WO2015108932A1 - Liposome compositions encapsulating modified cyclodextrin complexes and uses thereof - Google Patents
Liposome compositions encapsulating modified cyclodextrin complexes and uses thereof Download PDFInfo
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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
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/16—Amides, e.g. hydroxamic acids
- A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
- A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
- A61K31/525—Isoalloxazines, e.g. riboflavins, vitamin B2
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
- A61K47/40—Cyclodextrins; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
- A61K47/6951—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1277—Processes for preparing; Proliposomes
- A61K9/1278—Post-loading, e.g. by ion or pH gradient
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
- C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
Definitions
- Nanoparticles carrying drugs can increase this therapeutic ratio over that achieved with the free drug through several mechanisms.
- drugs delivered by nanoparticles are thought to selectively enhance the concentration of the drugs in tumors as a result of the enhanced permeability and retention (EPR) effect (Peer et al. (2007) Nat. Nanotech. 2:751- 760; Gubernator (2011) Exp. Opin. DrugDeliv. 8:565-580; Huwyler et al. (2008) Int. J. Nanomed. 3:21-29; Maruyama et al. (2011) Adv. Drug Deliv. Rev.
- EPR enhanced permeability and retention
- liposomal drug loading is achieved by either passive
- Passive loading involves dissolution of dried lipid films in aqueous solutions containing the drug of interest. This approach can only be used for water-soluble drugs, and the efficiency of loading is often low.
- active loading can be extremely efficient, resulting in high intraliposomal concentrations and minimal wastage of precious chemotherapeutic agents (Gubernator (2011) Exp. Opin. Drug Deliv. 8:565-580; Fenske and Cullis (2008) Liposome Nanomed. 5:25-44; and Barenholz (2003) J. Liposome Res. 13:1-8).
- active loading drug internalization into preformed liposomes is typically driven by a transmembrane pH gradient. The pH outside the liposome allows
- liposomal composition encapsulating therapeutic agents having various chemical properties (e.g. , nonionic and/or hydrophobic) at high efficiencies and large concentrations.
- the present invention is based in part on the discovery that cyclodextrins bearing ionizable functional groups (e.g., weakly basic and/or weakly acidic functional groups on their solvent-exposed surfaces, such as liposome internal phase surfaces) are able to efficiently encapsulate a therapeutic agent (e.g. , non-ionizable and/or hydrophobic compositions) at high concentrations and that the functionalized cyclodextrins containing the therapeutic agent can themselves be efficiently remotely loaded into liposomes at high concentrations to generate liposome compositions exhibiting unexpectedly reduced toxicity and enhanced efficacy properties when administered in vivo (see, for example, Figure 1C).
- Cyclodextrins are a family of cyclic sugars that are commonly used to solubilize hydrophobic drugs (Albers and Muller (1995) Crit. Rev. Therap. Drug Carrier Syst.
- a liposome composition comprising a cyclodextrin, a therapeutic agent, and a liposome, wherein the liposome encapsulates a cyclodextrin having at least one hydroxyl chemical group facing the liposome internal phase replaced with an ionizable chemical group and wherein the cyclodextrin encapsulates the therapeutic agent is provided.
- At least one a-D-glucopyranoside unit of the cyclodextrin has at least one hydroxyl chemical group selected from the group consisting of C2, C3, and C6 hydroxyl chemical groups that are replaced with an ionizable chemical group.
- at least one a-D-glucopyranoside unit of the cyclodextrin has at least two hydroxyl chemical groups selected from the group consisting of C2, C3, and C6 hydroxyl chemical groups that are replaced with ionizable chemical groups.
- the C2, C3, and C6 hydroxyl chemical groups of at least one a-D- glucopyranoside unit of the cyclodextrin that are replaced with ionizable chemical groups are replaced with ionizable chemical groups.
- the at least one ⁇ -D-glucopyranoside unit of the cyclodextrin is selected from the group consisting of two, three, four, five, six, seven, eight, and all a-D- glucopyranoside units of the cyclodextrin.
- the ionizable chemical group is the same at all replaced positions.
- the ionizable chemical group is a weakly basic functional group (e.g., a group X that has a pK a between 6.5 and 8.5 according to CH3-X) or a weakly acidic functional group (e.g., a group Y that has a pK a between 4.0 and 6.5 according to CH 3 -Y).
- the weakly basic or weakly acidic functional groups are selected from the group consisting of amino, ethylene diamino, dimethyl ethylene diamino, dimethyl anilino, dimethyl naphthylamino, succinyl, carboxyl, sulfonyl, and sulphate functional groups.
- the cyclodextrin has a pK a i of between 4.0 and 8.5.
- the composition is a liquid or solid pharmaceutical formulation.
- the therapeutic agent is neutrally charged or hydrophobic.
- the therapeutic agent is a chemotherapeutic agent.
- the therapeutic agent is a small molecule.
- the cyclodextrin is selected from the group consisting of ⁇ -cyclodextrin, a-cyclodextrin, and ⁇ - cyclodextrin. In another embodiment, the cyclodextrin is ⁇ -cyclodextrin, a-cyclodextrin.
- kits comprising a liposome composition described herein, and instructions for use, is provided.
- a method of treating a subject having a cancer comprising administering to the subject a therapeutically effective amount of a liposome composition described herein, is provided.
- the therapeutic agent is a
- the liposome composition is administered by injection subcutaneous ly or intravenously.
- the subject is a mammal.
- the mammal is a human.
- Figures 1A-1C show an embodiment of a schematic representation of active loading of a liposome.
- Figure 1A shows remote loading of an ionizable hydrophilic drug using a transmembrane pH gradient results in efficient incorporation.
- Figure IB shows that poorly soluble hydrophobic drug result in meager incorporation into pre-formed liposomes under the conditions shown in Figure 1A.
- R H, ionizable alkyl or aryl groups
- Figures 2A-2C show embodiments of synthesized ionizable cyclodextrins.
- Figure 2A shows an embodiment of a chemical reaction to form some of the presently disclosed synthesized ionizable cyclodextrins.
- Figure 2B shows some embodiments of the presently disclosed synthesized ionizable cyclodextrins bearing ionizable groups at their 6 '-position.
- Figure 2C depicts the toroidal shape of a cyclodextrin.
- Figures 3A-3D show the active loading of modified ⁇ -cyclodextrin using a transmembrane pH gradient.
- Figure 3A shows fluorescence of ⁇ -cyclodextrin V in relative fluorescence units (RLU) loaded into liposomes with a pH gradient (citrate liposomes) compared to that of the same compound loaded into liposomes in the absence of a pH gradient (PBS liposomes).
- Figure 3B shows dynamic light scattering measurements demonstrating a marginal increase in hydrodynamic radius, but no change in the polydispersity index (PDI) of liposomes remotely loaded with cyclodextrin V.
- RLU relative fluorescence units
- Figures 3C- 3D show cryoTEM images of dansylated ⁇ -cyclodextrin V loaded with a pH gradient (citrate liposomes; Figure 3C) or without a pH gradient (PBS liposomes; Figure 3D).
- Figure 4 shows the incorporation of dansylated cyclodextrins into citrate liposomes by analyzing fluorescence in relative fluorescence units (RLU) of dansylated I and cyclodextrin IV in citrate liposomes versus control (PBS) liposomes.
- RLU relative fluorescence units
- Figures 5A-5D show the remote loading of insoluble hydrophobic dyes into liposomes using modified ⁇ -cyclodextrins as seen by fluorescence intensity of remotely loaded coumarin 102 (Figure 5A), coumarin 314 ( Figure 5B), coumarin 334 ( Figure 5C), and cyclohexyl DNP ( Figure 5D).
- Insets show photographs of the vials containing the liposomes incubated with the cyclodextrin-encapsulated dye (top) or free dye (bottom).
- Figure 6 shows the ability of various cyclodextrins to transfer coumarin 314 into citrate liposomes. Fluorescence in relative fluorescence units (RLU) of uncomplexed coumarin 314 and coumarin 314 complexed with III (ionizable mono-6-ethylenediamino- 6'deoxy-cyclodextrin) and I (unionizable ⁇ -cyclodextrin) followed by remote loading into citrate liposomes is shown.
- RLU relative fluorescence units
- Figure 7 shows the structure and physical properites of BI-2536 and PD-0325901.
- Figures 8A-8C show the loading and activity of the PL 1 inhibitor, BI-2536L.
- Figure 8C shows the results of nude mice bearing HCT 116 xenografts treated with a single i.v. dose of (i) empty liposomes, (ii) BI-2536 (100 mg/kg), or (iii) CYCL-BI-2536 (100 mg/kg). Neutrophils were counted before any drug treatment and every 24 hours thereafter. Means and standard deviations (SD) of the neutrophil counts of five mice in each treatment arm are shown.
- SD standard deviations
- Figure 9 shows the tissue biodistribution of CYCL-coumarin 334 at the 2, 24, and 48 hour time points as histograms from left to right for each tissue, respectively, as indicated. Data are presented as the mean and standard deviation.
- Figures 10A-10B show the loading and activity of the MEK1 inhibitor, PD-
- Figure 10A shows survival curves of animals treated with a single dose of PD- 0325901 and CYCL-PD-0325901.
- Figure 10B shows the results of nude mice bearing RKO xenografts treated with 2 i.v.
- Figures 11A-11C show the anti-tumor activity of CYCL-BI-2536 and CYCL- PD0325901 in a second xenograft model. Liposomal formulations have been reported as equivalents of free drug. The relative tumor volumes and standard deviation of each experimental arm is shown.
- cyclodextrins bearing ionizable functional groups e.g., weakly basic and/or weakly acidic functional groups on their solvent-exposed surfaces, such as liposome internal phase surfaces
- a therapeutic agent e.g., non-ionizable and/or hydrophobic compositions
- the present invention provides, at least in part, liposome compositions and kits comprising such modified cyclodextrins and therapeutic agents, as well as methods of making and using such compositions and kits.
- cyclodextrin refers to a family of cyclic oligosaccharides composed of 6 or more -D-glucopyranoside units linked together by C1-C4 bonds having a toroidal topological structure, wherein the larger and the smaller openings of the toroid expose certain hydroxyl groups of the a-D-glucopyranoside units to the surrounding environment (e.g., solvent).
- inert cyclodextrin refers to a cyclodextrin containing a-D- glucopyranoside units having the basic formula C 6 Hi 2 0 6 and glucose structure without any additional chemical substitutions (e.g.
- cyclodextrin internal phase refers to the relatively less hydrophilic region enclosed within (i.e., encapsulated by) the toroid topology of the cyclodextrin structure.
- cyclodextrin external phase refers to the region not enclosed by the toroid topology of the cyclodextrin structure and can include, for example, the liposome internal phase when the cyclodextrin is encapsulated within a liposome.
- Cyclodextrins are useful for solubilizing hydrophobic compositions (see, for example, Albers and Muller (1995) Crit. Rev. Therap. Drug Carrier Syst. 12:311-337; Zhang and Ma (2013) Adv. Drug Delivery Rev. 65:1215-1233; Laza-Knoerr et al. (2010) J. Drug Targ. 18:645-656; Challa ei al. (2005) AAPS PharmSci. Tech. 6:E329-357; Uekama et al. (1998) Chem. Rev. 98:2045- 2076; Szejtli (1998) Chem. Rev. 98: 1743-1754; Stella and He (2008) Toxicol. Pathol.
- cyclodextrins there are no particular limitations on the cyclodextrin so long as the cyclodextrins (a) can encapsulate a desired therapeutic agent and (b) bear ionizable (e.g., weakly basic and/or weakly acidic) functional groups to facilitate encapsulation by liposomes.
- ionizable e.g., weakly basic and/or weakly acidic
- cyclodextrins can be selected and/or chemically modified according to the characteristics of the desired therapeutic agent and parameters for efficient, high-concentration loading therein.
- the cyclodextrin itself have high solubility in water in order to facilitate entrapment of a larger amount of the cyclodextrin in the liposome internal phase.
- the water solubility of the cyclodextrin is at least 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL or higher. Methods for achieving such enhanced water solubility are well known in the art.
- a large association constant with the therapeutic agent is preferable and can be obtained by selecting the number of glucose units in the cyclodextrin based on the size of the therapeutic agent (see, for example, Albers and Muller (1995) Crit. Rev. Therap. Drug Carrier Syst. 12:311-337; Stella and He (2008) Toxicol. Pathol. 36:30- 42; and Rajewski and Stella (1996) J. Pharm. Sci. 85: 1142-1169).
- the association constant depends on pH
- the cyclodextrin can be selected such that the association constant becomes large at the pH of the liposome internal phase.
- the solubility (nominal solubility) of the therapeutic agent in the presence of cyclodextrin can be further improved.
- the association constant of the cyclodextrin with the therapeutic agent can be 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, or higher.
- cyclodextrin hydroxyl groups e.g., those lining the upper and lower ridges of the toroid of an inert cyclodextrin
- cyclodextrin hydroxyl groups e.g., those lining the upper and lower ridges of the toroid of an inert cyclodextrin
- modifying hydroxyl groups such as those facing away from the cyclodextrin interior phase, can be replaced with ionizable chemical groups to facilitate loading into liposomes as well as loading of therapeutic agents, such as poorly soluble or hydrophobic agents, within the modified cyclodextrins.
- a modified cyclodextrin having at least one hydroxyl group substituted with an ionizable chemical group will result in a charged moiety under certain solvent (e.g. , pH) conditions.
- charged cyclodextrin refers to a cyclodextrin having one or more of its hydroxyl groups substituted with a charged moiety and the moiety bearing a charge.
- a moiety can itself be a charged group or it can comprise an organic moiety (e.g., a Ci-C6 alkyl or Ci-C6 alkyl ether moiety) substituted with one or more charged moieties.
- the "ionizable" or “charged” moieties are weakly ionizable.
- Weakly ionizable moieties are those that are either weakly basic or weakly acidic.
- Weakly basic functional groups (X) have a pKa of between about 6.0-9.0, 6.5-8.5, 7.0-8.0, 7.5-8.0, and any range in between inclusive according to CH 3 -X.
- weakly acidic functional groups (Y) have a log dissociation constant (pKa) of between about 3.0-7.0, 4.0- 6.5, 4.5-6.5, 5.0-6.0, 5.0-5.5, and any range in between inclusive according to CH3-Y.
- the pKa parameter is a well-known measurement of acid/base properties of a substance and methods for pKa determination are conventional and routine in the art.
- the pKa values for many weak acids are tabulated in reference books of chemistry and pharmacology. See, for example, IUPAC Handbook of Pharmaceutical Salts, ed. by P. H. Stahl and C. G Wermuth, Wiley-VCH, 2002; CRC Handbook of Chemistry and Physics, 82nd Edition, ed. by D. R. Lide, CRC Press, Florida, 2001, p. 8-44 to 8-56. Since cyclodextrins with more than one ionizable group have pKa of the second and subsequent groups each denoted with a subscript.
- anionic moieties include, without any limitation, carboxylate, carboxymethyl, succinyl, sulfonyl, phosphate, sulfoalkyl ether, sulphate carbonate, thiocarbonate, dithiocarbonate, phosphate, phosphonate, sulfonate, nitrate, and borate groups.
- Representative cationic moieties include, without limitation, amino, guanidine, and quarternary ammonium groups.
- the modified cyclodextrin is a "polyanion” or "polycation.”
- a polyanion is a modified cyclodextrin having more than one negatively charged group resulting in net negative ionic charger of more than two units.
- a polycation is a modified cyclodextrin having more than one positively charged group resulting in net positive ionic charger of more than two units.
- the modified cyclodextrin is a "chargeable amphiphile.”
- chargeable is meant that the amphiphile has a pK in the range pH 4 to pH 8 or 8.5.
- a chargeable amphiphile may therefore be a weak acid or base.
- amphoteric herein is meant a modified cyclodextrin having a ionizable groups of both anionic and cationic character wherein: 1) at least one, and optionally both, of the cation and anionic amphiphiles is chargeable, having at least one charged group with a pK between 4 and 8 to 8.5, 2) the cationic charge prevails at pH 4, and 3) the anionic charge prevails at pH 8 to 8.5.
- the "ionizable" or “charged” cyclodextrins as a whole, whether polyionic, amphiphilic, or otherwise, are weakly ionizable (i.e., have a p ai of between about 4.0-8.5, 4.5-8.0, 5.0-7.5, 5.5-7.0, 6.0-6.5, and any range in between inclusive).
- Any one, some, or all hydroxyl groups of any one, some or all a-D-glucopyranoside units of a cyclodextrin can be modified to an ionizable chemical group as described herein. Since each cyclodextrin hydroxyl group differs in chemical reactivity, reaction with a modifying moiety can produce an amorphous mixture of positional and optical isomers. Alternatively, certain chemistry can allow for pre-modified ⁇ -D-glucopyranoside units to be reacted to form uniform products.
- the aggregate substitution that occurs is described by a term called the degree of substitution.
- a 6-ethylenediamino- -cyclodextrin with a degree of substitution of seven would be composed of a distribution of isomers of 6-ethylenediamino - ⁇ -cyclodextrin in which the average number of ethylenediamino groups per 6- ethylenediamino- -cyclodextrin molecule is seven.
- Degree of substitution can be determined by mass spectrometry or nuclear magnetic resonance spectroscopy.
- the maximum degree of substitution is 18 for a-cyclodextrin, 21 for ⁇ , and 24 for ⁇ -cyclodextrin, however, substituents themselves having hydroxyl groups present the possibility for additional hydroxylalkylations.
- the degree of substitution can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more and can encompass complete substitution.
- Another parameter is the stereochemical location of a given hydroxyl substitution. In one embodiment, at least one hydroxyl facing away from the cyclodextrin interior is substituted with an ionizable chemical group.
- the C2, C3, C6, C2 and C3, C2 and C6, C3 and C6, and all three of C2-C3-C6 hydroxyls of at least one a-D- glucopyranoside unit are substituted with an ionizable chemical group. Any such combination of hydroxyls can similarly be combined with at least two, three, four, five, six, seven, eight, nine, ten, eleven, up to all of the a-D-glucopyranoside units in the modified cyclodextrin as well as in combination with any degree of substitution described herein.
- liposome refers to a microscopic closed vesicle having an internal phase enclosed by lipid bilayer.
- a liposome can be a small single-membrane liposome such as a small unilamellar vesicle (SUV), large single-membrane liposome such as a large unilamellar vesicle (LUV), a still larger single-membrane liposome such as a giant unilamellar vesicle (GUV), a multilayer liposome having multiple concentric membranes such as a multilamellar vesicle (MLV), or a liposome having multiple membranes that are irregular and not concentric such as a multivesicular vesicle (MVV).
- SUV small unilamellar vesicle
- LUV large unilamellar vesicle
- GUI giant unilamellar vesicle
- MLV multilamellar vesicle
- liposome internal phase refers to an aqueous region enclosed within (i.e., encapsulated by) the lipid bilayer of the liposome.
- liposome external phase refers to the region not enclosed by the lipid bilayer of the liposome, such as the region apart from the internal phase and the lipid bilayer in the case where the liposome is dispersed in liquid.
- the liposome so long as it can encapsulate the modified cyclodextrins harboring therapeutic agents.
- the liposome has a barrier function that prevents the modified
- the liposome exhibits in vivo stability and has a barrier function that prevents all of the modified cyclodextrin/therapeutic agent complexes from leaking to the liposome external phase in blood when the liposome is administered in vivo.
- the membrane constituents of the liposome include phospholipids and/or phospholipid derivatives.
- phospholipids and phospholipid derivatives include, without limitation, phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol, cardiolipin, sphingomyelin, ceramide phosphorylethanolamine, ceramide phosphoryl glycerol, ceramide phosphoryl glycerol phosphate, 1 ,2-dimyristoyl- 1,2-deoxyphosphatidyl choline, plasmalogen, and phosphatidic acid. It is also acceptable to combine one or more of these phospholipids and phospholipid derivatives.
- fatty-acid residues in the phospholipids and phospholipid derivatives can include saturated or unsaturated fatty-acid residues having a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or longer.
- acyl groups derived from fatty-acid such as lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, and linoleic acid.
- Phospholipids derived from natural substances such as egg-yolk lecithin and soy lecithin, partially hydrogenated egg-yolk lecithin, (completely) hydrogenated egg-yolk lecithin, partially hydrogenated soy lecithin, and (completely) hydrogenated soy lecithin whose unsaturated fatty-acid residues are partially or completely hydrogenated, and the like, can also be used.
- the mixing amount (mole fraction) of the phospholipids and/or phospholipid derivatives that are used when preparing the liposome there are no particular limitations on the mixing amount (mole fraction) of the phospholipids and/or phospholipid derivatives that are used when preparing the liposome. In one embodiment, 10 to 80% relative to the entire liposome membrane composition can be used. In another embodiment, a range of between 30 to 60% can be used.
- the liposome can further include sterols, such as cholesterol and cholestanol as membrane stabilizers and fatty acids having saturated or unsaturated acyl groups, such as those having a carbon number of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or longer.
- sterols such as cholesterol and cholestanol as membrane stabilizers
- fatty acids having saturated or unsaturated acyl groups such as those having a carbon number of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or longer.
- the mixing amount (mole fraction) of these sterols that are used when preparing the liposome but 1 to 60% relative to the entire liposome membrane composition is preferable, 10 to 50% is more preferable, and 30 to 50% is even more preferable.
- the mixing amount (mole fraction) of the fatty acids but 0 to 30% relative to the entire liposome membrane composition is preferable, 0 to 20% is more preferable, and 0 to 10% is even more preferable.
- the mixing amount (mole fraction) of the antioxidants it is sufficient if an amount is added that can obtain the antioxidant effect, but 0 to 15% of the entire liposome membrane composition is preferable, 0 to 10% is more preferable, and 0 to 5% is even more preferable.
- the liposome can also contain functional lipids and modified lipids as membrane constituents.
- functional lipids include lipid derivatives retained in blood (e.g., glycophorin, ganglioside GM1 , ganglioside GM3, glucuronic acid derivatives, glutaminic acid derivatives, polyglycerin phospholipid derivatives, polyethylene glycol derivatives (methoxypolyethylene glycol condensates, etc.) such as N-[carbonyl-methoxy polyethylene glyco 1-2000] -1 , 2-dipalmitoyl-sn-glycero-3- phosphoethanolamine, N-[carbonyl-methoxy polyethylene glycol-5000]-l,2-dipalmitoyl- sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxy polyethylene glycol-750]-l,2- distearoyl-sn glycero-3-phosphoethanolamine, N-[carbonyl-methoxy polyethylene
- phosphoethanolamine and methoxy polyethylene glycol phosphoethanolamine and methoxy polyethylene glycol
- temperature-sensitive lipid derivatives e.g., dipalmitoyl phosphatidylcholine
- pH-sensitive lipid derivatives e.g., dioleoyl phosphatidyl ethanolamine
- Liposomes containing lipid derivatives retained in blood are useful for improving the blood retention of the liposome, because the liposome becomes difficult to capture in the liver as a foreign impurity.
- liposomes containing temperature-sensitive lipid derivatives are useful for causing destruction of liposome at specific temperatures and/or causing changes in the surface properties of the liposome.
- Liposomes containing pH-sensitive lipid derivatives are useful for enhancing membrane fusion of liposome and endosome when the liposome is incorporated into cells due to the endocytosis to thereby improve transmission of the therapeutic agent to the cytoplasm.
- modified lipids include PEG lipids, sugar lipids, antibody-modified lipids, peptide-modified lipids, and the like. Liposomes containing such modified lipids can be targeted to desired target cells or target tissue. Also, there are no particular limitations on the mixing amount (mole fraction) of functional lipids and modified lipids used when preparing the liposome. In some embodiments, such lipids make up 0-50%, 0-40%, 0-30%, 0-20%, 0-15%, 0-10%, 0-5%, 0-1% or less of the entirety of liposome membrane constituent lipids.
- composition of the liposome membrane constituents having such membrane permeability at a level allowing practical application can be appropriately selected by those skilled in the art according to the therapeutic agent, target tissue, and the like.
- the therapeutic agent/cyclodextrin complex be released from the liposome after the liposome reaches the target tissue, cells, or intracellular organelles. It is believed that the liposome compositions described herein contain membrane constituents themselves are ordinarily biodegradable, and ultimately decompose in target tissue or the like and that the encapsulated therapeutic
- agent/cyclodextrin complex is thereby released through dilution, chemical equilibrium, and/or enzymatic cyclodextrin degradation effects.
- the particle size of the liposome can be regulated.
- liposome particle size when it is intended to transmit liposome to cancerous tissue or inflamed tissue by the Enhanced Permeability and Retention (EPR) effect as an injection product or the like, it is preferable that liposome particle size be 30-400 nm, 50-200 nm, 75- 1 0 nm, and any range in between.
- liposome particle size In the case where the intention is to transmit liposome to macrophage, it is preferable that liposome particle size be 30 to 1000 nm, and it is more preferable that the particle size be 100 to 400 nm.
- the particle size of liposome can be set at several microns.
- vascular walls serve as barriers (because the vascular walls are densely constituted by vascular endothelial cells), and microparticles such as supermolecules and liposome of specified size cannot be distributed within the tissue.
- vascular walls are loose (because interstices exist between vascular endothelial cells), increasing vascular permeability, and supermolecules and microparticles can be distributed to extravascular tissue (enhanced permeability).
- the lymphatic system is well developed in normal tissue, but it is known that the lymphatic system is not developed in diseased tissue, and that
- liposome particle size refers to the weight-average particle size according to a dynamic light scattering method (e.g., quasi-elastic light scattering method).
- a dynamic light scattering method e.g., quasi-elastic light scattering method
- liposome particle sizes can be measured using dynamic light scattering instruments (e.g. , Zetasizer Nano ZS model manufactured by Malvern Instruments Ltd. and ELS-8000 manufactured by Otsuka Electronics Co., Ltd.). The instruments measure Brownian motion of the particles and particle size is determined based on established dynamic light scattering methodological theory.
- exemplary buffer solutions include, without limitation, as phosphate buffer solution, citrate buffer solution, and phosphate-buffered physiological saline solution, physiological saline water, culture mediums for cell culturing, and the like.
- concentration of buffer agent be 5 to 300 mM, 10 to 100 mM, or any range in between.
- pH of the liposome internal phase In some embodiments, the liposome internal phase has a pH between 2 and 11, 3 and 9, 4 and 7, 4 and 5, and any range in between inclusive.
- -cyclodextrin has an internal phase pore diameter size of 0.45-0.6
- ⁇ - cyclodextrin has an internal phase pore diameter size of 0.6 to 0.8 nm
- ⁇ -cyclodextrin has an internal phase pore diameter size of 0.8 to 0.95 nm.
- the cyclodextrin can be chosen to match the size of the therapeutic agent to allow for encapsulation.
- modifications to the non-carbon cyclodextrin groups can be selected to modulate intermolecular interactions between the cyclodextrin and the therapeutic agent to thereby modulate encapsulation of the therapeutic agent by the cyclodextrin.
- any desired agent can be used, such as those useful in the fields of medicines (including diagnostic drugs), cosmetic products, food products, and the like.
- the therapeutic agent can be selected from a variety of known classes of useful agents, including, for example, proteins, peptides, nucleotides, anti-obesity drugs, nutriceuticals, corticosteroids, elastase inhibitors, analgesics, anti-fungals, oncology therapies, anti-emetics, analgesics, cardiovascular agents, anti-inflammatory agents, anthelmintics, anti-arrhythmic agents, antibiotics (including penicillins), anticoagulants, antidepressants, antidiabetic agents, antiepileptics, antihistamines, antihypertensive agents, antimuscarinic agents, antimycobacterial agents, antineoplastic agents,
- immunosuppressants antithyroid agents, antiviral agents, anxiolytic sedatives (hypnotics and neuroleptics), astringents, beta-adrenoceptor blocking agents, blood products and substitutes, cardiac inotropic agents, contrast media, corticosteroids, cough suppressants (expectorants and mucolytics), diagnostic agents, diagnostic imaging agents, diuretics, dopaminergics (antiparkinsonian agents), haemostatics, immunological agents, lipid regulating agents, muscle relaxants, parasympathomimetics, parathyroid calcitonin and biphosphonates, prostaglandins, radio-pharmaceuticals, sex hormones (including steroids), anti-allergic agents, stimulants and anoretics, sympathomimetics, thyroid agents, vasodilators and xanthines. With respect to therapeutic agents, it is acceptable to combine one or more agents.
- the therapeutic agents can be low-molecular compounds, such as small molecules.
- compounds used as antitumor agents, antibacterial agents, anti-inflammatory agents, anti-myocardial infarction agents, and contrast agents are suitable.
- the liposome membrane permeability of the therapeutic agent is generally satisfactory according to the compositions described herein.
- anti-neoplastic or anti-tumor agents include, without limitation, BI-2536, PD- 0325901, camptothecin; taxane; iphosphamide, nimstine hydrochloride, carvocon, cyclophosphamide, dacarbazine, thiotepa, busulfan, melfaran, ranimustine, estramustine phosphate sodium, 6-mercaptopurine riboside, enocitabine, gemcitabine hydrochloride, carmfur, cytarabine, cytarabine ocfosfate, tegafur, doxifluridine, hydroxycarbamide, fluorouracil, methotrexate, mercaptopurine, fludarabine phosphate, actinomycin D, aclarubicin hydrochloride, idarubicin hydrochloride, pirarubicin hydrochlor
- any salt is acceptable and free bodies are also acceptable.
- any salt is acceptable.
- antibacterial agents include, without limitation, amfotericine B, cefotiam hexyl, cephalosporin, chloramphenicol, diclofenac, and the like. With respect to compounds of the
- any salt is acceptable.
- anti-inflammatory agents there are no particular limitations on anti-inflammatory agents.
- Representative examples include, without limitation, prostaglandins (PGE1 and PGE2), dexamethasone, hydrocortisone, pyroxicam, indomethacin, prednisolone, and the like. With respect to compounds of the aforementioned anti-inflammatory agents, any salt is acceptable.
- Representative examples include, without limitation, adenosine, atenolol, pilsicamide, and the like. With respect to compounds of the aforementioned anti-myocardial infarction agents, any salt is acceptable.
- contrast agents there are also no particular limitations on contrast agents. Representative examples include, without limitation, iopamidol, ioxaglic acid, iohexyl, iomeprol, and the like. With respect to the contrast agents, any salt is acceptable.
- the therapeutic agent is "poorly water soluble" or
- hydrophobic which terms are used interchangeably to encompass therapeutic agents that are sparingly soluble in water, as evidenced by a room temperature water solubility of less than about 10 mg/mL, 9 mg/mL, 8 mg/mL, 7 mg/mL, 6 mg/mL, 5 mg/mL, 4 mg/mL, 3 mg/mL, 2 mg/mL, 1 mg/mL, 900 ⁇ g/mL, 800 ⁇ g mL, 700 ⁇ g/mL, 600 ⁇ g/mL, 500 ⁇ g/mL, 400 ⁇ g mL, 300 ⁇ g/mL, 200 ⁇ g/mL, 100 ⁇ g/mL, 95 ⁇ g/mL, 90 ⁇ g/mL, 85 ⁇ g/mL, 80 ⁇ g/mL, 75 ⁇ g/mL, 70 ⁇ g mL, 65 ⁇ g mL, 60 ⁇ g/mL, 55 ⁇ ,, and in some cases less than about 50 ⁇ g
- the term "slightly soluble" is applicable when one part of an agent can be solubilized by 100 to 1000 parts of solvent (e.g., water). It will be appreciated that the room temperature water solubility for any given compound can be easily determined using readily available chemistry techniques and tools, such as high performance liquid chromatography or spectrophotometry. D. Liposome Composition
- liposome composition refers to a composition that contains a liposome and that further contains cyclodextrin chemically modified from its inert form and a therapeutic agent in the liposome internal phase.
- Liposome compositions can include solid and liquid forms. In the case where the liposome composition is in a solid form, it can be made into a liquid form by dissolving or suspending it in a prescribed solvent. In the case where the liposome composition is frozen solid, it can be made into a liquid form by melting by leaving it standing at room temperature.
- the concentration of liposome and the concentration of the therapeutic agent in the liposome composition can be appropriately set according to the liposome composition objective, formulation, and other considerations well known to the skilled artisan.
- the concentration of liposome as the concentration of all lipids constituting the liposome may be set at 0.2 to 100 mM, and preferably at 1 to 30 mM.
- the concentration (dosage) of therapeutic agent in the case where the liposome composition is used as a medicine is described below.
- the quantity of cyclodextrin in the liposome composition it is preferable that it be 0.1 to
- the solvent of the liposome composition in the case where the liposome composition is a liquid formulation.
- Representative examples include, without limitation, buffer solutions such as phosphate buffer solution, citrate buffer solution, and phosphate-buffered physiological saline solution, physiological saline water, and culture mediums for cell culturing.
- buffer solutions such as phosphate buffer solution, citrate buffer solution, and phosphate-buffered physiological saline solution, physiological saline water, and culture mediums for cell culturing.
- the pH of the liposome external phase of the liposome composition In some embodiments, such as H is between 2 and 11, 3 and 10, 4 and 9, 7.4, 7.0, or any pH higher than that of the liposome internal phase.
- pharmaceutically excipients can be added, such as sugar, such as monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, and xylose; disaccharides such as lactose, sucrose, cellobiose, trehalose, and maltose;
- sugar such as monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, and xylose
- disaccharides such as lactose, sucrose, cellobiose, trehalose, and maltose
- trisaccharides such as raffmose and melezitose
- polysaccharides such as cyclodextrin
- sugar alcohols such as erythritol, xylitol, sortibol, mannitol and maltitol
- polyvalent alcohols such as glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkylether, diethylene glycol monoalkylether, 1,3-butylene glycol.
- Combinations of sugar and alcohol can also be used.
- the liposome that is dispersed in solvent For purposes of stable long-term storage of the liposome that is dispersed in solvent, from the standpoint of physical stability including coagulation and so on, it is preferable to eliminate the electrolyte in the solvent as much as possible. Moreover, from the standpoint of chemical stability of the lipids, it is preferable to set the pH of the solvent from acidic to the vicinity of neutral (pH 3.0 to 8.0), and to remove dissolved oxygen through nitrogen bubbling.
- Representative examples of liquid stabilizers include, without limitation, normal saline, isotonic dextrose, isotonic sucrose, Ringer's solution, and Hanks' solution.
- a buffer substance can be added to provide pH optimal for storage stability.
- pH between about 6.0 and about 7.5, more preferably pH about 6.5 is optimal for the stability of liposome membrane lipids, and provides for excellent retention of the entrapped entities.
- Histidine, hydroxyethylpiperazine-ethylsulfonate (HEPES), morpholipo-ethylsulfonate (MES), succinate, tartrate, and citrate are exemplary buffer substances.
- Other suitable carriers include, e.g., water, buffered aqueous solution, 0.4% NaCl, 0.3% glycine, and the like.
- Protein, carbohydrate, or polymeric stabilizers and tonicity adjusters can be added, e.g., gelatin, albumin, dextran, or polyvinylpyrrolidone.
- the tonicity of the composition can be adjusted to the physiological level of 0.25-0.35 mol/kg with glucose or a more inert compound such as lactose, sucrose, mannitol, or dextrin.
- These compositions can be sterilized by conventional, well known sterilization techniques, e.g., by filtration.
- the resulting aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous medium prior to administration.
- the concentration of the sugar contained in the liposome composition there are no particular limitations on the concentration of the sugar contained in the liposome composition, but in a state where the liposome is dispersed in a solvent, for example, it is preferable that the concentration of sugar be 2 to 20% (W/V), and 5 to 10% (W/V) is more preferable. With respect to the concentration of polyvalent alcohol, 1 to 5% (W/V) is preferable, and 2 to 2.5% (W/V) is more preferable.
- Solid formulations of liposome compositions can also include pharmaceutical excipients.
- Such components can include, for example, sugar, such as monosaccharides such as glucose, galactose, mannose, fructose, inositole, ribose, and xylose; disaccharides such as lactose, sucrose, cellobiose, trehalose, and maltose; trisaccharides such as raffmose and melezitose; polysaccharides such as cyclodextrin; and sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, and maltitol.
- sugar such as monosaccharides such as glucose, galactose, mannose, fructose, inositole, ribose, and xylose
- disaccharides such as lactose, sucrose, cellobiose, trehalose, and mal
- solid formulations can be stably stored over long periods.
- solid formulations contain polyvalent alcohols (aqueous solutions) such as glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkylether, diethylene glycol monoalkylether and 1 ,3-butylene glycol.
- glycerin, propylene glycol, and polyethylene glycol are preferable, and glycerin and propylene glycol are more preferable.
- glycerin and propylene glycol are more preferable.
- the liposome compositions described herein can further be characterized according to entity-to-lipid ratio.
- entity-to-lipid ratio e.g. , therapeutic agent load ratio obtained upon loading an agent depends on the amount of the agent entrapped inside the liposomes, the concentration of ions in active loading processes, and the physicochemical properties of the ions and the type of counter-ion used.
- the entity-to-lipid ratio for the entity entrapped in the liposomes is over 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more calculated on the basis of the amount of the entity and the liposome lipid taken into the loading process (the "input" ratio). It is also possible to achieve 100% (quantitative) encapsulation.
- the entity-to lipid ratio in the liposomes can be characterized in terms of weight ratio (weight amount of the entity per weight or molar unit of the liposome lipid) or molar ratio (moles of the entity per weight or molar unit of the liposome lipid).
- weight ratio weight amount of the entity per weight or molar unit of the liposome lipid
- molar ratio moles of the entity per weight or molar unit of the liposome lipid.
- One unit of the entity-to-lipid ratio can be converted to other units by a routine calculation, as exemplified below.
- the weight ratio of an entity in the liposome compositions described herein is typically at least 0.05, 0.1, 0.2, 0.35, 0.5, or at least 0.65 mg of the entity per mg of lipid.
- the entity-to-lipid ratio according to the present invention is at least from about 0.02, to about 5, preferably at least 0.1 to about 2, and more preferably, from about 0.15 to about 1.5 moles of the drug per mole of the liposome lipid.
- the entity-to-lipid ratio is at least 0.1 mole of therapeutic agent per mole of liposome lipid, and preferably at least 0.2, 0.3, 0.4, 0.5, or more.
- the liposome compositions of the present invention can further be characterized by their unexpected combination of high efficiency of the entrapped therapeutic agent and low toxicity.
- the activity of a therapeutic agent liposomally encapsulated according to the present invention e.g., the anti-neoplastic activity of an anti-cancer therapeutic agent in a mammal in a mammal, is at least equal to, at least 2, 2.5, 3, 3.5, 4, 4.5, 5 or more times higher, or at least such fold higher than the activity of the therapeutic entity if it is administered in the same amount via its routine non-liposome formulation, e.g., without using the liposome composition of the present invention, while the toxicity of the liposomally encapsulated entity does not exceed, is at least twice, at least three times, or at least four times lower than that of the same therapeutic entity administered in the same dose and schedule but in a free, non-encapsulated form.
- Representative examples include, without limitation, the lipid film method (Vortex method), reverse phase evaporation method, ultrasonic method, pre-vesicle method, ethanol injection method, French press method, cholic acid removal method, Triton X-100 batch method, Ca 2+ fusion method, ether injection method, annealing method, freeze-thaw method, and the like.
- the various conditions (quantities of membrane constituents, temperature, etc.) in liposome preparation can be suitably selected according to the liposome preparation method, target liposome composition, particle size, etc.
- cyclodextrin is known to have the effect of removing lipid (particularly, cholesterol, etc.) from liposomes. It is therefore preferable that the amount of lipid used in the liposome preparation be set in consideration of this effect.
- the therapeutic agent/cyclodextrin complexes can be obtained by agitating and mixing the cyclodextrin (e.g., a solution containing the cyclodextrin) upon dropwise addition of the therapeutic agent (e.g. , a solution containing the therapeutic agent) or vice versa. It is possible to use a substance dissolved in a solvent or a solid substance as the therapeutic agent according to the physical properties of the therapeutic agent. There are no particular limitations on the solvent, and one can use, for example, a substance identical to the liposome external phase.
- the amount of the therapeutic agent that is mixed with the cyclodextrin can be equimolar quantities or in different ratios depending on the desired level of incorporation.
- absolute amounts of therapeutic agent can range between 0.001 to 10 mol equivalents, 0.01 to 1 mol equivalent, or any range inclusive relative to the amount of cyclodextrin.
- heating temperature there are no particular limitations on the heating temperature. For example, 5°C or higher, room temperature or higher (e.g., 20°C or higher is also preferable) or the phase transition temperature of the lipid bilayer membrane of the liposome or higher, are all acceptable.
- the liposome particle size can be optionally adjusted as necessary.
- Particle size can be adjusted, for example, by conducting extrusion (extrusion filtration) under high pressure using a membrane filter of regular pore diameter. Particle size adjustment can be conducted at any timing during manufacture of the liposome composition. For example, particle size adjustment can be conducted before introducing the therapeutic
- agent/cyclodextrin complexes into the liposome internal phase or after the therapeutic agent/cyclodextrin complexes have been remotely loaded into the liposome internal phase.
- Representative examples include, without limitation, dialysis, centrifugal separation, and gel filtration.
- Dialysis can be conducted, for example, using a dialysis membrane.
- a dialysis membrane one may cite a membrane with molecular weight cut-off such as a cellulose tube or Spectra/Por.
- centrifugal separation centrifugal acceleration any be conducted preferably at 100,000 g or higher, and more preferably at 300,000 g or higher.
- Gel filtration may be carried out, for example, by conducting fractionation based on molecular weight using a column such as Sephadex or Sepharose.
- an active remote loading method can be used to encapsulate therapeutic agent/cyclodextrin complexes within a liposome.
- an ionic gradient e.g., titratable ammonium, such as unsubstituted ammonium ion
- an ionic gradient e.g., titratable ammonium, such as unsubstituted ammonium ion
- a mechanism of "active", “remote”, or “transmembrane gradient-driven” loading Haran, et al., Biochim. Biophys. Acta, 1993, v. 1152, p. 253-258; Maurer-Spurej, et al, Biochim. Biophys. Acta, 1999, v. 1416, p. 1-10).
- active remote loading can be achieved by using a transmembrane pH gradient.
- the liposome internal and external phases differ in pH by 1 -5 pH units, 2-4 pH units, 0.5 pH unit, 1 pH unit, 2 pH units, 3 pH units, 3.4 pH units, 4 pH units, 5 pH units, 6 pH units, 7 pH units, or any range inclusive.
- Either the liposome internal or external phase can have the higher pH according to the type of the therapeutic agent and the ionizable groups on the modified cyclodextrins.
- the pH gradient can be adjusted by using a compound conventionally known in the art used in pH gradient methods.
- Representative examples include, without limitation, amino acids such as arginine, histidine, and glycine; acids such as ascorbic acid, benzoic acid, citric acid, glutamic acid, phosphoric acid, acetic acid, propionic acid, tartaric acid, carbonic acid, lactic acid, boric acid, maleic acid, fumaric acid, malic acid, adipic acid, hydrochloric acid, and sulfuric acid; salts of the aforementioned acids such as sodium salt, potassium salt, and ammonium salt; and alkaline compounds such as tris- hydroxymethylamino methane, ammonia water, sodium hydride, potassium hydride, and the like.
- amino acids such as arginine, histidine, and glycine
- acids such as ascorbic acid, benzoic acid, citric acid, glutamic acid, phosphoric acid, acetic acid, propionic acid
- ions that can be used in the ion gradient method.
- Representative example include, without limitation, ammonium sulfate, ammonium chloride, ammonium borate, ammonium formate, ammonium acetate, ammonium citrate, ammonium tartrate, ammonium succinate, ammonium phosphate, and the like.
- the ion concentration of the liposome internal phase can be selected appropriately according to the type of the therapeutic agent. A higher ion concentration is more preferable and is preferably 10 mM or higher, more preferably 20 mM or higher, even more preferably 50 mM or higher.
- Either the liposome internal or external phase can have the higher ion concentration according to the type of the therapeutic agent. On the other hand, it is also acceptable if the liposome internal and external phases do not substantially have difference in ion concentration, i.e., the liposome external and internal phases have substantially the same ion concentration.
- the ion gradient can also be adjusted by substituting or diluting the liposome external phase.
- a step in which the membrane permeability of the liposome is enhanced can be added using well-known methods.
- Representative examples include, without limitation, heating liposome -containing compositions, adding a membrane fluidizer to liposome-containing compositions, and the like.
- the therapeutic agent/cyclodextrin complexes can generally be more efficiently introduced into the liposome internal phase by heating to higher temperatures.
- the temperature of heating taking into consideration the thermal stability of the therapeutic agent/cyclodextrin complexes and the employed liposome membrane constituents.
- the temperature of heating be set to the phase transition temperature of the lipid bilayer membrane of the liposome or higher.
- phase transition temperature of the lipid bilayer membrane of liposome refers to the temperature at which heat absorption starts (the temperature when endothermic reaction begins) in differential thermal analysis of elevated temperatures conditions.
- Differential thermal analysis is a technique enabling analysis of the thermal properties of specimens by measuring the temperature difference between a specimen and reference substance as a function of time or temperature while changing the temperature of the specimen and reference substance.
- the liposome membrane components fluidize as temperature increases, and endothermic reaction is observed.
- the temperature range in which endothermic reaction is observed greatly varies according to the liposome membrane components. For example, in the case where liposome membrane components consist of a pure lipid, the temperature range in which endothermic reaction is observed is extremely narrow, and endothermic reaction is often observed within a range of ⁇ 1°C relative to the endothermic peak temperature.
- the temperature range in which endothermic reaction is observed tends to widen, and endothermic reaction is observed, for example, within a range of ⁇ 5°C relative to the endothermic peak temperature (that is, a broad peak is observed).
- membrane permeability of the therapeutic agent/cyclodextrin complexes is increased by raising the temperature higher than the phase transition temperature of the liposome lipid bilayer membrane.
- the temperature ranges in some embodiments can be from the phase transition temperature of the liposome lipid bilayer membrane to +20°C, +10°C, +5°C, or less, or any range in between such as +5°C to +10°C of such a phase transition temperature.
- the heating temperature can ordinarily range between 20 to 100°C, 40 to 80°C, 45 to 65°C, and any range in between. It is preferable that the heating temperature is higher than or equal to the phase transition temperature.
- the heating step there are no particular limitations on the time during which the temperature is maintained at or above the phase transition temperature, and this may be properly set within a range, for example, of several seconds to 30 minutes. Taking into consideration the thermal stability of the therapeutic agent and lipids as well as efficient mass production, it is desirable to conduct the treatment within a short time. That is, it is preferable that the elevated temperature maintenance period be 1 to 30 minutes, and 2 minutes to 5 minutes is more preferable. However, these temperature maintenance times in no way limit the present invention.
- a membrane fluidizer to the obtained mixed solution (that is, adding it to the external phase side of the liposome).
- Representative examples include, without limitation, organic solvents, surfactants, enzymes, etc. that are soluble in aqueous solvents.
- Representative organic solvents include, without limitation, monovalent alcohols such as ethyl alcohol and benzyl alcohol; polyvalent alcohols such as glycerin and propylene glycol; aprotic polar solvents such as dimethyl sulfoxide (DMSO).
- Representative surfactants include, without limitation, anionic surfactants such as fatty acid sodium, monoalkyl sulfate, and monoalkyl phosphate; cationic surfactants such as alkyl trimethyl ammonium salt; ampho lytic surfactants such as alkyl dimethylamine oxide; and non-ionic surfactants such as polyoxyethylene alkylether, alkyl monoglyceryl ether, and fatty acid sorbitan ester.
- Representative enzymes include, without limitation, cholinesterase and cholesterol oxidase.
- Those skilled in the art can set the quantity of membrane fluidizer according to the composition of liposome membrane constituents, the membrane fluidizer, and the like, taking into consideration the degree of efficiency of entrapment of the therapeutic agent due to addition of the membrane fluidizer, the stability of the liposome, etc.
- Methods of making liposome compositions described herein can further include a step of adjusting the liposome external phase of the obtained liposome composition and/or a step of drying the obtained liposome composition before and/or after encapsulation of the therapeutic agent/cyclodextrin complexes.
- the liposome external phase in the liquid liposome composition can be adjusted (replaced, etc.) to make a final liposome composition if it is to be used as a liquid formulation.
- the liquid liposome composition obtained in the above-mentioned introduction step can be dried to make the final solid liposome composition. Freeze drying and spray drying are representative, non-limiting examples of methods for drying the liposome composition.
- the liposome composition is a solid preparation, it can be dissolved or suspended in a suitable solvent and used as a liquid formulation.
- the solvent for use can be appropriately set according to the purpose of use for the liposome composition.
- the solvent in the case of using the liposome composition as an injection product, can be sterile distilled water or other solvent compatible with injection.
- the physician or patient can inject the solvent into a vial into which the solid preparation is entrapped, for example, to make the preparation at the time of use.
- the liquid liposome composition is a frozen solid preparation, it can be stored in a frozen state, and put in use as a liquid formulation by returning it to a liquid state by leaving it to melt at room temperature or by rapidly melting it with heat at the time of use.
- the liposome compositions described herein can be used as a pharmaceutical composition such as a therapeutic composition or a diagnostic composition in the medical field.
- the liposome composition can be used as a therapeutic composition by incorporating an antineoplastic agent as the therapeutic agent and can be used as a diagnostic composition by incorporating contrast agent as the therapeutic agent.
- the liposome composition can also be used for any number of other purposes, such as a cosmetic product or as a food additive.
- the liposome pharmaceutical composition of the present invention is prepared as a topical or an injectable, either as a liquid solution or suspension.
- solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
- the composition can also be formulated into an enteric-coated tablet or gel capsule according to known methods in the art.
- the liposome composition of the present invention can be administered by injection (intravenous, intra-arterial, or local injection), orally, nasally, subcutaneously, pulmonarily, or through eye drops, and in particular local injection to a targeted group of cells or organ or other such injection is preferable in addition to intravenous injection, subcutaneous injection, intracutaneous injection, and intra-arterial injection.
- injection intravenous, intra-arterial, or local injection
- nasally nasally, subcutaneously, pulmonarily, or through eye drops
- local injection to a targeted group of cells or organ or other such injection is preferable in addition to intravenous injection, subcutaneous injection, intracutaneous injection, and intra-arterial injection.
- Tablet, powder, granulation, syrup, capsule, liquid, and the like may be given as examples of the formulation of the liposome composition in the case of oral administration.
- Injection product drip infusion, eye drop, ointment, suppository, suspension, cataplasm, lotion, aerosol, plaster, and the like can be given as examples of formulations of the liposome composition in the case of non- oral administration, and an injection product and drip infusion agent are particularly preferable.
- the liposome composition is used as a cosmetic product
- a cosmetic product as the form of the cosmetic product, one may cite, for example, lotions, creams, toners, moisturizers, foams, foundations, lipsticks, face packs, skin washes, shampoos, rinses, conditioners, hair tonics, hair liquids, hair creams, etc.
- administering a substance, such as a therapeutic entity to an animal or cell
- dispensing delivering or applying the substance to the intended target.
- the term “administering” is intended to refer to contacting or dispensing, delivering or applying the therapeutic agent to an animal by any suitable route for delivery of the therapeutic agent to the desired location in the animal, including in any way which is medically acceptable which may depend on the condition or injury being treated.
- Possible administration routes include injections, by parenteral routes such as intramuscular, subcutaneous, intravenous, intraarterial, intraperitoneal,
- intraarticular, intraepidural, intrathecal, or others as well as oral, nasal, ophthalmic, rectal, vaginal, topical, or pulmonary, e.g., by inhalation.
- a slow, sustained intracranial infusion of the liposomes directly into the tumor is of particular advantage (Saito et al. (2004) Cancer Res. 64:2572-2579; Mamot et al. (2004) J. Neuro-Oncology 68:1-9).
- the compositions can also be directly applied to tissue surfaces.
- Sustained release, pH dependent release, or other specific chemical or environmental condition mediated release administration is also specifically included in the invention, e.g., by such means as depot injections, or erodible implants.
- the dosage of the pharmaceutical composition upon administration can differ depending on the type of target disease, the type of the therapeutic agent, as well as the age, sex, and weight of the patient, the severity of the symptoms, along with other factors. It is to be understood that the determination of the appropriate dose regimen for any given therapeutic agent encapsulated within the liposomes and for a given patient is well within the skill of the attending physician. For example, the quantity of liposome pharmaceutical composition necessary to deliver a therapeutically effective dose can be determined by routine in vitro and in vivo methods, common in the art of drug testing (e.g., D. B. Budman, A. H. Calvert, E. K. Rowinsky (editors). Handbook of Anticancer Drug Development, LWW, 2003).
- the attending physician can rely on the recommended dose for the given drug when administered in free form.
- therapeutically effective dosages for various therapeutic entities are well known to those of skill in the art.
- the dosages for the liposome pharmaceutical composition of the present invention range between about 0.005 and about 500 mg of the therapeutic entity per kilogram of body weight, most often, between about 0.1 and about 100 mg therapeutic entity/kg of body weight.
- a kit for preparing the liposome composition.
- the kit can be used to prepare the liposome composition as a therapeutic or diagnostic, which can be used by a physician or technician in a clinical setting or a patient.
- the kit includes a liposome reagent.
- the liposome reagent can be either in a solid or a liquid form. If the liposome reagent is in a solid form, the liposome reagent can be dissolved or suspended in an appropriate solvent to obtain the liposome, and the above- mentioned liposome dispersion liquid can be dried to obtain the liposome reagent. Drying can be carried out similarly to the above-mentioned drying of the liposome composition.
- the liposome regent can be dissolved or suspended in an appropriate solvent to make the liposome dispersion liquid. When doing so, the solvent is similar to the liposome external phase in the above- mentioned liposome dispersion liquid.
- the kit of the present invention preferably further contains a therapeutic agent.
- the therapeutic agent can be either in a solid or liquid form (a state of dissolved or suspended in a solvent).
- a solvent can be appropriately set according to the physical properties and the like of the therapeutic agent, and may be made similar to the liposome external phase in the above- mentioned liposome dispersion liquid, for example.
- the liposome reagent and the therapeutic agent can be packaged separately, or they may be in solid forms and mixed together.
- the mixture of the liposome reagent and the therapeutic agent is appropriately dissolved or suspended in a solvent.
- the solvent is similar to the liposome external phase in the above-mentioned liposome dispersion liquid. It is thereby possible to form a state in which the liposome dispersion liquid and the therapeutic agent are mixed, after which use is made possible by carrying out other steps in the introduction of the therapeutic agent in the liposome internal phase of the liposome dispersion liquid in the manufacturing method of the above-mentioned liposome composition.
- the kit can comprise a liposome composition described herein including directions for use.
- Example 1 Materials and Methods for Examples 2-4
- ⁇ -Cyclodextrin (Sigma-Aldrich, St. Louis, MO) was monotosylated with 0.9 molar equivalent of tosyl chloride in pyridine at the primary 6' hydroxyl group to afford the corresponding tosylate, which was converted to the iodo-derivative by treatment with sodium iodide in acetone.
- the iodo derivative was converted to the desired aminated cyclodextrin by heating at 80°C for 8-12 h with the appropriate amine (Tang and Ng (2008) Nat. Protocol. 3:691-697).
- 6'-mono-succinyl-P-cyclodextrin was synthesized by treatment of parent ⁇ -cyclodextrin with 0.9 equivalents of succinic anhydride in DMF (Cucinotta et al. (2005) J. Pharmaceut. Biomed. Anal. 37: 1009-1014). The product was precipitated in acetone and purified by HPLC before use. 6',6',6',6',6',6',6',6'-heptakis-succinyl- - cyclodextrin was synthesized from ⁇ -cyclodextrin by treatment with excess succinic anhydride in DMF and precipitated with acetone. Fractional crystallization afforded the desired compound in -85% purity.
- Dansylated cyclodextrins I, IV, and V were synthesized from commercially available ⁇ -cyclodextrin and compounds II and III, respectively, by treatment with a 0.9 molar equivalent of dansyl chloride in pyridine.
- 6',6',6',6',6',6',6',6',6',6'-heptakis-amino- -cyclodextrin was purchased from CTD holdings and used without further purification.
- Hydrogenated egg phosphatidylcholine (Avanti Polar Lipids), cholesterol and 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (DSPE-PEG 2000) (Avanti Polar Lipids) (molar ratios, 50:45:5) were dissolved in chloroform (20 ml). The solvent was removed in vacuo to give a thin lipid film, which was hydrated by shaking in the appropriate buffer (PBS, pH 7.4; 200 mM citrate, pH4.0; or 80 mM Arg-HEPES, pH 9.0) at 50°C for 2 hours.
- PBS pH 7.4
- 200 mM citrate pH4.0
- 80 mM Arg-HEPES pH 9.0
- the vesicle suspension was sonicated for 30 minutes and then extruded successively through 0.4-, 0.2-, and 0.1- ⁇ polycarbonate membranes (Whatman, Nucleopore Track-Etch Membrane) at 50°C to obtain the final liposomes.
- the transmembrane gradient was then created by equilibrium dialysis of the liposomes against 300 mM sucrose or phosphate-buffered saline (PBS) overnight.
- PBS phosphate-buffered saline
- the average size and polydispersity index was then measure by Dynamic Light Scattering (DLS) on a Zetasizer Nano ZS90 (Malvern Instruments) at a wavelength of 633 nm and a 90° detection angle.
- DLS Dynamic Light Scattering
- Method 1 Encapsulation of BI-2 36 in the lipid layer. Hydrogenated egg phosphatidylcholine (Avanti Polar Lipids), cholesterol, and DSPE-PEG2000 (Avanti Polar Lipids) (molar ratios 50:45:5) were dissolved in chloroform (20 mL). Ten milligrams of BI-2536 (in 1 mL chloroform) was added and the solvent was evaporated to generate a thin film. One milliliter of PBS (pH 7.4) was added to hydrate the lipid layer, and the mixture was shaken at 50°C for 2 hours as described above.
- PBS pH 7.4
- the vesicle suspension was sonicated for 30 min and then extruded successively through 0.4-, 0.2-, and 0.1 - ⁇ polycarbonate membranes (Whatman; Nuclepore Track-Etched Membrane) at 50°C to obtain the final liposomes with low polydispersity at the desired size.
- the liposomes were then dialyzed in PBS overnight
- Method 2 Hydration of the lipid layer with an aqueous formulation of BI-2536.
- Hydrogenated egg phosphatidylcholine (Avanti Polar Lipids), cholesterol, and DSPE- PEG2000 (Avanti Polar Lipids) (molar ratios 50:45:5) were dissolved in chloroform (20 mL), and the solvent was evaporated in vacuo to generate a thin film.
- aqueous BI-2536 (4 mg; pH 5.5) was added to hydrate the lipid layer, and the mixture was shaken at 50°C for 2 hours as described above.
- the vesicle suspension was sonicated for 30 minutes and then extruded successively through 0.4-, 0.2-, and 0.1 - ⁇ polycarbonate membranes (Whatman; Nuclepore Track-Etched Membrane) at 50°C to obtain the final liposomes with
- the liposomes were then dialyzed against PBS overnight to remove unentrapped drug. The average size and polydispersity index were then measured by dynamic light-scattering experiments on a Malvern Z90. Drug content was calculated by rupturing the liposomes with an equal volume of methanol and measuring the UV-vis absorbance on a NanoDrop 1000.
- the lyophilized powder complex described above was pulverized and incubated with appropriate liposomal solutions (30-40 mg drug equivalent in 6 mL liposomal solution, to achieve loading ratios of 5-8 mg./mL concentrations) for 1 hour at 65°C. They were centrifuged at 1,000 x g for 3 minutes to remove particulate matter and then dialyzed against 300 mM sucrose or commercial PBS solution (pH 7.4) overnight to remove material that had not been loaded into the liposomes.
- the size distributions of the liposomal formulations were characterized using the Malvern ZS90 instrument described above.
- Concentrations of BI-2536 and PD-0325901 in liposomes were measured in triplicate using a Nanodrop 100 after disruption of the liposomal solutions with equal volumes of methanol at 367 nm for BI-2536 and 277 nm for PD-0325901.
- Coumarin 334 was used as a drug surrogate to assess biodistribution and pharmacokinetics of cyclodextrin-encapsulated liposomes.
- Coumarin 334 (3 mg) was dissolved in methanol (6 mL) and added dropwise to an aqueous solution of cyclodextrin VI (14 mg in 20 mL water). The solution was shaken at 55°C for 48 hours and lyophilized. The lyophilized powder was incubated with citrate liposomes (internal pH 4.0) at 65°C for 1 hour. The liposomal solution was dialyzed against PBS overnight. To assess loading efficiency, 100 liposomes was broken with 100 ⁇ ⁇ methanol and analyzed for fluorescence. The
- mice bearing HCT116 subcutaneous xenografts were used in the study following a modified protocol described in Macdiarmid et al. (2007) Cancer Cell 11 :431-445.
- 12 mice were treated intravenouslys (i.v.) with 200 cyclodextrin- encapsulated, liposomal (CYCL-)coumarin 334 (0.5 mg/mL).
- Posttreatment four mice were euthanized at time points 2, 24, and 48 hours and tumor, spleen, liver, kidneys, heart, and lungs were excised and weighed. Blood was also collected, and plasma was separated and stored at 4°C.
- each frozen tissue was homogenized and sonicated in 0.9% saline [3 x volume ( ⁇ ) of tissue mass (mg)].
- Methanol was added to a final volume of 33% (vol/vol) with vortexing.
- the samples were centrifuged (6,000 x g for 10 minutes) and the fluorescence in the supernatants was measured by a CytoFluor II fluorescence multiwall plate reader (Applied Biosystems) using excitation 485 nm/emission 530 nm.
- As a control for tissue autofluorescence tumor-bearing animals treated with equivalent volumes of empty liposomes were euthanized and their tissue and plasma were harvested.
- HCT116 p53 _/ ⁇
- HCT116 p53+/+
- RKO cells Five million HCT116 (p53 _/ ⁇ ), HCT116 (p53+/+), or RKO cells were injected subcutaneously (s.c.) into the flanks of female athymic nu/nu mice and allowed to grow for three weeks, reaching 300- 400 mm 3 in volume. In the case of CYCL-BI-2536, the animals were then randomly segregated into four arms. In all cases, liposomal formulations (CYCL-drug) have been reported as equivalents of free drug.
- the first arm received empty liposomes; the second arm received a single dose of 100 mg/kg formulation of the free drug twice using a formulation reported in the literature (at day 0 and day 7); the third and fourth arms received 100 mg/kg and 400 mg/kg, respectively, of the CYCL-BI-2536 liposomal formulation at the same time points.
- Tumor volume was recorded every 48 hours. The average tumor size for each respective group was normalized to the tumor volume at the first day of treatment.
- the first arm was treated twice with empty liposomes at day 0 and day 8
- the other two arms received two doses of free PD-0325901 and CYCL-PD-0325901, respectively, at the same time points.
- the data are presented as the average tumor size of each group normalized to the tumor volume on day 0.
- the tumor regression experiments in each case were terminated and the animals euthanized when the tumors on the control animals reached 2,000 mm 3 .
- Example 2 Remote Loading of Chemically-Modified 3-Cyclodextrins Into Liposomes
- a set of modified ⁇ -cyclodextrins bearing ionizable groups at their 6 '-position were designed and synthesized ( Figure 2).
- analogs II-V the 6' primary hydroxyl moiety was modified to an amino group, an ethylenediamino group, or a fluorescent version of either, whereas analog VIII involved introduction of a succinyl group in that position.
- the rest of the analogs (VI, VII and IX) had all seven primary hydroxyls replaced by amino, ethylene- diamino, or succinyl moieties. All analogs were purified by HPLC and characterized by MS and NMR spectra. Appropriate negative controls were synthesized by introducing similarly sized chemical modifications not containing ionizable groups.
- Two fluorescent (dansylated) cyclodextrins were tested for their ability to be loaded into liposomes.
- the liposomes were generated by hydrating lipid films with 200 mM citrate buffer, so that their internal pH was 4.0. These liposomes were then dialyzed in PBS (pH 7.4) to remove the citrate from outside the liposomes and were then incubated at 65°C for 1 hour with cyclodextrins that had been dissolved in PBS.
- PBS-loaded liposomes were generated by rehydration of the lipid film with PBS instead of citrate.
- the solubility of the coumarins increased at least 10- to 20-fold (from 100 ⁇ g/mL to 1-2 mg/mL) through this procedure.
- the cyclodextrin-coumarin complexes were incubated with pre-formed liposomes exactly as described above, using a pH gradient to drive the compounds across the bilayer. Following overnight dialysis to remove unincorporated complexes, the liposomes were subsequently disrupted with methanol and the coumarin fluorescence measured. As shown by fluorescence spectroscopy, all cyclodextrin-dye complexes were incorporated into liposomes with high efficiency (>95%; Figure 5).
- BI-2536 developed by Boehringer Ingelheim, is a highly selective inhibitor of polo-like kinase (PLK1), an enzyme required for the proper execution of mitosis (Steegmaierer al. (2007) Curr. Biol. 17:316-322; Lenart et al. (2007) Curr. Biol. 17:304-315; and Stewart et al. (20 ⁇ ⁇ ) Exp. Hematol. 39:330-338).
- PLK1 polo-like kinase
- BI-2536 has potent tumoricidal activity against cancer cells, particularly those harboring mutations in TP53 (Sur et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106:3964-3969; Sanhaji ei a/. (2013) Cell Cycle 12:1340-1351; Meng et al. (2013) Gynecol. Oncol. 128:461-469; and appi et al. (2009) Cane. Res. 69: 1916-1923).
- BI-2536 was the subject of several clinical trials in patients with cancers of the lung, breast, ovaries, and uterus (Mross et al. (2012) Br. J. Cane.
- aminated cyclodextrins V and VI dramatically improved the aqueous solubility of BI-2536.
- the BI-2536 complexes were determined to be reproducibly loadable into liposomes using compound VI, achieving stable aqueous solutions containing 10 mg/mL of drug.
- the maximum aqueous solubility of free BI-2536 was determined to be 0.5 mg/mL.
- CYCL cyclodextrin-encapsulated, liposomal
- mice Three weeks after HCT116 cells were subcutaneously injected into the mice, they were treated with empty liposomes, free BI-2536, or CYCL-BI-2536. At the initiation of treatment, the tumors were already relatively large, averaging -300 mm 3 and more closely mimicking clinical situations than small tumors. Severe acute toxicity was evident when the free drug was administered intravenously (iv) at 125 mg/kg: the mice became lethargic within minutes, their eyes turned glassy, they exhibited ruffled fur, and died a few hours later (Figure 8 A). Mice treated with a slightly lower dose of free BI-2536 (100 mg/kg) were somewhat lethargic immediately after drug administration, but survived. However, delayed toxicity, manifested as a drastic decrease in peripheral WBCs, was evident within 24-36 hours after free drug administration. This type of toxicity was identical to that observed in human clinical trials (Mross et al. (2008) J. Clin. Oncol.
- CYCL-BI-2 36 proved far superior to the free form, both with respect to toxicity and efficacy.
- CYCL-BI-2536 even at a dose of 500 mg/kg, did not cause any noticeable adverse reactions; this dose was 4-fold higher than the dose of free drug, which killed every animal ( Figure 8A).
- CYCL-BI-2536 induced a significantly improved tumor response, slowing tumor growth by nearly 80% after only two doses ( Figure 8B).
- Figure 8B At a dose of 400 mg/kg, the CYCL-BI-2536 resulted not only in slower growth, but also in partial regressions of tumors ( Figure 8B).
- liposomes loaded with coumarin 334 encapsulated in cyclodextrin VI were used to treat HCT116-bearing mice by intravenous (i.v.) injection.
- Samples from major tissues harvested at 2, 24, and 48 hours post-treatment were analyzed for their fluorescence.
- coumarin 334 was cleared from most of the tissues examined at 48 hours after treatment.
- the agent encapsulated in liposomes persisted in the blood and tumor, which is consistent with the typical pharmacokinetics of PEGylated liposomes ( Figure 9).
- BI-2536 was co-evaporated with hydrogenated egg phosphatidylcholine-cholesterol-1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (DSPE-PEG 2000) to prepare a thin film, which was subsequently hydrated with 1 mL PBS and extruded through a 100-nm polycarbonate membrane at 700 psi to prepare small, unilamellar vesicles (average particle size (Z avg ) 126 nm; PDI 0.09).
- the drug-containing liposomes Upon overnight dialysis against PBS to remove unentrapped drug, the drug-containing liposomes rapidly swelled (Z avg 539 nm; PDI 0.49) and released nearly 90% of the entrapped drug.
- the lipid film was hydrated with an aqueous formulation of BI-2536 (passive loading). Hydration of the lipid film, followed by extrusion and dialyses, led to stable liposomes.
- the lipid film was hydrated with an aqueous formulation of BI-2536 (passive loading). Hydration of the lipid film, followed by extrusion and dialyses, led to stable liposomes.
- the lipid film was hydrated with an aqueous formulation of BI-2536 (passive loading). Hydration of the lipid film, followed by extrusion and dialyses, led to stable liposomes.
- the lipid film was hydrated with an aqueous formulation of BI-2536 (passive loading). Hydration of
- PD-0325901 a mitogen-activated protein kinase kinase 1 (MEK1) inhibitor developed by Pfizer that was abandoned because it caused retinal vein occlusion (RVO) in Phase 2 trials, was evaluated (Brown et al. (2007) Cane. Chemo. Pharmacol. 59:671-679; LoRusso et al. (2010) Clin.
- CYCL-PD-0325901 proved far superior to the free drug. Even at a dose of 500 mg/kg, CYCL-PD-0325901 did not cause any noticeable adverse reactions; this dose was 2.5-fold higher than the dose of free drug which killed every animal ( Figure 10A). At a single dose of 250 mg/kg, the CYCL-PD-0325901 resulted not only in slower growth but also in partial regressions of tumors ( Figure 10B). Finally, CYCL-PD-0325901 was evaluated against two other human colorectal cancer models (HCT116 and its isogenic counterpart with genetically inactivated TP 53 alleles) and higher efficacy and lower toxicity compared with free drug were similarly observed (Figure 11).
- the drug to lipid ratios achieved through the approaches described herein ranged from 0.4 to 0.6, which is more than 1 ,000-fold higher than the drug to lipid ratios commonly achieved through passive loading (Zhu et al. (2013) J. Pharm. Pharmacol. 65(8): 1107-1117; Malaekeh- ikouei and Davies (2009) PDA J. Pharm. Sci. Technol. 63:139-148; Rahman et al. (2012) Drug Deliv. 19:346-353; Ascenso et al. (2013) J.
- the approaches described herein can be broadly applicable.
- the approaches not only increase water solubility, but also enhances the selectivity of drug delivery to tumors through an enhanced permeability and retention (EPR) effect (Wang et al. (2012) Annu. Rev. Med. 63: 185-198; Peer et al. (2007) Nat. Nanotech. 2:751-760; Gubernator (2011) Exp. Opin. Drug Deliv. 8:565-580; Huwyler et al. (2008) Int. J.
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CA2936963A CA2936963A1 (en) | 2014-01-14 | 2015-01-14 | Liposome compositions encapsulating modified cyclodextrin complexes and uses thereof |
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MX2019008533A (en) * | 2017-01-18 | 2019-12-02 | Temasek Life Sciences Laboratory Ltd | Hyperstabilized liposomes increase targeting of mitotic cells. |
CN108478533B (en) * | 2018-04-23 | 2020-12-08 | 滨州医学院 | Preparation method of beta cyclodextrin-LPC liposome and application of beta cyclodextrin-LPC liposome as drug carrier |
KR20210028639A (en) * | 2018-07-06 | 2021-03-12 | 마루다이 푸드 가부시키가이샤 | Plasmalogen-containing composition |
CN108853111B (en) * | 2018-08-07 | 2020-06-05 | 浙江大学 | Application of composition in preparation of medicine for treating liver toxicity of gefitinib |
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CN114113426B (en) * | 2021-12-27 | 2024-04-26 | 西安血氧生物技术有限公司 | Method for detecting phospholipid in hemoglobin oxygen carrier |
CN115386262B (en) * | 2022-09-26 | 2023-05-12 | 杭州海维特化工科技有限公司 | Gravure ink applied to slow-release essence and anions of automotive interior decorative plastic film and preparation method thereof |
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US10751306B2 (en) | 2015-11-06 | 2020-08-25 | The Johns Hopkins University | Methods of treating liver fibrosis by administering 3-bromopyruvate |
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CA2936963A1 (en) | 2015-07-23 |
CN106659683A (en) | 2017-05-10 |
EP3094313A1 (en) | 2016-11-23 |
US20180161274A1 (en) | 2018-06-14 |
US20190328665A1 (en) | 2019-10-31 |
AU2015206628A1 (en) | 2016-08-25 |
HK1231397A1 (en) | 2017-12-22 |
EP3094313A4 (en) | 2017-07-12 |
JP2017502985A (en) | 2017-01-26 |
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