WO2019083365A1 - Vecteurs d'administration - Google Patents

Vecteurs d'administration

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Publication number
WO2019083365A1
WO2019083365A1 PCT/NL2018/050709 NL2018050709W WO2019083365A1 WO 2019083365 A1 WO2019083365 A1 WO 2019083365A1 NL 2018050709 W NL2018050709 W NL 2018050709W WO 2019083365 A1 WO2019083365 A1 WO 2019083365A1
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WO
WIPO (PCT)
Prior art keywords
liposome
lipid
glycero
phosphocholine
disease
Prior art date
Application number
PCT/NL2018/050709
Other languages
English (en)
Inventor
Panagiota PAPADOPOULOU
Gabriela ARIAS ALPÍZAR
Frederick Campbell
Jeroen BUSSMANN
Alexander Kros
Original Assignee
Universiteit Leiden
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universiteit Leiden filed Critical Universiteit Leiden
Publication of WO2019083365A1 publication Critical patent/WO2019083365A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes

Definitions

  • Novel liposomes and liposome compositions are provided herein. Corresponding kits and methods of making the liposomes are also described.
  • the liposomes provide a useful means for selective delivery of a cargo such as an active pharmaceutical ingredient or an imaging agent to the blood brain barrier (BBB) of a subject.
  • BBB blood brain barrier
  • the liposomes may be used for therapeutic, diagnostic, or theranostic purposes.
  • CNS central nervous system
  • Systemic administration (e.g. by intravenous injection) is typically used to administer these types of medicament to a patient.
  • the medicament is effectively delivered to the blood brain barrier, there is an increased risk of off-target effects, and an increased need for high doses.
  • the percentage of injected dose (%ID) reaching the brain is rarely provided in the literature. Direct quantification as to the efficiency of targeting for each of these treatments is therefore, in general, hard to gauge. A few examples have been provided in the literature where the %ID within the brain (and inclusive of the brain endothelium) has been directly quantified.
  • Liposomes also known as lipid vesicles
  • Liposomes are colloidal particles that are prepared from polar lipid molecules derived either from natural sources or chemical synthesis. The lipids form a spherical, closed structure, wherein an external curved lipid bilayer forms around an aqueous core. Medicines and other cargo can be entrapped or embedded within the lipid bilayer, to reduce off-target toxicity, improve solubility and/or increase efficacy of the medicament.
  • Liposomes have become the most widely investigated nanoparticles for drug delivery applications (Sercombe, L. et al., Front. Pharmacol. 6, 286, (2015); Allen, T. M. et al., Adv.
  • the inventors have developed a novel liposome which selectively targets to the blood brain barrier (BBB) of embryonic zebrafish following systemic (intravenous) administration.
  • BBB blood brain barrier
  • the inventors have found selective targeting to the BBB requires liposomes that are made up of two lipids that undergo phase separation in the liposome (see Figure 14). When combinations of lipids were used that did not undergo phase separation in the resultant liposome, the liposomes did not demonstrate selective targeting to the BBB.
  • the inventors have tested a range of different lipid combinations and molar ratios and have identified the lipid characteristics required for production of a liposome that targets to the BBB after systemic administration. They have also demonstrated that cargoes such as small molecule drugs (e.g. the cytotoxic drug, doxorubicin) as well as larger cargoes (e.g. Au nanoparticles) can be encapsulated within the novel liposomes.
  • the inventors have therefore identified a new means for delivery of cargo to the blood brain barrier (BBB) of a subject.
  • BBB blood brain barrier
  • the components of the novel liposomes are cheap to make and the liposomes are easy to formulate on a large scale.
  • the invention provides a liposome comprising:
  • R and R 2 are each independently selected from H, Cio-C3o-alkyl, Cio-C3o-alkenyl, C(0)-C o-C 3 o-alkyl and C(0)-Cio-C 3 o-alkenyl;
  • R 3 is independently selected from H, Cio-C3o-alkyl and C io-C3o-alkenyl;
  • X 1 and X 2 are each independently selected from -0-, -S- and -NH-; wherein if R is H, X 1 is selected from -O- or -S-; wherein if R 2 is H, X 2 is selected from -O- or -S-;
  • X 3 is independently selected from a bond, -0-, -S-, -OC(O)-, and -NHC(O)-; wherein if R 3 is H, X 3 is not a bond;
  • each n is an integer independently selected from 1 to 4, preferably 1 or 2;
  • phase separation occurs between the first lipid and the second lipid in the liposome.
  • the lipid of formula I may be a lipid of formula II:
  • X 3 is -NHC(O)- or -OC(O)-;
  • R 1 and R 2 are each independently selected from H and C(0)-C io-C30-alkenyl
  • R 3 is independently selected from H and Cio-C 3 o-alkenyl
  • each n is an integer independently selected from 1 to 4, preferably 1 or 2.
  • R of lipid of formula II may be selected from C(0)-Ci4 . z-C26.z-alkenyl, wherein Z is selected from 1 to 10.
  • R 3 of lipid of formula II may be selected from Ci4 z-C26.z-alkenyl, wherein Z is selected from 1 to 10.
  • R 2 of lipid formula II may be H.
  • X 3 of lipid formula II may be -NHC(O)-
  • the first lipid may be:
  • the first lipid may be:
  • the first lipid may be:
  • the first lipid may be:
  • the second lipid may be a phospholipid, optionally having the following formula
  • R 5 and R 6 are each independently selected from C(0)-Cio-C3o-alkyl, C(0)-Cio-C3o-alkenyl and C(0)-Cio-C3o-alkynyl;
  • R 7 is independently at each occurrence selected from H or Ci-C4-alkyl.
  • the phospholipid may be selected from any one of phosphatidylcholine (PC), phos- phatidylglycerol (PG), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphati- dylglycerides, phosphatidic acid (PA), phospholsphingolipids, or any combination thereof.
  • the phospholipid may be a phosphatidylcholine (PC).
  • the phosphatidylcholine may be selected from the group consisting of 1 ,2-di- palmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-oleoyl-2-palmitoyl-sn-glycero-3-phospho- choline (OPPC), 1 ,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1 ,2-dierucoyl-sn- glycero-3-phosphocholine (DEPC), 1 ,2-dilinoleoyl-sn-glycero-3-phosphocholine
  • DLOPC 1 ,2-dilauroyl-sn-glycero-3-phosphocholine
  • DLPC 1 ,2-dimyristoyl-sn-glycero-3- phosphocholine
  • DMPC 1 ,2-dimyristoyl-sn-glycero-3- phosphocholine
  • DSPC 1 ,2-distearoyl-sn-glycero-3-phosphocholine
  • MPPC 1-myristoyl-2- palmitoyl-sn-glycero-3-phosphocholine
  • MSPC 1-myristoyl-2-stearoyl-sn-glycero-3-phos- phocholine
  • PMPC 1-palmitoyl-2-myristoyl-sn-3-phosphocholine
  • POPC 1-palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine
  • PSPC 1-palmitoyl-2-stearoyl-sn-glycero-3-phospho
  • the phospholipid may be 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
  • the liposome is not restricted to a first and second lipid but can contain further li- pidic/hydrophobic reagents.
  • the invention provides a liposome which comprises cholesterol.
  • the invention provides a liposome which comprises further lepidic/hydrophobic reagents selected from lipid-PEG, lipid-chelator, lipid-dye or lipid-conju- gates, wherein the targeting ligands are covalently attached to the lipid headgroup.
  • the liposome can comprise up to 20 % by weight of further lipidic/hydro- phobic reagents.
  • a lipid may form the external lipid bilayer of the liposome, surrounding an aqueous core (as shown in Figure 14).
  • a nanodomain of the first lipid may be encapsulated within part of the external lipid bilayer of the liposome (as shown in Figure 14).
  • the morphology of the liposome, specifically the nanodomain is encapsulated within at least part of the external lipid bilayer.
  • the liposome may have a diameter of the major axis of the liposome in the range of from about 50 nm to about 250 nm, optionally wherein the liposome has a diameter of the major axis of about 100 nm.
  • the molar ratio of first lipid:second lipid may be between about 1 :9 to about 3: 1 , optionally between about 1 :3 to about 1 :1 , preferably about 1 : 1.
  • the liposome may further comprise a cargo within the aqueous core.
  • the cargo may be an active pharmaceutical ingredient, optionally wherein the active pharmaceutical ingredient is selected from the group consisting of: a small molecule, peptide, protein, inorganic nanoparticle, oligonucleotide, or any combination thereof.
  • the cargo may be an imaging agent, optionally wherein the imaging agent is se- lected from: MR I contrast agents, PET/SPECT radioactive imaging agents, paramagnetic nanoparticles, fluorescent probes, bioluminescent probes, quantum dots, gold nanoparticles, optical coherence tomography agents, fluorescent proteins, fluorescent/radioactive latex beads/polymers, photoacoustic imaging agents (eg carbon nanotubes), Raman spectroscopy agents, nanobubbles, or any combination thereof.
  • the imaging agent is se- lected from: MR I contrast agents, PET/SPECT radioactive imaging agents, paramagnetic nanoparticles, fluorescent probes, bioluminescent probes, quantum dots, gold nanoparticles, optical coherence tomography agents, fluorescent proteins, fluorescent/radioactive latex beads/polymers, photoacoustic imaging agents (eg carbon nanotubes), Raman spectroscopy agents, nanobubbles, or any combination thereof.
  • the invention provides a composition comprising a liposome as defined herein.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a lipo- some as defined herein and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
  • the composition may further comprise at least one of: a preservative, an antioxidant, a buffering agent, an anionic polymer, or any combination thereof.
  • the invention provides a liposome as defined herein, or a composition comprising a liposome as defined herein, for use in therapy, diagnostics and/or theranostics.
  • the invention provides a liposome as defined herein, or a composition comprising a liposome as defined herein, for use in treating a brain disease in a subject in need thereof.
  • the brain disease may be selected from: Alzheimer's disease, Parkinson's disease, multiple sclerosis, brain cancer, brain tumour, amyotrophic lateral sclerosis (ALS), essential tremor, huntington's disease, Machado-Joseph disease, glaucoma, hereditary optic neuropathy (Leber), retinitis pigmentosa, meningitis, viral meningitis, inflammatory brain disease, psychotic disorder, narcolepsy, epilepsy/ seizure, and cranial nerve disorder.
  • ALS amyotrophic lateral sclerosis
  • Leber hereditary optic neuropathy
  • the subject may have been pre-treated with dextran sulphate.
  • the liposome or composition may be for separate, simultaneous or sequential ad- ministration with dextran sulphate to the subject.
  • the invention provides a method of treating a disease, wherein the method comprises administering a therapeutically effective amount of a liposome as defined herein, or a composition comprising a liposome as defined in herein, to a subject in need thereof.
  • the disease may be a brain disease.
  • the brain disease may be selected from: Alzheimer's disease, Parkinson's disease, multiple sclerosis, brain cancer, brain tumour, amyotrophic lateral sclerosis (ALS), essential tremor, huntington's disease, Machado-Joseph disease, glaucoma, hereditary optic neuropathy (Leber), retinitis pigmentosa, meningitis, viral meningitis, inflammatory brain disease, psychotic disorder, narcolepsy, epilepsy/ seizure, and cranial nerve disorder.
  • the method may comprise pre-treating the subject with dextran sulphate prior to administering the liposome or composition.
  • the method may comprise separate, simultaneous or sequential administration of the liposome or composition and dextran sulphate to the subject.
  • the invention provides for the use of a liposome as defined herein, or a composition comprising a liposome as defined herein, for the manufacture of a medicament for the treatment of a disease in a subject in need thereof.
  • the disease may be a brain disease.
  • the brain disease may be selected from: Alzheimer's disease, Parkinson's disease, multiple sclerosis, brain cancer, brain tumour, amyotrophic lateral sclerosis (ALS), essential tremor, huntington's disease, Machado-Joseph disease, glaucoma, hereditary optic neuropathy (Leber), retinitis pigmentosa, meningitis, viral meningitis, inflammatory brain disease, psychotic disorder, narcolepsy, epilepsy/ seizure, and cranial nerve disorder.
  • ALS amyotrophic lateral sclerosis
  • Leber hereditary optic neuropathy
  • retinitis pigmentosa meningitis
  • meningitis meningitis
  • viral meningitis inflammatory brain disease
  • psychotic disorder narcolepsy
  • epilepsy/ seizure and cranial nerve disorder.
  • the subject may have been pre-treated with dextran sulphate.
  • the liposome or composition may be for separate, simultaneous or sequential ad- ministration with dextran sulphate to the subject.
  • the invention provides a method for delivering a cargo to the blood brain barrier of a subject comprising administering a liposome as defined herein, or a composition comprising a liposome as defined herein, to the subject.
  • the subject may have been pre-treated with dextran sulphate.
  • the method may comprise separate, simultaneous or sequential administration of the liposome or composition and dextran sulphate to the subject.
  • the invention provides for the use of a liposome as defined herein, or a composition comprising a liposome as defined herein, for delivering a cargo to the blood brain barrier of a subject.
  • the subject may have been pre-treated with dextran sulphate.
  • the liposome or composition may be for separate, simultaneous or sequential administration with dextran sulphate to the subject.
  • the invention provides an in vitro use of a liposome as defined herein, or a composition comprising a liposome as defined herein, for delivering a cargo to an endothelial cell.
  • the invention provides a method of making a liposome as defined herein comprising the steps of:
  • R 1 and R 2 are each independently selected from H, Cio-C3o-alkyl, Cio-C3o-alkenyl, C(0)-Cio-C 3 o-alkyl and C(0)-Cio-C 3 o-alkenyl;
  • R 3 is independently selected from H, Cio-C3o-alkyl and Cio-C3o-alkenyl;
  • X 1 and X 2 are each independently selected from -0-, -S- and -NH-; wherein if R 1 is H, X 1 is selected from -O- or -S-; wherein if R 2 is H, X 2 is selected from -O- or -S-;
  • X 3 is independently selected from a bond, -0-, -S-, -OC(O)- and -NHC(O)-; wherein if R 3 is H, X 3 is not a bond;
  • each n is an integer independently selected from 1 to 4, preferably 1 or 2;
  • the invention provides a kit for preparing a liposome as defined herein, the kit comprising:
  • R and R 2 are each independently selected from H, Cio-C 3 o-alkyl, Cio-C 3 o-alkenyl, C(0)-Cio-C 3 o-alkyl and C(0)-C o-C 30 -alkenyl;
  • R 3 is independently selected from H, Cio-C3o-alkyl and Cio-C3o-alkenyl;
  • X 1 and X 2 are each independently selected from -0-, -S- and -NH-; wherein if R is H, X 1 is selected from -O- or -S-; wherein if R 2 is H, X 2 is selected from -O- or -S-;
  • X 3 is independently selected from a bond, -0-, -S-, -0C(0)- and -NHC(O)-; wherein if R 3 is H, X 3 is not a bond;
  • each n is an integer independently selected from 1 to 4, preferably 1 or 2;
  • the lipid of formula I may be a lipid of formula II:
  • X 3 is -N HC(O)- or -OC(O)-;
  • R 1 and R 2 are each independently selected from H and C(0)-Cio-C3o-alkenyl; R 3 is independently selected from H and Cio-Cso-alkenyl;
  • each n is an integer independently selected from 1 to 4, preferably 1 or 2.
  • R 1 of lipid of formula II may be selected from C(0)-C i4 z-C26 2-alkenyl, wherein Z is selected from 1 to 10.
  • R 3 of lipid of formula II may be selected from C i4 z-C26 z-alkenyl, wherein Z is selected from 1 to 10.
  • R 2 of lipid formula II may be H.
  • X 3 of lipid formula II may be -NHC(O)-.
  • the first lipid may be:
  • the first lipid may be:
  • the first lipid may be:
  • the first lipid may be:
  • R 5 and R 6 are each independently selected from C(0)-Cio-C3o-alkyl, C(0)-C io-C30-alkenyl and C(0)-Cio-C3o-alkynyl;
  • R 7 is independently at each occurrence selected from H or Ci-C4-alkyl.
  • the phospholipid may be selected from any one of phosphatidylcholine (PC), phos- phatidylglycerol (PG), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphati- dylglyceride, phosphatidic acid (PA), phospholsphingolipid, or any combination thereof.
  • PC phosphatidylcholine
  • PG phos- phatidylglycerol
  • PS phosphatidylserine
  • PE phosphatidylethanolamine
  • PA phosphatidic acid
  • phospholsphingolipid or any combination thereof.
  • the phospholipid may be a phosphatidylcholine (PC).
  • PC phosphatidylcholine
  • the phosphatidylcholine may be selected from the group consisting of 1 ,2-di- palmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-oleoyl-2-palmitoyl-sn-glycero-3-phospho- choline (OPPC), 1 ,2-didecanoyl-SA7-glycero-3-phosphocholine (DDPC), 1 ,2-dierucoyl-sn- glycero-3-phosphocholine (DEPC), 1 ,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLOPC), 1 ,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1 ,2-dimyristoyl-sn-glycero-3-phospho- choline (DMPC), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-myristo
  • FIG. 1 A shows a workflow for screening BBB-targeting liposomes in embryonic zebrafish.
  • 1 B shows that DSPC:PAPAP3 liposomes formulated in a 1 : 1 molar ratio exhibit very significant binding at the BBB of the embryonic zebrafish but also significant off- targeting to the venous endothelium.
  • 1 C shows BBB-targeting liposomes (+ 1 mol% DOPE-LR) accumulating at the BBB (dorsal, head only) of embryonic zebrafish at 3dpf (left) and control liposomes (100% DSPC) showing no targeting of the BBB (right).
  • FIG. 1 D shows lipid components of BBB-targeting liposomes - optimally in a 1 :1 molar ratio.
  • Figure 2 shows ApoE-targeted liposomes targeting the liver and kidney - where LDL receptors are highly expressed (Top panels). Transferrin receptor targeted liposomes targeting immature red blood cells (RBCs) - where transferrin receptor is highly expressed (Bottom panels).
  • Figure 3 shows the mixture of two regioisomers obtained from the synthesis of PAPAP3 and a 1 H-NMR spectrum of PAPAP3 (mixture of 2 regioisomers).
  • Figure 4 shows transmission electron microscopy images demonstrating that
  • DSPC:PAPAP3 liposomes can be formulated to encapsulate gold nanoparticles.
  • Figure 5 shows the results of different PAPAPs formulated 1 :1 with DSPC.
  • DSPC:PAPAP1 did not target the BBB in zebrafish (top left panel).
  • DSPC:PAPAP3 targeted the BBB in zebrafish (top right panel).
  • DSPC:PAPAP4 showed some binding to the BBB (middle left panel).
  • DSPC:PAPAP5 demonstrated strong off-targeting to venous endothelial cells (middle right panel).
  • DSPC:PAPAP6 could not be formulated into liposomes and DSPC:PAPAP7 shows low binding to the BBB, however the formulation of DSPC:PAPAP7 liposomes is not consistently reproducible (bottom panels).
  • Figure 6 shows the molecular structures of several first lipids used in the invention.
  • Figure 7 shows that DSPC:PAPAP3 liposomes formulated in a 3: 1 molar ratio accumulate at the BBB.
  • Figure 8 shows that DSPC:PAPAP3 liposomes formulated in a 9: 1 molar ratio also accumulate at the BBB.
  • Figure 9 shows that DSPG:PAPAP3 liposomes formulated in a 1 : 1 molar ratio exhibit low level binding at the BBB and strong off-target interaction with veins.
  • Figure 10 shows that DOPS:PAPAP3 liposomes formulated in a 1 :1 molar ratio exhibit no significant binding at the BBB and strong off-target interaction with veins.
  • Figure 11 shows that DPPC:PAPAP3 liposomes formulated in a 1 : 1 molar ratio exhibit binding at the BBB however is potently taken up by plasma exposed macrophages (large white spots).
  • Figure 12 shows that DMPC:PAPAP3 liposomes formulated in a 1 : 1 molar ratio exhibit low level binding at the BBB and some uptake by plasma exposed macrophages (mostly freely circulating).
  • Figure 13 shows that that DOPC:PAPAP3 liposomes formulated in a 1 : 1 molar ratio exhibit no significant binding at the BBB and strong off-target interactions with the veins.
  • Figure 14 shows cryoEM images of the liposomes of the invention (DSPC:PAPAP3 liposomes formulated in a 1 :1 molar ratio) and a schematic drawing of liposomes according to the invention.
  • Figure 15 shows the additional molecular structures of the lipids of this invention.
  • Figure 16 shows that the DPPC:PAPAP14 liposomes formulated in a 1 :1 molar ratio exhibit binding at the BBB.
  • Figure 17 shows that the DSPC:PAPAP1 1 liposomes formulated in a 1 :1 molar ratio exhibit binding at the BBB.
  • Figure 18 shows that the DSPC:PAPAP3 + 10 % cholesterol formulated in a 45:45:10 (DSPC:PAPAP3:cholesterol) molar ratio exhibit binding at the BBB.
  • a liposome is a colloidal particle that is prepared from polar lipid molecules derived either from natural sources or chemical synthesis.
  • the lipids form an spherical/oval, closed structure, wherein an external curved lipid bilayer forms around an aqueous core.
  • the liposome may include one or several lipid bilayers enclosing the aqueous core (e.g. see Figure 14).
  • a liposome typically serves as a carrier of an entity (i.e.
  • a cargo such as, without limitation, a chemical compound, a combination of compounds, a supramolecular complex of a synthetic or natural origin, a genetic material, a portion thereof, or a derivative thereof, that is capable of having a useful property or exerting a useful activity.
  • the liposomes described herein comprise two different lipids, described herein as a "first lipid” and a "second lipid".
  • the lipids may be of a natural and/or a synthetic/semi-synthetic origin. Mixtures of natural and synthetic/semi-synthetic lipids may also be employed.
  • the li osomes described include a first lipid of formula I:
  • R 1 and R 2 are each independently selected from H, Cio-C3o-alkyl, Cio-C3o-alkenyl, C(0)-Cio-C 3 o-alkyl and C(0)-Cio-C 3 o-alkenyl;
  • R 3 is independently selected from H, C o-C3o-alkyl and Cio-C3o-alkenyl;
  • X 1 and X 2 are each independently selected from -0-, -S- and -NH-; wherein if R 1 is H, X 1 is selected from -O- or -S-; wherein if R 2 is H, X 2 is selected from -O- or -S-;
  • X 3 is independently selected from a bond, -0-, -S-, -OC(O)- and -N HC(O)-; wherein if R 3 is H, X 3 is not a bond;
  • each n is an integer independently selected from 1 to 4, preferably 1 or 2.
  • the lipid of formula I is a lipid of formula II:
  • X 3 is -N HC(O)- or -OC(O)-;
  • R 1 and R 2 are each independently selected from H and C(0)-Cio-C3o-alkenyl
  • R 3 is independently selected from H and C o-C 3 o-alkenyl
  • each n is an integer independently selected from 1 to 4, preferably 1 or 2.
  • the following statements apply, where not mutually exclusive, to both the lipids of formula I and the lipids of formula II.
  • R and R 2 are each independently selected from H, C(0)-C io-C30-alkyl and C(0)-Cio-C3o-alkenyl. It may be that R 1 and R 2 are each independently selected from H and C(0)-Cio-C 3 o-alkenyl.
  • R 3 is independently selected from H and Cio-C3o-alkenyl.
  • R 1 and R 2 are each independently selected from H , Cio-C3o-alkenyl and C(O)- Cio-C 3 o-alkenyl; and that R 3 is independently selected from H and C io-C 3 o-alkenyl. It may be that R and R 2 are each independently selected from H and C(0)-C io-C3o-alkenyl; and that R 3 is independently selected from H and Cio-C3o-alkenyl.
  • the term C 10-C30 refers to a carbon chain length of 10 to 30.
  • R and R 2 may be H.
  • R is H. It may be that R 2 is H.
  • R , R 2 and R 3 are selected such that any alkyl or alkenyl group in R , R 2 and R 3 is from 12 to 25 carbons atoms in length. It may be that R , R 2 and R 3 are selected such that any alkyl or alkenyl group is from 15 to 20 carbons atoms in length.
  • R 1 , R 2 and/or R 3 are alkenyl it may be that that alkenyl group comprises more than one carbon-carbon double bond. Alternatively, it may be that the alkenyl group comprises a single carbon-carbon double bond. Where an alkenyl group comprises more than one carbon-carbon double bond it may be that at least two sp3 carbons separate any two carbon-carbon double bonds.
  • R 1 , R 2 and/or R 3 are alkenyl comprising a single carbon-carbon double bond
  • the carbon atom at one end of the double bond is situated x carbons away from X 1 , X 2 and X 3 and the carbon atom at the other end of the double bond is y carbons away from the end of the alkenyl group
  • x and y are selected dependent on the length of the alkenyl group and may each be an integer selected from 0 to 28, the sum of x and y being an integer from 8 to 28 where the alkenyl is a C io-C3o-alkenyl. It may be that x is not less than 2. It may be that y is not less than 2. It may be that x is not less than 5.
  • C 4 Z-C26.Z refers to a carbon chain length of from 14 to 26 carbon atoms, wherein there is from 1 to 10 points of saturation (Z).
  • the saturation degree is 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10. It may be that X 1 and X 2 are both -0-.
  • X 3 may be independently selected from -0-, -S-, -OC(O)- and -NHC(O)-.
  • X 3 is selected from -OC(O)- and -NHC(O)-. It may be that X 3 is -NHC(O)-.
  • alkyl refers to a linear saturated hydrocarbon chain.
  • alkenyl refers to a linear hydrocarbon chain containing at least one carbon- carbon double bond. The double bond(s) may be present as the E or Z isomer.
  • alkynyl refers to a linear hydrocarbon chain containing at least one carbon-carbon triple bond.
  • the first lipid may be:
  • lipids of formula I may mean, for example, that a double bond is in a different position along an alkyl chain. It may also mean that an acyl group is attached to an alternative heteroatom.
  • Many lipids of formula I comprise a chiral centre. Where the lipid of formula I comprises a chiral centre, the lipid may be in the form of the (R)-enantiomer or the (S)-enantiomer or a mixture thereof.
  • the lipid is a mixture, it may be that the (R)-enantiomer predominates or that the (S)-enantiomer predominates or it may a racemic mixture of the (R)-enantiomer or the (S)-enantiomer.
  • the liposome may comprise a mixture of more than one lipid of formula I.
  • the liposome may comprise a mixture of two regioisomers which differ in that in the first regioisomer R 1 is H and in the second regioisomer R 2 is H.
  • R 1 is H
  • R 2 is H
  • the R 2 group in the first isomer is the same as the R 1 group in the second isomer.
  • first lipid is also referred to as “Lipid 1” or “PAPAP”.
  • the liposomes described herein also comprise a second lipid (also described herein as the "co-formulant" lipid).
  • the first and second lipids undergo phase separation when mixed to form a liposome.
  • phase separation means that the two lipids within the liposome are present in two separate phases after mixing to form a liposome. It will also be understood that whilst the two lipids are in two separate phases, there is still interaction between the phases such that the first lipid may be protected by the second lipid from an aqueous environment.
  • the aqueous environment may, for example, either be the aqueous core of the liposome or an external aqueous environment.
  • An example of the phase separation is shown in Figure 14. Phase separation is caused by differences in packing parameters within lipid bilayers.
  • Phase separation usually refers to the formation of lipid rafts/domains (for example cholesterol rich domains), wherein the lipid raft/domain is enriched in a particular lipid (or subset of lipids) compared to other regions of the lipid bilayer.
  • phase separation is used to describe lipid droplet formation (also described as nanodomain formation herein) as shown in Figure 14, wherein a lipid droplet (or
  • phase separation i.e. first lipid droplet formation
  • the test is as follows: Mix individual lipids (lipid 1 and 2), stored as stock solutions (1-10 mM) in chloroform, to the desired molar ratios in a glass vial and dry to a film, first under a stream of N2 then >1 h under vacuum. Hydrate the lipid film with ddh O to a total lipid concentration of 5 mM (solution may require heating to 80°C and/or gentle vortexing. Do not sonicate).
  • lipid droplets To check for the presence of lipid droplets perform cryoEM imaging on this sample as described in the examples section below).
  • the majority of liposomes should appear as round or elipse structures (ranging from 50-250 nm longest axis) with a clearly defined outer membrane (dark contrast, approx. 5 nm in thickness) and a lipid droplet (dark contrast, approx. 10-50 nm in diameter) clearly visible and resembling a protrusion within the lipid bilayer (see Figure 14).
  • R 5 and R 6 are each independently selected from C(0)-Cio-C3o-alkyl, C(0)-Cio-C3o-alkenyl and C(0)-Ci o-C3o-alkynyl;
  • R 7 is independently at each occurrence selected from H or Ci-C4-alkyl.
  • the second lipid may be a phospholipid.
  • phospholipid refers to a lipid which has a hydrophilic portion comprising a phosphate group, and particularly includes lipids based on phosphatidylcholine (PC) , phosphatidylglycerol (PG), phosphatidylserine (PS),
  • PC phosphatidylcholine
  • PG phosphatidylglycerol
  • PS phosphatidylserine
  • PE phosphatidylethanolamine
  • PI phosphoinositide
  • PA phosphatidic acid
  • phospholsphingolipids e.g. sphingomyelin
  • the phospholipid is preferably of natural origin.
  • Natural phospholipids are preferably membrane lipids derived from various sources of both vegetable (e.g. rapeseed, sunflower, etc., or, preferably, soybean) and animal origin (e.g. egg yolk, bovine milk, etc.).
  • Phospholipids from soybean are normally obtained from the by-products (i.e. lecithins) in the refining of crude soybean oil by the degumming process.
  • the lecithins are further processed and purified using other physical unit operations, such as fractionation and/or chromatography.
  • Other phospholipids may be obtained, for example, by pressing various suitable seeds and grains, followed by solvent extraction and then further processing as described above.
  • Phospholipids derived from a natural source e.g. egg phosphatidylcholine (EPC), soy PC, hydro soy PC (HSPC), brain PC, heart PC, liver PC etc
  • EPC egg phosphatidylcholine
  • soy PC soy PC
  • HSPC hydro soy PC
  • brain PC i.e. may comprise some impurities
  • Phospholipids that are useful in forming the liposomes described herein may also be obtained from e.g. Avanti Polar Lipids, Lipoid GmbH, or Sigma-A
  • the phospholipid of choice may be a phosphatidylcholine (PC).
  • PC phosphatidylcholine
  • PC phosphatidylcholine
  • DPPC 1,2-dipalmitoyl-sn- glycero-3-phosphocholine
  • OPPC 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine
  • DDPC 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine
  • DEPC dierucoyl-sn-glycero-3- phosphocholine
  • DEPC dierucoyl-sn-glycero-3- phosphocholine
  • DLOPC 1- ,2-dilinoleoyl-sn-glycero-3-phosphocholine
  • DLOPC 1- ,2- dilauroyl-sn-glycero-3-phosphocholine
  • DLPC ,2-dimyristoyl-sn-glycero-3-phosphocholine
  • DOPC 1,2-dioleoyl-sn-glycero-3-phosphocho
  • EPC phosphatidylcholine
  • soy PC soy PC
  • hydro soy PC HSPC
  • brain PC heart PC
  • liver PC any combination thereof.
  • the phospholipid may be 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
  • the molar ratio of first lipid:second lipid in the liposome may be between about 1 :9 to about 3:1. In other words, the molar ratio may be about 1 :9, 1 :8, 1 :7, 1 :6, 1 :5, 1 :4, 1 :3, 1 :2, 1 : 1 , 1.5:1 , 2: 1 , 2.5: 1 , 3:1 , or any range there inbetween.
  • the molar ratio of first lipid:second lipid in the liposome may be between about 1 :3 to about 1 :1 , and is preferably about 1 : 1 (wherein about 1 : 1 encompasses 0.5: 1.4 to 1.4:0.5).
  • the external lipid bilayer of the liposome will predominantly be formed by either one (but not both) of the lipids.
  • the second lipid is typically more amphiphilic in character than the first lipid
  • the second lipid typically forms the external lipid bilayer of the liposome.
  • the second lipid may be the predominant lipid in the lipid bilayer that forms the liposome outer boundary, and interacts with the external environment. This does not exclude the first lipid being present in the external lipid bilayer, as described in more detail below.
  • phase separation of the first lipid and the second lipid in the liposome results in a nanodomain (also described as a lipid droplet) of the first lipid forming in the liposome.
  • a “nanodomain of the first lipid” refers to a lipid component of the liposome that is enriched with the first lipid (i.e. is predominantly made up of the first lipid).
  • the first lipid may form a lipid droplet in the liposome, wherein the lipid droplet is enriched in (i.e.
  • the nanodomain may comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% of the total amount of the first lipid in the liposome.
  • the nanodomain may therefore be formed substantially of the first lipid.
  • the nanodomain does not include an aqueous core.
  • the nanodomain or lipid droplet of the invention may have a diameter of a major axis in the range of about 10 to about 100 nm (e.g. about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 nm, or any range there inbetween).
  • the range may from any one of the specifically cited diameters as a starting point, to any one of the specifically cited diameters as an end point of the range.
  • the nanodomain (or lipid droplet) of the first lipid may become encapsulated (or embedded) within the external lipid bilayer of the liposome.
  • encapsulated within the external lipid bilayer means that the nanodomain may be located between the two lipid monolayers of the external lipid bilayer (see Figure 14).
  • the nanodomain or lipid droplet of the first lipid may form such that it is sandwiched between the two monolayers of the external lipid bilayer of the liposome.
  • the external lipid bilayer is predominantly made up of the second lipid. Therefore, in this context, a layer of second lipids (e.g. a monolayer of second lipids) may act as a barrier between the nanodomain and the external environment outside the liposome. In this example, the second lipid may also form a barrier between the nanodomain of the first lipid and the aqueous core of the liposome. As discussed above, the nanodomain (or lipid droplet) of the first lipid may also become embedded within the external lipid bilayer of the liposome. In this context, embedded within the external lipid bilayer means that the nanodomain (or lipid droplet) is predominantly (but not completely) surrounded by the two lipid monolayers of the external lipid bilayer.
  • a proportion of the nanodomain or lipid droplet of the first lipid may protrude from the external lipid bilayer of the liposome (e.g. into the aqueous core of the liposome, or into the external environment outside the liposome) or form part of the external lipid bilayer, and thus be in direct contact with the aqueous core or external environment.
  • the proportion of nanodomain or lipid droplet that may be in direct contact with the external environment or aqueous core may be less than 40%, less than 30%, less than 20%, less than 10%, less than 5% of the total amount of the first lipid in the nanodomain or lipid droplet.
  • the nanodomain or lipid droplet of the first lipid may also be encapsulated or embedded within two lipid bilayers formed by the second lipid e.g. the external bilayer and a second bilayer within the liposome that acts as a barrier between the nanodomain and the aqueous core of the liposome (see also Figure 14).
  • the liposome may take any shape, although the liposomes described herein are usually approximately ellipsoid.
  • the liposome described herein will typically have a diameter of a major axis of the liposome about 50 nm or above so that, once administered to a subject, the liposome is not filtered out of the bloodstream by the kidneys.
  • the liposome described herein will typically also have a diameter of the major axis about 250 nm or below so that once administered to a subject the rate of recognition and degradation/clearance within the liver and spleen is minimised. Therefore, the liposome defined herein may have a diameter of the major axis of between about 50 nm and about 250 nm for example between about 75 nm and about 200 nm, between about 90 nm and 150 mm, or about 100 nm.
  • the liposome may have a diameter of a major axis of about 100 nm.
  • the liposome may have a diameter of a major axis of about 50 nm to about 150 nm, or about 60 nm to about 140 nm, or about 70 nm to about 130 nm, or about 75 to nm to about 125 nm, or about 75 nm to about 100 nm, for example, about 100 nm.
  • the liposome may therefore have a diameter of a major axis of about 50 nm, above 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 1 15 nm, about 120 nm, about 125 nm, 5 about 130 nm, about 135 nm, about 140 nm, about 145 nm, about 150 nm.
  • the liposome may have a diameter of the major axis defined by a size range, with the lower end of the size range being any size selected from about 50 nm, 51 nm, 52 nm, 53 nm, 54 nm, 55 nm, 56 nm, 57 nm, 58 nm, 59 nm, 60 nm, 61 nm, 62 nm,63
  • diameter of the major axis refers to the largest dimension of the liposome.
  • compositions may contain liposomes that are a range of different sizes. Size of liposomes may be affected by a number of factors, such as, for example, lipid
  • composition and method of preparation can be determined by a number of techniques.
  • Reference to a diameter of the major axis of a liposome as used herein may also refer to a mean diameter of the major axis of the liposomes within said composition.
  • Sizes may be determined according to techniques that are known to the person skilled in the art, including electron microscopy techniques (such as transmission electron microscopy (TEM) and cryoTEM) and light scattering techniques (such as laser diffraction and dynamic light scattering). Laser diffraction or dynamic light scattering (as is described elsewhere herein) may be used to determine the size (and size distribution) of the liposomes. CryoTEM techniques are generally preferred over TEM as the sample does not need to be dried; this is particularly advantageous for liposomes because these particles can be kept in their native state. These techniques allow direct visualisation of liposomes and are often used as a qualitative tool to visualise liposome size and morphology. Quantification of size and polydispersity is possible if a sufficient number of particles are individually analysed.
  • electron microscopy techniques such as transmission electron microscopy (TEM) and cryoTEM
  • light scattering techniques such as laser diffraction and dynamic light scattering.
  • Laser diffraction or dynamic light scattering as is described elsewhere herein
  • the liposomes described herein may contain a plurality of different lipids, provided they contain a first lipid and a second lipid as described above, and phase separation occurs between the first lipid and the second lipid in the liposome.
  • the liposome typically also fulfils the size requirements described above. Additional lipids that may be present in the liposomes include those described above as well as others that are known to be useful in forming liposomes for clinical use.
  • the liposomes may contain one, two, three or more different lipids. It is preferred that the liposomes are formed from predominantly only a first lipid and a second lipid.
  • substantially all of the lipid molecules in the liposomes are either a first lipid or a second lipid as described herein.
  • At least 70% (e.g. at least 90%, e.g. at least 95%, e.g. at least 99%, e.g. 100%) by weight of the lipid molecules in the liposomes may be either a first lipid or a second lipid as described herein.
  • the liposomes described herein may further comprise lipidic/hydrophobic reagents.
  • the invention provides a liposome which comprises cholesterol.
  • the liposome can comprise at least 15 %, at least 20 %, at least 25 % by weight of further lipidic/hydrophobic reagents.
  • the liposomes described herein may further comprise a cargo.
  • the cargo may be any entity such as, without limitation, a chemical compound, a combination of compounds, a supramolecular complex of a synthetic or natural origin, a genetic material, a portion thereof, or a derivative thereof, that is capable of having a useful property or exerting a useful activity.
  • the cargo may be hydrophilic or hydrophobic.
  • any active pharmaceutical ingredient or any imaging agent may be used in the context of the invention. Non-limiting examples are provided below.
  • a liposome may contain a plurality of cargoes e.g. two or more active pharmaceutical ingredients, two or more imaging agents, or a mixture of active pharmaceutical ingredients and imaging agents.
  • the cargo is an active pharmaceutical ingredient such as a small molecule, peptide, protein, inorganic nanoparticle, oligonucleotide (or any combination thereof).
  • active pharmaceutical ingredient such as a small molecule, peptide, protein, inorganic nanoparticle, oligonucleotide (or any combination thereof).
  • the cargo may be an imaging agent, which includes MRI contrast agents (e.g. Gd), PET/SPECT radioactive imaging agents (e.g. 111 ln, 64 Cu), paramagnetic nanoparticles (e.g. iron oxide), fluorescent probes, bioluminescent probes, quantum dots, gold
  • MRI contrast agents e.g. Gd
  • PET/SPECT radioactive imaging agents e.g. 111 ln, 64 Cu
  • paramagnetic nanoparticles e.g. iron oxide
  • fluorescent probes e.g. iron oxide
  • bioluminescent probes e.g. gold
  • quantum dots gold
  • optical coherence tomography agents e.g. gold nanorods, fluorescent proteins, fluorescent/radioactive latex beads/polymers, photoacoustic imaging agents (eg carbon nanotubes), Raman spectroscopy agents (e.g. AuNPs), nanobubbles (or any combination thereof).
  • imaging agents which are suitable for radioimaging. This is because the liposomes have been found to target the BBB and information about the localisation of these liposomes may be useful to a clinician. Radioimaging techniques that may be mentioned include PET, SPECT and MRI, and imaging agents used in these methods would be known to the skilled person.
  • a cargo e.g. an imaging agent
  • a cargo may be bound to (or adsorbed onto, or tethered to) the outer surface of the liposome. This would be advantageous if, for example, they are being used in diagnostic methods.
  • a cargo may be incorporated into the liposome.
  • the cargo may be may be encapsulated within and/or covalently bound to said liposomes.
  • the cargo may be encapsulated by at least one lipid bilayer of the liposome such that it is located within the aqueous core.
  • the cargo may be located within or associated with the lipid bilayer of the liposome via covalent bonding, electrostatic or hydrophobic interactions.
  • a cargo e.g. a hydrophobic drug
  • references herein to "encapsulation" (or similar) of one or more ingredients within the liposome includes references to liposomes in which some or all of the active pharmaceutical ingredients and imaging agents are covalently bound to said liposome. It is to be expected that while a substantial proportion of that active pharmaceutical ingredient or imaging agent will be encapsulated within the liposome, a proportion may not be encapsulated therein.
  • substances being contained “within” liposomes we include that substances may be wholly encapsulated within the liposome structure (e.g. within the lipid bilayer wall(s), or within a region that is enclosed within that lipid bilayer wall(s)).
  • the substance may be covalently bound to one or more constituents of the liposome particle, e.g. the lipid bilayer structure in the case of liposomes.
  • Substances that are covalently bound in this way may become incorporated into the endothelial cells at the BBB as a result of the receptor- mediated endocytic process by which the liposomes themselves become taken into those cells.
  • at least a significant proportion (e.g. at least 75% by weight) of the active pharmaceutical ingredient or imaging agent is encapsulated within (or covalently bound to) the liposomes.
  • encapsulation efficiency is highly dependent on the choice of the active pharmaceutical ingredient and imaging agent. For example, passively encapsulating small molecules (during formulation) would result in approximately a 5% encapsulation efficiency. If the pharmaceutical ingredient and imaging agent is attached to a phospholipid, 100% encapsulation efficiency may be expected.
  • the liposomes may be prepared to contain the desired cargo in a liposome- incorporated form.
  • the process of incorporation of a desired cargo into a liposome is often referred to as "loading".
  • the liposome-incorporated cargo may be completely or partially located in the interior space of the liposome, within the bilayer membrane of the liposome, or within the nanodomain of the liposome, or associated with the exterior surface of the liposome membrane.
  • the incorporation of cargo into liposomes is also referred to as encapsulation or entrapment, and these three terms are used herein interchangeably with the same meaning.
  • the intent of the liposomal encapsulation of cargo is often to protect the cargo from the destructive environment while providing an opportunity for the encapsulated cargo to exert its activity mostly at the site or in the environment where such activity is advantageous but less so in other sites where such activity may be useless or undesirable.
  • a drug substance within the liposome can be protected from the destruction by enzymes in the body, but become released from the liposome and provide treatment at the site of disease.
  • such liposomes can be prepared to include the desired cargo (i) with high loading efficiency, that is, high percent of encapsulated cargo relative to the amount taken into the encapsulation process; (ii) high amount of encapsulated cargo per unit of liposome bilayer material; (iii) at a high concentration of encapsulated cargo, and (iv) in a stable form, i.e., with little release (leakage) of an encapsulated cargo upon storage or generally before the liposome appears at the site or in the environment where the liposome-entrapped cargo is expected to exert its intended activity.
  • the liposomes described herein may further comprise a coat, such as a steric shield coating, or an inert polymer coating.
  • a coat such as a steric shield coating, or an inert polymer coating.
  • a common form of coating involves derivatisation of the liposome with polyethylene glycol (PEG) as a steric shield. See, for example, Immordino M. L, et al., International Journal of Nanomedicine (2006) 1 (3), 297- 315.
  • PEG polyethylene glycol
  • liposomes that are useful in the methods of the invention may have a coating, e.g. one comprising a plurality of PEG groups. Suitable molecular weights for PEG groups may range from 100 to 5000 PEG units.
  • the liposomes described herein may form part of a composition i.e. the liposomes may be in a composition with other components.
  • the composition may be a pharmaceutical composition containing the liposomes described herein, which compositions are suitable for use in direct administration to mammals, and especially humans.
  • pharmaceutical composition is intended to encompass compositions that include only components that are regarded in the art as suitable for administration to mammalian, and especially human, patients. Compositions that are suitable for administration to such subjects will be known to the person skilled in the art. In this respect, reference may be made to the Draft Guidance for Industry provided by the U.S. Department of Health and Human Services, Food and Drug Administration (Center for Drug Evaluation and Research (CDER)), entitled "Liposome Drug Products, Chemistry, Manufacturing, and Controls;
  • the term may refer to formulations in which drugs are encapsulated within and/or covalently bound to liposomes requiring reconstitution shortly prior to administration in order to avoid leakage of drugs from liposomes into an aqueous carrier.
  • the compositions are in the form of a liquid that is ready-to- use, directly from the shelf, and not a formulation in which drugs are encapsulated within and/or covalently bound to liposomes requiring reconstitution prior to administration.
  • a pharmaceutical composition may comprise a liposome described herein along with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
  • Compositions may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents or compounds.
  • Excipients are natural or synthetic substances formulated alongside a cargo (e.g. an active pharmaceutical ingredient) as described herein, included for the purpose of bulking-up the formulation or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption or solubility. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. Pharmaceutically acceptable excipients are well known in the art. A suitable excipient is therefore easily identifiable by one of ordinary skill in the art.
  • suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, and the like.
  • Adjuvants are pharmacological and/or immunological agents that modify the effect of other agents in a formulation.
  • Pharmaceutically acceptable adjuvants are well known in the art. A suitable adjuvant is therefore easily identifiable by one of ordinary skill in the art.
  • Diluents are diluting agents. Pharmaceutically acceptable diluents are well known in the art. A suitable diluent is therefore easily identifiable by one of ordinary skill in the art.
  • Carriers are non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation.
  • the term "carrier” does not refer to liposomes, but instead denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the carrier may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use.
  • Pharmaceutically acceptable carriers are well known in the art. A suitable carrier is therefore easily identifiable by one of ordinary skill in the art.
  • suitable pharmaceutical carriers may be found in, for example, Remington: The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995).
  • the pharmaceutically acceptable excipient, adjuvant, diluent or may be selected with due regard to the intended route of administration and standard pharmaceutical practice.
  • the composition may also comprise at least one of: a preservative, an antioxidant, a buffering agent (e.g. dextrose), an anionic polymer (e.g. dextran sulfate or heparin), or any combination thereof.
  • a buffering agent e.g. dextrose
  • an anionic polymer e.g. dextran sulfate or heparin
  • compositions may therefore comprise an antioxidant, such as ascorbic acid, butylated hydroxyanisole, butylated hydroxytoluene, citric acid, fumaric acid, malic acid,
  • an antioxidant such as ascorbic acid, butylated hydroxyanisole, butylated hydroxytoluene, citric acid, fumaric acid, malic acid,
  • antioxidants include butylated hydroxytoluene, ascorbic acid and butylated hydroxyanisole.
  • a chelating agent may also be used to reduce the metal ion catalysed oxidation of phospholipid and/or active ingredient(s).
  • useful chelating agents are ethylenediaminetetraacetic acid (EDTA) and salts thereof (e.g. sodium or potassium EDTA), ethylenediaminetriacetic acid and diethylenetriaminepentaacetic acid (DTPA). It is also possible to use other agents that protect the compositions described herein and, in particular, any unsaturated fatty acid residues that may be present therein, from oxidation.
  • Preferred chelating agents include EDTA and salts thereof.
  • a chelating agent such as DOTA may also be used to chelate radioactive metal ions for PET or SPECT imaging in patients.
  • DOTA can be covalently bound to a phospholipid and included in formulations (e.g. DOPE-DOTA), typically at 1 to 2 mol% of the total lipids.
  • compositions described herein can also comprise one or more preservatives.
  • preservatives for liquid pharmaceutical compositions are benzalkonium chloride, benzoic acid, butylated hydroxyanisole, butylparaben, chlorbutanol, ethylparaben, methylparaben, propylparaben, phenoxyethanol or phenylethyl alcohol.
  • Preferred preservatives include benzalkonium chloride.
  • Other preservatives that may be mentioned include sorbic acid.
  • Optional additives including preservatives, antioxidants, and chelating agents should be selected, in terms of their identity and the amounts employed, keeping in mind that their detrimental effect on liposome stability should be kept at a minimum. For a given agent this can be ascertained by simple experiments, which are well within the understanding of the skilled person. Suitable amounts of such ingredients are however in the range of about 0.01 mg/mL to about 10 mg/mL. It is preferred that the compositions that are useful in the methods of the invention contain at least one preservative, antioxidant, chelating agent, buffering agent and/or viscosity-increasing agent. Suitable amounts of any/all of these optional additives include from about 0.02 to about 5 (e.g. about 3) mg/mL (e.g. from about 0.1 to about 2 mg/mL).
  • the (pharmaceutical) composition is sterile and has a purity level of, for example, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%.
  • the (pharmaceutical) composition comprises a liposome described herein present in the composition in a concentration of about 0.5 mg/mL to about 30 mg/mL (e.g., about 1 mg/mL to about 20 mg/mL (e.g., about 1 mg/mL, about 2 mg/mL, about 3 mg/mL, about 4 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL, about 8 mg/mL, about 10 mg/mL, about 1 1 mg/mL, about 12 mg/mL, about 13 mg/mL, about
  • the concentration of the liposome is about 13 mg/mL. In some embodiments, the liposome is present in the composition in an amount of about 1 mg/mL to about 10 mg/mL, or about 5 mg/mL to about
  • a liposome and/or composition described herein may be used in therapy, or diagnostics such as for theranostics.
  • the liposomes and/or composition described herein may be used in the treatment of a brain disease in a subject in need thereof.
  • treatment includes the therapeutic treatment, as well as the symptomatic treatment, the prophylaxis, or the diagnosis, of a condition.
  • treat By the term “treat”, “treating”, or “treatment of” (and grammatical variations thereof) it is also meant that the severity of the patient's condition is reduced, at least partially improved or ameliorated and/or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved and/or there is a delay in the progression of the disease or disorder.
  • subject refers to an individual, e.g., a mammal such as a human, having or at risk of having a specified condition, disorder or symptom.
  • the subject may be a patient i.e. a subject in need of treatment in accordance with the invention.
  • the subject may have received treatment for the condition, disorder or symptom. Alternatively, the subject has not been treated prior to treatment in accordance with the present invention.
  • the brain disease may be selected from: Alzheimer's disease, Parkinson's disease, multiple sclerosis, brain cancer, brain tumour, amyotrophic lateral sclerosis (ALS), essential tremor, huntington's disease, Machado-Joseph disease, glaucoma, hereditary optic neuropathy (Leber), retinitis pigmentosa, meningitis, viral meningitis, inflammatory brain disease, psychotic disorder, narcolepsy, epilepsy/ seizure, and cranial nerve disorder.
  • ALS amyotrophic lateral sclerosis
  • Huntington's disease Machado-Joseph disease
  • glaucoma glaucoma
  • Hereditary optic neuropathy (Leber) retinitis pigmentosa
  • meningitis meningitis
  • viral meningitis inflammatory brain disease
  • psychotic disorder narcolepsy
  • epilepsy/ seizure and cranial nerve disorder.
  • Brain and central nervous system (CNS) cancers and tumors that may be targeted include astrocytomas (including cerebellar and cerebral), brain stem glioma, brain tumors, malignant gliomas, ependymoma, glioblastoma, medulloblastoma, supratentorial primitive
  • neuroectodermal tumors visual pathway and hypothalamic gliomas, primary central nervous system lymphoma, ependymoma, brain stem glioma, visual pathway and hypothalamic glioma, extracranial germ cell tumor, medulloblastoma, myelodysplasia syndromes, oligodendroglioma, myelodysplastic/myeloproliferative diseases, myelogenous leukemia, myeloid leukemia, multiple myeloma, myeloproliferative disorders, neuroblastoma, plasma cell neoplasm/multiple myeloma, central nervous system lymphoma, intrinsic brain tumors, astrocytic brain tumors, gliomas, and metastatic tumor cell invasion in the central nervous system.
  • the liposomal delivery system described herein can be used, in principle, with any active pharmaceutical ingredient for the treatment of brain diseases.
  • Non-limiting examples of brain diseases, with suitable medication are discussed below.
  • AD Alzheimer's Disease
  • Active pharmaceutical ingredients for use in the treatment of AD include: i) Acetylcholinesterase inhibitors (AChEI) such as donepezil, galantamine, rivastigmine, tacrine ii) NMDA receptor antagonists such as memantine.
  • AChEI Acetylcholinesterase inhibitors
  • NMDA receptor antagonists such as memantine.
  • Parkinson's Disease Active pharmaceutical ingredients for use in the treatment of
  • Parkinson's disease include: i) dopamine precursors such as Levodopa, and combinations of levodopa with: carbidopa, COMT inhibitors (for example entacapone and tolcapone), amantadine ii) dopamine agonists such as bromocriptine, pergolide, pramipexole, ropinirole, piribedil, cabergoline, apomorphine and lisuride iii) MAO-B inhibitors such as
  • Anticholinergics such as trihexyphenidyl and benzatropine.
  • MS Multiple sclerosis
  • Active pharmaceutical ingredients for use in the management of MS include: i) glucocorticoids ( example prednisolone, prednisone, dexamethasone),
  • interferons such as interferon beta- 1 a, interferon beta-1 b, and peginterferon beta-1a.
  • interferons such as interferon beta- 1 a, interferon beta-1 b, and peginterferon beta-1a.
  • Glatiramer acetate immunosuppressants (such as dimethyl fumarate fingolimod, teriflunomide, natalizumab, mycophenolate mofetil), azathioprine inteleucin inhibitors (daclizumab), other monoclonal antibodies (ocrelizumab, alemtuzumab), immunostimulants (glatiramer), rituximab; iii) others : valacyclovir, dalfampridine, cladribine, mitoxantrone, cyclophosphamide.
  • Brain tumors may include: Anaplastic Astrocytoma, Anaplastic Oligodendroglioma, Angioblastoma, Glioblastoma Multiforme, Malignant Glioma,
  • Suitable active pharmaceutical ingredients for use in the treatment and/or management of brain tumors are the following: i) temozolomide, procarbazine, lomustine, everolimus, cyclophosphamide, carmustine, methotrexate, cisplatin, vincristine, interferon alfa-2b, bevacizumab, hydroxyurea, bromocriptine, octreotide, pegvisomant, lanreotide and pasireotide.
  • antiproliferative/antineoplastic drugs and combinations thereof as used in medical oncology such as alkylating agents (for example cis-platin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulphan and
  • nitrosoureas for example antifolates such as fluoropyrimidines like 5- fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside, hydroxyurea and gemcitabine); antitumour antibiotics (for example anthracydines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and
  • antifolates such as fluoropyrimidines like 5- fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside, hydroxyurea and gemcitabine
  • antitumour antibiotics for example anthracydines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,
  • antimitotic agents for example vinca alkaloids like vincristine, vinblastine, vindesine and vinorelbine and taxoids like paclitaxel and taxotere
  • topoisomerase inhibitors for example epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan and camptothecins
  • cytostatic agents such as antioestrogens (for example tamoxifen, toremifene, raloxifene, droloxifene and iodoxyfene), oestrogen receptor down regulators (for example fulvestrant), antiandrogens (for example bicalutamide, flutamide, nilutamide and cyproterone acetate), LHRH antagonists or LHRH agonists (for example goserelin, leuprorelin and buserelin), progestogens (for example megestro
  • inhibitors of growth factor function include growth factor antibodies and growth factor receptor antibodies (e.g.
  • such inhibitors also include, for example, tyrosine kinase inhibitors, e.g., tyrosine kinase inhibitors, e.g.
  • inhibitors of the epidermal growth factor family such as EGFR family tyrosine kinase inhibitors including A/-(3-chloro-4-fluorophenyl)-7-methoxy-6- (3-morpholinopropoxy)quinazolin-4-amine (gefitinib, ZD 1839), /V-(3-ethynylphenyl)-6,7- bis(2-methoxyethoxy)quinazolin-4-amine (erlotinib, OSI-774) and 6-acrylamido-/V-(3-chloro- 4-fluorophenyl)-7-(3-morpholinopropoxy)quinazolin-4-amine (CI 1033) and erbB2 tyrosine kinase inhibitors such as lapatinib), inhibitors of the hepatocyte growth factor family, inhibitors of the platelet-derived growth factor family such as imatinib, inhibitors of serine/threonine kinases (
  • demethylating agents such as 5' azacytidine and decitabine (5-aza-2'-deoxycytidine, dezocitidine) and deacetylase inhibitors such as vorinostat (suberoylanilide hydroxamic acid, Zolinza) and depsipeptide (romidepsin, Istodax); (ix) bisphosphonates, such as clodronate, aledronate, zoledronic acid, ibandronate and pamidronate; (x) aminoglycosides, such as streptomycin, kanamycin, neomycin and gentamicin. Pharmaceutically-acceptable salts or solvates of any of these compounds are also included.
  • brain diseases include:
  • ALS Amyotrophic lateral sclerosis
  • Essential tremor (beta blockers: propranolol, nadolol and timolol are active pharmaceutical ingredients that may be used in the treatment of essential tremors).
  • Huntington's disease suitable active pharmaceutical ingredients for use in the treatment of Huntington's disease are: chorea reducing drugs, such as
  • achado-Joseph disease symptomatic therapy using antispasmodic drugs (baclofen) may be undertaken.
  • the Parkinsonian symptoms can be treated with levodopa therapy
  • Wilson Disease may be treated with copper chelators
  • Glaucoma active pharmaceutical ingredients that may be used in the treatment of Glaucoma include: i) prostaglandin analogs, such
  • latanoprost bimatoprost and travoprost.
  • topical beta-adrenergic receptor antagonists such as timolol, levobunolol, and betaxolol
  • alpha2-adrenergic agonists such as timolol, levobunolol, and betaxolol
  • brimonidine and apraclonidine iv miotic agents such as brimonidine and apraclonidine iv miotic agents (parasympathomimetics), such
  • pilocarpine v) acetylcholinesterase inhibitors are used in chronic glaucoma, such as echothiophate and vi) carbonic anhydrase inhibitors, such as dorzolamide, brinzolamide, and acetazolamide.
  • Retinitis pigmentosa (supplements such as Vitamin A, DHA, and Lutein can be used to delay the progression of retinitis pigmentosa).
  • Meningitis 1) Bacterial meningitis can be treated with cephalosporins (such as
  • Fungal meningitis can be treated with antifungal agents such as amphotericin B and flucytosine; Antifungal agents that may be useful in treating infections include: fluconazole, flucytosine, voriconazole, clotrimazole, econazole n
  • Lymphomatous meningitis cytosine arabinoside (ara-C); III) Viral meningitis (acyclovir is used to treat Herpes Simplex Encephalitis, the only viral meningitis which can be treated to this date).
  • Inflammatory brain diseases include salicylates (such as aspirin, diflunisal, salicylic acid or salsalate), propionic acid derivatives (such as ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin or loxoprofen), acetic acid derivatives (such as indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, aceclofenac, or nabumetone), enolic acid (Oxicam) derivatives (such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam or phenylbutazone (Bute)), anthranilic acid derivatives (such as mefenamic acid, meclotriptyl, or phenylbutazone (Bute)),
  • Steroidal anti-inflammatory agents include:
  • Psychotic disorders including severe anxiety, severe depression, Asperger syndrome, bipolar disorder, borderline personality disorder, insomnia, mania, obsessive compulsive disorder, paranoia, post-traumatic stress disorder, tic disorder, Tourette's syndrome, schizophrenia etc.
  • atypical antipsychotics such as quetiapine olanzapine, aripiprazole and brexpiprazole
  • miscellaneous antipsychotic agents examples are pimozide, lithium, molindone, loxapine and haloperidol
  • antipsychotics such as trifluoperazine, chlorpromazine, perphenazine, fluphenazine, prochlorperazine and thioridazine; iv) thioxanthenes such as thiothixene; Other
  • tranylcypromine fluoxetine mirtazapine, nefazodone, trimipramine, vortioxetine, vilazodone, protriptyline, sertraline
  • Narcolepsy central nervous system stimulants such as methylphenidate, amphetamine, dextroamphetamine and modafinil can be used.
  • eslicarbazepine acetate ethosuximide, gabapentin, lacosamide, lamotrigine, levetiracetam, nitrazepam, oxcarbazepine, perampanel, piracetam, phenobarbital, phenytoin, pregabalin, primidone, rufinamide, sodium valproate, stiripentol, tiagabine, topiramate, vigabatrin, zonisamide, valproate can be used)
  • Cranial nerve disorders (Cranial nerve disorders such as trigeminal neuralgia can be treated with carbamazepine (anticonvulsant), baclofen, lamotrigine, oxcarbazepine, phenytoin, gabapentin and pregabalin).
  • brain diseases with no specific treatment include: sinal muscular-atrophy, Progressive supranuclear palsy (Steele Richardson-Olszewski syndrome), multi-system atrophy, shy- Drager syndrome, spinocerebellar ataxia (SCA), lewy-body disease, Hallervorden-Spatz disease, Creutzfeld-Jakob-disease, Pick's disease (frontotemporal dementia), Friedreich ataxia, Stargardt disease (macular degeneration), Keams- Sayre syndrome,
  • liposomes and/or compositions described herein can be administered to the subject by any conventional route, including injection or by gradual infusion over time.
  • administration may, for example, be by infusion or by intramuscular, intravascular, intracavity, intracerebral, intralesional, rectal, subcutaneous, intradermal, epidural, intrathecal, percutaneous administration.
  • compositions comprising liposomes may be in any form suitable for the above modes of administration.
  • compositions comprising liposomes may be in form suitable for injection (e.g. i.v. injection).
  • suitable forms for parenteral injection including, subcutaneous, intramuscular, intravascular or infusion
  • suitable forms for topical administration include an ointment or cream
  • suitable forms for rectal administration include a suppository.
  • the route of administration may be by direct injection into the target area, or by regional delivery or by local delivery.
  • the identification of suitable dosages of the compositions of the invention is well within the routine capabilities of a person of skill in the art.
  • the liposomes and/or compositions described herein are for administration in an effective amount.
  • An "effective amount” is an amount that alone, or together with further doses, produces the desired (therapeutic or non-therapeutic) response.
  • the effective amount to be used will depend, for example, upon the therapeutic (or non-therapeutic) objectives, the route of administration, and the condition of the patient/subject.
  • the suitable dosage for a given patient/subject will be determined by the attending physician (or person administering the composition), taking into consideration various factors known to modify the action of the composition of the invention for example severity and type of brain malignancy, body weight, sex, diet, time and route of administration, other medications and other relevant clinical factors.
  • the dosages and schedules may be varied according to the particular condition, disorder or symptom the overall condition of the patient/subject. Effective dosages may be determined by either in vitro or in vivo methods.
  • the amount of the cargo (e.g. active pharmaceutical ingredient) that is administered to the patient may be less than 50% (by weight) of the standard dose. Preferably the amount is less than 40%, less than 30%, less than 20%. More preferably, the amount of the cargo (e.g. active pharmaceutical ingredient) that is administered to the patient is less than 10% (by weight) of the standard dose. Still more preferably, the amount of the cargo (e.g. active pharmaceutical ingredient) that is administered to the patient is less than 5%, such as less than 2% or less than 1 %, (by weight) of the standard dose. In preferred embodiments of the methods of the invention, the amount of the cargo (e.g. active pharmaceutical ingredient) that is
  • Standard dose refers to the dose that is considered (e.g. by medical professionals) to be routinely suitable for use in treating the disease from which the patient is suffering. Standard doses are detailed in national formularies and
  • pharmacopoeia for example the US pharmacopoeia, the British pharmacopoeia and the European pharmacopoeia, as well as other sources such as Martindale "The Complete Drug Reference", Pharmaceutical Press, 38th Edition, 2014, London, UK. Each of these documents is hereby incorporated by reference. Dosages can also be found in the Summary of Product Characteristics that is associated with the marketing authorisations that are awarded by the European Medicines Agency or by many national competent authorities. Additional information is also available from various web-based sources, including www.drugs.com. In one embodiment, the term "standard dose” refers to the dose specified in the European pharmacopoeia for the disease or condition that is to be treated.
  • a "standard dose” may refer to the amount of the cargo (e.g. active pharmaceutical ingredient (or a prodrug thereof)) that is to be administered to a patient over a particular period of time.
  • a cargo e.g. active pharmaceutical ingredient
  • a cargo may be administered periodically (e.g. in one, two three or four portions) over the course of a 24-hour period, a 48-hour period or longer.
  • a cargo/drug may also be
  • methods involving administering to a patient an amount of cargo/drug over a given period of time may administer less than 50% by weight (e.g. less than 40%, 30%, 20%, 10%, 5%) of the standard dose that is typically administered over the same duration.
  • the standard dose may vary depending on the nature of the disease or on the patient to be treated, e.g. where the dose is reduced for paediatric use or due to limited tolerance by the patient
  • these methods involve administering to a patient an amount of cargo/drug that may be less than 50% by weight (e.g. less than 40%, 30%, 20%, 10%, 5%) of the lowest dose that is typically administered for such a patient.
  • the dose to be administered may be less than 50% by weight (e.g. less than 40%, 30%, 20%, 10%, 5%) of the lowest of those accepted doses. That is, in a preferred embodiment, the methods of the invention involve administering to a patient an amount of cargo/drug that may be less than 50% by weight (e.g. less than 40%, 30%, 20%, 10%, 5%) of the lowest standard dose.
  • compositions of the present invention are advantageously presented in unit dosage form.
  • cargo e.g. active pharmaceutical ingredients
  • novel low-dose formulations may be prepared for use by medical professionals.
  • a unit dosage form comprising a liposome composition as defined herein, wherein the amount of the cargo (e.g. active pharmaceutical ingredient) that is present in the unit dosage form may be less than 50% by weight (e.g. less than 40%, 30%, 20%, 10%, 5%) of the standard dose for that cargo (e.g. active pharmaceutical ingredient).
  • the cargo used in said unit dosage form may be any of the cargoes disclosed herein.
  • the unit dosage form may comprise any of the cargoes (e.g. active pharmaceutical ingredients) mentioned herein, but may typically contain a quantity of at least one of those cargoes (e.g. active pharmaceutical ingredients) which is greatly reduced as compared to the standard doses specified in national formularies and pharmacopoeia, as described hereinbefore.
  • a particular cargo e.g. active pharmaceutical ingredient
  • the amount of cargo (e.g. active pharmaceutical ingredient) in the unit dose form is lower than the lowest of the known doses, preferably at or below 50% of that lowest dose.
  • the liposomes When the liposomes (or compositions comprising the liposomes) are (systemically (e.g. i.v)) administered to a subject, the liposomes selectively target to the endothelial cells that make up the blood brain barrier.
  • the proportion of liposomes that target to the BBB will increase as the time after (systemic (e.g. i.v.)) administration increases.
  • the liposomes may target rapidly to the BBB such that at least 10% by weight of the liposomes (% injection dose (i.e. % ID)) reach the BBB within two hours (e.g. within 1 hour) of systemic administration of the liposomes to the subject.
  • At least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% weight percent of liposomes (% ID) reach the BBB within 2 hours (e.g. within 1 hour) post i.v. injection.
  • selective targeting to the BBB, it is intended to include at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% weight percent of liposomes (% ID) reaching the BBB within 2 hours (e.g. within 1 hour, within 30 minutes, within 20 minutes, within 10 minutes) post i.v. injection.
  • An alternative means for measuring "selective targeting" to the BBB is by comparing the targeting selectivity for the brain endothelium (the BBB) over systemic endothelium.
  • endothelium is at least >3-fold, at least >5-fold, at least >6-fold, at least >7-fold, at least >8- fold, at least >9-fold, or at least >10-fold within a time period (e.g. within 2 hours (e.g. within 1 hour, within 30 minutes, within 20 minutes, within 10 minutes) post i.v. injection.
  • a time period e.g. within 2 hours (e.g. within 1 hour, within 30 minutes, within 20 minutes, within 10 minutes) post i.v. injection.
  • the liposomes may undergo different fates once they reach the BBB, depending on their exact composition e.g. 1) they remain associated with the brain endothelium and are neither taken up by endothelial cells or transcytosed into the CNS; 2) they are internalised by endothelial cells, via e.g. receptor- mediated endocytosis or fuse with endothelial cells or 3) they are transcytosed (receptor mediated) across the BBB and released into the CNS.
  • the liposomes may reach the BBB, where they remain associated with the brain endothelium, without being taken up by the endothelial cells, and without crossing the BBB itself (option 1 above).
  • This may be advantageous in diagnostic methods, where the cargo is an imaging agent, and localization at the BBB provides important information to a clinician. Over time, liposomes localized at the BBB may be degraded and begin to "leak" their cargo, and its release would be localized to the BBB region.
  • the liposomes described herein may also be taken up by a brain endothelial cell (option 2 above).
  • taken up it is meant that the liposome is drawn through the walls of the cell to reach the interior of the cell. This allows the liposome to transport substances contained therein (or covalently bound thereto) into the cell.
  • the substances that are to be delivered may potentially be capable of interfering with the regular working of the cell, e.g. by killing the cell, modulating its function or affecting its ability to regulate its growth.
  • the liposome may contain one or more anti-cancer agents in order to reduce the growth, progression or spread of cancer in a subject suffering therefrom.
  • the liposomes are used to deliver substances which are membrane impermeable, and so are not transported across the cell membrane (in significant quantities) in the absence of a liposome delivery mechanism.
  • the liposomes may be transcytosed across the BBB and released into the CNS (option 3 above).
  • Transcytosis is a type of transcellular transport in which various
  • dextran sulfate i.v. injection of dextran sulfate.
  • a subject to be treated with the liposomes described herein may therefore benefit from an additional (separate, simultaneous or sequential) treatment with dextran sulfate.
  • the dextran sulfate may be administered as a pre-treatment (i.e. before the liposomes are administered) or may be co-administered with the liposomes.
  • a person of skill in the art is well aware of conventional methods for administering dextran sulfate to a subject.
  • timings for dextran sulfate and liposome administration (wherein the timings must be such that the benefits of the dextran sulfate treatment are not lost e.g. because the time between the two treatments is too long)).
  • a method for delivering a cargo to the blood brain barrier of a subject comprising
  • administering an effective amount of a liposome or composition as described herein is also provided.
  • the methods described herein do not need to be limited to methods of treatment per se, and also encompass e.g. diagnostic and theranostic methods.
  • the method may deliver an imaging agent to the BBB of the subject.
  • imaging agents have been described elsewhere herein.
  • the methods described above for administration of the liposome/composition during methods of treatment apply equally to this aspect of the invention.
  • a liposome / composition comprising a liposome for delivering a cargo to an endothelial cell is also described.
  • This aspect may be useful for delivery of cargo to endothelial cells in tissue culture (e.g. for increasing cargo uptake into the culture cells, or for increasing the amount of liposome/cargo at close proximity to the endothelial cell surface during cell culture).
  • tissue culture e.g. for increasing cargo uptake into the culture cells, or for increasing the amount of liposome/cargo at close proximity to the endothelial cell surface during cell culture.
  • suitable cargo applies equally to this aspect of the invention.
  • a method of making a liposome comprising the steps of: i) selecting a first lipid, ii) selecting a second lipid which combines with the first lipid to make a liposome, wherein phase separation occurs between the first lipid and the second lipid in the liposome; and iii) combining the first lipid and the second lipid, and optionally a cargo, to make the liposome as defined herein.
  • the first lipid, second lipid and cargo are as defined elsewhere herein.
  • liposomes may be prepared by various methods from dry lipid films of the desired lipid composition. Common methods include hydration then sonication, hydration then extrusion, detergent depletion, freeze-dried rehydration and reverse-phase evaporation. Suitable methods are known to those skilled in the art and are described, for instance, in Chapter 1 of the book: 'Liposomes: Second Edition' Vladamir Torchilin, Volkmar Weissig, OUP, 2003, the relevant disclosures in which document are hereby incorporated by reference. Liposome formation may be performed at above about 0°C (e.g. room
  • phase transition temperature if the phase transition temperature of the acyl chains (chain melting; gel-to- liquid crystals) is below the freezing point of water.
  • Liposomes containing an active pharmaceutical ingredient or imaging agent may be prepared by drying a lipid stock solution to form a thin film of lipid, and then hydrating that film with an aqueous solution containing the active pharmaceutical ingredient or imaging agent. This is preferably carried out in the presence of suitable agitation (e.g. stirring).
  • suitable agitation e.g. stirring
  • Purified (e.g. distilled) water is typically used to form the aqueous solution containing an active pharmaceutical ingredient or imaging agent.
  • saline or a buffer solution may be used.
  • Solutions/liquids may be purged with nitrogen or argon at a suitable stage in the above process, if and as appropriate.
  • Homogenisation of the liposomal composition may also be required. Homogenisation methods include vigorous mechanical mixing or high speed homogenisation, for instance by means of an Ultra Turrax® (Jankel & K ihnke, Germany). Shaking, vortexing and rolling may also be performed as part of the homogenisation step of the above process.
  • a homogeneous size distribution of the liposomes that are useful in the methods of the invention may be desirable and may be obtained by extrusion through a membrane filter, such as one made of polycarbonate, with a pore size of about 100 nm.
  • a membrane filter such as one made of polycarbonate, with a pore size of about 100 nm.
  • Membrane filters e.g. Nuclepore Track-Etch membranes, Whatman
  • Membrane filters may be procured from Sigma- Aldrich. Whichever lipid substance (or combination thereof) is used, suitable total
  • amounts/concentrations of lipid(s) that may be employed in the liposomal compositions that are useful in the methods of the invention are in the range of about 1 mg/mL to about 120 mg/mL.
  • Liposomal compositions that may be mentioned include those in which, when the second lipid comprises phospholipid (whether in combination with another lipid or otherwise), the amount of phospholipid(s) in the composition is from about 1 (e.g. 10 about 1) mg/mL to about 120 mg/mL. Typical ranges that may be mentioned include from about 5 mg/mL to about 50 mg/mL. Further, the total amount of phospholipid (when the polar lipid comprises phospholipid) is preferably in the range from about 10 mg to about 50 mg.
  • compositions described herein may be administered intravenously, intramuscularly, subcutaneously, transdermal ⁇ , topically, or by any other parenteral route, in the form of a pharmaceutical preparation comprising the composition in a pharmaceutically acceptable form.
  • Preferred modes of delivery include intravenous, subcutaneous, and intramuscular delivery.
  • the liposomal compositions described herein may be formulated using an ethanol injection method.
  • DSPC and PAPAP lipids were dissolved in absolute ethanol at high concentrations (about 50 mM), and rapidly injected into a warm (45 °C) stirred aqueous solution (e.g. ddhbO or buffer) at a ratio of 1 :70, ethanol to aqueous solution.
  • aqueous solution e.g. ddhbO or buffer
  • Freeze-dried formulations may themselves be prepared according standard techniques that are known to those skilled in the art, and are advantageous as they allow for convenient, long term storage of the formulation in a form which can be readily converted into one that may be administered to the patient.
  • Such freeze-dried formulations comprise a liposome and either an active pharmaceutical ingredient or an imaging agent, each as defined herein.
  • the amount of that active pharmaceutical ingredient/imaging agent may be less than 50% by weight (e.g. less than 40%, 30%, 20%, 10%, 5%) of the standard dose for that active pharmaceutical ingredient/imaging agent.
  • Freeze-dried compositions are reconstituted prior to use in order for the composition to be provided in a form which is suitable for administration to a patient.
  • freeze- dried compositions may be achieved using standard techniques that are known to those skilled in the art.
  • a kit comprising a freeze-dried liposomal composition as defined herein together with instructions for reconstituting said freeze-dried composition and administering it to a patient.
  • the freeze- dried compositions described herein may contain additional components besides the liposomes, for example lyoprotectants. Suitable lyoprotectants would be known to the skilled person and include disaccharides such as sucrose, lactose and trehalose.
  • the liposome compositions described herein may also be supplied to medical professionals in the form of solutions containing the liposomes and the active pharmaceutical ingredient and/or imaging agent, and may be provided in a form which is suitable for administration directly to the subject (e.g. via intravenous injection).
  • a kit is also provided for preparing a liposome described herein.
  • the kit comprises a first lipid and a second lipid, both of which are defined elsewhere herein.
  • the first lipid and the second lipid may be in separate containers in the kit, or they may be in the same container.
  • the kit may further comprise instructions for a user, e.g. to how to make a liposome as described herein.
  • the kit further comprises a container containing a cargo as described elsewhere herein.
  • the container with the cargo may be the same container that contains the first lipid (and/or the second lipid) or it may be a separate container.
  • a kit is also provided for preparing a liposome comprising a cargo, as described elsewhere herein.
  • the kit may contain a container with a liposome of the present invention, and, optionally, a container containing a cargo, and/or instructions for a user, e.g.
  • the instructions can be provided via any medium, e.g., hard paper copy, electronic medium, or access to a database or website containing the instruction.
  • Liposomes are the most widely investigated nanoparticles for drug delivery applications.
  • the only targeted, liposomal drug delivery technologies approved for clinical use remains the use of long-circulating, doxorubicin-filled liposomes passively targeting solid tumors via the enhanced permeability and retention (EPR) effect - eg. Doxil or Myocet.
  • EPR enhanced permeability and retention
  • Targeting efficiencies of these liposomes are typically ⁇ 1 % of the total injected dose (%I.D).
  • Targeting therapies to the BBB (and often onwards into the brain) specifically, following systemic administration, is currently investigated through receptor mediated ligand targeting.
  • Tf transferrin
  • insulin or mimics of
  • angiopeptide and ApoE fragments targeting low-density lipoprotein, LDL receptors
  • glutathione targeting low-density lipoprotein, LDL receptors
  • None of these receptors are exclusively expressed at the BBB.
  • rabies virus glycoprotein More recently components of neurotropic viruses (e.g. rabies virus glycoprotein) have also been implemented.
  • the current state-of-the-art in the targeted treatment of brain cancer is bevacizumab - a monoclonal antibody targeting vascular endothelial growth factor (VEGF).
  • VEGF vascular endothelial growth factor
  • %ID percentages of injected dose reaching the brain are rarely quoted, rather indirect effects of drug delivery (eg. reduction in tumor size). Direct quantification as to the efficiency of targeting is therefore hard to gauge. However, listed are some examples where %ID within the brain (and inclusive of the brain endothelium) is directly quantified:
  • cTfRMAb a chimeric mAb that binds to the mouse TfR - 1.4% ID— 1 hpi (i.v.)
  • HIRMAb - a fully humanized mAb for the human insulin receptor - approx. 2% ID - 2hpi (i.v.)
  • Targeting therapies to the BBB (and often onwards into the brain), following systemic administration, is currently investigated by others through receptor mediated ligand targeting.
  • the most commonly cited ligands to achieve this are transferrin (Tf) (or mimics of), insulin (or mimics of), angiopeptide and ApoE fragments (targeting low-density lipoprotein, LDL receptors) and glutathione, each targeting their respective named receptors. None of these receptors are exclusively expressed at the BBB.
  • the inventors have synthesized and tested many of the common ligands used to target the BBB in the embryonic zebrafish model (see Figure 2).
  • liposomes functionalized with these ligands accurately target organs in which their target receptors are highly expressed but none significantly accumulate at the brain endothelium.
  • a novel mechanism is required to selectively target drugs and other cargo such as imaging agents to the BBB.
  • the inventors have surprisingly identified specific lipids, which when mixed together with a naturally occurring phospholipid, such as DSPC, and formulated into approximately 100nm liposomes, result in selective accumulation of the liposomes at the BBB of embryonic zebrafish following systemic (i.v.) administration (see Figure 1).
  • the targeting selectivity for the brain endothelium (the BBB) over systemic endothelium is >10-fold.
  • An approximate quantification of the targeting efficiency of the described liposomes to the BBB is 50% I.D. at 2 hours post-injection in the embryonic fish.
  • the zebrafish genome is 70% homologous to humans and crucially brain morphology, organization and expression of key markers for BBB function and integrity is conserved these species.
  • the inventors have also demonstrated successful encapsulation of small molecule drugs (eg. the cytotoxic drug, doxorubicin) as well as larger cargoes (eg, Au nanoparticles) into these novel liposomes.
  • small molecule drugs eg. the cytotoxic drug, doxorubicin
  • cargoes eg, Au nanoparticles
  • lipid reagents can be purchased from Avanti Polar Lipids (Alabaster, AL, US).
  • 1 ,2-dioleoyl-sn-glycero-3-phosphocholine DOPC
  • DSPC 1,2-distearoyl-sn-glycero-3- phosphocholine
  • DOPG 1,2-dioleoyl-s/7-glycero-3-phospho-(1 '-rac-glycerol)
  • DOPG 1,2- distearoyl-sn-glycero-3-phospho-(1 '-rac-glycerol)
  • DOPS 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine
  • Rhodamine-PE 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)
  • Rhodamine-PE phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)
  • DOPC-PE phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)
  • DOPC and DSPC were purchased from Lipoid GmbH.
  • Additional POPC and cholesterol was purchased from Sigma-Aldrich.
  • Dextran sulfate (40kDa) was purchased from Sigma-Aldrich.
  • Doxorubicin hydrochloride (DOX.HCI) was purchased from Cayman
  • DLS For DLS, measurements were carried out at room temperature in ddH20 at a total lipid concentration of 100 ⁇ .
  • liposome solutions were first diluted in salt (NaCI) solution. Zeta potentials were measured at room temperature, at 500pM total lipid concentration and 10mM NaCI concentration. All reported DLS measurements and zeta potentials are the average of three measurements.
  • Size exclusion chromatography was carried out using illustraTM NAPTM SephadexTM G-25 DNA grade pre-made columns (GE Healthcare) and used according to the user instructions.
  • UV absorption spectra were measured using a Cary 3 Bio UV-vis spectrometer, scanning from 250nm to 600nm for doxorubicin and at 1 nm intervals. Scan rate: 120 nm/min.
  • Confocal microscopy Confocal z-stacks were captured on a Leica TCS SPE confocal microscope, using a 10x air objective (HCX PL FLUOTAR) or a 40x water-immersion objective (HCX APO L). For whole-embryo views, 3-5 overlapping z-stacks were captured to cover the complete embryo. Laser intensity, gain and offset settings were identical between stacks and sessions. Images were processed and quantified using the Fiji distribution of I mage J.
  • cryoEM was performed by using a CryoTitan (FEI Corp, Hillsboro, OR) operating at 300 kV and equipped with a field emission gun (FEG).
  • Cryo-samples were prepared from a 3 pL droplet of sample solution (5mM liposome as prepared above) placed on the grid inside the VitrobotTM chamber at 100% relative humidity and 20°C. Prior to use the TEM grids were glow discharged by a Cressington 208 carbon coater to render them hydrophilic. The samples were blotted to remove excess solution and vitrified by using an automated vitrification robot (VitrobotTM Mark III, FEI Corp).
  • Zebrafish husbandry and kdrl:GFP transgenic lines Zebrafish (Danio rerio, strain AB/TL) were maintained and handled according to the guidelines from the Zebrafish Model
  • Organism Database http://zfin.org
  • Fertilization was performed by natural spawning at the beginning of the light period and eggs were raised at 28.5°C in egg water (60 ug/ml Instant Ocean sea salts).
  • Tg(kdrl:GFP) s843 Jin, S.-W. Cellular and molecular analyses of vascular tube and lumen formation in zebrafish. Development 132, 5199-5209 (2005)).
  • EXAMPLE 1 Synthesis of PAPAP3 To a round bottom flask (100ml_) containing stirred solution of ( ⁇ )-3-Amino-1 ,2-propanediol (100mg, 1.1 mmol_, 1eq) in CH2CI2 ( ⁇ 15ml_), Oleic Acid (621.5mg, 2.2mmoL, 2eq), EDCI (527.1 mg, 2.75mmol_, 2.5eq), DMAP (336mg, 2.75mmoL, 2.5eq), DIPEA (479pL,
  • Liposomes (without encapsulated drugs) were formulated in ddH ⁇ 0 at a total lipid
  • lipids as stock solutions (1-10mM) in chloroform, were combined at the desired molar ratios and dried to a film, first under a stream of N2 then >1 h under vacuum.
  • Lipid films were hydrated in 1 mL ddH20 at >65°C (with gentle vortexing if necessary) to form a multilamellar vesicle solution.
  • Large unilamellar vesicles were formed through extrusion at >65°C (Mini-extruder with heating block, Avanti Polar Lipids, Alabaster, US).
  • PC polycarbonate
  • Lipid films (12.5mM total lipids) were hydrated with an aqueous solution of doxorubicin (5mg/mL, 8.6mM) at >65°C. Unilamellar liposomes were subsequently prepared with extrusion as stated above. Size exclusion chromatography was used as a method to separate liposomes from free doxorubicin. Triton 1 % v/v was used to disassemble the liposomes and the amount of encapsulated doxorubicin measured by absorbance of the solution at 495nm and fitting to a pre-determined calibration curve.
  • the final encapsulated concentration of doxorubicin in DSPC:PAPAP3 liposomes was 55pgmL (5.6% e.e.). Encapsulation efficiency could be increased to 1 1 % if the hydrated lipid mixture + doxorubicin (ie. prior to extrusion) was lyophilised, then rehydrated and extruded.
  • Cargo may also be loaded using active loading methods known in the art. Full details of such methods can be found in Fritze, A., Hens, F., Kimpfler, A., Schubert, R., & Peschka-Suss, R. (2006). Remote loading of doxorubicin into liposomes driven by a transmembrane phosphate gradient. Biochimica et Biophysica Acta (BBA)-Biomembranes, 7758(10), 1633-1640.
  • Lipid films (12.5mM total lipid) were hydrated with 1 mL of sodium citrate (10.2mM) at 85°C. The sample was then lyophilized and the formed powder was subsequently hydrated with 1 mL of aq. sol of HAuCL (2.5mM).
  • Liposomal formulations were injected into 2-day old zebrafish embryos (52-56hpf) using a modified microangraphy protocol (Weinstein, B. M., Stemple, D. L., Driever, W. & Fishman, M. C. Gridlock, a localized heritable vascular patterning defect in the zebrafish. Nat Med 1 , 1 143-1147 (1995)). Embryos were anesthetized in 0.01 % tricaine and embedded in 0.4% agarose containing tricaine before injection. To improve reproducibility of microangiography experiments, 1 nl volumes were calibrated and injected into the sinus venosus/duct of Cuvier.
  • a small injection space was created by penetrating the skin with the injection needle and gently pulling the needle back, thereby creating a small pyramidal space in which the liposomes and polymers were injected.
  • Successfully injected embryos were identified through the backward translocation of venous erythrocytes and the absence of damage to the yolk ball, which would reduce the amount of liposomes in circulation.
  • all PAPAPs were formulated 1 : 1 with DSPC, in addition to 45:45: 10 with PAPAP14:DPPC:cholesterol. All images show liposome associated fluorescence (B/W). Targeting to the BBB is shown in these 2 day-old fish for PAPAP3, PAPAP4, comparative example PAPAP5, PAPAP7, PAPAP1 1 and PAPAP14 containing liposomes, to varying extents.
  • comparative example PAPAP6 do not target the BBB (comparative example PAPAP6 also does not form stable liposomes).
  • Di-ester variant (PAPAP 4) shows some binding at the BBB.
  • Phosphate analogue (comparative example PAPAP 5) demonstrates a strong off- target to venous endothelial cells (almost certainly due to the negative charge) but also binds at the BBB (see Figure 5).
  • Saturated FA variant shows some binding at the BBB (see Figure 5), however DSPC:PAPAP7 (1 : 1 ) liposomes cannot be consistently formulated.
  • Figure 5 highlights comparative example PAPAP5 binding to venous endothelial cells. This is evident from what appears as a 'marbling' effect of liposome binding within these blood vessels (and continues through the trunk of the fish). This binding corresponds to an off- target interaction with endothelial cells primarily in the mammalian liver and spleen (ie. the RES - reticuloendothelial cell system).
  • Figure 17 shows that BBB targeting is evident with dichain variant PAPAP1 1 (C24:1) when formulated 1 : 1 with DSPC.
  • Figure 18 also shows that BBB targeting is evident with dichain variant PAPAP3 when formulated 45:45:10 with PAPAP3:DSPC:cholesterol.
  • the inventors have shown that different regioisomers (and a mixture of regioisomers) of PAPAP3 can be used with an appropriate co-formulant to generate liposomes that target to the BBB.
  • an 80:20 ratio of PAPAP3 regioisomers is routinely used herein and is shown to target to the BBB effectively.
  • the inventors have tested >90% pure 1° regioisomer of PAPAP3 and this also targets the BBB effectively (data not shown).
  • the data shown herein illustrates that a number of different lipid combinations may be used. For example, from the data shown herein, it appears that, for the "first lipid” (e.g. PAPAP), a di-chain is necessary (and a tri-chain is ineffective).
  • first lipid e.g. PAPAP
  • a di-chain is necessary (and a tri-chain is ineffective).
  • Second lipid herein
  • PAPAP first lipid
  • the inventors also tested liposome formations using different molar ratios of the two lipids.
  • the molar ratios of DSPC:PAPAP3 were varied as described below. It was found that liposomes generated from a 3: 1 molar ratio of DSPC:PAPAP3 (ie. 25% PAPAP3) accumulated at the BBB (see Figure 7). It was also found that liposomes generated from a 9: 1 molar ratio of DSPC:PAPAP3 (i.e. 10% PAPAP3) also accumulate at the BBB (see Figure 8). Conversely, the inventors found it difficult to successfully formulate liposomes using a 1 :3 molar ratio of DSPC:PAPAP3 (i.e. 75% PAPAP3) (data not shown). These data show an upper limit of PAPAP content of 75%, with an as yet undetermined lower limit of PAPAP.
  • a range of molar ratios of co-formulant: PAPAP may be used, from between 9:1 to 1 :3, preferably from 3: 1 to 1 :1 , with the most preferable ratio being 1 : 1.
  • second lipid A number of different co-formulant lipids (described herein as "second lipid”) were also tested. In these experiments, all liposomes formulated 1 :1 with PAPAP3 or PAPAP14. All images show liposome-associated fluorescence (B/W). Whole fish images are included to illustrate vein/macrophage targeting. Use of DSPG (C18:0, saturated, anionic) in a 1 : 1 ratio with PAPAP3 generated liposomes with low level binding at the BBB and mild off-target interaction with veins. Usually freely circulating (see Figure 9).
  • DOPS C18: 1 , unsaturated, anionic
  • DMPC C14:0, saturated, zwitterionic
  • the phase transition temperature of DMPC (below which the bilayer is rigid (gel state) and above which it is fluid (liquid crystalline) is 28°C.
  • the experimental temperature is 28°C.
  • DOPC DOPC
  • DPPC saturated, zwitterionic
  • second lipid different co-formulant lipids (described herein as "second lipid”) may be used.
  • the co-formulant lipid may be any (phospho)lipid with a phase transition temperature above 37°C, ideally with a zwitterionic headgroup. Most ideally, the co-formulant is phosphatidylcholine.
  • the inventors have therefore identified a number of different variations on the liposome formulation that will selectively target to the BBB, with different levels of efficiency/off target effects.
  • the optimal formulation tested is DSPC:PAPAP3.
  • the accumulation at the BBB for this formulation is very, very apparent compared to the other formulations tested.

Abstract

L'invention concerne de nouveaux liposomes et des compositions de liposomes. La présente invention concerne également des kits et des procédés de préparation de liposomes. Les liposomes constituent un moyen utile pour l'administration sélective d'une cargaison telle qu'un ingrédient pharmaceutique actif ou un agent d'imagerie à la barrière hématoencéphalique (BHE) d'un sujet. Les liposomes peuvent être utilisés à des fins thérapeutiques, diagnostiques ou théranostiques.
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CN112138166B (zh) * 2020-09-27 2022-11-25 中国人民解放军军事科学院军事医学研究院 一种纳米药物、其制备方法及用途

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