WO2001085131A2 - Compositions d'excipient a base de lipides pour retention medicamenteuse amelioree - Google Patents

Compositions d'excipient a base de lipides pour retention medicamenteuse amelioree Download PDF

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WO2001085131A2
WO2001085131A2 PCT/CA2001/000655 CA0100655W WO0185131A2 WO 2001085131 A2 WO2001085131 A2 WO 2001085131A2 CA 0100655 W CA0100655 W CA 0100655W WO 0185131 A2 WO0185131 A2 WO 0185131A2
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liposome
mol
liposomes
cholesterol
lipid
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WO2001085131A3 (fr
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Lawrence D. Mayer
Nancy Dos Santos
Marcel B. Bally
Murray Webb
Paul Tardi
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CELATOR TECHNOLOGIES Inc
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CELATOR TECHNOLOGIES Inc
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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—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant

Definitions

  • This invention is directed toward improving drug retention in lipid-based therapeutic carrier systems.
  • Liposomes and other lipid-based carrier systems have been extensively developed and analyzed for their ability to improve the therapeutic index of drags by altering the pharmacokinetic and tissue distribution properties of drags. This approach is aimed at reducing exposure of healthy tissues to therapeutic agents while increasing drug delivery to a diseased site.
  • lipid-based carriers such as liposomes for site-specific delivery of such drugs via the bloodstream presents possible means to improve the use of such drags.
  • the use of liposomes for site-specific delivery is limited by the rate of clearance of liposomes from the blood, for example by cells of the Mononuclear phagocytic system (MPS).
  • MPS Mononuclear phagocytic system
  • drags encapsulated into liposomes are often not retained in the liposome after intravenous adrninistration.
  • cholesterol increases bilayer thickness and fluidity while decreasing membrane permeability, protein interactions, and lipoprotein destabilization of the liposome.
  • Conventional approaches to liposome formulation dictate inclusion of substantial amounts (e.g. 30-45 mol %) cholesterol or equivalent membrane rigidification agents (such as other sterols) into liposomes.
  • a further advancement in the liposome field was the discovery that polymers such as polyethylene glycol (PEG) grafted to the surface of a liposome increases the circulation lifetime of a liposome by decreasing liposomal/protein interactions.
  • PEG polyethylene glycol
  • Thermosensitive liposomes are designed to have a phase transition temperature slightly above body temperature so that the liposomes remain in a gel state while in circulation but exceed the phase transition temperature upon application of heat to a patient's body or specific tissues. When heated, the liposome releases an encapsulated drag because the liposome bilayer becomes much more permeable above the transition temperature.
  • thermosensitive liposomes are often made without cholesterol. Further, to have a phase transition temperature sufficiently close to normal human body temperature (e.g. 40-45 °C), the lipid composition of the liposome is carefully tailored.
  • a preferred lipid for use in thermal-sensitive liposomes is DPPC, which has an acyl chain length of 16 carbon atoms. Incorporation of any substantial amount of lipids having longer acyl chain lengths will raise the phase transition temperature of the liposome beyond the point of usefulness in thermosensitive applications.
  • thermosensitive liposomes While circulation lifetime of a thermosensitive liposome may be enhanced by inclusion of PEG-conjugated lipids into the liposome just as in more conventional liposomes (see: United States Patent 5,843,473; Unezaki, S., et al. (1994) Pharm. Res. 11:1180-5; Marayama, K., et al. (1993) Biochimica et Biophysica Acta 1149:209-206; Blume, G. and Cevc, G. (1 & (2) Biochimica et Biophysica Acta (1990)1029:91-97 (1) & (1993) 1146:157-168 (2) ), thermosensitive liposomes exhibit poor drag retention in vivo. Summary of Invention
  • This invention is based on the discovery that liposomes having phase transition temperatures above that which is useful for thermosensitive applications will better retain certain drags, if the liposome is substantially free of cholesterol. In such cases, drag retention by the liposome is increased over an equivalent liposome formulation containing cholesterol. Appropriate circulation lifetime of such "cholesterol-free" liposomes may be achieved by incorporation of PEG-conjugated lipids.
  • this invention provides methods for determining whether retention of a particular drag is enhanced by elimination of cholesterol from a liposome.
  • This invention also provides improved drug retention in liposomes for specific drugs which previously exhibited poor retention in conventional cholesterol-containing liposomes.
  • novel cholesterol-free liposome formulations that are particularly suited for use in this invention. This invention provides a method for determining whether retention of a drug in a liposome may be improved, comprising the steps of:
  • the liposome at (a) will exhibit a phase transition temperature of at least 50°C.
  • the amount of cholesterol in the liposome at (b) will be about 30 to about 50 mol % .
  • the drug:lipid ratios will be determined in step (e) at a series of intervals subsequent to administration with the comparison at (f) being of the ratios determined over the series of intervals.
  • liposomes for use in step (a) of the preceding method will comprise at least 60 mol % of a phospholipid having two saturated fatty acids, the acyl chains of each having at least 18 carbon atoms.
  • a preferred phospholipid with acyl chains of 18 carbon atoms each is DSPC.
  • liposomes for use in step (a) in the method above will have at least about 80, at least about 85, and even more preferably, at least 90 mol % of such a phospholipid.
  • the remainder of the liposome may comprise one or more amphipathic lipids suitable for use in liposomes, but substantially no cholesterol.
  • such other lipids will include a hydrophilic polymer-conjugated lipid.
  • the amount of such polymer-conjugated lipids present in the liposome will be from about 2 to about 15 mol % .
  • Liposomes for use in the above method may be prepared using known and conventional techniques. Determination of phase transition temperatures, encapsulation of drag into liposomes (liposome loading), administration of liposomes, and deterrnining drag: lipid ratios from blood may be carried out according to known and conventional techniques.
  • this invention also provides a combination of a liposome and a drug wherein the liposome is a liposome as described above with respect to step (a) and the drug is an anti-neoplastic agent which exhibits greater retention in such a liposome, when the above-described method is performed.
  • “combination” it is meant that the drug is encapsulated in the liposome or is segregated but associated with the liposome (such as in a commercial package or kit comprising the liposome and the drug).
  • the liposome is one having the preferred characteristics of liposomes of step (a) as described above.
  • this aspect of the invention include the combination of the above-described liposome with a idarubicin-compound or a camptothecin-compound .
  • This invention also provides novel liposomes which are particularly suitable for use in this invention, which liposomes comprise: (a) at least 60 mol % of a phospholipid comprising two saturated fatty acids, the acyl chain of each being the same or different, at least one of said acyl chains having more than 18 carbon atoms;
  • the liposome contains substantially no cholesterol.
  • the liposome will contain essentially no cholesterol.
  • novel liposomes of this invention are those described above with respect to the liposome used in step (a) of the method, with the exception that the predominant phospholipid in the novel liposomes will have at least one acyl chain of greater than 18 carbon atoms.
  • This invention also provides the novel liposomes of this invention in combination with a drag and the use of such liposomes as a carrier for a drug encapsulated in the liposome.
  • Such drugs include anti-neoplastic agents.
  • Figure 1 A graph showing encapsulation of idarubicin into DSPC:DSPE-PEG2 ⁇ oo (95:5 mol %) liposomes as a function of time at 37 (filled squares), 50 (open, inverted triangles) and 65 °C (filled circles).
  • Figure 2A A histogram representing percent of lipid dose remaining in the blood for DSPC .-cholesterol (55: 45 mol %) and DSPC:DSPE-PEG2 ⁇ oo (95:5 mol %) liposomes in CD-I female mice after 1 hr.
  • Figure 2B A histogram representing percent of idarubicin dose remaining in the blood after intravenous administration of DSPCxholesterol (55:45 mol %) and DSPC:DSPE- PEG2000 (95:5 mol %) liposomes in CD-I female mice after 1 hr.
  • Figure 2C A histogram representing idarubicin: lipid ratio remaining in the blood for DSPCxholesterol (55:45 mol %) and DSPC:DSPE-PEG2ooo (95:5 mol %) liposomes in CD-I female mice after 1 hr.
  • Figure 3A A graph showing concentration of idarubicin ( ⁇ moles idarubicin/ mL plasma) recovered in the blood after intravenous injection of DSPC:cholesterol (55:45 mol %), DSPC:DSPE-PEG2 ⁇ oo (95:5 mol %), DSPC: cholesterol: DSPE-PEG2000 (50:45:5 mol %) liposomes and free idarubicin into Balb/c mice as a function of time represented by filled triangles, open circles, inverted filled triangles and open squares respectively.
  • Figure 3B A graph showing concentration of lipid ( ⁇ moles total lipid/ mL plasma) recovered in the blood after intravenous injection of DSPCxholesterol (55:45 mol %), DSPC:DSPE-PEG2ooo (95:5 mol %), DSPC:cholesterol:DSPE-PEG2 ⁇ oo (50:45:5 mol %) liposomes into Balb/c mice as a function of time represented by filled triangles, open circles, inverted filled triangles respectively.
  • Figure 3C A graph showing idarubicimlipid ratio remaining in the blood after intravenous injection of DSPCxholesterol (55:45 mol %), DSPC:DSPE-PEG2 ⁇ oo (95:5 mol %) and DSPCxholesterohDSPE-PEG ooo (50:45:5 mol %) liposomes into Balb/c mice as a function of time represented by represented by filled triangles, open circles, inverted filled triangles respectively.
  • Figure 4 A graph showing ratio of idarabicimlipid after intravenous administration of idarubicin: lipid by injection in Balb/c mice as a function of time for liposomes consisting of DSPC:PEG2000 (95:5 mol %), DAPC:PEG2000 (95:5 mol %) and DBPC:PEG2000 (95:5 mol %) represented by filled circles, inverted open triangles and upward open triangles respectively.
  • Figure 5 A graph showing percent survival of P388 murine lymphocytic leukemia model as a function of time after intravenous injection of HBS (HEPES Buffered Saline) (filled circles), free idarubicin (open circles), idarubicin encapsulated in DSPC:DSPE- PEG2000 (95:5 mol%) liposomes (downward, filled triangles) and idarubicin encapsulated in DAPC:DSPE-PEG2000 (95:5 mol%) liposomes (downward, open triangles).
  • HBS HBS Buffered Saline
  • Figure 6 A graph showing topotecamlipid ratio remaining in the blood after intravenous injection of DSPCxholesterol (55:45 mol %) and DSPC:DSPE-PEG2 ⁇ oo (95:5 mol %) liposomes into Balb/c mice as a function of time represented by filled circles and open circles respectively.
  • Figure 7 A graph showing ratio of daunorubicin:lipid remaining in the blood after intravenous injection of Balb/c mice as a function of time for liposomes consisting of DSPCxholesterol (CH) (55:45 mol %); DSPCDSPE-PEC ⁇ OOO (95:5 mol %); and, DSPC: CH:DSPE-PEGno ⁇ (50:45:5 mol %) represented by filled circles, open circles, and inverted closed triangles, respectively.
  • CH DSPCxholesterol
  • DSPE-PEC ⁇ OOO 95:5 mol %
  • DSPC CH:DSPE-PEGno ⁇
  • Figure 8 A graph showing doxorabicin: lipid ratio remaining in the blood after intravenous injection of Balb/c mice as a function of time for liposomes consisting of DSPCxholesterol (CHOL) (55:45 mol %); and, DSPGDSPE-PEGbooo (95:5 mol %) represented by filled circles and open circles, respectively.
  • PEG polyethylene glycol
  • PEG preceded or followed by a number the number is the molecular weight of PEG in Daltons
  • PEG-lipid polyethylene glycol-lipid conjugate
  • PE-PEG polyethylene glycol-derivatized phosphatidylethanolamine
  • PA phosphatidic acid
  • PE phosphatidylethanolamine
  • PC phosphatidylcholine
  • PI phosphatidylinositol
  • DSPC l,2-distearoyl-sra-glycero-3-phosphocholine
  • DSPE-PEG 2000 or 2000 PEG-DSPE or PE&ooo-DSPE
  • DSPE-PEG 750 or 750PEG-DSPE glycol 750]
  • DPPE-PEG2000 l
  • retention with respect to a drug or other agent encapsulated in a liposome refers to retention of the drug in a liposome while the liposome is present in the bloodstream of a mammal. This term does not refer to a measure of drag that may be loaded or incorporated into a liposome or the ability of a liposome to retain the drag in ex vivo conditions.
  • cholesterol-free as used herein with reference to a liposome means that a liposome is prepared in the absence of cholesterol, or that the liposome contains substantially no cholesterol, or that the liposome contains essentially no cholesterol.
  • substantially no cholesterol allows for the presence of an amount of cholesterol that is insufficient to significantly alter the phase transition characteristics of the liposome (typically less than 20 mol % cholesterol). 20 mol % or more of cholesterol broadens the range of temperatures at which phase transition occurs, with phase transition disappearing at higher cholesterol levels.
  • a liposome having substantially no cholesterol will have about 15 or less and more preferably about 10 or less mol % cholesterol.
  • phase transition temperature is the temperature or range of temperatures at which a liposome changes from a gel state to a liquid crystalline state. A convenient method for measuring phase transition temperature is to monitor energy absorption while heating a preparation of liposomes and noting the temperature or range in temperatures at which there is an energy absorbance.
  • liposome as used herein means vesicles comprised of one or more concentrically ordered lipid bilayers encapsulating an aqueous phase.
  • vesicle-forming lipids which are amphipathic lipids capable of either forming or being incorporated into a bilayer structure.
  • the latter term includes lipids that are capable of forming a bilayer by themselves or when in combination with another lipid or lipids.
  • An amphipathic lipid is incorporated into a lipid bilayer by having its hydrophobic moiety in contact with the interior, hydrophobic region of the membrane bilayer and its polar head moiety oriented toward an outer, polar surface of the membrane. Hydrophilicity arises from the presence of functional groups such as hydroxyl, phosphato, carboxyl, sulfato, amino or sulfhydryl groups. Hydrophobicity results from the presence of a long chain of alaphatic hydrocarbon groups.
  • hydrophilic polymer-lipid conjugate refers to a vesicle-fo ⁇ riing lipid covalently joined at its polar head moiety to a hydrophilic polymer, and is typically made from a lipid that has a reactive functional group at the polar head moiety in order to attach the polymer. Suitable reactive functional groups are for example, amino, hydroxyl, carboxyl or formyl.
  • the lipid may be any lipid described in the art for use in such conjugates other than cholesterol.
  • the lipid is a phospholipid such as PC, PE, PA or PI, having two acyl chains comprising between about 6 to about 24 carbon atoms in length with varying degrees of unsaturation.
  • the lipid in the conjugate is a PE, preferably of the distearoyl form.
  • the polymer is a biocompatible polymer characterized by a solubility in water that permits polymer chains to effectively extend away from a liposome surface with sufficient flexibility that produces uniform surface coverage of a liposome.
  • the polymer is a polyalkylether, including polymethylene glycol, polyhydroxy propylene glycol, polypropylene glycol, polylactic acid, polyglycolic acid, polyacrylic acid and copolymers thereof, as well as those disclosed in United States Patents 5,013,556 and 5,395,619.
  • a preferred polymer is polyethylene glycol (PEG).
  • the polymer has a molecular weight between about 1000 and 5000 daltons.
  • the conjugate may be prepared to include a releasable lipid-polymer linkage such as a peptide, ester, or disulfide linkage.
  • the conjugate may also include a targeting ligand. Mixtures of conjugates may be incorporated into liposomes for use in this invention.
  • PEG-conjugated lipid refers to the above-defined ' hydrophilic polymer-lipid conjugate in which the polymer is PEG.
  • the predominant vesicle-forming lipid in liposomes of this invention are responsible for achieving phase transition temperatures of greater than 45°C.
  • the lipid is a phospholipid such as PC, PE, PA or PE.
  • the preferred phospholipid is PC.
  • the lipid will have two saturated fatty acids, the acyl chains of which being independently selected from the group consisting of stearoyl (18:0), nonadecanoyl (19:0), arachidoyl (20:0), heniecosanoyl (21:0), behenoyl (22:0), tricosanoyl (23:0), lingnoceroyl (24:0) and cerotoyl (26:0).
  • at least one (and preferably both) of the acyl chains will be 19:0, or longer.
  • Liposomes of this invention may comprise amphipathic lipids in addition to those described above, but no substantial amount of cholesterol.
  • Such lipids include sphingomyelins, glycolipids, ceramides and phospholipids.
  • Such lipids may include lipids having therapeutic agents, targeting agents, ligands, antibodies or other such components which are used in liposomes, either covalently or non-covalently bound to lipid components.
  • drug and “therapeutic agent” as used herein refer to chemical moieties used in therapy and for which liposome-based drag delivery is desirable.
  • anti- neoplastic agent refers to chemical moieties having an effect on the growth, proliferation, invasiveness or survival of neoplastic cells or tumours.
  • Anti- neoplastic therapeutic agents include alkylating agents, antimetabolities, cytotoxic antibiotics and various plant alkaloids and their derivatives.
  • idarabicin-compound refers to idarubicin (e.g. 4- demethoxydaunorabicin) and related anthracyclines having the same core structure and hydrophobicity of idarubicin, including: daunorubicinol, 3'-hydroxyidarabicin, idarabicinol and WP619. This term includes salts of the aforementioned compounds.
  • a preferred idarabicin-compound for use in this invention is idarubicin hydrochloride, sold for example under the trademark IDAMYCIN.
  • camptothecin-compound refers to camptothecin and derivatized forms of this plant alkaloid having topoisomerase inhibition activity, including topotecan, ironotecan, lurtotecan, 9-aminocamptothecin, 9-nitrocamptothecin and 10- hydroxycamptothecin, including salts thereof.
  • a preferred camptothecin-compound is topotecan (hydrochloride salt) also known as hycamptamine.
  • Liposomes of the present invention or for use in the present invention may be generated by a variety of techniques including lipid filrn/hydration, reverse phase evaporation, detergent dialysis, freeze/thaw, homogenation, solvent dilution and extrusion procedures.
  • Drugs may be encapsulated inside these liposomes by passive or active loading techniques known in the art.
  • a particularly suitable encapsulation or liposome loading technique is pH gradient loading such as is described in the Examples herein.
  • liposomes are formed which encapsulate an aqueous phase of a selected pH. Hydrated liposomes are placed in an aqueous environment of a different pH selected to remove or n inimize a charge on the drug or other agent to be encapsulated. Once the drug moves inside the liposome, the pH of the interior results in a charged drag state, which prevents the drag from permeating the lipid bilayer, thereby entrapping the drag in the liposome.
  • Liposomes of this invention or for use in this invention may be from 50nm to about ljiim in diameter. However, preferred liposomes of this invention will be less than about 200 nm, preferably less than about 160 nm, and more preferably less than about 140 nm in diameter. 100-140 nm liposomes (cholesterol-free liposomes tend to be slightly larger than cholesterol containing ones) are employed in the Examples below. Liposomes are typically sized by extrusion through a filter (e.g. a polycarbonate filter) having pores or passages of the desired diameter. Liposomes of this invention may be formulated for parenteral administration in a suitable carrier such as a sterile aqueous solution.
  • a suitable carrier such as a sterile aqueous solution.
  • the carrier may comprise excipients known to be tolerated by warm-blooded animals.
  • a mammal such as a mouse will be injected with a liposome formulation and blood is removed from the mouse at fixed time intervals such as 1 or 4 or 24 hours post-admimstration.
  • a convenient means for obtaining blood at a fixed time interval is by cardiac puncture.
  • the plasma is isolated and subjected to suitable techniques for measuring the amount of lipid and drag present.
  • the lipid component may be radioactively labelled and the plasma subjected to liquid scintillation counting.
  • the amount of drag may be determined by a spectraphotometric assay.
  • DSPC and DSPE-PEG2000 were obtained from Northern Lipids.
  • DAPC and DBPC were obtained from Avanti Polar Lipids. Cholesterol and Sephadex G50 was from Sigma Aldrich. 3H-cholesteryl hexadecyl ether ( 3 CHE) was from Perkin Elmer.
  • Idarubicin hydrochloride (referred to below as idarubicin) was obtained from Pharmacia & Up John and topotecan from Smith Kline Beecham Pharma. Daunorubicin and doxorubicin were obtained from Rhone-Poulenc Rorer and Faulding Inc. respectively.
  • Example 1 Optimal uptake of idarubicin into cholesterol free liposomes occurs at 37°C
  • Average liposome size was determined by quasi-elastic light scattering using a NICOMP 370 submicron particle sizer at a wavelength of 632.8 nm.
  • the resulting liposomes were run down a Sephadex G50 column equilibrated with HBS (20 mM HEPES, 150 mM NaCl, pH 7.45) in order to create a transmembrane pH gradient by exchange of the exterior buffer.
  • Resulting pH gradient liposomes were combined with idarubicin to give a final concentration of 5 mM lipid and 1 mM idarubicin (0.2:1 drag:lipid ratio) in a final volume of 1 mL adjusted with HBS.
  • Fig. 1 shows that optimal loading of idarubicin occurred at 37°C, while no loading was observed at 50 and 65°C possibly due to collapse of the pH gradient at higher temperatures.
  • Example 2 shows that optimal loading of idarubicin occurred at 37°C, while no loading was observed at 50 and 65°C possibly due to collapse of the pH gradient at higher temperatures.
  • pH gradient liposomes consisting of DSPCxholesterol (55:45 mol %) and DSPC:
  • DSPE-PEG2000 (95:5 mol %) were prepared according to the materials and methods of Example 1.
  • the resulting liposomes were loaded as in Example 1 (drag:lipid ratio of 0.2 with 5 mM lipid and 1 mM idarubicin) at 37°C for 60 minutes.
  • a total lipid dose of 3.3 ⁇ moles (165 ⁇ moles/kg) was administered to CD-I female mice in a final volume of 200 ⁇ L immediately after preparation (within 1-2 hrs). Blood samples were removed at 1 hour after administration by cardiac puncture. Lipid concentration was determined by liquid scintillation counting.
  • Idarubicin was extracted from plasma samples and quantified as follows:
  • a defined volume of plasma was adjusted to 200 ⁇ L with distilled water followed by addition of 600 ⁇ L of distilled water, 100 ⁇ L of 10% SDS and 100 ⁇ L of 10 mM H2SO4. The resulting mixture was mixed and 2 mL of 1:1 isopropanol/chloroform was added followed by vortexing. The samples were frozen at -20°C overnight or -80°C for 1 hour to promote protein aggregation, brought to room temperature, vortexed and centrifuged at 3000 rpm for 10 minutes.
  • the bottom organic layer was removed and assayed for fluorescence intensity at 500 nm as the excitation wavelength (2.5 nm bandpass) and 550 nm as an emission wavelength (10 nm bandpass) and using an absorbance wavelength of 480 nm.
  • Fig. 2A, B and C show that although lipid concentrations after intravenous administration of DSPCxholesterol (55:45 mol %) and DSPC:DSPE-PEG2 ⁇ oo (95:5 mol %) were comparable, idarubicin levels were 5 fold higher in the cholesterol-free, PEGylated formulations after 1 hr. This is further exemplified in Fig. 3A, B and C at various time points after intravenous injection.
  • pH gradient liposomes consisting of DSPC:PEG2000 (95:5 mol %) DAPC (95:5 mol %) and DBPC (95:5 mol %) were prepared and loaded with idarubicin. Except during loading, concentrations of lipid and idarubicin were 16.5 mM and 2.2 mM respectively. Immediately following loading, (within 1-2 hrs) the liposomes were administered to Balb/C mice and blood samples were removed at 15 min, 30 min, 1, 2, 4 and 24 hours after intravenous injection and assayed for lipid and idarubicin concentrations as in Example 2.
  • Figure 4 shows that cholesterol-free liposomes exhibit enhanced retention of idarubicin as the length of the acyl chain is increased from 18 to 24 carbon atoms.
  • DSPC:DSPE-PEG2000 (95:5 mol%) and DAPC:DSPE-PEG2000 (95:5 mol%) liposomes were prepared and loaded with idarubicin as outlined in the materials and methods of Example 1 and Example 3 respectively.
  • P388/wt cells were maintained by passage in vivo (in the peritoneum) of BDF-1 female mice. Cells were only used for experiment between the 3 rd and 20 th passage. Cells were harvested 7 days post inoculation, diluted in Hepes Buffered Saline (HBS) to 2 x 10 6 cells/mL, and 0.5 mL was injected intraperitoneally into BDF-1 mice.
  • HBS Hepes Buffered Saline
  • BDFl female mice Two days after tumor cell inoculation, BDFl female mice were administered by intravenous administration one of the following: HBS; idarubicin (1 mg/kg); DSPC:DSPE-PEG2000 (95:5 mol%) and DAPC:DSPE-PEG2000 (95:5 mol%) liposomes (1 mg/kg) loaded with idarubicin. Percent survival was calculated based on 4 mice per group.
  • Figure 5 shows that percent survival was increased for idarubicin encapsulated in cholesterol-free DSPC and DAPC liposomes relative to administration of free drug.
  • DSPC/cholesterol (55:45 mol %) and DSPC/DSPE-PEG2000 (95:5 mol%) liposomes were prepared as in Example 1 except the dried lipid films were hydrated in 300 mM manganese sulfate and the external buffer was exchanged with 300 mM sucrose, 30 mM EDTA, and 20 mM HEPES pH 7.4. Topotecan was encapsulated into the liposomes using an ionophore-mediated proton gradient. The divalent cation ionophore A23187 (1 ⁇ g ionophore/ ⁇ mol lipid) was first added to the liposomes and the mixture was incubated at 60°C for 15 min.
  • topotecan was added at a drug:lipid ratio of 0.08: 1 and the mixture was incubated for 1 hr at 60° C. Unencapsulated topotecan and A23187 were removed from the preparation by dialyzing the sample at room temperature for 24 hrs against 100 volumes of 300 mM sucrose.
  • Liposomal topotecan (100 mg/kg lipid, 8 mg/kg topotecan) was injected into female Balb/C mice and the plasma elimination of both the lipid carrier and the drug were determined over a 24 hr. time course. Lipid concentrations remaining in the blood were measured by liquid scintillation counting. Topotecan was quantified by fluorescence spectroscopy. Plasma proteins were precipitated by the addition of 200 ⁇ L of methanol to 50 ⁇ L of plasma followed by centrifugation for 10 min. at 3500 rpm.
  • the supernatant was removed and assayed for fluorescence intensity using a Perkin-Elmer LS50 fluorescence spectrophotometer set at an excitation wavelength of 380 nm (2.5 nm slit width) and emission wavelength of 518 nm (2.5 nm slit width).
  • the methods employed in the preceding examples were performed to determine drug retention in cholesterol containing and cholesterol-free liposomes in cases where the drug is either daunorabicin (Fig. 7) or doxorabicin (Fig. 8).
  • the latter drugs are anthracycline anti-neoplastic agents which are similar in structure to idarubicin.
  • retention of daunorabicin and doxorabicin is decreased in cholesterol-free liposomes as compared to conventional liposomes containing cholesterol (with or without PEG-conjugated lipids).
  • Comparison of drag retention for the various drugs described above in cholesterol containing and cholesterol-free liposomes may be conveniently expressed by calculating the "area-under-the-curve" (AUC) of the graphs which plot drag:lipid ratios as a function of time.
  • AUC area-under-the-curve
  • the improvement in AUC over 0-4 hours post-administration that was observed in DSPC:DSPE-PEG liposomes as compared to DSPC:CHOL liposomes was +315% for idarubicin, +55% for topotecan, and -67% for daunorabicin.
  • this invention can provide significant improvements in drug retention (e.g. greater than 50%) for some drugs.

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Abstract

L'invention concerne des liposomes sensiblement exempts de cholestérol présentant une rétention médicamenteuse in vivo améliorée. Ces liposomes comprennent: (a) au moins 60 moles % d'un phospholipide comprenant deux acides gras saturés, la chaîne acyl de chacun étant identique ou différente, au moins une des deux chaînes acyl présentant plus de 18 atomes de carbone; (b) des lipides conjugués à des polymères hydrophiles représentant entre environ 2 et environ 15 moles %; et (c) jusqu'à 38 moles % d'au moins un lipide formant une vésicule. Des modes de réalisation spécifiques de la présente invention concerne des liposomes encapsulant de l'idarubicine ou du topotécan et présentant une rétention médicamenteuse améliorée. L'invention concerne également un procédé permettant de déterminer si une rétention d'un médicament particulier peut être améliorée par la présente invention.
PCT/CA2001/000655 2000-05-11 2001-05-11 Compositions d'excipient a base de lipides pour retention medicamenteuse amelioree Ceased WO2001085131A2 (fr)

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WO2004087104A1 (fr) * 2003-04-02 2004-10-14 Celator Pharmaceuticals, Inc. Compositions pharmaceutiques contenant des agents actifs ayant un groupe lactone et des ions de metaux de transition
WO2006052767A2 (fr) 2004-11-05 2006-05-18 Inex Pharmaceuticals Corporation Compositions et methodes destinees a stabiliser des preparations medicamenteuses liposomales
US7238367B2 (en) 2001-10-03 2007-07-03 Celator Pharmaceuticals, Inc. Liposome loading with metal ions
WO2009026427A3 (fr) * 2007-08-21 2009-09-24 Alza Corporation Compositions à base de liposomes pour une administration in vivo de composés d'acide boronique
US20110262524A1 (en) * 2004-10-06 2011-10-27 Bc Cancer Agency Liposomes with improved drug retention for treatment of cancer
WO2013067449A1 (fr) 2011-11-03 2013-05-10 Taiwan Liposome Company, Ltd. Compositions pharmaceutiques de dérivés de camptothécine hydrophobe
WO2014054026A1 (fr) * 2012-10-04 2014-04-10 University Of The Witwatersrand, Johannesburg Système d'administration de médicament liposomal
US10980798B2 (en) 2011-11-03 2021-04-20 Taiwan Liposome Company, Ltd. Pharmaceutical compositions of hydrophobic camptothecin derivatives

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US5013556A (en) * 1989-10-20 1991-05-07 Liposome Technology, Inc. Liposomes with enhanced circulation time
US5843473A (en) * 1989-10-20 1998-12-01 Sequus Pharmaceuticals, Inc. Method of treatment of infected tissues
US5997899A (en) * 1996-10-01 1999-12-07 Skyepharma Inc. Method for producing liposomes with increased percent of compound encapsulated
US5827533A (en) * 1997-02-06 1998-10-27 Duke University Liposomes containing active agents aggregated with lipid surfactants
BR9914601A (pt) * 1998-09-16 2001-10-23 Alza Corp Inibidor de topoisomerase capturado por lipossoma

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US7744921B2 (en) 2001-10-03 2010-06-29 Celator Pharmaceuticals, Inc. Liposome loading with metal ions
US7238367B2 (en) 2001-10-03 2007-07-03 Celator Pharmaceuticals, Inc. Liposome loading with metal ions
WO2004087104A1 (fr) * 2003-04-02 2004-10-14 Celator Pharmaceuticals, Inc. Compositions pharmaceutiques contenant des agents actifs ayant un groupe lactone et des ions de metaux de transition
US8709474B2 (en) 2004-10-06 2014-04-29 Bc Cancer Agency Liposomes with improved drug retention for treatment of cancer
US20110262524A1 (en) * 2004-10-06 2011-10-27 Bc Cancer Agency Liposomes with improved drug retention for treatment of cancer
US8349360B2 (en) * 2004-10-06 2013-01-08 Bc Cancer Agency Liposomes with improved drug retention for treatment of cancer
WO2006052767A2 (fr) 2004-11-05 2006-05-18 Inex Pharmaceuticals Corporation Compositions et methodes destinees a stabiliser des preparations medicamenteuses liposomales
WO2009026427A3 (fr) * 2007-08-21 2009-09-24 Alza Corporation Compositions à base de liposomes pour une administration in vivo de composés d'acide boronique
WO2013067449A1 (fr) 2011-11-03 2013-05-10 Taiwan Liposome Company, Ltd. Compositions pharmaceutiques de dérivés de camptothécine hydrophobe
US10391056B2 (en) 2011-11-03 2019-08-27 Taiwan Lipsome Company, LTD. Pharmaceutical compositions of hydrophobic camptothecin derivatives
KR20140057384A (ko) 2011-11-03 2014-05-12 타이완 리포솜 캄퍼니 리미티드 소수성 캄프토테신 유도체의 약제 조성물
CN103957912A (zh) * 2011-11-03 2014-07-30 台湾微脂体股份有限公司 疏水性喜树碱衍生物的药物组合物
US10980798B2 (en) 2011-11-03 2021-04-20 Taiwan Liposome Company, Ltd. Pharmaceutical compositions of hydrophobic camptothecin derivatives
KR101688898B1 (ko) 2011-11-03 2016-12-22 타이완 리포솜 캄퍼니 리미티드 소수성 캄프토테신 유도체의 약제 조성물
CN103957912B (zh) * 2011-11-03 2019-04-05 台湾微脂体股份有限公司 疏水性喜树碱衍生物的药物组合物
WO2014054026A1 (fr) * 2012-10-04 2014-04-10 University Of The Witwatersrand, Johannesburg Système d'administration de médicament liposomal
CN104812372A (zh) * 2012-10-04 2015-07-29 约翰内斯堡金山大学 脂质体药物递送系统

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