micelles in deformable lipo some (MiL) or drug in micelles in deformable liposomes (DiMiL), comprises deformable liposome, containing micelles in its aqueous core, free of cholesterol or other stabilizing materials in/on the bi layer, preferably characterized by a deformability constant not higher than 0.13 N/mm, more pref erably by a deformability constant in the range 0.01 - 0.13 N/mm.
  • Micelles are self-assembling aggregates of colloidal dimensions resulting from the interaction be tween the hydrophobic and hydrophilic moieties of amphiphilic monomers. Micelles can result from the self-aggregation of surfactants above the critical micelle concentration, such as the esters of fatty acids and PEGs, or of amphiphilic polymers, such as polaxamers.
  • the deformable liposome composition as micelles in Deformable liposome can be loaded with hydrophobic compounds selected from the group comprising neutral, cationic, anionic, zwitter- ionic compounds.
  • the Drug in micelles in deformable liposome compositions (DiMiL) can be load ed with neutral, cationic, anionic, zwitterionic compounds.
  • the deformable liposome composition as drug in micelles in Deformable liposome compositions are loaded with drug, such as a poorly permeable drug or a water-insoluble drug, prefera bly selected from the group comprising neutral, cationic, anionic, zwitterionic drugs, such as resveratrol, nifedipine, piroxicam or naproxene or tranexamic acid, retinol, arbutin, perfluordecalin, diclofenac, azelaic acid, caffeine, CBD (cannabidiol), curcumin, lidocaine, pycnogenol.
  • drug such as a poorly permeable drug or a water-insoluble drug
  • drug such as a poorly permeable drug or a water-insoluble drug
  • the deformable liposome composition as micelles in Deformable liposome (MiL) or deformable lip osome composition as drug in micelles in Deformable liposome compositions (DiMiL) can be pre pared by film hydration methods or solvent displacement or microfluidic or cross flow injection methods.
  • deformable liposome composition as micelles in deforma ble liposome (MiL), said deformable liposome composition comprising:
  • Non ionic Surfactants such as the esters of fatty acids or PEGs or Fatty alcohol ethoxylates; Nonoxynols; Fatty acid ethoxylates; Special ethox- ylated fatty esters and oils; Ethoxylated amines and/or fatty acid amides; Terminally blocked ethoxylates; Fatty acid esters of glycerol; Fatty acid esters of sorbitol; Fatty acid esters of sucrose; Alkyl polyglycoside; Ionic surfactants: such as Cetrimonium bromide; Cetylpyridinium chloride; Benzalkonium chloride; Benzethonium chloride; Dimethyldioc- tadecylammonium chloride; Dioctadecyldimethylammonium bromide; or o amphiphilic polymers,
  • Self assembling copolymers such as Amphiphilic block co polymers (i.e. Pluronics, poly(esters), hydrophobic poly(amino acids), copolymers of lac tic acid and glycolic acids, poly(caprolactone)), graft copolymers, polaxamers, semisyn thetic polysaccharides
  • said micelles encapsulated in the aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a de formable liposome free of cholesterol or other stabilizing materials in/on the bilayer, prefera bly characterized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm, said fluidizing agent preferably in a concentration of 5-20% w/w with respect to the phospholipid or lipid forming bilayer compo nent, said fluidizing agent preferably is selected from the group comprising non-ionic single chain surfactant, a skin penetration enhancer, ethanol or a combination thereof.
  • the fluidizing agents contained in the deformable liposome vesicle are in a concentration preferably of 5 %, 10 %, 15% or 20%, preferably 5- 20% or 5-15% or 5-10% all the percentage are % w/w with respect to the phospholipid or lipid forming bi layer component.
  • the micelles incorporate a hydrophobic compound.
  • the hydrophobic compound can be loaded in the micelles at its maximum solubility without affect ing the leakage thereof, said loading because the micelles are self-assembling aggregates of col loidal dimensions resulting from the interaction between the hydrophobic and hydrophilic moieties of amphiphilic monomers or amphiphilic polymers.
  • the hydrophobic moieties of Micelles entrapped the hydrophobic compound.
  • the hydrophobic compound is selected from the group comprising neutral, cationic, anionic, zwitterionic compounds.
  • the hydrophobic compound is a drug, such as poorly permeable drug or water-insoluble drug, preferably selected from the group comprising neutral, cationic, anionic, zwitterionic drugs, such as resveratrol, nifedipine, piroxicam or naproxene or tranexamic acid, retinol, arbutin, perfluordecalin, diclofenac, azelaic acid, caffeine, CBD (cannabidiol), curcumin, lidocaine, pycnogenol.
  • a drug such as poorly permeable drug or water-insoluble drug, preferably selected from the group comprising neutral, cationic, anionic, zwitterionic drugs, such as resveratrol, nifedipine, piroxicam or naproxene or tranexamic acid, retinol, arbutin, perfluordecalin, diclofenac, azelaic acid, caffeine, CBD (cannabidiol
  • the micelles have an av erage diameter no more than 30 nm, preferably an average diameter from 10 nm to 30 nm, either incorporating or non-incorporating a hydrophobic compound and/or the deformable liposome vesi cle has an average diameter no more than 300 nm, preferably an average diameter from 50 nm to 200 nm.
  • the phospholipid or lipid forming bilayer component of the deformable liposome vesicle is selected from the group comprising:
  • Phospholipids such as phosphatidylcholine, Phosphatidylethanolamine, phosphatidylserine, Phosphatidylglycerol and all derivatives of such phospholipids as well as all the combinations thereof;
  • - Cationic Lipids such as 1 ,2-di-0-octadecenyl-3-trimethylammonium propane, 1 ,2-dilauroyl-sn- glycero-3-ethylphosphocholine, Dimethyldioctadecylammonium, 1 ,2-dipalmitoyl-3- trimethylammonium-propane, 1 ,2-stearoyl-3-trimethylammonium-propane, 1 ,2-dimyristoyl-3- trimethylammonium-propane, 1 ,2-dioleoyl-3-trimethylammonium-propane, 1 ,2-dioleyloxy-3- dimethylaminopropane;
  • Sphingolipids such as Sphingomyelin (all sources) and derivatives.
  • the fluidizing agent contained in the deformable lip osome vesicle is selected from the group comprising non-ionic single chain surfactant, such as Tween, Span, sodium cholate etc.., a skin penetration enhancer, such as terpenes, unsaturated fatty acids, colic acid and its derivatives, ethylene glycol derivatives, propylene glycol, glycerine, or ethanol or a combination thereof.
  • non-ionic single chain surfactant such as Tween, Span, sodium cholate etc..
  • a skin penetration enhancer such as terpenes, unsaturated fatty acids, colic acid and its derivatives, ethylene glycol derivatives, propylene glycol, glycerine, or ethanol or a combination thereof.
  • deformable liposome composition comprising:
  • - micelles selected from the group comprising: amphiphilic monomers or amphiphilic polymers
  • a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deforma ble liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably charac terized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm,
  • - micelles selected from the group comprising: amphiphilic monomers or amphiphilic polymers
  • a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deforma ble liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably charac terized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm,
  • - micelles selected from the group comprising: amphiphilic monomers or amphiphilic polymers
  • a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deforma ble liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably charac- terized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm,
  • - micelles selected from the group comprising: amphiphilic monomers or amphiphilic polymers
  • a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deforma ble liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably charac terized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm,
  • (trans)dermal delivery system for a drug such as a poorly permeable drug or a water-insoluble drug.
  • said micelles selected from the group comprising: amphiphilic monomers or amphiphilic poly mers
  • a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deforma ble liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably charac terized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm,
  • the invention further provides a method of administering a hydrophobic compound to a target tis sue comprising the steps of:
  • a deformable liposome composition comprising a hydrophobic com pound incorporated in micelles, said micelles selected from the group comprising: amphiphilic monomers or amphiphilic polymers, said micelles encapsulated in aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deformable liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably characterized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm, and
  • the deformable liposome composition com prising a hydrophobic compound incorporated in micelles, said micelles encapsulated in aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deformable liposome free of cholesterol or other sta bilizing materials in/on the bilayer, preferably characterized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm,.
  • DiMiL drug in micelles in deformable liposome
  • a deformable liposome composition as drug in micelles in de- formable liposome (DiMiL), said deformable liposome composition comprising a drug, such as a poorly permeable drug or a water-insoluble drug, incorporated in micelles:
  • - micelles selected from the group comprising:
  • Non ionic Surfactants such as the esters of fatty acids or PEGs or Fatty alcohol ethoxylates; Nonoxynols; Fatty acid ethoxylates; Special ethox- ylated fatty esters and oils; Ethoxylated amines and/or fatty acid amides; Terminally blocked ethoxylates; Fatty acid esters of glycerol; Fatty acid esters of sorbitol; Fatty acid esters of sucrose; Alkyl polyglycoside; Ionic surfactants: such as Cetrimonium bromide; Cetylpyridinium chloride; Benzalkonium chloride; Benzethonium chloride; Dimethyldioc- tadecylammonium chloride; Dioctadecyldimethylammonium bromide; or o amphiphilic polymers, i.e.
  • Non ionic Surfactants such as the esters of fatty acids or PEG
  • Self assembling copolymers such as Amphiphilic block co polymers (i.e. Pluronics, poly(esters), hydrophobic poly(amino acids), copolymers of lac tic acid and glycolic acids, poly(caprolactone)), graft copolymers, polaxamers, semisyn thetic polysaccharides
  • said micelles encapsulated in the aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deforma ble liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably charac terized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm, said fluidizing agent preferably in a concentration of 5- 20% w/w with respect to the phospholipid or lipid forming bilayer component, said fluidizing agent preferably is selected from the group comprising non-ionic single chain surfactant, a skin penetra tion enhancer, ethanol or a combination thereof.
  • the fluidizing agent contained in the de formable liposome vesicle are in a concentration preferably of 5 %, 10 %, 15% or 20%, preferably 5- 20% or 5-15% or 5-10%; all the percentage are % w/w with respect to the phospholipid or lipid forming bilayer component.
  • the micelles incorporate a hydrophobic com pound.
  • the drug such as a poorly permeable drug or a water-insoluble drug
  • the hy drophobic moieties of Micelles entrapped the drug, such as a poorly permeable drug or a water- insoluble drug.
  • the drug such as poorly permeable drug or water-insoluble drug
  • the drug is preferably selected from the group comprising neutral, cationic, an ionic, zwitterionic drugs, such as resveratrol, nifedipine, piroxicam or naproxene or tranexamic acid, retinol, arbutin, perfluordecalin, diclofenac, azelaic acid, caffeine, CBD (cannabidiol), curcumin, li- docaine, pycnogenol.
  • neutral, cationic, an ionic, zwitterionic drugs such as resveratrol, nifedipine, piroxicam or naproxene or tranexamic acid, retinol, arbutin, perfluordecalin, diclofenac, azelaic acid, caffeine, CBD (cannabidiol), curcumin, li- docaine, pycnogenol.
  • the micelles have an average diameter no more than 30 nm, pref erably an average diameter from 10 nm to 30 nm, and/or the deformable liposome vesicle has an average diameter no more than 300 nm, preferably an average diameter from 50 nm to 200 nm.
  • the phospholipid or lipid forming bilayer component of the deformable liposome vesicle is selected from the group comprising:
  • Phospholipids such as phosphatidylcholine, Phosphatidylethanolamine, phosphatidylserine, Phosphatidylglycerol and all derivatives of such phospholipids as well as all the combinations thereof;
  • - Cationic Lipids such as 1 ,2-di-0-octadecenyl-3-trimethylammonium propane, 1 ,2-dilauroyl-sn- glycero-3-ethylphosphocholine, Dimethyldioctadecylammonium, 1 ,2-dipalmitoyl-3- trimethylammonium-propane, 1 ,2-stearoyl-3-trimethylammonium-propane, 1 ,2-dimyristoyl-3- trimethylammonium-propane, 1 ,2-dioleoyl-3-trimethylammonium-propane, 1 ,2-dioleyloxy-3- dimethylaminopropane;
  • Sphingolipids such as Sphingomyelin (all sources) and derivatives.
  • the fluidizing agent contained in the de formable liposome vesicle is selected from the group comprising non-ionic single chain surfactant, such as Tween, Span, sodium cholate etc.., a skin penetration enhancer, such as terpenes, unsaturated fatty acids, colic acid and its derivatives, ethylene glycol derivatives, propylene glycol, glycerine, or ethanol or a combination thereof.
  • non-ionic single chain surfactant such as Tween, Span, sodium cholate etc..
  • a skin penetration enhancer such as terpenes, unsaturated fatty acids, colic acid and its derivatives, ethylene glycol derivatives, propylene glycol, glycerine, or ethanol or a combination thereof.
  • a deformable liposome composition comprising a drug, such as a poorly permeable drug or a water-insoluble drug, incorporated in micelles:
  • said micelles selected from the group comprising: amphiphilic monomers or amphiphilic poly mers,
  • a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deforma ble liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably charac terized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm,
  • a drug such as a poorly permeable drug or a water-insoluble drug
  • said micelles selected from the group comprising: amphiphilic monomers or amphiphilic poly mers
  • a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deforma ble liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably charac terized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm, is (trans)dermally administered to said target tissue.
  • the invention further provides a method of administering a drug, such as a poorly permeable drug or a water-insoluble drug, to a target tissue comprising the steps of:
  • a deformable liposome composition comprising a drug, such as a poorly permeable drug or a water-insoluble drug, incorporated in micelles, said micelles selected from the group comprising: amphiphilic monomers or amphiphilic polymers, said micelles encap sulated in aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deformable liposome free of cho lesterol or other stabilizing materials in/on the bilayer, preferably characterized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm,
  • the deformable liposome composition com prising a drug, such as a poorly permeable drug or a water-insoluble drug, incorporated in mi
  • said micelles encapsulated in aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a de formable liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably characterized by a deformability constant not higher than 0.13 N/mm, more preferably by a de formability constant in the range 0.01 - 0.13 N/mm.
  • the poorly permeable drug or water-insoluble drug are selected from the group com prising neutral, cationic, anionic, zwitterionic drugs, such as resveratrol, nifedipine, piroxicam or naproxene or tranexamic acid, retinol, arbutin, perfluordecalin, diclofenac, azelaic acid, caffeine, CBD (cannabidiol), curcumin, lidocaine, pycnogenol.
  • the group com prising neutral, cationic, anionic, zwitterionic drugs such as resveratrol, nifedipine, piroxicam or naproxene or tranexamic acid, retinol, arbutin, perfluordecalin, diclofenac, azelaic acid, caffeine, CBD (cannabidiol), curcumin, lidocaine, pycnogenol.
  • a deformable liposome composition comprising a drug, such as a poorly permeable drug or a water-insoluble drug, incorporated in micelles:
  • Non ionic Surfactants such as the esters of fatty acids or PEGs or Fatty alcohol ethoxylates; Nonoxynols; Fatty acid ethoxylates; Special ethox- ylated fatty esters and oils; Ethoxylated amines and/or fatty acid amides; Terminally blocked ethoxylates; Fatty acid esters of glycerol; Fatty acid esters of sorbitol; Fatty acid esters of sucrose; Alkyl polyglycoside; Ionic surfactants: such as Cetrimonium bromide; Cetylpyridinium chloride; Benzalkonium chloride; Benzethonium chloride; Dimethyldioc- tadecylammonium chloride; Dioctadecyldimethylammonium bromide; or o amphiphilic polymers, i.e.
  • Non ionic Surfactants such as the esters of fatty acids or PEG
  • Self assembling copolymers such as Amphiphilic block copol ymers (i.e. Pluronics, poly(esters), hydrophobic poly(amino acids), copolymers of lactic acid and glycolic acids, poly(caprolactone)), graft copolymers, polaxamers, semisynthetic polysaccharides;
  • Amphiphilic block copol ymers i.e. Pluronics, poly(esters), hydrophobic poly(amino acids), copolymers of lactic acid and glycolic acids, poly(caprolactone)
  • graft copolymers such as graft copolymers, polaxamers, semisynthetic polysaccharides;
  • a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deformable liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably characterized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm,
  • the drug as a poorly permea ble drug or a water-insoluble drug, is selected from the group comprising neutral, cationic, anionic, zwitterionic drugs, such as resveratrol, nifedipine, piroxicam, naproxene.
  • a deformable liposome composition comprising a drug, such as a poorly permeable drug or a water-insoluble drug, incorporated in micelles:
  • Non ionic Surfactants such as the esters of fatty acids or PEGs or Fatty alcohol ethoxylates; Nonoxynols; Fatty acid ethoxylates; Special ethox- ylated fatty esters and oils; Ethoxylated amines and/or fatty acid amides; Terminally blocked ethoxylates; Fatty acid esters of glycerol; Fatty acid esters of sorbitol; Fatty acid esters of sucrose; Alkyl polyglycoside; Ionic surfactants: such as Cetrimonium bromide; Cetylpyridinium chloride; Benzalkonium chloride; Benzethonium chloride; Dimethyldioc- tadecylammonium chloride; Dioctadecyldimethylammonium bromide; or o amphiphilic polymers, i.e.
  • Non ionic Surfactants such as the esters of fatty acids or PEG
  • Self assembling copolymers such as Amphiphilic block copol ymers (i.e. Pluronics, poly(esters), hydrophobic poly(amino acids), copolymers of lactic acid and glycolic acids, poly(caprolactone)), graft copolymers, polaxamers, semisynthetic polysaccharides;
  • Amphiphilic block copol ymers i.e. Pluronics, poly(esters), hydrophobic poly(amino acids), copolymers of lactic acid and glycolic acids, poly(caprolactone)
  • graft copolymers such as graft copolymers, polaxamers, semisynthetic polysaccharides;
  • a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deformable liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably characterized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm,
  • the drug as a poorly permea ble drug or a water-insoluble drug, is selected from the group comprising neutral, cationic, anionic, zwitterionic drugs, such as tranexamic acid, retinol, arbutin, perfluordecalin, diclofenac, azelaic ac id, caffeine, CBD, curcumin, lidocaine, pycnogenol.
  • neutral, cationic, anionic, zwitterionic drugs such as tranexamic acid, retinol, arbutin, perfluordecalin, diclofenac, azelaic ac id, caffeine, CBD, curcumin, lidocaine, pycnogenol.
  • a deformable lipo some composition as micelles in deformable liposome (MiL) and a deformable liposome composi tion as drug in micelles in deformable liposome (DiMiL) the following are the preferred Micelles components, Phospholipid/Lipid components of the deformable liposome bilayer and Fluidizing agents in deformable liposomes bilayer.
  • Non ionic Surfactants Fatty alcohol ethoxylates; Nonoxynols; Fatty acid ethoxylates; Special ethoxylated fatty esters and oils; Ethoxylated amines and/or fatty acid amides; Terminally blocked ethoxylates; Fatty acid esters of glycerol; Fatty acid esters of sorbitol; Fatty acid esters of su crose; Alkyl polyglycoside.
  • Ionic surfactants Cetrimonium bromide; Cetylpyridinium chloride; Benzalkonium chloride; Benzethonium chloride; Dimethyldioctadecylammonium chloride; Dioctadecyldimethylammonium bromide.
  • Self assembling copolymers Amphiphilic block copolymers (i.e. Pluronics, poly(esters), hy drophobic poly(amino acids), copolymers of lactic acid and glycolic acids, poly(caprolactone)), graft copolymers, polaxamers, semisynthetic polysaccharides.
  • Amphiphilic block copolymers i.e. Pluronics, poly(esters), hy drophobic poly(amino acids), copolymers of lactic acid and glycolic acids, poly(caprolactone)
  • graft copolymers graft copolymers
  • polaxamers semisynthetic polysaccharides.
  • Phospholipid/Lipid components of the deformable liposome bilaver Phospholipid/Lipid components of the deformable liposome bilaver:
  • Phospholipids phosphatidylcholine, Phosphatidylethanolamine, phosphatidylserine, Phos- phatidylglycerol and all the derivatives of such lipids as well as all the combinations thereof.
  • Cationic Lipids 1 ,2-di-0-octadecenyl-3-trimethylammonium propane, 1 ,2-dilauroyl-sn- glycero-3-ethylphosphocholine, Dimethyldioctadecylammonium, 1 ,2-dipalmitoyl-3- trimethylammonium-propane, 1 ,2-stearoyl-3-trimethylammonium-propane, 1 ,2-dimyristoyl-3- trimethylammonium-propane, 1 ,2-dioleoyl-3-trimethylammonium-propane, 1 ,2-dioleyloxy-3- dimethylaminopropane.
  • Sphingolipids Sphingomyelin (all sources) and derivatives.
  • Fluidizing agents in deformable liposomes bilaver
  • single chain surfactants Teween, Span, sodium cholate etc..
  • skin penetration enhancers ter- penes, unsaturated fatty acids, colic acid and its derivatives, ethylene glycol derivatives, propylene glycol, glycerine
  • ethanol ethanol
  • a“matryoshka” system namely a deformable liposome composition as micelles in deformable liposome (MiL), i.e. micelles encapsulated in aqueous core of deformable liposome vesicle as a carrier of hydrophobic sub stance or a deformable liposome composition as a drug in micelles in deformable liposome (DiMiL), i.e. micelles incorporating a hydrophobic drug, said micelles encapsulated in aqueous core of de formable liposome vesicle.
  • the micelles were made of PEG-hydroxystearate (Kolliphor® HS15) while the deformable liposome vesicle components of: deformable liposome (DL), deformable liposome composition MiL and de formable liposome composition DiMiL were as phospholipid/lipid egg-phosphatidylcholine and fluid izing agent polisorbate 80 or Tween® 80 (T80) at 95:5 or 85:15 w/w ratios.
  • the drug leakage from DL was high after only one month of storage (almost 50% in the case of nifedipine and in the range of 39-79 % in the case of piroxicam loaded DL, depending on T80 content).
  • the deformable liposome composition Drug in micelles in deformable liposome DiMiL formulations retained instead the drug content up to two-month storage period.
  • deformable liposome composition as micelles in deformable liposome (MiL) and de formable liposome composition as drug in micelles in deformable liposome (DiMiL) reveals to be a suitable approach to avoid the leakage of hydrophobic compounds and an attractive (trans)dermal delivery system for hydrophobic compound in general, in particular a (trans)dermal delivery system for drug such as for a poorly permeable drug or a water-insoluble drug.
  • Deformable Liposome compositions the known Deformable liposome composition (DL), the micelles in deformable liposome composition (MiL) according to the present invention and the drug in micelles in deformable composition (DiMiL) according to the present invention were prepared by thin film hydration method.
  • deformable liposome vesicle components of:
  • the micelles were made of PEG-hydroxystearate (Kolliphor® HS15).
  • egg phosphatydilcholine (e-PC) and polisorbate 80 (Tween 80/T80) dissolved in a chloro form/methanol (2:1 v/v) were mixed in the proper weight ratios (Table 1 ) in a round bottom flask.
  • the organic solvent was evaporated for 1 hour at 40°C under reduced pressure using a rotatory evaporator (Rll, Buchi, Italy), to obtain a thin lipid film.
  • the film was rehydrated in milliQ® water for 1 h to obtain a final lipid concentration of 30 mg/mL.
  • Drug loaded micelles were prepared by adding a proper amount of drug to the kolliphor® HS15 so lution (Table 1 ).
  • the lipid film was re-hydrate with a 10% w/v solution of kolliphor® HS 15.
  • the organic solvent was evaporated for 1 hour at 40°C under reduced pressure using a rotatory evaporator (Rll, Buchi, Italy), to obtain a thin lipid film.
  • the film was rehydrated in milliQ® water for 1 h.
  • the lipid film was rehy drated with the micellar solution of PEG-hydroxystearate Kolliphor® HS 15 (10% w/v in ultrapure water) containing the hydrophobic drug.
  • the liposome dispersions were ex truded (Avanti® Mini-Extruder, Avanti Polar Lipids, Inc.) through sequential passages through 0.2 pm (5 passages) and 0.1 pm (6 passages) polycarbonate membranes.
  • formulations were purified from free materials (drug and/or micelles) by molecular size exclusion chromatography on Sepharose CL-4B columns, eluting with milliQ® water.
  • kolliphor® HS 15 in solution forms micelles having a mean particle size of 12.56 ⁇ 0.04 nm, and therefore they can be easily en trapped/encapsulated, either enclosing or not the hydrophobic drug, in the aqueous core of de formable liposome compositions according to the present invention: micelles in deformable lipo some composition (MiL) or drug in micelles in deformable liposome composition (DiMiL), such as during the film rehydration.
  • MiL deformable lipo some composition
  • DIMiL deformable liposome composition
  • the mean diameter of the vesicles was in all cases lower than 150 nm, therefore suitable for the application on the skin (Table 2).
  • the particle size distribution was determined both by dynamic light scattering (DLS), using a Zetasizer (Nano-ZS, Malvern Instrument, UK), and Nanoparticle Trafficking Analysis (NTA) using a Nanosight NS 300 (Malvern Instrument, UK).
  • DLS dynamic light scattering
  • NTA Nanoparticle Trafficking Analysis
  • particle size measurements were carried out inserting the sample in a disposable cuvette after a 1 :10 dilution in 0,22 pm filtered milMQ® water, with a detection angle of 173°.
  • z-potential was assessed on the diluted sample inserted in a capillary cell.
  • Drug encapsulation was measured by diluting the liposomes 1 :100 in methanol to break the vesi cles and release the free drug.
  • the amount of drug was then determined by a HPLC system equipped with a diode array detector (HLPC HP 1 100 Chemstations, Agilent Technologies, Waldbronn, Germany).
  • nifedipine quantification the samples were eluted through a lichrospher 100 RP-18E column (CPS Analitica, Milan, Italy) with a mixture of acetonitrile:methanol:Milli-Q® water (25:25:50 v/v), that was used as mobile phase; the flow rate was 1 .3 mL/min, the thermostat was set at 37°C and the UV lamp at 230 nm.
  • Nifedipine quantification was done on the basis of a calibration curve of drug in mobile phase in the concentration range 5-100 pg/mL.
  • Piroxicam quantification was performed using a 0.03 M phosphate pH 3.0 buffer/acetonitrile (60/40% v/v) mixture as mobile phase.
  • the flow rate was 1 .5 mL/min, and the UV lamp was set at two wavelengths (i.e., 248 nm, 360 nm). Sample concentrations were calculated basing on calibration curves built between 0.05 and 20 pg/mL.
  • the encapsulation efficiency (EE, %) was expressed as the percentage ratio between the amount of drug (mg/mL) encapsulated in the vesicles and the cumulative amount of the drug (mg/mL) loaded in the liposomes at the time of the preparation.
  • the deformability properties of the vesicles were determined using a dynanometer assisted extru sion assay previously developed (reference 3).
  • Lipid concentration was determined using an adaptation of the Rouser method, as described else where (see reference 4).
  • the syringe plunger was put in contact with a 50 N load cell of a dyna mometer (INSTRON 5965, ITW Test and Measurement Italia Sri, Italy) and forced to move at a constant speed of 1 mm/s, forcing the liposomal dispersion through a 50 nm polycarbonate mem brane fixed in an extruder casing.
  • Table 2 Physical-chemical properties of the: deformable liposome composition (DL); micelles in deformable liposome composition (MiL); drug in micelles in deformable liposome composition (DiMiL).
  • DL deformable liposome composition
  • MiL micelles in deformable liposome composition
  • DiMiL drug in micelles in deformable liposome composition
  • Placebo no drug
  • NIF nifediprine
  • PRX piroxicam
  • NPX naproxene
  • DL deformable liposome composition
  • MiL micelles in deformable liposome composition
  • DiMiL drug in micelles in deform- able liposome composition
  • d mean hydrodynamic diameter
  • EE encapsulation efficiency
  • NA not applicable
  • Pdl Polydispersity index.
  • Polymeric micelles were prepared using a polaxamer 407 (Kolliphor® P407) and resveratrol as neutral hydrophobic compound.
  • the solution was let under stirring for 3 hours at room temperature prior to be used for micelles preparation.
  • the suspension was let at the same temperature for 5 minutes and later for 2 hours at 37°C, under a constant stirring at 500 rpm.
  • the unentrapped drug was removed by low speed centrifugation (3000 rpm, 10 min, 23°C).
  • the polymeric micelles in deformable liposomes compositions were prepared according to the pro cedure reported for surfactant micelles in deformable liposomes.
  • the prepared compositions showed suitable physico-chemical properties (Table 4)
  • RES resveratrol
  • DiMiL drug in micelles in deformable liposome composition
  • d mean hydrody namic diameter
  • Pdl Polydispersity index.
  • Drug leakage from deformable liposomes composition (DL) in which the hydrophobic drug sits in the lipid bilayer vesicle thereof and drug in micelles in deformable liposome composition (DiMiL) in which the hydrophobic drug is entrapped in the aqueous core through micelles solubilization was determined by purifying the liposomal dispersions from leaked drug by low speed (5000 rpm) cen trifugation for 10 min at 25 °C (Universal 30RF, Hettich Zentrifugen, Germany).
  • Drug leakage % ((Et0-Etx))/Et0 X 100
  • Eto is the drug amount in mg/mL loaded in the lipid bilayer vesicle in the deformable liposome composition (DL) or encapsulated in Drug in micelles in deformable composition (DiMiL) at the time of preparation and Etx is the amount of drug still loaded/encapsulated after 1 or 2 months of storage.
  • NIF nifediprine
  • PRX piroxicam
  • DL deformable liposome composition
  • DiMiL drug in micelles in deformable liposome composition.
  • deformable liposome (DL) deformable liposome
  • MiL micelles in deformable liposome
  • DiMiL drug in micelles in deformable liposome
  • the syringe plunger was put in contact with a 50 N load cell of a dy namometer (INSTRON 5965, ITW Test and Measurement Italia Sri, Italy) and forced to move at a constant speed of 1 mm/s, forcing the liposomal dispersion through a 50 nm polycarbonate mem brane fixed in an extruder casing.
  • the force (N) required to move the syringe plunger (dependent on the resistance opposed to vesicle penetration through the pores) was registered as a function of the plunger displacement (mm).
  • the slope of this plot namely the constant of deformability (k), was then derived. The higher the k value, the lower the deformability of the carriers (see reference 3).
  • K constant of deformability (N/mm); the higher is the K value, the lower is the flexibility of the carri er; Placebo: no drug; NIF: nifediprine; PRX: piroxicam; NPX: naproxene; RES: resveratrol; DL: de formable liposome composition; MiL: micelles in deformable liposome composition; DiMiL: drug in micelles in deformable liposome composition.
  • the drug in micelles in deformable liposome compositions significantly slowed down the drug release kinetic with respect to deformable liposome composition (DL) as clearly shown in the percentage nifedipine (NIF) released diagram in figure 3A and in the percentage piroxicam (PRX) released diagram in figure 3B.
  • the skin samples are prepared according to an internal procedure (see reference 2).
  • Epidermis sheets are obtained through mechanical separation from the remaining tissue with for ceps, after skin immersion in water at 60 ⁇ 1 °C for 1 min.
  • the integrity of the skin is evaluated by measur ing the electrical impedance of the epidermis sheets (Agilent 4263B LCR Meter, Microlease, Italy).
  • the epidermis sheets are mounted on the lower half of the Franz diffusion cells with the stratum corneum facing upward.
  • the upper and lower parts of the cell are sealed with parafilm and fastened together with a clamp.
  • the receiver compartment was filled with an acetate buffer (pH 4.6)/polyethylene glycol 400 (PEG 400) mixture for nifedipine and physiologic solution for piroxicam to assure the sink conditions.
  • the receiver phase was continuously stirred by a magnetic bar at 1500 rpm.
  • micellar solutions were tested in parallel as control.
  • the composition was kept at 37 ⁇ 1 °C by means of a circulating water bath so that the epidermis surface temperature was at 32 ⁇ 1 °C throughout the experiment.
  • the cells were dismounted and the skin samples were recovered and washed on both sides with fresh methanol to remove any residues of formulation/solution.
  • Epidermis samples were let to dry and weighted prior to be cut in small pieces and immersed in 5 mL of methanol to extract the drug retained in the tissue.
  • Nifedipine is a good candidate for (trans)dermal delivery due to the low molecular weight and the logP value of 2.2. Nevertheless, its passive diffusion through the skin is negligible. Nevertheless, surprisingly, the drug in micelles in deformable liposome composition (DiMiL) according to the present invention led to a drastic increase of nifedipine permeation after 24 hours that moved from 2.73 ⁇ 0.06 pg/cm 2 when the nifedipine is loaded in the lipid bilayer vesicle of deformable liposome (DL) to 131 .27 ⁇ 48.1 1 pg/cm 2 when the nifedipine is (trans)dermally delivered by the drug in micelles in deformable liposome composition according to the present invention(DiMiLs).
  • the physical stability of DL and DiMiL systems was monitored over time by measuring the particle size distribution of the lipid vesicles after three and six months of storage to rule out the possibility of phenomena of fusion or aggregation of particles due to the fluidity of the bilayers containing two destabilizing agents, namely one in the bilayer and one as component of the micelles.
  • the particle size distribution d (nm) was monitored using DLS.
  • Table 7 Particle size distribution and polydispersity of DL and DiMiL systems at starting time after three and six months (mos) storage period.
  • DiMiL formulations DiMiL 85:15 formulations have been car ried out as reported in Table 8.

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Abstract

It is described a deformable liposome composition comprising: - micelles selected from the group comprising: amphiphilic monomers or amphiphilic polymers - said micelles encapsulated in the aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deformable liposome free of cholesterol or other stabilizing materials in/on the bilayer, said micelles can comprise a hydrophobic compound.

Description

Title:
“Deformable liposomes containing micelles”
DESCRIPTION
Technical field
Deformable liposomes (DL) are successfully exploited to enhance the skin penetration of several compounds. Nevertheless, the“soft” nature of the bilayer favors the drug leakage, mainly in the case of hydrophobic compounds. This disclosure relates to develop a suitable strategy to stabilize the lipid bilayer, without compromising the deformability properties of DL.
Background
The use of nanotechnology in transdermal drug delivery field is growing in interest and several nanocarries (e.i. polymeric and metallic nanoparticles, liposomes, dendrimers, nanogels etc) have been studied for breaching the skin barrier (Roberts, 2017 reference 6). This interest born with the hope to develop a shell able to maintain the drug payload whilst squeezing through the pores re sulting from the imperfect overlapping of the cell membranes of corneocytes. However, considering that each nanosystem has an its own structure and a diameter of 100-200 nm and that the pores of stratum corneum have a maximum opening of 36 nm in full hydration conditions, as a matter of fact almost all nanocarries used in drug delivery suffer of limited skin penetration. The only nanocarriers that seem to be able to cross the stratum corneum are the deformable vesicles. Among them lipo somes formed by the combination of phospholipids and single chain surfactants, that act as desta bilizing agents of the lipid bilayer, appear of particular interest. In fact, owing to their natural affinity for curved configurations, the surfactants may relocate in the areas of maximum curvature when the vesicles undergo an anisotropic stress. This behavior should allow the vesicle to reversibly modify its morphology elongating in the narrow pores of the stratum corneum. These deformable vesicles have been exploited to deliver in the skin several molecules and their efficiency has been widely proven both in in vitro and in vivo studies (Cevc et al, Biochimica et Biophisica Acta 1998,1368, 201 -205 reference 1 ).
One of the major drawbacks of deformable vesicle is the difficile balance between the flexibility, re quired for skin penetration, and the physico-chemical stability. In fact, mainly in the case of hydro- phobic compounds, the fluidity of the lipid bilayer of the deformable vesicle favors the leakage of the drug out of the bilayer. This decreases the shelf life of the product and therefore limits the pos sibility to reach the market. To avoid this instability issue some approaches can be used. The for mer consists in the development of particular active loading processes that, using peculiar charac teristics of a specific drug (such as acid/base equilibrium), create a gradient that determines the crystallization of the drug in the core of the liposome. As an example, the entrapment of doxorubi cin can be enhanced by using an ammonium solphate gradient. An alternative method, which in theory is not significantly influenced by the naive characteristics of the drug, may consist in the decoration of the surface of the deformable liposome with hydrophilic moieties. Nevertheless, this approach determines an increase of the stiffness of the lipid bilayer which slows down the permea tion of the vesicle through the stratum corneum.
Therefore, there is the need to use a different approach that allows to improve the chemical stabil ity of the systems without affecting the overall properties of deformable liposomes.
Summary of the invention The present invention deals with new deformable liposome compositions such as: micelles in de formable liposome (MiL) and drug in micelles in deformable liposome (DiMiL), in particular:
(MiL) a deformable liposome composition comprising:
- micelles selected from the group comprising: amphiphilic monomers or amphiphilic polymers
- said micelles encapsulated in the aqueous core of a deformable liposomal vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deformable liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably characterized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm,
or
(DiMiL) a deformable liposome composition comprising a drug, such as a poorly permeable drug or a water-insoluble drug, incorporated in micelles:
- said micelles selected from the group comprising: amphiphilic monomers or amphiphilic polymers,
- said micelles encapsulated in the aqueous core of a deformable liposomal vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deformable liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably characterized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm,
said deformable liposome compositions MiL and DiMiL being useful (trans)dermal delivery systems for a hydrophobic compound or for a poorly permeable drug or a water-insoluble drug.
Brief description of the drawings
Figure 1 shows graphical representation of: deformable liposome composition as micelles in de formable liposome (MiL) wherein deformable liposome vesicle, made of bilayer comprising phos pholipid or lipid forming bilayer and single chain surfactant as an example of fluidizing agent, en- capsules micelles according to the present invention;
Figure 2 shows graphical representation of deformable liposome composition as drug in micelles in deformable liposome (DiMiL) wherein deformable liposome vesicle, made of bilayer comprising phospholipid or lipid forming bilayer and single chain surfactant as an example of fluidizing agent, en-capsules micelles incorporating hydrophobic drug according to the present invention;
Figure 3 shows the graphics dealing with the in vitro drug release profile, as the percentage release vs time, of nifedipine (NIF) (A) and piroxicam (PRX) (B) loaded in the lipid bilayer of the deforma ble liposome composition (DL) system wherein the lipid vesicle is composed of phospholipid egg- phosphatidylcholine and fluidizing agent polisorbate 80 or Tween® 80 (T80) at 95:5 or 85:15 w/w ratios, and nifedipine (NIF) (A) and piroxicam (PRX) (B) encapsulated as drug in micelles in de formable liposome composition (DiMiL) system wherein the lipid vesicle is composed of phospho lipid egg-phosphatidylcholine and fluidizing agent polisorbate 80 or Tween® 80 (T80) at 95:5 or 85:15 w/w ratios, and the micelles component is PEG-hydroxystearate (Kolliphor® HS 15) incorpo rating nifedipine (NIF) and piroxicam (PRX), respectively;
Figure 4 shows the graphics dealing with the in vitro permeation profile of A) nifedipine and B) piroxicam through human epidermis after application thereto of: deformable liposome composition (DL) loading in the lipid bilayer of the vesicle thereof nifedipine and piroxicam, respectively, de formable liposome composition as drug in micelles in deformable liposome (DiMiLs) wherein the micelles component PEG-hydroxystearate (Kolliphor® HS 15) incorporates nifedipine (NIF) and piroxicam (PRX), respectively; and PEG-hydroxystearate (Kolliphor® HS 15) micelles incorporating nifedipine (NIF) and piroxicam (PRX), respectively.
Detailed description of the invention
The applicant surprisingly and unexpectedly developed a new deformable liposome composition as a micelles in deformable liposome (MiL) as a carrier system for hydrophobic substance and, ac cordingly, a new deformable liposome composition as a drug in micelles in deformable liposomes (DiMiL) to slow down the unwanted drug release from deformable liposomes, thus avoiding the drug leakage. Basically, micelles selected from the group comprising amphiphilic monomers or amphiphilic polymers, are used to solubilize hydrophobic compounds in the aqueous core of the deformable liposomes. This allows to accommodate hydrophobic drugs in the aqueous core of de formable liposomes through micelles entrapment, in order to avoid drug leakage.
The presence of micelles inside the deformable liposome vesicles does not affect the flexibility of the carriers and then, does not mine the skin penetration capability of the deformable liposome vesicles. In contrast, the deformable liposome composition as drug in micelles in deformable lipo some (DiMiLs) revealed to have superior properties of enhancing drug permeation with respect to both conventional deformable liposomes and surfactant based micelles alone.
The applicant found that it is possible to encapsulate drug-loaded-micelles having an average di ameter of 10-30 nm in deformable liposome vesicles having an average diameter of 50-300 nm. This is unexpected since it is generally recognized that the addition of self-assembling nanosys tems furtherly compromises the stability of deformable liposome. As a matter of fact, the addition of surfactants to liposomal dispersion is usually used for vesicles disruption. Indeed, in literature only a combination of polymeric micelles with liposome is reported (see reference 5). However, in this case very stiff conventional liposomes (made of phospholypids and a large amount of cholesterol) were used with the final goal to protect paclitaxel loaded micelles from the degradation in the gas trointestinal fluids.
Moreover, US 6 143 321 A discloses liposomes containing micelles wherein it is mandatory the use of stabilizing agents in the bilayer or on the bilayer of the liposome vescicle. Examples of these stabilizing agents are cholesterol and grafted PEG, respectively.
US 2012/231069 Al discloses the feasibility to allow the gastro-intestinal absorption of nanoparticu late drug delivery systems by using the active transport of vitamin B12 or a derivative thereof and, using the same targeting moiety or other molecules, to deliver the cargo of the carrier to diseased cells in the body [007] The vitamin B12 or a derivative thereof (hereinafter targeting moiety) is at tached to the surface of the nanoparticle [008] or to micelle [009] loaded in a nanoparticle. Fur thermore, the targeting moiety can be attached to the surface of liposomes. However, the stability of liposome is due to the fact that the vitamin B12 or a derivative thereof must be grafted on the lip osome surface.
A. GILLET ET AL: "Liposomes and parameters affecting their skin penetration behaviour", JOUR NAL OF DRUG DELIVERY SCIENCE AND TECHNOLOGY, vol. 21 , no. 1 , 1 January 201 1 , pages 35-42 does not disclose not even suggests the possibility to produce a deformable liposome con taining micelles in its aqueous core.
Surprisingly, the addition of drug loaded surfactant or polymeric micelles in deformable liposomes (DiMiL compositions) according to the present invention abolishes the drug leakage without affect ing the ability of deformable lipid vesicle to undergo a reversible deformation. And again, we found that Drug in micelles in deformable liposome compositions (DiMiL) are able to significantly enhance the skin permeation of drugs with respect to both the simple micelles and deformable liposomes. Therefore they represent a new tool for the (trans)dermal delivery of poor permeable drugs.
Liposomes are lipid vesicles composed of one or more lipid bilayers enclosing aqueous compart ments. The main components of liposomes are phospholipids or other lipids able to form bilayers, and cholesterol. The last is required to reduce bilayer fluidity and to allow the control of the drug re lease and the liposome stability. To further improve in vitro and in vivo stability, the surface of lipo somes can bear hydrophilic chains, generally, but not exclusively, of polyethylene glycol (PEG). Conversely, deformable liposomes, according to the present invention, are free of cholesterol or other stabilizing materials in/on the bilayer since they have to be“soft” structures to pass through the tight skin barrier. For this reason, deformable liposomes contain at least one fluidizing agent (generally in a concentration of 5-20% w/w with respect to the main bilayer forming lipid). The fluid izing agent is a substance able to confer flexibility to the lipid vesicles. For instance a non-ionic sin gle chain surfactant, a skin penetration enhancer, ethanol or a combination thereof can be used. The deformable liposome according to the present invention, such as: micelles in deformable lipo some (MiL) or drug in micelles in deformable liposomes (DiMiL), comprises deformable liposome, containing micelles in its aqueous core, free of cholesterol or other stabilizing materials in/on the bi layer, preferably characterized by a deformability constant not higher than 0.13 N/mm, more pref erably by a deformability constant in the range 0.01 - 0.13 N/mm.
Micelles are self-assembling aggregates of colloidal dimensions resulting from the interaction be tween the hydrophobic and hydrophilic moieties of amphiphilic monomers. Micelles can result from the self-aggregation of surfactants above the critical micelle concentration, such as the esters of fatty acids and PEGs, or of amphiphilic polymers, such as polaxamers.
The deformable liposome composition as micelles in Deformable liposome (MiL) can be loaded with hydrophobic compounds selected from the group comprising neutral, cationic, anionic, zwitter- ionic compounds. The Drug in micelles in deformable liposome compositions (DiMiL) can be load ed with neutral, cationic, anionic, zwitterionic compounds.
The deformable liposome composition as drug in micelles in Deformable liposome compositions (DiMiL) are loaded with drug, such as a poorly permeable drug or a water-insoluble drug, prefera bly selected from the group comprising neutral, cationic, anionic, zwitterionic drugs, such as resveratrol, nifedipine, piroxicam or naproxene or tranexamic acid, retinol, arbutin, perfluordecalin, diclofenac, azelaic acid, caffeine, CBD (cannabidiol), curcumin, lidocaine, pycnogenol.
The deformable liposome composition as micelles in Deformable liposome (MiL) or deformable lip osome composition as drug in micelles in Deformable liposome compositions (DiMiL) can be pre pared by film hydration methods or solvent displacement or microfluidic or cross flow injection methods.
MiL: micelles in deformable liposome
It is an object of the present invention a deformable liposome composition as micelles in deforma ble liposome (MiL), said deformable liposome composition comprising:
- micelles selected from the group comprising: o amphiphilic monomers, i.e. Non ionic Surfactants: such as the esters of fatty acids or PEGs or Fatty alcohol ethoxylates; Nonoxynols; Fatty acid ethoxylates; Special ethox- ylated fatty esters and oils; Ethoxylated amines and/or fatty acid amides; Terminally blocked ethoxylates; Fatty acid esters of glycerol; Fatty acid esters of sorbitol; Fatty acid esters of sucrose; Alkyl polyglycoside; Ionic surfactants: such as Cetrimonium bromide; Cetylpyridinium chloride; Benzalkonium chloride; Benzethonium chloride; Dimethyldioc- tadecylammonium chloride; Dioctadecyldimethylammonium bromide; or o amphiphilic polymers, i.e. Self assembling copolymers: such as Amphiphilic block co polymers (i.e. Pluronics, poly(esters), hydrophobic poly(amino acids), copolymers of lac tic acid and glycolic acids, poly(caprolactone)), graft copolymers, polaxamers, semisyn thetic polysaccharides
- said micelles encapsulated in the aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a de formable liposome free of cholesterol or other stabilizing materials in/on the bilayer, prefera bly characterized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm, said fluidizing agent preferably in a concentration of 5-20% w/w with respect to the phospholipid or lipid forming bilayer compo nent, said fluidizing agent preferably is selected from the group comprising non-ionic single chain surfactant, a skin penetration enhancer, ethanol or a combination thereof.
In a preferred embodiment of the deformable liposome composition as micelles in deformable lipo some (MiL) according to the present invention, the fluidizing agents contained in the deformable liposome vesicle are in a concentration preferably of 5 %, 10 %, 15% or 20%, preferably 5- 20% or 5-15% or 5-10% all the percentage are % w/w with respect to the phospholipid or lipid forming bi layer component.
In a preferred embodiment of the deformable liposome composition as micelles in deformable lipo some (MiL) according to the present invention, the micelles incorporate a hydrophobic compound. The hydrophobic compound can be loaded in the micelles at its maximum solubility without affect ing the leakage thereof, said loading because the micelles are self-assembling aggregates of col loidal dimensions resulting from the interaction between the hydrophobic and hydrophilic moieties of amphiphilic monomers or amphiphilic polymers. The hydrophobic moieties of Micelles entrapped the hydrophobic compound.
According to the present invention, the hydrophobic compound is selected from the group compris ing neutral, cationic, anionic, zwitterionic compounds.
According to a preferred embodiment of the present invention, the hydrophobic compound is a drug, such as poorly permeable drug or water-insoluble drug, preferably selected from the group comprising neutral, cationic, anionic, zwitterionic drugs, such as resveratrol, nifedipine, piroxicam or naproxene or tranexamic acid, retinol, arbutin, perfluordecalin, diclofenac, azelaic acid, caffeine, CBD (cannabidiol), curcumin, lidocaine, pycnogenol. In the deformable liposome composition as micelles in deformable liposome (MiL) according to the present invention the micelles have an av erage diameter no more than 30 nm, preferably an average diameter from 10 nm to 30 nm, either incorporating or non-incorporating a hydrophobic compound and/or the deformable liposome vesi cle has an average diameter no more than 300 nm, preferably an average diameter from 50 nm to 200 nm.
In a preferred embodiment of the deformable liposome composition as micelles in deformable lipo some (MiL) according to the present invention the phospholipid or lipid forming bilayer component of the deformable liposome vesicle is selected from the group comprising:
- Phospholipids: such as phosphatidylcholine, Phosphatidylethanolamine, phosphatidylserine, Phosphatidylglycerol and all derivatives of such phospholipids as well as all the combinations thereof;
- Cationic Lipids: such as 1 ,2-di-0-octadecenyl-3-trimethylammonium propane, 1 ,2-dilauroyl-sn- glycero-3-ethylphosphocholine, Dimethyldioctadecylammonium, 1 ,2-dipalmitoyl-3- trimethylammonium-propane, 1 ,2-stearoyl-3-trimethylammonium-propane, 1 ,2-dimyristoyl-3- trimethylammonium-propane, 1 ,2-dioleoyl-3-trimethylammonium-propane, 1 ,2-dioleyloxy-3- dimethylaminopropane;
- Sphingolipids: such as Sphingomyelin (all sources) and derivatives.
In a preferred embodiment of the deformable liposome composition as micelles in deformable lipo some (MiL) according to the present invention, the fluidizing agent contained in the deformable lip osome vesicle is selected from the group comprising non-ionic single chain surfactant, such as Tween, Span, sodium cholate etc.., a skin penetration enhancer, such as terpenes, unsaturated fatty acids, colic acid and its derivatives, ethylene glycol derivatives, propylene glycol, glycerine, or ethanol or a combination thereof.
It is a further object of the present invention a deformable liposome composition comprising:
- micelles selected from the group comprising: amphiphilic monomers or amphiphilic polymers
- said micelles encapsulated in the aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deforma ble liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably charac terized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm,
as (trans)dermal delivery system.
It is a further object of the present invention the use of a deformable liposome composition com prising:
- micelles selected from the group comprising: amphiphilic monomers or amphiphilic polymers
- said micelles encapsulated in the aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deforma ble liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably charac terized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm,
as (trans)dermal delivery system.
It is a further object of the present invention the use of a deformable liposome composition com prising:
- micelles selected from the group comprising: amphiphilic monomers or amphiphilic polymers
- said micelles encapsulated in the aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deforma ble liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably charac- terized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm,
as (trans)dermal delivery system for a hydrophobic substance.
It is a further object of the present invention the use of a deformable liposome composition com prising:
- micelles selected from the group comprising: amphiphilic monomers or amphiphilic polymers
- said micelles encapsulated in the aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deforma ble liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably charac terized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm,
as (trans)dermal delivery system for a drug, such as a poorly permeable drug or a water-insoluble drug.
It is a further object of the present invention a method of administering a hydrophobic compound to a target tissue wherein a deformable liposome composition comprising a hydrophobic compound incorporated in micelles,
- said micelles selected from the group comprising: amphiphilic monomers or amphiphilic poly mers
- said micelles encapsulated in the aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deforma ble liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably charac terized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm,
is (trans)dermally administered to said target tissue.
The invention further provides a method of administering a hydrophobic compound to a target tis sue comprising the steps of:
- preparing according to the methods of the invention, such as by film hydration methods or sol vent displacement methods a deformable liposome composition comprising a hydrophobic com pound incorporated in micelles, said micelles selected from the group comprising: amphiphilic monomers or amphiphilic polymers, said micelles encapsulated in aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deformable liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably characterized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm, and
- administering (trans)dermally to said target tissue the deformable liposome composition com prising a hydrophobic compound incorporated in micelles, said micelles encapsulated in aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deformable liposome free of cholesterol or other sta bilizing materials in/on the bilayer, preferably characterized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm,.
DiMiL: drug in micelles in deformable liposome
It is an object of the present invention a deformable liposome composition as drug in micelles in de- formable liposome (DiMiL), said deformable liposome composition comprising a drug, such as a poorly permeable drug or a water-insoluble drug, incorporated in micelles:
- micelles selected from the group comprising:
o amphiphilic monomers, i.e. Non ionic Surfactants: such as the esters of fatty acids or PEGs or Fatty alcohol ethoxylates; Nonoxynols; Fatty acid ethoxylates; Special ethox- ylated fatty esters and oils; Ethoxylated amines and/or fatty acid amides; Terminally blocked ethoxylates; Fatty acid esters of glycerol; Fatty acid esters of sorbitol; Fatty acid esters of sucrose; Alkyl polyglycoside; Ionic surfactants: such as Cetrimonium bromide; Cetylpyridinium chloride; Benzalkonium chloride; Benzethonium chloride; Dimethyldioc- tadecylammonium chloride; Dioctadecyldimethylammonium bromide; or o amphiphilic polymers, i.e. Self assembling copolymers: such as Amphiphilic block co polymers (i.e. Pluronics, poly(esters), hydrophobic poly(amino acids), copolymers of lac tic acid and glycolic acids, poly(caprolactone)), graft copolymers, polaxamers, semisyn thetic polysaccharides
- said micelles encapsulated in the aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deforma ble liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably charac terized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm, said fluidizing agent preferably in a concentration of 5- 20% w/w with respect to the phospholipid or lipid forming bilayer component, said fluidizing agent preferably is selected from the group comprising non-ionic single chain surfactant, a skin penetra tion enhancer, ethanol or a combination thereof.
In a preferred embodiment of the deformable liposome composition as drug in micelles in deforma ble liposome (DiMiL) according to the present invention, the fluidizing agent contained in the de formable liposome vesicle are in a concentration preferably of 5 %, 10 %, 15% or 20%, preferably 5- 20% or 5-15% or 5-10%; all the percentage are % w/w with respect to the phospholipid or lipid forming bilayer component.
In a preferred embodiment of the deformable liposome composition as drug in micelles in deforma ble liposome (MiL) according to the present invention, the micelles incorporate a hydrophobic com pound.
The drug, such as a poorly permeable drug or a water-insoluble drug, can be loaded in the micelles at its maximum solubility without affecting the leakage thereof, said loading because the micelles are self-assembling aggregates of colloidal dimensions resulting from the interaction between the hydrophobic and hydrophilic moieties of amphiphilic monomers or amphiphilic polymers. The hy drophobic moieties of Micelles entrapped the drug, such as a poorly permeable drug or a water- insoluble drug.
According to a preferred embodiment of the present invention, the drug, such as poorly permeable drug or water-insoluble drug, is preferably selected from the group comprising neutral, cationic, an ionic, zwitterionic drugs, such as resveratrol, nifedipine, piroxicam or naproxene or tranexamic acid, retinol, arbutin, perfluordecalin, diclofenac, azelaic acid, caffeine, CBD (cannabidiol), curcumin, li- docaine, pycnogenol.
In the deformable liposome composition as drug in micelles in deformable liposome (DiMiL) ac- cording to the present invention the micelles have an average diameter no more than 30 nm, pref erably an average diameter from 10 nm to 30 nm, and/or the deformable liposome vesicle has an average diameter no more than 300 nm, preferably an average diameter from 50 nm to 200 nm.
In a preferred embodiment of the deformable liposome composition as drug in micelles in deforma ble liposome (DiMiL) according to the present invention the phospholipid or lipid forming bilayer component of the deformable liposome vesicle is selected from the group comprising:
- Phospholipids: such as phosphatidylcholine, Phosphatidylethanolamine, phosphatidylserine, Phosphatidylglycerol and all derivatives of such phospholipids as well as all the combinations thereof;
- Cationic Lipids: such as 1 ,2-di-0-octadecenyl-3-trimethylammonium propane, 1 ,2-dilauroyl-sn- glycero-3-ethylphosphocholine, Dimethyldioctadecylammonium, 1 ,2-dipalmitoyl-3- trimethylammonium-propane, 1 ,2-stearoyl-3-trimethylammonium-propane, 1 ,2-dimyristoyl-3- trimethylammonium-propane, 1 ,2-dioleoyl-3-trimethylammonium-propane, 1 ,2-dioleyloxy-3- dimethylaminopropane;
- Sphingolipids: such as Sphingomyelin (all sources) and derivatives.
In a preferred embodiment of the deformable liposome composition as drug in micelles in deforma ble liposome (DiMiL) according to the present invention, the fluidizing agent contained in the de formable liposome vesicle is selected from the group comprising non-ionic single chain surfactant, such as Tween, Span, sodium cholate etc.., a skin penetration enhancer, such as terpenes, unsaturated fatty acids, colic acid and its derivatives, ethylene glycol derivatives, propylene glycol, glycerine, or ethanol or a combination thereof.
It is a further object of the present invention a deformable liposome composition comprising a drug, such as a poorly permeable drug or a water-insoluble drug, incorporated in micelles:
- said micelles selected from the group comprising: amphiphilic monomers or amphiphilic poly mers,
- said micelles encapsulated in the aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deforma ble liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably charac terized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm,
as (trans)dermal drug delivery system for a poorly permeable drug or a water-insoluble drug.
It is a further object of the present invention a method of administering a drug, such as a poorly permeable drug or a water-insoluble drug, to a target tissue wherein a deformable liposome com position comprising a drug, such as a poorly permeable drug or a water-insoluble drug, incorpo rated in micelles,
- said micelles selected from the group comprising: amphiphilic monomers or amphiphilic poly mers
- said micelles encapsulated in the aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deforma ble liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably charac terized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm, is (trans)dermally administered to said target tissue.
The invention further provides a method of administering a drug, such as a poorly permeable drug or a water-insoluble drug, to a target tissue comprising the steps of:
- preparing according to the methods of the invention, such as by film hydration methods or sol vent displacement methods a deformable liposome composition comprising a drug, such as a poorly permeable drug or a water-insoluble drug, incorporated in micelles, said micelles selected from the group comprising: amphiphilic monomers or amphiphilic polymers, said micelles encap sulated in aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deformable liposome free of cho lesterol or other stabilizing materials in/on the bilayer, preferably characterized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm,
and
- administering (trans)dermally to said target tissue the deformable liposome composition com prising a drug, such as a poorly permeable drug or a water-insoluble drug, incorporated in mi celles, said micelles encapsulated in aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a de formable liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably characterized by a deformability constant not higher than 0.13 N/mm, more preferably by a de formability constant in the range 0.01 - 0.13 N/mm.
In particular, the poorly permeable drug or water-insoluble drug are selected from the group com prising neutral, cationic, anionic, zwitterionic drugs, such as resveratrol, nifedipine, piroxicam or naproxene or tranexamic acid, retinol, arbutin, perfluordecalin, diclofenac, azelaic acid, caffeine, CBD (cannabidiol), curcumin, lidocaine, pycnogenol.
It is a further object of the present invention a deformable liposome composition comprising a drug, such as a poorly permeable drug or a water-insoluble drug, incorporated in micelles:
- said micelles selected from the group comprising:
o amphiphilic monomers, i.e. Non ionic Surfactants: such as the esters of fatty acids or PEGs or Fatty alcohol ethoxylates; Nonoxynols; Fatty acid ethoxylates; Special ethox- ylated fatty esters and oils; Ethoxylated amines and/or fatty acid amides; Terminally blocked ethoxylates; Fatty acid esters of glycerol; Fatty acid esters of sorbitol; Fatty acid esters of sucrose; Alkyl polyglycoside; Ionic surfactants: such as Cetrimonium bromide; Cetylpyridinium chloride; Benzalkonium chloride; Benzethonium chloride; Dimethyldioc- tadecylammonium chloride; Dioctadecyldimethylammonium bromide; or o amphiphilic polymers, i.e. Self assembling copolymers: such as Amphiphilic block copol ymers (i.e. Pluronics, poly(esters), hydrophobic poly(amino acids), copolymers of lactic acid and glycolic acids, poly(caprolactone)), graft copolymers, polaxamers, semisynthetic polysaccharides;
- said micelles encapsulated in the aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deformable liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably characterized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm,
for use in trans(dermal) therapeutic applications/treatments wherein the drug, as a poorly permea ble drug or a water-insoluble drug, is selected from the group comprising neutral, cationic, anionic, zwitterionic drugs, such as resveratrol, nifedipine, piroxicam, naproxene.
It is a further object of the present invention a deformable liposome composition comprising a drug, such as a poorly permeable drug or a water-insoluble drug, incorporated in micelles:
- said micelles selected from the group comprising:
o amphiphilic monomers, i.e. Non ionic Surfactants: such as the esters of fatty acids or PEGs or Fatty alcohol ethoxylates; Nonoxynols; Fatty acid ethoxylates; Special ethox- ylated fatty esters and oils; Ethoxylated amines and/or fatty acid amides; Terminally blocked ethoxylates; Fatty acid esters of glycerol; Fatty acid esters of sorbitol; Fatty acid esters of sucrose; Alkyl polyglycoside; Ionic surfactants: such as Cetrimonium bromide; Cetylpyridinium chloride; Benzalkonium chloride; Benzethonium chloride; Dimethyldioc- tadecylammonium chloride; Dioctadecyldimethylammonium bromide; or o amphiphilic polymers, i.e. Self assembling copolymers: such as Amphiphilic block copol ymers (i.e. Pluronics, poly(esters), hydrophobic poly(amino acids), copolymers of lactic acid and glycolic acids, poly(caprolactone)), graft copolymers, polaxamers, semisynthetic polysaccharides;
- said micelles encapsulated in the aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deformable liposome free of cholesterol or other stabilizing materials in/on the bilayer, preferably characterized by a deformability constant not higher than 0.13 N/mm, more preferably by a deformability constant in the range 0.01 - 0.13 N/mm,
for use in trans(dermal) therapeutic applications/treatments wherein the drug, as a poorly permea ble drug or a water-insoluble drug, is selected from the group comprising neutral, cationic, anionic, zwitterionic drugs, such as tranexamic acid, retinol, arbutin, perfluordecalin, diclofenac, azelaic ac id, caffeine, CBD, curcumin, lidocaine, pycnogenol.
According to the present invention for both deformable liposome compositions: a deformable lipo some composition as micelles in deformable liposome (MiL) and a deformable liposome composi tion as drug in micelles in deformable liposome (DiMiL) the following are the preferred Micelles components, Phospholipid/Lipid components of the deformable liposome bilayer and Fluidizing agents in deformable liposomes bilayer.
Micelles components:
I. Non ionic Surfactants: Fatty alcohol ethoxylates; Nonoxynols; Fatty acid ethoxylates; Special ethoxylated fatty esters and oils; Ethoxylated amines and/or fatty acid amides; Terminally blocked ethoxylates; Fatty acid esters of glycerol; Fatty acid esters of sorbitol; Fatty acid esters of su crose; Alkyl polyglycoside.
II. Ionic surfactants: Cetrimonium bromide; Cetylpyridinium chloride; Benzalkonium chloride; Benzethonium chloride; Dimethyldioctadecylammonium chloride; Dioctadecyldimethylammonium bromide.
III. Self assembling copolymers: Amphiphilic block copolymers (i.e. Pluronics, poly(esters), hy drophobic poly(amino acids), copolymers of lactic acid and glycolic acids, poly(caprolactone)), graft copolymers, polaxamers, semisynthetic polysaccharides.
Phospholipid/Lipid components of the deformable liposome bilaver:
1. Phospholipids: phosphatidylcholine, Phosphatidylethanolamine, phosphatidylserine, Phos- phatidylglycerol and all the derivatives of such lipids as well as all the combinations thereof.
2. Cationic Lipids: 1 ,2-di-0-octadecenyl-3-trimethylammonium propane, 1 ,2-dilauroyl-sn- glycero-3-ethylphosphocholine, Dimethyldioctadecylammonium, 1 ,2-dipalmitoyl-3- trimethylammonium-propane, 1 ,2-stearoyl-3-trimethylammonium-propane, 1 ,2-dimyristoyl-3- trimethylammonium-propane, 1 ,2-dioleoyl-3-trimethylammonium-propane, 1 ,2-dioleyloxy-3- dimethylaminopropane.
3. Sphingolipids: Sphingomyelin (all sources) and derivatives.
Fluidizing agents in deformable liposomes bilaver:
single chain surfactants (Tween, Span, sodium cholate etc..), skin penetration enhancers (ter- penes, unsaturated fatty acids, colic acid and its derivatives, ethylene glycol derivatives, propylene glycol, glycerine), ethanol.
The approach according to the present invention relied on the design of a“matryoshka” system, namely a deformable liposome composition as micelles in deformable liposome (MiL), i.e. micelles encapsulated in aqueous core of deformable liposome vesicle as a carrier of hydrophobic sub stance or a deformable liposome composition as a drug in micelles in deformable liposome (DiMiL), i.e. micelles incorporating a hydrophobic drug, said micelles encapsulated in aqueous core of de formable liposome vesicle.
In particular the performances (drug leakage, deformability and in vitro skin penetration profile) of Drug in micelles in deformable liposome compositions (DiMiL)s were tested using nifedipine and piroxicam as an example of hydrophobic compounds and compared to those of traditional deform able liposome (DL) loading in the lipid bilayer of the vesicle thereof said drugs.
The micelles were made of PEG-hydroxystearate (Kolliphor® HS15) while the deformable liposome vesicle components of: deformable liposome (DL), deformable liposome composition MiL and de formable liposome composition DiMiL were as phospholipid/lipid egg-phosphatidylcholine and fluid izing agent polisorbate 80 or Tween® 80 (T80) at 95:5 or 85:15 w/w ratios. As expected, the drug leakage from DL was high after only one month of storage (almost 50% in the case of nifedipine and in the range of 39-79 % in the case of piroxicam loaded DL, depending on T80 content).
The deformable liposome composition Drug in micelles in deformable liposome DiMiL formulations retained instead the drug content up to two-month storage period.
Moreover, the constant of deformability of micelles in deformable liposome MiL and of drug in mi celles in deformable liposome DiMiLs felt in the acceptance range for deformable liposome vesicles intended for cutaneous application and skin permeated amount of the delivered drugs was in creased of at least 4 times.
In conclusion, deformable liposome composition as micelles in deformable liposome (MiL) and de formable liposome composition as drug in micelles in deformable liposome (DiMiL) reveals to be a suitable approach to avoid the leakage of hydrophobic compounds and an attractive (trans)dermal delivery system for hydrophobic compound in general, in particular a (trans)dermal delivery system for drug such as for a poorly permeable drug or a water-insoluble drug.
Preparation of compositions All the Deformable Liposome compositions: the known Deformable liposome composition (DL), the micelles in deformable liposome composition (MiL) according to the present invention and the drug in micelles in deformable composition (DiMiL) according to the present invention were prepared by thin film hydration method.
The deformable liposome vesicle components of:
- the known Deformable liposome composition (DL): formulation DL 95:5 and formulation DL 85:15,
- the micelles in deformable liposome composition (MiL): formulation MiL 95:5 and formulation MiL 85:15
- the drug in micelles in deformable composition (DiMiL): formulation DiMiL 95:5 and formulation DiMiL 85:15
were as phospholipid/lipid egg-phosphatidylcholine and as fluidizing agent polisorbate 80 or Tween® 80 (T80) at 95:5 or 85:15 w/w ratios.
When present the micelles were made of PEG-hydroxystearate (Kolliphor® HS15).
Briefly, egg phosphatydilcholine (e-PC) and polisorbate 80 (Tween 80/T80) dissolved in a chloro form/methanol (2:1 v/v) were mixed in the proper weight ratios (Table 1 ) in a round bottom flask. The organic solvent was evaporated for 1 hour at 40°C under reduced pressure using a rotatory evaporator (Rll, Buchi, Italy), to obtain a thin lipid film.
The film was rehydrated in milliQ® water for 1 h to obtain a final lipid concentration of 30 mg/mL. Drug loaded micelles were prepared by adding a proper amount of drug to the kolliphor® HS15 so lution (Table 1 ).
Then, the mixture was let under stirring for 24 hours prior to be used for liposomes preparation.
In particular saturated solutions of nifediprine and piroxicam drugs in kolliphor® HS 15 at 2.5, 5 and 10% w/v in water were prepared and let under stirring at 25 ± 1 °C for 24 hours, prior to be filter with a 0,45 pm PVDF filter.
To prepare micelles in deformable liposome compositions (MiL), the lipid film was re-hydrate with a 10% w/v solution of kolliphor® HS 15.
To prepare DRUG in DEFORMABLE liposome compositions (DL),the drug was dissolved in a chloroform/methanol (2:1 v/v) solution and mixed with the lipid and the fluidizing agent in the proper amount (Table 1 ).
The organic solvent was evaporated for 1 hour at 40°C under reduced pressure using a rotatory evaporator (Rll, Buchi, Italy), to obtain a thin lipid film.
The film was rehydrated in milliQ® water for 1 h.
To prepare drug in micelles in deformable liposome compositions (DiMiL), the lipid film was rehy drated with the micellar solution of PEG-hydroxystearate Kolliphor® HS 15 (10% w/v in ultrapure water) containing the hydrophobic drug.
To homogenized the particle size distribution of the vesicles, the liposome dispersions were ex truded (Avanti® Mini-Extruder, Avanti Polar Lipids, Inc.) through sequential passages through 0.2 pm (5 passages) and 0.1 pm (6 passages) polycarbonate membranes.
Finally, the formulations were purified from free materials (drug and/or micelles) by molecular size exclusion chromatography on Sepharose CL-4B columns, eluting with milliQ® water.
Figure imgf000016_0001
Placebo: no drug; NIF: nifediprine; PRX: piroxicam; NPX: naproxene; DL: deformable liposome composition; MiL: micelles in deformable liposome composition; DiMiL: drug in micelles in deform- able liposome composition; e-PC: egg-phosphatydilcholine; T80: Polisorbate 80; Kolliphor® HS 15: PEG-hydroxystearate; Drug concentration is expressed in mg/mL.
According to Dynamic light scattering (DLS) analysis, kolliphor® HS 15 in solution forms micelles having a mean particle size of 12.56 ± 0.04 nm, and therefore they can be easily en trapped/encapsulated, either enclosing or not the hydrophobic drug, in the aqueous core of de formable liposome compositions according to the present invention: micelles in deformable lipo some composition (MiL) or drug in micelles in deformable liposome composition (DiMiL), such as during the film rehydration.
Both DLS and Nanoparticle Trafficking Analysis confirmed the absence of free micelles in DiMiL compositions after purification since any traces of 10 nm particles were revealed in the particle size distribution profile of the purified DiMiL.
The lack of free micelles is also confirmed by the similarity in the ^-potential values of the DL and DiMiL composition pairs (Table 2) that excludes also the absorption of the micelles on the liposome surface, being the ^-potential of the micelles significantly lower (-14.7 ± 0.0 mV).
The mean diameter of the vesicles was in all cases lower than 150 nm, therefore suitable for the application on the skin (Table 2).
Determination of the main physical properties
The particle size distribution was determined both by dynamic light scattering (DLS), using a Zetasizer (Nano-ZS, Malvern Instrument, UK), and Nanoparticle Trafficking Analysis (NTA) using a Nanosight NS 300 (Malvern Instrument, UK).
In the first case particle size measurements were carried out inserting the sample in a disposable cuvette after a 1 :10 dilution in 0,22 pm filtered milMQ® water, with a detection angle of 173°.
z-potential was assessed on the diluted sample inserted in a capillary cell.
Three measurements were taken for each sample and the results are expressed as the mean and standard deviation.
For NTA analysis the samples were diluted 5 x 105 times with milliQ® water and 6 sequential measurements at 25 °C were performed using a Blue488 laser.
Determination of the encapsulation efficiency
Drug encapsulation was measured by diluting the liposomes 1 :100 in methanol to break the vesi cles and release the free drug.
The amount of drug was then determined by a HPLC system equipped with a diode array detector (HLPC HP 1 100 Chemstations, Agilent Technologies, Waldbronn, Germany).
For nifedipine quantification, the samples were eluted through a lichrospher 100 RP-18E column (CPS Analitica, Milan, Italy) with a mixture of acetonitrile:methanol:Milli-Q® water (25:25:50 v/v), that was used as mobile phase; the flow rate was 1 .3 mL/min, the thermostat was set at 37°C and the UV lamp at 230 nm.
Nifedipine quantification was done on the basis of a calibration curve of drug in mobile phase in the concentration range 5-100 pg/mL.
Piroxicam quantification was performed using a 0.03 M phosphate pH 3.0 buffer/acetonitrile (60/40% v/v) mixture as mobile phase.
The flow rate was 1 .5 mL/min, and the UV lamp was set at two wavelengths (i.e., 248 nm, 360 nm). Sample concentrations were calculated basing on calibration curves built between 0.05 and 20 pg/mL.
The encapsulation efficiency (EE, %) was expressed as the percentage ratio between the amount of drug (mg/mL) encapsulated in the vesicles and the cumulative amount of the drug (mg/mL) loaded in the liposomes at the time of the preparation.
Deformability assay
The deformability properties of the vesicles were determined using a dynanometer assisted extru sion assay previously developed (reference 3).
Briefly, all liposomal formulations were diluted to the same lipid concentration (0.23 mM) and load ed in a gas tight syringe.
Lipid concentration was determined using an adaptation of the Rouser method, as described else where (see reference 4). The syringe plunger was put in contact with a 50 N load cell of a dyna mometer (INSTRON 5965, ITW Test and Measurement Italia Sri, Italy) and forced to move at a constant speed of 1 mm/s, forcing the liposomal dispersion through a 50 nm polycarbonate mem brane fixed in an extruder casing.
The force (N) required to move the syringe plunger (dependent on the resistance opposed to vesi cle penetration through the pores) was registered as a function of the plunger displacement (mm). The slope of this plot, namely the constant of deformability (k), was then derived.
The higher the k value, the lower the deformability of the carriers (see reference 3).
Table 2 - Physical-chemical properties of the: deformable liposome composition (DL); micelles in deformable liposome composition (MiL); drug in micelles in deformable liposome composition (DiMiL). Each data is the mean of at least three batches of formulations. Data are expressed as mean ± standard deviation.
Figure imgf000018_0001
Placebo: no drug; NIF: nifediprine; PRX: piroxicam; NPX: naproxene; DL: deformable liposome composition; MiL: micelles in deformable liposome composition; DiMiL: drug in micelles in deform- able liposome composition; d: mean hydrodynamic diameter; EE: encapsulation efficiency; NA: not applicable; Pdl: Polydispersity index.
Preparation of hydrophobic drug in polymeric micelle in deformable liposome composition
Polymeric micelles were prepared using a polaxamer 407 (Kolliphor® P407) and resveratrol as neutral hydrophobic compound.
P407 and resveratrol (RES) exactly weighted were dissolved in 2 ml_ of acetone (Table 3) and the mixture was sonicated until complete dissolutions of the ingredients in the solvent.
The solution was let under stirring for 3 hours at room temperature prior to be used for micelles preparation.
The solution of P407 and RES in acetone was dropped in a milliQ® water previously filtered with a 0.2 pm nylon filter, keeping the ratio organic solvent/aqueous medium equal to 1 :10 and the tem perature of the bath at 4°C.
The suspension was let at the same temperature for 5 minutes and later for 2 hours at 37°C, under a constant stirring at 500 rpm.
The unentrapped drug was removed by low speed centrifugation (3000 rpm, 10 min, 23°C).
Table 3- Quali-quantitative composition of the prepared polymeric micelles
Figure imgf000019_0001
The polymeric micelles in deformable liposomes compositions were prepared according to the pro cedure reported for surfactant micelles in deformable liposomes.
The prepared compositions showed suitable physico-chemical properties (Table 4)
Table 4- Physico-chemical properties of DL containing polymeric micelles of resveratrol as hydrophobic drug in polymeric micelle in deformable liposome composition
Drug
RES
Figure imgf000019_0002
RES: resveratrol; DiMiL: drug in micelles in deformable liposome composition; d: mean hydrody namic diameter; Pdl: Polydispersity index.
Drug leakage studies
Drug leakage from deformable liposomes composition (DL) in which the hydrophobic drug sits in the lipid bilayer vesicle thereof and drug in micelles in deformable liposome composition (DiMiL) in which the hydrophobic drug is entrapped in the aqueous core through micelles solubilization was determined by purifying the liposomal dispersions from leaked drug by low speed (5000 rpm) cen trifugation for 10 min at 25 °C (Universal 30RF, Hettich Zentrifugen, Germany).
In these conditions, the free drug precipitated in the crystalline form. The supernatant instead was recovered and diluted with pure methanol to break the vesicles. The amount of drug encapsulated in the liposomes was determined by high performance liquid chromatography (HPLC) after 1 and 2 months of storage and the percentage of drug leakage was calculated according to the following equation:
Drug leakage %=((Et0-Etx))/Et0 X 100
(Eli) Etx)
Drug leakage % X 10Q
EtQ
were Eto is the drug amount in mg/mL loaded in the lipid bilayer vesicle in the deformable liposome composition (DL) or encapsulated in Drug in micelles in deformable composition (DiMiL) at the time of preparation and Etx is the amount of drug still loaded/encapsulated after 1 or 2 months of storage.
Moreover, the growth of drug crystals in the formulation during storage was monitored by light mi croscopy using a stereomicroscope (Nikon, Italy). An aliquot of formulation was spread on a glass slide. The micrographs were acquired at 20X magnification with a digital camera of 3.1 Mpx (CCD 3, ToupView, ToupTek, China).
After one month of storage a high amount of drug crystals leaked out from the bilayer was ob served in all formulations of deformable liposomes, regardless of the amount of surfactant present in the bilayer and the drug leakage percentage was quite high (Table 5).
In contrast, any drug leakage was observed from Drug in micelles in deformable liposome compo sition (DiMiL) compositions after one month of storage and the entrapped drug amount in DiMiLs did not change also over a two-month storage period (data not shown).
In the case of piroxicam (PRX) both Drug in micelles in deformable composition (DiMiL) composi tions were able to improve the retention of the drug inside the vesicles but this time only Drug in micelles in deformable liposome (DiMiL) 95:5 composition allowed to completely abolish the drug leakage from the liposomes (Table 5), even after two months of storage.
Table 5- Drug leakage (%) of deformable liposome (DL) and drug in micelles in deformable liposome composition (DiMiL) according to the invention after 1 month of storage at 4 °C.
Figure imgf000020_0001
NIF: nifediprine; PRX: piroxicam; DL: deformable liposome composition; DiMiL: drug in micelles in deformable liposome composition.
Deformabilitv studies To verify that the presence of micelles would have not compromised the deformability of the vesi cles under stress, the constant of deformability, k, of each formulation for the different type of de formable liposome compositions: deformable liposome (DL), micelles in deformable liposome (MiL) according to the present invention and drug in micelles in deformable liposome (DiMiL) according to the present invention, was determined by a modified extrusion assay previously developed (see reference 3).
This method allows to study the forces involved in liposome penetration through narrow pores and resulted to be sensitive to discriminate also little changes in the deformability properties of lipid based compositions (see reference 3). Briefly, all the different types of deformable liposomal com positions with different formulations were diluted to the same lipid concentration (0.23 mM) and loaded in a gas tight syringe. The syringe plunger was put in contact with a 50 N load cell of a dy namometer (INSTRON 5965, ITW Test and Measurement Italia Sri, Italy) and forced to move at a constant speed of 1 mm/s, forcing the liposomal dispersion through a 50 nm polycarbonate mem brane fixed in an extruder casing. The force (N) required to move the syringe plunger (dependent on the resistance opposed to vesicle penetration through the pores) was registered as a function of the plunger displacement (mm). The slope of this plot, namely the constant of deformability (k), was then derived. The higher the k value, the lower the deformability of the carriers (see reference 3). Basing on our experience, values of k in the range 0.01 -0.13 N/mm are acceptable for topical ap plication of lipid vesicles. As can be depicted in Table 6, all the formulations felt in the acceptance range. This means that the encapsulation of the micelles (both surfactant and polymeric micelles) in deformable liposome vesicle does not change the overall mechanical properties of the carriers, that are considered as surrogate of the skin penetration ability.
Table 6- Deformability properties of deformable liposome composition (DL) micelles in deformable liposome composition (MiL) and Drug in micelles in deformable liposome composition (DiMiL) formulations determined by the dynanometer-assisted extrusion assay. The data are expressed as mean ± standard deviation (N = 4).
Figure imgf000021_0001
Figure imgf000022_0001
K: constant of deformability (N/mm); the higher is the K value, the lower is the flexibility of the carri er; Placebo: no drug; NIF: nifediprine; PRX: piroxicam; NPX: naproxene; RES: resveratrol; DL: de formable liposome composition; MiL: micelles in deformable liposome composition; DiMiL: drug in micelles in deformable liposome composition.
In vitro drug release
The drug in micelles in deformable liposome compositions (DiMiL) significantly slowed down the drug release kinetic with respect to deformable liposome composition (DL) as clearly shown in the percentage nifedipine (NIF) released diagram in figure 3A and in the percentage piroxicam (PRX) released diagram in figure 3B.
Drug skin permeation studies
In vitro skin permeability studies were carried out using Franz diffusion cells method and human skin as membrane, which is obtained from healthy, informed volunteers undergoing abdominoplas ty-
The skin samples are prepared according to an internal procedure (see reference 2).
Briefly, at the arrival of the tissue in the lab, the excess fat is carefully removed and full-thickness skin is cut into squares, sealed in evacuated plastic bags and stored at -20 °C until their use. Epidermis sheets are obtained through mechanical separation from the remaining tissue with for ceps, after skin immersion in water at 60 ± 1 °C for 1 min.
Prior to use the tissue samples for the experiment, the integrity of the skin is evaluated by measur ing the electrical impedance of the epidermis sheets (Agilent 4263B LCR Meter, Microlease, Italy). The epidermis sheets are mounted on the lower half of the Franz diffusion cells with the stratum corneum facing upward.
The upper and lower parts of the cell are sealed with parafilm and fastened together with a clamp. The receiver compartment was filled with an acetate buffer (pH 4.6)/polyethylene glycol 400 (PEG 400) mixture for nifedipine and physiologic solution for piroxicam to assure the sink conditions. The receiver phase was continuously stirred by a magnetic bar at 1500 rpm.
Three hundred pL of each formulation were loaded in the donor compartment under non-occlusive conditions. The micellar solutions were tested in parallel as control. The composition was kept at 37 ± 1 °C by means of a circulating water bath so that the epidermis surface temperature was at 32 ± 1 °C throughout the experiment.
At fixed time intervals (1 , 3, 5, 7 and 24 h) 200 pL of receiver phase was withdrawn and replaced with an equal volume of fresh medium.
At the end of the experiment, the cells were dismounted and the skin samples were recovered and washed on both sides with fresh methanol to remove any residues of formulation/solution.
Epidermis samples were let to dry and weighted prior to be cut in small pieces and immersed in 5 mL of methanol to extract the drug retained in the tissue.
Nifedipine is a good candidate for (trans)dermal delivery due to the low molecular weight and the logP value of 2.2. Nevertheless, its passive diffusion through the skin is negligible. Nevertheless, surprisingly, the drug in micelles in deformable liposome composition (DiMiL) according to the present invention led to a drastic increase of nifedipine permeation after 24 hours that moved from 2.73 ± 0.06 pg/cm2 when the nifedipine is loaded in the lipid bilayer vesicle of deformable liposome (DL) to 131 .27 ± 48.1 1 pg/cm2 when the nifedipine is (trans)dermally delivered by the drug in micelles in deformable liposome composition according to the present invention(DiMiLs). Moreover, the drug in micelles in deformable liposome composition (DiMiL) according to the present invention seemed to favor the partition of the drug in the skin since the lag time is significantly reduced and the drug permeation is observed already at the first hour of experiment (Figure 4A).
It is worth noting that after application of free micelles containing nifedipine on human skin any drug permeation was observed.
Therefore, the higher permeation pattern of nifedipine when carried in deformable liposomes con taining micelles that incorporate nifedipine has to be ascribed to the whole composition.
Similar findings were obtained in the case of piroxicam.
Also in this case it was observed a faster repartition of drug in the human skin with the decrease of lag time and the amount of drug permeated after 24 hours experiment was almost five folds higher in the case of the drug in micelles in deformable liposome composition (DiMiL), according to the present invention, than in that of conventional deformable liposome (DL) loading in the lipid bilayer vesicle thereof the piroxicam (figure 4B).
Physical stability of DiMiL
The physical stability of DL and DiMiL systems was monitored over time by measuring the particle size distribution of the lipid vesicles after three and six months of storage to rule out the possibility of phenomena of fusion or aggregation of particles due to the fluidity of the bilayers containing two destabilizing agents, namely one in the bilayer and one as component of the micelles.
The particle size distribution d (nm) was monitored using DLS.
Particle size distribution d (nm) and Polydispersity index (Pdl) of DL and DiMiL systems at starting time (t=0*) and after three (t=3 mos) and six (t=6 mos) months storage period were monitored and reported in Table 7.
Table 7- Particle size distribution and polydispersity of DL and DiMiL systems at starting time after three and six months (mos) storage period.
Figure imgf000023_0001
Figure imgf000024_0001
The overall data summarized in Table 7 evidence that although a partial distribution of micelles in the bilayer is plausible, the presence of micelles in the bilayer do not cause the rupture of the vesi cles in mixed micelles and large aggregate neither fusion/aggregation of the vesicles.
In fact, both the particle size and Pdl of the drug delivery systems did not undergo significant varia tions up to two months of storage and more.
Further embodiments
Following paragraph“Preparation of compositions” further deformable liposome composition ac cording to the present invention, i.e. DiMiL formulations: DiMiL 85:15 formulations have been car ried out as reported in Table 8.
Table 8
Figure imgf000024_0002
References:
(1 ) Cevc, G., Gebauer, D., Stieber, J., Scha'tzlein, A., Blume, G. 1998. Ultraflexible vesicles, Trans- fersomes, have an extremely low pore penetration resistance and transport therapeutic amounts of insulin across the intact mammalian skin. Biochim Biophys Acta 1368, 201-215.
(2) Cilurzo, F., Gennari, C.G.M., Selmin, F., Franze, S., Musazzi, U.M., Minghetti, P. 2015. On the characterization of medicated plasters containing NSAIDs according to novel indications of USP and EMA: adhesive property and in vitro skin permeation studies. Drug Dev. Ind. Pharm., 41 , 183-189.
(3) Franze, S., Donadoni, G., Podesta, A., Procacci, P., Orioli, M., Carini, M., Minghetti, P., Cilurzo, F. 2017. Tuning the extent and depth of penetration of flexible liposomes in human skin. Mol. Pharmaceutics, 14 (6), 1998-2009.
(4) Franze, S., Marengo, A., Stella, B., Minghetti, P., Arpicco, S., Cilurzo, F. 2018. Hyaluronan- decorated liposomes as drug delivery systems for cutaneous administration. Int J Pharm, 535, 333-339.
(5) Li Y, Chen Z, Cui Y, Zhai G, Li L. The construction and characterization of hybrid paclitaxel-in- micelle-in-liposome systems for enhanced oral drug delivery. Colloids Surf B Biointerfaces 2017, 160, 572-580.
(6) Roberts, M.S., Mohammed, Y, Pastore, M.N., Namjoshi, S., Yousef, S., Alinaghi, A., Haridass, I.N., Abd, E., Leite-Silva, V.R., Benson, H.A.E., Grice J.E., 2017. Topical and cutaneous delivery using nanosystems. J Control Release 247, 86-105.

Claims

1 . A deformable liposome composition comprising:
- micelles selected from the group comprising:
o amphiphilic monomers, i.e. Non ionic Surfactants: such as the esters of fatty acids or PEGs or Fatty alcohol ethoxylates; Nonoxynols; Fatty acid ethoxylates; Special ethox- ylated fatty esters and oils; Ethoxylated amines and/or fatty acid amides; Terminally blocked ethoxylates; Fatty acid esters of glycerol; Fatty acid esters of sorbitol; Fatty acid esters of sucrose; Alkyl polyglycoside; Ionic surfactants: such as Cetrimonium bromide; Cetylpyridinium chloride; Benzalkonium chloride; Benzethonium chloride; Dimethyldioc- tadecylammonium chloride; Dioctadecyldimethylammonium bromide; or o amphiphilic polymers, i.e. Self assembling copolymers: such as Amphiphilic block copol ymers (i.e. Pluronics, poly(esters), hydrophobic poly(amino acids), copolymers of lactic acid and glycolic acids, poly(caprolactone)), graft copolymers, polaxamers, semisynthetic polysaccharides;
- said micelles encapsulated in the aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deforma ble liposome free of cholesterol or other stabilizing materials in/on the bilayer.
2. The deformable liposome composition according to claim 1 , wherein the fluidizing agent is in a concentration of 5-20% w/w with respect to the phospholipid or lipid forming bilayer component.
3. The deformable liposome composition according to claim 1 , wherein the fluidizing agent is se lected from the group comprising non-ionic single chain surfactant, a skin penetration enhancer, ethanol or a combination thereof.
4. The deformable liposome composition according to claim 1 , wherein the micelles incorporate a hydrophobic compound.
5. A deformable liposome composition comprising:
- micelles selected from the group comprising: amphiphilic monomers or amphiphilic polymers,
- said micelles encapsulated in the aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deforma ble liposome free of cholesterol or other stabilizing materials in/on the bilayer,
as (trans)dermal delivery system.
6. A deformable liposome composition for use in a method of administering a hydrophobic com pound to a target tissue wherein a deformable liposome composition comprising a hydrophobic compound incorporated in micelles,
- said micelles selected from the group comprising: amphiphilic monomers or amphiphilic poly mers
- said micelles encapsulated in the aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deforma ble liposome free of cholesterol or other stabilizing materials in/on the bilayer,
is (trans)dermally administered to said target tissue.
7. A deformable liposome composition comprising a drug, such as a poorly permeable drug or a water-insoluble drug, incorporated in micelles: - said micelles selected from the group comprising:
o amphiphilic monomers, i.e. Non ionic Surfactants: such as the esters of fatty acids or PEGs or Fatty alcohol ethoxylates; Nonoxynols; Fatty acid ethoxylates; Special ethox- ylated fatty esters and oils; Ethoxylated amines and/or fatty acid amides; Terminally blocked ethoxylates; Fatty acid esters of glycerol; Fatty acid esters of sorbitol; Fatty acid esters of sucrose; Alkyl polyglycoside; Ionic surfactants: such as Cetrimonium bromide; Cetylpyridinium chloride; Benzalkonium chloride; Benzethonium chloride; Dimethyldioc- tadecylammonium chloride; Dioctadecyldimethylammonium bromide; or o amphiphilic polymers, i.e. Self assembling copolymers: such as Amphiphilic block copol ymers (i.e. Pluronics, poly(esters), hydrophobic poly(amino acids), copolymers of lactic acid and glycolic acids, poly(caprolactone)), graft copolymers, polaxamers, semisynthetic polysaccharides;
- said micelles encapsulated in the aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deforma ble liposome free of cholesterol or other stabilizing materials in/on the bilayer.
8. The deformable liposome composition according to claim 7, wherein the fluidizing agent is in a concentration of 5-20% w/w with respect to the phospholipid or lipid forming bilayer component.
9. The deformable liposome composition according to claim 7, wherein the fluidizing agent is se lected from the group comprising non-ionic single chain surfactant, a skin penetration enhancer, ethanol or a combination thereof
10. The deformable liposome composition according to claim 7, wherein the drug, as a poorly permeable drug or a water-insoluble drug, is selected from the group comprising neutral, cationic, anionic, zwitterionic drugs, such as resveratrol, nifedipine, piroxicam or naproxene.
1 1. A deformable liposome composition comprising a drug, such as a poorly permeable drug or a water-insoluble drug, incorporated in micelles:
- said micelles selected from the group comprising: amphiphilic monomers or amphiphilic poly mers,
- said micelles encapsulated in the aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deforma ble liposome free of cholesterol or other stabilizing materials in/on the bilayer,
as (trans)dermal drug delivery system for a poorly permeable drug or a water-insoluble drug.
12. A deformable liposome composition for use in a method of administering a drug, such as a poorly permeable drug or a water-insoluble drug, to a target tissue wherein a deformable lipo some composition comprising a drug, such as a poorly permeable drug or a water-insoluble drug, incorporated in micelles,
- said micelles selected from the group comprising: amphiphilic monomers or amphiphilic poly mers
- said micelles encapsulated in the aqueous core of a deformable liposome vesicle containing at least a phospholipid or lipid forming bilayer component and at least a fluidizing agent, a deforma ble liposome free of cholesterol or other stabilizing materials in/on the bilayer,
is (trans)dermally administered to said target tissue.
13. The deformable liposome composition according to claim 7, wherein the drug as a poorly permeable drug or a water-insoluble drug is selected from the group comprising neutral, cationic, anionic, zwitterionic drugs, such as tranexamic acid, retinol, arbutin, perfluordecalin, diclofenac, azelaic acid, caffeine, CBD, curcumin, lidocaine, pycnogenol.
14. The deformable liposome composition according to claim 1 , wherein the deformable liposome free of cholesterol or other stabilizing materials in/on the bilayer is characterized by a deformabil- ity constant not higher than 0.13 N/mm, preferably by a deformability constant in the range 0.01 - 0.13 N/mm.
15. The deformable liposome composition according to claim 5, wherein the deformable liposome free of cholesterol or other stabilizing materials in/on the bilayer is characterized by a deformabil ity constant not higher than 0.13 N/mm, preferably by a deformability constant in the range 0.01 - 0.13 N/mm.
16. The deformable liposome composition according to claim 6, wherein the deformable liposome free of cholesterol or other stabilizing materials in/on the bilayer is characterized by a deformabil ity constant not higher than 0.13 N/mm, preferably by a deformability constant in the range 0.01 - 0.13 N/mm.
17. The deformable liposome composition according to claim 7, wherein the deformable liposome free of cholesterol or other stabilizing materials in/on the bilayer is characterized by a deformabil ity constant not higher than 0.13 N/mm, preferably by a deformability constant in the range 0.01 - 0.13 N/mm.
18. The deformable liposome composition according to claim 7 for use in trans(dermal) therapeu tic applications/treatments wherein the drug, as a poorly permeable drug or a water-insoluble drug, is selected from the group comprising neutral, cationic, anionic, zwitterionic drugs, such as resveratrol, nifedipine, piroxicam, naproxene.
19. The deformable liposome composition according to claim 7 for use in trans(dermal) therapeu tic applications/treatments wherein the drug, as a poorly permeable drug or a water-insoluble drug, is selected from the group comprising neutral, cationic, anionic, zwitterionic drugs, such as tranexamic acid, retinol, arbutin, perfluordecalin, diclofenac, azelaic acid, caffeine, CBD, curcu min, lidocaine, pycnogenol.
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