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WO1994026253A1 - Liposome ayant une couche a composants multiples qui contient un agent bioactif en tant que composants integres dans la bicouche - Google Patents

Liposome ayant une couche a composants multiples qui contient un agent bioactif en tant que composants integres dans la bicouche Download PDF

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Publication number
WO1994026253A1
WO1994026253A1 PCT/US1994/005332 US9405332W WO9426253A1 WO 1994026253 A1 WO1994026253 A1 WO 1994026253A1 US 9405332 W US9405332 W US 9405332W WO 9426253 A1 WO9426253 A1 WO 9426253A1
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Prior art keywords
liposome
bilayer
taxol
lipid
multicomponent
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PCT/US1994/005332
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English (en)
Inventor
James L. Slater
Andrew S. Janoff
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The Liposome Company, Inc.
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Publication date
Application filed by The Liposome Company, Inc. filed Critical The Liposome Company, Inc.
Priority to AU68327/94A priority Critical patent/AU6832794A/en
Publication of WO1994026253A1 publication Critical patent/WO1994026253A1/fr

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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 to a liposome having a multicomponent bilayer containing a bioactive agent which is an integral component of the bilayer.
  • Liposomes are spontaneously self-assembling structures comprising one or more bilayers of amphipathic lipid molecules enclosing an internal aqueous volume.
  • the amphipathic lipid molecules which make up the bilayer comprise a polar (hydrophilic) headgroup region covalently linked to one or more non-polar (hydrophobic) acyl chains.
  • the energetically unfavorable contact between the hydrophobic acyl chains and the aqueous medium causes the molecules to rearrange such that the polar headgroups are facing the aqueous medium while the acyl chains reorient towards the interior of the bilayer.
  • the net result is an energetically stable structure in which the acyl chains are effectively shielded from coming into contact with the aqueous medium.
  • the size distribution, lipid composition, lamellarity, as well as the other properties of presently available liposomes, may be tailored to meet specific needs.
  • a variety of methods exist for producing liposomes see, e.g., Szoka and Paphadjopoulos, in: Liposomes: From Physical Structure to Therapeutic Applications (C.G. Knight, ed., Elsevier/North Holland, pp. 51-82 (1981); Cullis et al., in: Liposomes, From Biophysics to Therapeutics M. J. Ostro, ed.), Marcel Dekker, pp. 39-72 (1987)). Bangham's original preparation (J. Mol. Biol.
  • Liposomes can be loaded with bioactive agents passively, i.e., by solubilizing the molecule in the medium in which the liposomes are formed in the case of water-soluble agents or adding lipid-soluble agents to the lipid solutions from which the liposomes are made.
  • Bioactive agents can also be loaded into liposomes actively, e.g., by establishing a potential gradient across the liposomal membrane and then adding the agent to the external medium (see Bally et al., U.S. Patent No. 5,077,056).
  • Bioactive molecules entrapped within liposomes can have an enhanced therapeutic index and improved biodistribution. Liposomal drugs are gradually released in the circulation, thereby alleviating the toxic side effects associated with administration of the free drug and minimizing the amount of the drug that need be administered to maintain desired serum levels. Additionally, drug- lipid formulations may be directed to intracellular sites of infection. However, preparation of liposomal formulations requires the development of vesicles capable of entrapping the drugs and preserving them in a useful form. As described above, known liposomes are useful in connection with lipid- and water-soluble agents. However, they are not well suited for the preparation of formulations of drugs poorly soluble in liposomal aqueous compartments or drugs which are not incorporated in a physically stable, pharmaceutically useful fashion into the hydrophobic bilayer interior.
  • This relief valve comprises an asymmetric lipid, which allows the bilayer to undergo a structural transformation between one organization in the absence of a bioactive agent to a distinct structural organization in its presence.
  • These references note the different forms of interdigitated structure which lipids with asymmetric acyl chains can assume. However, none of these references teach or suggest that an asymmetric lipid can undergo a structural transformation from an interdigitated state in the absence of a bioactive agent to a noninterdigitated state in its presence, let alone that during this transformation, the bioactive agent can become an integral component of a bilayer of which the asymmetric lipid is a component. None of the available references teach or suggest that individual bioactive agents are shielded from coming into contact with each other by the acyl chains of multicomponent bilayers having an asymmetric lipid, which pack around individual bioactive agent molecules and shield them from their nearest neighbors.
  • Taxol is an antimitotic agent which binds to tubulin, blocking disassembly of microtubules and thereby, cell division (Schiff et al., Nature 277:665 ( 1979)).
  • Taxol has been found to have activity against ovarian and breast cancers in particular, as well as against other cancers such as malignant melanoma, colon cancer, leukemias and lung cancer (see, e.g., Borman, Chemical & Engineering News, September 2, 1991, pp. 11- 18; The Pharmacological Basis of Therapeutics (Goodman Gilman et al., eds.), Pergamon Press, New York (1990), p.
  • Taxol derived from the bark of the Pacific yew Taxus brevifolia, is highly insoluble in water and aqueous solvents, thus limiting its bioavailability when administered to animals in the free form and making preparation of liposomal formulations difficult.
  • the drug is currently supplied as a concentrated sterile emulsion of 6 mg taxol per ml of a 50:50 mixture of CremophorELTM (Bristol
  • Myers Squibb which is a combination of a polyoxyethylated derivative of castor oil and ethanol.
  • the concentrated emulsion is diluted to 0.6 mg/ml, for therapeutic use, with sterile sodium chloride or dextrose solutions.
  • Administration of this formulation entails premedi cation with other drugs and a slow infusion of a large volume over 24 hours to avoid toxicity associated with the cremophor vehicle, thereby requiring that patients receiving taxol be admitted to hospitals over night. Additionally, because of the poor solubility of taxol in aqueous solutions, care must be exercised that the taxol does not precipitate from solution.
  • This invention provides a liposome having a multicomponent bilayer comprising: a first amphipathic lipid which comprises an acyl chain of length A and an acyl chain of length B, wherein A and B are integers equal to the numbers of carbon atoms in the acyl chains and A is greater than B; a second amphipathic lipid which comprises an acyl chain of length C and an acyl chain of length D, wherein C and D are integers equal to the numbers of carbon atoms in the acyl chains and C is greater than or equal to D; and a bioactive agent, wherein the difference between A and B is preferably greater than, but may be equal to, the difference between C and D, and wherein the bioactive agent is an integral component of the bilayer.
  • the first amphipathic lipid is known as the pocket-forming component of the multicomponent bilayer; the second amphipathic lipid is known as the bilayer thickness component.
  • “A” equals "C” and “C” equals "D”.
  • the first and second amphipathic lipids are preferably phospholipids, most preferably, phosphatidylcholines .
  • the multicomponent bilayer of the liposome of this invention when necessary to prevent lateral phase separation of the pocket forming and bilayer thickness components, may further comprise a phase separation prevention component which comprises an amphipathic lipid.
  • the first and second amphipathic lipids comprise a single bipolar lipid, that is, a lipid with two polar headgroups, each of which is linked to the same bilayer-spanning acyl chain and both of which are separately linked to shorter acyl chains, which together do not span the entire width of the bilayer.
  • the bioactive agent may be a therapeutic agent, e.g., an anticancer, antifungal, antibacterial, antiviral, antiparasitic, anti-aging, anti-inflammatory or growth-promoting agent.
  • the therapeutic agent is an anticancer agent, more preferably, taxol, camptothecin or nogalamycin and most presently preferable, taxol.
  • the liposome of this invention may further comprise a second bioactive agent, which may also be an integral component of the multicomponent bilayer, or which may be otherwise entrapped in the liposome.
  • the liposome may also further comprising an amount of a cyclodextrin effective to inhibit crystallization of the bioactive agent.
  • the cyclodextrin is preferably a chemically modified cyclodextrin, e.g., hydroxypropyl gamma cyclodextrin.
  • the liposome of this invention may be dehydrated, according to known procedures, to allow it to be stored and thereby extend its shelf life.
  • the first amphipathic lipid, the pocket forming component is C(18):C(10)-PC (SCPC).
  • SCPC C(18:1):C(18:1)-PC
  • DEPC C(18:1):C(18:1)-PC
  • the multicomponent bilayer of the liposome further comprises a phase separation prevention component which comprises an amphipathic lipid.
  • the phase separation prevention component comprises C(14):C(14)- PC (DMPC).
  • the bioactive agent is an anticancer agent, more preferably taxol, camptothecin or nogalamycin and most preferably, taxol.
  • composition comprising a pharmaceutically acceptable carrier and the liposome of this invention.
  • This pharmaceutical composition may be used in a method of treating an animal, e.g., a mammal, and preferably a human, afflicted with a disease which comprises administering to the animal a therapeutically effective amount of the pharmaceutical composition.
  • the bioactive agent incorporated into the multicomponent bilayer of the liposome is an anticancer agent.
  • the preferred uses for the liposome and pharmaceutical composition provided herein are the treatment of animals afflicted with a cancer, e.g., brain, ovarian, lung, colon or breast cancer.
  • the disease treated is a cancer and the liposome comprises a multicomponent bilayer comprising DEPC, DMPC, SCPC, and taxol or camptothecin.
  • the liposome and pharmaceutical composition may also be used to treat diseases caused by bacterial, viral, fungal or parasitic infections, or due to steroidal, regenerative, growth-associated or other diseases, disorders or conditions.
  • a unit dosage form of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the liposome of this invention.
  • the therapeutically effective amount of the liposome is from about 1 milligram of the liposome per kg of the body weight of the animal to which the pharmaceutical composition comprising the liposome is administered to about 1000 milligrams per kg, desirably from about 100 milligrams per kg of body weight to about 400 mg per kg.
  • the ratio of bioactive agent to lipid in the liposome is typically from about 1:50 to about 1:5, but may be made higher or lower when it is necessary to do so.
  • Figure 1 Depiction of the non-interdigitated configuration of a lipid (SCPC/(C(18:C(10)-PC)) with acyl chains of unequal length.
  • Figure 3 Chemical structures of hydrophobic molecules incorporated into the multicomponent liposome.
  • A taxol; B: nogalamycin; C: itraconazole (Janssen R51211); D: camptothecin; E: pregnenolone.
  • Figure 4. Three-dimensional graphical representations of results derived from differential scanning calorimetry studies of a liposome having a multicomponent bilayer comprising SCPC, DEPC and DMPC.
  • the base of the cube contains the triangular three-component phase diagram plotted in the x-y plane.
  • the vertical (z) axis can represent any single experimental measurement; for these figures, the transition onset temperatures is plotted on the z axis.
  • a three-dimensional surface can be drawn through these data.
  • a minimum in the onset temperature corresponds with the optimal proportions of SCPC, DEPC and DMPC in the multicomponent bilayer (SCPC: 0.300; DEPC: 0.375; DMPC: 0.325).
  • SCPC lipid with asymmetric acyl chains
  • DM dimyristoyl phosphatidylcholine (DMPC)
  • DE dielaidoyl phosphatidylcholine (DEPC).
  • A- G different views of the three-dimensional plot.
  • DSC Differential scanning calorimetry
  • FIG. 6 Schematic depictions of a bipolar lipid.
  • a and B mirror image views of a bipolar lipid in which two polar headgroups are both esterified to the same bilayer-spanning acyl chain. Each of the headgroups is linked to a separate, shorter acyl chain. The combined lengths of these shorter acyl chains does not equal the length of the membrane-spanning acyl chain.
  • DMPC dimyristoyl phosphatidylcholine
  • DPPC dipalmitoyl phosphatidylcholine
  • DSPC distearoyl phosphatidylcholine
  • DMPG dimyristoyl phosphatidylglycerol
  • POPG l-palmitoyl-2-oleoyl-phosphatidylglycerol
  • POPC l-palmitoyl-2-oleoyl-phosphatidylcholine
  • SOPC l-stearoyl-2-oleoyl- phosphatidylcholine
  • DOPC dioleoyl phosphatidylcholine
  • DEPC dielaidoyl phosphatidylcholine
  • BPS EPC: brain phosphatidylserine: egg phosphatidylcholine
  • EPC egg phosphatidylcholine
  • CHS cholesteryl hematomadylcholine
  • Liposomes are spontaneously self-assembling structures comprising one or more lipid bilayers surrounding an internal aqueous volume.
  • Lipid bilayers comprise two opposing monolayers of amphipathic lipid molecules, each of which comprises a polar (hydrophilic) headgroup adjacent to an internal or external aqueous phase and hydrophobic acyl chains arrayed in the bilayer interior.
  • the formation of stable bilayers reflects an energy balance of hydrophobic effects from the interaction of acyl chains and the surrounding aqueous environment, steric packing constraints on the acyl chains, attractive and repulsive interactions at the interface of the bilayer with the aqueous environment, curvature elasticity of the bilayer, and the like.
  • Liposomes may have one lipid bilayer, i.e., they may be unilamellar vesicles, or multiple bilayers, i.e., they may be multilamellar vesicles (MLVs). Unilamellar vesicles may be small (SUVs) or large unilamellar vesicles (LUVs). LUVs are liposomes with average diameters of from about 50 nm to about 200 nm. MLVs may be prepared by dissolving lipids in an organic solvent, evaporating the solvent and then adding an aqueous medium to the resultant lipid film (see, e.g., Bangham, J. Mol. Biol. 13:238 (1965)).
  • Freeze-and-thaw multilamellar vesicles are prepared by the repeated freezing and thawing of multilamellar liposomes according to the procedure described in Cullis et al. (U.S. Patent No. 4,975,282). Lenk et al. (U.S. Patent Nos. 4,522,803, 5,030,453 and 5,169,637) and Fountain et al. (U.S. Patent No. 4,588,708) disclose methods for producing multilamellar liposomes with substantially equal interlamellar solute distribution. LUVs may be prepared by extruding MLVs, under pressure, through filters with defined pore sizes.
  • the filter pore size is adjusted according to the size of LUV desired (see Cullis et al., U.S. Patent No. 4,588,708 and Loughrey et al., U.S. Patent No. 5,059,421). The contents of these references are incorporated herein by reference to describe the state of the art with respect to liposome preparation.
  • Amphipathic lipids used in conjunction with the above-described methods to prepare liposomes, comprise a polar (hydrophilic) headgroup and one or two hydrophobic acyl chains.
  • the headgroups may be phosphate, sulfate, amino or other suitable polar moities;
  • the acyl chains may be between 1 and 24, or more, carbon atoms in length and may have one or more double bonds, i.e., may be saturated or unsaturated.
  • the amphipathic lipids used in accordance with the practice of this invention are phospholipids, most preferably, phosphatidylcholines.
  • This invention provides a liposome having a multicomponent bilayer comprising: a first amphipathic lipid which comprises an acyl chain of length A and an acyl chain of length B, wherein A and B are integers equal to the numbers of carbon atoms in the acyl chains and A is greater than B; a second amphipathic lipid which comprises an acyl chain of length C and an acyl chain of length D, wherein C and D are integers equal to the numbers of carbon atoms in the acyl chains and C is greater than or equal to D; and a bioactive agent, wherein the difference between A and B is greater than or equal to the difference between C and D and wherein the bioactive agent is an integral component of the bilayer.
  • Multicomponent as used herein describes lipid bilayers comprising a bioactive agent, a first amphipathic lipid denoted the pocket forming component and a second amphipathic lipid denoted the bilayer thickness component. Multicomponent bilayers of the liposomes of this invention may further comprise a phase separation prevention component which comprises an amphipathic lipid. Accordingly, a multicomponent bilayer of a liposome of this invention has more than one component.
  • Bioactive agent as used herein, means a chemical compound, whether synthetically produced or naturally derived, that exhibits biological activity. The terms “bioactive agent” and “bioactive agent molecules” are both used to denote such compounds.
  • Bioactive agents suitable for use in accordance with the practice of this invention include, but are not limited to: antiviral, antibacterial, antifungal, antiparasitic or tumoricidial compounds, sterols, proteins, dyes, toxins, enzymes, immunomodulators, immunoglobulins, hormones, neurotransmitters, glycoproteins, radiolabels, radiopaque compounds, fluorescent compounds, cell receptor proteins, cell receptor ligands, antiinflammatory compounds, antiglaucomic agents, mydriatic compounds, bronchodilators, local anaesthetics, growth promoting agents and regenerative agents.
  • the first amphipathic lipid of the multicomponent bilayer is its pocket forming component.
  • "Pocket forming component” as used herein describes an amphipathic lipid with asymmetric acyl chains, that is, acyl chains with an unequal number of carbon atoms and hence, of uneven length.
  • A denotes the longer acyl chain of the first amphipathic lipid, or pocket forming component, and "B," the shorter acyl chain.
  • the asymmetry of acyl chain length allows the pocket forming component to undergo a change in configuration, from an interdigitated state in the absence of a bioactive agent to a non-interdigitated state in its presence.
  • the pocket thereby created in the bilayer is filled by the bioactive agent which induces its formation.
  • the second amphipathic lipid of the multicomponent bilayer is its bilayer thickness component, with "C” and “D” denoting the lengths of its acyl chains. It is preferred, but not required, that the acyl chains of this component be of equal, symmetric, length, i.e., that "C” equals "D.” However, when the acyl chains are of unequal length, “C” is used to denote the longer, and “D” the shorter, acyl chain.
  • Bilayer thickness component denotes an amphipathic lipid that defines the thickness of the multicomponent bilayer.
  • Determining thickness means that the thickness of the multicomponent bilayer will be approximately the same as the thickness of a bilayer containing the bilayer thickness component, but not the other component(s). Except for the terminal methyl gap, the space between the methyl terminal ends of acyl chains in opposing monolayers normally found at the bilayer midplane, the acyl chains of the bilayer thickness component will span the entire width of the bilayer interior. It is preferred that the longer acyl chains of the first and second amphipathic lipids be of equal length, i.e, that "A" equals "C.” However, "C” may also be greater than "A".
  • the critical feature of the bilayer thickness component is that it comprise one, if not two, acyl chains of a length sufficient to induce the pocket forming component's acyl chains to adopt a non-interdigitated packing conformation in the presence of a bioactive agent, i.e., that the acyl chains do not cross the bilayer midplane and interact with acyl chains of a pocket forming component in the opposing monolayer.
  • a bioactive agent i.e., that the acyl chains do not cross the bilayer midplane and interact with acyl chains of a pocket forming component in the opposing monolayer.
  • the acyl chains of the amphipathic lipids of opposing monolayers do not normally cross the bilayer midplane.
  • Bilayers comprising interdigitated amphipathic lipids have acyl chains which cross the bilayer midplane and interact with acyl chains in the opposing monolayer.
  • the other acyl chain of the second amphipathic lipid i.e, the acyl chain of length "D”
  • the shorter acyl chain of the first amphipathic lipid i.e., the acyl chain of length "B”
  • the difference between "A” and “B” is greater than the difference between "C” and "D,” i.e., that the first amphipathic lipid have more asymmetry than the second.
  • "D" and "B” may also be equal.
  • the first and second amphipathic lipid comprise acyl chains of the same lengths, i.e., that "A” equals "C” and "B” equals "D.” Accordingly, when the headgroup of the first and second amphipathic lipids is the same, the first and second amphipathic lipid is the same lipid.
  • the first and second amphipathic lipids may comprise a single, bipolar lipid.
  • a "bipolar lipid” (see Figure 6) comprises two polar headgroups, both of which are linked to an acyl chain which spans the entire width of the bilayer interior (the "bilayer-spanning" acyl chain). This acyl chain has about twice the length of the longest acyl chain of the bilayer thickness component.
  • Bipolar lipids form bilayers, in the presence of a bioactive agent, whose thickness is defined by the length of the bilayer- spanning acyl chain.
  • This thickness is about the same as the thickness of a bilayer formed from amphipathic lipid molecules having symmetric acyl chains, each of which has a length equal to one half of that of the bilayer-spanning chain (taking into account the terminal methyl gap normally found between the terminal methyl groups of opposing acyl chains in ordinary bilayers, but not found in bilayers made from bipolar lipids).
  • Each polar headgroup is also linked to a second acyl chain, this second acyl chain not being linked to the other headgroup. The total number of carbon atoms in both of these two second acyl chains is less than the number of carbon atoms in the bilayer-spanning acyl chain.
  • bipolar lipids have acyl chains of unequal, asymmetric, length, which can lead to formation of a pocket in the presence of a bioactive agent.
  • the two shorter acyl chains of a bipolar lipid fill the role of the pocket forming component.
  • Bipolar lipids are preferably formed by: preparing a longer acyl chain of the desired langth, i.e., having a number of carbon atoms equal to "A" plus “C", this longer acyl chain defining the thickness of bilayers comprising the bipolar lipid; selecting two shorter acyl chains such that their combined size is shorter than the length of the longer acyl chain, thereby establishing the degree of asymmetry desired for the bioactive agent selected to be an integral component of the bilayer; and attaching the longer acyl chain to two separate headgroups while linking each of the shorter acyl chains to one of the headgroups.
  • the liposome of this invention is preferably a large unilamellar vesicle and the above-described extrusion technique is the preferred method of its preparation, the liposome of this invention is not limited to being prepared by any of the above-described techniques. Rather, the liposome may be made by any of those techniques presently available for producing liposomes.
  • Bioactive agents preferred for use in accordance with the practice of this invention are those chemical compounds having structures which limit their solubility in aqueous media, and which are poorly soluble in lipids.
  • a bioactive agent is a therapeutic agent, i.e., an agent capable of treating or preventing diseases or disorders in living organisms to which it is administered.
  • Therapeutic agents may be anticancer, antibacterial, antiviral, antifungal, antiparasitic, antiaging, antiinflammatory, growth promoting or other such agents capable of treating or preventing diseases, disorders or conditions.
  • the therapeutic agent in the liposome of this invention is an anticancer agent, e.g., taxol, camptothecin or nogalamycin.
  • the anticancer agent is taxol.
  • Taxol as used herein is meant to include the compound itself (C47H51NO 14, tax-ll-en-9-one (see Figure 3)) as well as taxol analogs, i.e., those compounds, whether synthetically produced or naturally derived, with similar structures and activities.
  • Taxotere which differs from taxol by having a te/ -butoxycarbonyl group instead of a benzoyl group on the C- 13 side chain, and a hydroxyl group instead of an acetoxyl group at C-10, is one such analog (see, e.g., Borman, supra).
  • Other analogs include those which are derivatives of a hydroxylated taxane compound found in yew needles, e.g., baccatin-based 14-hydroxy taxol analogs such as 14-hydroxy-10- deacetylbaccatin III (see, e.g., Borman, Chemical and Engineering News, April 12, 1993, pp. 36-37).
  • the liposome may further comprise a second bioactive agent, which may also be an integral component of the bilayer. Alternatively, the second bioactive agent may be entrapped in an aqueous compartment of the liposome.
  • the multicomponent bilayer provides a pocket into which such a bioactive agent may fit without disrupting acyl chain packing.
  • "ordinary bilayers” i.e., those without pocket-forming and bilayer thickness components, insufficient space exists to accomodat ' e the preferred bioactive agents without incurring disruption of acyl chain packing.
  • Each bioactive agent molecule which sequesters itself within the bilayer due to its hydrophobic nature, causes an increasing amount of acyl chain disorder as the chains readjust in their attempt to pack around the molecule.
  • Disorder in the bilayer may eventually reach the point at which stability is compromised. Restoration of bilayer stability may result in the ejection of an amount of the bioactive agent sufficient to lower chain disorder below the critical point.
  • the pocket forming and bilayer thickness components enable the bioactive agent to become an integral component of the multicomponent bilayer.
  • the pocket formed by addition of the bioactive agent to a mixture of the pocket forming and bilayer thickness components minimizes the increase in acyl chain disorder, and may actually decrease it. Minimizing the amount of disorder allows for more bioactive agent to be incorporated into the multicomponent bilayer than can be incorporated into ordinary bilayers. Up to one bioactive agent molecule may be incorporated into the multicomponent bilayer for each pocket forming component for which there is an available bilayer thickness component.
  • “Integral” describes a bioactive agent which induces a change in conformation of the pocket forming component, in the presence of the bilayer thickness component, from an interdigitated to noninterdigitated state, and which is incorporated into the pocket in the bilayer thereby created.
  • “Interdigitation” and “interdigitated” are used herein to denote lipid bilayers in which the acyl chains of the pocket forming component in each monolayer cross the bilayer midplane and penetrate into the opposing monolayer, where they interact with the asymmetric acyl chains of another pocket forming component. Lipids may be fully, mixed or partially interdigitated.
  • Full interdigitation describes lipid bilayers in which each acyl chain in the bilayer spans the entire width of the bilayer, i.e., where there are four acyl chains per headgroup surface area.
  • asymmetric lipids cannot be fully interdigitated.
  • Mixed interdigitation describes the association of lipids with asymmetric acyl chains where the longer acyl chains span the entire width of the bilayer interior and the shorter acyl chains meet end-to-end across the gap between their terminal methyl groups, i.e., the "terminal methyl gap.”
  • Partially interdigitated asymmetric lipids have two acyl chains per headgroup surface area, with the longer acyl chain of one lipid meeting end-to-end with the shorter acyl chain of the other across the gap between their terminal methyl groups.
  • their acyl chains do not ordinarily cross the bilayer midplane.
  • Optimal incorporation of bioactive agent into the multicomponent bilayer occurs when the pocket forming and bilayer thickness components are combined in proportions such that a eutectic combination is formed. The components are most miscible with each other when they are combined in eutectic proportions.
  • Maximal miscibility means that the maximum amount of the bilayer thickness component will be available to aid in transformation of the pocket forming component from an interdigitated to non-interdigitated state.
  • the bioactive agents fits into the bilayer pocket created when its addition induces the pocket forming component to undergo this conformational transformation.
  • the maximum amount of bioactive agent one molecule of the agent per pocket forming component, may be incorporated into the multicomponent bilayer.
  • the relative proportions of the components may be altered such that they do not form a eutectic composition, but nevertheless form a stable bilayer in the presence of a bioactive agent.
  • the eutectic composition of a combination of amphipathic lipids can readily be determined by ordinarily skilled artisans using differential scanning calorimetry (DSC) analysis of the liquid/gel phase behavior of mixtures of different proportions of the lipids.
  • DSC differential scanning calorimetry
  • the eutectic composition of a combination of amphipathic lipids is the point at which a bilayer containing the lipids has a lower gel-liquid transition temperature than does a bilayer formed from any of the individual lipids.
  • Raman spectroscopy can be used by ordinarily skilled artisans to determine the most probable mode of acyl chain packing of a combination of amphipathic lipids.
  • Pocket forming and bilayer thickness components will be misicble when they adopt compatible acyl chain packing. When they do not, the components may exhibit lateral phase separation when combined. Formation of multicomponent bilayers in such cases requires the presence of a third amphipathic lipid, the "phase separation prevention component.” This is an amphipathic lipid with symmetric or asymmetric, saturated or unsaturated, acyl chains that inhibits or prevents lateral phase separation of two or more amphipathic lipids by adopting compatible acyl chain packing with each lipid.
  • the pocket forming component comprises acyl chains of lengths "A” and "B", where "A” and “B” are asymmetric, i.e., unequal. Presently, it is preferred that "A” is equal to 18, “B” is equal to 10 and the amphipathic lipid is a phosphatidylcholine, i.e., that the pocket forming component is C(18):C(10)-PC (l-myristoyl-2-capryl-3-sn-phosphatidylcholine (SCPC)).
  • the bilayer thickness component comprises an amphipathic lipid with acyl chains of lengths "C” and "D". "C” is preferably, but not necessarily, equal to "A”.
  • the pocket forming component is C(18):C(10)-PC
  • the bilayer thickness component is C(18:1):C(18:1)-PC (l-elaidoyl-2-elaidoyl-3-sn- phosphatidylcholine (DEPC)).
  • SCPC and DEPC exhibit lateral phase separation as a binary system because of their inability to adopt compatible acyl chain packing. Accordingly, formation of SCPC/DEPC multicomponent bilayers requires the presence of a phase separation prevention component.
  • this component is C(14):C(14)-PC (l-myristoyl-2-myristoyl-3-sn-phosphatidylcholine (DMPC)).
  • DMPC l-myristoyl-2-myristoyl-3-sn-phosphatidylcholine
  • SCPC and DMPC form eutectic compositions when the relative proportion of DMPC in the mixture is 40 mole percent (see Lin and Huang, Biochim. Biophys. Acta 946: 178 (1988)).
  • the SCPC/DMPC and DEPC/DMPC binary systems can be combined to form a ternary lipid mixture in which the acyl chain packing incompatibilities between SCPC and DEPC are overcome by the favorable interactions of both with DMPC.
  • Combination of eutectic binary systems in equal proportions results in a ternary lipid system comprising 37.5 mole percent DEPC, 32.5 mole percent DMPC and 30.0 mole percent SCPC.
  • SCPC SCPC
  • DEPC DEPC
  • DMPC DEPC
  • DEPC DEPC
  • DMPC DEPC
  • DMPC DMPC
  • DEPC DEPC
  • DMPC DMPC
  • DEPC DEPC
  • DMPC DMPC
  • the bioactive agent that is an integral component of a SCPC/DEPC/DMPC multicomponent bilayer be an anticancer agent, e.g., taxol, camptothecin or nogalamycin. Most preferably, the anticancer agent is taxol.
  • the liposome comprising this multicomponent bilayer may further comprise a second bioactive agent, which may be an integral component of the bilayer, but may also be otherwise entrapped in the liposome.
  • Bioactive agents of differing dimensions may be incorporated into the multicomponent bilayer by adjusting the acyl chain lengths of the pocket forming component, i.e., adjusting its asymmetry, to fit the bioactive agent.
  • the bilayer thickness component should constrain the pocket forming component to adopt a non-interdigitated conformation in the presence of the bioactive agent.
  • the thickness of a bilayer containing the bilayer thickness component, but not the other components, will be approximately equal to the thickness of a bilayer formed from an amphipathic lipid having symmetrical acyl chains, each having a length about equal to that of the longer acyl chain of the pocket forming component (i.e., the acyl chain of length A of the first amphipathic lipid).
  • These bilayer thicknesses can be measured by x-ray diffraction or any other technique known in the art suitable for measuring bilayer thicknesses.
  • the phase separation prevention component if necessary, should force the pocket forming and bilayer thickness components to adopt compatible acyl chain packing, i.e., that pocket forming and bilayer thickness components be able to pack next to each other in non-interdigitated configurations.
  • the most probable mode of acyl chain packing can be determined by Raman spectroscopy or any other suitable technique.
  • the length of the acyl chains of the phase separation prevention component are such that a bilayer formed from it, without the other components, is approximately equal to the thickness of a bilayer formed from the pocket forming component arrayed in a partially interdigitated conformation.
  • the multicomponent liposome of this invention may be lyophilized, stored and rehydrated by known procedures (see, e.g., Janoff et al., U.S. Patent No. 4,880,635, the disclosure of which is incorporated herein by reference).
  • This invention provides a liposome comprising an amount of a cyclodextrin effective to enhance the solubility of, and thereby inhibit crystallization of, the bioactive agent.
  • the invention provides a liposome in which the bioactive agent is both an integral component of the bilayer and is otherwise entrapped in the liposome.
  • This latter form of the bioactive agent may be associated with a cyclodextrin to enhance its solubility.
  • the cyclodextrin may be the parent compound, but is preferably, a chemically modified cyclodextrin.
  • Naturally occurring cyclodextrins (CDs) alpha, beta and gamma are cyclic polymers with 6,7 or 8 glucopyranose units.
  • CMCDs Chemically modified cyclodextrins
  • CMCD complexes provide enhanced solubility in comparison to their parent cyclodextrins, and are therefore valuable for further enhancing the amount of poorly soluble compounds which may be incorporated into liposomes.
  • CMCD complexes may be especially useful in connection with a lyophilized liposome preparation wherein crystallization need only be inhibited between the time of rehydration and administration.
  • the liposome may be administered to animals such that the hydrophobic molecule/bioactive agent is presented to the animals in a therapeutically useful form.
  • the liposome may be administered alone, but will more commonly be given as part of a pharmaceutical composition comprising the liposome and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier means any of the standard carriers, diluents, excipients and the like generally intended for use in connection with the administration of biologically active agents to animals.
  • Such carriers are well known in the art and are generally chosen with regards to a number of factors, such as the particular drug being used and the intended route of administration, which are understood by the ordinarily skilled artisan.
  • compositions of this invention be administered intravenously.
  • pharmaceutical carriers presently preferred for use in accordance with the practice of this invention are those well known carriers suitable for use in connection with intravenous administration of liposomes and include, but are not limited to, sterile aqueous solutions such as physiological saline, 5% dextrose USP solutions and various aqueous buffers, e.g., aqueous phosphate buffers. The total solute concentration in such carriers should be controlled to keep the composition isotonic.
  • Pharmaceutically acceptable carriers may also contain additional components, such as anti-oxidants, preservatives and the like, which are compatible with the active agent. The choice of such additional components is well within the purview of the ordinarily skilled artisan.
  • Other carriers e.g., tablets for oral administration and oils for mucosal or topical administration, may be prepared employing general knowledge and used in accordance with the practice of this invention.
  • Such pharmaceutical compositions may be administered to animals, e.g., mammals, and preferably humans, for the diagnosis, treatment or pevcention of diseases, disorders or conditions.
  • the bioactive agent is a therapeutic agent and the pharmaceutical composition is used in a method of treating an animal for a disease, disorder or condition which comprises administering to the animal a therapeutically effective amount of the pharmaceutical composition.
  • “Therapeutically effective amount” as used herein means any amount of the pharmaceutical composition effective to treat, inhibit or prevent a disease, condition or disorder in an animal.
  • therapeutically effective amounts depend upon a number of factors within the purview of the ordinarily skilled artisan to determine including: the age, weight, size and general condition of the animal being treated; the type of disease being treated and the stage of its progression; and the type of liposome employed and the lipids used to prepare it.
  • the therapeutically effective amount of the pharmaceutical composition is an amount comprising from about 1 milligram of the liposome of this invention per kg of the body weight of the animal to which the pharmaceutical composition is administered to about 1000 milligrams per kg, desirably from about 100 milligrams per kg of body weight to about 400 mg per kg.
  • the ratio of bioactive agent to lipid in the liposome is typically from about 1:50 to about 1:5, but may be made higher or lower when it is necessary to do so.
  • the bioactive agent which is an integral component of the multicomponent bilayer is an anticancer agent.
  • the liposome provided is used in a pharmaceutical composition to treat an animal afflicted with a cancer. Cancers are treated, according to the practice presently preferred herein, with a liposome having a multicomponent bilayer comprising SCPC, DEPC, DMPC and taxol or camptothecin, but, most preferably, taxol.
  • this invention is not limited to one specific embodiment of disease treatment, or even to disease treatment. Rather, the multicomponent liposome may be employed for any diagnostic or therapeutic purpose for which a suitable bioactive agent may be found.
  • the liposome may be used to diagnose and/or treat diseases caused by bacterial, viral, fungal and parasitic infections or growth, aging or regenerative disorders.
  • a unit dosage form of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the liposome of this invention.
  • “Therapeutically effective amount” as used herein means any amount of the liposome effective to treat, inhibit or prevent a disease, condition or disorder in an animal.
  • the therapeutically effective amount of the liposome is from about 1 mg of the multicomponent liposome per kg of the body weight of the animal to which the pharmaceutical composition is administered to about 1000 mg per kg of body weight. Desirably, the therapeutically effective amount is from about 100 mg per kg of body weight to about 400 mg per kg.
  • Drug-to-lipid ratios in the liposome of this invention depend upon a number of factors, such as the relative proportions of the various lipid components, the nature of the bioactive agent and the type of liposome used, that are within the purview of the ordinarily skilled artisan to control.
  • the drug-to-lipid ratio (w/w) is from about 1:50 to about 1:5, but may be higher or lower when necessary.
  • Dispersion of taxol and a lipid were formulated, with the taxol and lipid concentrations (as well as the relative proportions of taxol and lipid) given in Table 1 (see below).
  • the ability of taxol to form stable dispersions with each lipid is indicated (a "Yes" in Table 1 indicates that a stable dispersion was formed).
  • the ability of taxol to form a dispersion with a particular lipid is an indicator of that lipid's potential suitability for formulation in a multicomponent bilayer.
  • the initial screening of lipid candidates was performed at a target taxol concentration of 30 micrograms taxol per milligram lipid, using a target lipid concentration of 20 milligrams per milliliter.
  • the actual lipid concentration was determined for phospholipids by using a modified Bartlett assay.
  • Taxol concentration was determined by UV absorbance, using the standard literature value of 29,800 liter mol "1 cm "1 at 228 nm for the molar extinction coefficient. In cases where lipid absorbed appreciably, a correction was made by subtracting the lipid contribution to the absorbance at that wavelength. Physical stability of the preparations were examined by light microscopy to determine the presence of taxol crystals, which appeared as needle-shaped aggregates.
  • Taxol crystals formed readily with gel phase saturated chain lipids; crystals also formed if the lipid headgroups had a negative charge or the acyl chains had transunsaturation. Taxol crystallization was observed in dispersions with most of the lipids tested within 24 hours to five days; crystallization was observed by seven months in all of the dispersions, indicating that taxol/lipid dispersions are of limited therapeutic utility.
  • Example 2
  • the solubility limit of taxol in EPC was examined (see Table 2, below).
  • the first study varied the taxoklipid ratio at low lipid (20 mg/ml) concentrations as follows: multilamellar liposomes were prepared, in triplicate, at target concentrations ranging from 50 micrograms taxol per milligram lipid to 300 micrograms taxol per milligram lipid. The target concentration of lipid was 20 milligrams per milliliter.
  • DSC Differential scanning calorimetry
  • Figures 4A-4G are a three dimensional graphical representation of a parameter derived from these DSC studies.
  • the base of the cube contains the triangular three component phase diagram, plotted in the x-y plane.
  • the vertical (z) axis can represent any single experimental measurement; for these figures, the transition onset temperature is plotted.
  • a three dimensional surface can be drawn through these data.
  • a minimum in the onset temperature corresponds with the chosen ternary composition (0.375 DEPC: 0.325 DMPC: 0.300 SCPC), indicating that with respect to the resolution of this experiment, the composition initially chosen is near an optimal point.
  • samples were prepared at target taxohlipid ratios of 0, 10, 20, 30, 40, 50, 60, 70, 80 micrograms taxol per milligram lipid. These samples were characterized by differential scanning calorimetry (see Figure 3).
  • Molecules of pharmaceutical interest other than taxol were examined for their ability to be an integral component of a multicomponent bilayer.
  • Nogalamycin (antitumor) (Fig 3B), Janssen R51211 [itraconazole] (antifungal) (Fig 3C); camptothecin (antitumor) (Fig 3D); and pregnenolone (steroid) (Fig 3E) were incorporated into a DEPC/DMPC/SCPC multicomponent bilayer by adding the compounds to a mixture of the lipids (at mole ratios of 0.375, 0.325 and 0.300 for DEPC, DMPC and SCPC, respectively) prior to their formation into liposomes.
  • the compounds were prepared at approximately 3 mole percent with respect to lipid, which corresponds roughly with one molecule per bilayer pocket. All of the compounds share the common trait of vanishingly low water solubility.
  • taxol is currently administered as a cremophor-based suspension. Typically, this suspension is given at 250 mg/sqm, intravenously over a 24-hour period. It is accompanied by administration of dexamethasone (20 mg, at 7 and 14 hours prior to the administration of taxol), diphenyhdramine and cimetidine (25 mg and 300 mg, respectively, intravenously 1 hour prior to taxol administration), and diphenydramine and epinephrine (on an "as needed" basis during infusion).
  • Other drug related toxicities observed to occur in subjects given taxol include mucositis, nausea and vomiting, alopecia and fever.
  • the subjects also exhibit vehicle-related toxicities such as anaphylactoid reaction, rashes, pruritus and flushing. Such toxicities are observed to occur at a rate about equal to the infusion rate.
  • Taxol Vehicle Dose (mg/kg) Predictor Equation 1
  • % Dead log dose + b; "m,” is the slope of a graphical representation of the predictor equation and "b,” its y intercept. 2 DEPC/DMPC/SCPC liposomes.
  • BDF1 C57BL6 x DBA/2
  • B16 melanoma cells 0.5 ml of 10% Brei
  • Administration of taxol was intraperitoneally at 1, 5 and 9 days after injection of the B16 cells.
  • Taxol was formulated into EPC liposomes, by adding it to the EPC prior to liposome formation.
  • Taxol was formulated into DEPC/DMPC/SCPC multilamellar liposomes by adding it to a mixture of these lipids (at mole ratios of 0.375, 0.325 and 0.300 for DEPC, DMPC and SCPC, respectively) prior to liposome formation.
  • the drug was administered as a dispersion with polysorbate base, in a Cremophor suspension, in EPC multilamellar vesicles and in DEPC/DMPC/SCPC multilamellar liposomes at the indicated dosages (6.25, 12.5, 25, 50 and 100 mg per kg of body weight per day of taxol, and 18.75, 37.5, 75, 150 and 300 mg per kg total taxol). Also administered were "vehicle controls" of polysorbate base or cremophor base alone, i.e., with no taxol associated. The animals were weighed at the first, fifth, ninth and thirteenth days of the treatment period, and were sacrificed on the sixtieth day.
  • Polysorbate Base 1 20.5 108 0
  • Cremophor Base 1 19.0 100 0
  • % T/C (100 x median day of death of treated animals)/(median day of death of untreated animals) [excludes survivors]. Long term survivors: tumor-free animals surviving to the end of the experimental period.
  • BDF1 C57BL/6 x DBA/2 female mice weighing 14-20 grams were used in this study. These mice were injected with P388 leukemia cells (1 x 10"), intraperitoneally, at day zero. Taxol was administered to the animals, intraperitoneally, at the first, fifth and ninth days of the treatment period at the dosages indicated in Table 10 (see below). The animals were sacrificed on the sixtieth day. The median day of death in the sample population, the % T/C, the number of long term survivors and the occurrence of toxic deaths were determined (see Table 10).
  • Table 11 shows the %T/C exhibited when mice were administered P388 leukemia cells and the optimal daily dose of taxol, either as a dispersion with polysorbate, in a Cremophor suspension, formulated in EPC liposomes or formulated in DEPC/DMPC/SCPC liposomes.
  • Table 9 presents data derived from publicly available sources indicating the known antitumor activities of taxol in the indicated tumor systems.
  • Cremophor 1 11.5 105 0 NO
  • taxol doses greater than 6. ⁇ mg/kg/day the median day of death was higher for animals administered the taxol-containing liposomes of this invention than for animals treated with taxol/cremophor.
  • %T/C a measure of the relative survival of treated and untreated populations, was higher for animals given DEPC/DMPC/DSPC liposomes containing taxol.
  • potency (effect unit mass) of the liposomal forms were comparable to the non- liposomal forms.
  • Regressions partial (part.): greater than or equal to a ⁇ 0% reduction in tumor size; complete: reduction to below palpable limits.
  • the DEPC/DMPC/SCPC liposomes containing taxol were better able to increase the proportion of survivors amongst treated animals and were also better able to inhibit tumor growth.
  • Stability studies involved formation of the various preparations indicated below; and the preparations were monitored for the formation of visible crystals using phase-contrast light microscopy at 400x magnification.
  • the target concentrations of drug and lipid in each set of experiments are indicated below.
  • A TAXOL CRYSTAL FORMATION IN EPC MLVs: The following samples were prepared at a lipid concentration of 20 mg/ml: ⁇ O micrograms taxol/mg lipid (A); 100 micrograms taxol/mg lipid (B); l ⁇ O micrograms taxol mg lipid (C); 200 micrograms taxol/mg lipid (D); 2 ⁇ 0 micrograms taxol/mg lipid (E). Samples B - E had visible crystals two days after sample hydration. Two out of three sample A's had crystals present ⁇ days after sample hydration. All samples had crystals present 2 weeks after hydration.
  • the following samples were prepared at a lipid concentration of 100 mg/ml: 20 micrograms taxol mg lipid (A); 30 micrograms taxol mg lipid (B); 40 micrograms taxol mg lipid (C); ⁇ O micrograms taxol mg lipid (D); 60 micrograms taxol/mg lipid (E); 70 micrograms taxol/mg lipid (F); and 80 micrograms taxol/mg lipid (G).
  • A micrograms taxol mg lipid
  • B micrograms taxol mg lipid
  • C ⁇ O micrograms taxol mg lipid
  • D 60 micrograms taxol/mg lipid
  • E 60 micrograms taxol/mg lipid
  • F 70 micrograms taxol/mg lipid
  • G micrograms taxol/mg lipid
  • the following sample was prepared at a lipid concentration of 20 mg/ml: 30 micrograms taxol/mg lipid. No crystals were visible 1, ⁇ , and 30 days after sample hydration. Crystals developed in the sample between day 30 and day 100.
  • the following sample was at a concentration of 187 mg/ml lipid; 21.3 micrograms taxol mg lipid: This sample was extruded to form 100-nm LUVETs. No crystals are visible after l ⁇ O days; some vesicle fusion to form larger structures was noted.
  • the following sample was at a lipid concentration of 200 mg/ml; 2 ⁇ .O micrograms taxol/mg lipid. Crystals were visible after 160 days.
  • the following sample was at a lipid concentration of 187 mg/ml; 21.3 micrograms taxol/mg lipid. No crystals were visible at 150 days.
  • the following sample was at a lipid concentration of 200 mg/ml; 20.0 micrograms taxol/mg lipid. No crystals were visible at 120 days.
  • sample C had visible crystals within two weeks; samples A and B did not exhibit taxol crystal formation by the 170th day of observation.
  • the stability of taxol in EPC liposomes is determined by a combination of taxoblipid ratio as well as the actual taxol (and lipid) concentrations.
  • the taxol:lipid ratio corresponding to a stable preparation is correspondingly less than the taxol:lipid ratio achievable at low taxol and lipid concentrations.
  • an infinitely dilute preparation of taxo EPC will be able to achieve the highest taxol:lipid ratio.
  • the liposomes of this invention where the stability is only a function of the taxol:lipid ratio. Therefore, in these liposomes, a high taxol:lipid ratio may be achieved even in a concentrated preparation.
  • mice Thirty female Balb/c mice weighing 20-22 grams each were injected subcutaneously with 1 x 10" C26 cells at day 0. The mice were randomly separated into control, liposomal taxol and cremophor taxol groups, each containing 10 mice. The groups treated with a taxol formulation were administered intravenously, at days 8, 9, 15, 16, 21 and 22, a taxol dose of 2 ⁇ mg per kg of body weight each day. Weight and tumor size in each mouse were measured twice weekly until the tumors reaches 2 cm ⁇ . Survival was assessed at day 120. The tumor volumes and survival in the groups were then compared. Results of these studies are presented in Table 14 (see below).
  • a Growth inhibition percent reduction in tumor size in comparison to controls, on stated days; D number of long term survivors (120 days) out of total number of mice treated (10) in each group.

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Abstract

L'invention concerne un liposome ayant une bicouche à composants multiples. Cette bicouche contient: un composant de formation de poche qui est un lipide amphipathique avec des chaînes acyle asymétriques, c'est-à-dire des chaînes acyle de longueur inégale; un composant d'épaisseur de la bicouche qui est un lipide amphipathique avec des chaînes acyle symétriques ou asymétriques, à condition que toute asymétrie ou inégalité de longueur soit inférieure ou égale à l'asymétrie des chaînes acyle du composant de formation de poche; et un agent bioactif qui est un composant faisant partie intégrante de la bicouche à composant multiple. Lorsqu'il est nécessaire d'empêcher la séparation de phase des composants de formation de poche et d'épaisseur de la bicouche, la bicouche à composants multiples contiendra également un lipide amphipathique appelé composant de prévention de séparation de phase. L'agent bioactif peut être un agent thérapeutique tel que des agents antibactériens, antifongiques, antiviraux et antiparasites, et vraisemblablement un agent anticancer tel que le taxol, la camptothécine ou la nogalamycine. Des compositions pharmaceutiques contenant le liposome de cette invention peuvent être administrées à des animaux pour le traitement ou la prévention de maladies telles que des infections antimicrobiennes et des cancers.
PCT/US1994/005332 1993-05-19 1994-05-16 Liposome ayant une couche a composants multiples qui contient un agent bioactif en tant que composants integres dans la bicouche WO1994026253A1 (fr)

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EP0785772A1 (fr) * 1994-10-14 1997-07-30 Pharmacia, Inc. Lyophilisat de complexe lipide de camptothecines non hydrosolubles
US5670536A (en) * 1994-04-25 1997-09-23 Rhone-Poulenc Rorer S.A. Pharmaceutical composition based on taxoids
EP1005326A1 (fr) * 1996-10-01 2000-06-07 SkyePharma Inc. Procede de production de liposomes comportant un pourcentage accru de compose encapsule
US6355268B1 (en) 1998-09-16 2002-03-12 Alza Corporation Liposome-entrapped topoisomerase inhibitors
WO2003039437A3 (fr) * 2001-11-08 2003-07-10 Max Delbrueck Centrum Preparation pharmaceutique d'administration orale contenant du paclitaxel encapsule dans des liposomes
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JP2022506612A (ja) * 2018-11-02 2022-01-17 テソリックス ファーマ， エルエルシー リポソーム強化された腹腔内化学療法
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US7074821B1 (en) 1992-12-09 2006-07-11 Aventis Pharma, S.A. Taxoids, their preparation and pharmaceutical composition containing them
EP0721328A4 (fr) * 1993-09-27 1997-09-17 Smithkline Beecham Corp Compositions de camptothecine
EP0721328A1 (fr) * 1993-09-27 1996-07-17 Smithkline Beecham Corporation Compositions de camptothecine
US5670536A (en) * 1994-04-25 1997-09-23 Rhone-Poulenc Rorer S.A. Pharmaceutical composition based on taxoids
EP0785772A1 (fr) * 1994-10-14 1997-07-30 Pharmacia, Inc. Lyophilisat de complexe lipide de camptothecines non hydrosolubles
EP0785772A4 (fr) * 1994-10-14 2005-12-21 Pharmacia & Upjohn Co Llc Lyophilisat de complexe lipide de camptothecines non hydrosolubles
EP1005326A1 (fr) * 1996-10-01 2000-06-07 SkyePharma Inc. Procede de production de liposomes comportant un pourcentage accru de compose encapsule
EP1005326A4 (fr) * 1996-10-01 2006-03-22 Skyepharma Inc Procede de production de liposomes comportant un pourcentage accru de compose encapsule
US9205052B2 (en) 1997-09-18 2015-12-08 Pacira Pharmaceuticals, Inc. Sustained-release liposomal anesthetic compositions
US9192575B2 (en) 1997-09-18 2015-11-24 Pacira Pharmaceuticals, Inc. Sustained-release liposomal anesthetic compositions
US8834921B2 (en) 1997-09-18 2014-09-16 Pacira Pharmaceuticals, Inc. Sustained-release liposomal anesthetic compositions
US7348025B2 (en) 1997-09-23 2008-03-25 Research Development Foundation Small particle liposome aerosols for delivery of anticancer drugs
US7341739B2 (en) 1997-09-23 2008-03-11 Research Development Foundation Small particle liposome aerosols for delivery of anti-cancer drugs
US7244449B2 (en) 1998-09-16 2007-07-17 Alza Corporation Liposome-entrapped topoisomerase inhibitors
US7279179B2 (en) 1998-09-16 2007-10-09 Alza Corporation Liposome-entrapped topoisomerase inhibitors
US6465008B1 (en) 1998-09-16 2002-10-15 Alza Corporation Liposome-entrapped topoisomerase inhibitors
US6355268B1 (en) 1998-09-16 2002-03-12 Alza Corporation Liposome-entrapped topoisomerase inhibitors
US6613352B2 (en) 1999-04-13 2003-09-02 Universite De Montreal Low-rigidity liposomal formulation
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US12178909B1 (en) 2021-01-22 2024-12-31 Pacira Pharmaceuticals, Inc. Manufacturing of bupivacaine multivesicular liposomes
US12285419B2 (en) 2021-10-14 2025-04-29 Pacira Pharmaceuticals, Inc. Bupivacaine multivesicular liposome formulations and uses thereof
US12156940B1 (en) 2024-05-20 2024-12-03 Pacira Pharmaceuticals, Inc. Manufacturing of bupivacaine multivesicular liposomes
US12246092B1 (en) 2024-05-20 2025-03-11 Pacira Pharmaceuticals, Inc. Manufacturing of bupivacaine multivesicular liposomes
US12251468B1 (en) 2024-05-20 2025-03-18 Pacira Pharmaceuticals, Inc. Manufacturing of bupivacaine multivesicular liposomes
US12251472B1 (en) 2024-05-20 2025-03-18 Pacira Pharmaceuticals, Inc. Manufacturing of bupivacaine multivesicular liposomes
US12280149B1 (en) 2024-05-20 2025-04-22 Pacira Pharmaceuticals, Inc. Manufacturing of bupivacaine multivesicular liposomes
US12296047B2 (en) 2024-11-11 2025-05-13 Pacira Pharmaceuticals, Inc. Manufacturing of bupivacaine multivesicular liposomes

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