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
• • 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
  • A61K9/1277—Processes for preparing; Proliposomes

Definitions

• This invention relates to liposome technology and in particular to methods of preparing liposomes.
• Liposomes are one of the most potential drug carriers available currently.
• Liposomes contain both a hydrophobic bilayer, which may encapsulate hydrophobic substances, and an aqueous core, which may encapsulate other substances (e.g. hydrophilic or amphiphatic compounds).
• Liposomal encapsulation of therapeutic compounds has shown significant promise in controlled drug delivery. For example, some lipid-based formulations provide a longer half-life in vivo, superior tissue targeting, or decreased toxicity. In efforts to develop more effective therapeutic treatments, attempts have been made to encapsulate a variety of therapeutic compounds in liposomes. For example, many anticancer or antineoplastic drugs have been encapsulated in liposomes.
• Liposomes are prepared by many methods and the obtained vesicles may vary significantly in terms of diameter and number of bilayers. Liposomes may be classified as small or large unilamellar vesicles (SUV 5 LUV), multilamellar vesicles (MLV) and multivesicular vesicles (MVV) or large multivesicular vesicles (LMVV) 5 which contain several vesicles and, consequently, several separate aqueous phases [Kulkarni, S.B., et al. J. Microencapsul 12:229-246 (1995)]. The vesicles-in- vesicles are formed during the preparation of multivesicular vesicles (MVV) [Szoka, F. and Papahadjopoulos, D. Proc.
• MVV multivesicular vesicles
• MVVs and FT MLVs encapsulate far more aqueous phase volume than SUVs and MLVs, but the structures of MVVs and FT MLVs (LMVV) are large, i.e. 0.5-15 ⁇ m in diameter.
• the majority of the liposome preparation methods are based on either dry lipid hydration or the evaporation of an organic solvent in which the thus dried lipids are added into an aqueous solution.
• the methods based on dry lipid hydration are typically multi-step processes (organic solvent evaporation from lipid solution, lipid drying, hydration, calibration, and possibly other steps).
• an aqueous phase is dispergated in an organic solvent (ethyl ether, halothane, chloroform, methylene chloride or other) to form a water-in-oil emulsion by sonicating the mixture of both of these phases.
• an organic solvent ethyl ether, halothane, chloroform, methylene chloride or other
• the emulsion is transferred to a rotary evaporator, and the solvent is removed under reduced pressure.
• some of the aqueous phase droplets combine and form the environment where buffer droplets, enveloped in lipid membrane, are suspended.
• the aggregates are centrifuged and the supernatant is filtered to obtain LUVs, smaller than the defined sizes of a filter's pores.
• Another method using double emulsion characteristics is based on aqueous emulsion formation in chloroform and ethyl ether solutions of liposomal lipids by mechanical agitation with a shaker. Constant bubbling of nitrogen agitates the combined emulsions, and consequently unilamellar or multivesicullar vesicles are formed [Kim S. and Martin, G.M. Biochim. Biophys. Acta 646:1-9 (1981), and US Patent No. 5,723,147].
• multivesicular liposomes containing biologically active substances, the multivesicular liposomes having a defined size distribution, adjustable average size, adjustable internal chamber size and number, and a modulated rate of the biologically active substance.
• the process used to form such multivesicular liposomes comprises dissolving a lipid component in volatile organic solvents, adding an immiscible aqueous component containing at least one biologically active substance to be encapsulated, and adding a hydrochloride effective to control the - A -
• release rate of the biologically active substance from the multivesicular liposome to either or both of the organic solvents and the lipid component making a water-in-oil emulsion from the two components, immersing the emulsion into a second aqueous component, dividing the emulsion into small solvent spherules which contain even smaller aqueous chambers, and then removing the solvents to give an aqueous suspension of multivesicular liposomes encapsulating biologically active substances.
• LMVV liposomal bupivacaine compositions are prepared using an ammonium salt (e.g. sulfate) gradient loading procedure, at a pH which prevents precipitation of the drug from the loading solution.
• the liposomes may be large multivesicular liposomes (referred to as GMV) which are prepared by vortexing a dry lipid film with an aqueous solution of ammonium salt, homogenizing the resulting suspension to form a suspension of SUV and repeatedly (at least 5 times) freeze- thawing the suspension of SUV in liquid nitrogen followed by water.
• GMV large multivesicular liposomes
• the present invention is aimed at providing a simplified and more cost-effective method for preparing liposomes.
• a method for preparing liposomes comprising: (a) providing a dry liposome-forming lipid or a dry mixture comprising a liposome-forming lipid;
• the invention also provides a method for preparing liposomes consisting of:
• the invention provides a method for preparing liposomes comprising: (a) providing a dry liposome-forming lipid or a dry mixture comprising a liposome-forming lipid;
• the liposomes may be used to carry active agents such as drugs.
• the invention provides a method for preparing agent- carrying liposomes comprising or consisting of:
• the method of the invention for preparing agent carrying liposomes excludes drying said solution or dispersion to form a dry lipid film; and/or down-sizing liposomes in said liposome suspension to form small unilamellar vesicles (SUV).
• SUV small unilamellar vesicles
• the invention also provides pharmaceutical compositions comprising a physiologically acceptable carrier and agent-carrying liposomes whenever prepared by any of the methods of the invention.
• the invention provides a method for treating a subject in need comprising administering to said subject an amount of agent-carrying liposomes whenever prepared by the method of the invention.
• the present invention concerns a method for preparing liposomes with a high agent-to-lipid ratio (by amount in moles of each), which, as appreciated by those versed in the art, may have benefits in terms of prolonged delivery, safety and other pharmacological and pharmacokinetic parameters.
• the simplicity of the proposed approach resides, inter alia, in the elimination of several steps which hitherto have been considered essential components in the preparation of liposomes of similar characteristics and loading capabilities.
• liposome and "vesicle” are well known in the art and are used interchangeably herein, except where otherwise specifically stated or required by context.
• the method of the present invention comprises: providing a dry liposome- forming lipid or a dry mixture comprising one or more liposome-forming lipids; dissolving the lipid or the dry mixture comprising the same with a protic organic solvent to form a solution or dispersion of the lipid(s); and adding the solution or dispersion thus formed to an ion-containing aqueous solution, resulting in the formation of a liposomal suspension.
• liposomes are capable of loading an agent at a high agent-to-lipid mole/mole ratio, a high ratio being greater than 1.0.
• the active agent may be loaded into the liposome by different methods, such as incubation of the liposome suspension with an aqueous solution of the agent.
• a "high agent-to-lipid ratio” is determined by the mole/mole or weight/weight ratio of the agent to the liposome-forming lipid(s).
• ratio denotes a mole/mole ratio.
• a high agent-to-lipid (agent/lipid) ratio in accordance with the invention is to be understood as any ratio being at least greater than 1.0, preferably greater than 1.5, more preferably greater than 1.8. It has been found that under suitable conditions which are within the scope of the present invention the ratio may even be greater than 2.0.
• SUV small unilamellar vesicles
• F&T freezing and thawing
• the method of the present invention provides agent-carrying liposomes with a high agent/lipid ratio without the need to perform any of the above steps in order to achieve the desired high loading.
• a much simpler, cost-effective method is provided.
• Such a simplified method may have many advantages, in particular with respect to large-scale production of agent-carrying liposomes.
• the liposomes of the present invention are formed from liposome-forming lipids.
• “Liposome-forming lipids” (or “vesicle-forming lipids”) are amphiphilic molecules essentially characterized by a packing parameter of 0.74 - 1.0, inclusive, or by a lipid mixture having an additive packing parameter (the sum of the packing parameters of each component of the liposome multiplied by the mole fraction of each component) in the range between 0.74 and 1, inclusive.
• liposome-forming lipids in accordance with the invention are lipids having a glycerol backbone wherein at least one, preferably two, of the hydroxyl groups at the head group is substituted by one or more of an acyl, an alkyl or alkenyl group, a phosphate group, preferably an acyl chain (to form an acyl or diacyl derivative), a combination of any of the above, and/or derivatives of the above, and may contain a chemically reactive group (such as an amine, acid, ester, aldehyde or alcohol) at the headgroup, thereby providing a polar head group.
• Sphingolipids, and especially sphingomyelins are good alternatives to glycerophospholipids.
• a substituting chain e.g. an acyl, alkyl and/or alkenyl chain
• a substituting chain is between about 14 to about 24 carbon atoms in length, and has varying degrees of saturation, thus resulting in fully, partially or non-hydrogenated liposome-forming lipids.
• the liposome-forming lipid may be of a natural source, semi-synthetic or a fully synthetic lipid, and may be neutral, negatively or positively charged.
• phospholipids which are preferred lipids in accordance with the invention
• phospholipids such as phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidyl glycerol (PG), dimyristoyl phosphatidyl glycerol (DMPG), egg yolk phosphatidylcholine (EPC), l-palmitoyl-2-oleoylphosphatidyl choline (POPC), distearoylphosphatidylcholine (DSPC), dimyristoyl phosphatidylcholine (DMPC), phosphatidic acid (PA), phosphatidylserine (PS), l-palmitoyl-2-oleoylphosphatidyl choline (POPC) 5 and sphingophospholipids such as sphingomyelins (SM) having 12- to
• lipids and phospholipids whose hydrocarbon chain (e.g., acyl/alkyl/alkenyl chains) have varying degrees of saturation, can be obtained commercially or prepared according to published methods.
• Other suitable lipids which may be included in the liposomes are glyceroglycolipids, . sphingoglycolipids and sterols (such as cholesterol or plant sterol).
• the liposomes formed in accordance with the invention may comprise a mixture of lipids.
• the above- described list of lipids for use in accordance with the invention is not exhaustive and non-limiting, and thus, other lipids not disclosed herein may be used in accordance with the invention.
• the liposome-forming lipids are selected from those having a T m (gel to liquid crystalline phase transition temperatures), above 45°C, such as, without being limited thereto, phosphatidylcholine (PC) and derivatives thereof having an acyl chain with 16 or more carbon atoms.
• PC phosphatidylcholine
• One preferred example of a PC derivative is hydrogenated soy PC (HSPC) having a Tm of 52°C.
• the liposome- forming lipids may additionally or alternatively comprise sphingomyelins of various N- acyl chains, such as N-stearoyl sphingomyelin.
• Cationic lipids are also suitable for use in the liposomes of the invention, where the cationic lipid can be included as a minor component of the lipid composition or as a major or sole component.
• Such cationic lipids typically have a lipophilic moiety, such as a sterol, an acyl or diacyl chain, and the lipid typically has an overall net positive charge.
• the head group of the lipid carries the positive charge.
• Monocationic lipids may include, for example: 1,2- dimyristoyl-3-trrmethylammonium propane (DMTAP); l,2-dioleyloxy-3-
• DMRIE N-hydroxyethylammonium bromide
• DORIE N-[l-(2,3,-dioleyloxy)propyl]-N,N- dimethyl-N-hydroxy ethyl ammonium bromide
• DORIE N-[I -(2,3 -dioleyloxy) propyl]-N,N,N-trimethylammonium chloride
• DC-Choi 3 ⁇ [N-(N',N'- dimethylaminoethane) carbamoyl] cholesterol
• DDAB dimethyl-dioctadecylammonium
• polycationic lipids may include a lipophilic moiety similar to those described for monocationic lipids, to which the polycationic moiety is attached.
• exemplary polycationic moieties include spermine or spermidine (as exemplified by DOSPA and DOSPER), or a peptide, such as polylysine or other polyamine lipids.
• DOPE neutral lipid
• Polycationic lipids 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]-l-propanaminium (DOSPA), and ceramide carbamoyl spermine (CCS).
• DOSPA 1,3-oxo-9- octadecenyl)oxy]-l-propanaminium
• CCS ceramide carbamoyl spermine
• the liposomes may also include a lipid derivatized with a hydrophilic polymer to form new entities known by the term lipopolymers.
• Lipopolymers preferably comprise lipids modified at their head group with a polymer having a molecular weight equal to or above 750 Da.
• the head group may be polar or apolar; however it is preferably a polar head group to which a large (>750 Da), highly hydrated (at least 60 molecules of water per head group), flexible polymer is attached.
• the attachment of the hydrophilic polymer head group to the lipid region may be a covalent or non-covalent attachment; however it is preferably via the formation of a covalent bond (optionally via a linker).
• the outermost surface coating of hydrophilic polymer chains is effective to provide a liposome with a long blood circulation lifetime in vivo.
• the lipopolymer may be introduced into the liposome in two different ways either by: (a) adding the lipopolymer to a lipid mixture, thereby forming the liposome, where the lipopolymer will be incorporated and exposed at the inner and outer leaflets of the liposome bilayer [Uster P.S. et al. FEBBS Letters 386:243 (1996)]; or (b) first preparing the liposome, and then incorporating the lipopolymers into the external leaflet of the pre-formed liposome either by incubation at a temperature above the Tm of the lipopolymer and liposome-forming lipids, or by short-term exposure to microwave irradiation.
• vesicles composed of liposome-forming lipids and lipids such as phosphatidylethanolamines (which are not liposome-forming lipids) and derivatization of such lipids with hydrophilic polymers (thereby forming lipopolymers) which in most cases are not liposome-forming lipids.
• lipids such as phosphatidylethanolamines (which are not liposome-forming lipids) and derivatization of such lipids with hydrophilic polymers (thereby forming lipopolymers) which in most cases are not liposome-forming lipids.
• the lipopolymers may be non-ionic lipopolymers (also referred to at times as neutral lipopolymers or uncharged lipopolymers) or lipopolymers having a net negative or a net positive charge.
• Polymers typically used as lipid modifiers include, without being limited thereto, polyethylene glycol (PEG), polysialic acid, polylactic acid (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, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.
• the polymers may be employed as honiopolymers or as block or random copolymers.
• lipids derivatized into lipopolymers may be neutral, negatively charged, or positively charged, i.e. there is no restriction regarding a specific (or no) charge
• the most commonly used and commercially available lipids derivatized into lipopolymers are those based on phosphatidyl ethanolamine (PE), usually distearylphosphatidylethanolamine (DSPE).
• a specific family of lipopolymers which may be employed by the invention includes monomethylated PEG attached to DSPE (with different lengths of PEG chains, the methylated PEG referred to herein by the abbreviation PEG), in which the PEG polymer is linked to the lipid via a carbamate linkage resulting in a negatively charged lipopolymer.
• Other lipopolymers are the neutral methyl polyethyleneglycol distearoylglycerol (mPEG-DSG) and the neutral methyl polyethyleneglycol oxycarbonyl-3 -amino- 1,2-propanediol distearoylester (mPEG-DS) [Garbuzenko O. et al, Langmuir.
• the PEG moiety preferably has a molecular weight of the PEG head group from about 750 Da to about 20,000 Da. More preferably, the molecular weight of the headgroup is from about 750 Da to about 12,000 Da, and it is most preferably between about 1,000 Da to about 5,000 Da.
• One specific PEG-DSPE employed herein is a PEG moiety with a molecular weight of 2,000 Da, designated herein 2000 PEG-DSPE or 2k PEG-DSPE. Preparation of liposomes including such derivatized lipids has also been described where typically between 1-20 mole percent of such a derivatized lipid is included in the liposome formulation.
• liposome-forming lipids such as PCs and sphingomyelins
• cholesterol and phopshatidylethanolamines can be included in the liposomal formulation (e.g. to decrease a membrane's free volume and thereby permeability and leakage of an agent encapsulated therein).
• the liposomes comprise cholesterol.
• the lipid/cholesterol mole/mole ratio is within the range of between about 80:20 to about 50:50. A more specific mole/mole ratio is about 60:40.
• the liposome may include other constituents.
• charge-inducing lipids such as phosphatidyl glycerol may also be incorporated into the liposome bilayer to decrease vesicle-vesicle fusion, and to increase interaction of the liposome with cells.
• Buffers at a pH suitable to make the pH of the surface of the liposomes close to neutral can decrease hydrolysis.
• Addition of an antioxidant, such as vitamin E, or chelating agents, such as Desferal or DTPA may be used.
• a protic organic solvent is typically an alcohol, preferably C2 to C4 alcohols.
• the solvent is preferably miscible in water.
• protic organic solvents include methanol, ethanol, and tertiary butanol (tert-butanol). Ethanol is a preferred solvent.
• the solvent dissolves the lipid or mixture of lipids to form a solution or dispersion.
• solvents or “solution” it is to be understood that the lipid is preferably homogeneously mixed within the solvent; nonetheless, non-homogenous mixtures may be formed and used in the context of the present invention (in a dispersion).
• ions-comprising aqueous solution is used herein to denote an aqueous solution which comprises a salt, such as ammonium sulfate, calcium acetate, etc., dissolved therein.
• a salt such as ammonium sulfate, calcium acetate, etc.
• the different types of ions and salts are discussed in detail below with respect to pH or ion gradient formation. •
• the method of the invention is also characterized in that a high lipid concentration may be employed.
• lipid concentration in the formed liposomes is above 20 mM, above 30 mM, above 50 mM, above 90 mM and even up to 97-100 mM, inclusive.
• the lipid concentration is between 30 mM and 100 mM, inclusive.
• Variations in ratios between these liposome constituents dictate the pharmacological properties of the liposome.
• stability of the liposomes which is a major concern for various types of vesicular applications, may be dictated by selecting specific liposome constituents.
• the stability of liposomes should meet the same standards as conventional pharmaceuticals.
• Chemical stability involves prevention of both the hydrolysis of ester bonds in the phospholipid bilayer and the oxidation of unsaturated sites in the lipid chain. Chemical instability can lead to physical instability or leakage of encapsulated drug from the bilayer and fusion and aggregation of vesicles. Chemical instability also results in short blood circulation time of the liposome, which affects the effective access to and interaction with the target.
• LMVV multilamellar vesicles
• SSL small unilamellar vesicles
• MVV multivesicular vesicles
• LMVV large multivesicular vesicles
• SUV may be obtained by any known technique to down- size liposomes (such as vortexing, ultrasonication, extrusion, etc.). Those versed in the art will know how to select the appropriate additional treatment step(s) in order to convert the liposomes formed by the method of the present invention to other liposomal forms and structures.
• the method of the invention preferably provides MLVs. Following one F&T cycle, the MLVs are converted to LMVVs. Additional F&T cycles may be performed. In accordance with an embodiment, at least one F&T cycle is performed. In accordance with another embodiment, between 5 to 9 F&T steps are performed.
• the F&T cycle(s) convert(s) the MLV to LMVV which are then incubated with the agent to be loaded therein.
• the liposome suspension thus formed comprises an amount of the protic organic solvent. The amount may vary, depending on the solvent and the type of the liposomes thus formed. The amount of the organic solvent will be such that the liposomes are not converted to micelles (or micellae).
• ethanol levels in the liposomal suspension may be as high as about 25% by volume. In accordance with one embodiment, ethanol level is about 10% by volume.
• An active agent to be loaded into the liposomes may be any substance, e.g., a low or high molecular weight compound, having a utility in therapy or diagnostics.
• the active substance is an amphiphatic weak acid or an amphiphatic weak base.
• the agent is an amphiphatic weak acid drug or amphipathic weak base drug.
• Amphiphatic weak base drugs include, among others, the following non-limiting list: tempamine (TMN) doxorubicin, epirubicin, daunorubicin, carcinomycin, N- acetyladriamycin, rubidazone, 5-imidodaunomycin, N-acetyldaunomycine, all anthracyline drugs, daunoryline, topotecan, irinotecan propranolol, pentamidine, dibucaine, bupivacaine, tetracaine, procaine, chlorpromazine, vinblastine, vincristine, mitomycin C, pilocarpine, physostigmine, neostigmine, chloroquine, amodiaquine, chloroguanide, primaquine, mefloquine, quinine, pridinol, prodipine, benztropinemesylate, trihexyphenidyl hydrochloride, prop
• Amphiphatic weak acid drugs include, without being limited thereto, ibuprofen, toluetin, indomethacin, phenylbutazone, mecloferamic acid, piroxicam, citrofloxacin, prostaglandins, fluoresgein, carboxyfluorescein, methyl perdnisolone hemisuccinate
• MMS metacetamol
• acetaminophen paracetamol
• aspirin acetyl salicylic acid
• other NSAIDs acetyl salicylic acid
• glucocorticosteroids as an agent loaded in liposomes and treating these liposomes prior to administration with empty liposomes.
• a non-limiting list of glucocorticoids may be found at the internet site http://www.steraloids.com/, incorporated herein in its entirety by reference.
• Non- limiting examples include: prednisolone hemisuccinate, methylprednisolone hemisuccinate, dexamethasone hemisuccinate, allopregnanolone hemisuccinate; beclomethasone 21 -hemisuccinate; betamethasone 21 -hemisuccinate; boldenone hemisuccinate; prednisolone hemisuccinate; sodium salt; prednisolone 21- hemisuccinate; nandrolone hemisuccinate; 19-nortestosterone hemisuccinate; deoxycorticosterone 21 -hemisuccinate; dexamethasone hemisuccinate; dexamethasone hemisuccinate: spermine; corticosterone hemisuccinate; cortexolone hemisuccinate.
• entrapment denotes any form of loading of the agent onto the liposomes, such that at least a substantial part of the agent is encapsulated within the interior aqueous core of the liposomes. Within the interior core the agent may be free or associated to the inner surface of the lipid bilayer.
• entrapment may at times be used interchangeably with the terms “encapsulation” or “carrying” or “loading”.
• the passive entrapment method is most suited for entrapment of lipophilic drugs in the liposome membrane and for entrapment of agents having high water solubility.
• agent- loading efficiency can be achieved by loading the agent into liposomes against a transmembrane pH or ion gradient [Nichols, J.W., et al., Biochim. Biophys. Acta 455:269-271 (1976); Cramer, J., et al., Biochemical and Biophysical Research Communications 75(2):295-301 (1977)].
• This loading method typically involves an agent that is amphiphatic in nature and has an ionizable group which is loaded by adding it to a suspension of liposomes having a higher inside/lower outside H + and/or ion gradient.
• the liposomes employed in the context of the present invention are preferably loaded by the remote loading principle.
• Remote loading occurs due to pH or ion gradient, such as ammonium or ammonium-like (with non-organic, organic or polymeric anions, e.g. alkylamine) gradient aggregation due to a high intra-liposome concentration of the agent and the formation of an agent-counter-ion salt within the liposome. Excess of the counter-ion occurs when the NH 3 is released from the liposomes.
• Remote loading via an ammonium salt is based on the large difference in permeability of the neutral ammonia gas molecule (1.3XlO "1 cm/s) and the charged anion ( ⁇ 10 "10 cm/s).
• the pH of the intra-liposome aqueous phase composed of an ammonium salt solution may be decreased by lowering the external concentration of ammonium and ammonia [Haran G., et al., Biochim Biophys. Acta 1151:201-215, (1993)].
• the decrease of intra-liposomal pH results from the release from the liposome of the unprotonated ammonia compound (NH 3 ) leaving within the liposome protons (H + ) and the counter-ion (e.g. HSO 4 " , SO 4 "2 ); thereby an excess of the counter-anions over NH 4 + is created within the liposome.
• Reduction of the pH inhibits ammonia formation and thereby inhibits its release from the liposome.
• an agent e.g., an amphiphatic weak base
• it freely crosses the lipid bilayer in its uncharged form and accumulates in its charged (having low permeability) form in the internal aqueous compartment (after being protonated by the free H + )
• an agent e.g., an amphiphatic weak base
• the liposome have a low mter-liposomal/high intra-liposomal trans-membrane gradient, such as ammonium salt gradient (e.g. ammonium sulfate).
• ammonium salt gradient e.g. ammonium sulfate
• the liposomes When the agent is a weak amphiphatic acid, it is preferable that the liposomes have a high inter-liposome/low intra-liposome transmembrane gradient. Such a gradient may be achieved using an aqueous solution of acetate salt such as calcium acetate. In this case the acetate ion gradient is the driving force while the Ca +2 ions, which have very low permeability through the liposome membrane, act as counter-ions of the weak amphiphatic acid within the aqueous phase, thereby stabilizing the loading and enabling better control over the release rate of the loaded weak amphiphatic acid. [Clerc, S. and Barenholz, Y., Loading of amphiphatic weak acids into liposomes in response to transmembrane calcium acetate gradients. Biochim. Biophys. Acta 1240:257-265 (1995)].
• the equilibrium between charged (protonated) and uncharged agents enables the slow leakage of the uncharged weak base from the liposomes at a rate which is dependent on the permeability coefficient. Shifting the equilibrium via formation of aggregates (formed between the loaded charged agent and the counter-ion within the liposome) further improves the retention of the agent inside the liposome, and as now being disclosed, may function as a tool for controlling the release of the agent from the liposome.
• the H + and/or ion gradient is formed by dissolving the liposome-forrning lipid or the mixture comprising a liposome-forming lipid and other lipids (not necessarily liposome-forming lipids, e.g., cholesterol) with a protic organic solvent to form a solution or suspension of said lipid and then adding the lipid solution to an ion-containing aqueous solution to form a liposome suspension.
• the liposome suspension is then incubated with a solution comprising the agent to be loaded and an H + or ion concentration suitable for achieving a respective H + or ion gradient between the inter-liposomal compartment and the intraliposomal surrounding.
• amphiphatic weak acids or amphiphatic weak bases as the active agent/drug
• the agent is loaded by adding it to a suspension of liposomes prepared so as to have an inside/outside pH gradient.
• the uncharged species of amphiphatic weak acid or amphiphatic weak base diffuses trough the liposome membrane.
• the agent is protonated or deprotonated, respectively, to become a charged species.
• the liposomes formed by the method of the invention may be prepared by using an aqueous buffer containing an ammonium salt, such as ammonium sulfate, ammonium phosphate, ammonium citrate, etc., typically about 0.1 M to 0.3 M ammonium salt, at a suitable pH, e.g., about 5.5 to 7.5.
• the gradient can also be produced by including sulfated polymers in the aqueous solution added to the lipid solution.
• such sulfated polymers may include dextran sulfate ammonium salt, heparin sulfate ammonium salt or sucralfate.
• the external medium may be exchanged for one lacking ammonium ions. In this approach, during loading, the amphiphatic weak base is exchanged with ammonium ion.
• An H + ZiOn gradient may also be achieved by including in the liposomes with a selected ionophore.
• liposomes prepared to contain valinomycin in the liposome bilayer are prepared in a potassium buffer, after which the external medium is then exchanged with a sodium buffer, creating a potassium inside/sodium outside gradient. Movement of potassium ions in an inside-to-outside direction in turn generates a lower inside/higher outside H + or ion gradient, presumably due to movement of protons into the liposomes in response to the net electronegative charge across the liposome membranes [Deamer, D. W., et al., Biochim. et Biophys. Acta 274:323
• a similar approach is to add the lipid to an aqueous solution having a high concentration of magnesium sulfate.
• the magnesium sulfate gradient is created by dialysis against 20 mM HEPES buffer, pH 7.4, in sucrose.
• an A23187 ionophore is added, resulting in outwards transport of the magnesium ion in exchange for two protons for each magnesium ion, plus establishing a inner liposome high/outer liposome low proton gradient [Senske DB et al. Biochim. Biophys. Acta 1414: 188-204 (1998)].
• the H + / ion gradient may be formed by using salts having a counter-ion selected from, without being limited thereto: hydroxide; sulfate; phosphate; glucuronate; citrate; carbonate; bicarbonate; nitrate; cyanate; acetate; benzoate; bromide; chloride; other inorganic or organic anions; an anionic polymer such as dextran sulfate, dextran phosphate, dextran borate, carboxymethyl dextran and the like; as well as polyphosphates.
• salts having a counter-ion selected from, without being limited thereto: hydroxide; sulfate; phosphate; glucuronate; citrate; carbonate; bicarbonate; nitrate; cyanate; acetate; benzoate; bromide; chloride; other inorganic or organic anions; an anionic polymer such as dextran sulfate, dextran phosphate, dextran borate,
• the counter-ion may be calcium, magnesium, sodium, ammonium and other inorganic and organic cations, or a cationic polymer such as dextran spermine, dextran spermidine, aminoethyl dextran, trimethyl ammonium dextran, diethylaminoethyl dextran, polyethyleneimine dextran and the like.
• the counter-ion may be present in the form of a free small ion or attached to a polymer, or in both forms simultaneously.
• a specific embodiment for liposomes carrying weak amphiphatic acids is those in which the high inter-liposomal/low intra- liposomal trans-membrane gradient is formed by using an acetate salt, such as calcium acetate, sodium acetate or potassium acetate. Ca 2+ acetate is a preferred acetate salt.
• the release rate of the loaded agent from liposomes was shown to be dependent on a variety of factors, including, without being limited thereto, the counter ion which forms a salt with the active agent (see in this connection WO03/032947, "A method for preparing liposome formulations with a predefined release profile", incorporated herein its entirety by reference), temperature, medium-related properties (medium composition, ionic strength, pH), liposome-related properties (membrane lipid composition, liposome type, number of lamellae, liposome size, physical state of phospholipid membrane i.e., liquid- disordered (LD), liquid-ordered (LO), solid-ordered (SO)), and loaded-molecule-related properties (lipophilicity, hydrophilicity, size) [Haran G., et ah, Biochim Biophys. Acta 1151:201-215, (1993)].
• LD liquid- disordered
• LO liquid-ordered
• SO solid-ordered
• the invention also provides pharmaceutical compositions comprising a physiologically acceptable carrier and an amount of the agent-carrying liposomes prepared in accordance with the invention, the amount being effective to treat or prevent a disease or disorder.
• the pharmaceutical composition may be provided as a single dose, however it may be preferably administered to a subject in need of treatment over an extended period or time (e.g. to produce a cumulative effective amount), in a single daily dose for several days, in several doses a day, etc.
• the treatment regimen and the specific formulation to be administered will depend on the type of disease to be treated and may be determined by various considerations, known to those skilled in the art of medicine, e. g. physicians.
• the term "effective amount” or “amount effective” is used herein to denote the amount of the agent which, when loaded in the liposome, is sufficient in a given therapeutic regimen to achieve a desired therapeutic effect with respect to the treated disease or disorder.
• the amount is determined by such considerations as may be known in the art and depends on the type and severity of the condition to be treated and the treatment regime.
• the effective amount is typically determined in appropriately designed clinical trials (dose range studies) and the person versed in the art will know how to properly conduct such trials in order to determine the effective amount.
• an effective amount depends on a variety of factors, including the mode of administration, type of vehicle carrying the amphipathic weak acid/base, the reactivity of the active agent (the weak amphiphatic acid or base), the liposome's distribution profile within the body, a variety of pharmacological parameters such as half-life in the body after being released from the liposome, undesired side effects, if any, factors such as age and gender of the treated subject, etc.
• administering is used to denote the contacting or dispensing, delivering or applying of the liposomal formulation to a subject by any suitable route of delivery thereof to the desired location in the subject, including oral, parenteral (including subcutaneous, intramuscular and intravenous, intra-arterial, intraperitoneal, etc.) and intranasal administration, as well as intrathecal and infusion techniques.
• the composition used in accordance with the invention is in a form suitable for injection.
• the requirements for effective pharmaceutical vehicles for injectable formulations are well known to those of ordinary skill in the art [See Pharmaceutics and Pharmacy Practice, J.B.
• the invention concerns a method of treating a subject for a disease or disorder, the method comprising administering to said subject an amount of agent-carrying liposomes prepared by the method of the invention.
• treatment denotes curing of an undesired pathological condition or prevention of a condition from developing.
• treatment includes ameliorating undesired symptoms associated with the condition, slowing down progression of the condition, delaying the onset of a progressive stage of the condition, slowing down deterioration of such symptoms, enhancing onset of a remission period of the condition, if existing, delaying onset of a progressive stage, improving survival rate or more rapid recovery from the condition, lessening the severity of or curing the condition, etc.
• Treatment also includes prevention of a disease or disorder.
• prevention includes, without being limited thereto, administering an amount of the composition to prevent the condition from developing or to prevent irreversible damage caused by the condition, to prevent the manifestation of symptoms associated with the condition before they occur, to inhibit the progression of the condition etc.
• lipid includes one or more, of the same or different lipids.
• the term “comprising” is intended to mean that the liposome includes the recited constituents, but does not exclude others which may be optional in the formation or composition of the liposome, such as antioxidants, cryoprotectants, etc.
• the term “consisting essentially of” is used to define a substance, e.g. liposome, that includes the recited constituents but excludes other constituents that may have an essential significant effect on a parameter of the substance (e.g., in the case 006/001229
• Hydrogenated soy phosphatidylcholine (hereinafter referred to by the abbreviation HSPC) was obtained from Lipoid, Ludwigsahfen, Germany.
• Cholesterol was obtained from Sigma.
• BUP Bupivacaine hydrochloride
• LID Lidocaine hydrochloride
• DMPC Dimyristoylphosphatidylcholine
• DPPC DPPC
• MLV liposomes were prepared by weighing 450 mg of dry HSPC and 154 mg of dry cholesterol (a 60:40 mole ratio). The dry phospholipid/cholesterol mixture was then dissolved in 1 ml ethanol at 80°C and the dissolved mixture was added to an aqueous solution of (NHU) 2 SO 4 (250 niM, prepared by adding 297 mg of ammonium sulfate to 9 ml of water), to obtain a preparation having a final phospholipid concentration of 60 mM. Ethanol volume was 10% of final volume. The thus obtained MLVs were heated at 65 0 C for 45 min.
• LMW large multi-vesicular vesicles
• MLV prepared as above were freeze-thawed either once or more (up to a total of
• Freezing was performed using liquid nitrogen (-196°C) and thawing was performed using a water bath (37°C). Freezing time was proportional to the volume of liposome preparation such that for each milliliter of preparation, one minute freezing was executed (i.e. for 10 ml, 10 minute freezing took place).
• the liposome preparation was centrifuged 4 times sequentially in normal saline (4 0 C, 1000 g, 5 min). This is effective to create an inside-to-outside ammonium ion gradient across the liposomal membrane.
• the ammonium ion concentration gradient provides the driving force for loading of amph philic weak bases such as Bupivacaine (BUP).
• BUP Bupivacaine
• the presence of a transmembrane pH gradient was verified by determining the distribution of amphiphatic weak base acridine orange (AO), as described in Haran, G. et al. Biochim. Biophys. Acta 1151:201-215 (1993) and Clerc S., Barenholz Y. Anal Biochem. 259(l):104-ll (1998).
• AO amphiphatic weak base acridine orange
• the drug, BUP or LID was remote-loaded into the liposomes by incubating the liposome preparation with 4.5% of appropriate drug solution (50 mg/ml solution of drug) at 60°C for 45 min.
• Non-entrapped drug was removed from LMVV suspension by centrifugation in normal saline (4°C, 1000 g, 5 min). The pH of the final medium was about 5.5. This pH was retained to ensure the drug's solubility and prevent precipitation.
• HPLC high performance liquid chromatography
• the amount of trapped and free drug after one day of storage and for at least one month after storage was measured using high performance liquid chromatography (HPLC) (Grant G. et al. Pharm. Res., 18 N3:336-343 (2001) and Grant G. et al. Anasthesiology, 101:133-137 (2004)); the amount of phospholipid in the liposomal formulation was determined using the Bartlett method (Shmeeda, H. et al. Methods Enzymol. 367:272-292 (2003)). The drug-to-lipid ratio was calculated from the parameters obtained.
• the drug-to-lipid ratio obtained in the liposomal formulation prepared as described above was greater than 2 (mole drug/mole lipid > 2).
• the size of the liposomes was determined using laser Fraunhofer diffraction (LS 13320 Laser Diffraction Particle Sizer Analyzer, Beckman Coulter UK). The instrument's Software expresses particle size as the volume median diameter. The mean size of LMVV was ⁇ 8.5 ⁇ 6.5 micron. The level of transmembrane pH gradient according to AO distribution was ⁇ 89% verifying a low inter-liposome /high intra- liposome transmembrane pH gradient, being larger than 3. Kinetics of Drug Loaded Liposome
• Table 1 Kinetics of BUP leakage from liposomes.
• Table 3 shows that the number of F&T had no significant effect on the drug-lipid ratio. Further, the size distribution of the liposomes was measured as a function of the number of F&T.
• Fraunhofer.rf780d for size distribution calculation
• a precision size standard diameter of 1.27 ⁇ m Cat No. 64035 of Polyscience, Inc
• PL denotes phospholipid

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Medicinal Chemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Epidemiology (AREA)
• Dispersion Chemistry (AREA)
• Animal Behavior & Ethology (AREA)
• General Health & Medical Sciences (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Medicinal Preparation (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Manufacturing Of Micro-Capsules (AREA)

Priority Applications (5)

Applications Claiming Priority (2)

Publications (2)

Family

ID=37968217

Family Applications (1)

Country Status (5)

Cited By (14)

Families Citing this family (1)

Citations (5)

• 2006
  • 2006-10-26 WO PCT/IL2006/001229 patent/WO2007049278A2/en active Application Filing
  • 2006-10-26 EP EP06809790A patent/EP1954243A2/en not_active Withdrawn
  • 2006-10-26 AU AU2006307460A patent/AU2006307460A1/en not_active Abandoned
  • 2006-10-26 CA CA002627657A patent/CA2627657A1/en not_active Abandoned
  • 2006-10-26 JP JP2008537312A patent/JP2009513621A/ja active Pending

Patent Citations (5)

Also Published As

Similar Documents

Legal Events