WO1992002208A1 - Stable doxorubicin/liposome composition - Google Patents
Stable doxorubicin/liposome compositionInfo
- Publication number
- WO1992002208A1 WO1992002208A1 PCT/US1991/005519 US9105519W WO9202208A1 WO 1992002208 A1 WO1992002208 A1 WO 1992002208A1 US 9105519 W US9105519 W US 9105519W WO 9202208 A1 WO9202208 A1 WO 9202208A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- doxorubicin
- composition
- suspension
- mole percent
- lipid
- Prior art date
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- the present invention relates to a stable, lyophilized doxorubicin/liposome composition.
- Gabizon, A., et al.. Cancer Research 43:473C 198 .
- Gabizon, A., et al., Brit J Cancer, 51:681 (j.985) .
- Doxorubicin is a potent chemotherapeutic agent effec ⁇ tive against a broad spectrum of neoplasms (Aubel-Sadron, Young) .
- the use of the drug is limited by serious side effects. Its acute toxicity includes malaise, nausea, vomiting, myelosuppression, and severe alopecia. In addi ⁇ tion, cumulative and irreversible cardiac damage occurs with repeated administration, which seriously limits the use of the drug in protracted treatment (Young) .
- doxorubicin When administered in liposomal form, doxorubicin re ⁇ tains its therapeutic effectiveness against animal tumors, but is significantly less toxic (Forssen, Gabizon, 1985) .
- the drug-protective effect of liposomes is due, at least in part, to a marked alteration in plasma pharmacokinetics and tissue disposition of the injected drug (Gabizon, 1982, 1983; Juliano) .
- liposomal doxo ⁇ rubicin preparations are relatively unstable on storage in liquid form, as evidenced by rapid breakdown of both doxo ⁇ rubicin and liposomal lipids.
- the invention includes a lyophilized doxorubicin/liposome (L-DOX) composition which is characte ⁇ rized by less than 15%, and preferably less than about 10%, doxorubicin breakdown after accelerated storage in lyophi- lized form at 40°C for 4 weeks.
- L-DOX lyophilized doxorubicin/liposome
- the composition is prepared by lyophilizing an aqueous liposome suspension having a pH between 3.0 and 4.4, and preferably between 3.5 and 4.0, and containing (i) liposomes whose dominant lipid compo ⁇ nents are neutral phospholipids, cholesterol, and a negatively charged lipid, (ii) doxorubicin, at a drug:lipid ratio of between 5-10 percent by weight, and a doxorubicin concentration of less than 10 mg/ml, and (iii) a bulking agent.
- Figure 1 is an HPLC chromatogram showing doxorubicin and breakdown products in a lyophilized L-DOX composition of the invention after 2 weeks at 50°C;
- Figure 2 is an HPLC chromatogram showing doxorubicin and breakdown products in the same L-DOX composition, but after storage in liquid suspension form for 2 weeks at 40°C;
- Figure 3 is an HPLC chromatogram showing doxorubicin and breakdown products in a lyophilized L-DOX composition at pH 4.8 in the presence of succinate after 2 weeks at 50°C.
- Liposomes produced by the method of the present inven ⁇ tion are formed from standard vesicle-forming lipids, typi ⁇ cally including neutral phospholipids, such as phosphati- dylcholine (PC) , negatively charged lipids, such as phosphatidylglycerol (PG) , phosphatidylserine (PS) , phosphatidylinositol (PI) , phosphatidic acid (PA) or negatively charged sterol lipids, such as cholesterol sulfate and cholesterol hemisuccinate, and cholesterol or neutral cholesterol analogs.
- PC phosphati- dylcholine
- PG phosphatidylglycerol
- PS phosphatidylserine
- PI phosphatidylinositol
- PA phosphatidic acid
- sterol lipids such as cholesterol sulfate and cholesterol hemisuccinate, and cholesterol or neutral cholesterol analogs.
- the phospholipid components may contain either satura ⁇ ted acyl chains, such as dipalmitoyl acyl chains, or unsaturated acyl chains, such as the mixture of unsaturated chains present in egg PC or egg PG.
- the liposomal lipids may also include a lipophilic free-radical quencher, such as alpha-tocopherol ( ⁇ - ⁇ ) , alpha-tocopherol succinate ( ⁇ -TS) , or butylatedhydroxytolu- ene (BHT) , at a preferred concentration of about 0.5-2 mole percent of total lipids.
- One preferred lipid composition contains 47.1 mg/ml egg PC (EPC) , 19.9 mg/ml egg PG (EPG) , 13.4 mg/ml cholesterol, and 0.91 mg/ml alpha-toco ⁇ pherol acid succinate ( ⁇ TS) , in prelyophilized liquid- suspension form.
- EPC egg PC
- EPG egg PG
- ⁇ TS alpha-toco ⁇ pherol acid succinate
- the L-DOX composition can be prepared by a variety of liposome-forming methods, such as have been reviewed (Szoka, 1980) .
- the lipid components are initially dissolved in a volatile non-polar solvent or solvent system, such as chloroform, or a chlorofluorocarbon solvent.
- a volatile non-polar solvent or solvent system such as chloroform, or a chlorofluorocarbon solvent.
- FreonTM 11 or a solvent system containing FreonTM 11 and 2-5 v/v percent ethanol are well suited for dissolv- ing lipid components for use in the present invention.
- the low boiling point of this solvent permits rapid, and substantially complete solvent removal under the solvent- removal conditions of the invention.
- the solvent poses minimal health and safety-hazard risks and can be readily reclaimed by condensation.
- the lipid solution is dried, under vacuum, in a suit ⁇ able drying vessel.
- drying and rehydration steps are preferably carried out in a planetary mixer partially filled with chemical-inert, preferably hydrophobic, spherical particles which become coated with the lipid during dehydration, as has been detailed in co-owned U.S. patent application for "Large-scale Liposome Production," filed March 30, 1990, Serial No. 502,222.
- One suitable particle is a 5/16 inch Teflon bead, preferably having a roughened surface, such as obtained from Clifton Plastics (Clifton Heights, PA) .
- the quantity of particles added to the vessels is preferably such as to produce an area of the particles of between about 0.02 and 0.04, and preferably about 0.025 and 0.03 cm 2 / / ⁇ t ⁇ ol total lipid added to the vessel.
- Planetary mixers having mixer volumes between about 1- 10 liters and greater are commercially available.
- One pre ⁇ ferred mixer is a 2- or 4- gallon mixer supplied by Charles Ross and Sons (Hauppauge, NY) . Suitable mixing speeds are given in Example 1. Drying with mixing is typically car ⁇ ried out for 3-4 hours for large lipid-solution volumes. After complete solvent removal, the particles in the mixer are coated with an irregular film of lipid, providing a high surface area of dried lipids.
- the lipids are hydrated with an aqueous volume to a final concentration of lipids of between about 100-300 //mol/ml.
- the aqueous medium contains doxorubicin, at a concentration of between about 10-20 mg/ml, and preferably about 15-16 mg/ml doxorubicin in a pyrogen-free aqueous medium.
- the mole concen ⁇ tration ratio of drug to phospholipid is between 1:10 and 1:50, depending on the drug being encapsulated.
- common doxorubicin/phospholipid concentration ratios are on the order of 1:15.
- the hydration medium may include desferioxamine, at a concentration of about 50 /M to 1 mM and preferably about 0.2 mM, and a physiological salt, such as NaCl, at a final concentration which gives a substan- tially isoosmolar solution.
- the hydration medium may include a bulking agent (Section B) , at a weight concentra ⁇ tion between about 1-10%, and preferably about 5%.
- the bulking agent may be added to the re- hydrated liposome suspension just prior to lyophilizati ⁇ n, as described in Section B.
- the pH of the aqueous medium is between about 3.0 and 4.4, and preferably between about 3.5-4.0.
- a pH of 4.4 or below significantly enhances the stability of doxorubicin against breakdown under accelerated storage conditions in lyophilized L-DOX form.
- maintaining the pH above 3.0 reduces the extent of phospholipid hydrolysis, which is promoted by low pH.
- One exemplary aqueous medium, used for producing doxo ⁇ rubicin liposomes is prepared by dissolving desferal in pyrogen-free water, then adding doxorubicin with stirring until the drug is dissolved. To this solution is added the bulking agent and NaCl solution, to a final concentration of components of 200 ⁇ M. desferal, 5 mg/ml doxorubicin, 0.45 % NaCl, and 5% (w/v) bulking agent. The solution is adjusted to pH 3.8 by addition of HC1 (Example 1). Hydration of the lipid-coated particles preferably occurs under mixing conditions similar to those used in preparing the lipid-coated particles.
- the hydration procedure is prefer ⁇ ably carried out in a planetary mixer, under the mixing condition described above.
- the thorough mixing action pro ⁇ vided by the bi-axial motion of the mixing blades breaks up particle-particle aggregates, and thus exposes more lipid- coated surface area for hydration.
- the greater degree of lipid surface ex p osure to the aqueous medium enhances the rate and final yield of liposome formation.
- the liposome suspension may be sized to achieve a selected size distribution of vesicles in a size range less than about 1 micron and preferably between about 0.05 to 0.5 microns, and most preferably between about 0.05 and 0.2 microns.
- the sizing serves to eliminate larger liposomes and to produce a defined size range having optimal pharma- cokinetic properties.
- Extrusion of liposomes through a small-pore polycarbonate membrane is an effective method for reducing liposome sizes down to a well-defined size distribution whose average is in the range between about 0.08 and 1 micron, depending on the pore size of the membrane.
- the suspension is cycled through the membrane several times until the desired liposome size distribution is achieved.
- the liposomes may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in liposome size.
- Free doxorubicin i.e., doxorubicin present in the bulk aqueous phase of the medium, is preferably removed to increase the ratio of liposome-entrapped to free drug.
- the drug removal is designed to reduce the final concentration of free doxorubicin to less than about 20% and preferably, less than about 10% of the total drug present in the composition.
- a sized liposome suspension can be pelleted by high-speed centrifugation, leaving free drug and very small liposomes in the supernatant.
- Another method involves concentrating the suspension by ultrafil- tration, then resuspending the concentrated liposomes in a drug-free replacement medium.
- gel filtra ⁇ tion can be used to separate larger liposome particles from solute (free drug) molecules.
- One preferred procedure for removing free doxorubicin, or analog thereof, utilizes an ion-exchange resin capable of binding drug in free, but not in liposome-entrapped, form.
- the preferred resin is a cation-exchanger, since the drug is positively charged at neutral pH.
- One preferred ion exchange resin is Dowex 50W-X4 50-100 mesh resin.
- the liposomes may be diluted by addition of a physiological buffer, or concentrated, e.g., by ultrafiltration, to a desired drug concentration. Bulking agent, if not already present, is added to the suspension at this stage. The liposomes are then lyophi ⁇ lized for storage, as will now be described.
- degradative changes in a liposome or cell suspension can be arrested, for long-term storage, by freezing or lyophilization, i.e., freezing followed by water removal by sublimation under vacuum.
- cryoprotectants such as glycerol, DMSO, polyethylene glycol, polypropylene glycol, 1,3,-butanediol, 2,3, butanediol, 1,3-propanediol, a variety of mono- and disaccharides, such as lactose, sucrose, and trehalose, and polysaccharides, such as dextran and hydroxyethyl starch (HES) which appear to interrupt or minimize ice crystal formation, when present both inside and outside the liposomes.
- cryoprotectants such as glycerol, DMSO, polyethylene glycol, polypropylene glycol, 1,3,-butanediol, 2,3, butanediol, 1,3-propanediol, a variety of mono- and disaccharides, such as lactose, sucrose, and trehalose, and polysaccharides, such as dextran and hydroxyethyl starch (HES) which appear to
- liposomes tend to show a pro ⁇ gressive size growth with freeze/thaw cycles.
- the liposome size growth may be due to a solute-exclusion effect in which the formation of ice crystals concentrates solutes and liposomes into microenvironments of very high lipid and salt concentrations, in effect, forcing liposome together.
- This effect can be reduced by including in the bulk (extra- liposomal) phase of a liposome suspension, a bulking agent which is effective to interrupt formation of large ice crystals.
- the bulking agent should also have the property of forming a solid, somewhat porous, non-crystalline matrix on drying, to allow escape of water by sublimation from the frozen sample, and to reduce additional solvent exclusion effects due to crystal formation in the bulking agent.
- a solid bulking agent excludes a variety of small viscous-liquid cryoprotectants such as DMSO, glycerol, ethylene glycol, and propylene glycol as the bulking agent in the present invention.
- Non-crystalline carbohydrates such as trehalose and lactose
- polysaccharides such as dextrans and hydroxyethylcellulose.
- cryoprotectants or bulking agents for lipo ⁇ somes, microsomal membranes or blood cells has been reported (e.g., Crowe; Anchordoguy, 1988; Strauss; Madden).
- Another general class of bulking agents includes a ino acids, and amino acid analogs, such as 2-aminobutyric acid,
- a third general class of bulking agents includes a variety of non-sugar glycolytic pathway compounds, such as sodium or potassium salts of tartrate, oxaloacetate, fuma- rate, malate, ketoglutarate, and pyruvate.
- the bulking agent is preferably a mixture of two or more of these com ⁇ pounds, to minimize crystal formation effects on freezing and dehydration.
- suc- cinate is not a suitable bulking agent since it enhances doxorubicin breakdown on storage in lyophilized form, nor are di- or tri-dicarboxylic acid compounds, such as ci ⁇ trate, since these may precipitate with doxorubicin, which is positively charged.
- a fourth class of bulking agents includes non-saccharide polymeric compounds, such as higher molecular weight polyethylene glycol (PEG) , which are (a) solids at room temperature, (b) readily soluble in water, and (b) pharmaceutically acceptable for parenteral adminis ⁇ tration.
- PEG polyethylene glycol
- PEG polymers with molecular weights above about 1,500-2,000 daltons are contemplated.
- the polymer is included in the bulk phase of the suspension, at a weight concentration between about 1-10 percent.
- the liposomes themselves are derivatized with a PEG or other polyalkyl oxide, for pur ⁇ poses of enhanced lifetime in the bloodstream when admini ⁇ stered intravenously, as described in co-owned U.S. patent application for "Liposomes with Enhanced Circulation Times," filed October 10, 1989, Serial No. 425,224.
- the surface-derivatized molecules act to prevent ice cry ⁇ stal growth in the region of the liposomes.
- the liposome suspension is preferably concentrated to insure a relative ⁇ ly high polymer concentration in the bulk phase.
- Ts liposome suspension may also contain a solution-phase bulking agent selected from one of the classes above, in addition to the liposome-bound agent.
- the bulking agents may be included in the aqueous hydration medium used in forming the liposomes, yielding a suspension with an equal concentration of agent in the bulk and encapsulated liposomal aqueous phases.
- the agent can be added to the final sized liposome suspen ⁇ sion, wherein the agent is present only in the bulk phase of the suspension.
- the final concen- tration of the bulking agent in the suspension is between about 1-10 weight percent and preferably about 4-6 weight percent.
- liposome fusion on freezing and dehydration is minimized by forming the initial liposome dispersion in a low-ionic-strength medium in which the doxorubicin/liposomes have a partially ordered gel structure, by virtue of the surface charge repulsion effects.
- This type of liposome gel has been described in co-owned U.S. application for "Liposome Gel Composition and Method," filed May 22, 1989, Serial No.356,262.
- the gel formulation requires an ionic strength comparable to that of NaCl, at a concentration of less than about 20 mM NaCl.
- the medium may contain non-ionic species, such as mono or disaccharide protective agents.
- the liposome dispersion is frozen and lyophilized as described below.
- the lyophilized material can be recon ⁇ stituted, for parenteral injection, by addition of a rehydration medium containing physiological salts or other ionic species.
- the suspension is first cooled to 4-5°C, then frozen to -45°C and main- tained at this temperature for 12 hours in a conventional lyophilizer.
- the lyophilizer chamber is pumped down to about 200 ⁇ pressure, after which the chamber temperature is raised, at about 10°C/hr, to -20°C.
- the chamber is maintained at this temperature until the lowest reading product thermocouple is within 3°C of shelf temperature.
- the chamber temperature is now allowed to rise, again at about 10°C/hour, to room temperature, and the material is held under vacuum at this temperature for an additional 15 hours.
- the chamber is backfilled with nitrogen to a pressure of about 2 inches Hg, and the sample vessels stoppered for storage.
- One suitable lyophilizer is a Edwards Lyoflex 08 lyophilizer supplied commercially from Edwards High Vacuum, Inc. (New York) .
- the stability of the lyophilized preparation can be examined by accelerated studies conducted at elevated temperatures, according to known principles.
- the storage conditions which were used in the studies described below were 40°C for 4 weeks, and 50°C for two weeks.
- the lyophilized sample in a stoppered vessel was placed in an incubator at the selected temperature and analyzed after the two- or four-week incubation period for doxorubicin breakdown, with the results discussed in Section C.
- Doxorubicin Stability on Storage Table I below shows composition and pH variables in several lyophilized L-DOX preparations which were studied under accelerated storage conditions. Each of the 20 pre ⁇ parations were prepared according to the methods outlined above, and detailed for Preparation No. 11 in Example 1.
- each preparation was reconstituted by addition of dis ⁇ tilled water to a final doxorubicin concentration of about 5 mg/ml.
- An aliquot of the material was diluted with mobile phase to a final dilution of 1:50 at a final liquid concentra- tion of about 2.5 /zmol/ml (1.75 mg/ml).
- the lipid and drug components of the L-DOX suspension are in solution.
- a typical example of this material, chroma- tographed by HPLC is detailed in Example 3.
- Figure l shows an HPLC profile of doxorubicin and doxoru ⁇ bicin breakdown products observed in a lyophilized preparation corresponding to composition No. 11 in Table 1, after acceler ⁇ ated storage for 2 weeks at 50°C.
- the peaks in the chromato ⁇ gram represent absorbance at the doxorubicin absorbance peak of at 480 nm.
- Three distinct peaks, with RT (retention time) values of 9.92, 12.75, and 14.4 (doxorubicin) were observed, with the measured relative peak areas shown.
- the vertical markers on either side of each peak indicate the portion of the curve that was integrated.
- the doxorubicin peak in the Figure 1 HPLC profile consti ⁇ tutes all but about 2% of the detected products, leaving approximately 7% of the breakdown products unaccounted for.
- One possible source of this discrepancy is that one or more of the breakdown products have lower absorbance coefficients at 480 nm, so that these products are either not seen or underes- timated in amount.
- Another possible source of discrepancy is that the breakdown products do not have well-defined peaks, and therefore are not included in the total peak area calcula ⁇ tions.
- it is possible that some of the breakdown products are not eluted from the column under the chromato- graphy conditions employed.
- Figure 2 shows an HPLC profile of the same L-DOX formula ⁇ tion as in Figure 1, but after accelerated storage at 40°C for two weeks in liquid suspension form.
- the relative peak areas of the four peaks are given in Table 3 below.
- the total amount of doxorubicin in the main peak in the figure repre ⁇ sents about 67% of the doxorubicin present prior to storage.
- storage in liquid suspension form substantially increa ⁇ ses the breakdown of doxorubicin in the a low-pH L-DOX formu- lation.
- Figure 3 shows an HPLC .profile of a lyophilized L-DOX formulation corresponding to composition No. 2 in Table l, after accelerated storage at 50°C for 2 weeks.
- the doxorubicin peak accounting for about 85% of the total peak area.
- the total amount of doxorubicin in the main peak in the figure represents about 74% of the doxo ⁇ rubicin present prior to storage. Peak#
- [DOX] was measured using HPLC. Triplicate vials were analyzed at each time point, with the exception that 4 weeks, 40°C samples 2-5, 19 and 20 were done in duplicate.
- Example #13 vs. sample #11 Adding lactate resulted in significantly more DOX degradation (sample #13 vs. sample #11) , although lactate had a less detrimental effect than succinate in this regard (sample #13 vs. sample #14) .
- Doxorubicin stability was compared in formations con- taining no Desferal (sample #7) , 200 ⁇ _ (sample #1) or 1 mM Desferal (sample #8) .
- the present invention is useful as an improved storagable form of doxorubicin/liposomes, for use in the treatment of a variety of tumor types. Reduced side effects have been observed in Phase I and Phase II clinical trials with doxoru ⁇ bicin liposome formulations prepared by reconstituting lyophilized preparations.
- the present L-DOX invention provides the additional advantage of long-term stability on storage, without significant loss of active drug or generation of undesired breakdown products.
- the preparation is easily prepared by a method that is suitable for large-scale manufac ⁇ ture.
- anthracene glycoside anti-neoplastic drugs such as daunomy- cin, carcinomycin, N-acetyladriamycin, N-acetydaunomycin, rubidazone, 5-imidodaunomycin, and epirubicin.
- Example 1 Preparation of Lyophilized L-DOX Composition (Preparation 11. from Table 1) A 300 ml chloroform solution containing 20.1 g EPC (Lipoid) , 8.5 g EPG (Asahi) , 5.7 g CH (Croda) , and 0.4 g ⁇ - tocopherol succinate (Henkel) was added to a 2-liter round- bottomed flask containing 180 g of 3 mm diameter glass beads and the whole dried in vacuo using a rotary evaporator. The lipid film was subsequently exposed to a vacuum of 50 Torr overnight to complete drying.
- the lipids were hydrated to a final total lipid concen ⁇ tration of about 240 ⁇ mole/ml by addition of a doxorubicin- containing solution and mechanically agitating the mixture for 2 hours at room temperature.
- This solution contained about 15 mg/ml DOX, 5% (w/v) lactose monohydrate, 0.4% (w/v) sodium chloride, 200 ⁇ M desferal in water and was prepared by dissol ⁇ ving the doxorubicin in water and adding the other excipients when complete dissolution was achieved.
- the pH of the mixture was adjusted to 3.8 with HC1.
- the resultant liposome suspen- sion was sized by passage five times through 0.4 ⁇ m and five times through 0.2 ⁇ m pore-size polycarbonate membranes. Unincorporated DOX was removed by passing the sized liposome suspension over a Dowex 50W-X4 cation exchange resin. Finally, the suspension was diluted to a DOX concentration of about 5 mg/ml prior to lyophilization, using a 5% lactose, 0.4% sodium chloride, 200 ⁇ M desferal solution, pH 3.8. The characteristics of this pre-lyophilization solution are given in Table 6. TABLE 6
- Doxorubicin HCl (mg/ml) 4.91 % encapsulation 96 Liposome diameter (nm) 290
- a lipid solution containing 1500 ml Freon 11TM, 151.1 g EPC (Lipoid) , 63.9 g EPG (Asahi) , 42.9 g cholesterol (Croda) , and 0.86 g of butylated hydroxytoluene (Penta) was added to a 2-gallon double planetary mixer (Ross model 130 LDM) which contained about 4,100 PTFE Teflon beads (Clifton Plastics) having a mean diameter of 1/4 inch.
- the beads had been washed in an aqueous solution containing a detergent (Alconox) to remove any oily residues from the surfaces of the beads, rinsed with ethanol, and dried, before placement into the mixer.
- the mixer was sealed and the pressure inside the mixer was reduced while the solution and beads were mixed at an orbital stirring rate of 20 rpm and an axial stirring rate of 26 rpm for about two hours, to transform the lipids to a solid state attached to the beads.
- a vacuum was maintained for an additional 3 hours without mixing to achieve additional sol ⁇ vent removal.
- an aqueous solution of doxorubicin HCl to a final lipid concentration of about 262 / /mol/ml was added to the lipid-coated particles in the planetary mixer.
- the drug solution was prepared to contain 0.132 mg/ml deferox- amine mesylate in pyrogen-free distilled water.
- Doxorubicin hydrochloride (Farmitalia Carlo Erba) was then added with stirring to a final concentration of about 12.5 mg/ml. After complete dissolution of the drug, lactose was added to a final concentration of 5 weight percent and NaCl 0.4 weight percent. The pH of the solution was then adjusted to 3.7 with HCl solution.
- lipid Hydration of the lipid was carried out at a temperature of 25° C. for 2 hours, with mixer stirring at about 25 rpm.
- the resulting liposome suspension was sized by passes through 0.4 and 0.2 micron polycarbonate filters.
- Free doxorubicin was reduced by treating the sized-liposome suspension with a Biorad AG 50w-X4 50-100 ion exchange resin.
- the preparation was then diluted with a solution containing deferoxamine mesylate, lactose, sodium chloride and hydrochloric acid in pyrogen-free distilled water.
- the preparation had the following properties: pH of approxi ⁇ mately 3.8, mean particle size of approximately 232 nm; approximately 96% of the total drug was liposome associated.
- Lyophilized L-DOX was reconstituted with water to a final doxorubicin concentration of about 5 mg/ml. A 0.25 ml aliquot of the rehydrated suspension was diluted to 5 ml with mobile phase.
- the separation of the doxorubicin and- its degradation products was carried out on a Waters HPLC using a Whatman Partisil ODS-3 column, 250 x 4.6 mm held at ambient tempera ⁇ ture.
- the mobile phase was 43% aqueous buffer in methanol.
- the aqueous buffer was 95 mM ammonium phosphate/ 5 mM tri- ethylamine, pH 4.0.
- 15 ⁇ l of sample was injected in the system at a 1 ml/min flow rate. Effluent was monitored at 480 nm.
Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP3514139A JPH06502623A (en) | 1990-08-08 | 1991-08-02 | Stable doxorubicin/liposomal compositions |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US56407090A | 1990-08-08 | 1990-08-08 | |
US564,070 | 1990-08-08 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1992002208A1 true WO1992002208A1 (en) | 1992-02-20 |
Family
ID=24253045
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1991/005519 WO1992002208A1 (en) | 1990-08-08 | 1991-08-02 | Stable doxorubicin/liposome composition |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0543871A1 (en) |
JP (1) | JPH06502623A (en) |
AU (1) | AU8329391A (en) |
CA (1) | CA2088975A1 (en) |
WO (1) | WO1992002208A1 (en) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
BE1006379A3 (en) * | 1992-11-26 | 1994-08-09 | Raymond Gilles | Method for preserving living cells, groups of cells and/or derivatives ofsaid cells by means of freeze-drying |
EP0655239A1 (en) * | 1993-11-25 | 1995-05-31 | Lipotec, S.A. | Liposomes encapsulating doxorubicine |
US7153490B2 (en) | 2000-10-10 | 2006-12-26 | Lipotec, Sa | Liposomes encapsulating anticancer drugs and use thereof in the treatment of malignant tumors |
WO2012117385A2 (en) | 2011-03-03 | 2012-09-07 | WROCŁAWSKIE CENTRUM BADAŃ EIT+ Sp z o.o. | Liposome formulation comprising an anti-tumour active substance, method for its preparation and pharmaceutical compositions comprising it |
US9526761B2 (en) | 2006-10-06 | 2016-12-27 | Scil Technology Gmbh | Dried reconstituted vesicle formation for pharmaceutical application |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU700833B2 (en) * | 1991-12-02 | 1999-01-14 | Merial | Medicinal products and pure preparations of melarsomine dihydrochloride, process for obtaining them and intermediate products obtained |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1988009168A1 (en) * | 1987-05-18 | 1988-12-01 | Liposome Technology, Inc. | Liposome/doxorubicin composition and method |
-
1991
- 1991-08-02 WO PCT/US1991/005519 patent/WO1992002208A1/en not_active Application Discontinuation
- 1991-08-02 CA CA002088975A patent/CA2088975A1/en not_active Abandoned
- 1991-08-02 JP JP3514139A patent/JPH06502623A/en active Pending
- 1991-08-02 EP EP91914508A patent/EP0543871A1/en not_active Ceased
- 1991-08-02 AU AU83293/91A patent/AU8329391A/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1988009168A1 (en) * | 1987-05-18 | 1988-12-01 | Liposome Technology, Inc. | Liposome/doxorubicin composition and method |
Non-Patent Citations (1)
Title |
---|
JOURNAL OF CONTROLLED RELEASE. vol. 9, no. 3, August 1989, AMSTERDAM NL pages 215 - 229; G.STORM ET AL.: 'LIPOSOME ENCAPSULATION OF DOXORUBICIN' * |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
BE1006379A3 (en) * | 1992-11-26 | 1994-08-09 | Raymond Gilles | Method for preserving living cells, groups of cells and/or derivatives ofsaid cells by means of freeze-drying |
EP0655239A1 (en) * | 1993-11-25 | 1995-05-31 | Lipotec, S.A. | Liposomes encapsulating doxorubicine |
WO1995014459A1 (en) * | 1993-11-25 | 1995-06-01 | Lipotec, S.A. | Liposomes for the encapsulation of doxorubicine |
US5605703A (en) * | 1993-11-25 | 1997-02-25 | Lipotec, S.A. | Liposomes encapsulating doxorubicin |
US7153490B2 (en) | 2000-10-10 | 2006-12-26 | Lipotec, Sa | Liposomes encapsulating anticancer drugs and use thereof in the treatment of malignant tumors |
US9526761B2 (en) | 2006-10-06 | 2016-12-27 | Scil Technology Gmbh | Dried reconstituted vesicle formation for pharmaceutical application |
WO2012117385A2 (en) | 2011-03-03 | 2012-09-07 | WROCŁAWSKIE CENTRUM BADAŃ EIT+ Sp z o.o. | Liposome formulation comprising an anti-tumour active substance, method for its preparation and pharmaceutical compositions comprising it |
Also Published As
Publication number | Publication date |
---|---|
EP0543871A1 (en) | 1993-06-02 |
JPH06502623A (en) | 1994-03-24 |
CA2088975A1 (en) | 1992-02-09 |
AU8329391A (en) | 1992-03-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US4927571A (en) | Preparation of injectable doxorubicin/liposome suspension | |
US5100591A (en) | Process for preparing lipid microparticles | |
US4766046A (en) | Stabilized liposome/amphotericin composition and method | |
JP2958774B2 (en) | Improved preparation of amphotericin B liposomes | |
US4857319A (en) | Method for preserving liposomes | |
US5194266A (en) | Amphotericin B/cholesterol sulfate composition and method | |
EP0437479B1 (en) | Method of making liposomes with improved stability during drying | |
EP3142656B1 (en) | Multivesicular liposome formulations of tranexamic acid | |
US5000959A (en) | Liposome composition and production thereof | |
CA2505520C (en) | Protein-stabilized liposomal formulations of pharmaceutical agents | |
US4877561A (en) | Method of producing liposome | |
CA2221341C (en) | Submicron liposome suspensions obtained from preliposome lyophilizates | |
JPH01502270A (en) | Amphotericin B/cholesterol sulfate composition and method for producing the same | |
US5032582A (en) | Method for treating fungal infections with amphotericin B/cholesterol sulfate composition | |
US6060080A (en) | Liposomal products | |
US5080904A (en) | Liposome composition and its production | |
CN104000783A (en) | Cefquinome liposome | |
CA2705031A1 (en) | Gel-stabilized liposome compositions, methods for their preparation and uses thereof | |
US20060030578A1 (en) | Pharmaceutically active lipid based formulation of irinotecan | |
US5180713A (en) | Stabilized liposome/amphotercin B composition and method | |
WO1992002208A1 (en) | Stable doxorubicin/liposome composition | |
US20050129750A1 (en) | Process for producing liposome suspension and product containing liposome suspension produced thereby | |
US20040175417A1 (en) | Amphotericin B liposome preparation | |
JPH0114A (en) | Liposome formulation and its manufacturing method | |
WO1993023016A1 (en) | Liposomal formulations containing rifamycins |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AU CA FI JP KR NO |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FR GB GR IT LU NL SE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2088975 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1991914508 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 1991914508 Country of ref document: EP |
|
WWR | Wipo information: refused in national office |
Ref document number: 1991914508 Country of ref document: EP |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1991914508 Country of ref document: EP |