US20110250266A1 - Liposomal systems comprising sphingomyelin - Google Patents

Liposomal systems comprising sphingomyelin Download PDF

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US20110250266A1
US20110250266A1 US13/123,112 US200913123112A US2011250266A1 US 20110250266 A1 US20110250266 A1 US 20110250266A1 US 200913123112 A US200913123112 A US 200913123112A US 2011250266 A1 US2011250266 A1 US 2011250266A1
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liposomes
spm
bup
liposomal system
lmvv
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Yechezkel Barenholz
Rivka Cohen
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Yissum Research Development Co of Hebrew University of Jerusalem
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Yissum Research Development Co of Hebrew University of Jerusalem
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P23/00—Anaesthetics
    • A61P23/02—Local anaesthetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • This invention relates to the field of liposome technology.
  • liposomes are used as carriers of drugs for delivery via a plurality of mechanisms.
  • various types of liposomes are used, from small unilamellar vesicles (SUV), large unilamellar vesicles (LUV), multilamellar vesicles (MLV), multivesicular vesicles (MVV), large multivesicular vesicles (LMVV, also referred to, at times, by the term giant multivesicular vesicles, “GMV”), oligolamellar vesicles (OLV), and others.
  • SUV small unilamellar vesicles
  • LUV multilamellar vesicles
  • MVV multivesicular vesicles
  • LMVV large multivesicular vesicles
  • GMV giant multivesicular vesicles
  • OSV oligolamellar vesicles
  • LMVV are somewhat different from unilamellar vesicles of various sizes and of the “onion like” MLV structure.
  • the amount of aqueous medium forming the aqueous phase per the amount of lipid is greater than that in MLV, this potentially allowing higher amount of drug to be loaded into the aqueous phase, namely, higher drug to lipid mole ratio in the LMVV when compared to MLV system of similar size distribution.
  • This difference was exemplified by Grant et al. 2004 [Anesthesiology 101(0:133-7, 2004] and in U.S. Pat. No. 6,162,462. It has been found that the difference in structure between MLV an LMVV not only allows higher loading of the drug into the liposomes but also a prolonged release of the drug from the LMVV system.
  • U.S. Pat. No. 6,162,462 discloses liposomal bupivacaine compositions in which the bupivacaine is loaded by a transmembrane ammonium sulfate gradient, the liposomes being giant multivesicular vesicles (GMV, a synonym for LMVV) having a molar ratio of encapsulated drug to lipid in said liposomal composition of at least 1.0.
  • GMV giant multivesicular vesicles
  • a specific drug encapsulated in the liposomes of U.S. Pat. No. 6,162,462 is the amphipathic analgesic drug bupivacaine (BUP).
  • the present disclosure is based on the finding that large multivesicular vesicles (LMVV) loaded with high amount of an amphipathic drug (bupivacaine, BUP) can be stabilized, in terms of reduced BUP leakage, if the liposomes' membranes comprise sphingomyelin and the LMVV are within an aqueous medium being in an iso-osmotic equilibrium with the intraliposomal aqueous medium.
  • LMVV large multivesicular vesicles
  • BUP amphipathic drug
  • the present disclosure provides, in accordance with a first of its aspects a liposomal system comprising an aqueous medium having dispersed therein liposomes encapsulating in their intraliposomal aqueous compartment at least one active agent, the aqueous medium being in iso-osmotic equilibrium with said intraliposomal aqueous compartment, the liposomes having a membrane comprising a liposome forming lipids, at least one of which being sphingomyelin (SPM), the liposomal system having increased stability as compared to the same liposomes free of SPM (namely, where there is no SPM in the liposome forming membrane).
  • the liposomal system is stable during long-term storage, said stability being characterized in that no more than 30% of the at least one active agent is present in the aqueous medium after said storage.
  • the present disclosure also provides, in accordance with a second of its aspects, a method for storage of liposomes encapsulating in their intraliposomal aqueous compartment at least one active agent, the liposomes having a membrane comprising liposome forming lipids, at least one liposome forming lipid being sphingomyelin (SPM), the method comprising forming a liposomal system where said liposomes are dispersed in an aqueous medium being in an iso-osmotic equilibrium with the intraliposomal aqueous compartment of said liposomes and storing said liposomal system, the liposomal system having increased stability as compared to the same liposomes free of SPM.
  • SPM sphingomyelin
  • a liposomal system as defined herein, for the preparation of a pharmaceutical or diagnostic composition; as well as the liposomal system as defined for use in the treatment of a medical condition or for the diagnostic of a medical condition.
  • an aspect of the present disclosure provides a pharmaceutical or diagnostic composition
  • a pharmaceutical or diagnostic composition comprising the liposomal system as defined herein and at least one physiologically acceptable carrier.
  • the present disclosure provides a method of treating or diagnosing of a medical condition comprising administering to a subject an amount of the liposomal system as defined herein.
  • the active agent is an amphipathic compound, being loaded into the liposomes by remote loading technique;
  • the SPM is synthetic or semi-synthetic C16 or C18 SPM and the liposomes are large multivesicular vesicles (LMVV).
  • a particular liposomal system in accordance with the present disclosure comprises LMVV formed from a combination of at least hydrogenated soy phosphatidylcholine (HSPC), C16SPM, cholesterol and encapsulating BUP.
  • HSPC soy phosphatidylcholine
  • C16SPM cholesterol
  • encapsulating BUP a particular liposomal system in accordance with the present disclosure
  • FIGS. 1A-1B are graphs showing the release of Bupivacaine (BUP), during storage at 4° C. ( FIG. 1A ) or at 37° C. ( FIG. 1B ), from large multivesicular vesicles (LMVV) of different lipid compositions (BUP to phospholipid mole ratio of each is given) which have been loaded with BUP using remote loading driven by trans-membrane ammonium sulphate (AS) gradient.
  • BUP Bupivacaine
  • LMVV large multivesicular vesicles
  • FIGS. 2A-2B are graphs showing the release of Bupivacaine (BUP), during storage at 4° C. ( FIG. 2A ) or at 37° C. ( FIG. 2B ), from large multivesicular vesicles (LMVV) of different lipid compositions (BUP to phospholipid mole ratio of each is given) which have been loaded with BUP using remote loading driven by trans-membrane calcium acetate (CA) gradient.
  • BUP Bupivacaine
  • LMVV large multivesicular vesicles
  • FIGS. 3A-3B are graphs showing the release of Bupivacaine (BUP), during storage at 4° C. ( FIG. 3A ) or at 37° C. ( FIG. 3B ), from LMVV of different lipid compositions (HSPC/CHOL 6/4 mole ratio; HSPC/C16SPM/CHOL 3/3/4 mole ratio; and HSPC100/CHOL 6/4 mole ratio, BUP to phospholipid mole ratio of each composition is given) which have been loaded with BUP using the passive loading approach.
  • BUP Bupivacaine
  • FIGS. 4A-4C are graphs showing the duration of analgesia in mice using various liposomal systems identified in Table 8 as formulations 1 to 8 (identified in the Figures with in the corresponding formulation number “x” as “lip x”), FIG. 4A showing the effect of injected volume of liposomal BUP or in free form, the amount of BUP being constant 6 mg/mouse; FIG. 4B showing the effect of 5 different LMVV formulations, the amount of BUP being constant 3 mg; and FIG. 4C which describes a comparison of the eight different LMVV formulations (Table 8) at a dose of 3 mg/mouse.
  • FIGS. 5A-5F are graphs comparing analgesia duration of two different doses of BUP (3 mg/mouse and 6 mg/mouse) for the five different LMVV formulations identified in Table 8 (“lip x” in FIGS. 5A-5E ) and 2 different amounts (0.375 and 0.75 mg/mouse) of non-encapsulated (free) BUP (in FIG. 5F );
  • FIG. 5A comparing the effect of lip 2 (3 and 6 mg BUP/mouse
  • FIG. 5B comparing the effect of lip 3 (3 and 6 mg BUP/mouse)
  • FIG. 5C comparing the effect of lip 4 (3 and 4.5 mg BUP/mouse
  • FIG. 5D comparing the effect of lip 5 (3 and 6 mg BUP), FIG.
  • FIG. 5E comparing the effect of lip 8 (3 and 6 mg BUP/mouse), and FIG. 5F comparing the effect of free (non liposomal) BUP at 0.375 mg/mouse using two volumes (150 and 300 ⁇ l) and 0.75 mg/mouse at a volume of 150 ⁇ l.
  • FIG. 6 is a graph showing in vivo analgesia after 20 hours of LMVV comprising HSPC:C16SPM:cholesterol [3/3/4] 3 mg BUP and LMVV as described by Grant et al. 2004 and free BUP 0.75 mg/mouse (the maximal tolerated dose, MTD).
  • FIG. 7 is a graph showing in vivo analgesia after 40 hours of LMVV comprising HSPC:C16SPM:cholesterol [3/3/4] 3 mg BUP LMVV and free BUP 0.75 mg/mouse (the maximal tolerated dose, MTD).
  • FIGS. 8A-8E are graphs comparing the change in level of free bupivacaine (as % in storage medium) during the indicated storage period, at 4° C. of HSPC100/C16SPM/CHOL (3/3/4 mole ratio) LMVV loaded with bupivacaine via the AS trans-membrane as is when stored in various storage media (Saline, 0.5% BUP or 2.0% BUP).
  • the present invention is based on the understanding that existing bupivacaine liposomal formulations such as those described in U.S. Pat. No. 6,162,462, and Grant et al. (Grant et al. 2004, ibid.) have a tendency to leak during long term storage at low temperatures which may impose a risk of toxicity when administered to subjects in need of the drug.
  • These bupivacaine liposomal formulations contained high drug to phospholipid ratio (>0.5 mole/mole) in large multivesicular vesicle (LMVV, referred to in U.S. Pat. No.
  • liposomes comprising in the liposome's bilayer sphingomyelin at the amount of up to 75% of the total phospholipids (or 50% of total lipids (which include ⁇ 33 mole % cholesterol) forming the liposome's bilayer decreased the amount of leakage without compromising the rate of bupivacaine release from the liposomes at 37° C. and without compromising the high loading of the drug into the liposomes.
  • the present disclosure provides a liposomal system comprising an aqueous medium having dispersed therein liposomes encapsulating in their intraliposomal aqueous compartment at least one active agent, the aqueous medium being in iso-osmotic equilibrium with said intraliposomal aqueous compartment, the liposomes having a membrane comprising liposome forming lipids, at least one of which being a sphingomyelin (SPM), the liposomal system being stable.
  • SPM sphingomyelin
  • the stability of the SPM containing liposomes is significantly greater than that of liposomes which do not contain SPM in their lipid membrane.
  • the stability of the liposomal system is also determined in terms of long-term storage, the stability being characterized in that no more than 30%, at times, not more than 20% and even not more than 10% of the at least one active agent of the system is present in the aqueous medium after said storage.
  • liposomal system denotes a system comprising an organized collection of lipids forming at least one type of liposomes, and enclosing at least one intraliposomal aqueous compartment.
  • the system comprises an aqueous medium in which the liposomes are dispersed or suspended.
  • the aqueous medium is any water based buffer solution having a desired osmolarity and ion concentration and is to be understood as encompassing a variety of physiologically acceptable buffers.
  • the buffer system is generally a mixture of a weak acid and a soluble salt thereof, e. g., sodium citrate/citric acid; or the monocation or dication salt of a dibasic acid, e. g., potassium hydrogen tartrate; sodium hydrogen tartrate, phosphoric acid/potassium dihydrogen phosphate, and phosphoric acid/disodium hydrogen phosphate.
  • a weak acid buffer is a buffer solution with constant pH values of between 4 and 7 and a weak base buffer is a buffer solution with constant pH values between 7 and 10.
  • the aqueous medium comprises an amount of free active agent
  • the presence of said free active agent in the aqueous medium allows or participates in the formation of said iso-osmotic equilibrium.
  • the amount of free active agent is determined such to form said iso-osmotic equilibrium.
  • the presence of the free agent in the aqueous medium also reduced the leakage of eth agent from the liposomes (this being comparable the same formulation without free drug in the aqueous medium).
  • dispersed liposomes In the aqueous medium are dispersed liposomes.
  • the term “dispersed” is used to denote the distribution or suspension of the liposomes in the aqueous medium.
  • liposomes are comprises of a lipid bilayer comprising liposome forming lipids, discussed hereinbelow, and an aqueous intraliposomal core.
  • the aqueous medium external to the liposomes and the intraliposomal aqueous compartment are in iso-osmotic equilibrium.
  • the iso-osmotic equilibrium should be understood as meaning that the aqueous medium and the medium of the intraliposomal aqueous compartment have similar osmolarities, the similarity being defined by a difference in osmolarity of not more than 50 mOsmole.
  • the osmolarity of the aqueous medium and of the liposomal aqueous phase are in the range of about 50 to about 600 mOsm/kg, or even between about 250 to about 550 mOsm/kg.
  • the iso-osmotic equilibrium may be obtained by washing the liposomes encapsulating the active agent with the buffer solution having an osmolarity similar to that of the intraliposomal aqueous compartment. Specifically, once the active agent is loaded into the liposomes, the non-encapsulated agent may be washed out by the selected buffer solution.
  • the liposomes' membrane is a bilayer membrane and may be prepared to include a variety of physiologically acceptable liposome forming lipids.
  • liposome forming lipids is used to denote primarily glycerophospholipids and sphingomyelins.
  • the glycerophospholipids have a glycerol backbone wherein at least one, preferably two, of the hydroxyl groups at the head group is substituted by one or two of an acyl, alkyl or alkenyl chain, a phosphate group, or combination of any of the above, and/or derivatives of same and may contain a chemically reactive group (such as an amine, acid, ester, aldehyde or alcohol) at the head group, thereby providing the lipid with a polar head group.
  • a chemically reactive group such as an amine, acid, ester, aldehyde or alcohol
  • the sphingomyelins consists of a ceramide unit with a phosphorylcholine moiety attached to position 1 and thus in fact is an N-acyl sphingosine
  • the phosphocholine moiety in sphingomyelin contributes the polar head group of the sphingomyelin.
  • the acyl chain(s) are typically between 14 to about 24 carbon atoms in length, and have varying degrees of saturation being fully, partially or non-hydrogenated lipids.
  • the lipid matrix may be of natural source, semi-synthetic or fully synthetic lipid, and neutral, negatively or positively charged.
  • liposome forming glycerophospholipids include, without being limited thereto, glycerophospholipid.
  • phosphatidylglycerols including dimyristoyl phosphatidylglycerol (DMPG); phosphatidylcholine (PC), including egg yolk phosphatidylcholine, dimyristoyl phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoylphosphatidyl choline (POPC), hydrogenated soy phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC); phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylserine (PS).
  • PG phosphatidylglycerols
  • DMPG dimyristoyl phosphatidylglycerol
  • PC phosphatidylcholine
  • POPC 1-palmitoyl-2-oleo
  • the liposome forming lipids may also include cationic lipids (monocationic or polycationic lipids).
  • Cationic lipids typically consist of a lipophilic moiety, such as a sterol or the same glycerol backbone to which two acyl or two alkyl, or one acyl and one alkyl chain contribute the hydrophobic region of the amphipathic molecule, to form a lipid having an overall net positive charge.
  • the headgroup of the lipid carries the positive charge.
  • Monocationic lipids may include, for example, 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP) 1,2-dioleyloxy-3-(trimethylamino) propane (DOTAP); N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE); N-[1-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethyl-ammonium bromide (DORIE); N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA); 3 ⁇ [N-(N′,N′-dimethylaminoethane) carbamoly] cholesterol (DC-Chol); and dimethyl-dioctadecylammonium (DDAB).
  • DMTAP 1,2-dimyristoy
  • Polycationic lipids may include a similar lipophilic moiety as with the mono cationic lipids, to which spermine or spermidine is attached. These include, without being limited thereto, N-[2-[[2,5-bis[3-aminopropyl)amino]-1-oxopentyl]amino]ethyl]-N,N-dimethyl-2,3-bis[(1-oxo-9-octadecenyl)oxy]-1-propanaminium (DOSPA), and ceramide carbamoyl spermine (CCS).
  • DOSPA 1-oxo-9-octadecenyl
  • the cationic lipids may form part of a derivatized phospholipids such as the neutral lipid dioleoylphosphatidyl ethanolamine (DOPE) derivatized with polylysine to form a cationic lipopolymer.
  • DOPE neutral lipid dioleoylphosphatidyl ethanolamine
  • the liposome forming lipid comprises at least a sphingomyelin.
  • sphingomyelin or “SPM” as used herein denotes any N-acetyl sphingosine conjugated to a phosphocholine group, the later forming the polar head group of the sphingomyelin (N-acetylsphingosylphosphorylcholines).
  • the acyl chain bound to the primary amino group of the sphingosine may be saturated or unsaturated, branched or unbranded. In one embodiment, the acyl chain comprises between 12 to 24 carbon atoms (C12-C24), at times between 14 to 20 carbon atoms.
  • the SPM is a C16:0 or C18:0 sphingomyelin, namely, saturated C16 or C18 SPM.
  • the SPM is preferably a synthetic or semi-synthetic SPM, i.e. a derivative of a naturally occurring SPM and may include the natural D-erythro (2S, 3R) isomer and the non naturally occurring L-threo (2S, 3S) isomer, although the former, i.e. the naturally occurring isomer is preferable.
  • sphingomyelin is also used to denote the corresponding dihydro species, namely, any dihydrosphingomyelins (DHSM) corresponding to the SPM defined herein above.
  • DHSM dihydrosphingomyelins
  • the liposomal system comprises SPM content in the liposomes membrane in an amount between 25 to 75 mole % of the total phospholipids (liposome forming lipid) in said membrane, or about 50 mole % of the total lipids when including cholesterol.
  • the mole ratio between the liposome forming lipids other than SPM and said SPM is typically in the range of 1:1 to 2:1, irrespective of the SPM used in accordance with the present disclosure.
  • the liposome forming lipids have when assembled into the liposome membranes have a solid ordered (SO) to liquid disordered (LD) phase transition temperature having a characteristic temperature defined as T m >37° C.
  • T m is the temperature within the range of the SO to LD phase transition temperatures in which the maximal change in the heat capacity of the phase transition occurs.
  • the combination HSPC having a solid ordered to liquid disordered with a T m at ⁇ 53° C. with C16SPM having its T m at ⁇ 41.4° C. led to the formation of a stable liposomal system, i.e. reduced drug leakage during 4° C. storage, as compared to a liposomal system lacking C16SPM which was less stable, namely, showing higher rate of drug leakage during 4° C. storage (i.e. same storing conditions).
  • stablility in the context of the present disclosure is used to denote that the resulting liposomes were more stable (less agent being leaked from the liposomes during or following storage, the difference in leakage being statistically significant) as compared to the same liposomes, albeit free of SPM, namely, the liposome's membrane does not comprise SPM as part of the liposome forming lipids.
  • the stability may also be defined that the drug loaded liposomes are chemically and physically unaltered when stored at 4° C. and for a period of at least 3 months. The stability is determined, for example, by measuring the amount of free active agent that present or was released (leaked) to the extra-liposome aqueous medium, i.e.
  • the amount indicative of stability being less than 30%, 20% and at times even less than 10% from the total amount of active agent in the liposomal system (the total amount including encapsulated and non-encapsulated agent).
  • the results presented herein show that when comparing a liposome formulation e.g. comprising HSPC and Cholesterol with the amount of leakage of an encapsulated agent from the same formulation, albeit with SPM in the lipid membrane, leakage of the agent was reduced.
  • the liposomes may also comprise other lipids typically used in the formation of liposomes, e.g. for stabilization, for affecting surface charge, membrane fluidity and/or assist in the loading of the active agents into the liposomes.
  • lipids may include sterols such as cholesterol, cholesteryl hemisuccinate, cholesteryl sulfate, or any other derivatives of cholesterol.
  • the liposomes may further comprise lipopolymers.
  • lipopolymer is used herein to denote a lipid substance modified at its polar headgroup with a hydrophilic polymer.
  • the polymer headgroup of a lipopolymer is typically water-soluble and may be covalently or non-covalently attached to a hydrophobic lipid region.
  • the hydrophilic polymer has a molecular weight equal or above 750 Da and may be polar or apolar.
  • Lipopolymers such as those that may be employed according to the present disclosure are known to be effective for forming long-circulating liposomes.
  • polymers which may be attached to lipids to form such lipopolymers, such as, without being limited thereto, polyethylene glycol (PEG), polysialic acid, polylactic (also termed polylactide), polyglycolic acid (also termed polyglycolide), apolylactic-polyglycolic acid, polyvinyl alcohol, polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxyethyloxazoline, polyhydroxypropyloxazoline, polyaspartamide, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polyvinylmethylether, polyhydroxyethyl acrylate, derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.
  • PEG polyethylene glycol
  • polysialic acid polylactic
  • polyglycolic acid also termed polyglycolide
  • apolylactic-polyglycolic acid polyvinyl alcohol,
  • the polymers may be employed as homopolymers or as block or random copolymers.
  • the lipids derivatized into lipopolymers may be neutral, negatively charged, as well as positively charged.
  • the most commonly used and commercially available lipids derivatized into lipopolymers are those based on phosphatidyl ethanolamine (PE), usually, distearylphosphatidylethanolamine (DSPE).
  • One particular family of lipopolymers that may be employed according to the present disclosure are the monomethylated PEG attached to DSPE (with different lengths of PEG chains, in which the PEG polymer is linked to the lipid via a carbamate linkage resulting in a negatively charged lipopolymer, or the neutral methyl polyethyleneglycol distearoylglycerol (mPEG-DSG) and the neutral methyl poly ethyleneglycoloxy carbonyl-3-amino-1,2-propanediol distearoylester (mPEG-DS) [Garbuzenko O. et al., Langmuir. 21:2560-2568 (2005)].
  • Another lipopolymer is the phosphatidic acid PEG (PA-PEG).
  • the PEG moiety has a molecular weight of the head group is from about 750 Da to about 20,000 Da, at times, from about 750 Da to about 12,000 Da and typically between about 1,000 Da to about 5,000 Da.
  • One specific PEG-DSPE commonly employed in liposomes is that wherein PEG has a molecular weight of 2000 Da, designated herein 2000 PEG-DSPE or 2k PEG-DSPE.
  • the liposomes of the liposomal system encapsulate at least one active agent.
  • Encapsulation includes the entrapment/enclosure, in the intraliposomal phase, of at least one active agent.
  • the entrapment is a non-covalent entrapment, namely in the liposomal aqueous phase the active agent is freely dispersed and may, under appropriate conditions, be released from the liposomes in a controlled manner.
  • the active agent may be a small molecular weight compound as well as a polymer (e.g. peptide, protein, nucleic acid sequence etc.).
  • a polymer e.g. peptide, protein, nucleic acid sequence etc.
  • the term “active agent” is used to denote that the encapsulated agent, once administered has a beneficial effect, e.g. as a therapeutic, as a contrasting agent (e.g. radionuclei dyes or dye-conjugates to carrier, chromophor or fluorophor producing agent etc.), as a nutraceutical compound etc.
  • the active agent may be a water soluble, hydrophilic compound as well as an amphipathic compound.
  • the active agent is an amphipathic compound.
  • amphipathic compound is used to denote a active agent possessing both hydrophilic and lipophilic properties.
  • doxorubicin e.g., DOXILTM
  • DOXILTM transmembrane ammonium sulfate gradient
  • the amphipathic active agent is an analgesic drug.
  • the analgesic drug would typically be for local analgesic.
  • a non-limiting group of analgesic drugs are selected from the group consisting of benzocaine, chloroprocaine, cocaine, cyclomethycaine, dimethocaine, propoxycaine, procaine, proparacaine, tetracaine, articaine, bupivacaine, carticaine, cinchocaine, etidocaine, levobupivacaine, lidocaine, mepivacaine, piperocaine, prilocaine, ropivacaine, trimecaine, saxitoxin and tetrodotoxin.
  • a preferred group of analgesic drugs include, without being limited thereof, bupivacaine, lidocaine, ropivacaine, levobupivacaine, procaine, chloroprocaine, benzocaine, etidocaine, mepivacaine, prilocaine, ciprocaine, tetracaine, dibucaine, heptacaine, mesocaine, propanocaine, carbisocaine, and butacaine.
  • a specific analgesic drug according to the present disclosure is bupivacaine (hereinafter referred to, at times, as “BUP”).
  • the active agent is a water soluble molecule such as a peptide, protein or nucleic acid sequences, including, for example, cytokines, antibodies, immunostimulating oligonucleotides (ISS-ODN), siRNA etc.
  • a water soluble molecule such as a peptide, protein or nucleic acid sequences, including, for example, cytokines, antibodies, immunostimulating oligonucleotides (ISS-ODN), siRNA etc.
  • liposomes in general may have various shapes and sizes.
  • the liposomes may be multilamellar liposomes (MLV) or multivesiclular vesicles (MVV).
  • MVV liposomes are known to have the form of numerous non-concentric, closely packed internal aqueous chambers separated by a network of lipid membranes and enclosed in a lipid membrane.
  • the MVV are referred to as large multivesicular vesicles (LMVV), also known in the art by the term giant multivesicular vesicles (GMV).
  • LMVV large multivesicular vesicles
  • GMV giant multivesicular vesicles
  • the liposomes typically have a diameter of at least 200 nm, typically in the range of about 200 nm and 25 ⁇ m, at times between about 250 nm and 25 ⁇ m.
  • the loading of the agent into the LMVV includes containment of the agent in more than one aqueous compartment formed by the lipid membranes, and typically also in the aqueous environment surrounding the non-concentric lipid membrane. At times, the agent may be entrapped (embedded) in the lipid membrane, e.g. when the active agent is lipophilic compound.
  • the liposomal system disclosed herein is characterized by a high active agent to lipid ratio, namely, high level of active agent per liposome.
  • a high loading would typically depend on the type of liposomes used, their size, the loading conditions etc.
  • a high loading is achieved by active loading (see below) of the active agent into LMVV under condition of high initial active agent concentration.
  • high loading is used to denote a loading with a active agent to lipid ratio in the resulting liposomal system of at least about 0.5 mole drug per mole liposome forming phospholipid ratio (mole/mole) (this being characteristic of the LMVV according to the present disclosure).
  • Loading of the active agent into the liposomes may be by any technique known in the art. Such techniques typically include passive loading or active (“remote loading”) loading of agents into the liposomes.
  • Passive loading techniques of encapsulating agents into liposomes typically involve loading of the agent during preparation of the liposomes, e.g. by hydrating dry liposome forming lipids with a solution of the active agent. By passive loading the agent may be associated to the liposomal membrane or encapsulated within the aqueous core.
  • One method for passive loading was described by Bangham, et al., [Bangham A D, Standish M M, Watkins J C (1965) Diffusion of univalent ions across the lamellae of swollen phospholipids. J MoI Biol.
  • liposomes may be loaded using an ion gradient or pH gradient as the pre-formed liposome loading driving force.
  • Loading using a pH gradient may be carried out according to methods described in U.S. Pat. Nos. 5,616,341, 5,736,155 and 5,785,987, U.S. Pat. No. 5,192,549, U.S. Pat. No. 5,316,771 and Haran et al., [Haran G, et al. (1993) Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases. Biochim Biophys Acta. 1151(2):201-15], incorporated herein by reference.
  • the pH gradient may be calcium citrate-based or ammonium sulphate-based gradient.
  • the liposomes have the form of multilamellar vesicles (MLV) or multivesicular vesicles (MVV), preferably, large multivesicular vesicles (LMVV).
  • MLV multilamellar vesicles
  • MVV multivesicular vesicles
  • LMVV large multivesicular vesicles
  • the present disclosure also provides a method for storage of liposomes as defined above, i.e. encapsulating in their intraliposomal aqueous compartment at least one active agent, the liposomes having a membrane comprising liposome forming lipids, at least one liposome forming lipid being sphingomyelin (SPM), the method comprising forming a liposomal system where said liposomes are dispersed in an aqueous medium being in an iso-osmotic equilibrium with the intraliposomal aqueous compartment of said liposomes and storing said liposomal system, whereby no more than 30%, at times no more than 20% and even no more than 10% of the at least one active agent is present in the aqueous medium after said storage.
  • SPM sphingomyelin
  • the method allows long term stable storage (at low temperatures, e.g. 4° C.) of the liposomes. While at minimum stable storage is for a period of 3 months, as will be shown in the following non-limiting examples, stable storage was also obtained for a period of four months (120 days), 4.5 months and even up to 6 months storing at 4° C. However, as indicated above, the stability would be retained at any other temperature that is lower than the physiological temperature of the body, namely, below 37° C. When referring to lower temperatures it is to be understood that the reasonable storage temperature should be at least 15° C. below body core temperature, i.e. below 22° C. According to one embodiment, storing is at a temperature between about 2° C. to 8° C.
  • the liposomal system may be administered to the subject in need thereof as is or may be combined with a physiologically acceptable additive.
  • the present invention also provides the use of the liposomal system as defined hereinabove for the preparation of a pharmaceutical or diagnostic composition, for, respectively, treatment of a medical condition or for diagnostic purposes.
  • the composition typically comprises, in addition to said liposomal system, at least one physiologically acceptable additive.
  • the present invention provides a method for the treatment or diagnostic of a medical condition, the method comprising administering to a subject in need of said treatment or diagnostic an amount of the liposomal system as defined hereinabove or physiologically acceptable composition comprising the same.
  • the liposomal system alone or in combination with physiologically acceptable additives may be administered by any route acceptable in the art.
  • the administration of the liposomal system is by parenteral injection or infusion. This would include, without being limited thereto, intravenous, intraarterial, intramuscular, intracerebral, intracerebroventricular, intracardiac, subcutaneous, intraosseous (into the bone marrow), intradermal, intratheacal, intraperitoneal, intravesical, and intracavernosal and epiduaral (peridural) injection or infusion. Pareneral administration may also include transdermal, e.g. by transdermal patches, transmucosal (e.g. by diffusion or injection into the peritoneum), inhalation and intravitreal (through the eye).
  • a preferred mode of administration is local administration by any acceptable route, as can be determined by a medical doctor or any other appropriate physician.
  • the amount of liposomal system administered, and thereby the amount of active agent encapsulated therein should be effective to achieve the desired effect by the active agent, at the target site.
  • the active agent is a drug
  • the amount of the liposomal systems should be determined so that at the target site the amount of the drug encapsulated therein is sufficient to achieve the desired therapeutic effect.
  • Such desired therapeutic effect may include, without being limited thereto, amelioration of symptoms associated with a medical condition, prevention of the manifestation of symptoms associated with a medical condition, slow down of a progression state of a medical condition, enhance of onset of a remission period, prevent or slow down irreversible damage caused by the medical condition, lessen the severity of the medical condition, cure the medical condition or prevent it from developing, etc.
  • the medical condition to be treated by the liposomal system may be any such condition treatable by the active agent encapsulated in the liposomes according to the present disclosure.
  • the active agent may be a diagnostic agent.
  • the amount of the liposomal system should be such that it would be possible to image the marker at the target site.
  • the amount of the liposomal systems will be determined by such considerations as may be known in the art, typically using appropriately designed clinical trials (dose range studies etc.).
  • a liposome forming lipid includes one or more lipids capable of forming a liposome.
  • the term “comprising” is intended to mean that the liposomal system include the recited constituents, i.e. the liposome forming lipid, SPM and the active agent, but not excluding other elements, such as physiologically acceptable carriers and excipients as well as other active agents.
  • the term “consisting essentially of” is used to define liposomal systems which include the recited elements but exclude other elements that may have an essential significance on the effect to be achieved by the liposomal system. “Consisting of” shall thus mean excluding more than trace amounts of other elements. Embodiments defined by each of these transition terms are within the scope of this invention.
  • Bupivacaine hydrochloride (B UP) USP XXIII (Orgamol, SA, Evionnaz, Switzerland).
  • Methylprednisolone sodium succinate (MPS) (PHARMACIA NV/SA Puurs-Belgium).
  • Cholesterol (CHOL) (NF; Solvay Pharmaceuticals (Veenedaal, Netherlands).
  • HSPC-100 Fully hydrogenated soy phosphatidylcholine (HSPC-100), Phospholipon® 100H batch no 50190 (Phospholipids GmbH Nattermannallee 1*D 50829 Koln, Germany).
  • HSPC100 is 99.5 pure, i.e. comprising lysoPC and fatty acid in an amount less than the detectable limit.
  • HSPC Fully hydrogenated soy phosphatidylcholine
  • Ammonium sulfate (AS, MERCK);
  • LMVV Large Multi Vesicular Vesicles
  • Powder mixtures of lipids at the desired mole ratio were dissolved in ethanol at 60-65° C. and added to an aqueous solution (ammonium sulfate (AS), calcium acetate (CA) or another buffer, as indicated below) to reach a final phospholipid (PL) concentration of 60 mM and final ethanol concentration of 10%.
  • AS ammonium sulfate
  • CA calcium acetate
  • PL phospholipid
  • MLV multilamellar vesicles
  • LMVV were prepared from the MLV with the desired aqueous phase (for example: ammonium sulfate 250 mM or 127 mM, calcium acetate 250 mM, or 200 mM; or a desired buffer) from the MLV by exposing the MLV to 10 cycles of freezing in liquid nitrogen and thawing in a water bath at 60° C. thereby forming the LMVV.
  • the desired aqueous phase for example: ammonium sulfate 250 mM or 127 mM, calcium acetate 250 mM, or 200 mM; or a desired buffer
  • Transmembrane AS or CA gradient were created by removal of AS or CA (respectively) from the extra liposome aqueous phase and replacing it with NaCl.
  • HSPC and C16SPM are the liposome-forming lipids of the LMVV described here.
  • An amount 0.5 ml of a wet LMVV pellet and 2 ml of BUP solution were used for the remote loading. The mixture was then cooled to 4° C. overnight.
  • Passive loading of BUP was performed by hydrating the ethanol lipid solution with aqueous solution of distilled water containing 4.5% (231 mOsm/kg), or 5.5% (285 mOsm/kg), or 6% (301 mOsm/kg) or 7% (346 mOsm/kg), or 8% (373 mOsm/kg) or 10% (454 mOsm/kg) BUP (W/V) at 65° C. for 30 min.
  • 0.5 ml ethanolic lipids solution containing 225 mg phospholipids and 77 mg CHOL were used. This solution was mixed with 5 ml of one of the above indicated BUP aqueous solutions.
  • the suspension was processed by 10 freezing and thawing cycles (as described above) and than kept overnight in a cold room (4-6° C.).
  • Non-encapsulated BUP was removed from LMVV by washing with saline (1 ml liposomes/4 ml saline) and centrifuging the dispersion at 1000 g for 5 min at 4-5° C. The washing process was repeated 7 times.
  • the final medium (referred to herein as the “aqueous medium”) used to replace extra-liposome from CA gradient loaded liposomes was PBS.
  • the use of PBS was preferred over saline.
  • AS and the medium used for passive loading of liposomes was replaced and LMVV were washed with un-buffered saline.
  • the LMVV was concentrated to a final solution of 2% BUP for the passive loading and AS gradient loading.
  • LMVV with 1% BUP final concentration was used, due to the large volume of these LMVV. These concentrations were close to the highest concentrations used for injection of BUP.
  • the stability of LMVV thus formed was measured with respect to the release rate of BUP from liposomes during storage at 4° C.
  • BUP was loaded into AS-LMVV using three types of BUP to lipid v/v ratios:
  • FIGS. 1A and 1B compare the loading stabilities of BUP-LMVV (prepared by similar procedure, albeit with H100), as measured with respect to release rate at 4° C. ( FIG. 1A ) and 37° C. ( FIG. 1B ).
  • the comparison relates to different lipid compositions of LMVV as follows:
  • FIGS. 1A and 1B show that the release rates of BUP during 60 days storage at 4° C. of the HSPC/CHOL liposomes was the highest, followed by the release rate from HSPC100/CHOL liposomes. The lowest release rate was achieved for HSPC100/C16SPM/CHOL liposomes. In 24 hours, the release at 37° C. reaches the level of 60% to 70% of the BUP from the liposome—this being without reaching a plateau. It was thus concluded that although a slight lower loading of BUP (lower BUP/PL ratio) reached with the LMVV composed of HSPC100/C16SPM/CHOL, the low release rate of BUP from this particular formulation at 4° C. rendered this combination a preferred formulation. It was thus further concluded that the presence of SPM reduced leakage as compared to the same formulation without SPM.
  • FIGS. 2A and 2B demonstrate the release rate at 4° C. ( FIG. 2A ) and 37° C. ( FIG. 2B ) of BUP from LMVV having the same lipid compositions as used in FIGS. 1A-1B , wherein BUP was remotely loaded using Ca acetate gradient.
  • the SPM used was C16 SPM, and comparison with HSPC/SPM/CHOL and HSPC100/SPM/CHOL was also made at 4° C.
  • the ratio BUP/PL for the CA gradient loading was lower than that obtained for the AS gradient loading. Stability was assessed from the release at 4° C. This ratio was also lower (i.e. higher release rate) than that obtained for LMVV remote loaded by AS gradient at 37° C.
  • the release rates are similar to those of the LMVV loaded BUP by AS gradient, except that rate of release is faster at the first 10 hours followed by an almost plateau. It is apparent from FIG. 2A that the HSPC100 LMVV has better stability (i.e. lower leakage at 4° C.) than HSPC based LMVV, and that C16 SPM effect on improving stability is much greater than the difference between the two HSPC preparations. C 16 SPM also reduces leakage rate for both HSPC compositions by a similar extent.
  • FIGS. 3A and 3B demonstrate the release rate at 4° C. ( FIG. 3A ) and 37° C. ( FIG. 3B ) of BUP loaded LMVV of the same lipid compositions used in FIGS. 1A and 1B , wherein LMVV were passively loaded with BUP.
  • the SPM used is C16 SPM, and a comparison of HSPC/SPM/CHOL and HSPC100/SPM/CHOL was also made at 4° C.
  • release rates at 4° C., for passively loaded LMVV of the 3 lipid compositions used were higher than for the remote loading via CA gradient and even higher when compared with AS remote loading LMVV.
  • pre-formed LMVV were centrifuged for 5 min at 4° C. at 2000 g to give packed LMVV.
  • the packed LMVV were suspended in various volumes of 5.7% BUP.
  • the volume ratio of BUP to LMVV or PL is given in Table 4.
  • All liposomal formulations were analyzed for free BUP and total BUP before the in vivo experiment and concentrated to reach the level of 2% (w/w) BUP (liposomes formulations #1, 2, 3, 5, 6, 8) or 1% (w/w) BUP (liposomes formulations #4, 7). BUP was loaded into the liposomes either by active loading (CA or AS gradient) or by passive loading.
  • Liposome composition analysis prior to in vivo experimentation Liposome # Lipids* Loading technique type % free BUP 1 H100/SPM/CHOL AS gradient MLV 3.88 2 H100/SPM/CHOL CA gradient MLV 3.95 3 H100/SPM/CHOL AS gradient LMVV 3.69 4 H100/SPM/CHOL CA gradient LMVV 4.52 5 H100/CHOL AS gradient LMVV 3.68 6 HSPC/CHOL AS gradient LMVV 7.80 7 H100/CHOL CA gradient LMVV 7.66 8 H100/SPM/CHOL 6% BUP passive LMVV 1.90 loading *with SPM the ratio is 3/3/4 and without SPM the ratio is 6/4
  • Testing for analgesia was done by electrical stimulation of the skin directly overlying the abdomen at the site of injection using a current generator (model S48, Grass Instruments).
  • mice Male Swiss-Webster, 26 ⁇ 3 gr were tested prior to injection to determine the vocalization threshold than were injected with encapsulation BUP liposomes than determine analgesic duration (G. J. Grant et al, pharmaceutical research, vol 18, no. 3, 336-343, 2001).
  • the duration of the main in vivo screening study was 2 days and started after a preliminary study using two different injection volumes of formulation #4 (referred to as the PILOT in Table 9A) was performed.
  • mice received 150 ⁇ L of the 2% formulation and 3 mice received 300 ⁇ L of a 1:1 diluted 2% formulation.
  • LMVV (GMV) encapsulated BUP provide an analgesic effect for approximately 75 minutes post injection.
  • analgesic efficacy of the various formulations 1 to 8, at different BIP concentration, different injection volume etc. is presented in Tables 9A to 9C.
  • an numeric score of “1” denotes full analgesia
  • a numeric score of “0” was given when there was no analgesic effect
  • a numeric value of “10” when there was partial analgesia.
  • the numeric value “10” is replaced by “0.5”.
  • mice injected with LMVV formulation #4 two mice with 300 ⁇ l and two mice with 150 ⁇ l are presented as “PILOT 1-4” Testing was done at 4, 17, and 21 hours following injection.
  • FIGS. 4A-4C , 5 A- 5 F, 6 and 7 show the duration of analgesia.
  • the difference in these figures is in the formulations used, FIGS. 4 and 5 making use of the various formulations identified in Table 8, and FIGS. 6 and 7 making use of HSPC100/C16SPM/CHOL (3/3/4).
  • the in vivo results show that SPM containing liposomes have a significantly greater analgesic effect as compared to free BUP. These results specifically show that the inclusion of SPM into the liposomes did not reduce the analgesic effect to the system, as compared to prior art formulations [Grant et al. 2004, ibid.].
  • the numerical score to the spreadsheet was introduced for the evaluation of the analgesic effect of various liposome preparations performance in vivo: For each time period (e.g. 4 hrs, 8 hrs etc) a numeric value of 1 was given if the anesthesia was complete; 10 or 0.5 was given when analgesia was partial (incomplete) and 0 for no anesthesia. The mean for each subgroup was calculated separately (i.e. 1% 300 ⁇ l, 2% 150 ⁇ g).
  • formulation 4 where BUP was actively loaded into LMVV with CA gradient and the iso-osmotic aqueous medium was saline provided the best analgesic effect, although the differences between the various formulations was not significant, when compared to the 10 fold increase in analgesia when compared to BUP formulations as the reference liposomal GMV formulation [Grant et al. 2004, ibid., Bolotin et al. 2000, ibid. and U.S. Pat. No. 6,162,462].

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US20110244029A1 (en) 2011-10-06
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EP2344132A2 (fr) 2011-07-20
US9713591B2 (en) 2017-07-25
US20210093566A1 (en) 2021-04-01
US10842745B2 (en) 2020-11-24
US11839685B2 (en) 2023-12-12
EP2344133A2 (fr) 2011-07-20
KR20120081938A (ko) 2012-07-20
CN102231978A (zh) 2011-11-02
AU2009302042A1 (en) 2010-04-15
CN102231978B (zh) 2016-01-20
US20170281540A1 (en) 2017-10-05
US20190151243A1 (en) 2019-05-23
WO2010041255A3 (fr) 2011-04-07
CN102223878A (zh) 2011-10-19
EP2344133B1 (fr) 2019-06-26
CA2739822A1 (fr) 2010-04-15
US20240058269A1 (en) 2024-02-22

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